JPWO2007141926A1 - Prolonged amyloid β production inhibitor - Google Patents

Abstract

The present invention is a compound represented by the formula (I): [Chemical Formula 1] (wherein R1 and R2 are each independently an optionally substituted aryloxy or an optionally substituted arylthio; R3 is a hydrogen atom or a lower alkyl group) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the solvate thereof as an active ingredient, in particular, the above-mentioned compound has an inhibitory effect on extended amyloid β production The present invention relates to a pharmaceutical composition that can be effectively used for the prevention or treatment of diseases involving extended amyloid β, particularly Alzheimer's disease. The present invention also relates to a reagent for screening for an extended amyloid β production inhibitor.

Description

  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 composition that can be effectively used for the prevention or treatment of diseases involving extended amyloid β, particularly Alzheimer's disease, based on the effect of inhibiting the production of extended amyloid β. Furthermore, the present invention relates to the use of a compound having an inhibitory action on extended amyloid β production other than the above-mentioned pharmaceuticals (for example, screening reagents and test agents).

  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.

  Alzheimer's disease is representative of a progressive neurodegenerative disease with significant neuronal loss. Some Alzheimer's disease (AD) is familial AD (FAD) with a genetic mutation, but the majority is sporadic AD (SAD) without a family history. Both are pathological findings characterized by senile plaques composed of amyloid β peptide (hereinafter also simply referred to as “Aβ”) deposited in the brain and neurofibrillary tangles caused by abnormal phosphorylation of tau protein. For this reason, it is thought that the increase in Aβ is deeply related to the pathological condition of Alzheimer's disease (amyloid cascade hypothesis) (Non-patent Document 1).

  Aβ, which is a major determinant of Alzheimer's disease, is a metabolite of β-amyloid precursor protein (βAPP), mainly 40, 42 or 43 amino acid residues (“Aβ40”, “Aβ42” and (Also referred to as “Aβ43”). Both are produced by βAPP undergoing enzymatic cleavage by the action of β-secretase and γ-secretase. The N-terminus of Aβ is formed by β-secretase cleavage between methionine residue 596 and aspartate residue 597 of βAPP (see, for example, Patent Document 1 or 2), and the C-terminus of Aβ is β-secretase cleaved. The βAPP fragment is further formed by cleaving at position 38, 40, or 43 with γ-secretase (for a review of γ-secretase, see, for example, Patent Document 3).

  In normal individuals, there are two major Aβs, Aβ40 and Aβ42, but the dominant one is Aβ40. However, in patients with familial Alzheimer's disease, Aβ42 increased significantly compared to normal, and in the neuritic plaques observed in the lesion area of Alzheimer's disease, Aβ42 was present at a very high rate compared to Aβ40. . It has also been reported that Aβ42 accumulates early and preferentially in soft tissue plaques, whereas Aβ40 is unrelated to the early deposition of amyloid plaques. For this reason, it is considered that Aβ42 plays a major role in amyloid plaque deposition in familial Alzheimer's disease patients (see, for example, Non-Patent Documents 2 to 5).

  As described above, Aβ, particularly extended Aβ typified by Aβ42, is deeply involved in the pathogenesis and development of Alzheimer's disease. Therefore, a substance that suppresses the production of extended Aβ is a prophylactic or therapeutic agent for Alzheimer's disease. It can be considered.

  For example, U.S. Patent No. 6,057,031 describes that certain non-steroidal anti-inflammatory drugs (NSAIDS) reduce the production and / or level of Aβ42 that results from cleavage of βAPP in cell culture. It has been suggested that drugs are effective in preventing, delaying or ameliorating the progression of Alzheimer's disease. However, in order to significantly reduce Aβ42 levels using these drugs, a large amount of drug is required, which causes serious gastrointestinal side effects such as hemorrhagic ulcers (Non-Patent Document 6).

Therefore, a substance having an action of suppressing the production of extended Aβ by a mechanism different from that of nonsteroidal anti-inflammatory drugs (NSAIDS) is required.
Hardy, JA & Selkoe, DJ, Science, 19, 353-356 Roher et al., 1993, Proc. Natl. Acad. Sci. USA 90: 10836 Iwatasubo, T. et al., 1994 Neuron 13:45 Yamaguchi et al., 1995, Amyloid Int. J. Clin. Invest. 2: 7-16 Mann et al., 1996 Am. J. Pathol. 148: 1257 Langman et al., 1994, Lancet 343: 1075-1078 U.S. Pat.No. 6,440,698 U.S. Pat.No. 5,744,346 US patent application, 20020025540 U.S. Patent Application No. 20020128319A1

  The present invention relates to a compound having an action of suppressing the production of extended Aβ causing Alzheimer's disease, an extended Aβ production inhibitor, and a disease caused by extended Aβ (extended Aβ-related disease), particularly Alzheimer's disease. An object is to provide a new use as a preventive or therapeutic agent for. More specifically, an object of the present invention is to provide a pharmaceutical composition effective as an extended Aβ production inhibitor or an extended Aβ-related disease preventive / therapeutic agent, particularly as a preventive or therapeutic agent for Alzheimer's disease.

  The present invention also provides an extended Aβ production inhibitor, particularly a prophylactic or therapeutic agent for the above-mentioned extended Aβ-related diseases, among test substances, using a compound having an action of suppressing the production of extended Aβ as a standard reagent. It is an object of the present invention to provide a method (screening method) for selecting candidate compounds useful as active ingredients.

  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 excellently extended. A compound having an inhibitory effect on type Aβ production, and a prophylactic or therapeutic agent for these compounds as prolonged Aβ production inhibitors and diseases caused by production of prolonged Aβ (prolonged Aβ-related diseases), In particular, we were convinced that it can be effectively used as an agent for preventing or treating Alzheimer's disease. Furthermore, the present inventors have confirmed that such a compound has no anti-inflammatory action, and the compound suppresses prolonged Aβ production by a mechanism different from NSAIDS, and thus, bleeding ulcers which are a concern with NSAIDS, etc. It was convinced that it was useful as an excellent preventive or therapeutic agent for extended Aβ-related diseases without worrying about side effects. 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 inhibitory action on extended Aβ production, so that extended Aβ can be selected from test substances. It was judged that it was possible to select a substance (prolonged Aβ production inhibitory substance) that had a production inhibitory action and could be an active ingredient of a prophylactic or therapeutic agent for extended Aβ-related diseases.

The present invention has been completed based on such findings and has the following aspects:
Item 1. Formula (I):

(In the formula, R ¹ and R ² are each independently an optionally substituted aryloxy or an optionally substituted arylthio, and R ³ is a hydrogen atom or a lower alkyl group. )
Or a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient.

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

  Item 3. In the general formula (I), the optionally substituted aryloxy or optionally substituted arylthio substituent is a nitro group, a carboxy group, a lower alkoxycarbonyl group, an acyl group, a sulfo group. Item 3. The pharmaceutical composition according to any one of Items 1 and 2, which is any one selected from the group consisting of: a sulfino group, a sulfeno group, a lower alkylsulfonyl group, and a cyano group.

Item 4. Item 4. The pharmaceutical composition according to any one of Items 1 to 3, wherein, in General Formula (I), R ¹ and R ² are nitrophenoxy groups, and R ³ is a hydrogen atom.

  Item 5. 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.

  Item 6. Item 6. The pharmaceutical composition according to Item 5, wherein the prolonged amyloid β-related disease is Alzheimer's disease.

  Item 7. Item 7. A method for treating or preventing an extended amyloid β-related disease, comprising administering the pharmaceutical composition according to Item 5 or 6 to a subject suffering from or likely to suffer from an extended amyloid β-related disease.

  Item 8. Formula (I):

(In the formula, R ¹ and R ² are each independently an optionally substituted aryloxy or an optionally substituted arylthio, and R ³ is a hydrogen atom or a lower 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.

  Item 9. 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.

Item 10. Investigate the amyloid β production inhibitory effect on the test substance,
Formula (I):

(In the formula, R ¹ and R ² are each independently an optionally substituted aryloxy or an optionally substituted arylthio, and R ³ is a hydrogen atom or a lower alkyl group. )
An extended amyloid comprising a step of selecting a test substance having an inhibitory action higher than the extended Aβ production inhibitory action of the compound or a salt thereof, or a solvate thereof, as an extended amyloid β production inhibitory substance A method for screening a β production inhibitor.

Item 11. Item 10. The screening method for an extended amyloid β production inhibitor described in Item 10 having the following steps:
(1) contacting a test substance with a cell capable of expressing and producing β-amyloid precursor protein (β-APP);
(2) measuring the amount of Aβ42 produced from the cells,
(3) Steps (1) and (2) above, wherein the production amount of Aβ42 is changed using a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, or a solvate thereof instead of the test substance. The test substance showing an Aβ42 production amount lower than the positive control value as compared with the control Aβ42 production amount (positive control value) obtained by performing the above is selected as a candidate substance for an extended amyloid β production inhibitory substance Process.

Item 12. The screening method according to Item 11, further comprising the following steps:
(4) Anti-inflammatory action is examined for the candidate substance for the extended amyloid β production inhibitor selected in step (3), and a substance having no or little anti-inflammatory action is produced as a non-anti-inflammatory extended amyloid β product. A process of selecting candidate substances for inhibitors.

  Item 13. 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.

  Item 14. Formula (I):

(In the formula, R ¹ and R ² are each independently an optionally substituted aryloxy or an optionally substituted arylthio, and R ³ is a hydrogen atom or a lower alkyl group. )
Or a salt thereof, or a solvate thereof, is applied to β-amyloid precursor protein or β-amyloid precursor protein-expressing cells.

  3,5-bis (4-nitrophenoxy) benzoic acid and its analog (I) provided by the present invention have an excellent extended Aβ production inhibitory action. Therefore, the compound (I) and a composition containing the compound (I) as an active ingredient can be effectively used as an extended Aβ production inhibitor, particularly an Aβ42 production inhibitor, for various purposes such as a reagent, a test agent or a medicine. . Further, as described above, it is known that the onset and development of Alzheimer's disease is deeply related to the production and deposition of Aβ in the brain, particularly in the central nerve cells, particularly extended Aβ typified by Aβ42. It has been. 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 extended Aβ production inhibitory action as described above. It is useful as an agent for preventing or improving the onset and progression of Alzheimer's disease. Furthermore, unlike NSAIDS, these compounds (I) are considered to be useful pharmaceutical compositions with few side effects because they are effective in small amounts and do not have anti-inflammatory effects.

(1) Pharmaceutical composition and use thereof The pharmaceutical composition provided by the present invention comprises the following formula (I):

(In the formula, R ¹ and R ² are each independently an optionally substituted aryloxy or an optionally substituted arylthio, and R ³ is a hydrogen atom or a lower alkyl group. )
Or a pharmaceutically acceptable salt thereof as an active ingredient.

In the formula (I), in the aryloxy or arylthio defined by R ¹ and R ² , examples of the aryl group include a phenyl group, a naphthyl group, an anthryl group, and a phenanthryl group. A phenyl group is preferred. R ¹ and R ² may be either aryloxy or arylthio. Aryloxy is preferable, and a phenoxy group is particularly preferable.

In addition, examples of the substituent defined by “aryloxy optionally having substituent” and “arylthio optionally having substituent” defined by R ¹ and R ² include a nitro group, a carboxy group, Lower alkoxycarbonyl group, acyl group, sulfo group, sulfino group, sulfeno group, lower alkylsulfonyl group, cyano group, halogen atom, hydroxy group, lower alkoxy group, acyloxy group, amino group, acylamino group, lower alkylamino group, lower Examples thereof include an alkylthio group, a carbamoyl group, a lower alkylcarbamoyl group, and aryl. Preferred are a nitro group, a carboxy group, a lower alkoxycarbonyl group, an acyl group, a sulfo group, a sulfino group, a sulfeno group, a lower alkylsulfonyl group and a cyano group, and more preferred is a nitro group. The position of the substituent is not particularly limited. For example, when the aryl group is a phenyl group, it is preferably a meta or para position, more preferably a para position with respect to an 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 group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, isopentyl group, neopentyl group, hexyl group, isohexyl group, An n-heptyl group, an isoheptyl group, an n-octyl group, an isooctyl group, an n-nonyl group, an n-decyl group, and the like can be given. In addition, the lower alkyl group moiety in the “lower alkylsulfonyl group”, “lower alkoxy group”, “lower alkylamino group”, “lower alkylthio group”, “lower alkylcarbamoyl group” and “lower alkoxycarbonyl group” is A group similar to the “lower alkyl group” can be exemplified. A methyl group, an ethyl group, and an n-propyl group are preferable.

  The “acyl group” mentioned as a substituent includes an aliphatic acyl group having 1 to 10 carbon atoms, a carbocyclic carbonyl group, and a heterocyclic carbonyl group. Specifically, formyl group, acetyl group, propionyl group, butyryl group, isobutyryl group, valeryl group, pivaloyl group, hexanoyl group, acryloyl group, propioyl group, methacryloyl group, crotonoyl group, benzoyl group, cyclohexanecarbonyl group, pyridinecarbonyl Group, furancarbonyl group, thiophenecarbonyl group, benzothiazolecarbonyl group, pyrazinecarbonyl group, piperidinecarbonyl and the like. Examples of the acyl group moiety 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, an iodine atom and a bromine atom.

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 means a linear or branched alkyl group having 1 to 10 carbon atoms as described above. Preferably it is a C1-C6 linear or branched alkyl group, More preferably, it is a C1-C3 alkyl group, specifically, a methyl group, an ethyl group, and n-propyl group.

In the formula (I), R ¹ and R ² are preferably both p-nitrophenoxy 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 (I ′) or lower thereof Mention may be made of alkyl esters.
Formula (I ′):

(In the formula, R ³ is a hydrogen atom or a lower alkyl group.)
The compound (I ′) is a known compound. For example, Gang Yang et al. (Macromolecules 1998, 31, 5964; Macromolecules 1999, 32, 2215-2220) and International Publication WO 01/02466 A1 (Example 4) ).

  The following method can be mentioned as an example of the manufacturing method of compound (I).

(In the formula, Z represents an oxygen atom or a sulfur atom, Ar represents an aryl which may have a substituent, R ^{3 ′} represents a lower alkyl group, and Hal represents a halogen atom. )
Specifically, the compound (Ia) in which R ^{3 ′} is a lower alkyl group is obtained by replacing 3,5-dihydroxybenzoic acid or 3,5-dimercaptobenzoic acid lower alkyl ester (III) with copper or a copper salt. It can be prepared by reacting with an aryl (II) halide in the presence or absence 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 or potassium hydroxide. And sodium carbonate and potassium carbonate. The compound (Ib) in which R ^{3 ′} is substituted with hydrogen is obtained by reacting the lower alkyl ester (Ia) obtained above with an acid such as sulfuric acid or hydrochloric acid in the presence of a base such as lithium hydroxide or sodium hydroxide. It can be produced by hydrolysis in the presence.

  These compounds may be either free or salt forms. 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 ammonium. Examples of the organic base include primary amines such as ethanolamine; secondary amines such as diethylamine, diethanolamine, dicyclohexylamine, N, N′-dibenzylethylenediamine; trimethylamine, triethylamine, pyridine, picoline, triethanolamine and the like. And tertiary amines.

  Further, the compound (I) targeted by the present invention may be a compound obtained by solvating a free substance or a salt thereof. Here, solvates include hydrates.

  In the present specification, the term “extended Aβ” means that the position opened by γ-secretase is from 1 amino residue to 8 amino residues, preferably from 1 amino residue to 3 amino residues, from the C-terminal side than Aβ40. Means amyloid β which has shifted to Specific examples include Aβ42 and Aβ43.

  The above-mentioned compound (I) has an action of inhibiting the production of extended Aβ, specifically, the action of inhibiting the production of Aβ42 by the degradation of βAPP by γ-secretase (see Example 1). In addition, the extended Aβ production inhibitory action of compound (I) can be evaluated based on measuring (quantifying) the amount of extended Aβ secreted from cells. Specifically, a cell that expresses and produces βAPP is reacted with compound (I) in the presence of an appropriate solvent such as DMSO, and the production of Aβ42 secreted outside the cell in a given time is measured. Can be evaluated. The production amount of Aβ42 can be measured by a usual method such as ELISA or Western blotting, and the amount of Aβ42 produced from the cells (preferably nerve cells) treated with the compound (I) is untreated. The Aβ42 production inhibitory action of the compound can be evaluated by being lower than the Aβ42 production amount of the cells.

  Therefore, the pharmaceutical composition of the present invention containing Compound (I) as an active ingredient can be effectively used as an extended Aβ 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 an effect of inhibiting the production of extended Aβ, and is appropriately selected from the range of, for example, 0.1 to 100% by weight. be able to. Preferably it is 1 to 60 weight%, More preferably, it is 3 to 40 weight%.

  Further, Compound (I) is a disease caused by the production, secretion or deposition of extended Aβ based on its inhibitory action on extended Aβ production (in the present invention, “prolonged amyloid β-related disease” or “extended Aβ-related”). It is possible to prevent the development and development of 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 extended Aβ-related diseases.

  Examples of such extended Aβ-related diseases include Alzheimer-type dementia (Alzheimer's disease, Alzheimer-type senile dementia, etc.), Down's syndrome, memory impairment, prion disease (Kreuzfeld-Jakob disease, etc.), mild cognitive impairment (MCI), Dutch genetics 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, dementia associated with corticobasal degeneration, Examples include diffuse Lewy body type Alzheimer's disease, age-related macular degeneration, Parkinson's disease, and amyloid angiopathy. Among them, Alzheimer type dementia, particularly Alzheimer's disease is preferable.

  In this case, the ratio of the compound (I) contained in the pharmaceutical composition may be any amount as long as it has an effect of suppressing the production of extended Aβ. For example, it is appropriately selected from the range of 0.1 to 100% by weight. You can choose. Preferably it is 1 to 60 weight%, More preferably, it is 3 to 40 weight%.

  The pharmaceutical composition of the present invention usually contains an effective amount of compound (I) that suppresses extended Aβ production, specifically, an effective amount of compound (I) that suppresses cleavage of βAPP by enzymatic degradation. It is prepared by blending the above acceptable carrier or additive.

  Examples of the administration method of the pharmaceutical composition of the present invention include oral administration and parenteral administration such as intravenous administration, intramuscular administration, subcutaneous administration, transmucosal administration, transdermal administration, and rectal administration. Oral administration and intravenous administration are preferable, and oral administration is more preferable.

  The pharmaceutical composition of the present invention can be prepared into various forms of preparations (dosage forms) depending on the administration method. The preparations (dosage forms) will be described below, but the dosage forms used in the present invention are not limited to these, and various dosage forms that are usually used in the pharmaceutical preparation field can be used.

  Examples of dosage forms for oral administration include powders, granules, capsules, pills, tablets, elixirs, suspensions, emulsions, and syrups, and can be appropriately selected from these. . Moreover, modifications such as sustained release, stabilization, easy disintegration, poor disintegration, enteric solubility, easy absorption and the like can be applied to these preparations.

  In addition, dosage forms for intravenous administration, intramuscular administration, or subcutaneous administration include injections and infusions (including dried products prepared at the time of use), and can be selected as appropriate.

  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 select here as appropriate. These formulations can also be modified such as sustained release, stabilization, easy disintegration, difficulty disintegration, and easy absorption.

  The pharmaceutical composition of the present invention can 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 can be mentioned. Specific examples of pharmaceutically acceptable carriers and additives are listed below, but the present invention is not limited thereto.

  Examples of the solvent include purified water, sterilized purified water, water for injection, physiological saline, peanut 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, talc, titanium oxide, trehalose, xylitol Etc.

  Examples of binders include starch and derivatives thereof, cellulose and derivatives thereof (for example, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose), gelatin, sodium alginate, tragacanth, gum arabic and other natural polymer compounds, polyvinyl pyrrolidone, polyvinyl alcohol, etc. Synthetic polymer compounds, dextrin, hydroxypropyl starch and the like.

  Lubricants include light anhydrous silicic acid, stearic acid and its salts (eg, magnesium stearate), talc, waxes, wheat denbun, macrogol, hydrogenated vegetable oil, sucrose fatty acid ester, polyethylene glycol, silicone oil, etc. be able to.

  Examples of disintegrants include starch and derivatives thereof, agar, gelatin powder, sodium bicarbonate, calcium carbonate, cellulose and derivatives thereof, hydroxypropyl starch, carboxymethylcellulose and salts thereof, and cross-linked products thereof, and low-substituted hydroxypropylcellulose. be able to.

  Examples of the solubilizer include cyclodextrin, ethanol, propylene glycol, polyethylene glycol and the like. Examples of the suspending agent include sodium carboxymethylcellulose, polypinyl pyrrolidone, gum arabic, tragacanth, sodium alginate, aluminum monostearate, citric acid, various surfactants and the like.

  Examples of the thickener include sodium carboxymethyl cellulose, polypinyl pyrrolidone, methyl cellulose, hydroxypropyl methyl cellulose, polyvinyl alcohol, tragacanth, gum arabic, sodium alginate and the like.

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

  Stabilizers include tocopherols, chelating agents (eg, EDTA, thioglycolic acid), inert gases (eg, nitrogen, carbon dioxide), reducing substances (eg, sodium bisulfite, sodium thiosulfate, ascorbic acid, Rongalite) and the like. be able to.

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

  Examples of isotonic agents include sodium chloride and glucose. Examples of soothing agents include local anesthetics (procaine hydrochloride, lidocaine), pendyl alcohol, glucose, sorbitol, amino acids and the like.

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

  Examples of the preservative include benzoic acid and its salts, paraoxybenzoic acid esters, chlorobutanol, reverse soap, benzyl alcohol, phenol, thimerosal, dehydroacetic acid, boric acid, and the like.

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

  Bases 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, witepsol, gelatin, stearyl Alcohol, hydrolanolin, cetanol, light liquid paraffin, hydrophilic petrolatum, simple ointment, white ointment, hydrophilic ointment, macrogol ointment, hard fat, oil-in-water emulsion base, water-in-oil emulsion glaze etc. Can do.

  In addition, about each said dosage form, the technique of a well-known drug delivery system (DDS) is employable. 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 Considering side effects and the like, it is a preparation in an optimal preparation form.

  The pharmaceutical composition of the present invention is administered to a subject suffering from or likely to suffer from a disease caused by prolonged Aβ production (prolonged Aβ-related disease) (preferably Alzheimer-type dementia, particularly Alzheimer's disease). Thus, it is possible to prevent, delay or improve the onset and progression of the extended Aβ-related disease in the subject.

  When the pharmaceutical composition of the present invention is used as a preventive or therapeutic agent for a disease caused by prolonged Aβ production (prolonged Aβ-related disease) (for example, a prophylactic or therapeutic agent for Alzheimer's disease), its oral dosage is a compound The range of 0.03 to 300 mg / kg body weight in terms of the amount of (I) is preferable, and more preferably 0.1 to 50 mg / kg body weight. In the case of intravenous administration, there can be mentioned such a dose that the effective blood concentration of compound (I) is in the range of 0.2 to 50 μg / mL, more preferably 0.5 to 20 μg / mL.

  Note that these doses may vary depending on age, sex, body type and the like.

(2) Method for screening extended Aβ production inhibitor and standard reagent used therein The present invention also provides 3,5-bis (4-nitrophenoxy) benzoic acid represented by the above general formula (I) and its analogs (compounds) (I)) or a salt thereof, or a solvate thereof is used as a standard substance (positive control reagent) having an inhibitory action on extended amyloid β production, and has an inhibitory action on extended amyloid β production among test substances. A method for screening for an extended Aβ production inhibitor is provided. 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.

  Here, the target compound (I) includes all the compounds (I) described in the above (1). Preferably, it is a lower alkyl ester of 3,5-bis (4-nitrophenoxy) benzoic acid represented by the following formula (I ′), more preferably 3,5-bis (4-nitrophenoxy) benzoic acid. .

(In the formula, R ³ is a hydrogen atom or a lower alkyl group.)
Here, as described 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. A methyl group, an ethyl group, and an n-propyl group are preferable.

  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. The solvate of compound (I) or a salt thereof includes a hydrate.

  The screening method provided by the present invention is a method for selecting a substance having an inhibitory action on prolonged amyloid β 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).

Specifically, the method examines the extended Aβ production inhibitory action of the test substance, and among them, the compound (I) or the salt, or a solvate thereof has a higher effect than the extended Aβ production inhibitory action. It can be performed by selecting a test substance having an inhibitory action as an extended amyloid β production inhibitory substance. 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 (β-APP) or a cell capable of expressing and producing the protein;
(2) a step of measuring the amount of β-APP or Aβ42 produced from the cells;
(3) The amount of Aβ42 produced is controlled by using the compound (I) or salt, or a solvate thereof instead of the test substance, and the control Aβ42 obtained by carrying out the steps (1) and (2) above. A step of selecting a test substance showing an Aβ42 production amount lower than the positive control value as a candidate substance for an extended amyloid β production inhibitory substance in comparison with the production amount (positive control value).

  Here, as test substances, synthetic compounds, natural products (animals and plants, microorganisms, soil, etc.) or compositions or isolates obtained therefrom, and those obtained by genetic engineering techniques are widely used without particular limitation. Can do.

  The cells capable of expressing and producing β-APP include βAPP swedish mutation and wild type PS1 constitutive expression HEK293 cells, βAPP swedish mutation and PS1 L166P mutation constitutive expression HEK293 cells, βAPP swedish mutation constitutive expression HEK293 cells, wild type βAPP constitutively expressing HEK293 cells, HEK293 cells, CHO cells, HeLa cells or the like in which wild-type βAPP or βAPP swedish mutations are transiently expressed can be used. Preferably, βAPP swedish mutation and wild-type PS1 constitutively expressed HEK293 cells, and βAPP swedish mutation and PS1 L166P mutation constitutively expressed HEK293 cells are used.

  In the step (1), a method for bringing a test substance into contact with a cell capable of expressing and producing β-APP or β-APP is not particularly limited. However, when a β-APP expressing cell is used, at least the cell is β-APP. It is necessary for the cell and the test substance to coexist under conditions that allow the expression and production of. For example, after treating the cells with methionine-depleted MEM, the cells can be left overnight using a medium containing 450 μCi [35S] methionine / cysteine (Redivue Promix; Amersham), 5% dialyzed FCS (without amino acids), etc. Examples thereof include a method of adding a test substance at an appropriate concentration to a culture solution during a pulse period at the same time as metabolic labeling.

  Examples of the method for measuring the amount of Aβ42 produced from β-APP or β-APP-expressing cells in the step (2) include a method of performing ELISA or Western blotting using an antibody against Aβ42.

  In addition, it obtained by performing the said process (1) and (2) using the compound (I) or salt which has an extended type amyloid (beta) production inhibitory effect, or those solvates instead of a test substance. The production amount of Aβ42 is used as a control production amount of Aβ42 (positive control value) for evaluating the extended amyloid β production inhibitory action of the test substance in the step (3).

  Specifically, in step (3), the amount of Aβ42 produced in the above (1) and (2) using the test substance was compared with the amount of Aβ42 produced in the control (positive control value), It is performed by selecting a test substance showing a production amount of Aβ42 lower than the positive control value as a candidate substance for an extended amyloid β production inhibitory substance. That is, in the step (3), the compound (I) or a salt, or a solvate thereof is used as a positive standard reagent, and a test substance exhibiting a higher extended amyloid β inhibitory action is added to the extended amyloid β. This is a step of selecting as a candidate substance for a production inhibitory substance. Here, 3,5-bis (4-nitrophenynoxy) benzoic acid is preferable as a positive standard reagent.

  As described above, compound (I) or a salt thereof or a solvate thereof is used as a positive standard reagent or the like in a method of screening for an extended Aβ production inhibitor using its extended Aβ production inhibitory action. Or it can use suitably in the basic field | area in pharmaceutical development.

  The reagent of the present invention may be composed of the compound (I) or a salt thereof, or a solvate thereof, and in addition to these compounds (I) and the like, various carriers and You may have a form of the composition formed by mix | blending an additive. In this case, the ratio of the compound (I) contained in the composition may be any amount as long as it has an effect of suppressing the production of extended Aβ, and is appropriately selected from the range of, for example, 0.1 to 100% by weight. can do.

  Compound (I) or a salt thereof or a solvate thereof expresses β-amyloid precursor protein (βAPP) or the same, for example, for the purpose of suppressing production of extended Aβ produced from βAPP, preferably Aβ42 It can be used for cells. Examples of cells that express βAPP include nerve cells, particularly neurons in the brain, and cells that are artificially constructed to express βAPP by genetic manipulation.

  Therefore, the present invention includes a method for suppressing the production of extended Aβ, preferably Aβ42, by applying Compound (I) or a salt thereof or a solvate thereof to βAPP or a cell expressing the same.

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

Experimental example 1
1. Antibody and Test Compound Anti-Aβ antibody 4G8 antibody was purchased from Signet Laboratories (Dedham, MA). Among the test compounds, non-steroidal anti-inflammatory drugs (NSAIDS) (sulindac sulfide, indomethacin, naproxen), peroxisome proliferator activated receptor alpha agonist (fenofibrate), farnesyl pyrophosphate ammonium salt (FPP), and geraylgeranyl pyrophosphate ammonium salt (GGPP) It was purchased from Sigma, and 3,5-bis (4-nitrophenoxy) benzoic acid (hereinafter referred to as “Compound W” or “CW”) was purchased from Tokyo Chemical Industry Co., Ltd.

2. Cell cultures and cell types βAPP swedish mutation and wild-type PS1 constitutive expression HEK293 cells, and βAPP swedish mutation and PS1 L166P mutation constitutive expression HEK293 cells are disclosed in known papers (Okochi, M., Steiner, H., Fukumori, A. , Tanii, H., Tomita, T., Tanaka, T., Iwatsubo, T., Kudo, T., Takeda, 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 culture medium supplemented with 200 μg / ml G418 and 200 μg / ml zeocin.

3. Pulse-chase experiment βAPP swedish mutation and wild-type PS1 constitutive expression HEK293 cells were treated with methionine-depleted MEM, and then the cells were treated overnight with 450 μCi [35S] methionine / cysteine (Redivue Promix; Amersham), 5% dialyzed FCS ( Metabolic labeling in a medium containing no amino acids) and at the same time Compound W (100 μM), fenofibrate (100 μM), farnesyl pyrophosphate ammonium salt (FPP) (100 μM), geraneryl pyrophosphate ammonium salt (GGPP) (100 μM), naproxen (100 μM) ), Indomethacin (100 μM) and sulfindac sulfide (100 μM) 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.

4). The culture supernatant collected by immunoprecipitation / autoradiography is diluted to a final concentration of 50 mM Tris HCl (pH 7.4), 1: 1000 amount of protease inhibitor cocktail (Sigma), and 5 mM EDTA (pH 8.0). It was. 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 / 10% acetic acid. Autoradiography was performed by performing fluorographic analysis after the gel was dried.

5). Immunoprecipitation / MALDI-TOF MS analysis The immunoprecipitated protein was used for precipitation using a trifluoroacetic acid / acetone / water mixture (mixing ratio 1:20:20) saturated with α-cyano-4-hydroxy cinnamic acid. It was eluted from the carrier, and a soluble fraction was obtained by centrifugation. 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 preparations.

  The results are shown in FIG. 1 and FIG.

  As a result, among the test compounds, sulindac sulfide (100 μM), indomethacin (100 μM), and Compound W (100 μM) significantly reduced production and secretion of Aβ42 in endogenous PS-expressing cells. Among these compounds, Compound W has the highest inhibitory effect on Aβ42 production and significantly decreased the amount of Aβ42 secreted. As can be seen from FIG. 2, Compound W exhibited Aβ43 production inhibitory action in addition to Aβ42 production inhibitory action.

  Compound W confirmed that the inhibitory activity against COX-1 and COX-2 enzymes at 10 μM was 0% and 11%, respectively. Therefore, Compound W is considered to exhibit an Aβ production inhibitory action by a mechanism different from anti-inflammatory agents such as non-steroidal anti-inflammatory drugs (NSAIDS).

It is a figure which shows the relative ratio (Aβ42 amount / total Aβ amount) of Aβ42 secreted when cells expressing endogenous PS are treated with various test compounds. * Indicates that the relative ratio is significantly different (P <0.001) compared to the control (DMSO is used instead of the test compound). It is the figure which analyzed by the immunoprecipitation / MALDI-TOF MS method the change of various A (beta) secreted when the PS1 L166P mutation constitutive expression HEK293 cell is processed by Compound W (100 micromol). The up and down arrows indicate a clear increase and decrease, respectively, compared to the control (using DMSO instead of test compound).

Claims (11)

Formula (I):

(In the formula, R ¹ and R ² are each independently an optionally substituted aryloxy or an optionally substituted arylthio, and R ³ is a hydrogen atom or a lower alkyl group. )
Or a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient.   The pharmaceutical composition according to claim 1, which is an extended amyloid β production inhibitor.   In the general formula (I), the optionally substituted aryloxy or optionally substituted arylthio substituent is a nitro group, a carboxy group, a lower alkoxycarbonyl group, an acyl group, a sulfo group. The pharmaceutical composition according to claim 1, which is any one selected from the group consisting of sulfino group, sulfeno group, lower alkylsulfonyl group and cyano group. The pharmaceutical composition according to claim 1, wherein in general formula (I), R ¹ and R ² are nitrophenoxy groups, and R ³ is a hydrogen atom.   The pharmaceutical composition according to claim 1, which is a prophylactic or therapeutic agent for an elongated amyloid β-related disease.   The pharmaceutical composition according to claim 5, wherein the prolonged amyloid β-related disease is Alzheimer's disease.   A method for treating or preventing an extended amyloid β-related disease, comprising administering the pharmaceutical composition according to claim 5 to a subject suffering from or likely to suffer from an extended amyloid β-related disease. Formula (I):

(In the formula, R ¹ and R ² are each independently an optionally substituted aryloxy or an optionally substituted arylthio, and R ³ is a hydrogen atom or a lower alkyl group. )
Or a salt thereof, or a solvate thereof, a reagent for screening an extended amyloid β production inhibitor. Investigate the amyloid β production inhibitory effect on the test substance,
Formula (I):

(In the formula, R ¹ and R ² are each independently an optionally substituted aryloxy or an optionally substituted arylthio, and R ³ is a hydrogen atom or a lower alkyl group. )
A test substance having an inhibitory action higher than that of the extended amyloid β production inhibitory compound possessed by the compound or a salt thereof, or a solvate thereof, as an extended amyloid β production inhibitory substance, A method of screening for an amyloid β production inhibitor.   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. Formula (I):

(In the formula, R ¹ and R ² are each independently an optionally substituted aryloxy or an optionally substituted arylthio, and R ³ is a hydrogen atom or a lower alkyl group. )
Or a salt thereof, or a solvate thereof, is applied to β-amyloid precursor protein or β-amyloid precursor protein-expressing cells.

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