JP6528238B2 - Inhibitor of cell death due to endoplasmic reticulum stress, endoplasmic reticulum stress control agent, and prophylactic / therapeutic agent containing the control agent as an active ingredient - Google Patents

Description

  The present invention relates to a cell death inhibitor due to endoplasmic reticulum stress, an endoplasmic reticulum stress regulator, and a preventive / therapeutic agent containing the regulator as an active ingredient.

  Conventionally, it is known that denatured protein is accumulated by stress applied to the endoplasmic reticulum of cells in vivo (hereinafter sometimes referred to as "endoplasmic reticulum stress") to induce cell death (mainly apoptosis). . In recent years, this series of phenomena is related to Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, cerebral infarction, neurodegenerative diseases such as polyglutamine disease, inflammatory neurological diseases such as multiple sclerosis, and depression such as depression. It has become clear that it is closely involved in the onset and pathogenesis of various diseases such as diseases, myocardial infarction, ischemic heart disease such as arteriosclerosis, diabetes, glomerulonephritis, renal diseases such as renal failure, and cancer. The Thus, controlling endoplasmic reticulum stress is important for preventing or treating these diseases.

  For example, Patent Document 1 discloses methoxychalcone, methoxyflavonoid and flavone compounds having a specific structure as compounds that control endoplasmic reticulum stress.

  Patent Document 2 discloses cyanidin, which is an extract derived from black rice, and cyanidin glycoside derived from the extract as a compound that reduces endoplasmic reticulum stress.

JP 2007-314468 A JP, 2013-151459, A

As a method for producing a compound described in Patent Document 1, a method of extracting from citrus peel or a method of using a derivative of the extracted compound is disclosed, but in the case of using for pharmaceutical use Low purity, poor yield. Furthermore, although the manufacturing method by chemical synthesis is also possible, the synthesis is difficult.
Moreover, although the method of extracting from black rice or the method of using the derivative of the extracted compound is disclosed as a manufacturing method also about the compound described in patent document 2, when using it for a pharmaceutical use, Low purity, poor yield. Furthermore, although the manufacturing method by chemical synthesis is also possible, the synthesis is difficult.

  This invention is made in view of the said situation, Comprising: The cell death inhibitor by the endoplasmic reticulum stress which uses a specific compound as an active ingredient, an endoplasmic reticulum stress control agent, and the prevention * containing the said control agent as an active ingredient Provide a therapeutic agent.

The present invention is as follows.
(1) The agent for suppressing cell death due to endoplasmic reticulum stress according to the present invention (except the hypoglycemic agent and the agent for preventing and treating diabetes) comprises the compound represented by the general formula [1] as an active ingredient It features.

(In the general formula [1], X represents a hydroxy group, m is table to .R 2 represents a hydrogen atom or a hydroxy group.)

(2) The endoplasmic reticulum stress control agent of the present invention (with the exception of antihyperglycemic agents and agents for preventing and treating diabetes) is characterized by containing the compound represented by the general formula [1] as an active ingredient. .

(In the general formula [1], X represents a hydroxy group, m is table to .R 2 represents a hydrogen atom or a hydroxy group.)
(3) The compound described in (2), wherein the compound represented by the general formula [1] is a compound represented by the formula [1-1-1] or a compound represented by the formula [1-1-2] Endoplasmic reticulum stress control agent .

( 4 ) The preventive / therapeutic agent of the present invention is a preventive / therapeutic agent for neurodegenerative diseases comprising the endoplasmic reticulum stress control agent according to (2) or (3) as an active ingredient .

  According to the present invention, a compound for suppressing or controlling cell death due to endoplasmic reticulum stress can be simply synthesized, and high industrial efficiency can be achieved. Furthermore, according to the present invention, cell death due to endoplasmic reticulum stress can be easily suppressed or controlled.

It is the graph which showed the evaluation result of the cell survival rate by MTT method in Experiment 1.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings as needed.

[Cell death suppressor due to endoplasmic reticulum stress]
In one embodiment, the present invention provides an inhibitor of cell death due to endoplasmic reticulum stress, which comprises the compound represented by the general formula [1] as an active ingredient.

  In the general formula [1], X represents a hydroxy group, an unsubstituted or substituted C1-6 alkyl group, or an unsubstituted or substituted C1-6 alkoxy group, and m is any of 0 to 4 When m is 2 or more, X may be the same or different. R represents a hydrogen atom or a hydroxy group.

  Furthermore, the present inventors have made the compound represented by the general formula [1] of the present embodiment ((2R, 3R) only) against endoplasmic reticulum stress-induced cell death (mainly apoptosis). It has become clear for the first time to exert a remarkable effect.

  In the present specification, “cell death suppression by endoplasmic reticulum stress” means suppressing cell death induced by endoplasmic reticulum stress. For example, MTT method using a compound represented by the general formula [1], a cell such as HT22 (hippocampal-derived cell line) cell, and a compound such as Tunicamycin as an endoplasmic reticulum stress inducer (Thiazolyl Blue Tetrazolium Bromide Assay) When evaluated in the above, it means that the cell viability is preferably 50% or more, more preferably 80% or more, and still more preferably 90% or more.

Moreover, a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group etc. are mentioned as "a C1-6 alkyl group which has no substitution or a substituent." Although it does not specifically limit as a substituent of the said C1-6 alkyl group, Halogen atoms, such as a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, etc. are mentioned.
Moreover, a methoxy group, an ethoxy group, a protoxy group, a butoxy group, a pentoxy group, a hexatoxy group etc. are mentioned as "a C1-6 alkoxy group which is unsubstituted or has a substituent." Although it does not specifically limit as a substituent of the said C1-6 alkoxy group, Halogen atoms, such as a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, etc. are mentioned.

  It is preferable that X in General Formula [1] is a hydroxy group from the viewpoint of high suppression effect of cell death due to endoplasmic reticulum stress. In addition, m is preferably 2. R is preferably a hydroxy group.

  As a specific example of a compound represented by General formula [1], the compound represented by Formula [1-1] or [1-2] is mentioned.

  In formulas [1-1] and [1-2], X and m have the same meanings as described above. It is preferable that X in Formula [1-1] and [1-2] is a hydroxyl group from a viewpoint with a high inhibitory effect of the cell death by endoplasmic reticulum stress. In addition, m is preferably 2.

  Furthermore, as a specific example of a compound represented by Formula [1-1] and [1-2], the compound represented by Formula [1-1-1] or [1-1-2] is mentioned.

[Method for producing cell death inhibitor due to endoplasmic reticulum stress]
Hereinafter, the method for producing the compound represented by the general formula [1] is described in detail.

The compound represented by the general formula [2] is protected by a large excess of chloromethyl methyl ether (MOMCl) in the presence of a large excess of hydrogenated sodium to obtain a compound represented by the general formula [3]. In the general formula [2] and the general formula [3], X and m have the same meanings as described above.
Similarly, the hydroxyl group of the compound represented by the general formula [4] which is a benzaldehyde derivative is protected with MOMCl in the presence of potassium carbonate to obtain a compound represented by the general formula [5]. In the general formula [4] and the general formula [5], R represents the same meaning as described above, and R ₁ represents a hydrogen atom or a -OMOM group.
The compound represented by the general formula [3] and the compound represented by the general formula [5] are aldol condensed in the presence of KOH to obtain the compound represented by the general formula [6]. Further, the compound represented by the general formula [6] is oxidized with an aqueous hydrogen peroxide solution in an alkaline aqueous solution to obtain a compound represented by the general formula [7] which is an epoxide. The compound represented by the general formula [7] is treated with hydrochloric acid to obtain a compound represented by the general formula [1 ′] which is a racemic mixture.
The production method of the present embodiment is not limited to the solvents and bases described above, and standard solvents and bases can be used as common solvents and bases as long as they meet the purpose of the reaction.

Furthermore, the method to refine | purify the compound represented by General formula [1] of this embodiment which is a (2R, 3R) body from the compound represented by General formula [1 '] is demonstrated.
The compound represented by the general formula [1 '], which is a racemic mixture, is dissolved in methanol and purified using an optically active support column mounted on a high performance liquid chromatography apparatus. (2R, 3R) and (2S, 3S) can be separated and fractionated by developing with ethanol-hexane (preferably, a ratio of 3: 7).
The separated enantiomer can be confirmed by measuring the optical rotation by sodium D line in ethanol.

[Endoplasmic reticulum stress regulator]
In one embodiment, the present invention provides an endoplasmic reticulum stress control agent containing a compound represented by the general formula [1] as an active ingredient.

  In the general formula [1], X, m and R have the same meanings as described above.

  Moreover, it became clear for the first time by the present inventors that the compound (only (2R, 3R) form) represented by General Formula [1] of this embodiment can effectively control endoplasmic reticulum stress.

  As used herein, “endoplasmic reticulum stress control” means suppressing accumulation of denatured protein in the endoplasmic reticulum, or suppressing cell death induced by endoplasmic reticulum stress. The endoplasmic reticulum stress control mechanism by the compound represented by the general formula [1] is not clarified in detail, but it is presumed to be to control the expression of endoplasmic reticulum stress-related gene or protein.

  It is preferable that X in general formula [1] is a hydroxy group from the viewpoint of high control effect of endoplasmic reticulum stress. In addition, m is preferably 2. R is preferably a hydroxy group.

  As a specific example of a compound represented by General formula [1], the compound represented by Formula [1-1] or [1-2] is mentioned.

  In formulas [1-1] and [1-2], X and m have the same meanings as described above. It is preferable that X in Formula [1-1] and [1-2] is a hydroxy group from a viewpoint with a high control effect of endoplasmic reticulum stress. In addition, m is preferably 2.

  Furthermore, as a specific example of a compound represented by Formula [1-1] and [1-2], the compound represented by Formula [1-1-1] or [1-1-2] is mentioned.

  The method for producing the compound represented by the general formula [1] is as described above.

  The endoplasmic reticulum stress control agent of the present embodiment may contain, as another component, a buffer such as PBS or Tris-HCl; and an additive such as sodium azide or glycerol.

In this embodiment, the endoplasmic reticulum stress control agent is used to cause neurodegenerative diseases such as Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, cerebral infarction, and polyglutamine disease, which are caused by endoplasmic reticulum stress, multiple sclerosis Used in the treatment of various diseases such as inflammatory neurological diseases such as hypertension, psychiatric diseases such as depression, ischemic heart disease such as myocardial infarction and arteriosclerosis, diabetes, glomerulonephritis, renal diseases such as renal failure and cancer can do.
The subject to be treated is not limited, and includes mammals including human or non-human animals, preferably human.

The dose of the endoplasmic reticulum stress control agent of this embodiment is the age, sex, body weight, symptoms, treatment method, administration method, treatment time of the test animal (various mammals including human or non-human animals, preferably human). The dose may be appropriately adjusted in consideration of, for example, in the case of adult humans, usually about 0.5 to 5000 mg as an active ingredient per day, and in children about 0.5 to 3000 mg.
The compounding ratio of the endoplasmic reticulum stress reducing agent can be appropriately changed depending on the dosage form, but it is about 0.3 to 15 when administered orally or by mucosal absorption to a test animal (preferably human). In the case of parenteral administration such as injection, it is recommended that the concentration be about 0.01 to 10 wt%. In addition, since the dose varies as described above under various conditions, it may be sufficient to use less than the dose, or it may be necessary to administer beyond the range.
The administration frequency is preferably once to several times per day on average.
Dosage forms include methods known to those skilled in the art, for example, intraarterial injection, intravenous injection, subcutaneous injection, intranasal, transbronchial, intramuscular, transdermal, or orally.
The injection can also be prepared as a non-aqueous diluent (for example, vegetable oil such as porene glycol and olive oil, alcohol such as ethanol, etc.), suspension, or emulsion. Such sterilization of the injection can be carried out by filter sterilization with a filter, blending of a bactericidal agent and the like. The injection can be manufactured as a form of topical preparation. That is, it can be used as a sterile solid composition by a lyophilization method etc. and dissolved in distilled water for injection or other solvent before use.

[Neurodegenerative disease prevention and treatment agent]
In one embodiment, the present invention provides an agent for preventing and / or treating neurodegenerative diseases, which comprises the above-mentioned endoplasmic reticulum stress control agent as an active ingredient.

  According to the agent for preventing and treating neurodegenerative diseases of the present embodiment, neurodegenerative diseases such as Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, cerebral infarction, and polyglutamine disease produced by endoplasmic reticulum stress are effectively It can be prevented or treated.

The neurodegenerative disease preventive / therapeutic agent of the present embodiment contains a therapeutically effective amount of an endoplasmic reticulum stress control agent, and a pharmaceutically acceptable carrier or diluent. Pharmaceutically acceptable carriers or diluents include excipients, diluents, bulking agents, disintegrants, stabilizers, preservatives, buffers, emulsifiers, fragrances, coloring agents, sweeteners, thickeners, taste masking agents Agents, solubilizers, additives and the like. By using one or more of these carriers, it is possible to prepare an agent for preventing or treating neurodegenerative diseases in the form of injection, solution, capsule, suspension, emulsion, syrup or the like.
Also, a colloidal dispersion system can be used as a carrier. The colloidal dispersion system is expected to have an effect of enhancing the in vivo stability of the endoplasmic reticulum stress control agent, and an effect of enhancing the transferability of the endoplasmic reticulum stress control agent to a specific organ, tissue or cell. Colloidal dispersion systems may include polyethylene glycol, polymer complexes, polymer aggregates, nanocapsules, microspheres, beads, oil-in-water emulsifiers, micelles, mixed micelles, lipids including liposomes, specific Liposomes and artificial membrane vesicles are preferred, which have the effect of efficiently transporting endoplasmic reticulum stress control agents to organs, tissues, or cells of

Examples of formulation in the agent for preventing or treating neurodegenerative diseases of the present embodiment include those orally used as tablets, capsules, elixirs and microcapsules coated with sugar coating, if necessary.
Or, those used parenterally in the form of injections of sterile solutions or suspensions with water or other pharmaceutically acceptable liquids. Furthermore, a pharmacologically acceptable carrier or diluent, specifically, sterile water or saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, Those formulated by blending in unit dose form required for generally accepted pharmaceutical practice by combining appropriately with preservatives, binders and the like are suitable.

  Examples of additives that can be incorporated into tablets and capsules include, for example, gelatin, corn starch, tragacanth gum, binders such as gum arabic, excipients such as crystalline cellulose, puffs such as corn starch, gelatin and alginic acid Agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavors such as peppermint, red mono oil or cherry are used. When the dosage unit form is a capsule, the above-mentioned materials can further contain a liquid carrier such as oil and fat. Sterile compositions for injection can be formulated according to a conventional formulation using a vehicle such as distilled water for injection.

  Aqueous solutions for injection include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and suitable solubilizers, such as, for example, It may be used in combination with alcohols such as ethanol, polyalcohols such as propylene glycol, polyethylene glycol, nonionic surfactants such as polysorbate 80 (TM), HCO-50.

  Examples of oily liquids include sesame oil and soybean oil, which may be used in combination with benzyl benzoate and benzyl alcohol as a solubilizing agent. It may also be formulated with a buffer such as phosphate buffer, sodium acetate buffer, a soothing agent such as procaine hydrochloride, a stabilizer such as benzyl alcohol, phenol, an antioxidant. The prepared injection is usually filled in a suitable ampoule.

[Diabetes prevention and treatment agent]
In one embodiment, the present invention provides an agent for preventing and / or treating diabetes comprising the above-mentioned endoplasmic reticulum stress control agent as an active ingredient.

  According to the agent for preventing and / or treating diabetes of the present embodiment, diabetes can be effectively prevented or treated.

  The diabetes prevention and treatment agent of the present embodiment contains a therapeutically effective amount of an endoplasmic reticulum stress control agent, and a pharmaceutically acceptable carrier or diluent. The carrier or diluent has the same meaning as described above. The form of orally or parenterally administering the agent for preventing or treating diabetes according to this embodiment is as described above.

  Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to the following examples.

Example 1 Synthesis of Compound Represented by Formula [1-1-1] (1) Synthesis of Racemic Mixture The following is 2,4,6-trihydroxyacetophenone (compound represented by Formula [8]) And a reaction formula showing a step of synthesizing a compound represented by the formula [1-1-1 '], which is a racemic mixture, from 3-hydroxybenzaldehyde (compound represented by the formula [10]).

  First, sodium hydride (60% liquid paraffin dispersion, 4. g) was added to 2,4,6-trihydroxyacetophenone (compound represented by the formula [8]) (5 g) dissolved in anhydrous dimethylformamide under ice-cooling. 8 g (4.5 equivalents) was added dropwise. After the temperature of the reaction solution sufficiently decreased, chloromethyl methyl ether (9.7 g, 4.5 equivalents) was added little by little under ice cooling so that the solution temperature was kept at 5 ° C. or less. . The reaction was then stirred at room temperature for 30 minutes. The reaction was quenched by adding ice-cold purified water, and the product was extracted with ethyl acetate. The ethyl acetate fraction was washed with water and then with brine, dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane-ethyl acetate) to give 2,4,6-tris (methoxymethoxy) acetophenone (compound represented by the formula [9]) as a colorless oil (7. 26 g, 90% yield).

  Next, under ice-cooling, 3-hydroxybenzaldehyde (compound represented by the formula [10]) (1.0 g) was dissolved in 20 mL of anhydrous acetone, and then potassium carbonate (11.3 g, 10 equivalents) was added. Chloromethyl methyl ether (1.0 g, 1.5 equivalents) was added in small portions, keeping the temperature of the reaction solution below 5 ° C. The reaction was then stirred at room temperature for 30 minutes. The reaction was quenched by adding ice-cold purified water, and the product was extracted with ethyl acetate. The ethyl acetate fraction was washed with water and then with brine, dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane-ethyl acetate) to give 3- (methoxymethoxy) benzaldehyde (compound represented by formula [11]) as a pale yellow oil (1.22 g, yield 90%).

  A solution of potassium hydroxide (0.28 g, 3 equivalents) in ethanol was added to 20 mL of an ethanol solution of 2,4,6-tris (methoxymethoxy) acetophenone (compound represented by the formula [9]) (0.5 g) . After stirring for 5 minutes, 3- (methoxymethoxy) benzaldehyde (compound represented by the formula [11]) (0.28 g, 1 equivalent) was added (room temperature). After reacting at room temperature for 3 hours, ice-cold purified water was added to stop the reaction, and the product was extracted with ethyl acetate. The ethyl acetate fraction was washed with water and then with brine, dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure. The residue is purified by silica gel column chromatography (hexane-ethyl acetate), and chalcone derivative 3- (3- (methoxymethoxy) phenyl) -1- (2,4,6-tris (methoxymethoxy) as a pale yellow oil Phenyl) prop-2-en-1-one (compound represented by the formula [12]) was obtained (0.52 g, yield 70%).

  3- (3- (methoxymethoxy) phenyl) -1- (2,4,6-tris (methoxymethoxy) phenyl) prop-2-en-1-one (compound represented by the formula [12]) (0 To 10 mL of a methanol solution of 0.5 g) was added 2 mL of 30% hydrogen peroxide water and 0.5 mL of 2 M aqueous sodium hydroxide solution, and the mixture was stirred at room temperature for 3 hours. After distilling off methanol under reduced pressure, purified water was added and the mixture was extracted with ethyl acetate. The extract was washed with purified water and saturated brine and then dried over anhydrous sodium sulfate. The solvent is removed under reduced pressure, and the epoxide compound (3- (3- (methoxymethoxy) phenyl) oxiran-2-yl) (2,4,6-tris (methoxymethoxy) phenyl) methanone (formula [13] Were obtained as a colorless oil (0.49 g, yield 95%).

(3- (3- (methoxymethoxy) phenyl) oxiran-2-yl) (2,4,6-tris (methoxymethoxy) phenyl) methanone (compound represented by the formula [13]) (0.5 g) The mixture was stirred at 55 ° C. for 25 minutes in 10 mL of 1 M hydrochloric acid-anhydrous methanol, and methanol was distilled off under reduced pressure. Purified water was added and extracted with ethyl acetate. The solvent was removed by evaporation under reduced pressure, and the remaining reddish-yellow oil was purified by silica gel column chromatography (hexane-ethyl acetate) to obtain flavanonol derivative 3,5,7-trihydroxy-2- (3-hydroxyphenyl). ) Chroman-4-one (compound represented by the formula [1-1-1 ']) was obtained as a white powder (0.15 g, yield 48%).
¹ H NMR (600 MHz, Acetone-d6) δ: 4.63 (d, 1 H, J = 11.4 Hz, H-3), 5.12 (d, 1 H, J = 11.4 Hz, H-2), 5.98 (d, 1 H, J = 1.8 Hz, H-6), 6.00 (d, 1 H, J = 1.8 Hz, H-8), 6.89 (dd, 1 H, J = 8. 4, 3.0 Hz, H-4 ', 7.06 (d, 1 H, J = 8.4 Hz, H-6'), 7.07 (d, 1 H, J = 3.0 Hz, H-2 ' ), 7.25 (dd, 1 H, J = 8.4, 8.4 Hz, H-5 ′). ¹³ C NMR (150 MHz, Acetone-d6) δ: 73.3 (3-C), 84.4 (2-C), 96.2 (6-C), 97.3 (8-C), 101.6 (10-C), 115.8 (2'-C), 116.6 (4'-) C), 120.0 (6'-C , 130.3 (5'-C), 139.8 (1'-C), 158.3 (3'-C), 164.1 (4-C), 165.1 (9-C), 167 .9 (7-C), 198.0 (4-C). ^{HR-EI-MS: m /} s 288.06321 [M] + (Calcd 288.06336 for C 15 H 12 O 6).

(2) Separation and purification of the compound represented by the formula [1-1-1] from a racemic mixture The compound (20 mg) represented by the formula [1-1-1 '], which is a racemic mixture, was dissolved in 200 μL of methanol . Load 50 μL in 200 μL onto the optically active support column (Daicell Corporation CHIRALPAK-IA, 10 mm × 250 mm) mounted on a high-performance liquid chromatography apparatus (JASCO PU-1580 pump, UV 1575 UV absorption detector), and The (2R, 3R) body and the (2S, 3S) body were separated and fractionated by developing with 5 mL / min of hexane (3: 7). Furthermore, the said operation was repeated 3 times about every 50 microliters about remaining 150 microliters. The retention time was 4 minutes and 25 seconds for the (2R, 3R) body and 2 minutes and 45 seconds for the (2S, 3S) body.
For the separated enantiomers, the optical rotation by sodium D line was measured in ethanol. The (2R, 3R) isomer was [α] ¹² _D = 9.4 °, and the (2S, 3S) isomer was [α] ¹² _D = -7.3 °.

Example 2 Synthesis of Compound Represented by Formula [1-1-2] (1) Synthesis of Racemic Mixture The following is 2,4,6-trihydroxyacetophenone (compound represented by Formula [8]) And a reaction formula showing a step of synthesizing a compound represented by the formula [1-1-2 '], which is a racemic mixture, from 3,5-dihydroxybenzaldehyde.

In the same manner as Example 1, 2,4,6-trihydroxyacetophenone (compound represented by the formula [8]) to 2,4,6-tris (methoxymethoxy) acetophenone (formula [9]) Compound) was obtained.
Next, under ice-cooling, 3,5-dihydroxybenzaldehyde (compound represented by the formula [14]) (1.0 g) was dissolved in 20 mL of anhydrous acetone, and then potassium carbonate (20 g, 20 equivalents) was added. Chloromethyl methyl ether (1.75 g, 1.5 equivalents) was added in small portions, keeping the temperature of the reaction solution below 5 ° C. The reaction was then stirred at room temperature for 30 minutes. The reaction was quenched by adding ice-cold purified water, and the product was extracted with ethyl acetate. The ethyl acetate fraction was washed with water and then with brine and dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane-ethyl acetate) to give 3,5-bis (methoxymethoxy) benzaldehyde (compound represented by the formula [15]) as a pale yellow oil (1.47 g) , Yield 90%).

  To 20 mL of an ethanol solution of 2,4,6-trihydroxyacetophenone (compound represented by the formula [8]) (0.5 g) was added an ethanol solution of potassium hydroxide (0.28 g, 3 equivalents). After stirring for 5 minutes, 3,5-bis (methoxymethoxy) benzaldehyde (compound represented by the formula [15]) (0.38 g, 1 equivalent) was added (room temperature). After reacting at room temperature for 3 hours, ice-cold purified water was added to stop the reaction, and the product was extracted with ethyl acetate. The ethyl acetate fraction was washed with water and then with brine and dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane-ethyl acetate) to give a chalcone derivative 3- (3,5-bis (methoxymethoxy) phenyl) -1- (2,4,6-) as a pale yellow oil. Tris (methoxymethoxy) phenyl) prop-2-en-1-one (compound represented by the formula [16]) was obtained (0.68 g, yield 80%).

  3- (3,5-bis (methoxymethoxy) phenyl) -1- (2,4,6-tris (methoxymethoxy) phenyl) prop-2-en-1-one (compound represented by the formula [16] 2) 30 mL of a 30% aqueous solution of hydrogen peroxide and 0.5 mL of a 2 M aqueous solution of sodium hydroxide were added to 10 mL of a methanol solution of 0.5 g of methanol and stirred at room temperature for 3 hours. After distilling off methanol under reduced pressure, purified water was added and the mixture was extracted with ethyl acetate. The extract was washed with purified water and saturated brine and then dried over anhydrous sodium sulfate. The solvent is removed under reduced pressure and the epoxide compound (3- (3,5-bis (methoxymethoxy) phenyl) oxirane-2-yl) (2,4,6-tris (methoxymethoxy) phenyl) methanone (formulae) The compound represented by [17] was obtained as a colorless oil (0.49 g, yield 95%).

(3- (3,5-bis (methoxymethoxy) phenyl) oxiran-2-yl) (2,4,6-tris (methoxymethoxy) phenyl) methanone (compound represented by the formula [17]) (0. 5 g) was stirred in 10 mL of 1 M hydrochloric acid-anhydrous methanol at 55 ° C. for 25 minutes, and methanol was distilled off under reduced pressure. Purified water was added and extracted with ethyl acetate. The solvent is removed by evaporation under reduced pressure, and the remaining reddish-yellow oil is purified by silica gel column chromatography (hexane-ethyl acetate) to obtain flavanonol derivative 3,5,7-trihydroxy-2- (3,5-). Dihydroxyphenyl) chroman-4-one (compound represented by the formula [1-1-2 ']) was obtained as a white powder (0.09 g, yield 32%).
¹ H NMR (600 MHz, Acetone-d6) δ: 4.57 (d, 1 H, J = 11.4 Hz, H-3), 5.02 (d, 1 H, J = 11.4 Hz, H-2), 5.97 (d, 1 H, J = 1.8 Hz, H-6), 5.99 (d, 1 H, J = 1.8 Hz, H-8), 6.38 (t, 1 H, J = 1. 8 Hz, H-4 '), 6.56 (d, 2 H, J = 1.8 Hz, H-2', H-6 '). ¹³ C NMR (150 MHz, Acetone-d6) δ: 73.3 (3-C), 84.6 (2-C), 96.1 (6-C), 97.2 (8-C), 101. 6 (10-C), 103.8 (4'-C), 107.4 (2 ', 6'-2C), 140.4 (1'-C), 159.4 (3', 5'-) 2C), 164.1 (5-C), 165.1 (9-C), 168.0 (7-C), 198.0 (4-C). ^{HR-EI-MS: m /} s 304.05804 [M] + (Calcd 304.05830 for C 15 H 12 O 7).

(2) Separation and purification of the compound represented by the formula [1-1-2] from a racemic mixture The compound (20 mg) represented by the formula [1-1-2 '] which is a racemic mixture was dissolved in 200 μL of methanol . Load 50 μL in 200 μL onto the optically active support column (Daicell Corporation CHIRALPAK-IA, 10 mm × 250 mm) mounted on a high-performance liquid chromatography apparatus (JASCO PU-1580 pump, UV 1575 UV absorption detector), and The (2R, 3R) isomer and the (2S, 3S) isomer were separated and fractionated by developing at 5 mL / min of hexane (45: 55). Furthermore, the said operation was repeated 3 times about every 50 microliters about remaining 150 microliters. The retention time was 7 minutes and 25 seconds for the (2R, 3R) body and 4 minutes and 25 seconds for the (2S, 3S) body.
For the separated enantiomers, the optical rotation by sodium D line was measured in ethanol. The (2R, 3R) form was [α] ¹² _D = 10.9 °, and the (2S, 3S) form was [α] ¹² _D = -9.0 °.

Test Example 1 Evaluation of Endoplasmic Reticulum Stress Control Agent by MTT Method The test was carried out as follows using the compounds synthesized in Examples 1 and 2 and cyanidin (manufactured by Wako) as a comparative example.
Cells such as HT22 (hippocampal-derived cell line) cells were seeded at a density of 4000 cells / well in 96-well plates and cultured in an incubator at 37 ° C. and 5% CO ₂ for 24 hours. After culture, the medium was discarded, and the compounds synthesized in Examples 1 and 2 together with fresh medium, and cyanidin as a comparative example were added to each well at 10, 30, 100 μM. After 10 minutes, 50 ng / mL Tunicamycin (manufactured by Sigma) was added to half of the wells as an endoplasmic reticulum stress inducer, and cultured in an incubator at 37 ° C. and 5% CO ₂ for 24 hours. The measurement wavelength of 570 nm was measured, and live cells were measured. As a control, cells to which no compound was added and cells to which only an endoplasmic reticulum stress inducer was added were prepared. Cell viability was calculated in the other wells, with 100% viability of cells without any compound added. The results are shown in FIG.

  It became clear from FIG. 1 that cell death due to endoplasmic reticulum stress of HT22 cells is suppressed depending on the concentration of the compounds synthesized in Example 1 and Example 2 and cyanidin. Furthermore, it was revealed that the compounds synthesized in Example 1 and Example 2 have a significantly higher inhibitory effect on cell death due to endoplasmic reticulum stress compared to cyanidin.

  From the above results, it has become clear that according to the present invention, cell death due to endoplasmic reticulum stress can be easily suppressed and controlled.

Claims (4)

An agent for suppressing cell death due to endoplasmic reticulum stress characterized by containing the compound represented by the general formula [1] as an active ingredient (with the exception of hypoglycemic agents and agents for preventing and treating diabetes) .

(In the general formula [1], X represents a hydroxy group, m is table to .R 2 represents a hydrogen atom or a hydroxy group.) An endoplasmic reticulum stress control agent characterized by containing the compound represented by the general formula [1] as an active ingredient (however, except for a hypoglycemic agent and an agent for preventing and treating diabetes) .

(In the general formula [1], X represents a hydroxy group, m is table to .R 2 represents a hydrogen atom or a hydroxy group.) The vesicle according to claim 2, wherein the compound represented by the general formula [1] is a compound represented by the formula [1-1-1] or a compound represented by the formula [1-1-2]. Stress control agent .

An agent for preventing or treating neurodegenerative diseases, which comprises the endoplasmic reticulum stress control agent according to claim 2 or 3 as an active ingredient.

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