JP7748726B2 - Thioredoxin-interacting protein expression inhibitors - Google Patents

Description

The present invention relates to an inhibitor of thioredoxin interacting protein expression.

Thioredoxin-interacting protein (TXNIP) binds to and inhibits the action of thioredoxin, an antioxidant. In humans, TXNIP expression increases with age, leading to a decrease in resistance to oxidative stress (Oberacker T et al. FEBS Letters 592:2297-2307, 2018). Oxidative stress is associated with many diseases, including sickle cell disease, atherosclerosis, Parkinson's disease, Alzheimer's disease, heart failure, myocardial infarction, schizophrenia, bipolar disorder, fragile X syndrome, and chronic fatigue syndrome.

In Drosophila, TXNIP deficiency extends lifespan (Oberacker T et al. FEBS Letters 592:2297-2307, 2018).

TXNIP is also deeply involved in the pathology of cancer and diabetes. For example, in diabetes, hyperglycemia-induced TXNIP activates NLRP3, causing an inflammatory response (Zhou R et al. Nature Immunology 11:136-141, 2010). TXNIP damages pancreatic beta cells. Knocking down TXNIP reduces beta cell damage and suppresses the progression of diabetes.

In other words, reducing TXNIP can lead to the treatment of various diseases related to oxidative stress, including diabetes. The reduction of TXNIP has been reported in Patent Document 1 and Non-Patent Document 1.

Patent Document 1 reports compounds that can reduce or inhibit (a) the biological activity of thioredoxin-interacting protein (TXNIP) or (b) the expression of the gene encoding TXNIP, for use in treating conditions that have the beneficial effect of improving resistance to oxidative stress. Examples of such compounds include antisense oligonucleotides or shRNAs that reduce or inhibit the expression of the gene encoding TXNIP.

Non-patent document 1 reports that the antihypertensive drug verapamil reduces TXNIP levels and enhances the effectiveness of insulin treatment in adult patients with newly diagnosed type 1 diabetes.

Furthermore, in Patent Document 2, the present inventors reported that curcumin derivatives containing F atoms have high binding specificity for amyloid beta protein and are useful as active ingredients in imaging diagnostic agents for Alzheimer's disease.

Japan Special Publication No. 2015-522251 International Publication No. 2010/098502

Nature Medicine 24:1108-1112, 2018

The present invention aims to provide a TXNIP expression inhibitor that uses a substance different from conventional substances as a new active ingredient and has excellent TXNIP expression inhibitory activity.

As a result of extensive research to achieve the above-mentioned objective, the inventors discovered that the curcumin derivative described in Patent Document 2 above strongly suppresses the expression of TXNIP at both the mRNA and protein levels. However, curcumin was not found to have any inhibitory effect on TXNIP expression. Similarly, compounds in which the fluorine atoms of the curcumin derivative described in Patent Document 2 are replaced with hydrogen atoms were also not found to have any inhibitory effect on TXNIP expression.

The present invention was completed based on these findings and through further research, and provides the following TXNIP expression inhibitors.

Item 1. Formula (I):

A thioredoxin interacting protein (TXNIP) expression inhibitor containing a curcumin derivative or its salt represented by the formula: (wherein ^{R1 's} are each independently a fluorine atom, _CH2F- , _CHF2- , _CF3- , _CH2FO- , _CHF2O- or _CF3O- , R2 ^'s are each independently a hydrogen atom or a fluorine atom, A1 ^'s are a hydrogen atom or methyl, A2's are alkyl, cyano, carboxy, alkoxycarbonyl or ^R3- ( _CH2 ) _m- , R3 ^'s are hydroxy, carboxy, cyano, alkylcarbonyloxy, alkoxycarbonyl, alkoxyalkoxy, hydroxyalkoxy or ^CONR4R5 , ^R4 and ^{R5 's} are each independently a hydrogen atom or alkyl, and m's an integer of 1 to 5).
Item 2. The TXNIP expression inhibitor according to Item 1, wherein A ¹ is a hydrogen atom.
Item 3. The TXNIP expression inhibitor according to Item 1 or 2, wherein A ² is R ³ —(CH ₂ ) _m —.
Item 4. The TXNIP expression inhibitor according to Item 3, wherein ^R3 is carboxy.

The present invention also provides the following:
Item 5. A method for suppressing the expression of TXNIP, comprising the step of administering the curcumin derivative represented by formula (I) or a salt thereof to a mammal in need thereof.
Item 6. Use of the curcumin derivative represented by the above formula (I) or a salt thereof in the manufacture of a TXNIP expression inhibitor.
Item 7. The method according to Item 5 or the use according to Item 6, wherein A ¹ is a hydrogen atom.
Item 8. The method according to item 5 or 7, or the use according to item 6 or 7, wherein A ² is R ³ —(CH ₂ ) _m —.
Item 9. The method according to Item 5, 7, or 8, or the use according to Item 6, 7, or 8, wherein ^R3 is carboxy.

The curcumin derivative or its salt represented by the above formula (I) has excellent inhibitory activity against TXNIP expression and is therefore useful as an active ingredient in a TXNIP expression inhibitor.

1 is a graph showing the results of the analysis of the inhibitory effect of curcumin, compounds 1, and 3 on TXNIP mRNA expression, performed in Test Example 1. The vertical axis shows the amount of TXNIP mRNA (ratio when the untreated amount is set to 1.0). Values are mean ± standard error, ^*** p<0.001, ^**** p<0.0001 vs. DMSO, ns: not significant, n=5-6 1 is a graph showing the results of an analysis of the inhibitory effect of curcumin, compounds 1, and 3 on TXNIP protein expression, performed in Test Example 1. The vertical axis shows the amount of TXNIP protein (ratio when the untreated amount is set to 100). Values are mean ± standard error, ^*** p<0.001, ^**** p<0.0001 vs. DMSO, ns: not significant, n=5-6 1 is a graph showing the results of an analysis of the inhibitory effect of curcumin, Compounds 1, and 2 on TXNIP mRNA expression, performed in Test Example 2. The vertical axis shows the amount of TXNIP mRNA (ratio when the untreated amount is set to 1.0). Values are mean ± standard error, ^** p<0.01, ^*** p<0.001, ^**** p<0.0001 vs. control, ns: not significant, n=3. 1 is a graph showing the results of an analysis of the inhibitory effect of curcumin and compounds 1 and 2 on TXNIP protein expression, performed in Test Example 2. The vertical axis shows the amount of TXNIP protein (ratio when the untreated amount is set to 100). Values are mean ± standard error, ^* p<0.05, ^** p<0.01, ^**** p<0.0001 vs. control, ns: not significant, n=3.

The following describes in detail the embodiments of the present invention.

In this specification, the term "comprise" includes the meanings of "essentially consist of" and "consist of only."

The thioredoxin interacting protein (TXNIP) expression inhibitor of the present invention has the formula (I):

The composition is characterized by containing a curcumin derivative or a salt thereof represented by the formula: (wherein ^{R1 's} are each independently a fluorine atom, _CH2F- , _CHF2- , _CF3- , _CH2FO- , _CHF2O- or _CF3O- , R2 ^'s are each independently a hydrogen atom or a fluorine atom, A1 ^'s are a hydrogen atom or methyl, A2's are alkyl, cyano, carboxy, alkoxycarbonyl or ^R3- ( _CH2 ) _m- , ^{R3 's} are hydroxy, carboxy, cyano, alkylcarbonyloxy, alkoxycarbonyl, alkoxyalkoxy, hydroxyalkoxy or ^CONR4R5 , ^R4 and R5 ^'s are each independently a hydrogen atom or an alkyl, and m's an integer of 1 to 5).

The alkyl of ^A2 , ^R4 , and ^R5 may be a linear or branched _C1-6 alkyl, preferably a linear or branched _C1-3 alkyl, which also applies to the alkyls constituting alkylcarbonyloxy, alkoxycarbonyl, alkoxyalkoxy, and hydroxyalkoxy in the curcumin derivatives of formula (I).

Specific examples of C _1-6 alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, and hexyl.

Specific examples of C _1-3 alkyl include methyl, ethyl, n-propyl, and isopropyl.

It is desirable that the curcumin derivative of formula (I) does not cross the blood-brain barrier to prevent side effects on the brain, and to have such properties, it is preferable that ^A2 is ^R3- ( _CH2 ) _m- and ^R3 is carboxy.

A ¹ is preferably a hydrogen atom, that is, the only substituent at the 4-position of 1,6-heptadiene is A ² .

When the curcumin derivative of formula (I) or its salt contains an asymmetric carbon, both the optical isomers separated by conventional methods and the racemate are included in the curcumin derivative of the present invention.

The curcumin derivative of formula (I) may be in the form of a salt. Such salts may be any pharmaceutically acceptable salt, including, for example, alkali metal salts such as potassium salts and sodium salts; alkaline earth metal salts such as calcium salts; and organic amine salts such as triethanolamine salts and tris(hydroxymethyl)aminomethane salts. Some of these salts contain water of crystallization.

The curcumin derivative of formula (I) in which A ¹ is a hydrogen atom can be produced according to known methods (for example, the method described in WO 2010/098502).

Furthermore, the curcumin derivative of formula (I) or a salt thereof in which A ¹ is methyl can be prepared by the method described below.

The compound of formula (IA) can be prepared by hydrolyzing the compound of formula (II).

(wherein R ¹ , R ² and A ² are as defined above.)

Solvents for this reaction include alcohols such as methanol, ethanol, n-propanol, iso-propanol, and butanol; ethers such as aqueous tetrahydrofuran and aqueous dioxane; acid amides such as aqueous dimethylformamide and aqueous dimethylacetamide; sulfoxides such as aqueous dimethyl sulfoxide, and mixed solvents of these.

It is desirable to add a mineral acid to promote this reaction. Examples of such mineral acids include hydrochloric acid, sulfuric acid, nitric acid, and perchloric acid. The mineral acid can be used in an amount of 3 to 10 times, preferably 4 to 6 times, the molar amount of the compound of formula (II).

This reaction can usually be carried out at 0 to 150°C, preferably 30 to 100°C, and the reaction time is usually about 1 to 150 hours.

Compound (II) can be prepared by reacting compound (III) with methyl iodide.

(wherein R ¹ , R ² and A ² are as defined above.)

Solvents for this reaction include aromatic hydrocarbons such as benzene, toluene, and xylene; aliphatic hydrocarbons such as pentane, hexane, petroleum ether, and ligroin; ethers such as diethyl ether, dipropyl ether, dibutyl ether, tetrahydrofuran, and dioxane; ketones such as acetone and 2-butanone; nitriles such as acetonitrile and propionitrile; acid amides such as dimethylformamide and dimethylacetamide; sulfoxides such as dimethyl sulfoxide, and mixed solvents of these.

To promote this reaction, it is desirable to add a base. Examples of such bases include organic bases such as triethylamine, pyridine, N-methylmorpholine, 1,8-diazabicyclo[5.4.0]-7-undecene, and N,N-dimethylaniline; alkali metal carbonates such as sodium carbonate and potassium carbonate; alkali metal bicarbonates such as sodium bicarbonate and potassium bicarbonate; alkali metal hydroxides such as sodium hydroxide and potassium hydroxide; and alkaline earth metal hydroxides such as barium hydroxide and calcium hydroxide. The base can be used in an amount of 3 to 20 times, preferably 5 to 10 times, the molar amount of the compound of formula (III).

This reaction can usually be carried out at 0 to 70°C, and the reaction time is approximately 1 to 48 hours.

It is also desirable to use methyl iodide in an amount of 2 to 20 times the molar amount of the compound of formula (III).

The compound of formula (III) can be produced by condensing the compound of formula (IV) with the compound of formula (V). However, the compound of formula (IV) must be reacted in a molar amount twice that of the compound of formula (V). The compound of formula (V) can be produced by known methods.

(wherein R ¹ , R ² and A ² are as defined above.)

To ensure efficient reaction, it is desirable to conduct the reaction in a solvent in the presence of a boron compound and a base. Examples of boron compounds that can be used in this reaction include boric acid, diboron trioxide, trimethyl borate, triethyl borate, tripropyl borate, tri-n-butyl borate, tri-tert-butyl borate, and mixtures of diboron trioxide with various boric acid esters. It is desirable to use 0.5 to 6 times the molar amount of the boric acid compound relative to the compound of formula (IV).

Examples of the base include primary amines such as n-butylamine, sec-butylamine, tert-butylamine, n-propylamine, n-hexylamine, and cyclohexylamine; and secondary amines such as morpholine, piperidine, and 1,2,3,4-tetrahydroquinoline. It is desirable to use a 1-fold molar amount of base relative to the compound of formula (V).

Examples of solvents that can be used include aromatic hydrocarbons such as benzene, toluene, and xylene; aliphatic hydrocarbons such as pentane, hexane, heptane, petroleum ether, and ligroin; ethers such as diethyl ether, dipropyl ether, dibutyl ether, tetrahydrofuran, and dioxane; esters such as methyl acetate, ethyl acetate, and methyl propionate; acid amides such as dimethylformamide and dimethylacetamide; sulfoxides such as dimethyl sulfoxide; phosphoric acid amides such as hexamethylphosphortriamide, and mixed solvents thereof.

The reaction temperature is usually 0 to 150°C, preferably 0 to 100°C, and the reaction time is usually about 0.5 to 24 hours.

After the reaction, the reaction mixture must be treated with an acid to decompose the resulting boron complex of the compound of formula (III). Examples of acids that can be used include mineral acids such as hydrochloric acid and sulfuric acid, and organic acids such as acetic acid and propionic acid.

The compound of formula (IV) can be produced by reacting the compound of formula (VI) with chlorodimethyl ether. The compound of formula (VI) can be produced by known methods.

(wherein ^R1 and ^R2 are as defined above.)

Examples of solvents include aromatic hydrocarbons such as benzene, toluene, and xylene; aliphatic hydrocarbons such as pentane, hexane, heptane, petroleum ether, and ligroin; ethers such as diethyl ether, dipropyl ether, dibutyl ether, tetrahydrofuran, and dioxane; ketones such as acetone and 2-butanone; nitriles such as acetonitrile and propionitrile; acid amides such as dimethylformamide and dimethylacetamide; sulfoxides such as dimethyl sulfoxide; halogenated hydrocarbons such as dichloromethane, carbon tetrachloride, and 1,2-dichloroethane, and mixed solvents thereof.

To promote this reaction, it is desirable to add a base. Examples of such bases include organic bases such as triethylamine, pyridine, N-methylmorpholine, N-methylpiperidine, 1,8-diazabicyclo[5.4.0]-7-undecene, and N,N-dimethylaniline; alkali metal carbonates such as sodium carbonate and potassium carbonate; alkali metal bicarbonates such as sodium bicarbonate and potassium bicarbonate; and alkali metal hydroxides such as sodium hydroxide and potassium hydroxide. The base can be used in an amount of 1 to 3 times the molar amount of the compound of formula (VI), preferably 1.4 to 1.8 times the molar amount.

This reaction can usually be carried out at 0 to 50°C, and the reaction time is usually around 1 to 48 hours.

The curcumin derivative of formula (I) obtained by the above-mentioned production method and its associated methods can be isolated and purified by known means, such as concentration, vacuum concentration, distillation, fractional distillation, solvent transfer, solvent extraction, crystallization, recrystallization, chromatography, etc.

When the curcumin derivative of formula (I) is obtained in its free form, a salt can be formed by conventional methods.

Specific examples of curcumin derivatives of formula (I) are shown in Table 1 below.

Many curcumin derivatives of formula (I) are hydrophobic compounds with low solubility in water. For compounds to be administered to living organisms, high water solubility is desirable, and among curcumin derivatives of formula (I), salts are more desirable.

The curcumin derivative of formula (I) or its salt has excellent inhibitory activity against TXNIP expression at both the mRNA level and the protein level, and can therefore be used as an active ingredient in a TXNIP expression inhibitor.

The nucleotide sequence of the TXNIP gene is registered on the NCBI website under RefSeq Accession No. NM_006472 (human), NM_001313972 (human), etc., and the amino acid sequence is registered under RefSeq Accession No. NP_006463 (human), NP_001300901 (human), etc.

In addition, suppression of TXNIP expression means reducing the expression of the TXNIP gene, and can be confirmed by a decrease in the amount of transcription products produced from the TXNIP gene, a decrease in the amount of translation products produced from the TXNIP gene, etc.

Inhibition of TXNIP expression improves resistance to oxidative stress. Diseases associated with oxidative stress include diabetes, atherosclerosis, Parkinson's disease, Alzheimer's disease, arteriosclerosis, stroke, heart failure, myocardial infarction, schizophrenia, bipolar disorder, sickle cell disease, fragile X syndrome, chronic fatigue syndrome, and cancer (e.g., gastric cancer, colorectal cancer (rectal cancer, colon cancer), small intestine cancer, liver cancer, pancreatic cancer, lung cancer, pharyngeal cancer, esophageal cancer, kidney cancer, gallbladder and bile duct cancer, head and neck cancer, bladder cancer, prostate cancer, breast cancer, uterine cancer (cervical cancer, endometrial cancer), ovarian cancer, brain tumor, thymoma, leukemia, malignant lymphoma, etc.), and the TXNIP expression inhibitors of the present invention can be used to treat and prevent these conditions.

The curcumin derivative of formula (I) is highly safe because its basic structure is curcumin, a food ingredient.

The TXNIP expression inhibitor of the present invention can be used as a pharmaceutical composition, food composition, etc.

The pharmaceutical composition of the present invention is administered to mammals, including humans. The pharmaceutical composition of the present invention may be administered locally or systemically. There are no particular limitations on the administration method, and the composition may be administered orally or parenterally. Parenteral administration routes include subcutaneous, intraperitoneal, intravenous, arterial, or spinal fluid injection or infusion, and transdermal administration.

The pharmaceutical compositions of the present invention are in a pharmaceutically acceptable form suitable for administration to humans and contain physiologically acceptable additives. Such pharmaceutical compositions may contain, as appropriate, pharmaceutically acceptable diluents, buffers, solubilizers (e.g., cyclodextrin, polyethylene glycol, or surfactants such as Tween™, Pluronic™, Cremophor™, and phospholipids), soothing agents, etc., and may further contain, as necessary, components such as pharmaceutically acceptable solvents, stabilizers, or antioxidants (e.g., ascorbic acid, etc.). The dosage of the pharmaceutical compositions of the present invention is selected appropriately depending on the method of administration, the patient's age, sex, and other conditions, and the severity of the disease.

The content of the curcumin derivative of formula (I) or its salt in the pharmaceutical composition of the present invention can be appropriately selected from the range of 0.01 to 100% by mass, preferably 0.1 to 100% by mass.

The food compositions of the present invention include any food compositions that can be ingested by animals (including humans). The food compositions of the present invention may contain, as needed, amino acids, nucleic acids, minerals, vitamins, flavonoids, quinones, polyphenols, binders, cooling agents, sweeteners, essential fatty acids, disintegrants, lubricants, flavorings, stabilizers, colorants, preservatives, surfactants, sustained-release regulators, solubilizers, humectants, etc.

The type of food composition of the present invention is not particularly limited, and examples include beverages (soft drinks such as coffee, juice, and tea drinks, dairy drinks, carbonated drinks, alcoholic beverages such as sake, Western liquor, and fruit liquor); spreads (custard cream, etc.); pastes (fruit paste, etc.); Western confectionery (chocolate, donuts, pies, cream puffs, gum, jelly, candy, cookies, cakes, puddings, etc.); Japanese confectionery (daifuku, mochi, manju, castella, anmitsu, yokan, etc.); frozen desserts (ice cream, popsicles, sorbet, etc.); foods (curry, beef bowls, rice porridge, miso soup, soup, meat sauce, pasta, pickles, jam, royal jelly, etc.); dairy products; fermented foods (yogurt, royal jelly, etc.); seasonings (dressings, furikake, umami seasonings, soup bases, etc.).

The food composition of the present invention can also be used as a health food, functional food, nutritional supplement, dietary supplement, food for specified health uses, or food with functional claims. When used as a supplement, the dosage unit form is not particularly limited and can be selected appropriately, and examples include tablets, granules, liquids, capsules, powders, etc.

The content of the curcumin derivative of formula (I) or its salt in the food composition of the present invention can be appropriately selected from the range of 0.01 to 100% by mass, preferably 0.1 to 100% by mass, of the total amount of the food composition.

The intake amount of the food composition of the present invention can be appropriately determined depending on various conditions such as the consumer's weight, age, sex, symptoms, etc.

Next, synthesis and test examples related to the present invention are described, but the present invention is not limited to these. The NMR spectra in the following synthesis examples were measured using a JEOL RESONANCE ECZ-400S.

Synthesis Example 1 Synthesis of 1,7-bis(4'-hydroxy-3'-trifluoromethoxy)phenyl-4-carboxypropyl-1,6-heptadiene-3,5-dione (Compound 2) 90 mg (0.156 mmol) of 1,7-bis(4'-hydroxy-3'-trifluoromethoxy)phenyl-4-methoxycarbonylpropyl-1,6-heptadiene-3,5-dione, synthesized with reference to the description in International Publication WO 2010/098502, was added to 4.7 mL (0.468 mmol) of 0.1 M aqueous sodium hydroxide solution and stirred at room temperature for 2 hours. The reaction mixture was adjusted to pH 2 with 1 M hydrochloric acid and then extracted with ethyl acetate. The extract was washed with water and saturated brine, dried over magnesium sulfate, and the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: ethyl acetate:hexane = 1:1) to give 35 mg (40.0%) of 1,7-bis(4'-hydroxy-3'-trifluoromethoxy)phenyl-4-carboxypropyl-1,6-heptadiene-3,5-dione having a melting point of 160-161°C.
¹⁹ FNMR (d ₆ DMSO): δ -58.34 (s), -58.47 (s), ¹ HNMR (d ₆ DMSO): δ1.49 (0.7H, m), δ1.63 (1.3H, m), δ1.84 (0.7H, m), δ2.24 (0.7H, m), δ2.33 (0.5H, m), δ2.49 (0.7H, m), δ2.5-2.7 (1.5H), δ4.58 (0.3H, t, J=7.0Hz), δ6.95 (0.7H, d, J=15.6Hz), δ7.04 (0.7H, d, J=9.0Hz), δ7.06 (1.3H, d, J=9.0Hz), δ7.28 (1.3H, d, J=15.6Hz), δ7.61 (0.7H, dd, J=2.0Hz, 9.0Hz), δ7.63 (1.3H, d, J=15.6Hz), δ7.64 (0.7H, d, J=15.6Hz), δ7.65 (0.7H, br.s), δ7.75 (1.3H, dd, J=2.0Hz, 9.0Hz), δ7.78 (1.3H, br.s), δ17.80 (0.7H, s).

Synthesis Example 2 Synthesis of 1,7-bis(4'-hydroxy-3'-methoxy)phenyl-4-carboxyethyl-1,6-heptadiene-3,5-dione (Compound 3) (1) A solution of 186 mg (1.0 mmol) of methyl 4-acetyl-5-oxohexanoate (Sigma-Aldrich) and 56 mg (0.8 mmol) of boron trioxide (Nacalai Tesque) in 2 mL of ethyl acetate was heated at 60°C for 30 minutes, followed by the addition of 304 mg (2.0 mmol) of vanillin (Nacalai Tesque) and 0.54 mL (2.0 mmol) of tri-n-butyl borate (Tokyo Chemical Industry Co., Ltd.), and heating at the same temperature for an additional 30 minutes. Next, 0.1 mL (1.0 mmol) of n-butylamine (Nacalai Tesque) was added, and the mixture was heated at the same temperature for 4 hours. After the reaction mixture was cooled to room temperature, 1M hydrochloric acid (2 mL) was added and the mixture was stirred vigorously for 15 minutes. The reaction mixture was extracted with ethyl acetate, and the extract was washed with water and then with saturated brine, and then dried over magnesium sulfate. The solvent was distilled off under reduced pressure, and the resulting residue was purified by silica gel column chromatography (eluent: ethyl acetate:hexane = 1:1). A small amount of dichloromethane was added to the resulting substance, and the mixture was allowed to stand at room temperature to yield 208 mg (45.8%) of 1,7-bis(4'-hydroxy-3'-methoxy)phenyl-4-methoxycarbonylethyl-1,6-heptadiene-3,5-dione with a melting point of 124-125°C.
¹ HNMR (d ₆ DMSO): δ2.07 (1.3H, m), δ2.31 (1.3H, m), δ2.48 (0.7H, m), δ2.98 (0.7H, m), δ3.54 (1H, s), δ3.59 (2H, s), δ3.80 (4H, s), δ3.85 (2H, s), δ4.58 (0.6H, m), δ6.80 (1.3H, d, J=8.0Hz), δ6.83 (0.7H, d, J=8.0Hz), δ6.91 (1.3H, d, J=15.6Hz), δ7.13 (0.7H, d, J=15.6Hz), δ7.16 (1.3H, dd, J=8.0Hz, 2.0Hz), δ7.23 (0.7H, dd, J=8.0Hz, 2.0Hz), δ7.32 (1.3H, d, J=2.0Hz), δ7.35 (0.7H, d, J=2.0Hz), δ7.60 (0.7H, d, J=15.6Hz), δ7.61 (1.3H, d, J=15.6Hz), δ18.03 (0.4H, s).

(2) 182 mg (0.4 mmol) of 1,7-bis(4'-hydroxy-3'-methoxy)phenyl-4-methoxycarbonylethyl-1,6-heptadiene-3,5-dione obtained in step (1) was added to 12 mL (1.2 mmol) of 0.1 M aqueous sodium hydroxide solution and stirred at room temperature for 1 hour. The reaction mixture was adjusted to pH 2 with 1 M hydrochloric acid and then extracted with ethyl acetate. The extract was washed with water and saturated brine, dried over magnesium sulfate, and the solvent was distilled off under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: ethyl acetate) to yield 126 mg (71.5%) of 1,7-bis(4'-hydroxy-3'-methoxy)phenyl-4-carboxyethyl-1,6-heptadiene-3,5-dione with a melting point of 150-151°C.
¹ HNMR (d ₆ DMSO): δ2.04 (1.3H, m), δ2.22 (1.3H, m), δ2.40 (0.7H, m), δ2.93 (0.7H, m), δ3.79 (4H, s), δ3.83 (2H, s), δ4.56 (0.6H, m), δ6.79 (0.7H, d, J=8.0Hz), δ6.81 (0.7H, d, J=15.6Hz), δ6.90 (1.3H, d, J=15.6Hz), δ7.1-7.25 (3.3H), δ7.31 (1.3H, d, J=2.0Hz), δ7.33 (0.7H, d, J=2.0Hz), δ7.59 (0.7H, d, J=15.6Hz), δ7.60 (1.3H, d, J=15.6Hz), δ18.00 (0.4H, s).

Compound 1 used in the following test examples was synthesized according to the description in Synthesis Example 2 of WO 2010/098502. Compound 1 has the following structure:

[Test Example 1] Analysis of the TXNIP expression inhibitory effect of compounds 1 and 3
ARPE-19 cells were seeded in 12-well plates at 2 × ¹⁰ cells/well in DMEM/F-12 (10% FBS) medium and allowed to adhere for 24 hours. Cells were then treated with medium containing or without curcumin, Compound 1, or Compound 3 (1 μM or 5 μM) for 24 hours. After treatment, cells were extracted for RNA or protein analysis. TXNIP mRNA levels were analyzed using real-time PCR, and TXNIP protein levels were analyzed using Western blotting.

The results are shown in Figures 1 and 2. TXNIP mRNA levels (Figure 1) were significantly reduced in cells treated with compound 1 at concentrations of 1 μM and 5 μM compared to untreated cells, but no difference was observed between cells treated with curcumin and compound 3 at concentrations of 1 μM and 5 μM compared to untreated cells. TXNIP protein levels (Figure 2) were also significantly reduced in cells treated with compound 1 at concentrations of 1 μM and 5 μM compared to untreated cells, but no difference was observed between cells treated with curcumin and compound 3 at concentrations of 1 μM and 5 μM compared to untreated cells. These results suggest that compound 1 has the effect of inhibiting TXNIP expression.

[Test Example 2] Analysis of the TXNIP expression inhibitory effect of Compounds 1 and 2
ARPE-19 cells were seeded in 12-well plates at 2 × ¹⁰ cells/well in DMEM/F-12 (10% FBS) medium and allowed to adhere for 24 hours. Cells were then treated with medium containing or without curcumin, Compound 1, or Compound 2 (0.5 μM, 1 μM, or 3 μM) for 24 hours. After treatment, cells were extracted for RNA or protein analysis. TXNIP mRNA levels were analyzed using real-time PCR, and TXNIP protein levels were analyzed using Western blotting.

The results obtained are shown in Figures 3 and 4. In Figure 3, a concentration-dependent decrease in TXNIP mRNA levels was observed in cells treated with compound 1 and compound 2 at concentrations of 0.5 μM, 1 μM, or 3 μM, but no difference was observed between cells treated with curcumin and untreated cells. Similarly, a concentration-dependent decrease in TXNIP protein levels (Figure 4) was observed in cells treated with compound 1 and compound 2, but no difference was observed between cells treated with curcumin and untreated cells. These results suggest that not only compound 1 but also compound 2 have the effect of suppressing TXNIP expression.

Claims (4)

Formula (I):
A thioredoxin interacting protein (TXNIP) expression inhibitor containing a curcumin derivative or its salt represented by the formula: (wherein ^{R1 's} are each independently a fluorine atom, _CH2F- , _CHF2- , _CF3- , _CH2FO- , _CHF2O- or _CF3O- , R2 ^'s are each independently a hydrogen atom or a fluorine atom, A1 ^'s are a hydrogen atom or methyl, A2's are alkyl, cyano, carboxy, alkoxycarbonyl or ^R3- ( _CH2 ) _m- , R3 ^'s are hydroxy, carboxy, cyano, alkylcarbonyloxy, alkoxycarbonyl, alkoxyalkoxy, hydroxyalkoxy or ^CONR4R5 , ^R4 and ^{R5 's} are each independently a hydrogen atom or alkyl, and m's an integer of 1 to 5). The TXNIP expression inhibitor according to claim 1, wherein ^A1 is a hydrogen atom. The TXNIP expression inhibitor according to claim 1 or 2, wherein ^A2 is ^R3- ( _CH2 ) _m- . The TXNIP expression inhibitor according to claim 3, wherein ^R3 is carboxy.