DNA methylation promoter, oral composition for promoting DNA methylation, and use of compound

Abstract

This DNA methylation promoter contains a compound represented by formula (I), a racemate thereof, or a salt thereof. [In formula (I), X represents a carbon atom or an oxygen atom, R1 represents a saturated aliphatic hydrocarbon group optionally including a ring structure, R2 represents a saturated or unsaturated aliphatic hydrocarbon group that has 10-30 carbon atoms and that optionally has a substituent group which is an alkyl group having 1-4 carbon atoms or an alkoxy group having 1-4 carbon atoms, a carbon atom of a glycerol skeleton optionally has a substituent group, * represents an oxygen atom bound to R3, and R3 represents an AA optionally having a substituent group.]

Description

Use of DNA methylation promoter, oral composition and compound for promoting DNA methylation

[0001] The present invention relates to the use of DNA methylation promoters, oral compositions and compounds for promoting DNA methylation.

[0002] Methylation of the 5th carbon atom of cytosine in DNA (5-cytosine) is a physiological DNA modification mechanism. Most DNA methylation in mammals occurs in sequences of consecutive cytosines and guanines (CpG sequences). 5-cytosine methylation in mammalian DNA plays an important role in many biological processes, including genomic imprinting, cell fate determination, organization of chromatin structure, regulation of gene expression, and suppression of retrotransposon activity (see Non-Patent Documents 1 to 3).

[0003] It has also been shown that increased autonomous transposition of retrotransposons due to exposure to chemicals during pregnancy or aging may cause changes in the function of other genes (see Non-Patent Document 4), and may cause chronic inflammation due to aging (see Non-Patent Document 5).

[0004] It is assumed that a decrease in methylation of genomic DNA is associated with various diseases. Patent documents 1 and 2 disclose that extracts of Rugosa rose and bottle gourd fruit, respectively, have an inhibitory effect on DNA demethylation induced by ultraviolet irradiation and an effect of promoting remethylation of DNA demethylation.

[0005] JP 2019-77621 A JP 2019-77622 A

[0006] Rudolf Jaenisch and Adrian Bird, “Epigenetic regulation of gene expression: how the genome integrates intrinsic and 'Environmental signals', Nature Genetics, 2003, 33, 245-254 Zachary D. Smith and Alexander Meissner, “DNA methylation: roles in mammalian development”, Nature Reviews Genetics, 2013, 14, 204-220 Keiko Nohara, 5 others, “Gestational arsenic exposure induces site-specific DNA hypomethylation in active retrotransposon subfamilies in offspring sperm in mice”, Epigenetics & Chromatin, 2020, 13, Article number: 53Christopher H. Switzer, 4 others, “NOS2 and S-nitrosothiol signaling induces DNA hypomethylation and LINE-1 retrotransposon "L1 drives IFN in senescent cells and "promotes age-associated inflation", Nature, 2019, 566, 73-78

[0007] In Patent Documents 1 and 2, the active ingredient in the extract of Rugosa rose or bottle gourd fruit that has the effect of inhibiting DNA demethylation or promoting remethylation of DNA demethylation is not identified. In the development of medicines and the like, it is desirable to identify the active ingredient.

[0008] The present invention has been made in view of the above-mentioned circumstances, and aims to provide a DNA methylation promoter that can promote methylation of genomic DNA, an oral composition for promoting DNA methylation, and use of the compound.

[0009] A DNA methylation promoter according to a first aspect of the present invention includes a compound represented by formula (I), a racemate thereof, or a salt thereof. [In formula (I), X represents a carbon atom or an oxygen atom, and R １ represents a saturated aliphatic hydrocarbon group which may contain a cyclic structure, and R ２ represents a saturated or unsaturated aliphatic hydrocarbon group having 10 to 30 carbon atoms which may have a substituent, which is an alkyl group having 1 to 4 carbon atoms or an alkoxy group having 1 to 4 carbon atoms, the carbon atoms of the glycerol skeleton may have a substituent, R ３ The oxygen atom bonded to is *, and R ３ may have a substituent It is.]

[0010] An oral composition for promoting DNA methylation according to a second aspect of the present invention comprises a compound represented by formula (I), a racemate thereof, or a salt thereof. [In formula (I), X represents a carbon atom or an oxygen atom, and R １ represents a saturated aliphatic hydrocarbon group which may contain a cyclic structure, and R ２ represents a saturated or unsaturated aliphatic hydrocarbon group having 10 to 30 carbon atoms which may have a substituent, which is an alkyl group having 1 to 4 carbon atoms or an alkoxy group having 1 to 4 carbon atoms, the carbon atoms of the glycerol skeleton may have a substituent, R ３ The oxygen atom bonded to is *, and R ３ may have a substituent It is.]

[0011] X is an oxygen atom, and R １ is an alkyl group having 18 carbon atoms.

[0012] R ２ is R ２ It may be a monovalent group which, when expressed as COOH, represents arachidonic acid, oleic acid, or palmitic acid.

[0013] Use of a compound according to a third aspect of the present invention is use of a compound represented by formula (I), its racemate, or a salt thereof for the manufacture of a DNA methylation promoter or an oral composition for promoting DNA methylation. [In formula (I), X represents a carbon atom or an oxygen atom, and R １ represents a saturated aliphatic hydrocarbon group which may contain a cyclic structure, and R ２ represents a saturated or unsaturated aliphatic hydrocarbon group having 10 to 30 carbon atoms which may have a substituent, which is an alkyl group having 1 to 4 carbon atoms or an alkoxy group having 1 to 4 carbon atoms, the carbon atoms of the glycerol skeleton may have a substituent, R ３ The oxygen atom bonded to is *, and R ３ may have a substituent It is.]

[0014] According to the present invention, methylation of genomic DNA can be promoted.

[0015] Fig. 1 is a diagram showing a fluorescent stained image according to Test Example 1. Fig. 2 is a diagram showing fluorescence intensity based on the fluorescent stained image shown in Fig. 1. Fig. 3 is a diagram showing a fluorescent stained image according to Test Example 2. Fig. 4 is a diagram showing the anti-5-methylcytosine (5-mC) antibody positive rate based on the fluorescent stained image shown in Fig. 3. Fig. 5 is a diagram showing the anti-5-mC antibody positive rate according to Test Example 3.

[0016] Embodiments of the present invention will be described with reference to the drawings. Note that the present invention is not limited to the following embodiments and drawings. Note that in the following embodiments, the expressions "have," "include," or "contain" also include the meaning of "consisting of" or "consisting of."

[0017] (Embodiment) A DNA methylation promoter according to this embodiment includes a compound represented by formula (I), a racemate thereof, or a salt thereof.

[0018]

[0019] In formula (I), X represents a carbon atom or an oxygen atom. Preferably, X is an oxygen atom. １represents a saturated aliphatic hydrocarbon group which may contain a cyclic structure. The number of carbon atoms in the saturated aliphatic hydrocarbon group is not particularly limited, but is, for example, 10 to 30, 10 to 25, or 10 to 20. The number of carbon atoms constituting the cyclic structure is, for example, 3 to 6. The cyclic structure is formed by the cycloalkylene group R １ The cycloalkyl group may be included in R １ The number of carbon atoms constituting the cyclic structure is preferably 3. The saturated aliphatic hydrocarbon group may be a cyclic structure represented by R １ Preferably, R １ is an alkyl group having 18 carbon atoms.

[0020] R ２ represents a saturated or unsaturated aliphatic hydrocarbon group having 10 to 30 carbon atoms which may have a substituent which is an alkyl group having 1 to 4 carbon atoms or an alkoxy group having 1 to 4 carbon atoms. For example, R ２ is R ２ It is a monovalent group that, when expressed as COOH, represents an ω-3 fatty acid, an ω-6 fatty acid, an ω-7 fatty acid, an ω-9 fatty acid, or an ω-10 fatty acid. Preferably, R ２ is R ２ When COOH is used, it is a monovalent group representing arachidonic acid, a monovalent group representing docosahexaenoic acid, a monovalent group representing oleic acid, a monovalent group representing palmitic acid, a monovalent group representing eicosapentaenoic acid, or a monovalent group representing linoleic acid. ２ is R ２ It is a monovalent group which, when expressed as COOH, represents arachidonic acid, oleic acid, or palmitic acid.

[0021] The carbon atoms of the glycerol skeleton in formula (I) may have a substituent. Examples of the substituent that the carbon atoms of the glycerol skeleton may have include a hydroxy group, a halogen, an aryl group, an alkyl group having 1 to 4 carbon atoms, and an alkoxy group having 1 to 4 carbon atoms. R ３ The oxygen atom bonded to is *, and R ３ may have a substituent It is. ３Examples of the substituent that may be contained in the alkyl group include a hydroxy group, a halogen atom, an aryl group, an alkyl group having 1 to 4 carbon atoms, and an alkoxy group having 1 to 4 carbon atoms.

[0022] The salts of the compound represented by formula (I) and the racemic salts of the compound are not particularly limited as long as they are pharmacologically acceptable salts, and may be either acidic salts or basic salts. Examples of the salts include alkali metal salts such as lithium salt, sodium salt, and potassium salt; alkaline earth metal salts such as magnesium salt and calcium salt; inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, oxalate, and phosphate; acetate, propionate, hexanoate, cyclopentanepropionate, glycolate, pyruvate, lactate, malonate, succinate, malate, fumarate, tartrate, citrate, benzoate, o-(4-hydroxybenzoyl)benzoate, cinnamate, mandelate, methanesulfonate, ethoxylated ... and organic acid salts such as benzenesulfonate, 1,2-ethanedisulfonate, 2-hydroxyethanesulfonate, benzenesulfonate, p-chlorobenzenesulfonate, 2-naphthalenesulfonate, p-toluenesulfonate, camphorsulfonate, glucoheptanoate, 3-phenylpropionate, trimethyl acetate, tert-butyl acetate, lauryl sulfate, gluconate, glutamate, hydroxynaphthoate, salicylate, stearate, trifluoroacetate (TFA) salt, maleate, and muconate.

[0023] For example, the DNA methylation promoter according to this embodiment includes a compound represented by formula (II), a racemate thereof, or a salt thereof. １ and R ２ respectively represent X and R in the above formula (I). １ and R ２ is the same as

[0024]

[0025] Preferably, the DNA methylation promoter according to this embodiment includes a compound represented by any one of the following formulas (i), (ii), and (iii), a racemate thereof, or a salt thereof.

[0026]

[0027]

[0028]

[0029] The compound according to this embodiment can be synthesized by a known method. For example, tert-butyl(2-((tert-butoxy((R)-2-hydroxy-3-(octadecyloxy)propoxy)phosphoryl)oxy)ethyl)carbamate is converted to R ２ COOH is reacted. The resulting product is deprotected by the action of a strong acid such as trifluoroacetic acid, to obtain the compound according to this embodiment. The condensing agent is not particularly limited, and examples that can be used include 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) hydrochloride, dicyclohexylcarbodiimide (DCC), HATU, HBTU, TATU, TBTU, and diphenylphosphoryl azide (DPPA). A nucleophile such as dimethylaminopyridine (DMAP) may be used to promote the dehydration condensation reaction using the condensing agent.

[0030] The DNA methylation promoter according to this embodiment contains the compound represented by formula (I), its racemate, or a salt thereof (hereinafter also referred to as "compound, etc.") as an active ingredient, and may also contain other pharmacologically acceptable ingredients. Examples of other pharmacologically acceptable ingredients include excipients, lubricants, binders, disintegrants, solvents, solubilizers, suspending agents, isotonicity agents, buffers, soothing agents, etc. Furthermore, additives such as preservatives, antioxidants, colorants, and sweeteners may be incorporated into the DNA methylation promoter as needed.

[0031] Examples of excipients include lactose, sucrose, D-mannitol, starch, crystalline cellulose, light anhydrous silicic acid, etc. Examples of lubricants include magnesium stearate, calcium stearate, talc, colloidal silica, etc. Examples of binders include crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, etc. Examples of disintegrants include starch, carboxymethylcellulose, carboxymethylcellulose calcium, croscarmellose sodium, carboxymethylstarch sodium, etc.

[0032] Examples of solvents include water for injection, alcohol, propylene glycol, macrogol, etc. Examples of solubilizing agents include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, etc. Examples of suspending agents include surfactants, hydrophilic polymers, etc., such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glycerin monostearate, polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, etc.

[0033] Examples of isotonic agents include sodium chloride, glycerin, D-mannitol, etc. Examples of buffers include phosphate, acetate, carbonate, citrate buffer solutions, etc. Examples of soothing agents include benzyl alcohol, etc. Examples of preservatives include paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, etc. Examples of antioxidants include sulfites, ascorbic acid, etc.

[0034] As shown in the examples below, the DNA methylation promoter according to this embodiment promotes methylation of genomic DNA compared to before administration of the DNA methylation promoter.

[0035] The DNA methylation promoter of this embodiment promotes the methylation of genomic DNA, and is therefore useful for preventing and treating symptoms or diseases caused by a decrease or suppression of methylation of genomic DNA or a decrease in methylated genomic DNA.

[0036] The DNA methylation promoter is produced by a known method and contains the above-mentioned compound, etc. as an active ingredient. The DNA methylation promoter contains, for example, 0.1 to 99 wt %, 1 to 50 wt %, and preferably 1 to 20 wt % of the compound, etc. as an active ingredient.

[0037] The DNA methylation promoter according to this embodiment is administered to humans or animals other than humans. The animals are preferably mammals, more specifically, dogs, cats, cows, pigs, horses, sheep, deer, etc. The route of administration of the DNA methylation promoter to humans, etc. is not particularly limited. The DNA methylation promoter is preferably used as an external preparation, an injection, or an oral preparation.

[0038] The DNA methylation promoter is provided in the form of, for example, a liquid, tablet, granule, fine granule, powder, tablet, capsule, or the like.

[0039] The dosage of the DNA methylation promoter is determined appropriately depending on the age, weight, symptoms, etc. of the human subject. The DNA methylation promoter is administered so that the compound or the like is in an effective amount. The effective amount is the amount of the compound or the like necessary to promote methylation of genomic DNA.

[0040] As described above in detail, the DNA methylation promoter according to this embodiment can promote methylation of genomic DNA.

[0041] In another embodiment, there is provided an oral composition for promoting DNA methylation, which comprises the compound or the like. Specific examples of the oral composition for promoting DNA methylation include supplements, food compositions, foods and beverages, functional foods, and food additives. The DNA methylation promoter may be used as a supplement or added to foods and beverages and functional foods.

[0042] The form of the supplement is not particularly limited and may be any form such as tablets, powders, granules, capsules, sugar-coated tablets, films, lozenges, chewable tablets, solutions, emulsions, suspensions, etc. The supplement may contain any ingredient typically used as a supplement.

[0043] The term "functional food" refers to food or beverages taken for the purpose of maintaining health, and includes foods with health claims, foods with specified health uses, foods with functional claims, foods with nutrient functions, health foods, and nutritional supplements. Functional foods are preferably foods with health claims or foods with nutrient functions. When commercializing a product as a functional food, various additives used in foods, specifically colorants, preservatives, thickening agents, antioxidants, bleaching agents, antibacterial and antifungal agents, acidulants, sweeteners, seasonings, emulsifiers, strengthening agents, manufacturing agents, flavorings, etc., may be added to the oral composition for promoting DNA methylation.

[0044] Functional foods may be either foods or beverages, and are not particularly limited as long as they can be taken orally. Examples of functional foods include beverages, confectioneries, processed grain products, paste products, dairy products, seasonings, etc. Examples of beverages include nutritional drinks, soft drinks, black tea, green tea, etc. Examples of confectioneries include candy, cookies, tablet candy, chewing gum, jelly, etc. Examples of processed grain products include bread, cooked rice, biscuits, etc. Examples of paste products include sausages, ham, kamaboko, etc. Examples of dairy products include butter, yogurt, etc.

[0045] The oral composition for promoting DNA methylation may be added to food as a food additive. In this case, the food additive may be in the form of a paste, gel, powder, liquid, suspension, emulsion, granules, or the like, so as to be easily added to food.

[0046] The oral composition for promoting DNA methylation may contain water, vitamins, minerals, organic acids, organic bases, fruit juice, flavors, functional ingredients, food additives, etc., within the range that maintains the effect of promoting genomic DNA methylation. The oral composition for promoting DNA methylation can be produced by a known method, adding ingredients other than the above-mentioned compounds, etc., as needed.

[0047] The oral composition for promoting DNA methylation may be divided and stored in one or more containers so that the daily intake amount is the above-mentioned intake amount. In this case, preferably, one container contains one day's worth of the oral composition for promoting DNA methylation.

[0048] The oral composition for promoting DNA methylation is provided in a form that can be distinguished from other products in that it is used to promote methylation of genomic DNA. For example, at least one of the packaging, instructions, and promotional materials for the oral composition for promoting DNA methylation indicates that the composition has the effect of promoting methylation of genomic DNA.

[0049] When an oral composition for promoting DNA methylation is provided as a food or beverage composition, it may be provided or sold as a food or beverage labeled with the use of promoting DNA methylation (including health uses). "Labeling" includes all acts intended to inform consumers of the above-mentioned use. Any expression that can recall or infer the above-mentioned use is considered to be "labeling," regardless of the purpose, content, object, or medium of the labeling.

[0050] It is preferable that the "labeling" be done in an expression that allows consumers to directly recognize the intended use. Specifically, this includes acts of transferring, delivering, displaying for the purpose of transferring or delivering, or importing food and beverage products or product packaging that lists the intended use, displaying or distributing advertisements, price lists, or transaction documents that list the intended use, or providing information containing the above-mentioned uses by electromagnetic means (such as the Internet).

[0051] "Labeling" includes labeling as health food, functional food, enteral nutritional food, special dietary food, health functional food, food for specified health uses, food with nutrient functions, food with functional claims, quasi-drugs, etc. Among these, labeling approved under systems related to foods for specified health uses, foods with nutrient functions, or foods with functional claims, or similar systems, is particularly included. Specific examples include labeling as a food for specified health uses, labeling as a conditional food for specified health uses, labeling that indicates an effect on the structure or function of the body, labeling that reduces disease risk, and labeling of functionality based on scientific evidence. More specifically, labeling as a food for specified health uses (especially labeling of health uses) and similar labeling are typical examples.

[0052] In another aspect of the present embodiment, there is provided use of the compound or the like for manufacturing a DNA methylation promoter or an oral composition for promoting DNA methylation. In another aspect of the present embodiment, there is provided a method for promoting DNA methylation, comprising the step of administering the compound or the like to a subject. In another aspect of the present embodiment, there is provided the compound or the like for use in promoting DNA methylation.

[0053] The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0054] Example 1 Synthesis of Compound KIT-13 KIT-13 was synthesized as a compound according to formula (I) as follows.

[0055]

[0056] To a dichloromethane solution (3 mL) of compound 1 (tert-butyl(2-((tert-butoxy((R)-2-hydroxy-3-(octadecyloxy)propoxy)phosphoryl)oxy)ethyl)carbamate, 187 mg, 0.30 mmol), EDC hydrochloride (115 mg, 0.60 mmol), DMAP (37 mg, 0.30 mmol), and arachidonic acid (100 mg, 0.33 mmol) were added at room temperature, and the mixture was stirred for 20 hours. The reaction solution was diluted with ethyl acetate and then washed with water. The ethyl acetate layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. The residue was purified by column chromatography to give the desired (2R)-1-((tert-butoxy(2-((tert-butoxycarbonyl)amino)ethoxy)phosphoryl)oxy)-3-(octadecyloxy)propan-2-yl(5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoate as a colorless liquid (260 mg, 95%).

[0057] 1 H NMR (500 MHz, CDCl3) δ 5.43-5.31 (m, 8H), 5.16 (quin, J = 4.9 Hz, 1H), 5.16 (br, 1H), 4.21 (m, 1H), 4.14-4.09 (m, 1H), 4.07-4.02 (m, 2H), 3.55 (d, J = 5.3 Hz, 2H), 3.47-3.38 (m, 5.3 Hz), 2.85-2.79 (m, 6H), 2.35 (t, J = 7.6 Hz), 2.12 (q, J = 7.0 Hz, 2H), 2.06 (q, J = 7.1 Hz, 2H), 1.74-1.67 (m, 2H), 1.56-1.54 (m, 2H), 1.502 (s, 9H, one diastereomer), 1.496 (s, 9H, the other diastereomer), 1.39-1.25 (m, 38H), 0.90-0.87 (m, 6H).

[0058]

[0059] ​To a dichloromethane solution (2 mL) of (2R)-1-((tert-butoxy(2-((tert-butoxycarbonyl)amino)ethoxy)phosphoryl)oxy)-3-(octadecyloxy)propan-2-yl(5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoate (260 mg, 0.29 mmol), 2 mL of trifluoroacetic acid was added at room temperature, and the mixture was stirred for 3 hours. The reaction mixture was concentrated under reduced pressure to give the desired 2-(hydroxy((R)-2-(((5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoyl)oxy)-3-(octadedecyloxy)propoxy)phosphoryl)oxy)ethane-1-ammonium 2,2,2-trifluoroacetate (KIT-13) as a colorless liquid (249 mg, quant.).

[0060] KIT-13: 1 H NMR (500 MHz, CDCl3) δ 11.31 (br, 1H), 5.41-5.31 (m, 8H), 5.16-5.15 (m, 1H), 4.17 (br, 2H), 4.07-4.00 (m, 2H), 3.54 (d, J = 4.9 Hz, 2H), 3.45-3.39 (m, 2H), 3.27 (br), 2.84-2.78 (m, 6H), 2.35 (t, J = 7.4 Hz, 2H), 2.10 (q. J = 7.1 Hz, 2H), 2.05 (q. J = 7.2 Hz, 2H), 1.69-1.66 (m, 2H), 1.54-1.51 (m, 2H), 1.39-1.25 (m, 38H), 0.90-0.86 (m, 6H).

[0061] Example 2: Synthesis of compound KIT-18 KIT-18 was synthesized as a compound according to formula (I) as follows.

[0062]

[0063] CHClO (338 mg, 1.20 mmol), DMAP (98 mg, 0.80 mmol), and Compound 1 (500 mg, 0.80 mmol) ２ Cl ２To the solution (8.0 ml) was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl, 230 mg, 1.20 mmol) at 0° C., and the mixture was stirred at room temperature for 13 hours. The reaction mixture was then diluted with ethyl acetate (40 ml) and washed with water. ２ SO ４ The extract was dried at 47°C, filtered, concentrated, and purified by column chromatography (hexane / ethyl acetate=1 / 2), yielding the target compound 2 ((2R)-1-((tert-butoxy(2-((tert-butoxycarbonyl)amino)ethoxy)phosphoryl)oxy)-3-(octadecyloxy)propan-2-yl oleate) in 42% yield (300 mg, 0.338 mmol) as a transparent liquid.

[0064] Compound 2: 1 H NMR (500 MHz, CDCl 3, a mixture of diastereomers) d 5.38-5.30 (2H, m), 5.18-5.12 (2H, m), 4.21-4.15 (1H, m), 4.15-4.09 (1H, m), 4.08-4.02 (2H, m), 3.55 (2H, d, J = 5.2 Hz), 3.47-3.37 (4H, m), 2.33 (2H, t, J = 7.6 Hz), 2.03-1.98 (4H, m), 1.65-1.59 (2H, m), 1.57-1.51 (2H, m), 1.50 (9H, d, J = 3.4 Hz), 1.44 (9H, s), 1.34-1.23 (50H, m), 0.88 (3H, t, J = 6.9 Hz), 0.88 (3H, t, J = 6.9 Hz).

[0065]

[0066] Compound 2 (300 mg, 0.338 mmol) was dissolved in TFA (3 ml) and CH ２ Cl ２(3 ml) was added and the mixture was stirred at room temperature for 3 hours. The mixture was concentrated under reduced pressure to quantitatively obtain the target product, KIT-18 (2-((hydroxy((R)-3-(octadecyloxy)-2-(oleoyloxy)propoxy)phosphoryl)oxy)ethane-1-aminium trifluoroacetate) as a yellow liquid (258 mg, 0.338 mmol).

[0067] KIT-18: 1 H NMR (500 MHz, CDCl3) d 10.42 (1H, brs), 8.05 (3H, brs), 5.38-5.30 (2H, m), 5.17-5.12 (1H, m), 4.18-4.10 (2H, m), 4.07-3.95 (2H, m), 3.53 (2H, d, J = 4.8 Hz), 3.47-3.39 (2H, m), 3.27-3.18 (2H, m), 2.32 (2H, t, J = 7.6 Hz), 2.04-1.98 (4H, m), 1.64-1.49 (4H, m), 1.34-1.23 (50H, m), 0.88 (3H, t, J = 6.9 Hz), 0.88 (3H, t, J = 6.9 Hz).

[0068] Example 3: Synthesis of compound KIT-24 KIT-24 was synthesized as a compound according to formula (I) as follows.

[0069]

[0070] Palmitic acid (38 mg, 0.15 mmol), DMAP (78 mg, 0.64 mmol), and Compound 1 (100 mg, 0.16 mmol) in CH ２ Cl ２ To the solution (1.6 ml) was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC, 0.06 ml, 0.32 mmol) at 0°C, and the mixture was stirred at room temperature for 13 hours. The reaction mixture was then diluted with ethyl acetate (10 ml) and washed with water. ２After drying over SO, the mixture was filtered and concentrated, and then crudely purified by column chromatography (hexane / ethyl acetate = 1 / 2). The resulting transparent liquid was diluted with TFA (1 ml) and CH ２ Cl ２ (1 ml) was added and stirred at room temperature for 3 hours. The reaction mixture was concentrated and purified by column chromatography (CHCl ３ This was then purified again with TFA (1 ml) and CH ２ Cl ２ (1 ml) was added and stirred for 1 hour, whereby the target product, KIT-24 (2-((hydroxy((R)-3-(octadecyloxy)-2-(palmitoyloxy)propoxy)phosphoryl)oxy)ethane-1-aminium trifluoroacetate) was obtained in 98% yield as a yellow liquid (40.6 mg, 0.057 mmol).

[0071] KIT-24: 1 H NMR (500 MHz, CDCl3) d 7.62 (3H, brs), 5.19-5.15 (1H, m), 4.32-3.94 (4H, m), 3.59-3.28 (6H, m), 2.35-2.29 (2H, m), 1.60-1.50 (4H, m), 1.32-1.22 (54H, m), 0.88 (3H, t, J = 7.0 Hz), 0.88 (3H, t, J = 7.0 Hz).

[0072] [Test Example 1] A vector (catalog number: TRCN0000304464, manufactured by Sigma) expressing shRNA targeting mouse MeCP2 (NM_010788) was used below as the "shMeCP2 vector." An amplification product was obtained by PCR for the shMeCP2 vector, and a vector was prepared from which the MeCP2 target sequence had been removed, and this vector was designated the "shCont. vector." The nucleotide sequences of the forward primer and reverse primer for the shMeCP2 vector used in PCR are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0073] Mouse glial cell-derived BV2 cells cultured in Dulbecco's Modified Eagle Medium (DMEM, Sigma-Aldrich) containing 10% fetal calf serum (FCS, Sigma-Aldrich) were transfected with shMeCP2 or shCont. vectors, and puromycin-resistant clones (puromycin concentration: 1 μg / ml) were isolated using the puromycin resistance gene contained in the vectors. The resulting cells were designated BV2 / shMeCP2 and BV2 / shCont., respectively.

[0074] BV2 / shMeCP2 cells were cultured for 2 days in DMEM (Sigma-Aldrich) containing 10% FCS (Sigma-Aldrich), 100 units / ml penicillin, and 100 μg / ml streptomycin (Nacalai) in the presence (shMeCP2+KIT-13) or absence (shMeCP2) of 5 μg / ml KIT-13. As a control, BV2 / shCont. cells were cultured for 2 days in the same medium in the absence of KIT-13 (shCont.).

[0075] The cells were fixed with 4% paraformaldehyde (PFA) solution and washed with phosphate-buffered saline (PBS). The obtained samples were treated with 2N HCl at 37°C for 15 minutes. After washing with PBS, the samples were treated with 1% Tx-100 / PBS at room temperature for 2 minutes. The samples were then washed with PBS and treated with 1% bovine serum albumin (BSA). The primary antibody used was an anti-5-mC antibody (#28692, Cell Signaling Technology) diluted 4000-fold with 1% BSA-PBS. The primary antibody was allowed to react with the samples for 2 hours at 25°C. After washing with PBS, the sample was reacted with a solution of Alexa Fluor 488 anti-rabbit IgG (Invitrogen) and 3 mM DAPI diluted 2000-fold in 1% BSA-PBS at room temperature for 1 hour in the dark. ２ The sections were rinsed with 0, embedded in a cover glass, and observed using a BZ-X810 (Keyence Corporation).

[0076] (Results) Fluorescent staining images of the samples are shown in Figure 1. Fluorescence intensities quantified based on the fluorescent staining images are shown in Figure 2. The fluorescence intensity derived from 5-methylcytosine was reduced in shMeCP2 compared to shCont. The fluorescence intensity derived from 5-methylcytosine was significantly increased in shMeCP2+KIT-13 compared to shMeCP2.

[0077] Test Example 2 TIG-107 cells (JCRB0532) and TIG-120 cells (JCRB0542) were obtained from the JCRB Cell Bank. TIG-107 cells were cultured for 4 days in DMEM (Sigma-Aldrich) containing 10% FCS (Sigma-Aldrich), 100 units / ml penicillin, and 100 μg / ml streptomycin (Nacalai) in the presence (TIG-107 + KIT-13) or absence (TIG-107) of 10 μg / ml KIT-13. As a control, TIG-120 cells were cultured for 4 days in the same medium in the absence of KIT-13 (TIG-120). Fluorescent staining images of samples prepared from the cultured cells were observed in the same manner as in Test Example 1.

[0078] (Results) Fluorescent staining images of the samples are shown in Figure 3. The anti-5mC antibody positive rate calculated from the fluorescent staining images is shown in Figure 4. In the TIG-107+KIT-13 group, the number of cells positive for 5-methylcytosine was significantly increased compared to TIG-107.

[0079] Test Example 3 TIG-107 cells were cultured for 4 days in DMEM (Sigma-Aldrich) containing 10% FCS (Sigma-Aldrich), 100 units / ml penicillin, and 100 μg / ml streptomycin (Nacalai) in the presence of 10 μg / ml KIT-18, 10 μg / ml KIT-24, or in the presence or absence (−) of KIT-24. As in Test Example 1, fluorescently stained images of samples prepared from the cultured cells were observed, and the anti-5mC antibody positivity rate was determined from the fluorescently stained images.

[0080] (Results) The anti-5mC antibody positive rate calculated from the fluorescent stained images of the samples is shown in Figure 5. KIT-18 or KIT-24 significantly increased the number of 5-methylcytosine positive cells.

[0081] The present invention allows various embodiments and modifications without departing from the broad spirit and scope of the present invention. Furthermore, the above-described embodiments are intended to illustrate the present invention and do not limit the scope of the present invention. That is, the scope of the present invention is defined by the claims, not the embodiments. Various modifications made within the scope of the claims and the meaning of the invention equivalent thereto are considered to be within the scope of the present invention.

[0082] This application is based on Japanese Patent Application No. 2024-009152, filed on January 25, 2024. The entire specification, claims, and drawings of Japanese Patent Application No. 2024-009152 are incorporated herein by reference.

[0083] The present invention is useful for promoting or inducing methylation of genomic DNA.

Claims

1. A DNA methylation promoter comprising a compound represented by formula (I), a racemate thereof, or a salt thereof. [In formula (I), X represents a carbon atom or an oxygen atom, and R １ represents a saturated aliphatic hydrocarbon group which may contain a cyclic structure, and R ２ represents a saturated or unsaturated aliphatic hydrocarbon group having 10 to 30 carbon atoms which may have a substituent which is an alkyl group having 1 to 4 carbon atoms or an alkoxy group having 1 to 4 carbon atoms, and the carbon atoms of the glycerol skeleton may have a substituent, and taking the oxygen atom bonded to R ３ as *, R ３ is which may have a substituent. ] 2. An oral composition for promoting DNA methylation, comprising a compound represented by formula (I), a racemate thereof, or a salt thereof. [In formula (I), X represents a carbon atom or an oxygen atom, and R １ represents a saturated aliphatic hydrocarbon group which may contain a cyclic structure, and R ２ represents a saturated or unsaturated aliphatic hydrocarbon group having 10 to 30 carbon atoms which may have a substituent which is an alkyl group having 1 to 4 carbon atoms or an alkoxy group having 1 to 4 carbon atoms, and the carbon atoms of the glycerol skeleton may have a substituent, and with the oxygen atom bonded to R ３ being denoted as *, R ３ is which may have a substituent. ] 3. X is an oxygen atom, and R １ is an alkyl group having 18 carbon atoms, the DNA methylation promoter according to claim 1, or the oral composition for promoting DNA methylation according to claim 2.

4. R ２ is a monovalent group representing arachidonic acid, oleic acid or palmitic acid when R ２ is COOH, the DNA methylation promoter or oral composition for promoting DNA methylation according to claim 3.

5. Use of a compound represented by formula (I), a racemate thereof, or a salt thereof for the production of a DNA methylation promoter or an oral composition for promoting DNA methylation. [In formula (I), X represents a carbon atom or an oxygen atom, and R １ represents a saturated aliphatic hydrocarbon group which may contain a cyclic structure, and R ２ represents a saturated or unsaturated aliphatic hydrocarbon group having 10 to 30 carbon atoms which may have a substituent which is an alkyl group having 1 to 4 carbon atoms or an alkoxy group having 1 to 4 carbon atoms, and the carbon atoms of the glycerol skeleton may have a substituent, and taking the oxygen atom bonded to R ３ as *, R ３ may have a substituent is. ]

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