Pharmaceutical composition for treating or preventing obesity

Abstract

To provide a pharmaceutical composition for treating or preventing the obesity which can inhibit the accumulation of neutral fat.SOLUTION: A pharmaceutical composition for treating or preventing obesity comprises a compound (1) represented by general formula (1) or a salt thereof, and a pharmaceutically acceptable carrier. In the formula, R1 and R2 each independently represent a substituent, and n1 represents an integer from 0 to 4, and n2 represents an integer from 0 to 7.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a pharmaceutical composition for treating or preventing obesity. [Background technology]

[0002] A balanced diet and moderate exercise are the most effective treatments for obesity. However, it is difficult to continue these in busy daily life. Drug therapy is one of the means of treating obesity. As a drug candidate for obesity treatment, a search for a component that specifically induces beige fat cells has been conducted, but a component that combines sufficient safety and efficacy has not yet been obtained.

[0003] Flavanones are a type of flavonoid contained in plants, and more than 450 kinds of compounds are known to exist widely in nature. In recent years, it has been reported that sakuranetin, a flavanone contained in Asteraceae plants and rice, has the effect of promoting the formation of fat cells that constitute adipose tissue (Non-Patent Document 1). In addition, naringenin, a flavanone contained in citrus fruits, has been reported to have the effect of suppressing fat cell differentiation (Non-Patent Document 2). Meanwhile, the present inventors have established an efficient chemical synthesis method for flavanones using a fluoride ion catalyst (Non-Patent Document 3). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Saito et al., Sakuranetin induces adipogenesis of 3T3-L1 cells through enhanced expression of PPARgamma2. 2008. Biochem Biophys Res Commun. 2008 Aug 8;372(4):835-9. [Non-Patent Document 2] Dayarathne et al., Restoration of the adipogenic gene expression by naringenin and naringin in 3T3-L1 adipocytes. J Vet Sci. 2021 Jul; 22(4): e55. [Non-Patent Document 3] Miura et al., Convenient synthesis of flavanone derivatives via oxa-Michael addition using catalytic amount of aqueous cesium fluoride. Tetrahedron Letters Volume 85, 23 November 2021, 153480. Summary of the Invention [Problem to be solved by the invention]

[0005] A compound capable of suppressing the accumulation of neutral fats is expected to be able to effectively treat and / or prevent obesity.

[0006] Therefore, an object of the present invention is to provide a pharmaceutical composition for treating or preventing obesity, which is capable of suppressing the accumulation of neutral fats. [Means for solving the problem]

[0007] The present invention includes the following aspects. [1] A pharmaceutical composition for treating or preventing obesity, comprising a compound (1) represented by the following general formula (1) or a salt thereof, and a pharma- ceutically acceptable carrier:

[0008] [ka] [In the formula, R 1 and R 2 each independently represents a substituent; n1 represents an integer of 0 to 4; and n2 represents an integer of 0 to 7.]

[0009] [2] The pharmaceutical composition for treating or preventing obesity according to [1], wherein the compound (1) is a compound represented by the following general formula (1-1):

[0010] [ka] [In the formula, R 11 , R 12 , and R 21 each independently represents a hydrogen atom or a substituent.

[0011] [3] R in the general formula (1-1) 11 , R 12 , and R 21 each independently represents a hydrogen atom, an alkyl group, an alkoxy group, an oxyalkylene structure-containing group, a hydroxyl group, or a halogen atom. [4] R in the general formula (1-1) 11 , R 12 , and R 21 are each independently a hydrogen atom, a methyl group, a methoxy group, a methoxymethoxy group, a hydroxyl group, or a halogen atom. [5] The pharmaceutical composition for treating or preventing obesity according to any one of [1] to [4], which induces differentiation of preadipocytes into beige adipocytes. [6] The pharmaceutical composition for treating or preventing obesity according to any one of [1] to [5], which induces transformation of white adipocytes into beige adipocytes. Effect of the Invention

[0012] According to the present invention, there is provided a pharmaceutical composition for treating or preventing obesity, which is capable of suppressing accumulation of neutral fat. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 is a schematic diagram illustrating adipocyte differentiation and transformation. [Diagram 2] 1 shows microscopic images illustrating the results of evaluating the effect of 2NC on the induction of differentiation of preadipocytes in Example 1. [Diagram 3] FIG. 2 shows the results of quantitatively evaluating the effect of 2NC on differentiation induction of preadipocytes based on the amount of accumulated fat in Example 1. [Figure 4] This is a diagram showing the results of evaluating the effect of 2NC on differentiation induction of preadipocytes by gene expression levels in Example 2. Differentiation markers for white adipocytes: Pparγ2, aP2, C / ebpα; differentiation marker for beige adipocytes: Ucp1; beige adipocyte-related genes: Elovl3, Pgc1α. [Diagram 5] FIG. 1 shows details of each treatment group in Example 3. [Figure 6A] FIG. 13 shows the results of examining factors involved in inducing beige adipocyte differentiation by 2NC in Example 3. [Figure 6B] FIG. 13 shows the results of examining factors involved in inducing beige adipocyte differentiation by 2NC in Example 3. [Figure 6C] FIG. 13 shows the results of examining factors involved in inducing beige adipocyte differentiation by 2NC in Example 3. [Figure 7] FIG. 13 shows the results of evaluating the change in the expression level of the Fgf9 gene during the beige process caused by 2NC in Example 4. [Figure 8] FIG. 13 shows the results of evaluating the change in mitochondrial amount during the beige process induced by 2NC in Example 5. [Figure 9] FIG. 13 shows the results of verifying the transformation of white adipocytes into beige adipocytes by 2NC in Example 6. [Figure 10] FIG. 13 shows the results of verifying transformation into beige adipocytes by 2NC in terms of mitochondrial amount in Example 7. [Figure 11] FIG. 13 shows changes in body weight of mice administered with 2NC in Example 8. [Figure 12]1 shows the change in tissue weight in mice administered with 2NC in Example 8. eWAT: weight of epididymal adipose tissue; sWAT: weight of subcutaneous adipose tissue. [Figure 13] 13 shows histopathological images of adipose tissue from mice administered 2NC in Example 9. [Figure 14] FIG. 13 shows the results of evaluating the UCP1 protein amount in subcutaneous adipose tissue of mice administered 2NC in Example 10. [Figure 15] FIG. 13 shows the results of evaluating changes in mitochondrial amounts in adipose tissue of mice administered 2NC in Example 11. [Figure 16] The 2NC derivatives used in Examples 12 and 13 are shown below. These 2NC derivatives are modified at the A ring of the flavanone skeleton. [Figure 17] 13 shows microscopic images illustrating the results of evaluating the effect of 2NC derivatives on the induction of differentiation of preadipocytes in Example 12. [Figure 18A] FIG. 13 shows the results of evaluating the effect of 2NC derivatives on induction of differentiation of preadipocytes based on gene expression levels in Example 12. [Figure 18B] FIG. 13 shows the results of evaluating the effect of 2NC derivatives on induction of differentiation of preadipocytes based on gene expression levels in Example 12. [Figure 19] FIG. 13 is a diagram showing the results of evaluating the effect of 2NC derivatives on the induction of differentiation of preadipocytes based on the amount of mitochondria in Example 12. [Figure 20] The 2NC derivatives used in Examples 12 and 13 are shown below. These 2NC derivatives are modified at the B ring of the flavanone skeleton. [Figure 21] 13 shows microscopic images illustrating the results of evaluating the effect of 2NC derivatives on the induction of differentiation of preadipocytes in Example 12. [Figure 22] FIG. 13 shows the results of evaluating the effect of 2NC derivatives on induction of differentiation of preadipocytes based on gene expression levels in Example 12. [Diagram 23]FIG. 13 is a diagram showing the results of evaluating the effect of 2NC derivatives on the induction of differentiation of preadipocytes based on the amount of mitochondria in Example 12. [Figure 24] FIG. 13 shows the results of evaluating the effect of 2NC derivatives in promoting the transformation of white adipocytes into beige adipocytes based on gene expression levels in Example 13. [Diagram 25] FIG. 13 shows the results of verifying the effect of 2NC derivatives in promoting the transformation of white adipocytes into beige adipocytes in terms of mitochondrial amount in Example 13. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The term "aliphatic" is a relative concept to aromatic, and is defined as meaning a group, compound, etc. that does not have aromaticity. Unless otherwise specified, the term "alkyl group" includes linear, branched and cyclic monovalent saturated hydrocarbon groups. The same applies to the alkyl group in an alkoxy group. The "halogen atom" includes a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. The phrase "may have a substituent" includes both the case where a hydrogen atom (-H) is replaced with a monovalent group and the case where a methylene group (-CH2-) is replaced with a divalent group.

[0015] In the present specification and claims, some structures represented by chemical formulas may have asymmetric carbons, and may have enantiomers or diastereomers. In such cases, a single chemical formula represents all of the isomers. These isomers may be used alone or as a mixture.

[0016] [Pharmaceutical composition for treating or preventing obesity] A first aspect of the present invention is a pharmaceutical composition for treating or preventing obesity. The pharmaceutical composition of this aspect contains a compound (1) represented by the following general formula (1) or a salt thereof as an active ingredient.

[0017] [ka] [In the formula, R 1 and R 2 each independently represents a substituent; n1 represents an integer of 0 to 4; and n2 represents an integer of 0 to 7.]

[0018] (Compound (1)) Compound (1) has an effect of suppressing neutral fat accumulation, and functions as a neutral fat accumulation inhibitor. The suppression of neutral fat accumulation by compound (1) may be by inducing beige coloration or by suppressing differentiation of preadipocytes into white adipocytes.

[0019] When compound (1) has a beige-formation inducing effect, compound (1) functions as a beige-formation inducer. "Beige-formation" is a concept that encompasses both the induction of beige adipocytes by differentiation of preadipocytes and the induction of beige adipocytes by transformation of white adipocytes. A beige-formation inducer is a drug that has the effect of promoting either or both of the induction of differentiation from preadipocytes to beige adipocytes and the induction of beige adipocytes by transformation of white adipocytes. FIG. 1 is a schematic diagram illustrating the differentiation and transformation of adipocytes. Preadipocytes differentiate into white adipocytes or beige adipocytes. Beige adipocytes can also be induced by reversible transformation of white adipocytes. White adipocytes are adipocytes that accumulate neutral fats and have a high neutral fat content. Beige adipocytes have a high mitochondria content and consume neutral fats to produce energy. Therefore, by inducing beigeification, it is possible to suppress the accumulation of neutral fats and treat or prevent obesity.

[0020] The compound (1) is a compound represented by the above general formula (1). In the general formula (1), R 1 and R 2 The substituent in R is a monovalent group that replaces a hydrogen atom on the aromatic ring. 1and R 2 Examples of the substituent in include a hydrocarbon group which may have a substituent, a hydroxy group, and a halogen atom.

[0021] The hydrocarbon group which may have a substituent may be an aliphatic hydrocarbon group or an aromatic hydrocarbon group, with an aliphatic hydrocarbon group being preferred. The aliphatic hydrocarbon group may be saturated or unsaturated, with a saturated group being preferred. The aliphatic hydrocarbon group preferably has 1 to 10 carbon atoms, more preferably has 1 to 6 carbon atoms, further preferably has 1 to 3 carbon atoms, and particularly preferably has 1 or 2 carbon atoms. The aliphatic hydrocarbon group may be a chain aliphatic hydrocarbon group or a cyclic aliphatic hydrocarbon group, with a chain aliphatic hydrocarbon group being preferred. The chain aliphatic hydrocarbon group may be a straight-chain aliphatic hydrocarbon group or a branched-chain aliphatic hydrocarbon group. The chain-like aliphatic hydrocarbon group may be a linear or branched alkyl group. The linear alkyl group preferably has 1 to 10 carbon atoms, more preferably has 1 to 6 carbon atoms, further preferably has 1 to 3 carbon atoms, and particularly preferably has 1 or 2 carbon atoms. The branched alkyl group preferably has 3 to 10 carbon atoms, more preferably has 3 to 6 carbon atoms, and further preferably has 3 carbon atoms.

[0022] The linear or branched alkyl group may or may not have a substituent. Examples of the substituent substituting the hydrogen atom of the alkyl group include a halogen atom, a hydroxyl group, and a carboxyl group. Examples of the substituent substituting the methylene group of the alkyl group include a carbonyl group (-CO-), an oxygen atom (-O-), an ester bond (-COO-), and an amide bond (-CONH-), with an oxygen atom being preferred. Examples of the group in which the methylene group of the alkyl group is substituted with an oxygen atom include an alkoxy group and an oxyalkylene structure-containing group.

[0023] The cyclic aliphatic hydrocarbon group may be polycyclic or monocyclic, but is preferably monocyclic. The monocyclic aliphatic hydrocarbon group is preferably a group in which one hydrogen atom has been removed from a monocycloalkane. The monocycloalkane preferably has 3 to 6 carbon atoms. Specific examples of the monocycloalkane include cyclopropane, cyclobutane, cyclopentane, and cyclohexane.

[0024] The aromatic hydrocarbon group is a hydrocarbon group having at least one aromatic ring. The aromatic ring is not particularly limited as long as it is a cyclic conjugated system having 4n+2 π electrons, and may be monocyclic or polycyclic. The aromatic ring preferably has 5 to 12 carbon atoms, more preferably 6 to 12 carbon atoms. Specific examples of the aromatic ring include benzene, naphthalene, anthracene, and phenanthrene. The aromatic ring may be an aromatic heterocycle in which a part of the carbon atoms constituting the aromatic hydrocarbon ring is substituted with a heteroatom (such as a sulfur atom, a nitrogen atom, or an oxygen atom).

[0025] R 1 and R 2 The substituent in is preferably an alkyl group, an alkoxy group, an oxyalkylene structure-containing group, a hydroxy group or a halogen atom. The alkyl group is preferably a linear or branched alkyl group, more preferably a linear alkyl group, further preferably a linear alkyl group having 1 to 3 carbon atoms, and more preferably a methyl group or an ethyl group. The alkoxy group is preferably a linear or branched alkoxy group, more preferably a linear alkoxy group, still more preferably a linear alkoxy group having 1 to 3 carbon atoms, and more preferably a methoxy group or ethoxy group. The oxyalkylene structure-containing group is an oxyalkylene structure (-O-(CH2) mIn the above formula, m is an integer of 1 or more, preferably an integer of 1 to 3, more preferably 1 or 2, and even more preferably 1. The number of oxyalkylene structures contained in the oxyalkylene structure-containing group is, for example, 1 to 5, preferably 1 to 3, and more preferably 1 or 2. The number of carbon atoms in the oxyalkylene structure-containing group is preferably 1 to 5, more preferably 1 to 3, and even more preferably 2. Examples of the oxyalkylene structure-containing group include -O-[(CH2) m -O] n Examples of the group include a group represented by -CH3 (m is an integer of 1 to 3, and n is an integer of 1 to 10). In the above formula, m is preferably 1 or 2, and more preferably 1. In the above formula, n is preferably an integer of 1 to 5, more preferably an integer of 1 to 3, further preferably 1 or 2, and more preferably 1. Among them, R 1 and R 2 The substituent in is preferably a methyl group, a methoxy group, a methoxymethoxy group, a hydroxy group, or a halogen atom.

[0026] R 1 The substituent in R is preferably an alkyl group, an alkoxy group, an oxyalkylene structure-containing group, or a halogen atom. As the halogen atom, a chlorine atom, a fluorine atom, and a bromine atom are preferred, and a chlorine atom and a fluorine atom are more preferred. 1 From the viewpoint of inducing beige formation, the substituent in is preferably an alkyl group, an alkoxy group, or a group containing an oxyalkylene structure, and more preferably an alkyl group or a group containing an oxyalkylene structure. R 2 The substituent in is preferably an alkoxy group, a hydroxy group, or a halogen atom, more preferably a halogen atom. As the halogen atom, a chlorine atom, a fluorine atom, or a bromine atom is preferred, more preferably a bromine atom.

[0027] In the formula (1), n1 is preferably an integer of 0 to 3, more preferably an integer of 0 to 2, and even more preferably 0 or 1. From the viewpoint of exerting a neutral fat suppressing effect at a low dose, n1 is preferably 1. From the viewpoint of increasing the mitochondrial content, n1 is preferably 0. In the formula (1), n2 is preferably an integer of 0 to 5, more preferably an integer of 0 to 3, further preferably an integer of 0 to 2, and particularly preferably 0 or 1. From the viewpoint of exerting a neutral fat suppressing effect at a low dose, n2 is preferably 1. From the viewpoint of increasing the mitochondrial content, n2 is preferably 0.

[0028] The compound (1) is preferably a compound represented by the following general formula (1-1).

[0029] [ka] [In the formula, R 11 , R 12 , and R 21 each independently represents a hydrogen atom or a substituent.

[0030] R in the above formula (1-1) 11 , R 12 , and R 21 The substituent in R is a monovalent group that replaces a hydrogen atom on the aromatic ring. 11 , R 12 , and R 21 The substituent in the formula (1) is R 1 and R 2 The substituents in the above formula (I) are the same as those in the above formula (I). Among them, R 11 , R 12 , and R 21 The substituent in is preferably an alkyl group, an alkoxy group, an oxyalkylene structure-containing group, a hydroxy group or a halogen atom, and more preferably a methyl group, a methoxy group, a methoxymethoxy group, a hydroxy group or a halogen atom.

[0031] R 11 is preferably a hydrogen atom, an alkoxy group, or a halogen atom, more preferably a hydrogen atom, a methoxy group, or a halogen atom. The halogen atom is preferably a chlorine atom or a fluorine atom. From the viewpoint of exerting a neutral fat suppressing effect at a low dose, R 11is preferably an alkoxy group or a halogen atom. From the viewpoint of inducing beige formation, R 11 is preferably a hydrogen atom or an alkoxy group. From the viewpoint of increasing the mitochondrial content, R 11 is preferably a hydrogen atom. R 12 is preferably a hydrogen atom, an alkyl group, an oxyalkylene structure-containing group, or a halogen atom, more preferably a hydrogen atom, a methyl group, a methoxymethoxy group, or a halogen atom. The halogen atom is preferably a chlorine atom or a fluorine atom. From the viewpoint of exerting a neutral fat suppressing effect at a low dose, R 12 is preferably an alkyl group, an oxyalkylene structure-containing group, or a halogen atom. 12 is preferably a hydrogen atom, an alkyl group, or an oxyalkylene structure-containing group. From the viewpoint of increasing the mitochondrial content, R 12 is preferably a hydrogen atom. R 21 is preferably a hydrogen atom, an alkoxy group, a hydroxy group, or a halogen atom, more preferably a hydrogen atom, a methoxy group, a hydroxy group, or a halogen atom, and even more preferably a hydrogen atom or a halogen atom. The halogen atom is preferably a bromine atom. From the viewpoint of exerting a neutral fat suppressing effect at a low dose, R 21 is preferably an alkoxy group, a hydroxy group, or a halogen atom, and more preferably a halogen atom. 21 is preferably a hydrogen atom, an alkoxy group, a hydroxyl group, or a halogen atom, and more preferably a halogen atom. From the viewpoint of increasing the mitochondrial content, R 21 is preferably a hydrogen atom.

[0032] The compound (1) is preferably a compound represented by any one of the following general formulas (1-1-1) to (1-1-3).

[0033] [ka] [In the formula, R 111 , R 121 , and R211 each independently represents a hydrogen atom or a substituent.

[0034] In the formulas (1-1-1) to (1-1-3), R 111 , R 121 , and R 211 The substituent in the formula (1) is R 1 and R 2 The same can be mentioned. R 111 , R 121 , and R 211 The substituent in is preferably an alkyl group, an alkoxy group, an oxyalkylene structure-containing group, a hydroxy group or a halogen atom, and more preferably a methyl group, a methoxy group, a methoxymethoxy group, a hydroxy group or a halogen atom.

[0035] R 111 is preferably a hydrogen atom, an alkoxy group, or a halogen atom, more preferably a hydrogen atom, a methoxy group, or a halogen atom. The halogen atom is preferably a chlorine atom or a fluorine atom. From the viewpoint of exerting a neutral fat suppressing effect at a low dose, R 111 is preferably an alkoxy group or a halogen atom. From the viewpoint of inducing beige formation, R 111 is preferably a hydrogen atom or an alkoxy group. From the viewpoint of promoting differentiation of preadipocytes into beige cells, R 111 is preferably an alkoxy group. From the viewpoint of increasing the mitochondrial content, R 111 is preferably a hydrogen atom. R 121 is preferably a hydrogen atom, an alkyl group, an oxyalkylene structure-containing group, or a halogen atom, more preferably a hydrogen atom, a methyl group, a methoxymethoxy group, or a halogen atom. The halogen atom is preferably a chlorine atom or a fluorine atom. From the viewpoint of exerting a neutral fat suppressing effect at a low dose, R 121 is preferably an alkyl group, an oxyalkylene structure-containing group, or a halogen atom. 121is preferably a hydrogen atom, an alkyl group, or an oxyalkylene structure-containing group. From the viewpoint of promoting differentiation of preadipocytes into beige adipocytes, the alkyl group or the oxyalkylene structure-containing group R 121 is preferably an alkoxy group. From the viewpoint of increasing the mitochondrial content, R 12 is preferably a hydrogen atom. R 211 is preferably a hydrogen atom, an alkoxy group, a hydroxy group, or a halogen atom, more preferably a hydrogen atom, a methoxy group, a hydroxy group, or a halogen atom, and even more preferably a hydrogen atom or a halogen atom. The halogen atom is preferably a bromine atom. From the viewpoint of exerting a neutral fat suppressing effect at a low dose, R 211 is preferably an alkoxy group, a hydroxy group, or a halogen atom, and more preferably a halogen atom. 211 is preferably a hydrogen atom, an alkoxy group, a hydroxyl group, or a halogen atom, and more preferably a halogen atom. From the viewpoint of inducing transformation from white adipocytes to beige adipocytes, R 211 is preferably an alkoxy group, a hydroxy group, or a halogen atom. From the viewpoint of increasing the mitochondrial content, R 211 is preferably a hydrogen atom.

[0036] The compound represented by the formula (1-1-1) or (1-1-2) tends to have a high ability to induce differentiation of preadipocytes into beige adipocytes. The compound represented by the formula (1-1-3) tends to have a high ability to induce transformation of white adipocytes into beige adipocytes.

[0037] Specific examples of compound (1) are shown below, but are not limited to these.

[0038] [ka]

[0039] [ka]

[0040] Among the above, 2NC, 2NC-A2, 2NC-A3, 2NC-A4, 2NC-B4, 2NC-B6, and 2NC-B7 are preferred as compound (1), and 2NC, 2NC-A2, 2NC-A4, and 2NC-B7 are more preferred. As beige-inducing agents, 2NC, 2NC-A2, 2NC-A3, 2NC-A4, 2NC-B4, 2NC-B6, and 2NC-B7 are preferred. As differentiation inducers from preadipocytes to beige adipocytes, 2NC, 2NC-A2, 2NC-A3, and 2NC-A4 are preferred. As transformation inducers from white adipocytes to beige adipocytes, 2NC, 2NC-B4, 2NC-B6, and 2NC-B7 are preferred.

[0041] Compound (1) may be in the form of a salt. Examples of the salt include pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" refers to a salt form that does not inhibit the pharmacological action of compound (1). Examples of the pharmacological function of compound (1) include neutral fat accumulation reducing action and beige inducing action. The pharmaceutically acceptable salt of compound (1) is not particularly limited, and examples thereof include salts with alkali metals (sodium, potassium, etc.); salts with alkaline earth metals (magnesium, calcium, etc.); salts with organic bases (pyridine, triethylamine, etc.), salts with amines, salts with organic acids (acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, etc.), and salts with inorganic acids (hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, nitric acid, etc.).

[0042] Compound (1) may be in the form of a solvate, or may be in the form of a solvate of a salt of compound (1). The solvate is not particularly limited, and examples thereof include a hydrate, an ethanol solvate, and the like.

[0043] The pharmaceutical composition of the present embodiment contains one or more compounds selected from the group consisting of compound (1) and salts thereof. The compounds selected from the group consisting of compound (1) and salts thereof may be used alone or in combination of two or more.

[0044] The pharmaceutical composition of this embodiment may contain a therapeutically effective amount of compound (1) and / or a salt thereof per unit dosage form. The term "therapeutically effective amount" means an amount of a drug effective for treating or preventing a target disease. In the pharmaceutical composition of this embodiment, the therapeutically effective amount of compound (1) and / or a salt thereof may be an amount effective for treating or preventing obesity. The therapeutically effective amount may be appropriately determined depending on the symptoms, body weight, age, sex, etc. of a patient, the dosage form of the pharmaceutical composition, the administration method, etc. The pharmaceutical composition of this embodiment may contain 0.01 to 2000 mg of the compound (1) and / or a salt thereof per unit dosage form. In the pharmaceutical composition of this embodiment, the content of compound (1) and / or a salt thereof may be 0.01 to 90% by mass, 0.05 to 80% by mass, or 0.1 to 60% by mass.

[0045] (optional ingredient) The pharmaceutical composition of the present embodiment may contain other ingredients in addition to compound (1) or a salt thereof, such as a pharma- ceutically acceptable carrier. The term "pharmacologically acceptable carrier" refers to a carrier that does not inhibit the physiological activity of an active ingredient and does not show substantial toxicity to the subject of administration. "Not substantially toxic" refers to a carrier that does not show toxicity to the subject of administration at a dose that is normally used. In the pharmaceutical composition of this embodiment, the pharma- ceutical acceptable carrier is a carrier that does not inhibit the pharmacological action of compound (1) or a salt thereof and does not show substantial toxicity to the subject of administration. The pharma- ceutical acceptable carrier includes any known pharma- ceutical acceptable ingredient that is typically considered to be an inactive ingredient. The pharma- ceutical acceptable carrier is not particularly limited, and examples thereof include solvents, diluents, vehicles, excipients, flow enhancers, binders, granulating agents, dispersing agents, suspending agents, wetting agents, lubricants, disintegrating agents, solubilizers, stabilizers, emulsifiers, fillers, etc. The pharma- ceutical acceptable carrier may be used alone or in combination of two or more kinds.

[0046] The pharmaceutical composition of the present embodiment may contain other ingredients in addition to the above ingredients. The other ingredients are not particularly limited, and those commonly used in the pharmaceutical field can be used without particular limitation. Examples of the other ingredients include pharmaceutical additives other than those mentioned above. Examples of pharmaceutical additives include, but are not limited to, preservatives (e.g., antioxidants), chelating agents, flavoring agents, sweeteners, thickeners, buffers, colorants, etc. The pharmaceutical composition of the present embodiment may contain an active ingredient other than the compound (1) or a salt thereof. Examples of the active ingredient include, but are not limited to, antibiotics, anti-inflammatory agents, antipyretics, analgesics, etc. The other ingredients may be used alone or in combination of two or more.

[0047] (Dosage form, method of administration, etc.) The dosage form of the pharmaceutical composition of this embodiment is not particularly limited, and may be a dosage form generally used as a pharmaceutical preparation. The pharmaceutical composition of this embodiment may be an oral preparation or a parenteral preparation. Examples of oral preparations include tablets, coated tablets, pills, powders, granules, capsules, syrups, fine granules, liquids, drops, and emulsions. Examples of parenteral preparations include injections, suppositories, nasal drops, enteral preparations, and inhalants. Pharmaceutical compositions of these dosage forms can be formulated according to standard methods (for example, methods described in the Japanese Pharmacopoeia).

[0048] The administration route of the pharmaceutical composition of the present embodiment is not particularly limited, and it can be administered orally or parenterally. Parenteral administration includes intravenous administration, intranasal administration, subcutaneous administration, intradermal administration, intramuscular administration, intraperitoneal administration, enteral administration, etc.

[0049] The pharmaceutical composition of this embodiment can be administered in a therapeutically effective amount of compound (1) and / or a salt thereof. For example, the pharmaceutical composition of this embodiment can be administered in a single dose of 0.01 to 2000 mg of compound (1) and / or a salt thereof per kg of the subject's body weight. The dose may be 0.05 to 1500 mg / kg, 0.1 to 1000 mg / kg, 0.2 to 500 mg / kg, or 0.3 to 300 mg / kg.

[0050] The pharmaceutical composition of the present embodiment may be administered once or repeatedly. In the case of repeated administration, the administration interval may be appropriately determined depending on the symptoms, body weight, age, sex, etc. of the patient, the dosage form of the pharmaceutical composition, the administration method, etc. The administration interval may be, for example, every several hours, 2 to 3 times a day, once a day, once every 2 to 3 days, once a week, once a month, once every several months, etc.

[0051] The subject of administration of the pharmaceutical composition of this embodiment is not particularly limited. The subject of administration of the pharmaceutical composition is preferably a mammal, and may be a human or a non-human mammal. Examples of non-human mammals include non-human primates (monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, chimpanzees, etc.), rodents (mouse, rats, hamsters, guinea pigs, etc.), lagomorphs (rabbits, etc.), ungulates (pig, cow, goat, horse, sheep, etc.), and felines (dogs, cats, etc.).

[0052] The pharmaceutical composition of this embodiment can be used to treat or prevent obesity. Compound (1) has an effect of suppressing accumulation of neutral fat, and is therefore effective in treating or preventing obesity. Compound (1) can promote fat burning by inducing beigeification, thereby exhibiting an anti-obesity effect. Therefore, the pharmaceutical composition of this embodiment can treat or prevent obesity by a mechanism of action different from that of conventional anti-obesity drugs, which have an action mechanism of suppressing food intake or inhibiting fat absorption. Compound (1) has partial agonist activity of PPARγ, and therefore may have an insulin resistance improving effect. Compound (1) is an analogue of flavanone, a natural compound, and therefore is considered to be excellent in terms of safety. The pharmaceutical composition of the present embodiment contains compound (1) or a salt thereof as an active ingredient, and therefore is expected to have an insulin resistance improving effect and is considered to be highly safe.

[0053] [Other aspects] In one aspect, the present invention provides a method for treating or preventing obesity, comprising administering to a subject an effective amount of compound (1) or a salt thereof. The effective amount may be a therapeutically effective amount of compound (1) or a salt thereof. The subject is one in need of treatment or prevention of obesity. In one aspect, the present invention provides the use of compound (1) or a salt thereof in the manufacture of a pharmaceutical composition for treating or preventing obesity. In one aspect, the present invention provides compound (1) or a salt thereof for use in the treatment or prevention of obesity. In one aspect, the present invention provides use of compound (1) or a salt thereof for treating or preventing obesity. In one aspect, the present invention provides a neutral fat-lowering agent comprising compound (1) or a salt thereof. In one aspect, the present invention provides a beige induction agent comprising compound (1) or a salt thereof. In one aspect, the present invention provides a method for inducing beige formation, comprising contacting preadipocytes and / or white adipocytes with compound (1) or a salt thereof. In one aspect, the present invention provides a method for inducing differentiation of preadipocytes into beige adipocytes, comprising contacting preadipocytes with compound (1) or a salt thereof. In one aspect, the present invention provides a method for inducing transformation of white adipocytes into beige adipocytes, comprising contacting white adipocytes with compound (1) or a salt thereof. EXAMPLES

[0054] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0055] (Synthesis Example 1: Synthesis of 2NC) <Synthesis of chalcone (3Aa)> 2'-Hydroxyacetophenone (1A) (1.20 mL, 0.01 mol) and 1-naphthaldehyde (2a) (1.07 g, 0.01 mol) were dissolved in 30 mL of ethanol, and 1.20 g (0.03 mol) of sodium hydroxide was added thereto and stirred at room temperature for 20 hours. The resulting yellow suspension was added to 10 mL of concentrated hydrochloric acid and 200 g of ice and stirred for 2 hours, after which the resulting yellow solid was suction filtered. The resulting solid was recrystallized from boiling ethanol to obtain 2.60 g (95% yield) of chalcone (3Aa) with a naphthalene skeleton. The obtained compound data was consistent with the literature (Tetrahedron Letters 2021, Vol. 85, p. 153480).

[0056] [ka]

[0057] <Synthesis of 2NC> In a pressure-resistant glass container, chalcone (3Aa) (2.60 g, 0.095 mol), 100 mL of acetonitrile, and 1 mL of 1.0 mol / L CsF aqueous solution were added and sealed, and the mixture was heated and stirred in a warm bath at 80 ° C for 2 hours. The reaction solution was returned to room temperature, the solvent was removed using an evaporator, and the resulting yellow residue was recrystallized from boiling ethanol to obtain 2NC (4Aa) as a slightly yellow solid in 2.21 g (yield 85%). The obtained compound data was consistent with the literature (Tetrahedron Letters 2021, Vol. 85, p. 153480).

[0058] [ka]

[0059] (Synthesis example 2: Synthesis of 2NC-A2~A7) <Synthesis of chalcone (3Ba~3Ga)> 1-Naphthaldehyde (2a) (0.54g, 0.005mol) was added to each of the 2'-hydroxyacetophenone derivatives (1B-1G) (each 0.005mol) and dissolved in 15mL of ethanol. 0.6g (0.015mol) of sodium hydroxide was added thereto and stirred at room temperature for 20 hours. The resulting yellow suspension was added to 5mL of concentrated hydrochloric acid and 100g of ice and stirred for 2 hours, after which the resulting yellow solid was filtered by suction. The resulting solid was recrystallized from boiling ethanol to obtain chalcones (3Ba-3Ga) with a naphthalene skeleton.

[0060] [ka]

[0061] (E)-1-(2-Hydroxy-4-methylphenyl)-3-(naphthalen-1-yl)prop-2-en-1-one:3Ba Yield: 0.82g, 57% 1 H-NMR(600MHz:CDCl3)δppm:8.78(d,J=15.2Hz,1H),8.29(d,J=8.3Hz,1H),7.91-7.97(m,3H),7.7 6(d,J=15.2Hz,2H),7.58-7.64(m,3H),7.56(d,J=7.6Hz,1H),6.97(d,J=8.9Hz,1H),2.36(s,3H).

[0062] (E)-1-(2-Hydroxy-5-methoxyphenyl)-3-(naphthalen-1-yl)prop-2-en-1-one:3Ca Yield: 1.06g, 70% 1H-NMR (600 MHz, CDCl3) δ ppm: 12.4 (s, 1H), 8.79 (d, J = 15.1 Hz, 1H), 8.28 (d, J = 8.2 Hz, 1H), 7.91 - 7.97 (m, 3H), 7.70 (d, J = 15.1 Hz, 1H), 7.54 - 7.64 (m, 3H), 7.40 (d, J = 2.1 Hz, 1H), 7.17 (dd, J = 9.0, 2.8 Hz, 1H), 7.01 (d, J = 9.7 Hz, 1H), 3.84 (s, 3H).

[0063] (E)-1-(2-Hydroxy-4-(methoxymethoxy)phenyl)-3-(naphthalen-1-yl)prop-2-en-1-one: 3Da Yield · Yield rate: 1.22 g · 73% 1 H-NMR (600 MHz, CDCl3) δ ppm: 8.75 (d, J = 15.1 Hz, 1H), 8.28 (d, J = 8.2 Hz, 1H), 7.88 - 7.95 (m, 4H, 7.67 (d, J = 15.1 Hz, 1H), 7.53 - 7.62 (m, 3H), 6.68 (d, J = 2.7 Hz, 1H), 6.61 (dd, J = 8.9, 2.1 Hz, 1H), 5.24 (s, 2H), 3.50 (s, 3H).

[0064] (E)-1-(2-Hydroxy-5-chlorophenyl)-3-(naphthalen-1-yl)prop-2-en-1-one: 3Ea Yield · Yield rate: 0.43 g · 28% 1 H-NMR (600 MHz, CDCl3) δ ppm: 8.83 (d, J = 15.1 Hz, 1H), 8.28 (d, J = 8.3 Hz, 1H), 7.91 - 7.97 (m, 4H), 7.57 - 7.69 (m, 4H), 7.47 (d, J = 8.9 Hz, 1H), 7.02 (d, J = 9 Hz, 1H)

[0065] (E)-1-(2-Hydroxy-4-chlorophenyl)-3-(naphthalen-1-yl)prop-2-en-1-one: 3Fa Yield · Yield rate: 1.07 g · 69% 1 H-NMR(600MHz:CDCl3)δppm:8.51(d,J=15.1Hz,1H),8.28(d,J=9Hz,1H),7.97(d,J=8.2Hz,1H),7.89-7.93(m,3H),7.82(d,J=19.2 Hz,1H),7.62-7.64(m,1H),7.54-7.58(m,2H),7.08(d,J=2.1Hz,1H),6.94(dd,J=2.1,8.9Hz,1H)

[0066] (E)-1-(2-Hydroxy-5-fluorophenyl)-3-(naphthalen-1-yl)prop-2-en-1-one:3Ga Yield: 0.50g, 34% 1 H-NMR(600MHz:CDCl3)δppm:8.83(d,J=15.1Hz,1H),8.28(d,J=8.2Hz,1H),7.98(d,J=8.3Hz,1H), 7.91-7.95(m,2H),7.62-7.66(m,3H),7.59(q,J=8.2Hz,2H),7.26-7.29(m,3H),7.02-7.04(m,1H)

[0067] <Synthesis of 2NC-A2~A7 (4Ba~4Ga)> The obtained chalcones (3Ba-3Ga) (all 0.001 mol) were placed in a pressure-resistant glass container, and 5 mL of acetonitrile and 30 μL of 1.0 mol / LCsF aqueous solution were added to each chalcone, sealed, and heated and stirred in a warm bath at 60° C. for 2 hours. The reaction solution was returned to room temperature, and the solvent was removed using an evaporator. The resulting residue was purified by silica gel column chromatography (elution solvent: 10% ethyl acetate / hexane solution) to obtain 2NC-A2-A7 (4Ba-4Ga).

[0068] [ka]

[0069] 7-Methyl-2-(naphthalen-1-yl)chroman-4-one:4Ba White solid Yield·yield 0.23g·80% 1 H-NMR(600MHz:CDCl3)δppm:8.06(d,J=8.3Hz,1H),7.88-7.93(m,3H),7.77(d,J=6.9Hz,1H),7.52-7.57(m,3H),6 .91(d,J=10.3Hz,2H),6.21(dd,J=13.1,2.7Hz,1H),3.22-3.27(m,1H),3.08(dd,J=17.1,2.8Hz,1H),2.39(s,3H). 13 C-NMR(150MHz:CDCl3)δppm:192.2,162.0,148.0,137.5,134.5,134.1,130.4,129.6,129.3,127.3, 127.0,126.9,126.2,125.6,124.1,123.4,123.1,119.1,118.4,118.2,76.8,44.2,44.1,22.2,20.7.

[0070] 6-Methoxy-2-(naphthalen-1-yl)chroman-4-one:4Ca White solid Yield·yield 0.14g·46% 1 H-NMR(600MHz:CDCl3)δppm:8.05(d,J=8.3Hz,1H),7.89-7.93(m,2H),7.78(d,J=7.6Hz,1H),7.52-7.57(m,3H),7.43(d,J=3.4Hz,1H),7.1 6(dd,J=8.9,2.9Hz,1H),7.04(d,J=9.0Hz,1H),6.19(dd,J=13.8,2.8Hz,1H),3.85(s,3H),3.23-3.28(m,1H),3.09(dd,J=17.1,2.8Hz,1H). 13C-NMR (150MHz: CDCl3) δppm192.6,156.7,154.6,134.5,134.1,130.4,129.6,12 9.3,126.9,126.2,125.6,125.6,124.1,123.0,121.1,119.7,107.7,56.1,44.1.

[0071] 7-(Methoxymethoxy)-2-(naphthalen-1-yl)chroman-4-one:4Da White solid Yield·yield 0.14g·42% 1 H-NMR(600MHz:CDCl3)δppm:8.05(d,J=8.3Hz,1H),7.89-7.96(m,3H),7.78(d,J=6.9Hz,1H),7.53-7.58(m,3H) ,6.73-6.77(m,2H),6.22(dd,J=13.1,2.7Hz,1H),3.49(s,3H),3.19-3.24(m,1H),3.05(dd,J=17.2,2.8Hz,1H). 13 C-NMR (150MHz, CDCl3) δppm: 191.2, 163.9, 163.7, 134.4, 134.1, 130.4, 129.6, 129. 3,129.1,126.9,126.2,125.6,124.1,123.0,116.0,111.5,104.0,94.3,56.6,44.0.

[0072] 6-Chloro-2-(naphthalen-1-yl)chroman-4-one:4Ea White solid Yield·yield 0.22g·72% 1H-NMR(600MHz:CDCl3)δppm:8.02(d,J=7.7 Hz,1H),7.90-7.96(m,3H),7.76(d,J=7.5Hz,1H),7.53-7.58(m,3H),7.44(dd,J=2.8,8.2Hz,1H), 7.06(d,J=9Hz,1H),6.22(dd,J=2.8,13.0Hz,1H),3.23-3.28(m,1H),3.12(dd,J=2.7,17.2Hz,1H)

[0073] 7-Chloro-2-(naphthalen-1-yl)chroman-4-one:4Ca White solid Yield·yield 0.11g·37% 1 H-NMR(600MHz:CDCl3)δppm:8.03(d,J=8.2Hz,1H),7.89-7.94(m,3H),7.74(d,J=6.9Hz,1H),7.54-7.59(m,3H),7.1 2(d,J=2.1Hz,1H),7.08-7.09(m,1H),6.25(dd,J=2.8,13.1Hz,1H),3.24-3.29(m,1H),3.12(dd,J=2.8,17.2Hz,1H)

[0074] 6-Fluoro-2-(naphthalen-1-yl)chroman-4-one:4Da White solid Yield·yield 0.29g·99% 1 H-NMR(600MHz:CDCl3)δppm:8.04(d,J=8.2Hz,1H),7.90-7.94(m,2H),7.77(d,J=7.6Hz,1H),7.64-7.66(m,1H),7.53-7.58 (m,3H),7.25-7.28(m,4H),7.07-7.09(m,1H),6.22(dd,J=3.1,13.1Hz,1H),3.24-3.29(m,1H),3.12(dd,J=2.7,17.2Hz,1H)

[0075] (Synthesis Example 3: Synthesis of 2NC-B2, B4, and B7) <Synthesis of chalcones (3Ab-3Ad)> 2'-Hydroxyacetophenone (1A) (0.6mL, 0.005mol) and 1-naphthaldehyde derivatives (2b-2d) (0.005mol) were added and dissolved in 15mL of ethanol. 0.6g (0.015mol) of sodium hydroxide was added thereto and stirred at room temperature for 20 hours. The reaction solution was added to 5mL of concentrated hydrochloric acid and 100g of ice, then transferred to a separatory funnel and extracted twice with 100mL of ethyl acetate. The organic layer was washed with saturated saline, dehydrated by adding anhydrous sodium sulfate to the organic layer, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (elution solvent: toluene for 3Ab and 3Ac, 50% ethyl acetate / hexane for 3Ad) to obtain chalcones with a naphthalene skeleton (3Ab-3Ad).

[0076] [ka]

[0077] (E)-1-(2-Hydroxyphenyl)-3-(2-methoxynaphthalen-1-yl)prop-2-en-3-one:3Ab Yield: 0.82g, 54% 1 H-NMR(600MHz:CDCl3)δppm:8.63(d,J=15.1Hz,1H),8.28(d,J=8.9Hz,1H),8.07(d,J=15.1Hz,1H),7.94-7.92(m,2H),7.83(d,J=8.2Hz,1H),7.5 7(t,J=7.2Hz,1H),7.50(t,J=6.9Hz,1H),7.42(t,J=7.9Hz,1H),7.35(d, J=8.9Hz,1H),7.05(d,J=8.3Hz,1H),6.95(t,J=7.6Hz,1H),4.09(s,3H).

[0078] (E)-1-(2-Hydroxyphenyl)-3-(4-methoxymethoxynaphthalen-1-yl)prop-2-en-3-one:3Ac Yield: 0.75g 45% 1 H-NMR(600MHz:CDCl3)δppm:(d,J=15.2Hz,1H),8.36(d,J=8.2Hz,1H),8.28(d,J=8 .3Hz,1H),7.97(d,J=6.2Hz,1H),7.92(d,J=8.2Hz,1H),7.70(d,J=15.1Hz,1H),7. 65(dd,J=6.2,1.4Hz,1H),7.57(t,J=7.6Hz,1H),7.51(t,J=6.9Hz,1H),7.17(d,J= 8.2Hz,1H),7.05(d,J=7.5Hz,1H),6.96(t,J=7.6Hz,1H),5.47(s,2H),3.57(s,3H).

[0079] (E)-1-(2-Hydroxyphenyl)-3-(2-bromonaphthalen-1-yl)prop-2-en-3-one:3Da Yield: 1.10g, 63% 1 H-NMR(600MHz:CDCl3)δppm:8.27(d,J=15.8Hz,1H),8.14(d,J=9.6Hz,1H),7.89-7.84(m,2H), 7.72(dd,J=11.0,8.9Hz,2H),7.60-7.52(m,4H),7.07(d,J=8.2Hz,1H),6.93(t,J=7.2Hz,1H).

[0080] <Synthesis of 2NC-B4, B7 (4Ab, 4Ad), and 4Ac> The obtained chalcones (3Ab to 3Ad) (all 0.001 mol) were placed in a pressure-resistant glass container, and 5 mL of acetonitrile and 100 μL of 1.0 mol / LCsF aqueous solution were added to each chalcone, sealed, and heated and stirred in a warm bath at 80° C. for 3 hours. The reaction solution was returned to room temperature, and the solvent was removed using an evaporator. The resulting residue was purified by silica gel column chromatography (elution solvent: toluene) to obtain 2NC-B4, B7 (4Ab, 4Ad), and 4Ac.

[0081] <Synthesis of 2NC-B6(5Ac)> 4Ac (0.20g, 0.0006mol) was placed in a 50mL eggplant-shaped flask, 5mL of 10% hydrogen chloride-methanol solution was added, and the mixture was heated to reflux at 80℃ for 1 hour. The mixture was returned to room temperature and concentrated under reduced pressure, after which 50mL of ethyl acetate and 20mL of saturated aqueous sodium bicarbonate solution were added and the organic layer was dried over anhydrous sodium sulfate. After the solvent was distilled off under reduced pressure, the resulting residue was purified by silica gel column chromatography (elution solvent 50% ethyl acetate / hexane) to obtain the target product 2NC-B6(5Ac) in 37mg (yield 21%).

[0082] [ka]

[0083] 2-(2-Methoxynaphthalen-1-yl)chroman-4-one:4Ab white solid Yield: 0.13g, 42% 1 H-NMR(600MHz:CDCl3)δppm:8.46(d,J=8.9Hz,1H),8.03(dd,J=6.2,1.7Hz,1H),7.90(d,J= 9.6Hz,1H),7.84(d,J=8.3Hz,1H),7.52(t,J=6.9Hz,1H),7.48(t,J=7.2Hz,1H),7.39(t,J= 7.9Hz,1H),7.31(d,J=8.9Hz,1H),7.09(t,J=6.9Hz,1H),7.06(d,J=8.2Hz,1H),6.59(dd,J =11.7,3.1Hz1H),3.95(s,3H),3.74(dd,J=14.4,2.8Hz,1H),2.76(dd,J=14.4,3.1Hz,1H). 13 C-NMR(150MHz:CDCl3)δppm:193.2,162.4,154.8,136.2,132.3,131.5,130.0,129. 2,127.5,127.0,124.7,123.9,121.7,121.4,118.6,118.3,113.2,74.2,56.7,42.5.

[0084] 2-(2-Methoxymethoxynaphthalen-1-yl)chroman-4-one:4Ac White solid Yield·yield 0.20g·59% 1 H-NMR(600MHz:CDCl3)δppm:8.48(d,J=8.9Hz,1H),8.04(dd,J=6.2,1.7Hz,1H ),7.86(t,J=9.3Hz,2H),7.55-7.46(m,3H),7.42(t,J=7.6Hz,1H),7.11(t,J= 7.6Hz,1H),7.07(d,J=8.2Hz,1H),6.62-6.59(dd,J=11.6,3.1Hz,1H),5.30(s ,2H),3.77(dd,J=2.8,14.4Hz,1H),3.49(s,3H),2.79(dd,J=14.5,3.1Hz,1H). 13 C-NMR(150MHz:CDCl3)δppm:193.1,162.3,152.7,136.3,132.1,131.5,130.6,129.2,1 27.6,127.0,124.9,124.4,121.7,121.3,119.5,118.5,116.1,95.5,74.3,56.7,42.5.

[0085] 2-(2-Bromonaphthalen-1-yl)chroman-4-one:4Ad White solid Yield·yield 0.19g·55% 1 H-NMR(600MHz:CDCl3)δppm:8.62(d,J=8.9Hz,1H),8.05(d,J=8.3Hz,1H),7.89(t,J=4.8Hz,1H),7.73(d,J=8.9Hz,1H),7.66(d,J=8.9Hz,1H),7 .58-7.52(m,3H),7.14(t,J=7.6Hz,1H),7.08(d,J=8.2,1H),6.58(dd,J=11.7,3.4Hz,1H),3.63(t,J=16.1Hz,1H),2.89(dd,J=14.4,3.5Hz,1H). 1313C-NMR (150 MHz, CDCl3) δ ppm: 191.8, 161.5, 136.5, 133.9, 132.1, 132.0, 131.1, 130.5, 129.5, 127.7, 127.2, 126.5, 125.4, 122.2, 122.1, 121.3, 118.6, 41.8.

[0086] 1 1H-NMR (600 MHz, DMSO-d6) δ ppm: 10.42 (s, 1H), 8.22 (d, J = 8.2 Hz, 1H), 8.16 (d, J = 8.9 Hz, 1H), 7.85 (dd, J = 6.2, 1.7 Hz, 1H), 7.61 - 7.49 (m, 4H), 7.12 (t, J = 7.6 Hz, 1H), 7.07 (d, J = 8.2 Hz, 1H), 6.91 (d, J = 7.6 Hz, 1H), 6.32 (dd, J = 11.0, 2.1 Hz, 1H), 3.46 (dd, J = 13.1, 4.1 Hz, 1H), 2.90 (dd, J = 13.7, 2.8 Hz, 1H). 13 13C-NMR (150 MHz, DMSO-d6) δ ppm: 192.1, 161.3, 153.9, 136.0, 131.7, 126.7, 126.3, 125.3, 124.6, 124.5, 124.2, 123.3, 122.5, 121.3, 120.8, 117.9, 106.9, 75.7, 42.5.

[0087] 2-(2-Hydroxynaphthalen-1-yl)chroman-4-one: 5Ac White solid Yield 37 mg, 21% 1 1H-NMR (600 MHz, DMSO-d6) δ ppm: 10.42 (s, 1H), 8.22 (d, J = 8.2 Hz, 1H), 8.16 (d, J = 8.9 Hz, 1H), 7.85 (dd, J = 6.2, 1.7 Hz, 1H), 7.61 - 7.49 (m, 4H), 7.12 (t, J = 7.6 Hz, 1H), 7.07 (d, J = 8.2 Hz, 1H), 6.91 (d, J = 7.6 Hz, 1H), 6.32 (dd, J = 11.0, 2.1 Hz, 1H), 3.46 (dd, J = 13.1, 4.1 Hz, 1H), 2.90 (dd, J = 13.7, 2.8 Hz, 1H). 13 C-NMR(150MHz,DMSO-d6)δppm:192.1,161.3,153.9,136.0,131.7,126.7,126.3,1 25.3,124.6,124.5,124.2,123.3,122.5,121.3,120.8,117.9,106.9,75.7,42.5.

[0088] (Example 1: Verification of adipocyte differentiation of preadipocytes by flavanone derivative 2-(Naphthalen-1-yl)chroman-4-one (2NC)) 3T3-L1 preadipocytes are usually differentiated into white adipocytes through differentiation induction. In recent years, it has been reported that preadipocytes in white adipose tissue differentiate into beige adipocytes and white adipocytes transform into beige adipocytes by cold stimulation or dietary intake. Therefore, to evaluate the effect of 2NC in inducing beige-formation of preadipocytes, we first performed a test using 3T3-L1 cells, which are mouse-derived preadipocytes.

[0089] The flavanone derivative 2NC was dissolved in dimethyl sulfoxide (DMSO, Fujifilm Wako Pure Chemical Industries, Ltd.) at a concentration of 10 mM or 20 mM and used in the experiments. The cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Nissui Pharmaceutical Co., Ltd.) containing 10% calf serum (Calf Serum, Thermo Fisher Scientific Co., Ltd.) and penicillin-streptomycin-L-glutamine solution (PSG, Thermo Fisher Scientific Co., Ltd.) at 37°C in a 5% CO2 environment. The cells were cultured until they became 80% confluent and were used in the experiments.

[0090] Differentiation into adipocytes was carried out as follows. After confirming under a microscope that the cells had reached 100% confluence, the medium was removed and 10% fetal bovine serum (Fetal Bovine Serum, Cosmo Bio Co., Ltd.)-containing DMEM·Ham's F12 (Nissui Pharmaceutical Co., Ltd.)·PSG·triiodotyrosine (Sigma-Aldrich Japan Co., Ltd., T3) medium containing 1.6 μM insulin (Fujifilm Wako Pure Chemical Industries, Ltd.), 0.25 μM dexamethasone (Fujifilm Wako Pure Chemical Industries, Ltd.), and 500 μM 3-isobutyl-1-methylxanthine (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to induce differentiation into adipocytes. This point was designated as day 0 of culture, and on day 2 of culture, the medium was replaced with 10% fetal bovine serum-containing DMEM·Ham's F12·PSG·T3 medium containing insulin, and the medium was replaced every 2 days thereafter.

[0091] To evaluate the effect on 3T3-L1 preadipocytes, Oil Red O staining was performed using a Lipid Assay Kit (Cosmo Bio Co., Ltd.) as follows. 1 × 10 5 The cells seeded at a density of 100 cells / well were cultured as described above. 2NC was diluted with medium to a final concentration of 10 μM or 20 μM and added to the cell culture solution on days 0, 2, and 4. Note that a solution containing only 0.1% DMSO, the solvent, was used as a control and treated in the same manner.

[0092] After the end of cell culture (day 6), the cells were washed once with 1x phosphate buffer (1xPBS) and fixed overnight with a fixative (4% formaldehyde solution). After washing the fixative with purified water, the cells were stained with oil red O solution, dried, and observed under a microscope (Olympus IX71 microscope, Olympus Life Sciences Co., Ltd.). The lipid droplets were quantified by adding a cell extract (accessory of the Lipid Assay Kit) to the dried well to elute the dye, and measuring the absorbance at 540 nm using a microplate reader (FLUO star Omega, BMG LAB TECH Co., Ltd.). The dye elution time was 60 minutes. Comparison between control cells and each treatment group was performed by one-way analysis of variance (One way ANOVA), and Dunnett's test was used as a post hoc test (statistically significant when the respective significance levels (p) were *p<0.01 compared to the control).

[0093] The results are shown in Figure 2. In cells treated with 2NC for 6 days, along with induction of differentiation into adipocytes, it was observed under a microscope that the number of cells that had accumulated neutral fat was smaller than that of control cells treated with DMSO. Furthermore, a significant decrease in the amount of lipid droplet accumulation was quantitatively confirmed in cells treated with 2NC for 6 days (Figure 3). In other words, it was shown that 2NC suppresses the accumulation of neutral fat associated with the differentiation of preadipocytes into adipocytes.

[0094] (Example 2: Measurement of gene expression levels in cells treated with 2NC) From the results of Example 1, it is unclear whether the decrease in neutral fat accumulation in 3T3-L1 adipocytes treated with 2NC is directly due to the inhibition of differentiation from preadipocytes to white adipocytes, or whether it is dependent on the increase in lipid burning ability due to the beige-induced effect. Therefore, in order to clarify the fat accumulation inhibitory effect of 2NC, gene expression levels were measured in cells treated with 2NC. The expression levels of the Peroxisome proliferator-activated receptor γ2 (Pparγ2) gene, CCAAT / Enhancer-binding protein α (C / ebpα) gene, and adipocyte Protein 2 (aP2) gene, which are differentiation markers of white adipocytes, as well as the Uncoupling Protein 1 (Ucp1) gene, which is a molecular marker for beige adipocyte formation, the Elongation of very long chain fatty acid (Elovl3) gene, which is a beige adipocyte-related gene, and the Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc1α) gene, were measured.

[0095] The experiment was carried out as follows. 1 × 10 5 The cells seeded at a density of 10000 cells were subjected to differentiation induction treatment by the method described above. 2NC was diluted with differentiation medium to a final concentration of 20 μM and added to the cell culture solution on days 0, 2, and 4. The control was a solution containing only 0.1% DMSO, the solvent.

[0096] Total RNA was extracted and purified from the cells on days 0, 2, 4, and 6 using RNAiso Plus (Takara Bio Inc.). cDNA was synthesized by reverse transcription using 1 μg of total RNA as a template, ReverTra Ace RT (Toyobo Co., Ltd.), Oligo dT primer (Sigma-Aldrich Japan Co., Ltd.), and dNTP mix (Nippon Gene Co., Ltd.). 5×RT Buffer 4 μL (accessory of ReverTra Ace RT), 2 μL of sterile purified water, 2 μL of 10 mM dNTP mix, 1 μL of 10 μM Oligo dT primer, ReverTra Ace 1 μL (accessory of ReverTra Ace RT), and 10 μL equivalent to 1 μg of total RNA were mixed to make a total volume of 20 μL, which was used as the reaction solution. A reverse transcription reaction was carried out at 42°C x 40 minutes, followed by 99°C x 5 minutes using a PCR thermal cycler (trade name: Applied Biosystems (registered trademark) thermal cycler, manufactured by Applied Biosystems Japan, Inc.). The reverse transcription reaction solution after the reaction was diluted 3-fold with sterile purified water and used for real-time RT-PCR analysis.

[0097] For real-time RT-PCR analysis, the Mx3000 quantitative RT-PCR system (Agilent Technologies) was used. The primers included Pparγ2 forward primer (5'-GCTGTTATGGGTGAAACTCTG-3') (SEQ ID NO: 1), Pparγ2 reverse primer (5'-ATAATAAGGTGGAGATGCAGG-3') (SEQ ID NO: 2), aP2 forward primer (5'-ATGAAATCACCGCAGACGACAGGA-3') (SEQ ID NO: 3), aP2 reverse primer (5'-TGTGGTCGACTTTCCATCCCACTT-3') (SEQ ID NO: 4), C The following primers were used: / ebpα forward primer (5'-TGGACAAGAACAGCAACGAG-3') (sequence number 5), C / ebpα reverse primer (5'-TCACTGGTCAACTCCAGCAC-3') (sequence number 6), Ucp1 forward primer (5'-GGGCCCTTGTAAACAACAAA-3') (sequence number 7), Ucp1 reverse primer (5'-CTCGGTCCTTCCTTGGTGTA-3') (sequence number 8), Elovl3 forward primer (5'-ATGAACTTTGGCGTCCATTC-3') (sequence number 9), Elovl3 reverse primer (5'-CTTTCTCCTGCCTCCAGATG-3') (sequence number 10), Pgc1α forward primer (5'-AATCAGACCTGACACAACGC-3') (sequence number 11), and Pgc1α reverse primer (5'-GCATTCCTCAATTTCACCAA-3') (sequence number 12). As an internal standard, 36b4 forward primer (5'-AAGCGCGTCCTGGCATTGTCT-3') (SEQ ID NO: 13) and 36b4 reverse primer (5'-CCGCAGGGGCAGCAGTGGT-3') (SEQ ID NO: 14) were used. Both primers were manufactured by Sigma-Aldrich Japan Co., Ltd.

[0098] Go Taq qPCR Master mix (Promega Corporation) was used for the real-time RT-PCR reaction. 10 μL of Go-Taq qPCR Master Mix, 0.1 μL of 0.1 mM forward primer, 0.1 μL of 0.1 mM reverse primer, 2 μL of cDNA solution, and 7.8 μL of sterile purified water were mixed and placed in a 0.2 mL cap-integrated PCR 8-tube flat 120 (Nihon Genetics Co., Ltd.) to prepare a reaction solution. PCR reaction was performed using the program: {95°C x 30 seconds (pretreatment) → "denaturation: 95°C x 30 seconds → annealing: 58°C x 30 seconds → extension reaction 72°C x 30 seconds" x 40 cycles (amplification), 95°C x 15 seconds → 60°C x 30 seconds → 95°C x 15 seconds x 1 cycle (melting curve analysis)}.

[0099] The relative expression levels of each gene were calculated by the delta-delta Ct (ΔΔCt) method from the Ct values ​​of 36b4 (Threshold Cycle: the number of cycles at which a certain amount of amplification (threshold) is reached) and Pparγ2, aP2, C / ebpα, Ucp1, Elovl3, and Pgc1α. The gene expression levels of cells treated with 2NC and control cells on the same days were compared using Student's T-test (statistically significant when the significance level (p) was *p<0.05 compared to control cells).

[0100] The results are shown in Figure 4. It was shown that the expression levels of Pparγ2 gene, aP2 gene, and C / ebpα gene in cells treated with 2NC for 6 days were significantly increased compared to control cells. In addition, the expression levels of Ucp1 gene and Elovl3 gene were significantly increased in cells treated with 2NC. On the other hand, the induction of Pgc1α gene expression accompanying differentiation induction was suppressed in cells treated with 2NC. These results suggest that the inhibition of lipid droplet accumulation by 2NC is not due to the inhibition of differentiation induction from preadipocytes to white adipocytes, but due to the promotion of differentiation from preadipocytes to beige adipocytes.

[0101] (Example 3: Search for factors involved in the induction of beige adipocyte differentiation by 2NC) In Example 2, the effect of 2NC in promoting differentiation into beige adipocytes was demonstrated. Factors that promote the beigeification of adipocytes have been reported to include activation of PPARγ, thyroid hormone receptor (TR), and β-adrenergic receptor, as well as fibroblast growth factors (FGFs). Therefore, in Example 3, the factors involved in the induction of beige adipocyte differentiation by 2NC were examined.

[0102] As in Example 2, 3T3-L1 preadipocytes were seeded and cultured until the cells became confluent. In Example 3, the differentiation medium used in Example 2 was used as a basic differentiation induction medium, but without triiodotyrosine (T3), a thyroid stimulating hormone (1.6 μM insulin, 0.25 μM dexamethasone, 500 μM 3-isobutyl-1-methylxanthine, containing 10% FBS, DMEM·Ham's F12·PSG). Pioglitazone hydrochloride (Fujifilm Wako Pure Chemical Industries, Ltd., Pio), an agonist of PPARγ, was dissolved in DMSO to a concentration of 5 mM and used in the experiment.

[0103] The experiment was carried out as follows. Cells were cultured in the same manner as in Example 1, and 2NC, Pio, and T3 were added on day 0 to final concentrations of 20 μM, 5 μM, and 0.02 nM, respectively, and were also added on days 2 and 4. Note that the control was a solution containing only 0.1% DMSO, the solvent. Details of each treatment group are shown in FIG. 5.

[0104] Total RNA was extracted from the cells on day 6 of differentiation in the same manner as in Example 2, cDNA was prepared by reverse transcription reaction, and real-time PCR analysis was performed. In addition to the Pparγ2 gene, C / ebpα gene, aP2 gene, Ucp1 gene, and Elovl3 gene measured in Example 2, the gene expression levels of the Adrenoceptor Beta 1-3 (Adrb1-3) gene, the Fgf6 gene, the Fgf9 gene, and the Fgf receptor 1 (Fgfr1) gene were analyzed. The analysis was performed using Adrenoceptor Beta 1 (Adrb1) forward primer (5'-CAGCATTGAGACCCTGTGTG-3') (SEQ ID NO: 15), Adrenoceptor Beta 1 (Adrb1) reverse primer (5'-AGCACTTGGGGTCGTTGTAG-3') (SEQ ID NO: 16), Adrenoceptor Beta 2 (Adrb2) forward primer (5'-GAGCACAAAGCCCTCAAGAC-3') (SEQ ID NO: 17), Adrenoceptor Beta 2 (Adrb2) reverse primer (5'-GCTCTTGAAAGGCAATCCTG-3') (SEQ ID NO: 18), Adrenoceptor Beta 3 (Adrb3) forward primer (5'-GGCAACCTGCTGGTAATCAT-3') (SEQ ID NO: 19), Adrenoceptor Beta 3 (Adrb3) reverse primer (5'-TCCACTGACGTCCACAGTTC-3') (sequence number 20), Fgf6 forward primer (5'-GCATCAGTGGAACACACGAGGA-3') (sequence number 21), Fgf6 reverse primer (5'-CAGTCTTCCTTTACTGTTCATGGC-3') (sequence number 22), Fgf9 forward primer (5'-GGGGAGCTGTATGGATCAGA-3') (sequence number 23), Fgf9 reverse primer (5'-CTTTGTCAGGGTCCACTGGT-3') (sequence number 24), Fgf receptor 1 (Fgfr1) forward primer (5'-ATGGTTGACCGTTCTGGAAG-3') (sequence number 25), Fgf receptor 1 (Fgfr1) reverse primer (5'-GGAAGTCGCTCTTCTTGGTG-3') (sequence number 26) were used. All primers used were manufactured by Sigma-Aldrich Japan Co., Ltd.

[0105] From the measured Ct values ​​of 36b4 in each cell and the Ct values ​​of Ucp1, Elovl3, Pparγ2, aP2, C / ebpα, Adrb1, Adrb2, Adrb3, Fgf6, Fgf9, and Fgfr1, the relative expression levels of each gene were calculated in the same manner as in Example 2. Comparisons between control cells and each treatment group were performed using one-way analysis of variance (One way ANOVA), with Dunnett's test used as a post hoc test (statistically significant when each significance level (p) was *p<0.01 compared to control cells).

[0106] The results are shown in Figures 6A to 6C. Comparing the expression levels of the Ucp1 gene in cells treated with 2NC (2), pioglitazone (3), and T3 (4), a significant induction of expression was observed in the 2NC treatment. In addition, comparing the expression levels in cells co-treated with 2NC + T3 (5), pioglitazone + T3 (6), and 2NC + pioglitazone (7), a significant increase in the expression level of the Ucp1 gene in cells treated with 2NC was shown. However, no additive or synergistic effect of pioglitazone and T3 was observed on the increase in the expression level of the Ucp1 gene by 2NC. A significant increase in the expression level of the Ucp1 gene was observed in cells treated with pioglitazone alone (3) or co-treated with T3 (6), which shows a beigeification-inducing effect, but the degree of the increase was weaker than that of the expression induction by 2NC. This indicates that 2NC may be a compound with an excellent effect of promoting beige adipocyte differentiation. On the other hand, an increase in the expression level of Elovl3 gene was observed in cells treated with 2NC, but this increase was also observed in cells treated with pioglitazone. In addition, the expression levels of Pparγ2, C / ebpα, and aP2 genes, which are markers of adipocyte differentiation, were measured, and it was found that the expression level of Pparγ2 gene increased or tended to increase with 2NC treatment alone (2) and with T3 (5). Furthermore, the expression level of aP2 gene increased in all cells compared to control cells, and the expression level of C / ebpα gene increased with 2NC treatment (2). Next, the expression level of adrenergic receptor genes was examined, and no increase in the expression levels of Adrb1 gene and Adrb2 gene was observed in cells treated with 2NC. The expression level of Adrb3 gene was increased by 2NC treatment (2), but the increase disappeared with cotreatment with pioglitazone or T3. Finally, we examined the expression of fibroblast growth factor-related genes. Although no significant change was observed in the expression of Fgf6 gene in cells treated with 2NC, the expression of Fgf9 gene was significantly increased (2,5,7). The expression of Fgf9 gene was increased in cells treated with pioglitazone alone or with pioglitazone + T3, but the induction was milder than that with 2NC treatment. The expression of Fgf1R gene, which is an FGF receptor, was increased by 2NC treatment (2), but the increase disappeared when co-treated with pioglitazone or T3. These results suggest that the beige-colored induction of preadipocytes by 2NC involves the induction of Fgf9 gene expression.

[0107] (Example 4: Changes in the expression level of Fgf9 gene during beige formation by 2NC) In Example 3, an increase in the expression level of Fgf9 was confirmed in cells treated with 2NC. Therefore, the expression change of the Fgf9 gene during the beige process by 2NC was analyzed by real-time RT-PCR using the cDNA prepared in Example 2. The gene expression level of cells treated with 2NC and control cells on the same days was compared using Student's T-test (a statistically significant level (p) of *p<0.05 compared to control cells was considered to be statistically significant).

[0108] The results are shown in Figure 7. The results shown in Figure 6 indicate that the expression level of the Fgf9 gene was significantly increased on days 4 and 6 after the start of differentiation induction in 2NC-treated cells. These results suggest that FGF9 is involved in the beige-color-inducing effect of 2NC on preadipocytes.

[0109] (Example 5: Changes in mitochondrial content associated with beige formation of adipocytes by 2NC) It has been reported that beige adipocytes have a higher mitochondrial content and a higher fat burning capacity than white adipocytes, so we examined the mitochondrial content in cells treated with 2NC.

[0110] As in Example 1, cells treated with 2NC until the 6th day and control cells were collected and then treated with Proteinase K (Nippon Gene Co., Ltd.). Protein precipitation solution (Takara Bio Inc.) was added to precipitate proteins, and genomic DNA was purified and extracted using isopropanol and 70% ethanol solution (Fujifilm Wako Pure Chemical Industries, Ltd.). Using 10 ng of genomic DNA, the amount of the mitochondrial specific gene Cytochrome c oxidase subunit 2 (Cox2) gene and the amount of the β-actin gene present in the nucleus were measured by real-time PCR as in Example 2. The primers used were Cox2 forward primer (5'-AATTGCTCTCCCCTCTCTACG-3') (SEQ ID NO: 27), Cox2 reverse primer (5'-GTAGCTTCAGTATCATTGGTGC-3') (SEQ ID NO: 28), β-actin forward primer (5'-GACCCAGATCATGTTTGAGACC-3') (SEQ ID NO: 29), and β-actin reverse primer (5'-ATCAGAATGCCTGTGGTACGAC-3') (SEQ ID NO: 30). All primers were manufactured by Sigma-Aldrich Japan Co., Ltd. Statistical analysis was performed using Student's T test (statistically significant when the significance level (p) was *p<0.05 compared to control cells).

[0111] The results are shown in Figure 8. The amount of mitochondria in cells treated with 2NC for 6 days was significantly increased compared to control cells. These results indicate that the beige formation-promoting effect of 2NC not only increases the expression of the Ucp1 gene, but also increases the amount of mitochondria.

[0112] (Example 6: Verification of transformation of white adipocytes into beige adipocytes by 2NC) The effect of 2NC in promoting the beige formation of adipocytes in preadipocytes was shown in Examples 1 to 5. Next, the transformation ability of 2NC (the effect of transforming white adipocytes into beige adipocytes) was examined.

[0113] Cell culture was carried out as follows: 1 × 10 5 The cells were seeded at a density of 10 ...

[0114] Total RNA was extracted from the cells on day 18 in the same manner as in Example 2, and reverse transcription was performed, followed by real-time PCR analysis. The gene expression levels measured in Example 2 were analyzed in the same manner. Comparisons between control cells and each treatment group were performed using one-way ANOVA, with Dunnett's test used as a post hoc test (statistical significance was determined when each significance level (p) compared to control cells was *p<0.01).

[0115] The results are shown in Figure 9. The expression levels of the genes Ucp1, Elovl3, and Pgc1α in white adipocytes treated with 2NC for 8 days were significantly increased compared to control cells. On the other hand, no difference was observed in the expression levels of the genes Pparγ2 and Fgf9 in cells treated with 2NC compared to control cells. In cells treated with pioglitazone (Pio), which has the ability to transform into beige adipocytes, the expression levels of the genes Ucp1, Elovl3, Pgc1α, Pparγ2, and Fgf9 were increased. These results indicate that 2NC has the effect of transforming white adipocytes into beige adipocytes.

[0116] (Example 7: Verification of transformation into beige adipocytes by 2NC from the viewpoint of mitochondrial content) After differentiation into white adipocytes in the same manner as in Example 6, the cells were treated with 2NC for 8 days. DNA was purified and extracted from the cells in the same manner as in Example 5, and the amount of the Cox2 and β-actin genes was analyzed by real-time PCR. Comparison between the cells treated with 2NC and the control cells was performed using Student's T-test (statistical significance was determined when the respective significance levels (p) compared to the control cells were *p<0.05).

[0117] The results are shown in Figure 10. The mitochondrial content in cells treated with 2NC was significantly increased compared to control cells. This indicated that 2NC increases the amount of mitochondria by transforming cells into beige adipocytes. These results suggest that 2NC not only promotes differentiation of preadipocytes into beige adipocytes, but also has the ability to transform white adipocytes into beige adipocytes.

[0118] (Example 8: Verification analysis of changes in body weight and tissue weight of mice administered 2NC) The above examples using cultured cells demonstrated that 2NC promotes beige adipocyte differentiation and transformation. Therefore, we investigated the beige-forming effect of 2NC in an animal model (in vivo).

[0119] Four-week-old C57BL / 6J male mice (Japan SLC Co., Ltd.) were kept in accordance with the ethical regulations for experimental animals at Nihon University College of Pharmacy. The mice were distributed in groups of three per cage and kept under conditions of 23±1°C temperature, 50±10% relative humidity, and a 12-hour light-dark cycle. The mice were fed normal food (NMF, Oriental Yeast Co., Ltd.) ad libitum. 2NC was suspended in 20% 2-hydroxypropyl-β-cyclodextrin solution (HPCD, Fujifilm Wako Pure Chemical Industries Co., Ltd.) to prepare a 1mg / mL solution, and administered at 10mg / kg / mice. The solvent, 20% HPCD solution, was administered as a vehicle. 2NC was administered every 2 days for 6 weeks using an oral probe (Fuchigami Kikai Co., Ltd.). Body weight was measured every week from the start of administration (animal experiment approval number: AP22PHA019-1).

[0120] The changes in mouse body weight are shown in Figure 11. In addition, at the time of dissection 6 weeks after the start of administration, the adipose tissues around the epididymis and the subcutaneous adipose tissues were collected and their weights were measured. Comparisons between mice administered 2NC and mice administered vehicle were performed using Student's T-test (statistical significance was determined when the significance level (p) was *p<0.05 compared with each tissue of control mice). The results of tissue weight are shown in Figure 12.

[0121] The weight change of mice administered 2NC was not significantly different from that of mice administered vehicle, but a tendency for weight gain to be suppressed was observed in the sixth week of administration (Fig. 11). A significant decrease in subcutaneous adipose tissue weight (sWAT) was also observed in mice administered 2NC, but no difference was observed in the weight of epididymal adipose tissue (eWAT) (Fig. 12).

[0122] (Example 9: Histopathological analysis of adipose tissue in mice administered 2NC) In the above, mice administered 2NC showed a tendency to suppress weight gain and a decrease in subcutaneous adipose tissue weight. It has been reported that beige adipocytes contain many multilocular lipid droplets and many mitochondria expressing UCP1. Therefore, pathological sections of adipose tissue were prepared and histological analysis was performed.

[0123] Pathological sections were prepared using parts of each adipose tissue (epididymal adipose tissue, subcutaneous adipose tissue, brown adipose tissue) collected at the time of dissection in Example 7. The tissue fragments were placed in a Unicassette (Sakura Finetech Japan Co., Ltd.), infiltrated with 4% formaldehyde solution (Fujifilm Wako Pure Chemical Industries, Ltd.), and fixed for 18 hours. Paraffin blocks were prepared by treating with 50% alcohol solution (3 hours), 70% alcohol solution (24 hours), 80% alcohol solution (1 hour), 90% alcohol solution (1 hour), and 100% alcohol solution (2 hours) using Tissue-Tek (registered trademark) VIPTM 6AI (Sakura Finetech Japan Co., Ltd.), treating with xylene (Muto Chemical Co., Ltd.) for 2 hours, and infiltrating (16 hours) with paraffin solution (Sakura Finetech Japan Co., Ltd.) dissolved at 65°C. The paraffin block was humidified and sliced ​​into 5 μm thin sections using a microtome (Sakura Finetech Japan Co., Ltd.) and a feather microtome blade S35 (Tokyo Glass Instruments Co., Ltd.) to prepare paraffin sections, and a T-72 paraffin water bath extender (Takashima Co., Ltd.) was set to 45°C, and the paraffin sections were floated on the T-72 paraffin water bath extender (Takashima Co., Ltd.) to extend the tissue. The paraffin sections were fixed on fine frost slides (Matsunami Glass Industry Co., Ltd.) and dried on a Tissue-tek (registered trademark) Slide Warmer PS-53 (Sakura Finetech Japan Co., Ltd.) at 40°C for 12 hours.

[0124] The slide glass with the pathological section attached was deparaffinized by sequentially immersing it in xylene for 3 minutes x 3 times, 100% alcohol solution for 1 minute x 3 times, 90% alcohol solution for 1 minute, 80% alcohol solution for 1 minute, and 70% alcohol for 2 minutes. The deparaffinized sample was washed with running water, then immersed in distilled water and stained as follows: Mayer's hematoxylin solution (Sakura Finetech Japan Co., Ltd.) for 1 minute, washed with running water, then distilled water, eosin solution (Sakura Finetech Japan Co., Ltd.) for 45 seconds. The stained sample was immersed in 70% alcohol solution for 2 minutes, 80% alcohol solution for 1 minute, 90% alcohol solution for 1 minute, 100% alcohol solution for 1 minute x 3 times, and xylene three times, and the slide glass was mounted using mounting medium NEW MX (Matsunami Glass Industry Co., Ltd.) and cover glass (Matsunami Glass Industry Co., Ltd.).

[0125] The hematoxylin-eosin stained and mounted slides were observed using an upright microscope BX53 (Olympus Life Sciences Corp.), and histopathological images were taken using a digital camera for microscopes DP28 (Olympus Life Sciences Corp.) (Scale bar: 100 μm). LPF (Low power field: low magnification field (100x)) is a high power field (HPF (a magnified image of a part of the 100x field).

[0126] The results are shown in Figure 13. Beige adipocytes are characterized by the formation of multilocular lipid droplets within the cells. Many multilocular adipocytes were observed in the subcutaneous adipose tissue (sWAT) of mice treated with 2NC. On the other hand, multilocular adipocytes were hardly observed in the epididymal adipose tissue (eWAT) of any mice. No pathological differences were observed in the brown adipose tissue (BAT).

[0127] (Example 10: Verification of beige coloration of adipocytes in subcutaneous adipose tissue of mice administered 2NC) Since a decrease in tissue weight and the formation of multilocular lipid droplets were observed in the subcutaneous adipose tissue of mice administered 2NC, it is inferred that the beige color of adipocytes was promoted. Therefore, we examined the protein expression level of UCP1 and the amount of mitochondria in the adipose tissue of mice administered 2NC.

[0128] Protein extraction was performed as follows. The subcutaneous adipose tissue (about 10 mg) collected at the time of dissection in Example 7 was placed in 300 μL of tissue extraction solution (RIPA: 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 0.1% SDS, 1 mM EDTA, 1% TritonX-100, 0.1% deoxycholic acid, protease inhibitor cocktail (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)), and protein was extracted using a Dounce homogenizer (manufactured by AS ONE Co., Ltd.) and Bioruptor (ultrasonic device, manufactured by Cosmo Bio Co., Ltd.). The extracted protein was reacted using BCA Protein Assay Reagent (manufactured by Takara Bio Co., Ltd.), and the absorbance at 562 nm was measured using a microplate reader (FLUO star Omega, manufactured by BMG LAB TECH Co., Ltd.) to measure the protein concentration. The obtained protein extract was used for Western blotting.

[0129] Protein extracts equivalent to 20 μg obtained from subcutaneous adipose tissue of mice administered 2NC or vehicle were used for Western blotting. Western blotting was performed by polyacrylamide gel electrophoresis (SDS-PAGE). ExcelBand 3-color Regular Range Protein Marker (Cosmo Bio Co., Ltd.) was used as a protein molecular weight marker. Polyacrylamide gel electrophoresis was performed using an electrophoresis tank AE-6500 Rapidus Mini Slab (ATTO Co., Ltd.), and transfer to a PVDF membrane (Millipore Co., Ltd.) was performed using a semi-dry transfer device (Horize Blot 2M-R, ATTO Co., Ltd.). After electrophoresis, the PVDF membrane transferred from the polyacrylamide gel was reacted as follows. Staining was performed by 5% skim milk solution (Fujifilm Wako Pure Chemical Industries, Ltd.) (1 hour at room temperature) → primary antibody solution (1 hour at room temperature) → 1xPBST washing (1xPBS with 0.05% Tween 20 added, 10 minutes x 3 times) → secondary antibody solution (1 hour at room temperature) → 1xPBST washing (10 minutes x 3 times). The primary antibodies were UCP antibody 1 (ab10983, Abcam), β-ACTIN antibody (60008-1-Ig, Proteintec), and the secondary antibodies were Anti-Rabbit IgG (H&L) HRP-linked Antibody (7074S, Cell Signaling Technology Japan, Inc.), Anti-mouse IgG (H&L) HRP-linked Antibody (#7076, Cell Signaling Technology Japan, Inc.), which were diluted with 1xPBST solution and reacted. Proteins were detected by staining using Amersham ECL Prime (GE Healthcare Japan, Ltd.) and photographed using a CCD imager (Amersham ImageQuant 800, Cytiva). The protein amounts of UCP1 and the internal standard protein β-ACTIN are shown in Figure 14. Protein extracts obtained from different mice were electrophoresed in lanes 1-4.

[0130] Increased amounts of UCP1 protein were observed in the subcutaneous adipose tissue of mice administered 2NC, indicating that administration of 2NC promotes beige coloration in the subcutaneous adipose tissue.

[0131] (Example 11: Changes in mitochondrial content in adipose tissue of mice administered 2NC) DNA was extracted from the adipose tissues (periepididymal adipose tissue, subcutaneous adipose tissue, and brown adipose tissue) collected at the time of dissection in Example 7 by the same method as in Example 5, and the amount of mitochondria was measured by real-time PCR. Comparisons between mice administered with 2NC and mice administered with the vehicle were performed using Student's T-test (statistical significance was determined when the respective significance levels (p) were *p<0.05 compared to control mice).

[0132] The results are shown in Figure 15. The amount of mitochondria was significantly increased in the subcutaneous adipose tissue (sWAT) of mice administered 2NC. On the other hand, no significant difference was observed in the amount of mitochondria in the epididymal adipose tissue (eWAT) and brown adipose tissue (BAT). These results indicate that 2NC is a compound that promotes the beige coloring of mouse subcutaneous adipose tissue.

[0133] (Example 12: Verification of the effect of 2NC derivatives in promoting differentiation into beige adipocytes) The above experiments demonstrated that 2NC is a compound that exhibits beige color-promoting activity. 2NC is a compound with a phenylchroman structure (C6-C3-C6). Therefore, by synthesizing derivatives with modified A or B rings of the flavanone skeleton, we verified the structure-activity relationship of the beige color-promoting activity of 2NC derivatives with the aim of optimizing NC compounds (biological activity, stability, etc.).

[0134] First, the beige-promoting effect of derivatives in which the A ring of the flavanone skeleton was changed was examined. The structural formula of the derivative is shown in FIG. 16. The differentiation-promoting effect of the 2NC-A derivative from preadipocytes to beige adipocytes was evaluated using the change in the amount of accumulated neutral fat as an index. The 2NC-A derivatives were dissolved in DMSO to 5 mM. The 2NC-A derivatives were diluted with differentiation-inducing medium to a final concentration of 5 μM, and added at the time of medium exchange on the 0th, 2nd, and 4th days of differentiation induction as in Example 1. Note that cells similarly treated with differentiation culture medium containing 0.1% DMSO as a solvent were used as Control (Con), and pioglitazone (5 μM), which shows a beige-promoting effect, was used as a positive control. The cells on the 6th day of differentiation induction were washed once with 1xPBS, fixed overnight with a fixative, stained with Oil Red O, and observed under a microscope.

[0135] The results are shown in Figure 17. When cells were treated with 2NC-A derivatives for 6 days in conjunction with induction of adipocyte differentiation, the amount of neutral fat accumulated in cells treated with 2NC-A2, A3, A4, A5, A6, and A7 was suppressed.

[0136] 2NC-A2 to A7, which showed an inhibitory effect on neutral fat accumulation, were examined from the viewpoint of gene expression. As described above, 2NC-A2 to A7 (5 μM) were treated for 6 days during differentiation induction. Total RNA was extracted from the cells on the 6th day by the method of Example 2, reverse transcription reaction was performed, cDNA was prepared, and gene expression was analyzed by real-time PCR. Gene expression analysis was performed on genes (Ucp1, Fgf9, Elovl3, Pparγ2, C / ebpα, and aP2) that were increased by 2NC treatment in Example 2. Comparison between control (C) cells and cells treated with 2NC-A2 to A7 derivatives was performed by one-way analysis of variance (One way ANOVA), and Dunnett's test was used as a post hoc test (statistically significant when the respective significance levels (p) were *p<0.01 compared to control cells).

[0137] The results are shown in Figures 18A and 18B. The expression levels of the Ucp1, Elovl3, Pparγ2, and aP2 genes in cells treated with 2NC-A2, 2NC-A3, and 2NC-A4 were significantly increased compared to those in the control cells (C). Furthermore, the expression level of the Fgf9 gene, which was shown to be correlated with the induction of Ucp1 expression by 2NC treatment in Example 2, was significantly increased in cells treated with 2NC-A2 and 2NC-A4.

[0138] Among the 2NC-A derivatives, 2NC-A2 and 2NC-A4 enhanced the expression induction effect of beigeification-related genes such as Ucp1 and Fgf9, so the amount of mitochondria in cells treated with 2NC-A2 and 2NC-A4 was measured in the same manner as in Example 5. Comparisons between control (C) cells and cells treated with 2NC-A2 and 2NC-A4 were performed using one-way analysis of variance (One way ANOVA), with Dunnett's test used as a post hoc test (statistically significant when the respective significance levels (p) compared to control cells were *p<0.01).

[0139] The results are shown in Figure 19. The amount of mitochondria in cells treated with 2NC-A2 and 2NC-A4 was significantly increased compared to control cells. These results suggest that 2NC-A2 and 2NC-A4 are compounds that promote differentiation of preadipocytes into beige adipocytes at a lower concentration (5 μM) than 2NC (20 μM). These results indicate that 2NC-A2 and 2NC-A4 derivatives are involved in regulating the amount of mitochondria as well as increasing the expression of the Ucp1 gene.

[0140] Next, the beige-promoting effect of 2NC derivatives modified on the B ring of the flavanone skeleton was examined. The structural formula of the derivative is shown in FIG. 19. The differentiation-promoting effect of the 2NC-B derivatives from preadipocytes to beige adipocytes was evaluated using the change in the amount of accumulated neutral fat as an index, as described above. The 2NC-B derivatives were dissolved in DMSO to a concentration of 5 mM. The 2NC-B derivatives were diluted with differentiation-inducing medium to a final concentration of 5 μM, and added at the time of medium exchange on the 0th, 2nd, and 4th days of differentiation induction as in Example 1. The cells were washed once with 1xPBS, fixed overnight with a fixative, stained with Oil Red O, and observed under a microscope.

[0141] The results are shown in Figure 21. As a result, suppression of neutral fat accumulation was observed in cells treated with 2NC-B4, B6 and B7. In particular, suppression of neutral fat accumulation was significantly observed in cells treated with 2NC-B7.

[0142] 2NC-B4, B6 and B7, which showed inhibitory effects on neutral fat accumulation, were examined from the viewpoint of gene expression. As described above, 2NC-B4, B6 and B7 (5μM) were treated for 6 days during differentiation induction. Total RNA was extracted from the cells on the 6th day, cDNA was prepared by reverse transcription reaction, and gene expression was analyzed by real-time PCR as described above. Comparison between control (C) cells and cells treated with 2NC-B derivatives was performed by one-way analysis of variance (One way ANOVA), and Dunnett's test was used as a post hoc test (statistical significance was determined when the significance level (p) was *p<0.01 compared to control cells).

[0143] The results are shown in Figure 22. The expression levels of beige formation-related genes such as Ucp1, Elovl3, and Fgf9, and the expression levels of Pparγ2, aP2, and C / ebpα in cells treated with 2NC-B4, B6, and B7 were significantly increased compared to those in control cells (C).

[0144] The beige-promoting effect of 2NC-B derivatives (B4, B6, and B7) was examined from the viewpoint of mitochondrial amount. As described above, 2NC-B derivatives (B4, B6, and B7) were treated from the time of differentiation induction, and DNA was extracted from the cells on the 6th day of differentiation induction in the same manner as in Example 5, and the amount of mitochondrial DNA was measured by real-time PCR. Comparisons between control (C) cells and cells treated with 2NC-B4, B6, and 2NC-B7 were examined by one-way analysis of variance (One way ANOVA), and Dunnett's test was used as a post hoc test (statistically significant when the respective significance levels (p) compared to control cells were *p<0.01).

[0145] The results are shown in Figure 23. No increase in mitochondrial mass was observed in cells treated with any of the derivatives 2NC-B4, B6, and B7, which showed induction of expression of the Ucp1 gene and other genes. These results indicate that the 2NC-B derivatives have the effect of promoting differentiation of preadipocytes into beige adipocytes, as they show the effect of increasing the expression of the Ucp1 gene, but that these compounds have little effect on regulating mitochondrial mass.

[0146] (Example 13: Verification of the transformation-promoting effect of 2NC derivatives into beige adipocytes) Example 12 showed the effect of 2NC derivatives (A and B) in promoting the beige formation of adipocytes in preadipocytes. Next, the transformation ability (the effect of transforming white adipocytes into beige adipocytes) of the 2NC derivatives (A and B), which showed the effect of promoting the beige formation, was examined.

[0147] Cell culture and differentiation induction were carried out in the same manner as in Example 6. 1 × 10 5The cells were seeded at a density of 10 ...

[0148] Total RNA was extracted from white adipocytes treated with 2NC derivatives for 8 days in the same manner as above, and cDNA was prepared by reverse transcription. The expression levels of Ucp1 and Fgf9 genes, which are beige markers, were then measured by real-time PCR. Comparisons between control (C) cells and cells treated with 2NC-A derivatives (A2, A3, A4) and 2NC-B derivatives (B4, B6, B7) were examined by one-way ANOVA, with Dunnett's test used as a post hoc test (statistically significant when the significance level (p) was *p<0.01 compared to control cells).

[0149] The results are shown in Figure 24. The expression level of Ucp1 gene in cells treated with 2NC-A derivatives (A2, A3, A4) and 2NC-B derivatives (B4, B6, B7) was significantly increased compared to that in control (C) cells. On the other hand, the induction of Ucp1 gene expression was mild compared to cells treated with pioglitazone (Pio). No change was observed in the expression level of Fgf9 gene in white adipocytes treated with 2NC and 2NC-A derivatives.

[0150] Since the expression of Ucp1 was also induced in white adipocytes, the transformation effect of 2NC-A derivatives (A2, A3, A4) and 2NC-B derivatives (B4, B6, B7) was examined from the viewpoint of mitochondrial amount. As described above, 2NC-A derivatives (A2, A3, A4) and 2NC-B derivatives (B4, B6, B7) were diluted with differentiation medium to a final concentration of 5 μM, and added to white adipocytes on the 10th day of differentiation induction, and similarly added at the time of medium replacement on the 12th, 14th, and 16th days. Note that cells similarly treated with differentiation culture medium containing 0.1% DMSO as a solvent were used as Control (Con), and pioglitazone (5 μM), which shows a beige-promoting effect, was used as a positive control.

[0151] DNA was extracted from the cells on day 18 of differentiation induction, and the amount of mitochondrial DNA was measured by real-time PCR in the same manner as in Example 5. Comparisons between control (C) cells and cells treated with 2NC-A derivatives (A2, A3, A4) and 2NC-B derivatives (B4, B6, B7) were performed by one-way ANOVA, with Dunnett's test used as a post hoc test (statistical significance was determined when the respective significance levels (p) were *p<0.01 compared to control cells).

[0152] The results are shown in Figure 25. In the 2NC derivatives in which the expression of the Ucp1 gene and other genes was induced, the amount of mitochondria in the cells treated with 2NC-B7 was significantly increased compared to that in the control cells. These results indicate that 2NC-B7 has a transformation-promoting effect that increases the amount of mitochondria as well as inducing the expression of the Ucp1 gene.

[0153] The results of the above in vitro experiments using cultured cells and in vivo experiments using mice showed that the flavanone derivative 2NC is a potent compound that promotes the differentiation of preadipocytes into beige adipocytes and the transformation of white adipocytes into beige adipocytes. In addition, the promotion of differentiation into beige adipocytes by 2NC was correlated with the induction of FGF9 expression, which has been reported to promote beige formation. In addition, the derivatization of 2NC induced the expression of the Ucp1 gene even at lower concentrations than 2NC. The derivatives with functional groups introduced into the A ring (2NC-A2, A4) showed increased promotion of differentiation from preadipocytes into beige adipocytes, and the derivative with functional groups introduced into the B ring (2NC-B7) enhanced the transformation of white adipocytes into beige adipocytes. With further elucidation of the structure-activity relationship, 2NC is expected to provide a new molecular basis for obesity treatment drugs via the promotion of beige formation and to become a potent seed compound. [Industrial Applicability]

[0154] According to the present invention, there is provided a pharmaceutical composition for treating or preventing obesity, which is capable of suppressing accumulation of neutral fat.

Claims

1. A pharmaceutical composition for treating or preventing obesity, comprising a compound (1) represented by the following general formula (1) or a salt thereof, and a pharma- ceutical acceptable carrier: 【Chemistry 1】 [In the formula, R １ and R ２ each independently represents a substituent; n1 represents an integer of 0 to 4; and n2 represents an integer of 0 to 7.

2. The pharmaceutical composition for treating or preventing obesity according to claim 1, wherein the compound (1) is a compound represented by the following general formula (1-1): 【Chemistry 2】 [In the formula, R １１ , R １２ , and R ２１ each independently represents a hydrogen atom or a substituent.

3. R in the general formula (1-1) １１ , R １２ , and R ２１ The pharmaceutical composition for treating or preventing obesity according to claim 2 , wherein each of the is independently a hydrogen atom, an alkyl group, an alkoxy group, an oxyalkylene structure-containing group, a hydroxyl group, or a halogen atom.

4. R in the general formula (1-1) １１ , R １２ , and R ２１ The pharmaceutical composition for treating or preventing obesity according to claim 3 , wherein each of the is independently a hydrogen atom, a methyl group, a methoxy group, a methoxymethoxy group, a hydroxy group, or a halogen atom.

5. The pharmaceutical composition for treating or preventing obesity according to claim 1 , which induces differentiation of preadipocytes into beige adipocytes.

6. The pharmaceutical composition for treating or preventing obesity according to claim 1 , which induces transformation of white adipocytes into beige adipocytes.

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