Dehydroabietic acid derivative or pharmaceutically acceptable salt thereof, and fat accumulation inhibitor

Abstract

The present invention addresses the problem of providing a novel dehydroabietic acid derivative that can exhibit an excellent fat accumulation inhibitory effect in adipose cells.　Provided is a dehydroabietic acid derivative represented by formula (I) or a pharmaceutically acceptable salt thereof.

Description

Dehydroabietic acid derivatives or pharmaceutically acceptable salts thereof and fat accumulation inhibitors

[0001] The present invention relates to derivatives of dehydroabietic acid or pharmaceutically acceptable salts thereof, fat accumulation inhibitors, pharmaceutical compositions for treating or preventing fat-related diseases, and the like.

[0002] Obesity is considered the most important risk factor for lifestyle-related diseases such as diabetes, cardiovascular disease, arteriosclerosis, dyslipidemia, and hypertension. Managing obesity is of great importance not only for disease prevention but also for cosmetic reasons.

[0003] Preventing or treating obesity primarily requires changes in diet and exercise habits, but drug administration can also be used. Most anti-obesity drugs currently available reduce energy intake and act on the central nervous system to suppress appetite and fat absorption in the body. However, anti-obesity drugs such as orlistat and sibutramine have the problem of causing side effects such as gastrointestinal disorders, heart attacks, and hypertension. Therefore, there is a need for new drugs with fewer side effects.

[0004] The use of natural substances or their derivatives is attracting attention as an anti-obesity drug with fewer side effects. For example, rosin is an abundant natural resin and contains a mixture of abietane (abietic-type acid) and pimaran (pimaric-type acid). In particular, abietic acid (hereinafter sometimes abbreviated as "AA") is the main component of the rosin fraction of oleoresin produced by coniferous tree species, and these plants also contain AA derivatives such as dehydroabietic acid (hereinafter sometimes abbreviated as "DAA").

[0005] Non-patent document 1 reports that DAA has a greater effect on PPARα and PPARγ compared to AA. Furthermore, non-patent document 2 reports that DAA exhibits anti-obesity effects in obese diabetic mice, such as weight gain and hyperglycemia.

[0006] Non-patent document 3 reports that methyl dehydroabietate (mDA), a DAA derivative, has the effect of promoting adipocyte differentiation of 3T3-L1 preadipocytes. Furthermore, in a study of the effects of mDA on male C57BL / 6J mice fed a high-fat diet (HFD), it was reported that mDA reduced HFD-induced adipocyte hypertrophy and hepatic lipid droplet formation, and induced the expression of PPARα in the liver and PPARγ in adipose tissue.

[0007] Kang MS et al., Biochemical and Biophysical Research Communications, 2008, 369(2):333-338.Kang MS et al., Biofactors, 2009, 35(5):442-448.Yoshioka H. et al., Biomedicine & Pharmacotherapy, 2018, 99: 214-219.

[0008] The objective is to provide further derivatives of dehydroabietic acid that can exhibit excellent fat accumulation inhibitory effects.

[0009] To solve the above problems, the inventors synthesized various compounds as further derivatives of dehydroabietic acid. When undifferentiated or differentiated adipocytes were cultured in the presence of these compounds, and intracellular lipid droplets were stained with oil red, their effect on lipid accumulation was verified. As a result, several compounds exhibiting excellent lipid accumulation inhibitory effects were discovered. The present invention is based on these findings and provides the following.

[0010] (1) A derivative of dehydroabietic acid represented by the following formula (I) or a pharmaceutically acceptable salt thereof. (In the formula, X represents a carbamate group, an ester group, or a ketone group; R1 represents a hydrogen atom, an optionally substituted C1-C3 alkyl group, an optionally substituted C6-C12 aryl group, an optionally substituted C7-C10 arylalkyl group, or an optionally substituted C8-C11 arylalkylene group; and R2 represents a hydrogen atom or an optionally substituted C1-C3 alkyl group, except when X is an ester group or a ketone group and R1 is an unsubstituted methyl group.) (2) The derivative described in (1) or a pharmaceutically acceptable salt thereof, wherein X in formula (I) is a carbamate group. (3) The derivative described in (2) or a pharmaceutically acceptable salt thereof, wherein the derivative represented by formula (I) is represented by any of the following formulas (Ia) to (Ii). (4) The derivative of (1) or a pharmaceutically acceptable salt thereof, wherein X in formula (I) is an ester group. (5) The derivative of (4) or a pharmaceutically acceptable salt thereof, wherein the derivative represented by formula (I) is represented by any of the following formulas (Ij) to (Il). (6) The derivative represented by formula (I) is represented by any of the following formulas (Im) to (It) as described in (4) or a pharmaceutically acceptable salt thereof. (7) The derivative of (1) or a pharmaceutically acceptable salt thereof, wherein X in formula (I) is a ketone group. (8) The derivative of (7) or a pharmaceutically acceptable salt thereof, wherein the derivative represented by formula (I) is represented by the following formula (Iu). (9) A fat accumulation inhibitor comprising as an active ingredient a derivative described in any of (1) to (8) or a pharmaceutically acceptable salt thereof, or a derivative of dehydroabietic acid represented by the following formula (Iv) or formula (Iw) or a pharmaceutically acceptable salt thereof. (10) The fat accumulation inhibitor according to (9), wherein the fat accumulation inhibition comprises inhibition of adipocyte differentiation and / or fat reduction. (11) A pharmaceutical composition for treating or preventing a fatty disease, comprising the fat accumulation inhibitor according to (10). (12) The pharmaceutical composition according to (11), wherein the fatty disease is obesity, diabetes mellitus, dyslipidemia, fatty liver, or metabolic disorder-related steatohepatitis. (13) The pharmaceutical composition according to (12) for oral administration to the derivative or a pharmaceutically acceptable salt thereof. (14) The pharmaceutical composition according to (12) for administration to the derivative or a pharmaceutically acceptable salt thereof in a dose of 50 μg / kg body weight to 250 mg / kg body weight. This specification incorporates the disclosures of Japanese Patent Application Nos. 2025-051025 and 2025-150919, which form the basis of the priority of this application.

[0011] The present invention provides further derivatives of dehydroabietic acid that can exhibit excellent fat accumulation inhibitory effects in adipocytes.

[0012] Figure 1 schematically illustrates the function of PPARγ in the differentiation and hypertrophy / minimization of adipocytes. Figure 2 shows the effects of DAA (compound XI) and mDA (compound V) on fat accumulation. Figure 2A shows the amount of fat accumulation (A540 / A450) in adipocytes given DAA (compound XI) for 7 days (n=6). Figure 2B shows the amount of fat accumulation (A540 / A450) in adipocytes given mDA (compound V) for 7 days (n=6). Figure 3 shows the amount of fat accumulation (A540 / A450) in adipocytes given compound Ia for 5 days (n=5 or 6). In the figures, "**" indicates the result of testing the difference from the control (DMSO) using Dunnett's test, and P<0.01. Figure 4 shows the effects of various compounds on fat accumulation. Figure 4A shows the amount of fat accumulation (A540 / A450) in adipocytes treated with methyl piciferate (compound VI) for 6 days (n=6). Figure 4B shows the amount of fat accumulation (A540 / A450) in adipocytes treated with methyl 12-methoxypiciferate (compound VII) for 7 days (n=6). Figure 4C shows the amount of fat accumulation (A540 / A450) in adipocytes treated with unsubstituted phenylcarbamate methyl piciferate (compound VIII) for 8 days (n=6). In the figures, "*" and "**" indicate the results of testing the difference from the control (DMSO) using Dunnett's test, with P<0.05 and P<0.01, respectively. Figure 5 shows the effect of various compounds on fat accumulation. Figure 5A shows the amount of fat accumulation (A540 / A450) in adipocytes treated with p-methylcarbamate (compound Ib) for 5 days (n=6). Figure 5B shows the amount of fat accumulation (A540 / A450) in adipocytes treated with NO2 carbamate (compound Ie) for 5 days (n=6). In the figure, "**" indicates the result of testing the difference from the control (DMSO) using Dunnett's test, with P<0.01. Figure 6 shows the effect of various compounds on fat accumulation. Figure 6A shows the amount of fat accumulation (A540 / A450) in adipocytes treated with m-methylcarbamate (compound If) for 4 days (n=6). Figure 6B shows the amount of fat accumulation (A540 / A450) in adipocytes treated with CF3 carbamate (compound Id) for 5 days (n=6).Figure 6C shows the amount of fat accumulation (A540 / A450) in adipocytes treated with 3,5-dimethylcarbamate (compound Ig) for 4 days (n=6). In the figure, "**" indicates the result of testing the difference from the control (DMSO) using Dunnett's test, with P<0.01. Figure 7 shows the effect of various compounds on fat accumulation. Figure 7A shows the amount of fat accumulation (A540 / A450) in adipocytes treated with Bocphenylalanine (compound I-l') for 4 days (n=6). Figure 7B shows the amount of fat accumulation (A540 / A450) in adipocytes treated with trans-cinnamic acid (compound Im) for 4 days (n=6). Figure 7C shows the amount of fat accumulation (A540 / A450) in adipocytes treated with 3-methylcinnamic acid (compound In) for 4 days (n=6). In the figures, "**" indicates the result of testing the difference from the control (DMSO) using Dunnett's test, with P<0.01. Figure 8 shows the amount of fat accumulation (A540 / A450) in adipocytes treated with dimethoxycinnamic acid (compound Ir) for 4 days (n=6). In the figures, "**" indicates the result of testing the difference from the control (DMSO) using Dunnett's test, with P<0.01. Figure 9 shows the results of detecting perilipin 1 by Western blotting in cells cultured in the presence of 0 μM (DMSO Control), 25 μM, or 50 μM methyl piciferate (compound VI). Figure 10 shows the results of detecting perilipin 1 and PPARγ by Western blotting in cells cultured in the presence of 0 μM (DMSO Control), 25 μM, or 50 μM compound Ia. Figure 11 shows the results of detecting perilipin 1 and PPARγ by Western blotting in 3T3-L1 cells differentiated into adipocytes in the presence of 0 μM (DMSO Control), 25 μM, or 50 μM of compound Ia. Figure 12 shows the results of detecting perilipin 1 by Western blotting in cells cultured in the presence of 0 μM (DMSO Control), 25 μM, or 50 μM methyl 12-methoxypisiferate (compound VII).Figure 13 shows the results of Western blotting detection of perilipin 1 in cells cultured in the presence of 0 μM (DMSO Control), 25 μM, or 50 μM unsubstituted phenylcarbamate methyl piciferate (compound VIII). Figure 14 shows the results of Western blotting detection of perilipin 1 and PPARγ in cells cultured in the presence of 0 μM (DMSO Control), 25 μM, or 50 μM p-methylcarbamate (compound Ib). Figure 15 shows the results of Western blotting detection of perilipin 1 and PPARγ in cells cultured in the presence of 0 μM (DMSO Control), 25 μM, or 50 μM NO2 carbamate (compound Ie). Figure 16 shows the results of Western blotting detection of perilipin 1, PPARγ, and FABP4 in cells cultured in the presence of 0 μM (DMSO Control), 25 μM, or 50 μM m-methylcarbamate (compound If) and 3,5-dimethylcarbamate (compound Ig). FABP4, also known as fatty acid-binding protein or aP2, is a target protein of PPARγ present in differentiated adipocytes. It is involved in the uptake of fatty acids into cells as a carrier and serves as an indicator of fatty acid metabolic activity. Figure 17 shows the results of Western blotting detection of perilipin 1 and PPARγ in cells cultured in the presence of 0 μM (DMSO Control), 25 μM, or 50 μM dimethoxycinnamic acid (compound Ir). Figure 18 shows the results of Western blotting detection of perilipin 1 and PPARγ in cells cultured in the presence of 0 μM (DMSO Control), 25 μM, or 50 μM trans-cinnamic acid (compound Im). Figure 19 shows the weight changes of each treatment group from week 0 to week 8. The vertical axis represents weight (g). Figure 20 shows the weight change of each treatment group from week 6 to week 8. The vertical axis represents the weight change (g) relative to the weight at week 6. Figure 21 shows the changes in food intake of each treatment group from week 1 to week 8. Figure 22 shows the liver weight of each treatment group at week 8. Figure 23 shows the kidney weight of each treatment group at week 8. Figure 24 shows the fat weight around the testes of each treatment group at week 8.Figure 25 shows the blood glucose levels of each treatment group at 8 weeks. Figure 26 shows the insulin levels of each treatment group at 8 weeks. Figure 27 shows micrographs of white adipocytes around the testes of each treatment group at 8 weeks. Figure 28 shows micrographs of liver morphology observed in each treatment group at 8 weeks. Figure 29 shows the Pparγ2 expression level in white adipocytes of each treatment group at 8 weeks. Figure 30 shows the C / ebpα expression level in white adipocytes of each treatment group at 8 weeks. Figure 31 shows the Fabp4 expression level in white adipocytes of each treatment group at 8 weeks. Figure 32 shows the Il-6 expression level in white adipocytes of each treatment group at 8 weeks. Figure 33 shows the Tnfα expression level in white adipocytes of each treatment group at 8 weeks. Figure 34 shows the Mcp-1 expression level in white adipocytes of each treatment group at 8 weeks. Figure 35 shows the F4 / 80 expression level in white adipocytes of each treatment group at 8 weeks. Figure 36 shows the Il-6 expression level in the liver of each treatment group at 8 weeks. Figure 37 shows the Tnfα expression level in the liver of each treatment group at 8 weeks. Figure 38 shows the Mcp-1 expression level in the liver of each treatment group at 8 weeks. Figure 39 shows the F4 / 80 expression level in the liver of each treatment group at 8 weeks. Figure 40 shows the Fasn expression level in the liver of each treatment group at 8 weeks. Figure 41 shows the Scd1 expression level in the liver of each treatment group at 8 weeks. Figure 42 shows the Acc expression level in the liver of each treatment group at 8 weeks. Figure 43 shows the Srebf1 expression level in the liver of each treatment group at 8 weeks. Figure 44 shows the Fabp1 expression level in the liver of each treatment group at 8 weeks. Figure 45 shows the Cd36 expression level in the liver of each treatment group at 8 weeks. Figure 46 shows the triglyceride levels in the liver of each treatment group at 8 weeks.

[0013] 1. Derivatives of dehydroabietic acid or pharmaceutically acceptable salts thereof 1-1. Overview The first aspect of the present invention is a derivative of dehydroabietic acid or a pharmaceutically acceptable salt thereof. The derivative of dehydroabietic acid or a pharmaceutically acceptable salt thereof of the present invention can exhibit an excellent fat accumulation inhibitory effect.

[0014] 1-2. Definitions of Terms The terms frequently used in this specification are defined below.

[0015] Abietic acid (sometimes abbreviated as "AA" in this specification) is a carboxylic acid belonging to the tricyclic diterpene represented by the following formula (X). Also known as sylvic acid, it is a yellow or resinous powder obtained from pine resin by alcohol extraction or the like.

[0016] Dehydroabietic acid (sometimes abbreviated as "DAA" in this specification) has the structure represented by formula (XI) above. Dehydroabietic acid is found in the needles, bark, and roots of many coniferous trees, and is particularly abundant in resins. Methyl dehydroabietate (sometimes abbreviated as "mDA" in this specification) is a derivative obtained by methylation of dehydroabietic acid and is represented by formula (V) above. In dehydroabietic acid and its derivatives, the stereochemistry at position 5 of the abietane skeleton is α-configuration, but since this stereochemistry is often omitted in the art, it is also omitted in this specification when showing the structure.

[0017] "Pisiferic acid" has the structure represented by the following formula (XII). Pisiferic acid is a diterpene found in large quantities in Japanese cypress (sawara), and is a chemically stable crystal that can be extracted from Japanese cypress.

[0018] In this specification, "derivative" refers to a compound having the same or similar basic skeleton and similar structure and properties. Derivatives include, for example, substituted compounds, biosynthetic intermediates, metabolites, etc. Whether a compound is a derivative of another compound can be determined by common technical knowledge for those skilled in the art. Derivatives are not limited and include any structure, but substitution with a tert-butoxycarbonyl (Boc) group is also included as a derivative.

[0019] In this specification, “pharmaceutically acceptable salts” are not particularly limited and include, for example, metal salts, inorganic acid salts, and organic acid salts. Metal salts may be sodium salts, potassium salts, calcium salts, magnesium salts, or strontium salts. Inorganic acid salts may be hydrochloric acid, bromate, phosphoric acid, sulfuric acid, or disulfuric acid salts. Organic acid salts may be formic acid, acetic acid, propionic acid, lactic acid, oxalic acid, tartaric acid, malic acid, maleic acid, citric acid, fumaric acid, besilicic acid, cansilicic acid, edicylic acid, trichloroacetic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, or aspartic acid salts.

[0020] 1-3. Composition The derivatives of dehydroabietic acid of the present invention are represented by the following formula (I) or formula (I'). In further embodiments, the derivatives of dehydroabietic acid of the present invention are represented by the following formula (I).

[0021] The derivative of dehydroabietic acid of the present invention may be a compound represented by the above formula (I). In the above formula (I), X represents a carbamate group, an ester group, or a ketone group; R1 represents a hydrogen atom, an optionally substituted C1-C3 alkyl group, an optionally substituted C6-C12 aryl group, an optionally substituted C7-C10 arylalkyl group, or an optionally substituted C8-C11 arylalkylene group; and R2 represents a hydrogen atom or an optionally substituted C1-C3 alkyl group. However, this excludes the case where X is an ester group or a ketone group and R1 is an unsubstituted methyl group. When R1 is an arylalkyl group or an arylalkylene group, one end of the alkyl group or alkylene group is linked to the aryl group and the other end is linked to X.

[0022] Furthermore, the derivative of dehydroabietic acid of the present invention may be a compound represented by the above formula (I'). That is, the compound represented by the above formula (I') may be a compound in which the R1-X group at position 12 in the compound represented by the above formula (I) is substituted at position 11. In the above formula (I'), X represents a carbamate group, an ester group, or a ketone group; R1 represents a hydrogen atom, an optionally substituted C1-C3 alkyl group, an optionally substituted C6-C12 aryl group, an optionally substituted C7-C10 arylalkyl group, or an optionally substituted C8-C11 arylalkylene group; and R2 represents a hydrogen atom or an optionally substituted C1-C3 alkyl group. However, this excludes the case where X is an ester group or a ketone group and R1 is an unsubstituted methyl group. Furthermore, if R1 is an arylalkyl group or an arylalkylene group, one end of the alkyl group or alkylene group is linked to the aryl group and the other end is linked to X. In addition, the R1-X group at position 12 in the compound represented by formula (I) above may be substituted at position 11 or at position 14.

[0023] In this specification, "alkyl" or "alkyl group" means a saturated hydrocarbon having one or more carbon atoms. Specific examples of alkyl groups having 1 to 3 carbon atoms include methyl, ethyl, and propyl groups.

[0024] In this specification, "alkylene" or "alkylene group" means a linear, branched, or cyclic divalent aliphatic saturated hydrocarbon group. Examples of alkylenes include methylene, ethylene, and propylene.

[0025] In this specification, "aryl group" means an aromatic monocyclic or polycyclic hydrocarbon group, and includes aryl rings formed by the condensation or covalent bonding of monocyclic or polycyclic elements. Specific examples include the phenyl group, naphthyl group, and biphenyl group. Specific examples of aryl groups having 6 to 12 carbon atoms include the phenyl group, naphthyl group, and biphenyl group.

[0026] In this specification, "arylalkyl group" means an alkyl group substituted with one or more aryl groups. Examples include benzyl group, phenethyl group, phenylpropyl group, benzhydryl group, trityl group, naphthylmethyl group, etc. Specific examples of arylalkyl groups having 7 to 10 carbon atoms include benzyl group, phenethyl group, and phenylpropyl group.

[0027] In this specification, "arylalkylene group" means an alkylene group substituted with one or more aryl groups. Examples include the phenylethylene group (phenylmethylmethylene group), phenylpropylene group, phenylbutene group, and diphenylmethylene group. Specific examples of arylalkylene groups having 8 to 11 carbon atoms include the phenylethylene group (phenylmethylmethylene group), phenylpropylene group, and phenylbutene group.

[0028] In this specification, examples of hydrogen group substitution in alkyl groups, aryl groups, arylalkyl groups, and arylalkylene groups include substitution with hydroxyl, carboxyl, cyano, nitro, halogen (e.g., Cl, F, Br, or I), thiol, alkyl, alkoxy, ester, thioether, thioester, nitro, or amine. Furthermore, when hydrogen group substitution is made with an amine, this amine may be protected by a protecting group such as tert-butoxycarbonyl group (Boc group), benzyloxycarbonyl group (Cbz group), 9-fluorenylmethyloxycarbonyl group (Fmoc group), allyloxycarbonyl group (Alloc group), 2,2,2-trichloroethoxycarbonyl group (Troc group), or 2-nitrobenzenesulfonyl group (Ns group). The number of hydrogen group substitutions in alkyl groups, aryl groups, arylalkyl groups, and arylalkylene groups is not limited and may be, for example, one, two, or three or more substitutions. In this specification, the number of carbon atoms in alkyl groups, aryl groups, arylalkyl groups, and arylalkylene groups refers to the number of carbon atoms excluding those contained in substituents substituted with hydrogen groups.

[0029] In one embodiment, X in the above formula (I) is a carbamate group. In a further embodiment of the present embodiment, the derivative represented by formula (I) is represented by any of the following formulas (I-a) to (I-i). In the present specification, the derivatives represented by formulas (I-a) to (I-i) may be referred to as "Compound I-a" to "Compound I-i", respectively.

[0030] In one embodiment, X in the above formula (I) is an ester group. In a further embodiment of the present embodiment, the derivative represented by formula (I) is represented by any of the following formulas (I-j) to (I-l). In the present specification, the derivatives represented by formulas (I-j) to (I-l) may be referred to as "Compound I-j" to "Compound I-l", respectively.

[0031] In a further embodiment where X in the above formula (I) is an ester group, the derivative represented by formula (I) is represented by any of the following formulas (I-m) to (I-t). In the present specification, the derivatives represented by formulas (I-m) to (I-t) may be referred to as "Compound I-m" to "Compound I-t", respectively.

[0032] In one embodiment, X in the above formula (I) is a ketone group. In a further embodiment of the present embodiment, the derivative represented by formula (I) is represented by the following formula (I-u). In the present specification, the derivative represented by formula (I-u) may be referred to as "Compound I-u".

[0033] 1-4. Effects The dehydroabietic acid derivative or pharmaceutically acceptable salt thereof of the present invention can exert an excellent fat accumulation inhibitory effect. For example, the dehydroabietic acid derivative or pharmaceutically acceptable salt thereof of the present invention can reduce the size of enlarged adipocytes, reduce fat mass by increasing the expression level of PPARγ, or inhibit the differentiation of undifferentiated preadipocytes into adipocytes by reducing the expression level of PPARγ, and as a result, can exert an excellent fat accumulation inhibitory effect.

[0034] 2. Fat accumulation inhibitor 2-1. Summary The second aspect of the present invention is a fat accumulation inhibitor. The fat accumulation inhibitor of the present invention comprises, as an active ingredient, a dehydroabietic acid derivative or a pharmaceutically acceptable salt thereof, for example, the dehydroabietic acid derivative or a pharmaceutically acceptable salt thereof according to any one of the first aspect, and is capable of suppressing fat accumulation.

[0035] 2-2. Definition of Terms As used herein, the term "Adipocyte" refers to a cell containing lipid droplets including fatty acids in the cytoplasm. Adipocytes can be classified into unilocular adipocytes (white adipocytes) that accumulate fat and multilocular adipocytes (brown adipocytes) that burn fat to generate heat, and any type of adipocyte is acceptable in the present specification.

[0036] As used herein, the term "Pre-adipocyte" refers to a progenitor cell at a stage before maturation into an adipocyte. Pre-adipocytes have higher proliferative activity than adipocytes, and accumulate less lipid droplets in cells.

[0037] As used herein, the term "lipid droplet" refers to an intracellular organelle that stores lipids such as triglycerides (neutral fats) and cholesterol. Lipid droplets have a droplet-like shape formed when the aforementioned lipids are covered by a single phospholipid membrane. Expression of perilipin (e.g., Perilipin-1) is observed as an adipose tissue-specific protein on the surface of the phospholipid. Intracellular lipid droplets can be stained by Oil Red O staining using Oil Red O or the like. The size of lipid droplets is used as an indicator showing the maturity of adipocytes and the hypertrophy / shrinkage of adipocytes.

[0038] As used herein, the term "hypertrophy" of adipocytes refers to that adipocytes store neutral fat and become hypertrophied. Generally, the cell diameter of normal adipocytes is about 70 μm to 90 μm, while the cell diameter of hypertrophied adipocytes is about 100 μm to 150 μm or about 130 μm to 140 μm.

[0039] As used herein, the term "shrinkage" of adipocytes refers to a decrease in the size of adipocytes. Examples include a decrease in the size of hypertrophied adipocytes or normal adipocytes.

[0040] In this specification, “fat increase” or “fat accumulation” is used as a term encompassing an increase in adipocytes containing lipid droplets, an increase in lipid droplets within adipocytes, and / or hypertrophy of adipocytes.

[0041] In this specification, “fat reduction” is used as a term encompassing a reduction in adipocytes containing lipid droplets, a reduction in lipid droplets within adipocytes, and / or miniaturization of adipocytes.

[0042] In this specification, "inhibition of fat accumulation" is used as a term that encompasses not only the reduction of fat as described above, but also any action that inhibits the increase or accumulation of fat as described above, such as the inhibition of differentiation of mesenchymal stem cells and preadipocytes into adipocytes, as well as any action that leads to the inhibition of fat accumulation, such as inhibition of insulin resistance. Specific examples of fat accumulation inhibition include the inhibition of differentiation of stem cells or preadipocytes into adipocytes based on the suppression of PPARγ expression, and / or the miniaturization of hypertrophied adipocytes based on the increase in PPARγ expression.

[0043] In this specification, "perilipin" refers to a protein localized on the surface of lipid droplets in mature adipocytes. Perilipin is used as a marker of adipocyte differentiation. In mammals such as humans, perilipin-1 is known to be absent in small lipid droplets, but its expression increases in large lipid droplets as hypertrophy progresses.

[0044] In this specification, "peroxisome proliferator-activated receptor (PPAR)" refers to a protein belonging to the nuclear receptor superfamily that functions as a transcription factor. Among PPARs, PPARγ is highly expressed in adipose tissue and is involved in regulating adipocyte differentiation and fat accumulation. PPARγ is known to have the activity to induce differentiation of stem cells or preadipocytes into adipocytes, and to induce miniaturization of adipocytes (especially hypertrophied adipocytes) (Figure 1), and is also known to have the function of inducing the expression of perilipin 1 as described above.

[0045] 2-3. Composition The fat accumulation inhibitor of the present invention contains, as an active ingredient, a derivative of dehydroabietic acid represented by the following formula (I) or a pharmaceutically acceptable salt thereof.

[0046] In the fat accumulation inhibitor of the present invention, the derivative of dehydroabietic acid may be a compound represented by the above formula (I). In the above formula (I), X represents a carbamate group, an ester group, or a ketone group; R1 represents a hydrogen atom, an optionally substituted C1-C3 alkyl group, an optionally substituted C6-C12 aryl group, an optionally substituted C7-C10 arylalkyl group, or an optionally substituted C8-C11 arylalkylene group; and R2 represents a hydrogen atom or an optionally substituted C1-C3 alkyl group. When R1 is an arylalkyl group or an arylalkylene group, one end of the alkyl group or alkylene group is linked to the aryl group and the other end is linked to X.

[0047] In the fat accumulation inhibitor of the present invention, in formula (I) representing a derivative of dehydroabietic acid, X may be a carbamate group. In this case, the derivative represented by formula (I) may be any of the above formulas (Ia) to (Ii).

[0048] In the fat accumulation inhibitor of the present invention, in formula (I) representing a derivative of dehydroabietic acid, X may be an ester group. In this case, the derivative represented by formula (I) may be any of the above formulas (Ij) to (Il) or (Im) to (It).

[0049] In the fat accumulation inhibitor of the present invention, in formula (I) representing a derivative of dehydroabietic acid, X may be a ketone group. In this case, the derivative represented by formula (I) may be the one represented by formula (Iu) described above.

[0050] In the fat accumulation inhibitor of the present invention, the derivative of dehydroabietic acid represented by formula (I) may be a derivative represented by the following formulas (Iv) or (Iw). In this specification, the derivatives represented by formulas (Iv) and (Iw) may be referred to as "compound Iv" and "compound Iw," respectively.

[0051] The dosage form, method of use, method of administration, and target diseases of the fat accumulation inhibitor of the present invention shall be in accordance with the description of the pharmaceutical composition of the third embodiment or the food composition of the fourth embodiment described below. Therefore, a detailed explanation is omitted here.

[0052] 2-4. Effects The fat accumulation inhibitor of the present invention can exert an excellent fat accumulation inhibitory effect. For example, the fat accumulation inhibitor of the present invention can reduce the size of hypertrophied adipocytes or decrease fat by increasing the expression level of PPARγ, or it can suppress the differentiation of undifferentiated preadipocytes into adipocytes by decreasing the expression level of PPARγ, and as a result it exerts an excellent fat accumulation inhibitory effect.

[0053] 3. Pharmaceutical Composition 3-1. Overview The third aspect of the present invention is a pharmaceutical composition for treating or preventing fat-related diseases. The pharmaceutical composition of the present invention contains the fat accumulation inhibitor of the second aspect as an active ingredient and can treat or prevent fat-related diseases such as obesity, diabetes, dyslipidemia, fatty liver, or metabolic disorder-related steatohepatitis (MASH).

[0054] 3-2. Composition The pharmaceutical composition of this embodiment includes an active ingredient as an essential component, and a pharmaceutically acceptable carrier or other drug as an optional component.

[0055] (1) Active ingredient The active ingredient in the pharmaceutical composition of the present invention is the fat accumulation inhibitor described in the second embodiment. More specifically, in the second embodiment, it is a derivative of dehydroabietic acid or a pharmaceutically acceptable salt thereof that can be the active ingredient of the fat accumulation inhibitor.

[0056] The content of a derivative of dehydroabietic acid or a pharmaceutically acceptable salt thereof in the pharmaceutical composition of the present invention is not particularly limited. For example, the pharmaceutical composition of the present invention may contain as an active ingredient 0.001% to 99% by weight, 0.01% to 10% by weight, or 0.1% to 10% by weight of a derivative of dehydroabietic acid or a pharmaceutically acceptable salt thereof, such as 90% by weight, 50% by weight, 10% by weight, 5% by weight, 3% by weight, 2% by weight, 1% by weight, 0.1% by weight, or 0.01% by weight of a derivative of dehydroabietic acid or a pharmaceutically acceptable salt thereof.

[0057] (2) Pharmaceutically acceptable carriers "Pharmaceutically acceptable carriers" means solvents and / or additives that can be used in the field of pharmaceutical technology and that have little to no harmful effects on living organisms.

[0058] Examples of pharmaceutically acceptable solvents include water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters.

[0059] Pharmaceutically acceptable additives include, for example, excipients, binders, disintegrants, fillers, emulsifiers, flow additive regulators, and lubricants.

[0060] Examples of excipients include sugars such as monosaccharides, disaccharides, cyclodextrins, and polysaccharides (more specifically, but not limited to glucose, sucrose, lactose, raffinose, mannitol, sorbitol, inositol, dextrin, maltodextrin, starch, and cellulose), metal salts (e.g., sodium chloride, sodium phosphate or calcium phosphate, calcium sulfate, magnesium sulfate, calcium carbonate), citric acid, tartaric acid, glycine, low, medium, or high molecular weight polyethylene glycol (PEG), pluronic acid, kaolin, silicic acid, or combinations thereof.

[0061] Examples of binders include starch paste made from corn, wheat, rice, or potato starch, simple syrup, glucose solution, gelatin, tragacanth, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone.

[0062] Examples of disintegrants include the aforementioned starch, lactose, carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, laminaran powder, sodium bicarbonate, calcium carbonate, alginic acid or sodium alginate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, monoglyceride stearate, or salts thereof.

[0063] Examples of fillers include the aforementioned sugars and / or calcium phosphate (e.g., tricalcium phosphate or calcium hydrogen phosphate).

[0064] Examples of emulsifiers include sorbitan fatty acid esters, glycerin fatty acid esters, sucrose fatty acid esters, and propylene glycol fatty acid esters.

[0065] Examples of fluid additive regulators and lubricants include silicates, talc, stearates, or polyethylene glycol.

[0066] In addition to the above additives, the product may also contain, as needed, flavoring and deodorizing agents, solubilizers, suspending agents, diluents, surfactants, stabilizers, absorption enhancers (e.g., quaternary ammonium salts, sodium lauryl sulfate), bulking agents, humectants (e.g., glycerin, starch), adsorbents (e.g., starch, lactose, kaolin, bentonite, colloidal silicic acid), disintegration inhibitors (e.g., sucrose, stearin, cocoa butter, hydrogenated oil), coating agents, colorants, preservatives, antioxidants, fragrances, flavoring agents, sweeteners, buffering agents, etc.

[0067] A pharmaceutically acceptable carrier may be a component of biocompatible particles that encapsulate a derivative of dehydroabietic acid or a pharmaceutically acceptable salt thereof, in order to improve the pharmacokinetics of the derivative or pharmaceutically acceptable salt thereof. The biocompatible particles can be manufactured, for example, from a polymer. The polymer is preferably a biocompatible polymer that has low irritation and toxicity to living organisms and is biocompatible, being degraded and metabolized after administration. Preferably, polylactic acid, polyglycolic acid, lactic acid / glycolic acid copolymer, or lactic acid / aspartic acid copolymer is used as the biocompatible polymer. Preferably, the biocompatible particles include polylactide glycolide copolymer (PLGA) or polyethylene glycol / chitosan modified-PLGA (PEG / CS-PLGA) as the biocompatible polymer.

[0068] (3) Dosage Form The dosage form of the pharmaceutical composition of the present invention is not particularly limited as long as it does not inactivate or has little effect inactivating the active ingredient, dehydroabietic acid derivative or a pharmaceutically acceptable salt thereof, and can fully exert its pharmacological effect in the body after administration.

[0069] Dosage forms can be classified into liquid or solid forms depending on their shape, but the pharmaceutical composition of the present invention may be either. Furthermore, dosage forms can be broadly classified into oral and parenteral forms depending on the method of administration, but either is acceptable.

[0070] Specific dosage forms include, for oral dosage forms, liquid forms such as suspensions, emulsions, and syrups, and solid dosage forms such as powders (including powders and fine powders), granules, tablets, capsules, sublingual preparations, and lozenges. Parenteral dosage forms include, for example, liquid forms such as injections, suspensions, and emulsions.

[0071] (4) Method of administration The pharmaceutical composition of the present invention can be administered to a living organism in an effective amount by any method known in the art, as long as it is a method that can administer a derivative of dehydroabietic acid, which is the active ingredient, or a pharmaceutically acceptable salt thereof, to the living organism for the prevention or treatment of adipose-related diseases.

[0072] In this specification, "effective amount" means the amount necessary for the active ingredient to exert its function; that is, in this invention, the amount of dehydroabietic acid derivative or a pharmaceutically acceptable salt thereof, which is the active ingredient of the pharmaceutical composition, that is necessary for preventing or treating adipose-related diseases and that does not impart little to no harmful side effects to the organism to which it is applied. This effective amount may vary depending on conditions such as the subject's information, the route of administration, and the number of administrations.

[0073] The term "target person" refers to an individual to whom the pharmaceutical composition of this embodiment is applied, particularly a human individual.

[0074] The target population for the pharmaceutical composition of the present invention is individuals who are suffering from or are expected to suffer from adipose-related diseases in the future.

[0075] In this specification, "fat-related disease" refers to any disease that may develop or worsen in association with fat accumulation, or any disease that exhibits fat accumulation as a symptom. Specific examples of fat-related diseases include obesity such as subcutaneous fat obesity and visceral fat obesity, diabetes mellitus, dyslipidemia, fatty liver, metabolic disorder-related steatohepatitis (MASH), atherosclerosis, abdominal aortic aneurysm, coronary artery disease, macrovascular disease, cerebral infarction, or ischemic heart disease. In one embodiment, the disease targeted by the pharmaceutical composition of the present invention is obesity, diabetes mellitus, dyslipidemia, fatty liver, or metabolic disorder-related steatohepatitis.

[0076] "Subject information" refers to various individual information about the subject, including, for example, the subject's age, weight, sex, overall health status, drug sensitivity, and whether or not they are currently taking any medications. The effective dose, and the dosage calculated based on it, are determined according to the individual subject's information, etc. If it is necessary to administer a large amount of the preventive or therapeutic agent of the present invention in order to obtain sufficient effects for the prevention or treatment of adipose-related diseases, it may be administered in several divided doses to reduce the burden on the subject.

[0077] The pharmaceutical composition of the present invention may be administered by systemic or topical administration. Examples of systemic administration include oral administration and intravascular injection such as intravenous injection. For example, the pharmaceutical composition of the present invention is administered orally to the target.

[0078] The pharmaceutical composition of the present invention is administered, for example, one to four times a day. Alternatively, the preventive or therapeutic agent of the present invention may be administered at various frequencies, such as daily, every other day, once a week, every other week, or once a month.

[0079] As an example of specific dosages, the pharmaceutical composition of the present invention is administered in such a way that the amount of a derivative of dehydroabietic acid, which is the active ingredient in the pharmaceutical composition of the present invention, or a pharmaceutically acceptable salt thereof, is 0.1 mg / kg to 100 g / kg, 1 mg / kg to 10 g / kg, or 10 mg / kg to 1 g / kg per day. However, amounts outside the above ranges may be used as needed, and the dosage is not limited to these levels. For example, a derivative of dehydroabietic acid, which is the active ingredient in the pharmaceutical composition of the present invention, or a pharmaceutically acceptable salt thereof, may be administered at a dose of 1 μg / kg body weight or more, 5 μg / kg body weight, 10 μg / kg body weight, 20 μg / kg body weight, or 50 μg / kg body weight or more, and / or 1000 mg / kg body weight or less, 500 mg / kg body weight or less, or 250 mg / kg body weight or less, preferably 50 μg / kg body weight to 250 mg / kg body weight to the target.

[0080] 3-3. Effects: Based on its fat accumulation inhibitory effect, the pharmaceutical composition of the present invention can treat or prevent fat-related diseases such as obesity, diabetes, dyslipidemia, fatty liver, or metabolic disorder-related steatohepatitis.

[0081] Also provided are methods for treating and / or preventing diseases such as fat-related diseases, as well as methods for reducing fat, comprising the step of administering a derivative of dehydroabietic acid of the present invention or a pharmaceutically acceptable salt thereof, or a fat accumulation inhibitor of the present invention, to a target.

[0082] Derivatives of dehydroabietic acid or pharmaceutically acceptable salts thereof, or fat accumulation inhibitors of the present invention, are also provided for use in the treatment and / or prevention of diseases such as adipose-related diseases.

[0083] The use of a derivative of dehydroabietic acid or a pharmaceutically acceptable salt thereof, or a fat accumulation inhibitor of the present invention, in the manufacture of a pharmaceutical for treating and / or preventing diseases such as fat-related disorders is also provided.

[0084] 4. Food Composition 4-1. Overview The fourth aspect of the present invention is a food composition for the prevention or treatment of fat-related diseases. The food composition of this aspect contains the fat accumulation inhibitor of the second aspect as an active ingredient. The food composition of the present invention is consumed (eaten) as, for example, food, beverage, or functional food.

[0085] 4-2. Composition (1) Active ingredient The active ingredient in the food composition of the present invention is the fat accumulation inhibitor described in the second embodiment. More specifically, it is a derivative of dehydroabietic acid or a pharmaceutically acceptable salt thereof that can be the active ingredient of the fat accumulation inhibitor in the second embodiment.

[0086] (2) Food Composition The "food composition" of the present invention is not particularly limited, but includes foods, beverages, functional foods, etc. The types of food compositions according to the present invention are not particularly limited, but examples of foods include confectionery such as candy, cookies, tablets, chewing gum and jelly, processed grain products such as noodles, bread, rice and biscuits, processed fish products such as sausages, ham and kamaboko, dairy products such as butter and yogurt, instant foods, and seasonings. Examples of beverages include tea-based beverages (green tea, black tea, oolong tea, etc.), fruit or vegetable-based beverages, alcoholic beverages, oral rehydration solutions, carbonated beverages, soft drinks, nutritional drinks, water, etc.

[0087] The food composition of the present invention may also be a functional food. In the present invention, "functional food" means a food that has functionality for living organisms. For example, it includes all so-called health foods, such as Foods for Specified Health Uses and Foods with Nutrient Function Claims, Foods with Function Claims, Foods for Special Dietary Uses, Nutritional Supplements, Health Supplements, Supplements (e.g., liquids, powders, tablets or capsules), Beauty Foods, Diet Foods, etc. Furthermore, the packaging, accompanying leaflet, or sales brochure of the food composition of the present invention may state that it is intended to be consumed for the purpose of preventing or treating fat accumulation such as obesity.

[0088] The functional food of the present invention may be a solid formulation (e.g., tablets, granules, powders, pills, capsules, etc.), a liquid formulation (e.g., liquids, suspensions, syrups, etc.), or a gel or paste, or it may be in the form of a regular food or beverage (e.g., a beverage, confectionery, etc.).

[0089] The amount of a derivative of dehydroabietic acid or a pharmaceutically acceptable salt thereof added to the food composition of the present invention is not particularly limited. A person skilled in the art can determine the specific amount of a derivative of dehydroabietic acid or a pharmaceutically acceptable salt thereof, taking into consideration the type of food composition and the desired taste and texture. Typically, the amount of a derivative of dehydroabietic acid or a pharmaceutically acceptable salt thereof added is 0.001% to 99% by weight, 0.01% to 10% by weight, or 0.1% to 10% by weight relative to the total weight of the food, for example, 90% by weight, 50% by weight, 10% by weight, 5% by weight, 3% by weight, 2% by weight, 1% by weight, 0.1% by weight, or 0.01% by weight.

[0090] The food composition of the present invention may further contain any food components. The food composition of the present invention may contain water, protein, carbohydrates, lipids, vitamins, minerals, amino acids, organic acids, organic bases, fruit juice, flavors, etc. Furthermore, the food composition of the present invention may contain additives such as sweeteners, flavorings, and colorings.

[0091] (3) Method of intake The target persons, number of intakes, amount, and timing of intake of the food composition of the present invention shall be in accordance with the target persons, number of intakes, and amount described in "(4) Method of administration" in "3-2. Composition".

[0092] 4-3. Effects: The food composition of the present invention can prevent or treat fat-related diseases, such as obesity, in subjects who ingest it. Since the active ingredients in the food composition of the present invention are derivatives derived from natural substances, the food composition of the present invention can be safely ingested.

[0093] The present invention will be described in more detail below using examples. However, the technical scope of the present invention is not limited to these examples.

[0094] <Example 1: Synthesis of Derivatives of Dehydroabietic Acid or Pisiferic Acid> (Objective) To synthesize various derivatives of dehydroabietic acid or pisiferic acid.

[0095] (Methods and Results) The synthesis of Methyl 12-Phenylcarbamatedehydroabietate (compound Ia) was carried out by carbamate in a reaction vessel under a nitrogen atmosphere with Methyl 12-hydroxydehydroabietate and phenylisocyanic acid, followed by silica gel column chromatography to obtain a white powder.

[0096] The detailed synthesis methods for each derivative are as follows.

[0097] (1) Dehydroabietic acid (2.5 g, 8 mmol) and potassium carbonate (2.8 g, 15 mmol) were added to a flask for the synthesis of Methyl 12-hydroxydehydroabietate (compound II-a), methyl dehydroabietate (compound V), methyl 12-acetoxydehydroabietate (compound Iv), and methyl 12-acetyldehydroabietate (compound I-w). Under a nitrogen atmosphere, 30 mL of DMF was added and dissolved. A total of 1 mL of iodomethane was added dropwise, and after 2 hours, the reaction was stopped by adding 5% hydrochloric acid. The mixture was extracted with diethyl ether, and the organic layer was washed with pure water, saturated sodium bicarbonate aqueous solution, and saturated brine. After drying, it was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane-ethyl acetate) to obtain compound V, shown by the following formula (V) (hereinafter sometimes referred to as "methyl dehydroabietate" or "mDA"). The amount synthesized was 2.3 g (7.3 mmol), and the yield was 88%. In the following examples, the stereochemistry at position 5 of all abiethane skeletons is α-configuration, similar to that of the natural abiethane skeleton.

[0098] mDA (compound V) (2 g, 6.4 mmol) was dissolved in 40 mL of dichloromethane, and 1.6 mL of acetyl chloride was added dropwise under ice cooling, followed by aluminum chloride (2.63 g, 19.2 mmol). The mixture was stirred under ice cooling for 30 minutes, then stirred at room temperature for 15 hours. The mixture was poured into ice water, extracted with ethyl acetate, and the organic layer was washed with saturated brine, dried, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane / diethyl ether) to obtain compound Iw (hereinafter sometimes referred to as "12-acetyl mDA") represented by the above formula (Iw). The amount synthesized was 1.6 g (4.4 mmol), and the yield was 73%.

[0099] Compound Iw (1.5 g, 4.4 mmol) and p-toluenesulfonic acid (0.16 g, 0.9 mmol) were dissolved in 40 mL of dichloroethane, and 3-chloroperbenzoic acid (1.63 g, 7.3 mmol) was added. The mixture was stirred under reflux conditions under a nitrogen atmosphere for 5 hours. After returning to room temperature, 10% sodium sulfate aqueous solution and saturated sodium bicarbonate aqueous solution were added, and the mixture was stirred for 20 minutes. The mixture was extracted with ethyl acetate, the organic layer was washed with saturated brine, dried, and concentrated under reduced pressure. The residue containing compound Iv was dissolved in methanol, and 10% sodium bicarbonate aqueous solution was added dropwise under ice cooling. After all of the reaction mixture was dissolved, the mixture was stirred at room temperature for 2 hours. 5% hydrochloric acid aqueous solution was added dropwise until the pH reached 3 using pH test paper, extracted with ethyl acetate, the organic layer was washed with saturated brine, dried, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane-ethyl acetate) to obtain compound II-a (hereinafter sometimes referred to as "Methyl 12-hydroxydehydroabietate") shown in the following formula (II-a). The amount synthesized was 1.0 g (3.2 mmol), and the yield was 75%.

[0100] (2-1) Carbamate system: Compound II-a (1.5 g, 4.6 mmol) was placed in a flask and dissolved in 150 mL of dichloromethane under a nitrogen atmosphere. Triethylamine (1.6 g, 16.4 mmol) and phenyl isocyanate (0.8 g, 6.8 mmol) were added to this solution and stirred at room temperature for 19 hours. The mixture was poured into ice water and extracted with ethyl acetate. The organic layer was washed with 5% hydrochloric acid aqueous solution, saturated sodium bicarbonate, and saturated brine, dried, and concentrated under reduced pressure. The crude product was subjected to silica gel column chromatography (toluene-ethyl acetate) to obtain compound Ia (hereinafter sometimes referred to as "Methyl 12-Phenylcarbamatedehydroabietate") shown in the above formula (Ia). The amount synthesized was 0.8 g (1.7 mmol), and the yield was 39%.

[0101] (2-2) Carbamate type: Synthesis of compound Ib Compound I-b was obtained in the same manner as compound Ia, except that 4-Methylphenyl isocyanate was used instead of phenyl isocyanate. The amount synthesized was 0.019 g (0.041 mmol), and the yield was 67%.

[0102] (2-3) Carbamate type: Synthesis of compound Ic Compound I-c was obtained in the same manner as compound Ia, except that 4-Chlorophenyl isocyanate was used instead of phenyl isocyanate. The amount synthesized was 0.024 g (0.05 mmol), and the yield was 81.3%.

[0103] (2-4) Carbamate type: Synthesis of compound Id Compound Id was obtained in the same manner as compound Ia, except that 4-(Trifluoromethyl)phenyl isocyanate was used instead of phenyl isocyanate. The amount synthesized was 0.024 g (0.046 mmol), and the yield was 76%.

[0104] (2-5) Carbamate type: Synthesis of compound Ie Compound Ie was obtained in the same manner as compound Ia, except that 4-Nitrophenyl Isocyanate was used instead of Phenyl Isocyanate. The amount synthesized was 0.014 g (0.028 mmol), and the yield was 57%.

[0105] (2-6) Carbamate system: Synthesis of compound If Compound If was obtained in the same manner as compound Ia, except that m-tolyl isocyanate was used instead of phenyl isocyanate. The amount synthesized was 0.18 g (0.4 mmol).

[0106] (2-7) Carbamate type: Synthesis of compound Ig Compound Ig was obtained in the same manner as compound Ia, except that 3,5-DimethylPhenyl isocyanate was used instead of Phenyl isocyanate. The amount synthesized was 0.16 g (0.34 mmol).

[0107] (2-8) Carbamate system: Synthesis of compound Ih Compound Ih was obtained in the same manner as compound Ia, except that Benzyl isocyanate was used instead of Phenyl isocyanate. The amount synthesized was 0.13 g (0.28 mmol), and the yield was 92%.

[0108] (2-9) Carbamate system: Synthesis of compound Ii Compound Ii was obtained in the same manner as compound Ia, except that naphthyl isocyanate was used instead of phenyl isocyanate. The amount synthesized was 0.06 g (0.13 mmol), and the yield was 42%.

[0109] (3-1) Cinnamic acid system: Compound II-a (0.1 g, 0.3 mmol), trans-Cinnamic Acid (0.15 g, 1.0 mmol), WSC (0.20 g, 1.1 mmol), and DMAP 0.03 g (catalytic amount) were placed in a flask and dissolved in 10 mL of chloroform under a nitrogen atmosphere, and stirred at room temperature for 5 hours. The mixture was extracted with ethyl acetate, and the organic layer was washed with saturated sodium bicarbonate and saturated brine, dried, and concentrated under reduced pressure. The crude product was passed through silica gel column chromatography (hexane-ethyl acetate) to obtain compound Im shown in the above formula (Im). The amount synthesized was 0.08 g (0.19 mmol), and the yield was 61%.

[0110] (3-2) Cinnamic acid type: Synthesis of compound In Compound In was obtained in the same manner as compound Im, except that 3-Methylcinnamic Acid was used instead of trans-Cinnamic Acid. The amount synthesized was 0.087 g (0.18 mmol), and the yield was 69%.

[0111] (3-3) Cinnamic acid system: Synthesis of compound Io Compound Io was obtained in the same manner as compound Im, except that 4-Methylcinnamic Acid was used instead of trans-Cinnamic Acid. The amount synthesized was 0.094 g (0.20 mmol), and the yield was 73%.

[0112] (3-4) Cinnamic acid system: Synthesis of compound Ip Compound Ip was obtained in the same manner as compound Im, except that caffeic acid was used instead of trans-cinnamic acid. Since caffeic acid did not dissolve in chloroform, DMF was used as the solvent.

[0113] (3-5) Cinnamic acid system: Synthesis of compound Iq Compound Iq was obtained in the same manner as compound Im, except that trans-p-Coumaric Acid was used instead of trans-Cinnamic Acid. Since trans-p-Coumaric Acid did not dissolve in chloroform, DMF was used as the solvent.

[0114] (3-6) Cinnamic acid system: Synthesis of compound Ir Compound Ir was obtained in the same manner as compound Im, except that 3,4-Dimethoxycinnamic acid was used instead of trans-Cinnamic Acid. The amount synthesized was 0.13 g (0.25 mmol), and the yield was 86%. Since 3,4-Dimethoxycinnamic Acid did not dissolve in chloroform, DMF was used as the solvent.

[0115] (3-7) Cinnamic acid system: Synthesis of compound Is Compound Is was obtained in the same manner as compound Im, except that trans-4-Methoxycinnamic Acid was used instead of trans-Cinnamic Acid. The amount synthesized was 0.13 g (0.27 mmol), and the yield was 95%. Since trans-4-Methoxycinnamic Acid did not dissolve in chloroform, DMF was used as the solvent.

[0116] (4-1) Amino acid system: Compound II-a (0.1 g, 0.3 mmol), N-(tert-Butoxycarbonyl)glycine (0.15 g, 1.0 mmol), WSC (0.20 g, 1.0 mmol), and DMAP 0.05 g (catalytic amount) were placed in a flask for the synthesis of compound Ij. The mixture was dissolved in 10 mL of chloroform under a nitrogen atmosphere and stirred at room temperature for 5 hours. After washing with pure water, saturated sodium bicarbonate aqueous solution, and saturated saline solution, the mixture was dried and concentrated under reduced pressure to obtain compound Ij with a Boc group added, as shown in the above formula (Ij) (0.11 g, 0.28 mmol, 91%).

[0117] (4-2) Amino acid system: Synthesis of compound I-k Compound Ik was obtained in the same manner as compound Ij, except that N-(tert-Butoxycarbonyl)-L-tyrosine was used instead of N-(tert-Butoxycarbonyl)glycine, to which a Boc group was added.

[0118] (4-3) Amino acid type: Synthesis of compound Il Except for using N-(tert-Butoxycarbonyl)-L-phenylalanine instead of N-(tert-Butoxycarbonyl)glycine, compound I-l', i.e., compound Il with a Boc group added, was obtained in the same manner as compound Ij above, with a Boc group added to compound Il. The amount synthesized was 0.09 g (0.16 mmol), and the yield was 52%.

[0119] (5) Vilsmeier: Vilsmeier-Haack reaction synthesis of compound It To carry out the Vilsmeier reaction, compound II-a (0.1 g, 0.3 mmol) was dissolved in 10 mL of DMF in a flask, and 1 g of the Vilsmeier reagent (0.51 g of thionyl chloride dissolved in 10 mL of DMF) was added dropwise. The mixture was stirred at room temperature under a nitrogen atmosphere for 24 hours. The mixture was extracted with ethyl acetate, and the organic layer was washed with pure water, saturated sodium bicarbonate aqueous solution, and saturated brine, then dried and concentrated under reduced pressure. The crude product was passed through silica gel column chromatography (hexane-ethyl acetate) to obtain compound It shown in formula (It) above. The amount synthesized was 0.04 g (0.13 mmol), and the yield was 43%.

[0120] (6) Bromo group: Synthesis of compound Iu For the synthesis of the bromo group at position 12, compound Iw (0.2 g, 0.56 mmol), NBS (0.12 g, 0.68 mmol), and p-toluenesulfonic acid (0.16 g, 0.84 mmol) were added to a flask and dissolved in 40 mL of acetonitrile. The mixture was stirred under reflux conditions under a nitrogen atmosphere for 7 hours. The acetonitrile was removed by distillation, and after extraction with dichloromethane, the organic layer was washed with saturated brine and pure water. After drying, the mixture was concentrated under reduced pressure, and the crude product was subjected to silica gel column chromatography (hexane-ethyl acetate) to obtain compound Iu, shown in formula (Iu) above. The amount synthesized was 0.13 g (0.31 mmol), and the yield was 55%.

[0121] (7) Synthesis of Compounds Used as Comparative Examples Furthermore, compound VI represented by formula (VI) below (hereinafter sometimes referred to as "methyl piciferate"), compound VII represented by formula (VII) below (hereinafter sometimes referred to as "methyl 12-methoxypiciferate"), compound VIII represented by formula (VIII) below (hereinafter sometimes referred to as "methyl 12-phenylcarbamatepiciferate"), and compound XI represented by formula (XI) below (hereinafter sometimes referred to as "dehydroabietic acid" or "DAA") were synthesized. All of these compounds are known compounds and were synthesized using synthesis methods similar to those described in previous literature.

[0122] (8) 1H-NMR The following 1H-NMR tests were performed on the various compounds synthesized in (1) to (7) above and the compounds used in the subsequent examples. The results of the 1H-NMR tests (J values) are shown below. (i) Methyl 12-Phenylcarbamatedehydroabietate (Compound Ia) 1 H NMR(400 MHz CDCl3): δ = 7.46 (d, J = 7.4 Hz, 2H), 7.32 (t, J = 7.4 Hz, 2H), 7.09 (t, J = 7.4 Hz, 1H), 6.96 (s, 2H), 3.67 (s, 3H), 3.07 (sept, J = 6.6 Hz, 1H), 2.91-2.86 (m, 2H), 2.23 (t, J = 10.1 Hz, 2H), 1.91-1.61 (m, 5H), 1.55-1.41 (m, 3H), 1.35-1.00 (m, 12H)

[0123] (ii) Cinnamic acid type (compound Im) 1H NMR(400 MHz CDCl3): δ7.87 = (d, J = 16.0 Hz, 1H), 7.58 (d, J = 2.6 Hz, 2H), 7.41 (d, J = 2.1 Hz, 3H), 6.96 (s, 1H), 6.90 (s, 1H), 6.65 (d, J = 16.0 Hz, 1H), 3.66 (s, 3H), 2.97 (sept, J = 6.8 Hz, 1H), 2.91-2.85 (m, 2H), 2.28-2.13 (m, 2H), 1.88-1.67 (m, 5H), 1.66-1.38 (m, 2H), 1.28-1.16 (m, 12H)

[0124] (iii) Amino acid series (compound I-l') 1 H NMR(400 MHz CDCl3): δ7.37-7.24 (m, 5H), 6.92 (s, 1H), 6.66 (s, 1H), 5.07 (d, J = 7.9 Hz, 1H), 4.84 (q, J = 6.8 Hz, 1H), 3.66 (s, 3H), 3.24 (dd, J = 6.0 Hz, 2H), 2.88-2.33 (m, 3H), 2.24-2.09(m, 2H), 2.04 (s, 1H), 1.86-1.68 (m, 6H), 1.43 (s, 9H), 1.29-1.08 (m, 12H)

[0125] (vv) Fructus Fructus Fructus Acid Fructus (Compound VIII) 1¹H-NMR (500 MHz, 300 K, CDCl₃) δ 7.44 (2H, br d), 7.33 (2H, dd, J 7.6, 7.6), 7.10 (1H, br dd), 7.03 (1H, s), 7.02 (1H, s), 6.93 (1H, br s), 3.57 (3H, s), 3.06 (1H, sep, J 6.9), 2.99 (1H, dd, J 17.2, 6.0) 2.91-2.83 (2H, m, overlapped), 2.50 (1H, m), 1.96-1.86 (2H, m, overlapped), 1.60 (1H, m), 1.50 (1H, dd, J 13.0, 3.1), 1.45 (1H, m), 1.32-1.26 (2H, m, overlapped), 1.21 (3H, d, J 6.9), 1.20 (3H, d, J 6.9), 0.97 (3H, s), 0.78 (3H, s). 13 ¹³C-NMR (125 MHz, 300 K, CDCl₃) δ 175.9 (s), 145.9 (s), 139.2 (s), 139.0 (s), 137.8 (s), 135.1 (s), 129.3 (d), 127.7 (d), 123.97 (d), 123.95 (d), 119.9 (d), 118.9 (d), 118.8 (d), 52.1 (d), 51.8 (q), 47.9 (s), 42.0 (t), 37.0 (t), 34.1 (s), 32.2 (q), 29.9 (s), 29.8 (t), 27.3 (d), 23.3 (q), 23.1 (q), 20.5 (t), 20.3 (q), 18.8 (t).

[0126] (v) mDA (Compound V) 1H NMR (400 MHz CDCl3): δ = 7.18 (d, J=8.1Hz, 1H), 7.01 (d, J=8.0Hz, 1H), 6.89 (s, 1H), 3.67 (s, 3H), 2.92-2.78 (m, 3H), 2.34-2.22 (m, 2H), 1.91-1.58 (m, 5H), 1.55-1.41 (m,2H), 1.43-1.20 (m,12H)

[0127] (vi) Methyl 12-hydroxydehydroabietate (compound II-a) 1 H NMR(400 MHz CDCl3): δ = 6.82 (s, 1H), 6.64 (s, 1H), 4.88 (s, 1H), 3.67 (s, 3H), 3.13 (sept, J = 6.8 Hz, 1H), 2.84-2.79 (m, 2H), 2.25-2.15 (m, 2H), 1.88-1.61 (m,4H), 1.5-1.37 (m, 3H), 1.29-1.18 (m, 12H)

[0128] (vii) Trichosanthyl acid (Compound VI) 1 H-NMR (500MHz, 300K, CDCl3) δ = 6.89 (1H, s), 6.67 (1H, s), 3.11 (1H, sep, 6.9), 2.92-2.87 (1H, m), 2.83-2.76 (2H, m, overlapped), 2.50-2.42 (1H, m), 1.95 (1H, m), 1.90-1.85 (1H, m), 1.62-1.56 (1H, m), 1.50 (1H, dd, 13.0, 2.9), 1.47-1.43 (1H, m), 1.30-1.17 (2H, m, overlapped), 1.21 (3H, d, 6.9), 1.20 (3H, d, 6.9), 0.96 (3H, s), 0.89 (1H, dd, 6.7, 6.7), 0.82 (3H, s)

[0129] <Example 2: Verification of effects of various compounds / Evaluation of fat accumulation amount by Oil Red staining> (Objective) Undifferentiated adipocytes or differentiated adipocytes are cultured in the presence of various compounds synthesized in Example 1, and the effect on the amount of fat accumulation is verified by Oil Red staining using Oil Red O, which can stain intracellular lipid droplets.

[0130] (Method) (1) Culture of 3T3-L1 cells The mouse embryonic fibroblast cell line 3T3-L1 (hereinafter referred to as "3T3-L1 cells") was purchased from ATCC. As the growth medium used for proliferation of 3T3-L1 cells, a medium obtained by adding 10% Fetal calf Serum (FCS), Penicillin / Streptomycin, and Glutamic acid to Dulbecco's Modified Eagle's medium (DMEM) high glucose was used. 3T3-L1 cell stock in a 75 cm 2 ) flask, and cultured in growth medium. After proliferation, the cells were further subcultured in a 75 cm 2 ) flask or 24-well plate. After culturing the cells until they reached 100% confluence, differentiation induction into adipocytes was started using an induction medium. Hereinafter, the start day of differentiation is defined as Day 0, and the number of culture days after the start of differentiation is indicated as "differentiation Day". In addition, the number of culture days in an undifferentiated state before the start of differentiation is indicated as "undifferentiation Day". Culture in an undifferentiated state before the start of differentiation and culture after the start of differentiation were performed in the presence of various compounds (0 μM, 10 μM, 25 μM, or 50 μM) synthesized in Example 1. As the induction medium, a medium obtained by adding 1 μM Dexamethasone (DEX), 0.5 mM IBMX (3-isobutyl-1-methylxanthine), and 5 μg / mL Insulin (differentiation induction cocktail) to the growth medium was used. After induction for 2 days in the induction medium, the cells were replaced with growth medium and cultured for 2 days. Thereafter, the medium was replaced with a maintenance medium, which was replaced every 3 to 4 days, and the cells were cultured until the target number of experimental days. As the maintenance medium, a medium obtained by adding 5 μg / mL Insulin to the growth medium was used.

[0131] (2) Oil Red O staining 3T3-L1 cells were cultured in a 24-well plate and differentiated into adipocytes using the differentiation induction method described in (1) above. On the last day of culture, the medium was changed to growth medium, and 5 μL / well of CCK8 solution (Cell Counting Kit, Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the medium. The cells were incubated at 37°C and 5% CO2 for 1.5 hours. This incubation was performed in the absence of the various compounds synthesized in Example 1. After incubation, the color change was confirmed, and 120 μL of the medium was collected in 96-well plates. The absorbance (A450) at a wavelength of 450 nm was measured using a plate reader (Bio-Rad). The 24-well plates were then washed once with PBS, and 500 μL / well of 10% neutral buffered formalin solution (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to each plate and left overnight to fix the cells. Fixed cells were mixed with 250 μL / well of Oil Red O solution and allowed to stand for 15 minutes. The plates were then thoroughly washed with purified water until the washing solution ran clear, and the plates were dried. 500 μL / well of extract (99% isopropanol) was added to the dried plates, and the dye was extracted for 30 minutes to 2 hours. The absorbance of 100 μL of the extract at a wavelength of 540 nm (A540) was measured using a plate reader (A540). The absorbance of CCK8 was used as the relative cell number value (A540 / A450) to correct the absorbance of the Oil Red O staining. For each condition, the mean value was calculated from n=5 to n=6 results, and the difference from the control (DMSO) was tested using Dunnett's test. In the figure, "*" indicates P<0.05, and "**" indicates P<0.01.

[0132] (Results) Figures 2 to 8 show the results of oil red staining in adipocytes treated with various compounds.

[0133] In adipocytes treated with DAA (compound XI) at 50 μM, no reduction in fat accumulation was observed (Figure 2A). On the other hand, in adipocytes treated with mDA (compound V) at 50 μM, a reduction of approximately 25% in fat accumulation was observed. However, no significant difference was observed compared to the control group (Figure 2B).

[0134] In adipocytes treated with compound Ia, the amount of fat accumulation was significantly and substantially reduced (Figure 3). In particular, when compound Ia was administered to adipocytes at concentrations of 25 μM and 50 μM, a reduction of approximately 90% in fat accumulation was observed in both cases.

[0135] Furthermore, no decrease in fat accumulation was observed in adipocytes treated with 10 μM or 25 μM methyl piciferate (compound VI) (Figure 4A). However, adipocytes treated with 50 μM methyl piciferate (compound VI) died, so the effect of 50 μM on fat accumulation could not be measured. Adipocytes treated with 12-methoxymethyl piciferate (compound VII) showed a significant increase in fat accumulation (Figure 4B). In particular, when administered to adipocytes at 25 μM and 50 μM, an increase of approximately 30% in fat accumulation was observed in both cases. In contrast, adipocytes treated with unsubstituted phenylcarbamate methyl piciferate (compound VIII) showed a significant decrease in fat accumulation (Figure 4C). Fat accumulation decreased in a concentration-dependent manner with compound VIII, and when administered to adipocytes at 25 μM and 50 μM, decreases of approximately 19% and 33%, respectively, were observed.

[0136] Figures 5-8 show the results for p-methylcarbamate (compound Ib) (Figure 5A), NO2 carbamate (compound Ie) (Figure 5B), m-methylcarbamate (compound If) (Figure 6A), CF3 carbamate (compound Id) (Figure 6B), 3,5-dimethylcarbamate (compound Ig) (Figure 6C), Boc phenylalanine (compound I-l') (Figure 7A), trans-cinnamic acid (compound Im) (Figure 7B), 3-methylcinnamic acid (compound In) (Figure 7C), and dimethoxycinnamic acid (compound Ir) (Figure 8). In all cases, the amount of fat accumulation decreased in a concentration-dependent manner.

[0137] <Example 3: Verification of the effects of various compounds / Evaluation of protein expression by Western blotting> (Objective) Undifferentiated adipocytes or differentiated adipocytes are cultured in the presence of the various compounds synthesized in Example 1, and their effects on protein expression are verified by Western blotting.

[0138] Perilipin-1 is a protein localized on the surface of lipid droplets in mature adipocytes and is used as a marker of adipocyte differentiation. Perilipin-1 is absent in small lipid droplets, but its expression increases in large lipid droplets as hypertrophy progresses. Perilipin-1 is a target protein of PPARγ. PPARγ (Peroxisome Proliferator-Activated Receptors γ) is a protein belonging to the nuclear receptor superfamily and functions as a transcription factor. It is a key factor that induces differentiation into adipocytes. β-actin is a non-muscular actin protein belonging to the actin family. It is detected as a 42 kDa protein and is used as an internal control in Western blotting to confirm that there is no difference in the total amount of protein.

[0139] (Methods) (1) Recovery of Western protein samples from cells 3T3-L1 cells were cultured in a 6-well plate and differentiated into adipocytes using the differentiation induction method described in (1) of Example 2. The cells were cultured for the desired number of days, the culture medium was removed, and the cells were washed with PBS. Culture from undifferentiated Day 2 to undifferentiated Day 10, and from differentiated Day 2 to differentiated Day 10, was carried out in the presence of the various compounds synthesized in Example 1. 250 μL / well of Sample buffer (Bio-Red) with DTT warmed to 100°C was added and collected in a 1.5 mL Eppendorf tube to obtain the protein sample. The sample was heated at 100°C for 5 minutes, vortexed, and then stored at -20°C.

[0140] (2) Western blotting Protein samples stored at -20°C were dissolved in a 50°C bath for 5 minutes. Using a 10% polyacrylamide gel (e-PAGEL, ATTO), 2 μL / Lane of molecular weight marker (Precision Plus Protein Dual Color Standards, BioRad) and 10 μL / Lane of each sample were applied. ATTO's AE6530 was used for electrophoresis. First, electrophoresis was performed at 20 mA for 20 minutes, then, after confirming that the samples were moving parallel to the gel, electrophoresis was performed at 30 mA until the leading edge of the electrophoresis strip was below the gel. After that, blotting was performed on a PVDF membrane at 100 mA for 60 minutes. After blotting, the PVDF membrane was removed and immersed in blocking solution (5% skim milk (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) / PBST), and blocked by shaking at room temperature for 1 hour. The antibodies were dissolved in 0.5% skim milk / PBST solution. The primary antibody was reacted overnight at 4°C, and the secondary antibody was reacted at room temperature for 1 hour. After the antibody reaction was complete, each was immersed in PBST and shaken for 10 minutes, and this was repeated three times for washing. After washing, plastic wrap was placed on a flat workbench, and the drained PVDF film was placed on top with the protein side facing up. Amersham ECL Prime (GE Healthcare) was used as the chromogenic agent. 1 mL each of solution A and solution B were mixed and placed on top of the PVDF film. After standing at room temperature for 5 minutes, the PVDF film was wrapped in plastic wrap and developed using a LAS1000 plus (GE) developer.

[0141] The compositions of the reagents used for Western blotting are as follows: 10× electrophoresis buffer (1 L): Tris base 30.3 g (0.25 M), glycine 144 g (1.92 M), SDS 10 g (1%) (volume adjusted to 1 L with MilliQ) Transcription buffer (1 L): Tris base 3.0 g (25 mM), glycine 14.4 g (192 mM), methanol 200 mL (volume adjusted to 1 L with MilliQ) 1×PBST (1 L): 1×PBS 1 L, Tween20 1 mL (0.1%)

[0142] The antibodies used in Western blotting are listed below. Primary antibody: Anti-β actin (manufacturer: Santa Cruz). The immunized animals were mice, and the primary antibody was diluted at ×4000, and the secondary antibody at ×4000. Primary antibody: Anti-Perilipin 1 (manufacturer: PROGEN). The immunized animals were Guinea pigs, and the primary antibody was diluted at ×4000, and the secondary antibody at ×4000. Primary antibody: Anti-PPARγ (manufacturer: Santa Cruz). The immunized animals were mice, and the primary antibody was diluted at ×2000, and the secondary antibody at ×4000.

[0143] (Results) Figures 9 to 18 show the results of Western blotting on adipocytes treated with various compounds.

[0144] Figure 9 shows the results of Western blotting to detect perilipin 1 and β-actin in ADSC cells cultured in differentiation induction medium to which 25 μM or 50 μM methyl piciferate (compound VI) was added, and the cells were harvested on differentiation Day 14. Perilipin 1 expression was suppressed in cells treated with piciferate (compound VI) compared to control (DMSO) cells. In particular, almost no perilipin 1 expression was observed at 50 μM.

[0145] Figure 10 shows the results of Western blotting to detect perilipin 1, PPARγ, and β-actin in cells cultured with 3T3-L1 cells, after replacing the culture medium with growth medium or differentiation induction medium containing 25 μM or 50 μM of compound Ia, and harvesting the cells on undifferentiated Day 7 or differentiated Day 5. In cells given 25 μM or 50 μM of compound Ia, the expression of perilipin 1 and PPARγ was decreased on differentiation Day 5 compared to control (DMSO) cells, and their expression was almost absent.

[0146] Figure 11 shows the results of Western blotting to detect perilipin 1, PPARγ, and β-actin in cells treated with a differentiation induction medium containing 25 μM or 50 μM of compound Ia, after 5 days of treatment. Cells treated with 25 μM or 50 μM of compound Ia showed decreased expression of perilipin 1 and PPARγ compared to control (DMSO) cells.

[0147] Figure 12 shows the results of detecting perilipin 1 and β-actin by Western blotting after replacing the culture medium for 3T3-L1 cells with growth medium or differentiation induction medium containing 25 μM or 50 μM methyl 12-methoxypisiferate (compound VII), and harvesting the cells on undifferentiated Day 9 or differentiated Day 7. Perilipin 1 expression was increased in cells given 25 μM or 50 μM methyl 12-methoxypisiferate (compound VII) compared with control (DMSO) cells.

[0148] Figure 13 shows the results of detecting perilipin 1 and β-actin by Western blotting after replacing the culture medium for 3T3-L1 cells with growth medium or differentiation induction medium containing 25 μM or 50 μM unsubstituted phenylcarbamate methyl piciferate (compound VIII), and harvesting the cells on undifferentiated Day 10 or differentiated Day 8. No change in perilipin 1 expression was observed in cells given 25 μM or 50 μM unsubstituted phenylcarbamate methyl piciferate (compound VIII) compared to control (DMSO) cells.

[0149] Similarly, Figures 14–18 show the results for p-methylcarbamate (compound Ib) (Figure 14), NO2 carbamate (compound Ie) (Figure 15), m-methylcarbamate (compound If) (Figure 16, left side), 3,5-dimethylcarbamate (compound Ig) (Figure 16, right side), dimethoxycinnamic acid (compound Ir) (Figure 17), and trans-cinnamic acid (compound Im) (Figure 18).

[0150] Cells treated with p-methylcarbamate (compound Ib) showed decreased expression of perilipin 1 and PPARγ compared to control (DMSO) cells (Figure 14). Cells treated with NO2 carbamate (compound Ie) showed decreased expression of perilipin 1 and PPARγ compared to control (DMSO) cells (Figure 15). Cells treated with m-methylcarbamate (compound If) or 3,5-dimethylcarbamate (compound Ig) showed decreased expression of perilipin 1 and PPARγ compared to control (DMSO) cells (Figure 16). In cells treated with dimethoxycinnamic acid (compound Ir), no change in perilipin 1 and PPARγ expression was observed compared to control (DMSO) cells at a 25 μM level, but expression was decreased at a 50 μM level (Figure 17). In cells treated with trans-cinnamic acid (compound Im), there was almost no change in the expression of perilipin 1 and PPARγ compared to control (DMSO) cells at a 25 μM level, but expression was almost completely eliminated at a 50 μM level (Figure 18).

[0151] The effects on fat accumulation in Example 2 and on protein expression in Example 3 are shown in Table 1 below.

[0152]

[0153] The results described above showed that the lipid accumulation inhibitory effect of compound Ia was significantly higher than that of unsubstituted phenylcarbamate methyl piciferate (compound VIII), which has a carbamate group in the same position as the carbamate group in compound Ia (Figures 3C and 4C). Furthermore, the lipid accumulation inhibitory effect of compound Ia was also superior to that of mDA (Figures 2B and 3C). Compound Ia showed the effect of reducing the expression levels of perilipin 1 and PPARγ, whereas no such effect was observed with unsubstituted phenylcarbamate methyl piciferate (compound VIII) (Figures 10 and 13).

[0154] <Example 4: Conducting Animal Experiments Using Mice> (Purpose) In this example, animal experiments will be conducted using mice for a representative compound among the various compounds synthesized in Example 1. Furthermore, in Examples 5 to 29 below, the various effects of the representative compound (compound Ia) will be verified in comparison with existing drugs (dapagliflozin), etc. Hereafter, compound Ia will be referred to as the "carbamate," and compound V will be referred to as "mDA."

[0155] (Experimental Method) Four-week-old male C57BL / 6JJmsSlc mice were purchased from Nippon SLC Co., Ltd. and allowed to acclimate to the rearing environment for one week before being studied. The mice were reared in an environment with a 12-hour light-dark cycle (8:00-20:00: light period, 20:00-8:00: dark period), room temperature of 23±2℃, and humidity of 55±5%. Solid feed ("CE-2", manufactured by Nippon Crea Co., Ltd.), high-fat feed with 60% fat content ("HFD-60", manufactured by Oriental Yeast Co., Ltd.), and water were provided free of charge.

[0156] After acclimatizing the mice to the rearing environment for one week on a normal diet, they were divided into two groups: a control group that continued to receive the normal diet, and a high-fat diet group that switched to a high-fat diet (HFD group). The combinations of diet and medication given to the control and HFD groups are shown in the following administration groups (1), (3), (4), (5), (7), (8), and (9). Each administration group below used 5 to 9 mice. (1) Normal diet + vehicle (hereinafter sometimes referred to as the "group given only the normal diet") (3) Normal diet + carbamate compound (compound Ia) (4) Normal diet + mDA (compound V) (5) High-fat diet + vehicle (hereinafter sometimes referred to as the "group given only the high-fat diet") (7) High-fat diet + carbamate compound (compound Ia) (8) High-fat diet + mDA (compound V) (9) High-fat diet + dapagliflozin (DAP) (existing drug; positive control)

[0157] Drug administration began in the 6th week of the experiment and continued for two weeks until the end of the experiment (8th week). Each drug was administered orally daily via gastric tube (9-11 am / pm) to separate drug administration groups. For each drug administration group, excluding the positive control group, the dosage was adjusted to 111 μmol / kg for the carbamate (50 mg / kg) and mDA (35 mg / kg). Dapagliflozin (5 mg / kg), an SGLT2 inhibitor, was administered as a positive control. The carbamate was dissolved in 5% DMSO, followed by the addition of 95% olive oil solution. mDA and dapagliflozin were dissolved in 10% ethanol, followed by the addition of 90% olive oil solution. Furthermore, the solvent administration group (vehicle) was administered a mixture of 5% DMSO and 95% olive oil solution.

[0158] On the final day of the eighth week of the experiment, after fasting for 16-18 hours, dissection was performed, and fat cells and blood around the liver, kidneys, and testes were excised and collected, respectively. After weighing each tissue, they were either immersed in 10% neutral buffered formalin solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) or approximately 50 mg was placed in a 2 mL tube, frozen with dry ice, and stored at -80°C. Blood was collected by cardiac sampling, added to a heparin-coated 0.6 mL tube, and then centrifuged (900 G, 4°C, 10 min: "Micro-Chilled Centrifuge Model 3740", manufactured by Kubota Shoji Co., Ltd.). The supernatant (plasma) was stored at -80°C.

[0159] <Example 5: Verification of the effects of the representative compound in animal experiments / Evaluation of weight changes> (Objective) In the animal experiment using mice conducted in Example 4, the weight changes of mice administered the representative compound were compared with the weight changes of mice administered existing drugs, etc., to verify the effect of the representative compound on weight reduction.

[0160] (Methods) The body weight of the mice was measured weekly in the morning (9-11 am) from the start to the end of the experiment, and the average value for each treatment group was calculated.

[0161] (Results) Figures 19 and 20 show the weight changes in mice in each treatment group. The group given a high-fat diet and carbamates (group (7): HFD + carbamates) showed a weight reduction effect compared to the group given a high-fat diet only (group (5): HFD) and the group given a high-fat diet and mDA (group (8): HFD + mDA) (weight comparison results at 8 weeks (Dunnett test performed on the four groups (1), (5), (7), and (8) with group (7) as the baseline): P=0.128 for group (7) vs (5); P=0.356 for group (7) vs (8)).

[0162] <Example 6: Verification of the effects of representative compounds in animal experiments / Evaluation of food intake> (Objective) In the animal experiment using mice conducted in Example 4, the changes in food intake of mice in each administration group administered with representative compounds etc. will be compared to verify whether or not it is necessary to consider the effect of food intake between each administration group.

[0163] (Methods) The amount of food given to mice was measured weekly for each administration group. The average daily food intake per mouse was calculated weekly from the first week to the eighth week of the experiment, based on the number of mice and the number of days. (Results) Figure 21 shows the changes in food intake in mice of each administration group. No difference in food intake was observed among the groups. This result indicates that the weight loss effect observed in Example 5, etc., is not based on differences in food intake.

[0164] <Example 7: Verification of the effect of representative compounds in animal experiments / Evaluation of organ weight> (Objective) In the animal experiment using mice conducted in Example 4, the organ weight of mice after administration of the representative compound (8 weeks later) will be compared with the organ weight of mice after administration of existing drugs, etc. (8 weeks later) to verify the effect of the representative compound on suppressing the increase in organ weight.

[0165] (Methods) As described above, the organ weights of the mice were measured after excision of each tissue (liver, kidney, and adipocytes around the testes), and the average value for each treatment group was calculated.

[0166] (Results) Figures 22 to 24 show the measured organ weights (liver weight, kidney weight, and peritesticular fat weight) in mice of each treatment group after 8 weeks. As shown in Figures 22 and 23, there were no significant differences in liver and kidney weight between the treatment groups (Dunnett test, no statistically significant difference). On the other hand, as shown in Figure 24, the group given a high-fat diet and carbamates (group (7): HFD + carbamates) showed a decrease in peritesticular fat weight compared to the group given a high-fat diet only (group (5): HFD + vehicle) and the group given a high-fat diet and mDA (group (8): HFD + mDA) (weight comparison results at 8 weeks (Dunnett test performed on groups (1), (5), (7), and (8) with group (7) as the baseline): P=0.007 for group (7) vs (5); P=0.074 for group (7) vs (8)).

[0167] <Example 8: Verification of the effect of the representative compound in animal experiments / Evaluation of blood glucose levels> (Objective) In the animal experiment using mice conducted in Example 4, the blood glucose levels of mice after administration of the representative compound (8 weeks later) will be compared with the blood glucose levels of mice after administration of existing drugs, etc. (8 weeks later) to verify the effect of the representative compound on suppressing the rise in blood glucose levels.

[0168] (Methods) As described above, the blood glucose levels of mice were measured in plasma obtained by centrifuging the blood, and the average value for each treatment group was determined.

[0169] (Results) Figure 25 shows the blood glucose levels measured at 8 weeks in mice of each treatment group. The group given a high-fat diet and carbamates (Group (7): HFD + carbamates) showed a significant decrease in blood glucose levels compared to the group given a high-fat diet only (Group (5): HFD + vehicle) and the group given a high-fat diet and mDA (Group (8): HFD + mDA) (Dunnett's test was performed on the four groups (1), (5), (7), and (8) with Group (7) as the baseline: P<0.001 for Group (7) vs. Group (5); P<0.001 for Group (7) vs. Group (8)).

[0170] <Example 9: Verification of the effect of representative compounds in animal experiments / Evaluation of insulin levels> (Objective) In the animal experiment using mice conducted in Example 4, the insulin levels of mice after administration of the representative compound (8 weeks later) will be compared with the insulin levels of mice after administration of existing drugs, etc. (8 weeks later) to verify the effect of the representative compound on suppressing the rise in insulin levels.

[0171] (Methods) As described above, the insulin levels of mice were determined by measuring the insulin levels in plasma obtained by centrifuging the blood, and the average value was calculated for each treatment group.

[0172] (Results) Figure 26 shows the results of measuring insulin levels at 8 weeks in mice of each treatment group. The group given a high-fat diet and carbamates (Group (7): HFD + carbamates) showed a significant decrease in insulin levels compared to the group given a high-fat diet only (Group (5): HFD + vehicle) and the group given a high-fat diet and mDA (Group (8): HFD + mDA) (Dunnett's test was performed on the four groups (1), (5), (7), and (8) with Group (7) as the baseline: P<0.001 for Group (7) vs. Group (5); P<0.001 for Group (7) vs. Group (8)).

[0173] The results above clearly show that the carbamate metabolite exhibits superior fat accumulation inhibitory effects in mice, and that this effect is superior to that of mDA. Furthermore, in the high-fat diet group, the carbamate metabolite showed fat accumulation inhibitory effects comparable to those of the existing drug (dapagliflozin) in mice, and the results suggested an increase in PPARγ expression. Dapagliflozin is a drug that exerts its fat accumulation inhibitory effect by inhibiting SGLT2.

[0174] <Example 10: Verification of the effect of the representative compound in animal experiments / Evaluation of white adipocytes> (Objective) In the animal experiment using mice conducted in Example 4, the size of white adipocytes in mice after administration of the representative compound (8 weeks later) will be compared with the size of white adipocytes in mice after administration of existing drugs, etc. (8 weeks later) to verify the inhibitory effect of the representative compound on the growth of white adipocytes.

[0175] (Methods) As described above, the size of white adipocytes in mice was measured by examining white adipocytes under a microscope after sectioning and staining with hematoxylin and eosin, using the method described below. The size was then compared between mice administered with a representative compound (8 weeks later) and mice administered with existing drugs (8 weeks later).

[0176] <Section Formation> First, paraffin blocks were prepared by washing adipose tissue (white adipose tissue) from around the testis, which had been preserved in 10% neutral formalin buffer (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), overnight with water. Then, using an automated embedding machine ("Sakura Vacuum Rotary", manufactured by Sakura FineTech Japan Co., Ltd.), the tissue was embedded under the following reaction conditions, and the paraffin was cooled and solidified using a Tissue-Tec TEC Plus dispensing console (manufactured by Sakura FineTech Japan Co., Ltd.).

[0177] Reaction conditions: (1) 70% ethanol; 20 minutes, (2) 100% ethanol; 30 minutes, (3) 100% ethanol; 30 minutes, (4) 100% ethanol; 30 minutes, (5) 100% ethanol; 30 minutes, (6) 100% ethanol; 30 minutes, (7) 100% ethanol; 30 minutes, (8) U・I sol; 20 minutes, (9) U・I sol; 30 minutes, (10) U・I sol; 30 minutes, (11) paraffin; 30 minutes, (12) paraffin; 30 minutes, (13) paraffin; 30 minutes, (14) paraffin; 30 minutes.

[0178] Next, thin sections of 10 μm thickness were prepared using a riding microtome (manufactured by Yamato Koki Kogyo Co., Ltd.).

[0179] Next, the sections were spread on a 37°C warm bath, and then dried on a hot plate (manufactured by AS ONE Corporation) set to 37°C while placed on a glass slide (manufactured by Matsunami Glass Industry Co., Ltd.).

[0180] <Hematoxylin and eosin staining> First, the sections obtained on a glass slide during the sectioning process described above were immersed in U-I sol for at least one hour before staining, and then deparaffinized as shown below.

[0181] Specifically, the paraffin was melted by reacting it in a vat containing U-I sol for 5 minutes each, twice. After that, the paraffin was removed by reacting it with 100% ethanol for 1-3 minutes each, seven times.

[0182] Next, for hematoxylin and eosin (H&E) staining, the samples were stained with Meyer's hematoxylin for 6 minutes, followed by washing with tap water for 30 minutes. After washing with distilled water, they were stained with 0.1% eosin containing acetic acid for 3 minutes.

[0183] Next, the following seven steps were performed: (1) washing with water for 10 seconds, (2) 100% ethanol for 10 seconds, (3) 100% ethanol for 10 seconds, (4) 100% ethanol for 10 seconds, (5) 100% ethanol for 1 minute, (6) 100% ethanol for 2 minutes, and (7) 100% ethanol for 3 minutes. After that, the tissue was reacted with U-I sol for 1-3 minutes seven times. Finally, the sections on the glass slide were covered with coverslips by mounting.

[0184] (Results) Figure 27 shows microscopic images of adipocytes (white adipocytes) around the testes of mice in each treatment group after 8 weeks. As shown in Figure 27, the group given a high-fat diet and carbamates (Group (7): HFD + carbamates) showed a reduction in white adipocytes compared to the group given a high-fat diet only (Group (5): HFD + vehicle) and the group given a high-fat diet and mDA (Group (8): HFD + mDA) (in particular, the group given a high-fat diet only (Group (5): HFD + vehicle)).

[0185] <Example 11: Verification of the effect of a representative compound in animal experiments / Evaluation of lipid droplets in the liver> (Objective) In the animal experiment using mice conducted in Example 4, the presence or absence of lipid droplets in the liver of mice after administration of the representative compound (8 weeks later) will be compared with the presence or absence of lipid droplets in the liver of mice after administration of existing drugs, etc. (8 weeks later) to verify the inhibitory effect of the representative compound on the generation of lipid droplets in the liver.

[0186] (Methods) As described above, the presence or absence of lipid droplets in the livers of mice was examined by microscopic observation of the excised livers after sectioning and hematoxylin and eosin staining using the method described below. The presence or absence of lipid droplets was compared between mice administered with a representative compound (8 weeks later) and mice administered with existing drugs (8 weeks later).

[0187] <Section Formation> First, the paraffin blocks were prepared by washing the liver, which had been preserved in 10% neutral formalin buffer (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) overnight, with water, and then embedding it using an automated embedding machine (Sakura Vacuum Rotary, manufactured by Sakura FineTech Japan Co., Ltd.) under the following reaction conditions. Finally, the paraffin was cooled and solidified using a Tissue-Tec TEC Plus dispensing console (manufactured by Sakura FineTech Japan Co., Ltd.). Reaction conditions: (1) 70% ethanol; 30 minutes, (2) 100% ethanol; 45 minutes, (3) 100% ethanol; 45 minutes, (4) 100% ethanol; 45 minutes, (5) 100% ethanol; 45 minutes, (6) 100% ethanol; 45 minutes, (7) 100% ethanol; 45 minutes, (8) U・I sol; 30 minutes, (9) U・I sol; 45 minutes, (10) U・I sol; 45 minutes, (11) paraffin; 45 minutes, (12) paraffin; 45 minutes, (13) paraffin; 45 minutes, (14) paraffin; 45 minutes.

[0188] Next, thin sections of 4 μm thickness were prepared using a riding microtome (manufactured by Yamato Koki Kogyo Co., Ltd.).

[0189] Next, the sections were spread on a 45°C warm bath, and then dried on a hot plate (manufactured by AS ONE Corporation) set to 48°C while placed on a glass slide (manufactured by Matsunami Glass Industry Co., Ltd.).

[0190] <Hematoxylin and eosin staining> Using the same method as described for staining white adipocytes with hematoxylin and eosin, hematoxylin and eosin-stained liver sections were obtained on glass slides with coverslips attached.

[0191] (Results) Microscopic images of the liver morphology observed in mice from each treatment group after 8 weeks are shown in Figure 28. As shown in Figure 28, the group given a high-fat diet and carbamates (Group (7): HFD + carbamates) showed a reduction in lipid droplets observed in the liver compared to the group given a high-fat diet only (Group (5): HFD + vehicle) and the group given a high-fat diet and mDA (Group (8): HFD + mDA) (especially the group given a high-fat diet only (Group (5): HFD + vehicle)).

[0192] <Example 12: Verification of the effect of a representative compound in animal experiments / Evaluation of Pparγ2 expression in white adipocytes> (Objective) In the animal experiment using mice conducted in Example 4, the amount of Pparγ2 expression in mouse white adipocytes was compared with the concentration of PPARγ-coding mRNA in mouse white adipocytes 8 weeks after administration of the representative compound, and the concentration of PPARγ-coding mRNA in mouse white adipocytes 8 weeks after administration of existing drugs, etc., to verify the effect of the representative compound on promoting Pparγ2 expression in white adipocytes.

[0193] (Methods) The concentration of mRNA encoding PPARγ in mouse white adipocytes was measured, as described above, by performing mRNA extraction, reverse transcription, and real-time PCR in the following order on adipocytes from the excised testes. The measured mRNA concentrations were then used as the Pparγ2 expression level in mouse white adipocytes and compared between mice administered with a representative compound (8 weeks later) and mice administered with existing drugs (8 weeks later).

[0194] <mRNA Extraction> mRNA extraction from adipocytes around the excised testes, i.e., white adipocytes (10-20 mg), was performed using the FastGene RNA Basic Kit (manufactured by NIPPON Genetics Co., Ltd.) as shown below.

[0195] First, 350 μL of buffer RL solution containing 1% 2-mercaptoethanol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to a tube, and sonication was performed by moving ice up and down. Then, 350 μL of 70% ethanol was added to the tube, mixed well, and transferred to an RNA binding column. After standing at room temperature for 2-3 minutes, centrifugation was performed using a micro-refrigerated centrifuge ("Model 3740," manufactured by Kubota Shoji Co., Ltd.) at 20,000 g, 15 °C, 1 min.

[0196] Next, after discarding the liquid remaining in the collection tube, 600 μL of buffer RW1 was added, and the mixture was centrifuged for 1 minute (20,000 g, 15 ℃, 1 min), after which the liquid was discarded.

[0197] Next, 700 μL of buffer RW2 was added, and the column was centrifuged for 1 minute (20,000 g, 15 °C, 1 min). After that, it was air-centrifuged under the same conditions, and then the column was transferred to a new collection tube.

[0198] Next, mRNA extraction was performed by adding Buffer RE (20-100 μL), centrifuging for 1 minute, and then storing on ice.

[0199] <Reverse Transcription Reaction> The reverse transcription reaction was performed on the mRNA extracted as described above using ReverTraAce qPCR RT Master Mix (manufactured by TOYOBO Corporation) as shown below.

[0200] Specifically, 5×RT Master MIX (1 μL) was mixed with mRNA (100-500 ng), then nuclear-free water was added to adjust the total volume to 5 μL. Reverse transcription was then performed at 37 °C for 15 minutes and then at 98 °C for 5 minutes. After that, the generated cDNA was diluted with nuclear-free water (15-20 μL).

[0201] <Real-time PCR> The expression level of the Pparγ2 gene was measured using Thermo Scientific SYBR Green qPCR Master Mix on the cDNA obtained by the reverse transcription reaction described above, as shown below.

[0202] First, 9 μL of PCR reaction mixture (5 μL of SYBR Green qPCR Master Mix, 0.5 μL each of 10 μM primers (two types: forward primer / reverse primer), and 3 μL of nuclease-free water) was dispensed into a 96-well PCR plate, and then 1 μL of cDNA was added to each well.

[0203] Next, with the top sealed, air bubbles were removed by centrifugation, and then the sample was placed in a measuring instrument ("CFX Duet Real-Time PCR System," manufactured by Bio-Rad Laboratories, Inc.) to relatively quantify the expression level of the PPARγ gene using β-actin as an endogenous control. The PCR conditions were 95°C for 2 minutes, followed by 40 cycles (95°C for 5 seconds, 60°C for 30 seconds), and a dissociation step (abduction curve analysis: 95°C for 15 seconds, 60°C for 60 seconds, 95°C for 15 seconds). The oligonucleotide sequences of the primers used (Pparγ2 and β-actin) are as shown in Table 2 below.

[0204] (Results) Figure 29 shows the results of measuring Pparγ2 expression levels (concentration ratio of mRNA encoding PPARγ) in white adipocytes at 8 weeks in mice of each treatment group. The group given a high-fat diet and carbamate (group (7): HFD + carbamate) showed a significant increase in Pparγ2 expression levels in white adipocytes compared to the group given a high-fat diet only (group (5): HFD + vehicle) and the group given a high-fat diet and mDA (group (8): HFD + mDA) (Dunnett's test was performed on the four groups (1), (5), (7), and (8) with group (7) as the baseline: P<0.001 for group (7) vs (5); P<0.01 for group (7) vs (8)).

[0205]

[0206] <Example 13: Verification of the effect of representative compounds in animal experiments / Evaluation of C / ebpα expression in white adipocytes> (Objective) In the animal experiment using mice conducted in Example 4, the amount of C / ebpα expression in mouse white adipocytes was compared with the concentration of mRNA encoding C / EBPα in mouse white adipocytes after administration of the representative compound (8 weeks later), and the concentration of mRNA encoding C / EBPα in mouse white adipocytes after administration of existing drugs, etc. (8 weeks later), thereby verifying the effect of the representative compound on promoting C / ebpα expression in white adipocytes.

[0207] (Method) The concentration of mRNA encoding C / EBPα in mouse white adipocytes was measured in the same manner as in Example 12, except that in the real-time PCR step, primers for measuring the expression level of the C / EBPα gene (see Table 2) were used instead of primers for measuring the expression level of the PPARγ gene in the real-time PCR step, as described above, for adipocytes around the excised testis. The measured mRNA concentrations were then used as the C / ebpα expression level in mouse white adipocytes and compared between mice administered with a representative compound (8 weeks later) and mice administered with existing drugs (8 weeks later).

[0208] (Results) Figure 30 shows the results of measuring C / ebpα expression levels (concentration ratio of mRNA encoding C / EBPα) in white adipocytes after 8 weeks in mice of each treatment group. The group given a high-fat diet and a carbamate (group (7): HFD + carbamate) showed a significant increase in C / ebpα expression levels in white adipocytes compared to the group given a high-fat diet only (group (5): HFD + vehicle) (Dunnett's test was performed on the four groups (1), (5), (7), and (8) with group (7) as the baseline: P<0.05 between group (7) and group (5)).

[0209] <Example 14: Verification of the effect of representative compounds in animal experiments / Evaluation of Fabp4 expression in white adipocytes> (Objective) In the animal experiment using mice conducted in Example 4, the amount of Fabp4 expression in mouse white adipocytes was compared with the concentration of FABP4-encoding mRNA in mouse white adipocytes after administration of the representative compound (8 weeks later), and the concentration of FABP4-encoding mRNA in mouse white adipocytes after administration of existing drugs, etc. (8 weeks later), thereby verifying the effect of the representative compound on promoting Fabp4 expression in white adipocytes.

[0210] (Method) The concentration of mRNA encoding FABP4 in mouse white adipocytes was measured in the same manner as in Example 12, except that in the real-time PCR step, primers for measuring the expression level of the FABP4 gene (see Table 2) were used instead of primers for measuring the expression level of the PPARγ gene in the real-time PCR step, using adipocytes around the excised testis as described above. The measured mRNA concentrations were then used as the Fabp4 expression level in mouse white adipocytes and compared between mice administered with a representative compound (8 weeks later) and mice administered with existing drugs (8 weeks later).

[0211] (Results) Figure 31 shows the results of measuring Fabp4 expression levels (concentration ratio of mRNA encoding FABP4) in white adipocytes at 8 weeks in mice of each treatment group. The group given a high-fat diet and carbamate (group (7): HFD + carbamate) showed a significant increase in Fabp4 expression levels in white adipocytes compared to the group given a high-fat diet only (group (5): HFD + vehicle) and the group given a high-fat diet and mDA (group (8): HFD + mDA) (Dunnett's test was performed on the four groups (1), (5), (7), and (8) with group (7) as the baseline: P<0.001 for group (7) vs (5); P<0.01 for group (7) vs (8)).

[0212] <Example 15: Verification of the effect of representative compounds in animal experiments / Evaluation of Il-6 expression in white adipocytes> (Objective) In the animal experiment using mice conducted in Example 4, the amount of Il-6 expression in mouse white adipocytes was compared with the concentration of IL-6 encoding mRNA in mouse white adipocytes 8 weeks after administration of the representative compound, and the concentration of IL-6 encoding mRNA in mouse white adipocytes 8 weeks after administration of existing drugs, etc., to verify the inhibitory effect of the representative compound on Il-6 expression as an inflammation-related factor in white adipocytes.

[0213] (Method) The concentration of IL-6 encoding mRNA in mouse white adipocytes was measured in the same manner as in Example 12, except that in the real-time PCR step, primers for measuring IL-6 gene expression (see Table 2) were used instead of primers for measuring PPARγ gene expression in the real-time PCR step, using adipocytes around the excised testis as described above. The measured mRNA concentrations were then used as the IL-6 expression level in mouse white adipocytes and compared between mice administered with a representative compound (8 weeks later) and mice administered with existing drugs (8 weeks later).

[0214] (Results) Figure 32 shows the results of measuring Il-6 expression levels (concentration ratio of mRNA encoding IL-6) in white adipocytes at 8 weeks in mice of each treatment group. The group given a high-fat diet and a carbamate (group (7): HFD + carbamate) showed a significant decrease in Il-6 expression levels in white adipocytes compared to the group given a high-fat diet only (group (5): HFD + vehicle) (Dunnett's test was performed on the four groups (1), (5), (7), and (8) with group (7) as the baseline: P<0.05 for group (7) vs. group (5)).

[0215] <Example 16: Verification of the effects of representative compounds in animal experiments / Evaluation of Tnfα expression in white adipocytes> (Objective) In the animal experiment using mice conducted in Example 4, the amount of Tnfα expression in mouse white adipocytes was compared with the concentration of TNFα-encoding mRNA in mouse white adipocytes 8 weeks after administration of the representative compound, and the concentration of TNFα-encoding mRNA in mouse white adipocytes 8 weeks after administration of existing drugs, etc., to verify the inhibitory effect of the representative compound on Tnfα expression as an inflammation-related factor in white adipocytes.

[0216] (Method) The concentration of TNFα-encoding mRNA in mouse white adipocytes was measured in the same manner as in Example 12, except that in the real-time PCR step, primers for measuring TNFα gene expression (see Table 2) were used instead of primers for measuring PPARγ gene expression in the real-time PCR step, using adipocytes from the excised testicular region. The measured mRNA concentrations were then used as the Tnfα expression level in mouse white adipocytes and compared between mice administered with a representative compound (8 weeks later) and mice administered with existing drugs (8 weeks later).

[0217] (Results) Figure 33 shows the results of measuring Tnfα expression levels (concentration ratio of mRNA encoding TNFα) in white adipocytes at 8 weeks in mice of each treatment group. The group given a high-fat diet and carbamate (group (7): HFD + carbamate) showed a significant decrease in Tnfα expression levels in white adipocytes compared to the group given a high-fat diet only (group (5): HFD + vehicle) and the group given a high-fat diet and mDA (group (8): HFD + mDA) (Dunnett's test was performed on the four groups (1), (5), (7), and (8) with group (7) as the baseline: P<0.05 for group (7) vs (5); P<0.05 for group (7) vs (8)).

[0218] <Example 17: Verification of the effect of a representative compound in animal experiments / Evaluation of McP-1 expression in white adipocytes> (Objective) In the animal experiment using mice conducted in Example 4, the amount of McP-1 expression in mouse white adipocytes was compared with the concentration of mRNA encoding MCP-1 in mouse white adipocytes after administration of the representative compound (8 weeks later), and the concentration of mRNA encoding MCP-1 in mouse white adipocytes after administration of existing drugs, etc. (8 weeks later), thereby verifying the inhibitory effect of the representative compound on McP-1 expression as an inflammation-related factor in white adipocytes.

[0219] (Method) The concentration of mRNA encoding MCP-1 in mouse white adipocytes was measured in the same manner as in Example 12, except that in the real-time PCR step, primers for measuring MCP-1 gene expression (see Table 2) were used instead of primers for measuring PPARγ gene expression in the real-time PCR step, using adipocytes around the excised testis as described above. The measured mRNA concentrations were then used as the Mcp-1 expression level in mouse white adipocytes and compared between mice administered with a representative compound (8 weeks later) and mice administered with existing drugs (8 weeks later).

[0220] (Results) Figure 34 shows the results of measuring McP-1 expression levels (concentration ratio of mRNA encoding MCP-1) in white adipocytes at 8 weeks in mice of each treatment group. The group given a high-fat diet and carbamate (group (7): HFD + carbamate) showed a significant decrease in McP-1 expression levels in white adipocytes compared to the group given a high-fat diet only (group (5): HFD + vehicle) and the group given a high-fat diet and mDA (group (8): HFD + mDA) (Dunnett's test was performed on the four groups (1), (5), (7), and (8) with group (7) as the baseline: P<0.05 for group (7) vs (5); P<0.05 for group (7) vs (8)).

[0221] <Example 18: Verification of the effect of a representative compound in animal experiments / Evaluation of F4 / 80 expression in white adipocytes> (Objective) In the animal experiment using mice conducted in Example 4, the amount of F4 / 80 expression in mouse white adipocytes was compared with the concentration of mRNA encoding F4 / 80 in mouse white adipocytes after administration of the representative compound (8 weeks later), and the concentration of mRNA encoding F4 / 80 in mouse white adipocytes after administration of existing drugs, etc. (8 weeks later), thereby verifying the inhibitory effect of the representative compound on F4 / 80 expression as an inflammation-related factor in white adipocytes.

[0222] (Method) The concentration of mRNA encoding F4 / 80 in mouse white adipocytes was measured in the same manner as in Example 12, except that in the real-time PCR step, primers for measuring the expression level of the F4 / 80 gene (see Table 2) were used instead of primers for measuring the expression level of the PPARγ gene in the real-time PCR step, as described above, for adipocytes around the excised testis. The measured mRNA concentrations were then used as the F4 / 80 expression level in mouse white adipocytes and compared between mice administered with a representative compound (8 weeks later) and mice administered with existing drugs (8 weeks later).

[0223] (Results) Figure 35 shows the results of measuring the F4 / 80 expression level (concentration ratio of mRNA encoding F4 / 80) in white adipocytes at 8 weeks in mice of each treatment group. The group given a high-fat diet and carbamate (group (7): HFD + carbamate) showed a significant decrease in F4 / 80 expression in white adipocytes compared to the group given a high-fat diet only (group (5): HFD + vehicle) and the group given a high-fat diet and mDA (group (8): HFD + mDA) (Dunnett's test was performed on the four groups (1), (5), (7), and (8) with group (7) as the baseline: P<0.01 for group (7) vs (5); P<0.05 for group (7) vs (8)).

[0224] <Example 19: Verification of the effect of representative compounds in animal experiments / Evaluation of Il-6 expression in the liver> (Objective) In the animal experiment using mice conducted in Example 4, the amount of Il-6 expression in the liver of mice was compared with the concentration of IL-6 encoding mRNA in the liver of mice after administration of the representative compound (8 weeks later), and with the concentration of IL-6 encoding mRNA in the liver of mice after administration of existing drugs, etc. (8 weeks later), thereby verifying the inhibitory effect of the representative compound on Il-6 expression as an inflammation-related factor in the liver.

[0225] (Methods) The concentration of IL-6 encoding mRNA in mouse liver was measured in the same manner as in Example 12, except that in mRNA extraction, reverse transcription, and real-time PCR, liver was used instead of adipocytes around the testes for mRNA extraction, and in real-time PCR, primers for measuring IL-6 gene expression (see Table 2) were used instead of primers for measuring PPARγ gene expression. The measured mRNA concentrations were then used as the IL-6 expression level in the liver of mice, and compared between mice administered with a representative compound (8 weeks later) and mice administered with existing drugs (8 weeks later).

[0226] (Results) Figure 36 shows the results of measuring Il-6 expression levels (concentration ratio of mRNA encoding IL-6) in the liver of mice in each treatment group after 8 weeks. The group given a high-fat diet and a carbamate (group (7): HFD + carbamate) showed a significant decrease in Il-6 expression levels in the liver compared to the group given a high-fat diet only (group (5): HFD + vehicle) (Dunnett's test was performed on the four groups (1), (5), (7), and (8) with group (7) as the baseline: P<0.05 for group (7) vs. group (5)).

[0227] <Example 20: Verification of the effects of representative compounds in animal experiments / Evaluation of Tnfα expression in the liver> (Objective) In the animal experiment using mice conducted in Example 4, the amount of Tnfα expression in the liver of mice was compared with the concentration of TNFα-coding mRNA in the liver of mice 8 weeks after administration of the representative compound, and with the concentration of TNFα-coding mRNA in the liver of mice 8 weeks after administration of existing drugs, etc., to verify the inhibitory effect of the representative compound on Tnfα expression as an inflammation-related factor in the liver.

[0228] (Method) The concentration of TNFα-encoding mRNA in the liver of mice was measured in the same manner as in Example 12, except that the liver was used instead of adipocytes around the testes for mRNA extraction, and primers for measuring TNFα gene expression (see Table 2) were used instead of primers for measuring PPARγ gene expression in real-time PCR. The measured mRNA concentrations were then used as the Tnfα expression level in the liver of mice, and compared between mice administered with a representative compound (8 weeks later) and mice administered with existing drugs (8 weeks later).

[0229] (Results) Figure 37 shows the results of measuring Tnfα expression levels (ratio of TNFα encoding mRNA concentration) in the liver of mice in each treatment group after 8 weeks. The group given a high-fat diet and a carbamate (group (7): HFD + carbamate) showed a significant decrease in Tnfα expression levels in the liver compared to the group given a high-fat diet only (group (5): HFD + vehicle) (Dunnett's test was performed on the four groups (1), (5), (7), and (8) with group (7) as the baseline: P<0.001 for group (7) vs. group (5)).

[0230] <Example 21: Verification of the effect of representative compounds in animal experiments / Evaluation of McP-1 expression in the liver> (Objective) In the animal experiment using mice conducted in Example 4, the amount of McP-1 expression in the liver of mice was compared with the concentration of MCP-1 encoding mRNA in the liver of mice 8 weeks after administration of the representative compound, and with the concentration of MCP-1 encoding mRNA in the liver of mice 8 weeks after administration of existing drugs, etc., to verify the inhibitory effect of the representative compound on McP-1 expression as an inflammation-related factor in the liver.

[0231] (Methods) The concentration of mRNA encoding MCP-1 in the liver of mice was measured in the same manner as in Example 12, except that in mRNA extraction, reverse transcription, and real-time PCR, liver was used instead of adipocytes around the testes in mRNA extraction, and in real-time PCR, primers for measuring the expression level of the MCP-1 gene (see Table 2) were used instead of primers for measuring the expression level of the PPARγ gene. The measured mRNA concentrations were then used as the expression level of Mcp-1 in the liver of mice, and compared between mice administered with a representative compound (8 weeks later) and mice administered with existing drugs (8 weeks later).

[0232] (Results) Figure 38 shows the results of measuring McP-1 expression levels (concentration ratio of mRNA encoding MCP-1) in the liver of mice in each treatment group after 8 weeks. The group given a high-fat diet and carbamate (group (7): HFD + carbamate) showed a significant decrease in McP-1 expression levels in the liver compared to the group given a high-fat diet only (group (5): HFD + vehicle) and the group given a high-fat diet and mDA (group (8): HFD + mDA) (Dunnett's test was performed on the four groups (1), (5), (7), and (8) with group (7) as the baseline: P<0.01 for group (7) vs (5); P<0.01 for group (7) vs (8)).

[0233] <Example 22: Verification of the effect of a representative compound in animal experiments / Evaluation of F4 / 80 expression in the liver> (Objective) In the animal experiment using mice conducted in Example 4, the amount of F4 / 80 expression in the liver of mice was compared with the concentration of mRNA encoding F4 / 80 in the liver of mice after administration of the representative compound (8 weeks later), and with the concentration of mRNA encoding F4 / 80 in the liver of mice after administration of existing drugs, etc. (8 weeks later), thereby verifying the inhibitory effect of the representative compound on F4 / 80 expression as an inflammation-related factor in the liver.

[0234] (Methods) The concentration of mRNA encoding F4 / 80 in the liver of mice was measured in the same manner as in Example 12, except that in mRNA extraction, reverse transcription, and real-time PCR, liver was used instead of adipocytes around the testes in mRNA extraction, and in real-time PCR, primers for measuring the expression level of the F4 / 80 gene (see Table 2) were used instead of primers for measuring the expression level of the PPARγ gene. The measured mRNA concentrations were then used as the F4 / 80 expression level in the liver of mice, and compared between mice administered with a representative compound (8 weeks later) and mice administered with existing drugs (8 weeks later).

[0235] (Results) Figure 39 shows the results of measuring the F4 / 80 expression level (concentration ratio of mRNA encoding F4 / 80) in the liver of mice in each treatment group after 8 weeks. The group given a high-fat diet and a carbamate (group (7): HFD + carbamate) showed a significant decrease in F4 / 80 expression in the liver compared to the group given a high-fat diet only (group (5): HFD + vehicle) (Dunnett's test was performed on the four groups (1), (5), (7), and (8) with group (7) as the baseline: P<0.001 for group (7) vs. group (5)).

[0236] <Example 23: Verification of the effect of representative compounds in animal experiments / Evaluation of Fasn expression in the liver> (Objective) In the animal experiment using mice conducted in Example 4, the amount of Fasn expression in the liver of mice was compared with the concentration of FASN-encoding mRNA in the liver of mice after administration of the representative compound (8 weeks later), and with the concentration of FASN-encoding mRNA in the liver of mice after administration of existing drugs, etc. (8 weeks later), thereby verifying the inhibitory effect of the representative compound on Fasn expression as a factor involved in lipid synthesis in the liver.

[0237] (Method) The concentration of mRNA encoding FASN in the liver of mice was measured in the same manner as in Example 12, except that in mRNA extraction, reverse transcription, and real-time PCR, liver was used instead of adipocytes around the testes in mRNA extraction, and in real-time PCR, primers for measuring the expression level of the FASN gene (see Table 2) were used instead of primers for measuring the expression level of the PPARγ gene. The measured mRNA concentrations were then used as the Fasn expression level in the liver of mice, and compared between mice administered with a representative compound (8 weeks later) and mice administered with existing drugs (8 weeks later).

[0238] (Results) Figure 40 shows the results of measuring Fasn expression levels (concentration ratio of mRNA encoding FASN) in the liver of mice in each treatment group after 8 weeks. The group given a high-fat diet and carbamate (Group (7): HFD + carbamate) showed a tendency for decreased Fasn expression levels in the liver compared to the group given a high-fat diet only (Group (5): HFD + vehicle) and the group given a high-fat diet and mDA (Group (8): HFD + mDA).

[0239] <Example 24: Verification of the effect of representative compounds in animal experiments / Evaluation of Scd1 expression in the liver> (Objective) In the animal experiment using mice conducted in Example 4, the amount of Scd1 expression in the liver of mice was compared with the concentration of SCD1-coding mRNA in the liver of mice 8 weeks after administration of the representative compound, and with the concentration of SCD1-coding mRNA in the liver of mice 8 weeks after administration of existing drugs, etc., to verify the inhibitory effect of the representative compound on Scd1 expression as a factor involved in lipid synthesis in the liver.

[0240] (Methods) The concentration of mRNA encoding SCD1 in the liver of mice was measured in the same manner as in Example 12, except that in mRNA extraction, reverse transcription, and real-time PCR, liver was used instead of adipocytes around the testes in mRNA extraction, and in real-time PCR, primers for measuring the expression level of the SCD1 gene (see Table 2) were used instead of primers for measuring the expression level of the PPARγ gene. The measured mRNA concentrations were then used as the Scd1 expression level in the liver of mice and compared between mice administered with a representative compound (8 weeks later) and mice administered with existing drugs (8 weeks later).

[0241] (Results) Figure 41 shows the results of measuring Scd1 expression levels (concentration ratio of mRNA encoding SCD1) in the liver of mice in each treatment group after 8 weeks. The group given a high-fat diet and carbamate (group (7): HFD + carbamate) showed a significant decrease in liver Scd1 expression levels compared to the group given a high-fat diet only (group (5): HFD + vehicle) and the group given a high-fat diet and mDA (group (8): HFD + mDA) (Dunnett's test was performed on the four groups (1), (5), (7), and (8) with group (7) as the baseline: P<0.001 for group (7) vs (5); P<0.05 for group (7) vs (8)).

[0242] <Example 25: Verification of the effect of representative compounds in animal experiments / Evaluation of Acc expression in the liver> (Objective) In the animal experiment using mice conducted in Example 4, the amount of Acc (Acaca) expression in the liver of mice was compared with the concentration of mRNA encoding ACC in the liver of mice after administration of the representative compound (8 weeks later), and with the concentration of mRNA encoding ACC in the liver of mice after administration of existing drugs, etc. (8 weeks later), thereby verifying the inhibitory effect of the representative compound on Acc expression as a factor involved in lipid synthesis in the liver.

[0243] (Method) The concentration of mRNA encoding ACC in the liver of mice was measured in the same manner as in Example 12, except that in mRNA extraction, reverse transcription, and real-time PCR, liver was used instead of adipocytes around the testes in mRNA extraction, and in real-time PCR, primers for measuring the expression level of the ACC gene (see Table 2) were used instead of primers for measuring the expression level of the PPARγ gene. The measured mRNA concentrations were then used as the Acc expression level in the liver of mice and compared between mice administered with a representative compound (8 weeks later) and mice administered with existing drugs, etc. (8 weeks later).

[0244] (Results) Figure 42 shows the results of measuring Acc expression levels (concentration ratio of mRNA encoding ACC) in the liver of mice in each treatment group after 8 weeks. The group given a high-fat diet and a carbamate (group (7): HFD + carbamate) showed a significant decrease in Acc expression levels in the liver compared to the group given a high-fat diet only (group (5): HFD + vehicle) (Dunnett's test was performed on the four groups (1), (5), (7), and (8) with group (7) as the baseline: P<0.01 between group (7) and group (5)).

[0245] <Example 26: Verification of the effect of representative compounds in animal experiments / Evaluation of Srebf1 expression in the liver> (Objective) In the animal experiment using mice conducted in Example 4, the amount of Srebf1 expression in the liver of mice was compared with the concentration of mRNA encoding SREBF1 in the liver of mice after administration of the representative compound (8 weeks later), and with the concentration of mRNA encoding SREBF1 in the liver of mice after administration of existing drugs, etc. (8 weeks later), thereby verifying the inhibitory effect of the representative compound on Srebf1 expression as a factor involved in lipid synthesis in the liver.

[0246] (Methods) The concentration of mRNA encoding SREBF1 in the liver of mice was measured in the same manner as in Example 12, except that in mRNA extraction, reverse transcription, and real-time PCR, liver was used instead of adipocytes around the testes in mRNA extraction, and in real-time PCR, primers for measuring the expression level of the SREBF1 gene (see Table 2) were used instead of primers for measuring the expression level of the PPARγ gene. The measured mRNA concentrations were then used as the expression level of Srebf1 in the liver of mice, and compared between mice administered with a representative compound (8 weeks later) and mice administered with existing drugs, etc. (8 weeks later).

[0247] (Results) Figure 43 shows the results of measuring Srebf1 expression levels (concentration ratio of mRNA encoding SREBF1) in the liver of mice in each treatment group after 8 weeks. The group given a high-fat diet and carbamate (Group (7): HFD + carbamate) showed a tendency for decreased Srebf1 expression levels in the liver compared to the group given a high-fat diet only (Group (5): HFD + vehicle) and the group given a high-fat diet and mDA (Group (8): HFD + mDA).

[0248] <Example 27: Verification of the effect of representative compounds in animal experiments / Evaluation of Fabp1 expression in the liver> (Objective) In the animal experiment using mice conducted in Example 4, the amount of Fabp1 expression in the liver of mice was compared with the concentration of FABP1-encoding mRNA in the liver of mice after administration of the representative compound (8 weeks later), and with the concentration of FABP1-encoding mRNA in the liver of mice after administration of existing drugs, etc. (8 weeks later), thereby verifying the inhibitory effect of the representative compound on Fabp1 expression as a factor involved in lipid transport in the liver.

[0249] (Methods) The concentration of mRNA encoding FABP1 in the liver of mice was measured in the same manner as in Example 12, except that in mRNA extraction, reverse transcription, and real-time PCR, liver was used instead of adipocytes around the testes in mRNA extraction, and in real-time PCR, primers for measuring the expression level of the FABP1 gene (see Table 2) were used instead of primers for measuring the expression level of the PPARγ gene. The measured mRNA concentrations were then used as the Fabp1 expression level in the liver of mice, and compared between mice administered with a representative compound (8 weeks later) and mice administered with existing drugs (8 weeks later).

[0250] (Results) Figure 44 shows the results of measuring Fabp1 expression levels (concentration ratio of mRNA encoding FABP1) in the liver of mice in each treatment group after 8 weeks. The group given a high-fat diet and carbamate (group (7): HFD + carbamate) showed a significant decrease in Fabp1 expression levels in the liver compared to the group given a high-fat diet only (group (5): HFD + vehicle) (Dunnett's test was performed on the four groups (1), (5), (7), and (8) with group (7) as the baseline: P<0.05 for group (7) vs. group (5)).

[0251] <Example 28: Verification of the effect of representative compounds in animal experiments / Evaluation of Cd36 expression in the liver> (Objective) In the animal experiment using mice conducted in Example 4, the amount of Cd36 expression in the liver of mice was compared with the concentration of CD36-encoding mRNA in the liver of mice after administration of the representative compound (8 weeks later), and with the concentration of CD36-encoding mRNA in the liver of mice after administration of existing drugs, etc. (8 weeks later), thereby verifying the inhibitory effect of the representative compound on Cd36 expression as a factor involved in lipid transport in the liver.

[0252] (Method) The concentration of CD36-encoding mRNA in the liver of mice was measured in the same manner as in Example 12, except that the liver was used instead of adipocytes around the testes for mRNA extraction, and the primers for measuring the expression level of the CD36 gene (see Table 2) were used instead of the primers for measuring the expression level of the PPARγ gene for real-time PCR. The measured mRNA concentrations were then used as the Cd36 expression level in the liver of mice, and compared between mice administered with a representative compound (8 weeks later) and mice administered with existing drugs (8 weeks later).

[0253] (Results) Figure 45 shows the results of measuring Cd36 expression levels (concentration ratio of mRNA encoding CD36) in the liver of mice in each treatment group after 8 weeks. The group given a high-fat diet and carbamate (Group (7): HFD + carbamate) showed a tendency for decreased Cd36 expression levels in the liver compared to the group given a high-fat diet only (Group (5): HFD + vehicle) and the group given a high-fat diet and mDA (Group (8): HFD + mDA).

[0254] <Example 29: Verification of the effect of a representative compound in animal experiments / Evaluation of triglyceride levels in the liver> (Objective) In the animal experiment using mice conducted in Example 4, the effect of the representative compound on suppressing the increase in triglyceride levels in the liver was verified by comparing the triglyceride levels in the liver of mice after administration of the representative compound (8 weeks later) with the triglyceride levels in the liver of mice after administration of existing drugs, etc. (8 weeks later).

[0255] (Methods) As described above, the presence or absence of lipid droplets in the liver of mice was determined by measuring the triglyceride levels in the excised liver using the method shown below. The effect of suppressing the increase in triglyceride levels in the liver was compared between mice administered with a representative compound (8 weeks later) and mice administered with existing drugs (8 weeks later).

[0256] <Measurement of Triglyceride Levels> Triglyceride levels in the liver were measured using Lab Assay Triglycerides (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) as shown below.

[0257] First, 500 μL of RIPA buffer (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to liver (40-60 mg), and sonication was performed using ice. Then, centrifugation (20,000 g, 4 ℃, 20 min) was performed using a micro-refrigerated centrifuge ("Model 3740," manufactured by Kubota Shoji Co., Ltd.), and the protein content of the resulting supernatant was quantified using a protein assay BCA kit (manufactured by Nacalai Tesque Co., Ltd.).

[0258] Next, 0.5 μL of 100% trichloroacetic acid (manufactured by Nacalai Tesque Co., Ltd.) was added to 19.5 μL of the supernatant (protein extract), mixed thoroughly, and then centrifuged (900 g, 4 ℃, 10 min) to obtain the protein-reduced supernatant (protein-reduced supernatant).

[0259] Next, 2 μL of the deproteinized supernatant was added to each well of a 96-well plate, followed by the addition of 200 μL of chromogenic reagent. The mixture was then stirred and reacted at 37°C for 5 minutes. Subsequently, the triglyceride levels in the deproteinized supernatant were measured by measuring the absorbance using a microplate reader (primary wavelength 600 nm / secondary wavelength 700 nm). Furthermore, the liver triglyceride levels per 1 mg of protein were obtained by correcting for the previously measured protein content in the supernatant.

[0260] (Results) Figure 46 shows the results of measuring triglyceride levels in the liver of mice in each treatment group after 8 weeks. The group given a high-fat diet and carbamates (group (7): HFD + carbamates) showed a significant decrease in triglyceride levels in the liver compared to the group given a high-fat diet only (group (5): HFD + vehicle) (Dunnett's test was performed on the four groups (1), (5), (7), and (8) with group (7) as the baseline: P<0.001 for group (7) vs. group (5)).

[0261] From these results, it has become clear that the carbamate derivative has the effect of increasing or decreasing the expression level of PPARγ, showing a particularly strong reduction effect on hypertrophied white adipocytes, and also showing an effect of reducing lipid droplets in the liver. All publications, patents and patent applications cited herein are incorporated herein by direct reference in their entirety.

Claims

1. A derivative of dehydroabietic acid represented by the following formula (I), or a pharmaceutically acceptable salt thereof. (In the formula, X represents a carbamate group, an ester group, or a ketone group; R1 represents a hydrogen atom, an optionally substituted C1-C3 alkyl group, an optionally substituted C6-C12 aryl group, an optionally substituted C7-C10 arylalkyl group, or an optionally substituted C8-C11 arylalkylene group; and R2 represents a hydrogen atom or an optionally substituted C1-C3 alkyl group. Except when X is an ester group or a ketone group and R1 is an unsubstituted methyl group.) 2. The derivative according to claim 1, or a pharmaceutically acceptable salt thereof, wherein X in formula (I) is a carbamate group.

3. The derivative according to claim 2, or a pharmaceutically acceptable salt thereof, wherein the derivative represented by formula (I) is represented by any of the following formulas (Ia) to (Ii).

4. The derivative according to claim 1, or a pharmaceutically acceptable salt thereof, wherein X in formula (I) is an ester group.

5. The derivative represented by the formula (I) is represented by any one of the following formulas (I-j) to (I-l), the derivative according to claim 4 or a pharmaceutically acceptable salt thereof.

6. The derivative according to claim 4 or a pharmaceutically acceptable salt thereof, wherein the derivative represented by formula (I) is represented by any of the following formulas (Im) to (It).

7. The derivative according to claim 1, or a pharmaceutically acceptable salt thereof, wherein X in formula (I) is a ketone group.

8. The derivative according to claim 7 or a pharmaceutically acceptable salt thereof, wherein the derivative represented by formula (I) is represented by the following formula (Iu).

9. A fat accumulation inhibitor comprising as an active ingredient a derivative according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, or a derivative of dehydroabietic acid represented by the following formula (Iv) or formula (Iw) or a pharmaceutically acceptable salt thereof.

10. The fat accumulation inhibitor according to claim 9, wherein the fat accumulation inhibition includes inhibition of adipocyte differentiation and / or fat reduction.

11. A pharmaceutical composition for treating or preventing adipose-related disease, comprising the fat accumulation inhibitor described in claim 10.

12. The pharmaceutical composition according to claim 11, wherein the fat-related disease is obesity, diabetes, dyslipidemia, fatty liver, or metabolic disorder-related steatohepatitis.

13. The pharmaceutical composition according to claim 12 for oral administration of the derivative or a pharmaceutically acceptable salt thereof.

14. The pharmaceutical composition according to claim 12 for administration to a subject in a dose of 50 μg / kg body weight to 250 mg / kg body weight of the derivative or a pharmaceutically acceptable salt thereof.

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