JP5211322B2 - Novel use of pyrimidine-5-carboxylic acid derivatives as anti-obesity agents - Google Patents

Description

Background of the Invention

The present invention relates to 2- [N-cyclopropylmethyl-N- (5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphthalen-2-yl) amino] pyrimidine-5. -It relates to a novel use of carboxylic acid (compound A) as an anti-obesity agent.

Background Art Obesity is the proliferation, enlargement, and accumulation of adipocytes. Recent research has revealed that fat cells are not just energy reservoirs that store fat, but have physiological effects such as secretion of hormone-like substances that regulate appetite, blood pressure, blood sugar, blood flow, immunity, etc. ing.

  Obesity is classified into primary obesity (simple obesity) and secondary obesity (symptomatic obesity). Most of those clinically determined to be obese are primary obesity. The causes include excessive intake of energy, insufficient use of energy, and a decrease in the amount of released heat. On the other hand, secondary obesity is obesity caused by some underlying disease, and includes endocrine obesity, hypothalamic obesity, hereditary obesity and the like.

  Obesity is a risk factor for health and causes various diseases. In particular, there is a strong correlation between obesity and diabetes, and when it becomes obese, diabetes is induced at a 10-fold higher probability. In obesity, particularly visceral fat type obesity, a large amount of fatty acid is released into the blood, and sugar utilization as an energy source is reduced in skeletal muscle and liver, leading to insulin resistance that lowers the efficacy of insulin. In visceral fat obesity, adiponectin that increases insulin sensitivity decreases, TNF-α that decreases insulin sensitivity increases, and insulin resistance increases. Therefore, since obesity increases the necessary amount of insulin, the β cells of the pancreas increase insulin production, resulting in hyperinsulinemia. When insulin exceeds a certain amount, the amount of receptor is relatively decreased, the utilization efficiency of blood sugar is lowered, leading to hyperglycemia, and further production of insulin continues to normalize blood sugar. This increase in insulin production also causes abnormalities in β-cells and insufficient insulin secretion, resulting in diabetes.

  In addition, obesity is also a central symptom of metabolic syndrome that has become a serious problem in developed countries in recent years. If obesity occurs due to lifestyle, high-fat diet, or lack of exercise, lifestyle-related diseases such as diabetes, hyperlipidemia, and hypertension are likely to occur. Furthermore, when these risk factors are combined, the mortality due to arteriosclerotic diseases such as myocardial infarction and cerebral infarction increases synergistically.

  Epidemiological studies have pointed out mild inflammatory reactions throughout the body as the underlying pathology of metabolic syndrome. In the obese state, macrophages infiltrate the enlarged adipose tissue and adipocytes are activated. This activation causes the adipose tissue to undergo an inflammatory reaction, producing various inflammatory mediators such as leptin, resistin, angiotensinogen, CRP, PAI-1, TNF-α, IL-1β, and IL-6. These inflammatory mediators play an important role in the pathogenesis of metabolic syndrome such as insulin sensitivity and arteriosclerosis.

  In other words, in the metabolic syndrome, pathological conditions such as lipoprotein abnormalities, hypertension, adipocytokines, and exacerbation of inflammatory response interact with each other and contribute to the development of atherosclerotic disease against the backdrop of visceral obesity and insulin resistance. . Therefore, prevention of obesity is an important factor in preventing metabolic syndrome.

  The tissue most changed by obesity is white adipose tissue. Therefore, there is a great expectation for the clinical development of an anti-obesity agent that has the effect of converting the properties of adipose tissue that has proliferated and enlarged to produce various mediators into a normal state.

  2- [N-cyclopropylmethyl-N- (5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphthalen-2-yl) amino] pyrimidine-5-carboxylic acid (hereinafter "compound A ”) is synthesized as a pharmaceutical used for the prevention or treatment of diabetes (Patent Document 1: WO 00/66595 pamphlet). Compound A has an action as a retinoid X receptor (RXR) agonist (Non-patent Document 1: Chem. Pharm. Bull. 48, 1504-1513, 2000).

  Retinoid X receptor (RXR) is a type of nuclear receptor that exists mainly in the nucleus and acts as a transcription factor that regulates gene expression in a ligand-dependent manner. Retinoid X receptors (RXR) are peroxisome proliferator-activated receptor (PPAR), liver X receptor (LXR), farnesoid X receptor (FXR), Forming heterodimers with various receptors such as retinoic acid receptor (RAR), thyroid hormone receptor (Thyroid Hormone Receptorm, TR), vitamin D receptor (Vitamin D Receptor, VDR), It regulates gene transcription.

  Retinoid X receptor (RXR) heterodimers have been reported to be classified into sensitive and non-sensitive types (Non-Patent Document 2: AIShulman et. Al, N Engl J Med 2005; 353; 604-15 ). Sensitive forms include heterodimers of RXR and PPAR, LXR and FXR, which are activated by both RXR agonists and their respective partner receptor agonists. On the other hand, non-sensitive types include heterodimers of RXR and VDR, TR, etc., which are activated only by their respective agonists.

  LXR, FXR, and PPAR, which form a sensitive heterodimer with retinoid X receptor (RXR), are nuclear receptors that act as lipids as endogenous ligands, and in recent years, their association with metabolic syndrome has attracted attention.

  In particular, PPARγ is a transcription factor essential for adipocyte differentiation, increases small adipocytes in white adipose tissue, and suppresses the production of inflammatory mediators. In addition, it has the effect of suppressing gluconeogenesis in the liver and activating glucose uptake in muscles to enhance the insulin sensitivity of tissues, and is attracting attention as one of the targets for diabetes treatment. Thiazolidine derivatives such as rosiglitazone and pioglitazone are currently used clinically as antidiabetic drugs.

  It has been reported that a retinoid X receptor (RXR) agonist activates a PPARγ heterodimer, enhances insulin sensitivity, and exhibits an antidiabetic action (Non-patent Document 3: Stumvoll et al., Horm. Res. 2001, 55: 3-13).

  On the other hand, PPARγ agonists significantly increase the number of fat cells by promoting the differentiation of fat cells, and in order to strongly promote fat accumulation, fat cells are enlarged under a high fat diet and promote obesity.

  Since compound A has an action as a retinoid X receptor (RXR) agonist, it activates a heterodimer of RXR and PPARγ similarly to a PPARγ agonist, and therefore, the same effect as a PPARγ agonist is expected. That is, like a PPARγ agonist, Compound A is expected to enlarge fat cells under a high fat diet and promote obesity.

  Although there are reports in the literature that anti-obesity action was obtained by administration of other compounds that act on the retinoid X receptor (RXR) (Non-patent Document 4: Endocrinology 145: 565-573, 2004; Metabolism 49: 1610 , 2000), this is an effect associated with a decrease in food consumption. Therefore, it has not been reported that an RXR agonist exhibits an anti-obesity effect without being affected by food intake.

International Publication No. 00/66595 Pamphlet Chem. Pharm. Bull. 48, 1504-1513, 2000 A.I.Shulman et.al, N Engl J Med 2005; 353; 604-15 Stumvoll et al., Horm. Res. 2001, 55: 3-13 Endocrinology 145: 565-573, 2004; Metabolism 49: 1610, 2000

Summary of the Invention

  The inventors have surprisingly found 2- [N-cyclopropylmethyl-N- (5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphthalen-2-yl) amino. It was found that pyrimidine-5-carboxylic acid (Compound A) has a useful anti-obesity effect. The present invention is based on this finding.

  Accordingly, an object of the present invention is to provide a pharmaceutical composition useful for the treatment of obesity.

  The pharmaceutical composition according to the present invention is a pharmaceutical composition for use in the prevention or treatment of obesity, and 2- [N-cyclopropylmethyl-N- (5,6,7,8) as an active ingredient. -Tetrahydro-5,5,8,8-tetramethylnaphthalen-2-yl) amino] pyrimidine-5-carboxylic acid, or a pharmaceutically acceptable salt or solvate thereof.

  According to the present invention, it becomes possible to treat obesity efficiently. In particular, the treatment of obesity according to the present invention has the advantageous effect that it is not necessary to limit the amount of food (especially a high fat diet) consumed by the subject.

Detailed description of the invention

  The active ingredient of the pharmaceutical composition according to the present invention is 2- [N-cyclopropylmethyl-N- (5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphthalen-2-yl) amino. ] Pyrimidine-5-carboxylic acid (hereinafter referred to as “Compound A”). This compound is disclosed in the pamphlet of International Publication No. WO 00/66595 together with its production method, and the structural formula is as shown below.

  Compound A is a retinoid X receptor (RXR) agonist and exerts an action of activating the heterodimer of RXR and PPARγ. However, unlike the PPARγ activator, it has a novel action of suppressing obesity caused by a high fat diet. That is, Compound A exerts a strong anti-obesity effect on the administered subject without lowering the intake of food (particularly a high fat diet). For many obese patients, the limitation of food intake is particularly stressful, so it is particularly beneficial that there is no need to limit food intake.

  In connection with the anti-obesity effect described above, it has been demonstrated herein that Compound A miniaturizes white adipose tissue and further converts gene expression in white adipose tissue to a form characteristic of brown adipose tissue. Yes. Specifically, as the action of Compound A, the action of reducing the size of white adipocytes, the action of suppressing the expression of inflammatory mediators by white fat cells, and the action of enhancing the expression of energy consumption-related genes by white fat cells Has been demonstrated.

  Therefore, according to the present invention, a pharmaceutical composition for reducing the size of white adipocytes in a subject, a pharmaceutical composition for suppressing the expression of inflammatory mediators by white adipocytes in a subject, and a subject Also provided are pharmaceutical compositions for enhancing expression of energy expenditure related genes by white adipocytes. These pharmaceutical compositions also contain Compound A as an active ingredient.

  As used herein, “obesity” means a pathological state in which adipocytes proliferate, enlarge, and accumulate. Obesity generally includes primary obesity (simple obesity) and secondary obesity (symptomatic obesity). In the present invention, any pathological condition is a target of treatment, but primary obesity is preferable. More preferably, it is obesity caused by intake of a high fat diet.

  As used herein, “inflammatory mediator” means a substance that is expressed by white adipocytes and causes inflammation in the body of a subject. Such substances are known in the art and are not particularly limited herein, but are preferably leptin, TNFα, IL-6 and / or IL-1β.

  As used herein, “energy consumption-related gene” refers to a gene that is expressed by white adipocytes and is involved in energy consumption in the body of a subject. Such genes are known in the art and are not particularly limited herein, but are preferably aconitase, aconitase 2 (mitochondrion), Cyp2e1, Cyp2f2, hexokinase, P450 oxidoreductase, protein phosphatase 1, NADH dehydrogenase 1α5 subcomplex, NADH dehydrogenase 1α7 subcomplex, NADH dehydrogenase 1β8 subcomplex, and / or ATPase gene.

  As used herein, “subject” means any animal having fat cells or adipose tissue in the body, and is not particularly limited, but is preferably a mammal, more preferably a human. .

  Compound A used in the present invention may be in the form of a pharmaceutically acceptable salt or solvate. As used herein, “pharmaceutically acceptable” means that when a salt or solvate of Compound A is administered to a subject, it does not exhibit toxicity to the subject and retains its function as an active ingredient. Means that As the pharmaceutically acceptable salt, any salt known in the art may be used, for example, metal salts such as sodium salt, potassium salt, magnesium salt, calcium salt, organic amine salt such as ammonium salt, etc. Can be mentioned. As the pharmaceutically acceptable solvate, any solvate known in the art may be used, and examples thereof include hydrates and other solvates.

  The pharmaceutical composition according to the present invention can be manufactured as a dosage form suitable for various administration routes such as topical, intravenous, subcutaneous, intramuscular, oral, rectal and mucosal. According to a preferred embodiment of the invention, the pharmaceutical composition according to the invention is intended for oral administration. The pharmaceutical composition for oral administration may be any oral formulation known in the art, such as tablets, powders, capsules, foaming agents, liquids, troches and the like. Alternatively, the pharmaceutical composition according to the present invention may be formulated as a food additive, and the food additive containing Compound A can be suitably ingested by the subject by adding it to food.

  The pharmaceutical composition according to the present invention may further comprise a pharmaceutically acceptable carrier. Here, “pharmaceutically acceptable” means that when the pharmaceutical composition according to the present invention is administered to a subject, it is not toxic to the subject and does not inhibit the function of Compound A as an active ingredient. Means that. Such carriers include vehicles, excipients, diluents and the like, and are appropriately selected by those skilled in the art depending on the administration route and the like.

  According to another aspect of the present invention, in a subject, a method for preventing or treating obesity, a method for reducing the size of white adipocytes, a method for suppressing the expression of inflammatory mediators by white adipocytes, and white fat Methods are provided for enhancing expression of energy expenditure-related genes by cells. These methods comprise administering to the subject an effective amount of Compound A, or a pharmaceutically acceptable salt or solvate thereof.

  Compound A can be administered by any suitable route, including topical, intravenous, subcutaneous, intramuscular, oral, rectal, mucosal. According to a preferred embodiment of the invention, Compound A is orally administered to a subject in the form of an oral formulation known in the art, such as tablets, powders, capsules, foams, solutions, troches and the like. Alternatively, Compound A can be suitably ingested by a subject by adding it to food. The therapeutically effective amount is appropriately determined by a doctor or veterinarian according to the severity of symptoms, the age of the subject, the route of administration, the frequency of administration, and the like. Generally, the effective amount is from about 0.001 to about 1000 mg / kg body weight, preferably about 0.01 to about 10 mg / kg body weight, more preferably about 0.01 to about 1 mg / kg body weight per day. It is.

  According to another aspect of the present invention there is provided the use of Compound A or a pharmaceutically acceptable salt or solvate thereof in the manufacture of a medicament. This drug is used for prevention or treatment of obesity, a drug for reducing the size of white adipocytes in a subject, a drug for suppressing the expression of inflammatory mediators by white adipocytes in a subject, Alternatively, it is a drug for enhancing the expression of energy consumption-related genes by white adipocytes in a subject.

  Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto.

Example 1: 2- [N-cyclopropylmethyl-N- (5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphthalen-2-yl) amino] pyrimidine-5-carboxylic acid Production of (Compound A) In this example, Compound A was produced according to the method described in WO 00/66595.

(I) Preparation of 2- [N- (5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphthalen-2-yl) amino] pyrimidine-5-carboxylate 2-chloropyrimidine A mixture of 100 mg ethyl-5-carboxylate, 108 mg 5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphthalen-2-amine, and 400 mg K ₂ CO ₃ was heated at 110 ° C. After confirming disappearance of the raw material by TLC, the reaction solution was poured into water and extracted with CH ₂ Cl ₂ . The organic layer was dried over Na ₂ SO ₄ and the solvent was distilled off. The residue was purified by flash silica gel column chromatography (n-hexane: AcOEt = 20: 1) and 2- [N- (5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphthalene). There were obtained 170 mg (91%) of white crystals of ethyl-2-yl) amino] pyrimidine-5-carboxylate.

Colorless cottons (n-hexane-AcOEt);
¹ H-NMR (400 MHz, CDCl ₃ ) δ 8.95 (s, 2H), 7.56 (br s, 1H), 7.44 (dd, J = 2.4, 9.0 Hz, 1H), 7.44 (d, J = 2.4 Hz, 1H), 7.31 (d, J = 9.2 Hz, 1H), 4.37 (q, J = 7.0 Hz, 2H), 1.69 (s, 4H), 1.39 (t, J = 7.1 Hz, 3H), 1.30 (s, 6H), 1.28 (s, 6H).

(Ii) ethyl 2- [N-cyclopropylmethyl-N- (5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphthalen-2-yl) amino] pyrimidine-5-carboxylate Preparation of 2- [N- (5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphthalen-2-yl) amino] pyrimidine-5-carboxylate 100 mg was dissolved in 2 ml of DMF. Then, a suspension obtained by suspending 30 mg of NaH in 1.5 ml of DMF was added thereto. To the obtained mixture, 0.3 ml of cyclopropylmethyl bromide was added and stirred. After confirming disappearance of the raw material by TLC, the reaction solution was poured into water and extracted with CH ₂ Cl ₂ . The organic layer was dried over Na ₂ SO ₄ and the solvent was distilled off. The residue was purified by flash silica gel column chromatography (n-hexane: AcOEt = 20: 1) and 2- [N-cyclopropylmethyl-N- (5,6,7,8-tetrahydro-5,5,8). , 8-Tetramethylnaphthalen-2-yl) amino] pyrimidine-5-carboxylate 63 mg (55%) was obtained.

¹ H-NMR (400 MHz, CDCl ₃ ) δ 8.85 (s, 2H), 7.33 (d, J = 8.4 Hz, 1H), 7.18 (d, J = 2.2 Hz, 1H), 7.01 (dd, J = 2.2 , 8.3 Hz, 1H), 4.33 (q, J = 7.2 Hz, 2H), 3.86 (d, J = 7.0 Hz, 2H), 1.70 (s, 4H), 1.35 (t, J = 7.0 Hz, 3H), 1.30 (s, 6H), 1.27 (s, 6H), 1.17 (br m, 1H), 0.46 (dd, J = 5.9, 12.6 Hz, 2H), 0.19 (dd, J = 5.0, 10 Hz, 2H).

(Iii) 2- [N-cyclopropylmethyl-N- (5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphthalen-2-yl) amino] pyrimidine-5-carboxylic acid Preparation 2-ethyl [N-cyclopropylmethyl-N- (5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphthalen-2-yl) amino] pyrimidine-5-carboxylate 63 mg Dissolved in 4 ml of ethanol, 0.5 ml of 20% aqueous KOH solution was added and refluxed. After confirming disappearance of the raw material by TLC, the reaction solution was poured into 2N HCl and extracted with ethyl acetate. The organic layer was dried over Na ₂ SO ₄ , the solvent was distilled off, and the title compound 2- [N-cyclopropylmethyl-N- (5,6,7,8-tetrahydro-5,5,8, There was obtained 55 mg (85.5%) of white crude crystals of 8-tetramethylnaphthalen-2-yl) amino] pyrimidine-5-carboxylic acid (Compound A).

Colorless needles (n-hexane-AcOEt); mp 232 ° C;
¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 8.73 (s, 2H), 7.36 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 1.2 Hz, 1H), 7.02 (dd, J = 1.2, 8.4 Hz, 1H), 3.84 (d, J = 6.8 Hz, 2H), 1.67 (s, 4H), 1.28 (s, 6H), 1.24 (s, 6H), 1.18 (br m, 1H), 0.42 (dd, J = 5.1, 13 Hz, 2H), 0.14 (dd, J = 5.1, 10 Hz, 2H);
_{. Anal Calcd for C 23 H 28} N 3 O 2 · 1 / 10H 2 O, C: 72.64%, H: 7.47%, N: 11.05%; Found C: 72.35%, H: 7.67%, N: 10.81%.

Example 2: Evaluation of RXR heterodimer activation ability of compound A In this example, the RXR heterodimer activation ability of compound A was evaluated by a reporter gene assay.

(1) Plasmid used The human PPARγ expression vector and human RXRα expression vector were prepared by PCR cloning of full-length cDNAs of human PPARγ and RXRα and inserting them into pcDNA3.1 (Invitrogen). A reporter construct having a firefly luciferase gene linked to a PPAR response region (PPRE) was made by inserting two copies of acyl CoA oxidase PPRE into firefly luciferase tk-pGL3 (Promega) (Suzuki S, et al., Arterioscler Thromb Vasc Biol 2004; 24: 519-525). As a control plasmid having LacZ contrast bound to SV40, pSV-beta-galactosidase control vector (Promega) was used.

(2) Test method The reporter gene assay was performed by transient transfection of COS1 cells, and the ability of Compound A to activate the heterodimer of human PPARγ and human RXRα was evaluated. The COS1 cells seeded in the 48-well plate the previous day were subjected to polyfect (QIAGEN) using a PPARγ expression vector, an RXRα expression vector, a reporter construct having a firefly luciferase gene to which a nuclear receptor response region was bound, and SV40 A control plasmid with a LacZ construct bound to was transfected. Three hours later, the cells were washed, and a medium containing 0.1% fatty acid-free BSA containing 1 nM troglitazone which is an agonist of PPARγ and Compound A was added and incubated for 24 hours. Cells were harvested and nuclear receptor activation was quantified by luciferase assay system and β-galactosidase activity by hydrolysis of chlorophenol red β-galactopyranoside (CRPG). Transcriptional activity has the following formula:
(True transcriptional activity) = (measured value of luciferase activity) / (measured value of β-galactosidase activity)
Evaluated according to.

(3) Results As shown in FIG. 1, Compound A, which is an RXR agonist, activated PPARγ alone in a concentration-dependent manner. In addition, Compound A synergistically increased the action of the PPARγ agonist in the presence of a small amount (1 nM) of a PPARγ synthetic agonist (troglitazone) that had little effect by itself. Thus, it was revealed that Compound A activates PPARγ and increases the action of PPARγ agonist.

Example 3: Examination of anti-arteriosclerotic and anti-obesity effects of Compound A In this example, ApoE knockout mice that spontaneously develop hyperlipidemia and arteriosclerosis were used, and the anti-arteriosclerotic effect of Compound A according to the following method. And anti-obesity effect was examined.

(1) Test method (i) Animal Male ApoE knockout mice (C57BL / 6J-ApoE Jackson Laboratory) and male C57BL / 6Cr mice (Japan SLC) were bred under normal breeding conditions, and individuals aged 8 weeks were Used for testing. A high fat diet (Research Diet) was given as a bait.

(Ii) Method and period of administration of Compound A Compound A was mixed in a powder diet (high fat diet) so as to be 30 mg / kg / day and orally administered to ApoE knockout mice (9 cases in the drug treatment group). The first control group (drug non-administration group) was a mouse (10 cases) reared with a powder diet (high fat diet) alone, and the second control group (drug non-administration group) was a mouse reared with a normal powder diet ( 5 cases). All mice included in each group were bred continuously for 10 weeks (up to 18 weeks of age). Body weight and food intake were measured once a week, and blood and organs were removed after 1 week, 4 weeks and 10 weeks, and various experiments were performed.

  Male C57BL / 6Cr mice were used for oxygen consumption experiments. Compound A was mixed with powdered diet (high fat diet) so as to be 30 mg / kg / day and orally administered (6 cases in the drug treatment group). The control group (drug non-administered group) was a mouse (6 cases) reared with a powder diet (high fat diet) alone. All mice included in each group were bred continuously for 12 weeks (up to 20 weeks of age). Once a week, body weight and food consumption were measured.

(Iii) Observation and inspection methods Blood chemical mediator concentration After the final administration, mice were fasted. The next morning, mice were euthanized by cervical dislocation under ether anesthesia, laparotomy was performed to expose the abdominal vena cava, and 0.5-1.0 ml of blood was collected. The collected blood was centrifuged at 3000 rpm for 15 minutes to collect plasma.

  Leptin and adiponectin levels in plasma were detected by ELISA. For the measurement of leptin, a mouse leptin measurement kit (Morinaga Bioscience Institute) was used. For the measurement of adiponectin, mouse / rat adiponectin ELISA kit (Otsuka Pharmaceutical Co., Ltd.) was used.

B. Histological evaluation a. Observation of adipose tissue After blood collection, white fat was extracted, the total weight was measured, and then a part was embedded for frozen sections. Serial sections were prepared, adhered to a slide glass, immediately fixed with PFA, washed, and then stained with hematoxylin and eosin.

b. Analysis of mRNA changes in adipose tissue (real-time PCR)
After blood collection, white fat was collected, and total RNA was extracted using RNAeasy mini kit (QIAGEN). The expression of specific genes was measured by real-time RT-PCR. In this experiment, an oligonucleotide having the following sequence was used as a target gene-specific Taqman primer probe set. All data were corrected with a value of 18S.

Uncoupled protein UCP1:
Forward primer: 5'-AAGCTGTGCGATGTCCATGTAC-3 '(SEQ ID NO: 1);
Reverse primer: 5'-AGAAGCCACAAACCCTTTGAAA-3 '(SEQ ID NO: 2);
TaqMan probe: 5′-CCAAGGAAGGACCGACGGCCT-3 ′ (SEQ ID NO: 3).

Adrenergic β3 receptor (ARbeta3):
Forward primer: 5'-CCCAGCCAGCCCTGTTG-3 '(SEQ ID NO: 4);
Reverse primer: 5'-GCACCTTCATAGCCATCAAACC-3 '(SEQ ID NO: 5);
TaqMan probe: 5′-CCAGGCAGAGTCCACCGCTCAAC-3 ′ (SEQ ID NO: 6).

c. Analysis of mRNA change in adipose tissue (DNA chip)
After blood collection, white fat was collected, and total RNA was extracted using RNAeasy mini kit (QIAGEN). Using a mouse DNA chip (U74AVS), chip analysis was performed on each of three cases of the control group and the drug treatment group, genes with a significantly doubled variation were extracted by t-test, and Ontology analysis was performed.

(Iv) Measurement of oxygen consumption (VO2) of mice Oxygen consumption of mice (drug-treated group: 6 cases) in which Compound A was orally administered at 30 mg / kg / day for 12 weeks and control group mice (drug-untreated group: 6 cases) The amount was measured using a small animal metabolic measurement system MK-5000RQ (MUROMACHI KIKAI CO., LTD.). The average oxygen consumption VO2 (oxygen consumption value / mouse body weight) for 2 hours in the resting period was calculated.

(V) Statistical analysis and data processing method The results obtained were expressed as mean ± standard error. A significant difference test between the control group and the drug-treated group was performed by t-test and multiple test.

(2) Results FIG. 2 shows a graph of weight change in each group. Mice in the first control group fed the high fat diet gained weight compared to the mice in the second control group fed the normal diet. In contrast, the body weight of the mice in the drug-treated group fed with compound A in a high-fat diet was strongly suppressed compared to the mice in the first control group. Moreover, it was shown from the graph of the food intake on the right side of FIG.

  FIG. 3 shows the white fat weight, leptin value and adiponectin value in the drug-treated group (T), the first control group (Cont1), and the second control group (Cont2). When the amount of white adipose tissue around the testis was measured, a marked decrease in fat mass was observed in the drug-treated group, and it was revealed that Compound A has an anti-obesity effect. The amount of leptin secreted from adipocytes was low in the drug treated group. On the other hand, the adiponectin level secreted from small fat cells and decreased during obesity did not change.

  FIG. 4 shows a photomicrograph of white adipose tissue stained with hematoxylin and eosin. In the first control group, lipid droplets were collected and the cells were enlarged, but in the drug-treated group, it was observed that the fat cells were downsized.

  Panel A of FIG. 5 shows the effect of Compound A on adrenergic β3 receptor (ARbeta3) mRNA expression. The adrenergic β3 receptor is mainly present in white adipocytes and brown adipocytes, and promotes the decomposition of white fat and heat production in brown adipocytes by adrenergic stimulation. It has been pointed out that an abnormality in β3 receptor suppresses energy metabolism and is likely to become obese. It was revealed that white adipose tissue adrenergic β3 receptor mRNA was increased by Compound A treatment.

  Panel B of FIG. 5 shows the effect of Compound A on uncoupled protein UCP1 mRNA expression. UCP1 is an uncoupling protein that exists specifically in brown adipose tissue and has attracted attention as a target molecule for obesity because it uncouples mitochondrial oxidative phosphorylation and releases energy as heat. It was found that UCP1 mRNA, which is not normally in white adipose tissue, is increased by Compound A treatment.

  Tables 1 and 2 show the results of DNA chip analysis of genes that vary due to the action of Compound A in white adipose tissue. In these tables, chip analysis is performed for each of three cases of the control group and the drug treatment group, and a part of genes having a significantly doubled variation by t-test is listed.

  According to Table 1 and Table 2, gene groups of energy production systems such as ATP synthase and NAD dehydrogenase are generally increased by the action of Compound A. Furthermore, the expression of a specific gene group of constituent components of the mitochondrial inner membrane was increased, and it was shown that compound A increased the number of mitochondria in the brown adipose tissue in white adipose tissue.

  As described above, Compound A reduces the size of white adipocytes. Furthermore, Compound A induces expression of adrenergic β3 receptor, UCP1 gene, energy production gene group, and mitochondrial specific gene in white adipose tissue. That is, it was shown that Compound A exerts a characteristic action of increasing gene expression characteristic of brown adipose tissue in white adipose tissue.

  Adipose tissue secretes a variety of adipocytokines, and abnormal production of these causes aggravation of lesions in arteriosclerotic lesions. Adipocytokines contain many inflammatory cytokines, which have been reported to migrate monocytes in blood, adhere to vascular endothelium, become macrophages, and participate in the formation of arteriosclerotic foci.

  FIG. 6 shows inflammatory cytokine mRNA expression in adipose tissue of the drug-treated group (T), the first control group (Cont1), and the second control group (Cont2). In the drug-treated group, mRNA expression of TNFα (TNF alpha), IL-6 and IL-1β (IL-1 beta) in adipose tissue was strongly suppressed.

  FIG. 7 shows the oxygen consumption of the drug-treated group and the control group. In the drug-treated group, a significant increase in oxygen consumption of around 20% was shown.

  As described above, Compound A suppresses the expression of inflammatory mediators in white adipose tissue. Compound A also makes fat cells smaller. Furthermore, Compound A increases energy consumption due to the characteristic action that induces energy consumption-related gene expression induction in white adipose tissue. Therefore, Compound A is useful as an effective antiobesity agent.

FIG. 1 is a graph showing the effect of Compound A on PPARγ activity. FIG. 2 is a graph showing the effect of Compound A on body weight suppression and food intake in mice. FIG. 3 is a graph showing the effect of Compound A on white adipose tissue mass and the effect on blood leptin concentration and adiponectin concentration. FIG. 4 is a photomicrograph showing HE-stained images of white adipose tissue obtained from mice in the drug-treated group and the first control group. FIG. 5 is a graph showing the effect of Compound A on ADRβ3 and UCP1 mRNA expression in white adipose tissue. FIG. 6 is a graph showing the effect of Compound A on cytokine mRNA expression in adipose tissue. FIG. 7 is a graph showing the effect of Compound A on oxygen consumption in mice.

Claims (7)

  A pharmaceutical composition for use in the prevention or treatment of obesity, wherein 2- [N-cyclopropylmethyl-N- (5,6,7,8-tetrahydro-5,5,8,8) is used as an active ingredient. -Tetramethylnaphthalen-2-yl) amino] pyrimidine-5-carboxylic acid, or a pharmaceutically acceptable salt or solvate thereof.   The pharmaceutical composition according to claim 1, wherein obesity is caused by intake of a high fat diet.   2. A pharmaceutical composition according to claim 1 for oral administration. A pharmaceutical composition for suppressing fat cell hypertrophy caused by a high fat diet in a subject, wherein 2- [N-cyclopropylmethyl-N- (5,6,7,8-tetrahydro- A pharmaceutical composition comprising 5,5,8,8-tetramethylnaphthalen-2-yl) amino] pyrimidine-5-carboxylic acid, or a pharmaceutically acceptable salt or solvate thereof.   The pharmaceutical composition according to claim 4 for oral administration. A pharmaceutical composition for enhancing expression of energy consumption-related genes by white adipocytes in a subject, comprising 2- [N-cyclopropylmethyl-N- (5,6,7,8-tetrahydro 5,5,8,8-tetramethyl-2-yl) amino] pyrimidine-5-carboxylic acid Ri name or comprise a pharmaceutically acceptable salt or solvate thereof,
The energy consumption-related genes are aconitase, aconitase 2 (mitochondria), Cyp2e1, Cyp2f2, hexokinase, P450 oxidoreductase, protein phosphatase 1, NADH dehydrogenase 1α5 subcomplex, NADH dehydrogenase 1α7 subcomplex, NADH dehydrogenase 1β8 subcomplex, and A pharmaceutical composition which is selected from the group consisting of genes .   The pharmaceutical composition according to claim 6 for oral administration.