Ligand acting on adenosine receptor and composition for preventing, ameliorating or treating obesity comprising same

Abstract

The present invention relates to a dual-acting ligand which acts on both an A2A adenosine receptor and / or an A3 adenosine receptor, and a composition for preventing, ameliorating or treating obesity comprising the same. In addition, the invention relates to a method for treating diseases related to the A2A adenosine receptor (AR) and / or the A3 adenosine receptor (AR) by using the ligand, and application of the ligand in preparation of medicines for treating diseases related to the A2A adenosine receptor (AR) and / or the A3 adenosine receptor (AR). The ligand LJ-4378 acting on the adenosine receptor and the derivative of the ligand LJ-4378 have the dual effects of serving as an agonist of A2AAR and an antagonist of A3AR, mitochondrial protein, UCP1 and COXIV are remarkably increased respectively, and the ligand LJ-4378 and the derivative of the ligand LJ-4378 have an excellent anti-obesity effect on high-fat diet mice. Therefore, the ligand acting on the adenosine receptor according to the present invention can be effectively used in the preparation of a composition for preventing, ameliorating or treating obesity, a treatment method, and a medicament.

Description

Technical Field

[0001] The present invention claims priority to Korean Patent Application No. 10-2022-0091348 filed on Jul. 22, 2022, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to a 2A A dual-acting ligand that acts on both adenosine receptors and / or A3 adenosine receptors and a composition comprising the ligand for preventing, improving or treating obesity.

[0003] In addition, the present invention relates to a method of using the ligand 2A Methods for treating diseases related to adenosine receptor (AR) and / or A3 adenosine receptor (AR), and methods ...) 2A Use of the present invention in drugs for treating diseases related to adenosine receptor (AR) and / or A3 adenosine receptor (AR).

[0004] This invention was made with the support of the Ministry of Health and Welfare of South Korea, project number HN22C0687 (1465037037). Background Art

[0005] Obesity is defined as excessive fat accumulation, which is a major risk factor for metabolic diseases such as type 2 diabetes. Adipose tissue can be classified into brown adipose tissue (BAT) and white adipose tissue (WAT). BAT contains a large number of mitochondria and is responsible for non-shivering thermogenesis, while WAT is specialized for fat storage and mobilization. Therefore, activating and recruiting BAT is a strategy to increase energy expenditure and can ultimately be developed as a potential anti-obesity target.

[0006] As an inducer of brown adipocytes, pharmacological stimulation of adenosine receptor signaling has attracted much attention as a potential target for improving metabolic health. Adenosine receptors can be classified into four types of G-coupled receptors: A1, A 2A , A 2B A1 adenosine receptor (A1AR) and A3 adenosine receptor (A3AR) and G i / o Protein binds to and inhibits cAMP production and cAMP-dependent protein kinase (PKA) signaling. 2A Adenosine receptors (A 2A AR) and A 2B Adenosine receptors (A 2B AR) and G s / olf The protein binds to and promotes the activity of adenylate cyclase (AC) and the production of cAMP.

[0007] In adenosine receptors, A 2A AR is more abundantly expressed in BAT than in WAT, and its anti-obesity effects have been supported by several studies. 2A AR activation stimulates AC activity and promotes PKA-dependent lipolysis, A 2A AR signaling is required for the complete physiological function of BAT. In addition, a recent study showed that AR signaling 2A The AR agonist CGS21680 significantly increased the expression of thermogenic genes including UCP1 both in vitro and in vivo. 2A AR stimulation has an inhibitory effect. A3AR expression is abundant in white adipose tissue, but the regulatory role of A3AR in adipocyte metabolism is not fully understood.

[0008] Prior Art Problem Literature

[0009] Patent Literature

[0010] Patent Document 1KR10-2013-0028732 (2013-03-19)

[0011] Non-patent literature

[0012] Non-patent literature 1Jeong, LS; Lee, HW; Jacobson, KA; Kim, HO; Shin, DH; Lee, JA; Gao, Z.-G.; Lu, C.; Duong, HT; Gunaga, P.; Lee, SK; Jin, DZ; Chun, MW; Moon, HR Structure activity relationships of 2-chloro-N6-substituted-40-thioadenosine-50-uronamides as highly potent and selective agonists at the human A3 adenosine receptor. J. Med. Chem. 2006, 49, 273-281. Summary of the invention

[0013] Technical Problems to be Solved by the Invention

[0014] The present inventors are committed to providing 2A As a result of studies on dual-acting ligands that act on both adenosine receptors (AR) and / or A3 adenosine receptors (AR), LJ-4378 and its derivatives have been confirmed to be effective as A 2AThe present invention was completed by finding that an AR agonist and an A3AR antagonist can function and provide an excellent anti-obesity effect.

[0015] Therefore, an object of the present invention is to provide a ligand acting on adenosine receptors and a composition comprising the ligand for preventing, improving or treating obesity.

[0016] Another object of the present invention is to provide a method for treating a 2A Methods for treating diseases related to adenosine receptor (AR) and / or A3 adenosine receptor (AR), and methods ...) 2A Use of the present invention in drugs for treating diseases related to adenosine receptor (AR) and / or A3 adenosine receptor (AR).

[0017] Technical Solution

[0018] The present invention provides a method for preventing or treating A 2A A pharmaceutical composition for adenosine receptor (AR) and / or A3 adenosine receptor (AR)-related diseases, the pharmaceutical composition comprising a compound represented by the following [Chemical Formula 1], an isomer or a pharmaceutically acceptable salt thereof:

[0019] [Chemical formula 1]

[0020]

[0021] R1 is H, C1-C10 alkyl or aryl-C1-C10 alkyl,

[0022] R2 is a C1-C10 alkyl group.

[0023] According to a preferred embodiment of the present invention, R1 is H, methyl or aryl-C1-C10 alkyl,

[0024] R2 is a C1-C4 alkyl group.

[0025] According to a preferred embodiment of the present invention, the compound represented by [Chemical Formula 1], its isomers or pharmaceutically acceptable salts are selected from one or more of the group consisting of the following compounds:

[0026] (1) 2-(6-amino-2-(hex-1-yn-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol;

[0027] (2) (2R,3R,4S)-2-(6-amino-2-(prop-1-yn-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol;

[0028] (3) (2R,3R,4S)-2-(6-((3-fluorobenzyl)amino)-2-(prop-1-yn-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol;

[0029] (4) (2R,3R,4S)-2-(6-((3-chlorobenzyl)amino)-2-(prop-1-yn-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol;

[0030] (5) (2R,3R,4S)-2-(6-((3-bromobenzyl)amino)-2-(prop-1-yn-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol;

[0031] (6) (2R,3R,4S)-2-(6-((3-iodobenzyl)amino)-2-(prop-1-yn-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol;

[0032] (7) (2R,3R,4S)-2-(6-amino-2-(but-1-yn-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol;

[0033] (8) (2R,3R,4S)-2-(2-(but-1-yn-1-yl)-6-((3-fluorobenzyl)amino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol;

[0034] (9) (2R,3R,4S)-2-(2-(but-1-yn-1-yl)-6-((3-chlorobenzyl)amino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol;

[0035] (10) (2R,3R,4S)-2-(6-((3-bromobenzyl)amino)-2-(but-1-yn-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol; and

[0036] (11) (2R,3R,4S)-2-(2-(But-1-yn-1-yl)-6-((3-iodobenzyl)amino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol.

[0037] According to a preferred embodiment of the present invention, the compound represented by [Chemical Formula 1], its isomer or pharmaceutically acceptable salt is A 2A It is an agonist of adenosine receptor (AR) and also an antagonist of A3 adenosine receptor (AR).

[0038] According to a preferred embodiment of the present invention, the 2AAn adenosine receptor (AR) and / or A3 adenosine receptor (AR) related disease is obesity.

[0039] In addition, the present invention provides a health functional food composition for preventing or improving obesity, the health functional food composition comprising a compound represented by [Chemical Formula 1], an isomer thereof or a pharmaceutically acceptable salt thereof:

[0040] [Chemical formula 1]

[0041]

[0042] R1 is H, C1-C10 alkyl or aryl-C1-C10 alkyl,

[0043] R2 is a C1-C10 alkyl group.

[0044] In addition, the present invention provides a 2A A method for treating a disease associated with adenosine receptor (AR) and / or A3 adenosine receptor (AR), the method comprising the steps of: administering to adenosine receptor (AR) 2A A patient with adenosine receptor (AR) and / or A3 adenosine receptor (AR)-related diseases is administered a compound represented by the following [Chemical Formula 1], an isomer or a pharmaceutically acceptable salt thereof:

[0045] [Chemical formula 1]

[0046]

[0047] R1 is H, C1-C10 alkyl or aryl-C1-C10 alkyl,

[0048] R2 is a C1-C10 alkyl group.

[0049] In addition, the present invention provides a compound represented by the following [Chemical Formula 1], an isomer or a pharmaceutically acceptable salt thereof in the preparation of A 2A Use in drugs for treating adenosine receptor (AR) and / or A3 adenosine receptor (AR) related diseases:

[0050] [Chemical formula 1]

[0051]

[0052] R1 is H, C1-C10 alkyl or aryl-C1-C10 alkyl,

[0053] R2 is a C1-C10 alkyl group.

[0054] Extracellular adenosine is a powerful endogenous signaling molecule that regulates a variety of physiological and pathological events through four types of adenosine receptors. Studies using transgenic mice with altered adenosine receptor expression have further verified the fact that adenosine exerts multiple effects on energy metabolism. Therefore, targeting adenosine receptors may have translational potential for the treatment of metabolic diseases. The present invention confirms that A 2A Effects of dual-acting ligands with AR agonist and A3AR antagonist activity on adipocyte metabolism.

[0055] G αs- Conjugated A 2A Stimulation of adenosine receptors can activate adenylate cyclase and the subsequent cAMP-PKA signaling pathway, while G αi- Activation of the coupled A3AR can oppose A by inhibiting adenylate cyclase. 2A AR signal transduction effect. Therefore, the inventors hypothesize that: 2A Dual ligands with AR agonist and A3AR antagonist activity can synergistically activate PKA-dependent lipolysis and thermogenesis in adipocytes.

[0056] The present invention confirms that LJ-4378 treatment increases the mitochondrial content and activity in adipocytes, and the effect of LJ-4378 is greater than that of A 2A AR agonists or A3AR antagonists were more potent. In vivo LJ-4378 treatment increased energy expenditure and expression of the brown adipocyte marker CIDEA as determined by non-invasive imaging of bioluminescent signals in CIDEA reporter mice. In vivo LJ-4378 treatment reduced body weight and fat mass and exhibited anti-obesity effects by improving glucose tolerance in mice fed a HFD.

[0057] In the present invention, qPCR analysis showed that A 2A AR and A3AR are the main subtypes of adenosine receptors expressed in adipocytes. 2B The expression level of AR is lower than that of other adenosine receptors. 2B AR is mainly expressed in muscle and BAT. 2B Activation of AR has anti-aging and anti-obesity effects. Interestingly, A 2B AR in A 2A play a broad role in mediating lipolysis, forming heterodimers of the two receptors. αi-The coupled A1 receptor inhibits adenylate cyclase activity and is highly expressed in human white adipocytes. Thus, the A1AR is characterized as a lipolysis inhibitor that promotes insulin sensitivity in adipose tissue by reducing circulating FFA and triglyceride levels. Furthermore, as shown in studies of A1AR knockout mice, A1AR signaling promotes adipogenesis and regulates inflammation.

[0058] In addition to its effect on lipolysis, A 2A AR activation also exerts anti-inflammatory effects in a mouse model of diet-induced obesity.

[0059] In summary, the present invention is highly favored by patients because it can be orally administered, and its mechanism of action is not to stimulate the central nervous system, but to promote and consume fat decomposition via adenosine receptors in fat cells, so the probability of nervous system side effects is very low. As an obesity treatment agent with a new mechanism, it can provide an option for patients who are not effective in existing treatment methods.

[0060] In addition, in the case of existing GLP-1 derivatives and central nervous system stimulant obesity treatment agents, obesity is treated by reducing appetite, but unlike these, the present invention can reduce fat by using a mechanism that directly targets brown fat cells, thereby increasing patient preference, and it can be expected that the effect can be enhanced and the side effects can be reduced by combined administration.

[0061] Therefore, the present invention can provide a method for preventing or treating A 2A A pharmaceutical composition for adenosine receptor (AR) and / or A3 adenosine receptor (AR)-related diseases, the pharmaceutical composition comprising a compound represented by the following [Chemical Formula 1], an isomer or a pharmaceutically acceptable salt thereof:

[0062] [Chemical formula 1]

[0063]

[0064] R1 is H, C1-C10 alkyl or aryl-C1-C10 alkyl,

[0065] R2 is a C1-C10 alkyl group.

[0066] According to a preferred embodiment of the present invention, R1 may be H, methyl or aryl-C1-C10 alkyl,

[0067] R2 may be a C1-C4 alkyl group.

[0068] In the present invention, the pharmaceutically acceptable salts are acid addition salts formed based on pharmaceutically acceptable free acids. Acid addition salts are obtained from inorganic acids (such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid), aliphatic monocarboxylic acids and dicarboxylates, phenyl-substituted alkanoic acid esters, hydroxyalkanoic acid esters and alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids and other non-toxic organic acids. Such pharmaceutically nontoxic salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, fluorides, acetates, propionates, decanoates, octanoates, acrylates, formates, isobutyrates, decanoates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates , hexane-1,6-dioate, benzoate, chlorobenzoate, methyl benzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, beta-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, or mandelate.

[0069] The acid addition salt of the present invention can be prepared by conventional methods, for example, by dissolving the compound represented by [Chemical Formula 1] in an aqueous solution of an excess of an acid, and precipitating the salt using an organic solvent miscible with water (e.g., methanol, ethanol, acetone or acetonitrile). In addition, it can also be prepared by the following method: after evaporating the solvent or excess acid in the mixture, drying or suction filtering the precipitated salt.

[0070] In addition, pharmaceutically acceptable metal salts can be prepared by using a base. Alkali metal salts or alkaline earth metal salts are obtained by, for example, the following steps: dissolving the compound in an excess of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the salt of the insoluble compound, and evaporating and drying the filtrate. At this time, for the metal salt, sodium salt, potassium salt or calcium salt is prepared and is suitable for pharmaceutical use. The corresponding silver salt is obtained by reacting an alkali metal salt or alkaline earth metal salt with a suitable silver salt (such as silver nitrate).

[0071] According to a preferred embodiment of the present invention, the compound represented by [Chemical Formula 1], its isomer or pharmaceutically acceptable salt may be selected from one or more of the group consisting of the following compounds:

[0072] (1) 2-(6-amino-2-(hex-1-yn-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol;

[0073] (2) (2R,3R,4S)-2-(6-amino-2-(prop-1-yn-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol;

[0074] (3) (2R,3R,4S)-2-(6-((3-fluorobenzyl)amino)-2-(prop-1-yn-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol;

[0075] (4) (2R,3R,4S)-2-(6-((3-chlorobenzyl)amino)-2-(prop-1-yn-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol;

[0076] (5) (2R,3R,4S)-2-(6-((3-bromobenzyl)amino)-2-(prop-1-yn-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol;

[0077] (6) (2R,3R,4S)-2-(6-((3-iodobenzyl)amino)-2-(prop-1-yn-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol;

[0078] (7) (2R,3R,4S)-2-(6-amino-2-(but-1-yn-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol;

[0079] (8) (2R,3R,4S)-2-(2-(but-1-yn-1-yl)-6-((3-fluorobenzyl)amino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol;

[0080] (9) (2R,3R,4S)-2-(2-(but-1-yn-1-yl)-6-((3-chlorobenzyl)amino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol;

[0081] (10) (2R,3R,4S)-2-(6-((3-bromobenzyl)amino)-2-(but-1-yn-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol; and

[0082] (11) (2R,3R,4S)-2-(2-(but-1-yn-1-yl)-6-((3-iodobenzyl)amino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol;

[0083] According to a preferred embodiment of the present invention, the compound represented by [Chemical Formula 1], its isomer or pharmaceutically acceptable salt may be A 2AIt is an agonist of adenosine receptor (AR) and also an antagonist of A3 adenosine receptor (AR).

[0084] According to a preferred embodiment of the present invention, the 2A The adenosine receptor (AR) and / or A3 adenosine receptor (AR) related disease may be obesity.

[0085] The term "prevention" in the present invention means that the compound represented by [Chemical Formula 1], its isomer or pharmaceutically acceptable salt of the present invention is used to inhibit the occurrence of 2A All actions that affect adenosine receptor (AR) and / or A3 adenosine receptor (AR) related diseases, or delay their onset.

[0086] The “improvement” or “treatment” of the present invention refers to the use of the compound represented by [Chemical Formula 1], its isomer or pharmaceutically acceptable salt of the present invention to improve the 2A All actions that improve or are beneficial to parameters related to adenosine receptor (AR) and / or A3 adenosine receptor (AR)-related diseases (eg, severity of symptoms).

[0087] The pharmaceutical composition of the present invention can be oral or parenteral multiple dosage forms. When the composition is formulated, more than one buffer (e.g., saline or PBS), antioxidant, antibacterial agent, chelating agent (e.g., EDTA or glutathione), filler, extender, binding agent, adjuvant (e.g., aluminum hydroxide), suspending agent, thickening agent, wetting agent, disintegrating agent or surfactant, diluent or excipient can be used to prepare.

[0088] Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc., and such solid preparations may be prepared by mixing at least one excipient in at least one compound, such as starch (including corn starch, wheat starch, rice starch and potato starch, etc.), calcium carbonate, sucrose, lactose, glucose, sorbitol, mannitol, xylitol, erythritol, maltitol, cellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose or gelatin, etc. For example, after the active ingredient is mixed with the solid excipient, it is pulverized, and after adding appropriate adjuvants, it is processed into a granular mixture, so that tablets or sugar-coated tablets can be obtained.

[0089] In addition, in addition to simple excipients, lubricants such as magnesium stearate and talcum powder can also be used. Liquid preparations for oral administration can include suspensions, oral solutions, emulsions or syrups, and liquid preparations can include various excipients, such as wetting agents, sweeteners, aromatics, preservatives, etc., in addition to water and liquid paraffin as commonly used simple diluents. In addition, according to circumstances, cross-linked polyvinyl pyrrolidone, agar, alginic acid or sodium alginate, etc. can be added as disintegrating agents, and anticoagulants, spices, emulsifiers, solubilizers, dispersants, flavoring agents, antioxidants, packaging agents, pigments and preservatives, etc. can also be included.

[0090] Preparations for parenteral administration may include sterile aqueous solvents, non-aqueous solvents, suspensions, emulsions, lyophilized preparations or suppositories, etc. Non-aqueous solvents and suspensions may use plant oils such as propylene glycol, polyethylene glycol and olive oil, injectable esters such as ethyl oleate, etc. Suppositories may use fatty acid esters (witepsol), polyethylene glycol, Tween 61, cocoa butter, laurel oil, glycerin and gelatin, etc. as bases.

[0091] The composition of the present invention can be administered orally or parenterally. When administered parenterally, it can be formulated in the form of a topical, intraperitoneal, rectal, intravenous, subcutaneous or transdermal administration agent according to methods well known in the art.

[0092] When used as the injection, it must be sterilized to prevent contamination by microorganisms such as bacteria and fungi. For example, a suitable carrier for the injection can be water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.), mixtures thereof and / or solvents or dispersion media containing vegetable oils, but are not limited thereto. More preferably, suitable carriers can use Hanks solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, or isotonic solution (sterile water for injection, 10% ethanol, 40% propylene glycol and 5% glucose), etc. In order to protect the injection from contamination by microorganisms, a variety of antibacterial and antifungal agents may also be included, such as parabens, chlorobutanol, phenol, sorbic acid and thimerosal. In addition, in most cases, the injection may also contain sugar or sodium chloride isotonic agents.

[0093] Transdermal administration includes ointments, creams, emulsions, gels, external solutions, pastes, liniments and aerosols. As mentioned above, transdermal administration refers to the topical administration of a pharmaceutical composition to the skin, where an effective amount of active ingredients contained in the pharmaceutical composition is delivered to the skin.

[0094] The composition of the present invention can be administered in a pharmaceutically effective amount. A pharmaceutically effective amount refers to a sufficient amount for treating a disease at a reasonable benefit / risk ratio that is pharmaceutically suitable for treatment, and the effective dosage level can be determined according to the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the administration time, the route of administration, the excretion rate, the treatment time, the factors including the drugs used at the same time, and other factors known in the medical field. The composition of the present invention can be administered as a single therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered in a single dose or multiple doses. That is, the total effective amount of the composition of the present invention can be administered to the patient in a single dose (singledose), or in a fractionated treatment method (fractionated treatment protocol) of long-term administration of multiple doses (multipledose). Taking all of the above factors into account, it is important that the administration can achieve the maximum effect with the minimum amount and the amount without side effects, and this can be easily determined for those of ordinary skill in the art.

[0095] The dosage range of the pharmaceutical composition of the present invention may vary depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of the disease.

[0096] The composition of the present invention can be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy and methods using biological response modifiers.

[0097] The pharmaceutical composition of the present invention can also be provided in the form of an external preparation containing the compound represented by [Chemical Formula 1], its isomer or pharmaceutically acceptable salt as an active ingredient. 2A When the pharmaceutical composition for adenosine receptor (AR) and / or A3 adenosine receptor (AR)-related diseases is used as an external skin preparation, it may also contain adjuvants commonly used in the field of dermatology, such as fatty substances, organic solvents, solubilizers, concentrators and gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic emulsifiers, nonionic emulsifiers, fillers, metal ion chelators, chelating agents, preservatives, vitamins, retardants, wetting agents, essential oils, dyes, pigments, hydrophilic active agents, lipophilic active agents or lipid vesicles, which are the same as any other ingredients commonly used in external skin preparations. In addition, the ingredients can be introduced in an amount commonly used in the field of dermatology.

[0098] When the present invention is used for preventing or treating A 2AWhen the pharmaceutical composition for adenosine receptor (AR) and / or A3 adenosine receptor (AR)-related diseases is provided as a skin external preparation, its dosage form may be, but is not limited to, an ointment, a patch, a gel, a cream or a spray.

[0099] In addition, the present invention can provide a health functional food composition for preventing or improving obesity, wherein the health functional food composition comprises a compound represented by the following [Chemical Formula 1], an isomer thereof, or a pharmaceutically acceptable salt thereof:

[0100] [Chemical formula 1]

[0101]

[0102] R1 is H, C1-C10 alkyl or aryl-C1-C10 alkyl,

[0103] R2 is a C1-C10 alkyl group.

[0104] The compound represented by [Chemical Formula 1], its isomer or pharmaceutically acceptable salt is the same as that used in the pharmaceutical composition, and therefore its description is replaced by the above description.

[0105] The health functional food composition of the present invention can be prepared into various forms according to conventional methods known in the art. As for general food, although not limited to this, it can be prepared by adding the compound represented by [chemical formula 1] of the present invention, its isomer or pharmaceutically acceptable salt in beverages (including alcoholic beverages), fruits and processed foods thereof (for example: canned fruit, bottled canned food, jam, marmalade, etc.), fish, meat and processed foods thereof (for example: ham, sausage and corned beef, etc.), bread and noodles (for example: udon noodles, buckwheat noodles, instant noodles, Italian noodles, macaroni, etc.), fruit juice, various beverages, biscuits, starch syrups, dairy products (for example: butter, cheese, etc.), edible vegetable oils and fats, margarine, vegetable protein, packaged food, frozen food, various seasonings (for example: miso, soy sauce, sauce, etc.), etc., its isomer or pharmaceutically acceptable salt. In addition, nutritional supplements are not limited to this, but can be prepared by adding the compound represented by [chemical formula 1] of the present invention, its isomer or pharmaceutically acceptable salt in capsules, tablets, pills, etc. In addition, as for health functional foods, although not limited thereto, for example, the compound represented by [Chemical Formula 1] of the present invention, its isomer or pharmaceutically acceptable salt itself is made into the form of tea, juice, or beverage for drinking (health beverage), and can be taken by liquid phase, granulation, encapsulation, and powderization. In addition, in order to use the compound represented by [Chemical Formula 1] of the present invention, its isomer or pharmaceutically acceptable salt as a food additive, it can be prepared in the form of powder or concentrated liquid for use. In addition, the compound represented by [Chemical Formula 1] of the present invention, its isomer or pharmaceutically acceptable salt can be mixed with a known compound that is compatible with A 2A Active ingredients having preventive or improving effects on adenosine receptor (AR) and / or A3 adenosine receptor (AR)-related diseases are mixed together to prepare a composition.

[0106] When the compound represented by [Chemical Formula 1] of the present invention, its isomer or pharmaceutically acceptable salt is used as a health drink, the health drink composition may contain various sweeteners or natural carbohydrates as the same as conventional beverages as additional ingredients. The above-mentioned natural carbohydrates may be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; sugar alcohols such as xylitol, sorbitol and erythritol. Sweeteners may use natural sweeteners such as thaumatin and stevia extract; and synthetic sweeteners such as saccharin and aspartame.

[0107] In addition, the compound represented by [Chemical Formula 1] of the present invention, its isomer or pharmaceutically acceptable salt can be used as a method for preventing or improving A 2A The effective ingredients of the health functional food composition for adenosine receptor (AR) and / or A3 adenosine receptor (AR) related diseases include, and the content thereof is not particularly limited to, 2AThe effective amount of the compound represented by [Chemical Formula 1], its isomer or pharmaceutically acceptable salt thereof, and a known inhibitor of A3 adenosine receptor (AR) and / or an A3 adenosine receptor (AR)-related disease prevention or improvement effect is preferably 0.01 wt % to 100 wt % relative to the total weight of the entire composition. The health functional food composition of the present invention can be combined with a compound represented by [Chemical Formula 1], an isomer or a pharmaceutically acceptable salt thereof, and a known inhibitor of A3 adenosine receptor (AR)-related disease prevention or improvement effect. 2A The invention is prepared by mixing other active ingredients having effects on adenosine receptor (AR) and / or A3 adenosine receptor (AR)-related diseases.

[0108] In addition to the above, the health functional food of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectin acid, pectate, alginic acid, alginate, organic acid, protective colloid thickener, pH regulator, stabilizer, preservative, glycerol, alcohol or carbonating agent, etc. In addition, the health functional food of the present invention may contain pulp for preparing natural fruit juice, fruit juice beverage or vegetable beverage. Such ingredients can be used alone or in combination.

[0109] In addition, the present invention can provide a 2A A method for treating a disease associated with adenosine receptor (AR) and / or A3 adenosine receptor (AR), the method comprising the steps of: administering to adenosine receptor (AR) 2A A patient with adenosine receptor (AR) and / or A3 adenosine receptor (AR)-related diseases is administered a compound represented by the following [Chemical Formula 1], an isomer or a pharmaceutically acceptable salt thereof:

[0110] [Chemical formula 1]

[0111]

[0112] R1 is H, C1-C10 alkyl or aryl-C1-C10 alkyl,

[0113] R2 is a C1-C10 alkyl group.

[0114] The compound represented by [Chemical Formula 1], its isomer or pharmaceutically acceptable salt is the same as that used in the pharmaceutical composition, and therefore its description is replaced by the above description.

[0115] According to a preferred embodiment of the present invention, the 2A The adenosine receptor (AR) and / or A3 adenosine receptor (AR) related disease may be obesity.

[0116] In addition, the present invention can provide a compound represented by the following [Chemical Formula 1], an isomer or a pharmaceutically acceptable salt thereof in the preparation of A 2A Use in drugs for treating adenosine receptor (AR) and / or A3 adenosine receptor (AR) related diseases:

[0117] [Chemical formula 1]

[0118]

[0119] R1 is H, C1-C10 alkyl or aryl-C1-C10 alkyl,

[0120] R2 is a C1-C10 alkyl group.

[0121] The compound represented by [Chemical Formula 1], its isomer or pharmaceutically acceptable salt is the same as that used in the pharmaceutical composition, and therefore its description is replaced by the above description.

[0122] According to a preferred embodiment of the present invention, the 2A The adenosine receptor (AR) and / or A3 adenosine receptor (AR) related disease may be obesity.

[0123] Effects of the Invention

[0124] The ligand LJ-4378 and its derivatives acting on adenosine receptors of the present invention have the following properties: 2A The dual effects of AR agonist and A3AR antagonist significantly increased mitochondrial protein, UCP1 and COXIV, and showed excellent anti-obesity effect on high-fat diet mice. Therefore, the ligands acting on adenosine receptors of the present invention can be effectively used in the preparation of compositions, treatment methods and drugs for preventing, improving or treating obesity. BRIEF DESCRIPTION OF THE DRAWINGS

[0125] Figure 1a The synthesis process of LJ-4378 (Compound 6) is shown (a: Silylated 2-amino-6-chloropurine, TMSOTf, DCE, room temperature to 80°C for 3h; b: CuI, isoamyl nitrite, I2, CH2I2, THF, 110°C, 45 minutes; c: NH3 / MeOH, 80°C for 2h; d: 1-Hexyne, CuI, TEA, DMF, bis(triphenylphosphine)palladium dichloride, room temperature, 3h; e: 1N HCl, THF, room temperature, 15h).

[0126] Figure 1bThe synthesis process of LJ-4378 derivatives (Compounds 9a to 9j) is shown (a: 2-iodo-6-chloropurine, BSA, TMSOTf, CH3CN, room temperature to 80°C, 3h; b: propyne / butyne, CuI, Cs2CO3, Pd(PPh3)4, DMF, room temperature, 70 minutes; c: 1N HCl, THF, room temperature, 15h; d: NH3 / tBuOH, 100°C, 12h, or R1NH2, Et3N, EtOH, room temperature, 24-48h).

[0127] Figure 1c The drug profile of LJ-4378 is shown.

[0128] Figure 2 LJ-4378 treatment is shown to increase lipolysis and mitochondrial content in adipocytes. Part (a) shows the cytotoxicity evaluation of brown adipocytes (BA) treated with LJ-4378 for 24 hours under the indicated concentration conditions (n=5, mean ± SEM, ****p<0.0001). Part (b) shows immunoblot analysis of hormone-sensitive lipase (HSL) and phosphorylated HSL (p-HSL, serine 660) in BA treated with LJ-4378 for 4 hours (n=5, mean ± SEM, *p<0.05, ***p<0.001, ****p<0.0001). Part (c) shows the level of free fatty acids (FFA) in the conditioned medium of BA treated with 0.1 μM LJ-4378 for 24 hours (n=5, mean ± SEM, ****p<0.0001). Part (d) shows immunoblot analysis of BA treated with LJ-4378 for 48 hours at the indicated concentrations (n=5, mean ± SEM, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Part (e) shows the levels of mitochondrial proteins after treatment with LJ-4378 for 48 hours at the indicated concentrations (n=5, mean ± SEM, *p<0.05, ***p<0.001, ****p<0.0001).

[0129] Figure 3 The lipolysis effect of LJ-4378 is shown to be better than that of A 2A Agonists or A3 antagonists are stronger. Part (a) shows that the A1, A 2A , A 2BqPCR analysis of the expression levels of A3 adenosine receptors (n=5, mean±SEM, *p<0.05, **p<0.01, ***p<0.001). (b) and (c) show that the expression levels of A3 adenosine receptors were significantly higher in the presence of LJ-4378 (0.1 μM), A 2A Levels of glycerol and FFA in the conditioned medium of BA treated with an agonist (CGS21680, 0.1 μM) or an A3 antagonist (LJ-4433, 0.1 μM) for 24 hours (n=5, mean±SEM, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). 2A Immunoblot analysis of BA treated with agonist (CGS21680, 0.1 μM) or A3 antagonist (LJ-4433, 0.1 μM) for 24 h (n=5, mean±SEM, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0130] Figure 4 It is shown that in vitro LJ-4378 treatment increases mitochondrial content and activity in brown adipocytes. Part (a) shows immunoblot analysis of BA treated with 0.1 μM LJ-4378 for 24 hours (n=5, mean±SEM, ***p<0.001, ****p<0.0001). Part (b) shows representative images of mitochondrial fluorescent needle (MitoTracker) staining of BA treated with vehicle (CTL) and 0.1 μM LJ-4378 (n=5, size bar=20 μm). Part (c) shows OCR measurement of BA treated with 0.1 μM LJ-4378 for 24 hours (n=3, mean±SEM, *p<0.05, **p<0.01). Part (d) shows OCR measurement of BA treated with LJ-4378 (0.1 μM), A 2A Immunoblot analysis of BA treated with agonist (CGS21680, 0.1 μM) or A3 antagonist (LJ-4433, 0.1 μM) for 24 h (n=5, mean±SEM, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0131] Figure 5It is shown that in vivo LJ-4378 treatment increases mitochondrial activity and energy expenditure in adipose tissue. Part (a) shows 2,3,5-triphenyltetrazolium chloride (TTC) staining of BAT, iWAT and gWAT (n=5, mean ± SEM, **p<0.01, ***p<0.001, ****p<0.0001). Part (b) shows indirect calorimetric analysis of EE (energy expenditure), VO2 (oxygen consumption rate), VCO2 (carbon dioxide production rate), RER (respiratory exchange ratio), activity times and food consumption (FEED) (n=5, mean ± SEM, **p<0.01).

[0132] Figure 6 The browning effect of LJ-4378 based on the CIDEA reporter mouse study is shown. 2A Agonist (CGS21680, 1 mg kg-1 / day) or A3 antagonist (LJ-4433, 1 mg kg-1 / day) was treated for 10 days. (a) and (b) show representative bioluminescence at the dorsal and lateral positions (n ​​= 3, mean ± SEM, **p < 0.01, ****p < 0.0001). (c) shows representative in vitro fluorescence images of BAT and iWAT and quantification of fluorescence intensity (n = 3, mean ± SEM, **p < 0.01, ****p < 0.0001). (d) shows qPCR analysis of UCP1 and CIDE AmRNA expression levels in brown adipose tissue (BAT) (n = 3, mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001).

[0133] Figure 7 In vivo LJ-4378 treatment is shown to protect mice from obesity caused by a high-fat diet. Mice fed a normal diet (NCD) and a high-fat diet (HFD) were treated with LJ-4378 (LJ, 1 mg kg-1 / day) or a vehicle control group (CTL) for 10 days. Part (a) shows the change in body weight (n=6). Part (b) shows the composition of the obesity rate and the leanness rate (n=6, mean ± SEM, **p<0.01). Part (c) shows the tissue weight (mg) per g body weight of BAT, iWAT, and gWAT (n=6, mean ± SEM, *p<0.05, **p<0.01). Parts (d) and (e) show hematoxylin and eosin (H / E) staining of paraffin sections of BAT, iWAT, and gWAT (size bar = 10 μm). Part (f) shows the glucose tolerance test and the area under the curve (AUC) (n=5, mean±SEM, *p<0.05).

[0134] Figure 8 The anti-obesity effect of LJ-4378 is shown to include activation of the PKA signaling pathway in adipose tissue. Mice fed a normal diet (NCD) and a high-fat diet (HFD) were treated with LJ-4378 (LJ, 1 mg kg-1 / day) for 10 days. Part (a) shows immunoblot analysis of BAT (n=6, mean ± SEM, **p<0.01, ****p<0.0001). Part (b) shows immunoblot analysis in iWAT. Tubulin is a load control group (n=6, mean ± SEM, *p<0.05, ***p<0.001, ****p<0.0001). DETAILED DESCRIPTION

[0135] The present invention is described in more detail below by way of examples. These examples are only used to illustrate the present invention, and it is obvious to those skilled in the art that the scope of the present invention is not limited to these examples.

[0136] [Example 1]

[0137] <1-1> Cell culture

[0138] Adipocyte precursor cells that can differentiate into brown adipocytes are isolated from brown adipose tissue of mice. The cells are cultured in Dulbecco's modified Eagle's medium (Welgene, LM001-07) containing 10% fetal bovine serum (FBS, Gibco, 16000044) and 1% penicillin streptomycin (Welgene, LS202-02) at 37°C and in a humidified atmosphere of 5% CO2. When the cells reached about 100% confluency, for differentiation, the cells were exposed to differentiation medium supplemented with 2.5 mM isobutylmethylxanthine (IBMX, Cayman, I5879), 0.125 mM indomethacin (Cayman, 70270), 1 μM dexamethasone (Cayman), 1 μg / mL insulin (Sigma, I9278) and 1 nM triiodothyronine (T3, Cayman, 6028) for 3 days. Then, the cells were maintained in a growth medium containing 1 μg / mL insulin and 1 nM T3 (triiodothyronine).

[0139] <1-2> Animals

[0140] For in vivo experiments, 6-week-old C57BL / 6 male mice and CIDEA reporter mice were used according to protocols approved by the Animal Care Committee of Seoul National University (SNU-201107-1, SNU-201221-3). Mice were housed under 12h-light / 12h-dark cycle conditions and had free access to normal diet (NCD, Purina Lab, 38057, protein: 24.52% calories, carbohydrates: 63.07% calories, fat: 12.41% calories) and water at 22±1°C. LJ-4378 (1 mg kg-1 day-1) dissolved in 0.2% DMSO (diluted in PBS) was administered intraperitoneally for 10 days.

[0141] For the diet-induced obesity model, mice were fed a high-fat diet (HFD) (Research Diets, D12492, protein: 20% kcal, carbohydrate: 20% kcal, fat: 60% kcal) for 8 weeks, and then injected with LJ-4378 (1 mg kg-1) once a day for 10 days.

[0142] [Example 2]

[0143] Synthesis and overview of LJ-4378 and its derivatives

[0144] <2-1> Synthesis of LJ-4378

[0145] D-mannose was converted into a glycosyl donor (Compound 1) according to [Non-Patent Document 1]. The glycosyl donor (Compound 1) was condensed with 2-amino-6-chloropurine (2-amino-6-chloropurine) as a Lewis acid in the presence of TMS OTf to provide a single stereoisomer β-anomer (Compound 2, 30%) ( Figure 1a ). The anomer distribution is in the nucleus Overhauser effect between H-8 and 3'-H. 1It is easy to realize in H NMR experiment. In the presence of CuI, the 2-amino-6-chloro derivative (compound 2) is treated with isoamyl nitrite, iodine and diiodomethane to obtain a 2-iodo-6-chloro derivative (compound 3), and due to methanolic ammonia treatment, it is converted into a 2-iodo-6-amino derivative (compound 4). In the presence of bis(triphenylphosphine)palladium dichloride, the Sonogashira coupling reaction of compound 4 and 1-hexyne (1-hexyne) is used to obtain a 2-hexynyl derivative (compound 5). Finally, 1N HCl was used to remove the isopropylidene group of compound 5, and finally the 2-hexynyl-4'-thioadenosine derivative LJ-4378 (2-(6-amino-2-(hex-1-yn-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol (2-(6-amino-2-(hex-1-yn-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol), compound 6) was synthesized.

[0146] <2-2> Synthesis of LJ-4378 derivatives

[0147] D-mannose was converted into a glycosyl donor (compound 1) according to [Non-patent document 1]. The glycosyl donor (compound 1) as a Lewis acid was glycosylated (Vorbruggen condensed) with silylated 2-iodo-6-chloropurine (2-iodo-6-chloropurine) in the presence of TMSOTf to provide a trace amount of α-anomer and β-anomer (compound 3, 60%) ( Figure 1b ). The end group isomer type of compound 3 is 1 H NMR NOE experiments confirmed that a strong NOE effect was observed between H-8 and 3'-H of compound 3, confirming the β configuration, while the corresponding α-anomer did not show such NOE effect.

[0148] Next, in the presence of tetrakis(triphenylphosphine)palladium and cesium carbonate, using propyne and butyne, compound 3 was subjected to Sonogaoka coupling reaction to obtain C2-propynyl derivative (compound 7a) and C2-butynyl derivative (compound 7b), respectively. Compound 7a and compound 7b were treated with 1N HCl to generate 2-propynyl-6-chloro derivative (compound 8a) and 2-butynyl-6-chloro derivative (compound 8b), respectively. 2-propynyl-6-chloro derivative (compound 8a) was treated in tert-butyl alcohol containing ammonia and 3-halobenzylamine to obtain 2-propynyl-4'-thioadenosine derivative (compound 9a) and 2-propynyl-N6-3-halobenzyl-4'-thioadenosine derivative (compound 9b-e), respectively. Similarly, the 2-butynyl-6-chloro intermediate (Compound 8b) is converted into a 2-butynyl-4'-thioadenosine derivative (Compound 9f) and a 2-butynyl-N6-3-halogenated benzyl-4'-thioadenosine derivative (Compound 9g-j). The specific preparation methods are shown in the following [Preparation Method 1] and [Preparation Method 12].

[0149] [Preparation method 1]

[0150] (2R,3R,4S)-2-(6-Chloro-2-(prop-1-yn-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol (Compound 8a)

[0151] Tetrakis(triphenylphosphine)palladium (248mg, 0.21mmol), copper iodide (49mg, 0.25mmol) and cesium carbonate (700mg, 2.14mmol) were added to a stirred solution of compound 3 (943mg, 2.14mmol) in anhydrous N, N-dimethylformamide (25mL). The stirred reaction mixture was bubbled with propyne gas for 10 minutes at room temperature and further stirred for 1 hour at room temperature. The reaction mixture was evaporated under reduced pressure, and the crude product compound 7a was not further purified, but directly used for the next step of reaction. 1N HCl (5mL) was added to the stirred ice-cooled solution of the crude product compound 7a in tetrahydrofuran (5mL), and the mixture was stirred for 15 hours at room temperature. The reaction mixture was neutralized with 1N NaOH solution and evaporated carefully under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol=24:1) to give compound 8a (0.301 g, 45% from compound 3) as a white solid: mp 101-103° C.; UV (MeOH) λ max284 nm; 1 H NMR (500MHZ, CD3OD): δ8.85 (s, 1H), 6.08 (d, J=6.6Hz, 1H), 4.71-4.69 (m, 1H), 4.46 (q, J= 3.6Hz, 1H), 3.55 (dd, J=4.4, 11.0Hz, 1H), 2.96 (dd, J=3.4, 11.0Hz, 1H), 2.11 (s, 3H); [a] 20 D =-45.0 (c 0.10, MeOH); HRMS (FAB+): m / z calculated for C 12 H 12 ClN4O2S(M+H) + 311.0369, Actual measured value (Found): 311.0374. Theoretical value (Anal. Calcd for) C 12 H 12 ClN4O2S: C, 46.38; H, 3.57; N, 18.03. Actual measured value (Found): C, 46.78; H, 3.24; N, 18.04.

[0152] [Preparation method 2]

[0153] (2R,3R,4S)-2-(2-(But-1-yn-1-yl)-6-chloro-9H-purin-9-yl)tetrah ydrothiophene-3,4-diol (Compound 8b)

[0154] Tetrakis (triphenylphosphine) palladium (429mg, 0.37mmol), copper iodide (85mg, 0.44mmol) and cesium carbonate (1.21g, 3.71mmol) were added to the stirred solution of compound 3 (1.63g, 3.71mmol) in anhydrous N, N-dimethylformamide (25mL). The stirred reaction mixture was bubbled with butyne gas at room temperature for 10 minutes, and further stirred for 1 hour at room temperature. The reaction mixture was evaporated under reduced pressure, without further purification, and directly used in the next step reaction. 1NHCl (5mL) was added to the stirred ice-cooled solution of the crude product compound 7b in tetrahydrofuran (5mL), and the mixture was stirred for 15 hours at room temperature. The reaction mixture was neutralized with 1N NaOH solution, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol=24:1) to give compound 8b (530 mg, 44% from compound 3) as a white solid: mp 105-107° C.; UV (MeOH) λ max 284 nm; 1 H NMR (500MHz, CD3OD): δ8.86(s,1H),6.08(d,J=6.5Hz,1H),4.69(m,1H),3.54(dd,J=4.4,11.0Hz ,1H),3.30(bs,1H),2.96(dd,J=3.5,11.0Hz,1H),2.50(q,J=7.5Hz,2H),1.27(t,J=7.5Hz,3H); 13 C NMR (125MHz, CD3OD): δ154.2,151.7,148.7,147.8,132.6,93.4,81.6,80.5,75.1,65.3,36.1,14.3,14.2; [a] 20 D =-38.20 (c 0.10, MeOH); HRMS (FAB+): m / z calculated for (calculated for) C 13 H 14 ClN4O2S(M+H) + 325.0526, Actual measured value (Found): 325.0528. Theoretical value (Anal. Calcd for) C 13 H 14 ClN4O2S: C, 48.08; H, 4.03; N, 17.25. Actual measured value (Found): C, 48.09; H, 4.23; N, 17.01.

[0155] [Preparation method 3]

[0156] (2R,3R,4S)-2-(6-Amino-2-(prop-1-yn-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol (Compound 9a)

[0157] A solution of compound 8a (50 mg, 0.16 mmol) in NH3 / tBuOH (5 mL) was placed in a steel bomb and stirred at 100°C for 12 hours. The reaction mixture was evaporated and the residue was purified by silica gel column chromatography (dichloromethane / methanol = 24:1) to give 9a (45 mg, 85%) as a white solid: mp 247-249°C; UV (MeOH) λ max 271 nm; 1 H NMR (500MHz, DMSO-d6): δ8.46(s,1H),7.35(bs,2H),5.84(d,J=7.2Hz,1H),5.54(d,J=6.2Hz,1H),5.35(d,J=4.1Hz,1H),4 .60(dt,J=3.2,6.8Hz,1H),4.34-4.32(m,1H),3.40(dd,J=4.1,10.8Hz,1H),2.78(dd,J=2.8,10.8Hz,1H),2.01(s,3H); 13C NMR (125MHz, DMSO-d6): δ155.4,149.9,145.3,140.5,118.3,82.0,80.2,78.4,72.1,61.3,34.3,3.40; [a] 20 D =-38.0 (c 0.10, MeOH); HRMS (FAB+): m / z calculated for C 12 H 14 N5O2S(M+H) + 292.0868, actual measured value (Found): 292.0867. The analytical theoretical value is (Anal. Calcd for) C12H13N5O2S: C, 49.47; H, 4.50; N, 24.04. The actual measured value (Found): C, 49.29; H, 4.21; N, 23.98.

[0158] *General steps for the synthesis of compounds 9b to 9e

[0159] Triethylamine (3 equivalents) and 3-halogenated benzylamine (1.5 equivalents) were added to a stirred solution of diol compound 8a (50 mg, 0.16 mmol) in ethanol (5 mL) at room temperature, and the mixture was stirred at room temperature for 24 hours. The solvent was evaporated for 48 hours, and the residue was purified by silica gel column chromatography (dichloromethane / methanol=35:1) to obtain compounds 9b to 9e as white solids.

[0160] [Preparation method 4]

[0161] (2R,3R,4S)-2-(6-((3-Fluorobenzyl)amino)-2-(prop-1-yn-1-yl)-9H-purin-9-yl)tetrahydro thiophene-3,4-diol (Compound 9b)

[0162] Yield: 81%; white solid; mp 237-239°C; UV (MeOH) λ max 275nm; 1 H NMR (500MHz, DMSO-d6): δ8.52-8.45(m,2H),7.34(q,J=7.7Hz,1H),7.15-7.03(m,3H),5.86(d,J=7.2Hz,1H),5.55(d,J=6.2Hz,1H) ,5.36(d,J=4.0Hz,1H),4.68-4.61(m,3H),4.34(bs,1H),3.39(dd,J=3.9,10.7Hz,1H),2.80(dd,J=2.6,10.8Hz,1H),2.01(s,3H); 13 C NMR (125MHz, DMSO-d6): δ163.1,161.1,153.9,149.3,145.5,142.9,140.6,130 .2,122.9,118.7,113.5(CF),81.9,80.7,78.4,72.1,61.3,42.3,34.3,3.4;[a] 20 D =-32.0 (c 0.10, MeOH); HRMS (FAB+): m / z calculated for C 19 H 19 FN5O2S(M+H) +400.1244, Actual measurement value (Found): 400.1249. Theoretical value (Anal.Calcd for) C 19 H 18 FN5O2S: C, 57.13; H, 4.54; N, 17.53. Actual measured value (Found): C, 57.24; H, 4.94; N, 17.13.

[0163] [Preparation method 5]

[0164] (2R,3R,4S)-2-(6-((3-Chlorobenzyl)amino)-2-(prop-1-yn-1-yl)-9H-purin-9-yl)tetrahydro thiophene-3,4-diol (Compound 9c)

[0165] Yield: 78%; white solid; mp 224-226°C; UV (MeOH) λ max 275nm; 1 H NMR (500MHz, DMSO-d6): δ8.51(s,1H),8.47(bs,1H),7.37-7.28(m,4H),5.86(d,J=7.2Hz,1H),5.54(d,J=6.2Hz,1H),5.34(d,J =4.0Hz,1H),4.67(bs,2H),4.61(s,1H),4.34(s,1H),3.40(dd,J=3.9,10.7Hz,1H),2.80(dd,J=2.6,10.8Hz,1H),2.02(s,3H); 13 C NMR (125MHz, DMSO-d6): δ153.9,149.3,145.5,142.4,140.6,132.8,130.1,12 6.8,126.5,125.6,118.7,81.9,80.7,78.4,72.1,61.3,42.2,34.3,3.47;[a] 20 D =-42.0 (c 0.10, MeOH); HRMS (FAB+): m / z calculated for C 19 H 19 ClN5O2S(M+H) +416.0948, Actual measured value (Found): 416.0952. Theoretical value (Anal. Calcd for) C 19 H 18 ClN5O2S: C, 54.87; H, 4.36; N, 16.84. Actual measured value (Found): C, 54.76; H, 4.46; N, 16.45.

[0166] [Preparation method 6]

[0167] (2R,3R,4S)-2-(6-((3-Bromobenzyl)amino)-2-(prop-1-yn-1-yl)-9H-purin-9-yl)tetrahydro thiophene-3,4-diol (Compound 9d)

[0168] Yield: 79%; white solid; mp 240-242°C; UV (MeOH) λ max 275nm; 1 H NMR (500MHz, DMSO-d6): δ8.51(s,1H),8.47(bs,1H),7.52(s,1H),7.42(d,J=7.6Hz,1H)7.32-7.25(m,2H),5.86(d,J=7.2Hz,1H),5.53(d,J=6.2H z,1H),5.35(d,J=4.0Hz,1H),4.67(bs,2H),4.61(bs,1H),4.34(s,1H),3.40(dd,J=3.9,10.7Hz,1H),2.80(dd,J=2.6,10.8Hz,1H),2.02(s,3H); 13 C NMR (125MHz, DMSO-d6): δ153.9,149.3,145.5,142.7,140.6,130.4,129.7,12 9.5,126.1,121.5,118.7,81.9,80.7,78.4,72.1,61.3,42.1,34.3,3.45;[a] 20 D =1.40 (c 0.10, MeOH); HRMS (FAB+): m / z calculated for C 19 H 19 BrN5O2S(M+H) +462.0424, Actual measured value (Found): 462.0446. Theoretical value (Anal. Calcd for) C 19 H 18 BrN5O2S: C, 49.57; H, 3.94; N, 15.21. Actual measured value (Found): C, 49.17; H, 4.12; N, 15.43.

[0169] [Preparation method 7]

[0170] (2R,3R,4S)-2-(6-((3-Iodobenzyl)amino)-2-(prop-1-yn-1-yl)-9H-purin-9-yl)tetrahydro thiophene-3,4-diol (Compound 9e)

[0171] Yield: 77%; white solid; mp 253-255°C; UV (MeOH) λ max 275nm; 1 H NMR (500MHz, DMSO-d6): δ8.50(s,1H),8.45(bs,1H),7.71(s,1H),7.58(d,J =7.2Hz,1H),7.33(d,J=7.6Hz,1H),7.11(t,J=7.7Hz,1H),5.86(d,J=7.1Hz ,1H),5.54(d,J=6.1Hz,1H),5.34(d,J=3.8Hz,1H),4.63(bs,3H),4.34(s,1 H),3.41(dd,J=3.8,10.6Hz,1H),2.80(dd,J=2.6,10.8Hz,1H),2.02(s,3H); 13 C NMR (125MHz, DMSO-d6): δ153.9,149.3,145.5,142.5,140.6,135.7,135.4,1 30.4,126.5,118.7,94.7,81.9,80.7,78.4,72.1,61.3,40.0,34.3,3.4;[a] 20 D =107.0 (c 0.10, MeOH); HRMS (FAB+): m / z calculated for C 19 H 19 IN5O2S(M+H) +508.0304, Actual measured value (Found): 508.0312. Theoretical value (Anal. Calcd for) C 19 H 18 IN5O2S: C, 44.98; H, 3.58; N, 13.80. Actual measured value (Found): C, 45.13; H, 3.18; N, 14.12.

[0172] [Preparation method 8]

[0173] (2R,3R,4S)-2-(6-Amino-2-(but-1-yn-1-yl)-9H-purin-9-yl)tetra hydrothiophene-3,4-diol (Compound 9f)

[0174] Following the same procedure used in the preparation of compound 9a, compound 8b (50 mg, 0.15 mmol) was converted to white solid compound 9f (40 mg, 84%): mp 202-204°C; UV (MeOH) λ max 272 nm; 1 H NMR (500 MHz, DMSO-d6): δ8.47 (s, 1H), 7.36 (s, 1H), 5.86 (d, J = 7.2 Hz, 1H), 5.55 (d, J = 5.2 Hz, 1H), 5.37 (bs, 1H), 4.59-4.60 (m, 1H), 4.34 (bs, 1H), 3.41-3.35 (m, 2H; merged with water peak), 2.79 (dd, J = 2.4, 10.0 Hz, 1H), 2.40 (q, J = 7.5 Hz, 2H), 1.15 (t, J = 7.5 Hz, 3H); 13 C NMR (125MHz, DMSO-d6): δ155.6,149.9,145.7,140.4,118.2,86.7,80.6,78.5,72.1,61.1,34.3,13.3,11.9; [a] 20 D =90.00 (c 0.12, MeOH); HRMS (FAB+): m / z calculated for C 13 H 16 N5O2S(M+H) +306.1025, Actual measured value (Found): 306.1011. Theoretical value (Anal.Calcd for) C 13 H 15 N5O2S: C, 51.13; H, 4.95; N, 22.94. Actual measured value (Found): C, 51.42; H, 4.55; N, 23.10.

[0175] *General steps for the synthesis of compounds 9g to 9j

[0176] Triethylamine (3 equivalents) and 3-halogenated benzylamine (1.5 equivalents) were added to a stirred solution of diol compound 8b (50 mg, 0.15 mmol) in ethanol (5 mL) at room temperature, and the mixture was stirred at room temperature for 24 hours. The solvent was evaporated for 48 hours, and the residue was purified by silica gel column chromatography (dichloromethane / methanol=20:1) to obtain compounds 9g to 9j as white solids.

[0177] [Preparation method 9]

[0178] (2R,3R,4S)-2-(2-(But-1-yn-1-yl)-6-((3-fluorobenz yl)amino)-9H-purin-9-yl)tetrahydrothio-phene-3,4-diol (Compound 9g)

[0179] Yield: 80%; white solid; mp 230-231°C; UV (MeOH) λ max 275nm; 1 H NMR (500MHz, DMSO-d6): δ8.52(s,1H),8.46(bs,1H),7.35(q,J=7.7Hz,1H),7.16- 7.11(m,2H),7.06-7.02(m,1H),5.88(d,J=7.3Hz,1H),5.55(d,J=6.2Hz,1H),5.36 (d,J=3.8Hz,1H),4.69(bs,2H),4.60(bs,1H),4.34(s,1H),3.41(dd,J=3.9,10.7H z,1H),2.80(dd,J=2.6,10.8Hz,1H),2.40(q,J=7.5Hz,2H),1.15(t,J=7.5Hz,3H); 13C NMR (125MHz, DMSO-d6): δ163.1,161.2,153.9,149.4,145.6,142.9,140.5,130.1, 123.0,118.6,113.5(CF),86.9,80.9,78.5,72.1,61.1,42.3,34.3,13.2,11.9;[a] 20 D =-15.10 (c 0.10, MeOH); HRMS (FAB+): m / z calculated for C 20 H 21 FN5O2S(M+H) + 414.1400, Actual measurement value (Found): 414.1396. Theoretical value (Anal.Calcd for) C 20 H 20 FN5O2S: C, 58.10; H, 4.88; N, 16.94. Actual measured value (Found): C, 57.99; H, 5.12; N, 16.44.

[0180] [Preparation method 10]

[0181] (2R,3R,4S)-2-(2-(But-1-yn-1-yl)-6-((3-chlorobenz yl)amino)-9H-purin-9-yl)tetrahydrothio-phene-3,4-diol (Compound 9h)

[0182] Yield: 78%; white solid; mp 236-238°C; UV (MeOH) λ max 275nm; 1 H NMR (500MHz, DMSO-d6): δ8.52(s,1H),8.47(bs,1H),7.38-7.27(m,4H),5.88(d,J=7.2Hz,1H),5.56(d,J=6.1Hz,1H),5.37(d,J=3.5Hz,1H),4.6 8(bs,2H),4.60(s,1H),4.34(bs,1H),3.41(dd,J=3.8,10.6Hz,1H),2.80(dd,J=2.4,10.6Hz,1H),2.40(q,J=7.5Hz,2H),1.15(t,J=7.5Hz,3H); 13C NMR (125MHz, DMSO-d6): δ153.9,149.4,145.6,142.4,140.5,132.8,130.1,126. 9,126.6,125.7,118.6,86.9,80.8,78.5,72.1,61.1,42.2,34.3,13.2,11.9; [a] 20 D =-28.0 (c 0.10, MeOH); HRMS (FAB+): m / z calculated for C 20 H 21 ClN5O2S(M+H) + 430.1104, Actual measurement value (Found): 430.1111. Theoretical value (Anal.Calcd for) C 20 H 20 ClN5O2S: C, 55.88; H, 4.69; N, 16.29. Actual measured value (Found): C, 55.54H, 4.25; N, 16.29.

[0183] [Preparation method 11]

[0184] (2R,3R,4S)-2-(6-((3-Bromobenzyl)amino)-2-(but-1-yn-1-yl)-9H-purin-9-yl)tetrahydrothio-phene-3,4-diol (Compound 9i)

[0185] Yield: 77%; white solid; mp 222-224°C; UV (MeOH) λ max 275nm; 1H NMR (500MHz, DMSO-d6): δ8.52(s,1H),8.48(bs,1H),7.53(s,1H),7.42(d,J=7. 6Hz,1H),7.33-7.25(m,2H),5.87(d,J=5.2Hz,1H),5.54(d,J=5.2Hz,1H),5.36 (bs,1H),4.67(bs,1H),4.60(bs,2H),4.34(bs,1H),3.40(dd,J=3.9,10.7Hz,1 H), 2.80 (dd, J = 2.5, 10.7Hz, 1H), 2.41 (q, J = 7.4Hz, 2H), 1.16 (t, J = 7.5Hz, 3H); 13 C NMR (125MHz, DMSO-d6): δ153.9,149.4,145.6,142.7,140.5,130.4,129.8,129. 5,126.1,121.5,118.6,87.0,80.8,78.5,72.1,61.1,42.2,34.3,13.2,11.9;[a] 20 D =-9.90 (c 0.10, MeOH); HRMS (FAB+): m / z calculated for C 20 H 21 BrN5O2S(M+H) + 476.0580, Actual measured value (Found): 476.0594. Theoretical value (Anal. Calcd for) C 20 H 20 BrN5O2S: C, 50.64; H, 4.25; N, 14.76. Actual measured value (Found): C, 50.76; H, 4.32; N, 14.98.

[0186] [Preparation method 12]

[0187] (2R,3R,4S)-2-(2-(But-1-yn-1-yl)-6-((3-iodobenzyl)amino)-9H-purin-9-yl)tetrahydrothio-phene-3,4-diol (Compound 9j)

[0188] Yield: 77%; white solid; mp 239-241°C; UV (MeOH) λ max275nm; 1 H NMR (500MHz, DMSO-d6): δ8.52(s,1H),8.45(bs,1H),7.72(s,1H),7.58(d,J=7.6Hz,1 H),7.33(d,J=7.3Hz,1H),7.11(t,J=7.7Hz,1H),5.88(d,J=7.3Hz,1H),5.55(d,J=6. 1Hz,1H),5.36(d,J=3.9Hz,1H),4.63-4.60(m,3H),4.30(bs,1H),3.41(dd,J=3.9,10 .7Hz,1H),2.81(dd,J=2.7,10.8Hz,1H),2.41(q,J=7.4Hz,2H),1.16(t,J=7.5Hz,3H); 13 C NMR (125MHz, DMSO-d6): δ153.8,149.3,145.6,142.5,140.5,135.8,135.3,130. 4,126.5,118.6,94.6,86.9,80.8,78.5,72.1,61.1,42.1,34.3,13.2,11.9;[a] 20 D =-13.60 (c 0.10, MeOH); HRMS (FAB+): m / z calculated for C 20 H 21 IN5O2S(M+H) + 522.0461, Actual measured value (Found): 522.0468. Theoretical value (Anal. Calcd for) C 20 H 20 IN5O2S: C, 46.07; H, 3.87; N, 13.43. Actual measured value (Found): C, 46.23; H, 3.47; N, 13.95.

[0189] <2-1>Profile of LJ-4378 and its derivatives

[0190] LJ-4378 is a 2A AR and A3AR have excellent binding ability, which shows very high subtype selectivity. In addition, the most important A 2AThe results of the evaluation of AR cAMP activity showed EC50 and efficacy similar to those of the full agonist CGS21680. For A3AR, the results of the experiment using the A3AR full agonist Cl-IB-MECA showed that high concentrations of LJ-4378 acted as a full agonist that completely inhibited cAMP production, and the KB value showed high efficacy at the nM level ( Figure 1c ). That is, they act strongly on A as agonists and antagonists, respectively. 2A LJ-4378 of AR and A3AR can effectively increase the production of cAMP.

[0191] LJ-4378 and its derivatives 2A The binding affinities of hAR and A3AR are shown in Table 1 below. All binding affinity experiments were performed using adherent HEK293 cells stably transfected with cDNA encoding the appropriate hAR binding site and A3AR was tested. 2A The radioligands for A3AR and A3AR were 1 nM [3H]17, 10 nM [3H]18, or 0.2 nM [3H]16, respectively. The values ​​were normalized to the nonspecific binder 5'-N-ethylcarboxamidoadenosine (NECA, 10 μM).

[0192] Table 1

[0193]

[0194] [Example 3]

[0195] Capacity-dependent effects of LJ-4378 on lipolysis and mitochondrial content

[0196] <3-1> Cytotoxicity

[0197] LJ-4378 is a 2A A dual-acting ligand with AR agonist and A3AR antagonist activity. Cell viability was evaluated using EZ-CYTOX (DoGEN, EZ-3000) according to the manufacturer's instructions. The absorbance was measured at 450 nm using an ELISA reader (full wavelength ELISA reader (Thermo MULTISKAN GO), 8816-2015).

[0198] Cytotoxicity analysis of LJ-4378 showed that concentrations below 100 μM did not affect cell viability of brown adipocytes ( Figure 2(a)).

[0199] <3-2> Lipolysis effect of LJ-4378

[0200] Because A 2A AR increases cAMP levels and activates protein kinase A signaling in adipocytes, so the lipolytic effect of LJ-4378 was investigated in a dose-response relationship.

[0201] Table 2

[0202]

[0203] For protein immunoblotting, the primary antibodies used for protein immunoblotting are briefly shown in the table 2. Anti-rabbit horseradish peroxidase antibody (1:3000, Thermo Fisher, 31460) and anti-mouse horseradish peroxidase antibody (1:3000, Jackson, 115-035-174) are used as secondary antibodies. All antibodies are diluted in blocking buffer (TBST containing 5% skim milk or 5% bovine serum albumin). Fusion Solo chemiluminescent imaging system (Vilber Lourmat) is used to obtain protein immunoblotting images, and EvolutionCapt software (version 17.03) is used for analysis. NIH ImageJ software is used for quantification.

[0204] The free fatty acid (FFA) level in the culture medium was measured using NEFA reagent (WAKO, 436-91693) according to the manufacturer's protocol.

[0205] Immunoblotting analysis showed that LJ-4378 upregulated the expression level of phosphorylated hormone-sensitive lipase (P-HSL) at serine 660 in a dose-dependent manner ( Figure 2 Part (c): Increase (10 -11 M) 1.55 ± 0.11 times; (10 -9 M) 3.03 ± 0.21 times, (10 -7 )6.64±0.34 times, (10 -5 )6.87±0.69-fold). Consistently, free fatty acid (FFA) levels were also increased by LJ-4378 ( Figure 2 Part (d): increased by 1.92±0.03 times).

[0206] Furthermore, mitochondrial protein increased according to the dose of LJ-4378 ( Figure 2Part (e): ATP5A (ATP synthase alpha-subunit): increased (10 -11 M) 2.20 ± 0.08 times, (10 - 9M)3.51±0.05 times, (10 -7 )3.94±0.06, (10 -5 )3.72±0.04 times, ubiquinol-cytochrome c reductase core protein Ⅱ (UQCRC2): increased (10 -11 M) 2.15 ± 0.03 times, (10 -9 M)3.96±0.(10 -7 )4.21±0.1 times, (10 -5 )4.02±0.08 times, succinate dehydrogenase complex iron sulfur subunit B (SDHB): increased (10 -11 M) 1.87 ± 0.28 times, (10 -9 M) 3.47 ± 0.18 times, (10 -7 )3.41±0.47 times, (10 -5 )3.71±0.25 times, MCAD: increased (10 -11 M) 1.53 ± 0.03 times, (10 -9 M) 1.79 ± 0.03 times, (10 -7 )1.79±0.09 times, (10 -5 Based on this dose-response evaluation, a concentration of 0.1 μM was used in subsequent experiments.

[0207] [Example 4]

[0208] LJ-4378 and A 2A Comparison of AR agonists or A3AR antagonists

[0209] <4-1> Confirmation of adenosine receptor expression levels

[0210] The expression level of adenosine receptors in differentiated brown adipocytes from immortalized brown adipocyte precursor cells was investigated by qPCR analysis. The primers used in the qPCR analysis are listed in Table 3 below.

[0211] Table 3

[0212]

[0213]

[0214] A 2A AR showed the highest expression level ( Figure 3 (a): 343.7±68.04), A3AR( Figure 3 Part (a): 0.19 ± 0.03) had the second highest level among the adenosine receptors investigated.

[0215] <4-2>LJ-4378, A 2A Comparison of the lipolytic effects of AR agonists or A3AR antagonists

[0216] The lipolysis effect of LJ-4378 was compared with that of A 2A CGS21680 (Cayman Company, 124431-80-7) and LJ-4433, which are AR agonists and A3AR antagonists, were compared.

[0217] Glycerol and free fatty acid (FFA) levels in the culture medium were measured using glycerol reagent (Sigma Aldrich, F6428) and NEFA reagent (WAKO, 436-91693) according to the manufacturer's protocols.

[0218] In the analysis of glycerol and FFA, it is comparable to CGS21680 ( Figure 3 Part (b): 1.51±0.08 times, Figure 3 (c): 1.29±0.09 times) and LJ-4433 ( Figure 3 Part (b): 1.22±0.07 times, Figure 3 Compared with (c): 0.92±0.09 times), LJ-4378 treatment showed a maximum fold change of 1.61±0.07 and 1.59±0.1 ( Figure 3 Part (b) of Figure 3 The additional effect of LJ-4378 as described above was further confirmed by immunoblot analysis of up-regulated levels of PKA downstream proteins including phosphorylated cAMP response element binding protein (P-CREB) (increased by 2.32 ± 0.05-fold by LJ-4378; 2.00 ± 0.11-fold by CGS21680; and 1.40 ± 0.11-fold by LJ-4433) and P-HSL (increased by 2.10 ± 0.12-fold by LJ-4378; 1.54 ± 0.05-fold by CGS21680; and 1.22 ± 0.07-fold by LJ-4433). Figure 3 (d) of the Regulations.

[0219] [Example 5]

[0220] Effects of LJ-4378 on brown adipocytes

[0221] The effects of LJ-4378 on mitochondrial content and metabolic activation energy in brown adipocytes were evaluated.

[0222] Oxygen consumption rate (OCR) was measured using an XFp analyzer. Cells were cultured in XF DMEM basal medium (Agilent, 103575-100, pH 7.4) supplemented with 4 mM L-glutamine (Sigma, G8540) and 25 mM D-glucose (Sigma, G7021) at 37°C. XFp Cell Mitochondrial Stress Test Kit (Agilent, 103010-100) was prepared and calculated with optimal final concentrations of 2.5 μM oligomycin, 0.5 μM FCCP, and 0.5 μM rotenone / antimycin A, and basal, maximum, and proton leak. OCR was then normalized to protein concentration.

[0223] At 37°C, cells were exposed to the mitochondrial red fluorescent probe (MitoTracker TM RedCMXRos) (1:3000, Invitrogen, M7512), and after staining the mitochondria, they were fixed with 4% paraformaldehyde (PFA, Sigma, 158127). Images were acquired using a LSM800 confocal microscope (Zeiss, Germany) and analyzed using Zen software (version 3.0).

[0224] The expression levels of brown adipocyte marker UCP1 and mitochondrial protein COXIV were significantly increased by 2.93±0.23-fold and 2.18±0.15-fold, respectively, after LJ-4378 treatment. Figure 4 MitoTracker staining also showed that LJ-4378 increased mitochondrial membrane potential by 2.12±0.06 times in brown adipocytes ( Figure 4 In addition, the inventors compared these effects of LJ-4378 on mitochondrial content with A 2A AR agonist (CGS21680) and A3AR antagonist (LJ-4433) were compared. Both CGS21680 and LJ-4433 increased UCP1, COXIV and MCAD ( Figure 4 However, a higher effect was observed in LJ-4378 ( Figure 4 (d): 2.67±0.38-fold increase in UCP1, 2.53±0.08-fold increase in COXIV, and 4.91±0.02-fold increase in MCAD), indicating an additive effect.

[0225] Investigation of oxygen consumption rate for functional analysis showed that LJ-4378 increased OCR in relation to basal (30.5%), maximal (49.1%), and ATP production (29.3%). Figure 4 (c) of the Regulations).

[0226] [Example 6]

[0227] Effects of LJ-4378 on mitochondrial activity of adipose tissue and energy expenditure in vivo

[0228] In vitro electron transport activity associated with mitochondrial oxidative phosphorylation was evaluated by monitoring the reduction of 0.1% triphenyltetrazolium chloride (TTC, Sigma, T8877).

[0229] Indirect calorimetry was performed using the PhenoMaster (TSE Systems) to measure energy expenditure (EE), VO2, VCO2, activity, and dietary intake. Body composition was measured by magnetic resonance imaging (EchoMRI-700) (Echo Medical Systems).

[0230] In situ staining using triphenyltetrazolium chloride (TTC) for redox indices showed that LJ-4378 upregulated mitochondrial activity in all adipose tissue depots ( Figure 5 Part (a): 2.86±0.24-fold increase in BAT, 2.07±0.17-fold increase in iWAT, and 1.72±0.16-fold increase in gWAT).

[0231] Indirect calorimetry analysis showed that LJ-4378 treatment increased oxygen consumption by 17.8%, thereby increasing energy expenditure by 17.0% ( Figure 5 (b) of the Regulations).

[0232] [Example 7]

[0233] Effects of LJ-4378 on browning of adipose tissue in vivo

[0234] The synergistic effect of LJ-4378 on adipose tissue browning was investigated in CIDEA reporter mice, which reflect the expression of CIDEA, a marker of brown adipocytes, by fluorescent and luminescent signals.

[0235] In vivo bioluminescence was detected by using an optical imaging device (Ami-X, Spectral Instruments Imaging). In order to detect the in vivo bioluminescent signal, mice were injected intraperitoneally with D-luciferin (150 mg kg-1, Goldbio). Aura software (Software) (Spectral Instruments Imaging, version 2.2.1.1) was used for quantification. In addition, ex vivo imaging tissues were collected from CIDEA reporter mice treated with D-luciferin (150 mg kg-1, Goldbio, ip). During imaging, the separated tissues were maintained in 12-well plates containing D-luciferin in PBS (300 μg / ml).

[0236] LJ-4378 significantly increased the bioluminescence intensity of BAT and iWAT ( Figure 6 Part (a) and Figure 6 Part (b) of Figure 2). In particular, compared with CGS21680 and LJ-4433, iWAT showed a significant increase, and in particular, LJ-4378 showed an additional effect on browning in white adipose tissue. According to the in vivo bioluminescence results, among the in vitro luminescence signals in BAT and iWAT, LJ-4378 treatment resulted in the most significant increase ( Figure 6 (c) of the Regulations).

[0237] Investigation of mRNA levels of brown adipocyte markers showed that LJ-4378 brought about the following consistent results: the expression of genes associated with browning including Ucp1 (increased 2.13±0.21-fold by LJ-4378, 1.72±0.17-fold by CGS21680, and 1.74±0.08-fold by LJ-4433) and Cidea (increased 1.72±0.06-fold by LJ-4378, 1.29±0.12-fold by CGS21680, and 1.12±0.04-fold by LJ-4433) were increased the most.

[0238] [Example 8]

[0239] Anti-obesity effect of LJ-4378

[0240] We investigated whether the lipolysis and browning effects of LJ-4378 have anti-obesity effects.

[0241] Mice fed with HFD were used as a model of diet-induced obesity. Body weight changes were monitored during treatment, and fat and body composition of mice were measured after treatment.

[0242] For histological analysis, adipose tissue was fixed with 10% formalin and embedded in paraffin blocks. Paraffin sections were stained with hematoxylin / eosin (H&E).

[0243] For glucose tolerance test (GTT), 20% D-glucose (1 gkg-1, Sigma Aldrich, G7021) was injected intraperitoneally into mice. Blood glucose meter A blood glucose meter (GlucoDr. Top, allmedicus, AGM-4100) was used to measure blood glucose concentration.

[0244] Mice treated with LJ-4378 and fed a HFD had an 8.24% reduction in body weight and a 24.18% reduction in fat mass ( Figure 7 Part (a) and Figure 7 In addition, in HFD-fed mice, the weights of iWAT and gWAT were significantly reduced by LJ-4378 treatment ( Figure 7 (c): 30.1% reduction in iWAT and 18.6% reduction in gWAT). The reduced adipocyte size in iWAT and gWAT was further supported by H&E staining ( Figure 7 Part (d) of Figure 7 In addition, LJ-4378 improved glucose tolerance in mice fed a HFD ( Figure 7 (f) of the Regulations).

[0245] The present inventors then investigated the expression levels of mitochondrial proteins and PKA downstream markers to confirm whether these anti-obesity and anti-diabetic effects were caused by increased mitochondrial content and activation of PKA signaling. The results showed that LJ-4378 upregulated COXIV (BAT: 5.91 ± 0.23 times, iWAT: 1.24 ± 0.04 times), UCP1 (BAT: 1.33 ± 0.06 times, iWAT: 3.34 ± 0.36 times), P-CREB (BAT: 1.54 ± 0.03 times, iWAT: 3.86 ± 0.12 times), and P-HSL (BAT: 2.31 ± 0.18 times, iWAT: 1.72 ± 0.04 times) in BAT and iWAT of diet-induced obese mice ( Figure 8 ).

[0246] [Data and statistical analysis]

[0247] Statistical analysis was performed using Prism7 software (GraphPad Software, USA). Data are presented as mean ± SEM. Unpaired t-test was used to determine statistical significance between two groups.

[0248] Industrial Applicability

[0249] The ligand LJ-4378 and its derivatives acting on adenosine receptors of the present invention are used as A 2A The AR agonist and A3AR antagonist exert dual effects, significantly increasing mitochondrial protein, UCP1 and COXIV, respectively, and exhibiting excellent anti-obesity effects on mice fed a high-fat diet. Therefore, the ligand acting on adenosine receptors of the present invention can be effectively used as a composition for preventing, improving or treating obesity, and therefore has industrial applicability.

[0250] Sequence Listing Directory

[0251] SEQ ID NO. 1 is a forward primer used in qPCR analysis of Ucp1.

[0252] SEQ ID NO. 2 is a reverse primer used in qPCR analysis of Ucp1.

[0253] SEQ ID NO. 3 is a forward primer used in qPCR analysis of Cidea.

[0254] SEQ ID NO. 4 is a reverse primer used in qPCR analysis of Cidea.

[0255] SEQ ID NO. 5 is a forward primer used in qPCR analysis of Ppia.

[0256] SEQ ID NO. 6 is a reverse primer used in qPCR analysis of Ppia.

[0257] SEQ ID NO. 7 is a forward primer used in qPCR analysis of Adora1.

[0258] SEQ ID NO. 8 is a reverse primer used in qPCR analysis of Adora1.

[0259] SEQ ID NO. 9 is a forward primer used in qPCR analysis of Adora2a.

[0260] SEQ ID NO. 10 is a reverse primer used in qPCR analysis of Adora2a.

[0261] SEQ ID NO. 11 is a forward primer used in qPCR analysis of Adora2b.

[0262] SEQ ID NO. 12 is a reverse primer used in qPCR analysis of Adora2b.

[0263] SEQ ID NO. 13 is a forward primer used in qPCR analysis of Adora3. SEQ ID NO. 14 is a reverse primer used in qPCR analysis of Adora3.

Claims

1. A pharmaceutical composition for preventing or treating 2A Adenosine receptor and / or A3 adenosine receptor related disease, characterized in that Contains a compound represented by the following [Chemical Formula 1], an isomer or a pharmaceutically acceptable salt thereof: [Chemical formula 1] R1 is H, C1-C10 alkyl or aryl-C1-C10 alkyl, R2 is a C1-C10 alkyl group.

2. The pharmaceutical composition according to claim 1, characterized in that R1 is H, methyl or aryl-C1-C10 alkyl, R2 is a C1-C4 alkyl group.

3. The pharmaceutical composition according to claim 1, characterized in that The compound represented by [Chemical Formula 1], its isomer or pharmaceutically acceptable salt is selected from one or more of the group consisting of the following compounds: (1) 2-(6-amino-2-(hex-1-yn-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol; (2) (2R,3R,4S)-2-(6-amino-2-(prop-1-yn-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol; (3) (2R,3R,4S)-2-(6-((3-fluorobenzyl)amino)-2-(prop-1-yn-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol; (4) (2R,3R,4S)-2-(6-((3-chlorobenzyl)amino)-2-(prop-1-yn-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol; (5) (2R,3R,4S)-2-(6-((3-bromobenzyl)amino)-2-(prop-1-yn-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol; (6) (2R,3R,4S)-2-(6-((3-iodobenzyl)amino)-2-(prop-1-yn-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol; (7) (2R,3R,4S)-2-(6-amino-2-(but-1-yn-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol; (8) (2R,3R,4S)-2-(2-(but-1-yn-1-yl)-6-((3-fluorobenzyl)amino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol; (9) (2R,3R,4S)-2-(2-(but-1-yn-1-yl)-6-((3-chlorobenzyl)amino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol; (10) (2R,3R,4S)-2-(6-((3-bromobenzyl)amino)-2-(but-1-yn-1-yl)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol; and (11) (2R,3R,4S)-2-(2-(But-1-yn-1-yl)-6-((3-iodobenzyl)amino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol.

4. The pharmaceutical composition according to claim 1, characterized in that The compound represented by [Chemical Formula 1], its isomer or pharmaceutically acceptable salt is A 2A It is an agonist of adenosine receptors and an antagonist of A3 adenosine receptors.

5. The pharmaceutical composition according to claim 1, characterized in that Said and A 2A The adenosine receptor and / or A3 adenosine receptor related disease is obesity.

6. A health functional food composition for preventing or improving obesity, characterized in that: Contains a compound represented by the following [Chemical Formula 1], an isomer or a pharmaceutically acceptable salt thereof: [Chemical formula 1] R1 is H, C1-C10 alkyl or aryl-C1-C10 alkyl, R2 is a C1-C10 alkyl group.

7. A and A 2A Methods for treating diseases related to adenosine receptors and / or A3 adenosine receptors, It is characterized in that The following steps are involved: Xiang and A 2A A patient suffering from a disease related to adenosine receptor and / or A3 adenosine receptor is administered a compound represented by the following [Chemical Formula 1], an isomer or a pharmaceutically acceptable salt thereof, [Chemical formula 1] R1 is H, C1-C10 alkyl or aryl-C1-C10 alkyl, R2 is a C1-C10 alkyl group.

8. A compound, an isomer or a pharmaceutically acceptable salt thereof in the preparation of 2A Use of the invention in a drug for treating adenosine receptor and / or A3 adenosine receptor related diseases, characterized in that: The compound is represented by the following [Chemical Formula 1], [Chemical formula 1] R1 is H, C1-C10 alkyl or aryl-C1-C10 alkyl, R2 is a C1-C10 alkyl group.

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