Melanocortin-4 receptor agonist

Abstract

To provide a compound which has excellent selective agonist activity against melanocortin receptors, specifically melanocortin-4 receptors (MC4R).SOLUTION: The invention provides a compound represented by Formula (1), or a pharmaceutically acceptable salt or isomer thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a compound that exhibits excellent agonist activity against a melanocortin receptor. More specifically, the compound is represented by the following formula (1): [ka] (wherein R1, R2, R3, R4 and n are as defined in the present specification), a pharmaceutical composition containing the same as an active ingredient, and use thereof. The compound of the present invention exhibits excellent agonist activity against the melanocortin-4 receptor and is particularly useful for the prevention or treatment of obesity, diabetes, inflammation, and erectile dysfunction. [Background technology]

[0002] Leptin is a hormone secreted by fat cells (adipocytes), and its secretion increases as body fat percentage increases. Leptin regulates various physiological functions, including appetite, body fat mass, and energy metabolism, by regulating the functions of various neuropeptides produced in the hypothalamus (Non-Patent Document 1). Leptin signaling for appetite and body weight control is carried out postnatally through the activation of many downstream factors, the most representative of which are melanocortin, AgRP (agouti-related protein), and neuropeptide Y (NPY) hormone.

[0003] When blood leptin levels increase due to excessive caloric intake in the body, the pituitary gland increases the secretion of the proopiomelanocortin (POMC) protein hormone and decreases the production of AgRP and NPY. The small peptide hormone α-MSH (melanocyte-stimulating hormone) is produced by POMC neurons. This hormone acts as a melanocortin-4 receptor (MC4R) agonist on second-order neurons, ultimately inducing appetite reduction. On the other hand, when leptin levels decrease due to a caloric deficit, the expression of AgRP, an MC4R antagonist, increases, as does the expression of NPY, ultimately increasing appetite. Thus, in response to changes in leptin, the hormones α-MSH and AgRP are involved in appetite regulation as MC4R agonists and antagonists.

[0004] The α-MSH hormone induces various physiological responses by binding to three MCR subtypes in addition to MC4R. Five MCR subtypes have been identified to date. Of these subtypes, MC1R is primarily expressed in skin cells and is involved in melanin pigmentation. MC2R is primarily expressed in the adrenal gland and is involved in the production of glucocorticoid hormones. Its only ligand is known to be ACTH (adrenocorticotropic hormone) derived from POMC. MC3R and MC4R, primarily expressed in the central nervous system, are involved in regulating appetite, energy metabolism, and the efficiency of fat accumulation in the body. MC5R, expressed in various tissues, is known to regulate exocrine function (Non-Patent Document 2). Specifically, activation of the MC4R receptor effectively reduces weight by reducing appetite and increasing energy metabolism, and has been proven to be a key target of action in the development of obesity treatments (Non-Patent Documents 2, 3, 4, 5).

[0005] The role of MC4R in appetite and weight control was primarily established through experiments using an animal model (agouti mice) that overexpresses agouti protein. In agouti mice, a genetic mutation results in high levels of agouti protein in the central nervous system, where it acts as an agonist of MC4R in the hypothalamus, inducing obesity (Non-Patent Documents 6 and 7). Subsequent studies have revealed that Agouti-related protein (AgRP), which resembles the actual agouti protein, is expressed in hypothalamic neurons. AgRP is also known to be involved in appetite regulation as an antagonist of MC4R (Non-Patent Documents 8 and 9).

[0006] In vivo, intracerebral administration of the MC4R agonist α-MSH in animals reduced appetite, and subsequent administration of the MC4R antagonists SHU9119 (peptide) or HS014 (peptide) was observed to stimulate appetite (Non-Patent Document 10). Furthermore, animal studies using melanotan II (MTII, Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2) and its related agonist HP228 demonstrated appetite suppression, weight loss, and increased energy metabolism following intracerebral, intraperitoneal, or subcutaneous administration (Non-Patent Documents 11, 12, 13). Meanwhile, administration of the representative MC4R agonist SHU9119 in animals resulted in significant and sustained increases in food intake and weight gain, providing pharmacological evidence that MCR agonists can be used to treat obesity. The appetite-reducing effect that is clearly observed when MTII is administered is not observed in MC4RKO (knockout) mice, and these experimental results further demonstrated that the appetite-reducing effect is achieved mainly through activation of MC4R (Non-Patent Document 14).

[0007] The majority of obesity treatments developed to date are appetite suppressants that act on the central nervous system, many of which are drugs that modulate the action of neurotransmitters. Examples include noradrenergic agents (phentermine and mazindol) and serotonergic agents such as fluoxetine and sibutramine. However, these neurotransmitter modulators exert a wide range of effects on various physiological functions in addition to suppressing appetite through multiple subtype receptors. Therefore, these modulators have the major drawback of poor selectivity for each type of sweet and sour pork, and various side effects can occur when administered over a long period of time.

[0008] On the other hand, considering that melanocortin is a neuropeptide, not a neurotransmitter, and that MC4R gene KO mice have normal functions other than energy metabolism, melanocortin agonists have the advantage of being able to induce weight loss by suppressing appetite without affecting other physiological functions. In particular, the receptor is a G-protein-coupled receptor (GPCR), which distinguishes them from conventional receptors in that it is relatively easy to ensure selectivity for subtype receptors.

[0009] As examples of utilizing such melanocortin receptors as the site of action, Patent Documents 1 and 2 disclose compounds that act as melanocortin receptor agonists. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] WO 2008 / 007930 [Patent Document 2] WO 2010 / 056022 [Non-patent literature]

[0011] [Non-Patent Document 1] Schwartz, et al., Nature 404, 661-671 (2000) [Non-licensed document 2] Wikberg, et al., Pharm Res 42 (5) 393-420 (2000) [Non-licensed document 3] Wikberg, Eur. J. Pharmacol 375, 295-310 (1999)

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

[0012] An object of the present invention is to provide a novel compound represented by formula (1), or a pharmaceutically acceptable salt or isomer thereof, which has excellent selective agonist activity against melanocortin receptors, particularly against the melanocortin-4 receptor (MC4R).

[0013] Another object of the present invention is to provide a method for producing the compound represented by the formula (1).

[0014] Yet another object of the present invention is to provide a melanocortin receptor agonist pharmaceutical composition comprising the compound represented by the above formula (1) or a pharmaceutically acceptable salt or isomer thereof as an active ingredient.

[0015] Yet another object of the present invention is to provide use of the compound represented by formula (1) or a pharmaceutically acceptable salt or isomer thereof in the prevention or treatment of obesity, diabetes, inflammation and erectile dysfunction. [Means for solving the problem]

[0016] In order to achieve the above object, the present invention provides a compound represented by the following formula (1): [ka] (Wherein R1 is C2-C5 alkyl, R2 is a halo; R3 is hydrogen or halo; R4 is C1-C3 alkyl; n is an integer of 1 or 2, with the proviso that when R2 is chlorine and R3 is hydrogen, then n is 2.) or a pharmaceutically acceptable salt or isomer thereof.

[0017] The compounds of formula (1) according to the present invention can form pharmaceutically acceptable salts.

[0018] Additionally, compounds according to the present invention may have asymmetric carbon centers and asymmetric axes or planes and may therefore exist as cis or trans isomers, R or S isomers, racemates, diastereomeric mixtures, and individual diastereomers, and all of these isomers and mixtures are included within the scope of the present invention.

[0019] In this specification, unless otherwise specified, the compound of formula (1) is used to mean the compound of formula (1) and all of its pharmaceutically acceptable salts and isomers.

[0020] The term "halo" or "halogen" as used herein means a radical of fluorine (F), chlorine (Cl), bromine (Br), or iodine (I).

[0021] As used herein, the term "alkyl" means a straight-chain or branched hydrocarbon group.

[0022] In one embodiment according to the present invention, R1 in formula (1) is C2-C4 alkyl. In another embodiment according to the present invention, R1 in formula (1) is linear or branched C2-C4 alkyl, such as ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl.

[0023] In another embodiment according to the present invention, R1 in formula (1) is C3 or C4 alkyl. In another embodiment according to the present invention, R1 in formula (1) is branched C3 or C4 alkyl, for example, isopropyl or tert-butyl.

[0024] In another embodiment according to the present invention, the compound of formula (1) is represented by the following formula (2): [ka] N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)pivalamide represented by the formula:

[0025] In another embodiment of the present invention, the compound of formula (1) is represented by the following formula (3): [ka] N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide represented by the formula:

[0026] In another embodiment of the present invention, the compound of formula (1) is represented by the following formula (4): [ka] N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide represented by the formula:

[0027] In another embodiment of the present invention, the compound of formula (1) is represented by the following formula (5): [ka] N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)pivalamide represented by the formula:

[0028] In another embodiment of the present invention, the compound of formula (1) is represented by the following formula (6): [ka] The compound is N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-(4,4-dimethylcyclohexyl)isobutyramide, represented by the formula:

[0029] In another embodiment of the present invention, the compound of formula (1) is represented by the following formula (7): [ka] The compound is N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-(4,4-dimethylcyclohexyl)pivalamide.

[0030] In another embodiment according to the present invention, the pharmaceutically acceptable salt includes, but is not limited to, an acid addition salt formed with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, etc.; organic carboxylic acids such as tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, etc.; sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, or naphthalenesulfonic acid, etc.

[0031] In another embodiment according to the present invention, said pharmaceutically acceptable salt is a hydrochloride salt.

[0032] In another embodiment according to the present invention, the compound of formula (1) is represented by the following formula (8): [ka] N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)pivalamide hydrochloride, represented by the formula:

[0033] In another embodiment according to the present invention, the compound of formula (1) is represented by the following formula (9): [ka] N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide hydrochloride, represented by the formula:

[0034] In another embodiment of the present invention, the compound of formula (1) is represented by the following formula (10): [ka] N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide hydrochloride, represented by the formula:

[0035] In another embodiment according to the present invention, the compound of formula (1) is represented by the following formula (11): [ka] N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)pivalamide hydrochloride, represented by the formula:

[0036] In another embodiment according to the present invention, the compound of formula (1) is represented by the following formula (12): [ka] The compound is N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-(4,4-dimethylcyclohexyl)isobutyramide hydrochloride, represented by the formula:

[0037] In another embodiment according to the present invention, the compound of formula (1) is represented by the following formula (13): [ka] N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-(4,4-dimethylcyclohexyl)pivalamide hydrochloride, represented by the formula:

[0038] In another embodiment of the present invention, there is provided a compound represented by the formula (8): N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)pivalamide hydrochloride

[0039] N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide hydrochloride represented by the formula (9),

[0040] N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide hydrochloride of the formula (1)0,

[0041] N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)pivalamide hydrochloride of the formula (11) can be produced according to the following reaction scheme 1. <Reaction Scheme 1> [ka] (In the formula, R1, R2, and R3 are defined as above.) In another embodiment of the present invention, there is provided a compound according to the present invention, wherein N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-(4,4-dimethylcyclohexyl)isobutyramide hydrochloride of formula (12) is

[0042] N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-(4,4-dimethylcyclohexyl)pivalamide hydrochloride of the formula (13) can be produced according to the following reaction scheme 2. <Reaction Scheme 2> [ka] (R1 has the same meaning as defined above.)

[0043] The compound of formula (1) according to the present invention exhibits excellent agonistic activity against melanocortin receptors, particularly against the melanocortin-4 receptor (MC4R). Therefore, the present invention also provides a pharmaceutical composition for melanocortin receptor agonists, comprising the compound of formula (1) or a pharmaceutically acceptable salt or isomer thereof as an active ingredient together with a pharmaceutically acceptable carrier. In particular, the composition according to the present invention exhibits excellent effects in the prevention or treatment of obesity, diabetes, inflammation, and erectile dysfunction, but is not limited thereto.

[0044] As used herein, the term "carrier" refers to a compound that facilitates the infusion of a compound into cells or tissues.

[0045] When the compounds of the present invention are administered clinically, the total daily dose to a host in a single or divided dose is preferably in the range of 0.01 to 10 mg per kg of body weight, but the specific dose for an individual patient will vary depending on the specific compound used, the patient's body weight, sex, health condition, diet, drug administration time, administration method, excretion rate, drug mixture, and disease severity.

[0046] The compound of the present invention can be administered by any route depending on the purpose. For example, the compound of the present invention can be administered by injection or oral administration.

[0047] Injectable preparations can be prepared according to known techniques by using suitable dispersing agents, wetting agents or suspending agents.

[0048] Examples of solid dosage forms for oral administration include capsules, tablets, pills, powders and granules, and the solid dosage forms can be prepared by mixing the active compound of formula (1) according to the present invention with one or more carriers such as inert diluents, lubricants, disintegrants, binders, etc. [Effects of the Invention]

[0049] The compound of formula (1) according to the present invention exhibits excellent agonist activity against melanocortin receptors, particularly melanocortin-4 receptor (MC4R), and can therefore be conveniently and effectively used in the prevention or treatment of obesity, diabetes, inflammation, and erectile dysfunction.

[0050] The compound of formula (1) according to the present invention exhibits an on-target effect on the melanocortin-4 receptor, exhibits weight loss and diet effects, does not affect anxiety and depression, and can be administered without safety issues such as side effects of human ether-a-go-go related gene (hERG) inhibition and mutagenesis. Furthermore, the compound of formula (1) according to the present invention is not cytotoxic or hepatotoxic, and can be administered safely. DETAILED DESCRIPTION OF THE INVENTION

[0051] The present invention will now be described in more detail by the following examples, but it should be understood that the scope of protection of the present invention is not limited to these examples.

[0052] Preparation Example 1: Preparation of N-((1s,4R)-4-methylcyclohexyl)-N-((3S,5S)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)pivalamide hydrochloride [ka]

[0053] The title compound was obtained via the following steps A, B and C. Step A: Preparation of (2S,4S)-1-(tert-butoxycarbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)pivalamido)pyrrolidine-2-carboxylic acid [ka]

[0054] The title compound was obtained by the method disclosed in International Publication No. WO 2008 / 007930. MS [M+Na] = 433.4 (M+23) 1 H NMR (400 MHz, CD3OD) δ 4.25 (m, 1H), 3.86 (m, 2H), 3.42 (m, 2H), 2.80 (m, 1H), 2.27 (m, 1H), 2.00-1.80 (m, 3H), 1.66 (m, 4H), 1.43 (m, 11H), 1.26 (m, 9H), 1.05 (d, 3H)

[0055] Step B: Preparation of tert-butyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)pivalamido)-2-(morpholine-4-carbonyl)pyrrolidine-1-carboxylate [ka]

[0056] (2S,4S)-1-(tert-butoxycarbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)pivalamido)pyrrolidine-2-carboxylic acid (0.81 g, 1.97 mmol) obtained in Step A above was dissolved in 10 mL of dimethylformamide, and morpholine (0.19 mL, 2.17 mmol), 1H-benzo[d][1,2,3]triazol-1-ol monohydrate (0.36 g, 2.36 mmol), 3-(((ethylimino)methylene)amino)-N,N-dimethylpropan-1-amine hydrochloride (0.45 g, 2.36 mmol), and N,N-diisopropylethylamine (1.0 mL, 5.92 mmol) were added, followed by stirring at room temperature for 16 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the filtrate was washed with aqueous sodium bicarbonate and extracted with ethyl acetate. The organic layer was collected, washed with water, dried over anhydrous magnesium sulfate, and filtered to remove the solids. The filtrate was concentrated under reduced pressure and purified by column chromatography to give the title compound (0.62 g, 66%). MS [M+H] = 480.5 (M+1) 1H NMR (400 MHz, CD3OD) δ 4.65 (m, 1H), 4.00-3.40 (m, 12H), 2.82 (m, 1H), 2.22 (m, 1H), 2.00-1.80 (m, 3H), 1.70-1.60 (m, 4H), 1.50-1.41 (m, 11H), 1.24 (s, 9H), 1.04 (d, 3H)

[0057] Step C: Preparation of N-((1s,4R)-4-methylcyclohexyl)-N-((3S,5S)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)pivalamide hydrochloride [ka]

[0058] The tert-butyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)pivalamido)-2-(morpholine-4-carbonyl)pyrrolidine-1-carboxylate (0.62 g, 1.3 mmol) obtained in Step B above was dissolved in 4 mL of dichloromethane, and a 4 M solution of hydrochloric acid in 1,4-dioxane (1.3 mL, 5.1 mmol) was added, followed by stirring at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure, and the resulting solid was washed with diethyl ether and dried to obtain the title compound (0.55 g, 99%). MS [M+H] = 380.6 (M+1) 1 H NMR (400 MHz, DMSO-d6) δ 10.00-8.00 (brs, 2H), 4.46 (m, 1H), 4.14 (m, 1H), 3.76 (m, 1H), 3.65-3.25 (m, 10H), 2.47 (m, 1H), 1.99 (m, 1H), 1.91 (m, 1H), 1.80-1.50 (m, 6H), 1.39 (m, 2H), 1.19 (s, 9H), 1.00 (d, 3H)

[0059] Preparation Example 2: Preparation of N-((1s,4R)-4-methylcyclohexyl)-N-((3S,5S)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)isobutyramide hydrochloride [ka]

[0060] The title compound was obtained via the following steps A, B, C, D, E, F and G. Step A: Preparation of 1-(tert-butyl) 2-methyl (2S,4S)-4-azidopyrrolidine-1,2-dicarboxylate [ka]

[0061] 1-(tert-Butyl) 2-methyl(2S,4R)-4-((methylsulfonyl)oxy)pyrrolidine-1,2-dicarboxylate (48.5 g, 150 mmol) was dissolved in 250 mL of dimethylformamide, and sodium azide (19.5 g, 300 mmol) was added. The mixture was stirred at 80°C for 16 hours, and then the solution was concentrated under reduced pressure. Water was added thereto, and the mixture was extracted with ethyl acetate. The organic layer was washed with aqueous sodium chloride and water, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give the title compound (39.59 g, 98%), which was used in the next step without further purification. MS [M+H] = 271 (M+1) 1 H NMR (400 MHz, CD3OD) δ 4.43-4.37 (m, 1H), 4.35-4.27 (br, 1H), 3.77 (s, 1.8H), 3.76 (s, 1.2H), 3.73-3.66 (m, 1H), 3.44-3.38 (m, 1H), 2.63-2.49 (m, 1H), 2.19-2.11 (m, 1H), 1.50 (s, 4.5H), 1.44 (s, 4.5H)

[0062] Step B: Preparation of 1-(tert-butyl) 2-methyl (2S,4S)-4-aminopyrrolidine-1,2-dicarboxylate [ka]

[0063] 1-(tert-butyl) 2-methyl(2S,4S)-4-azidopyrrolidine-1,2-dicarboxylate (24.6 g, 91.0 mmol) obtained in Step A above was dissolved in 180 mL of tetrahydrofuran, and 1 M trimethylphosphine tetrahydrofuran solution (109 mL, 109 mmol) was slowly added at 0°C. After stirring at the same temperature for 1 hour, the reaction mixture was stirred at room temperature for 3 hours. The reaction solvent was concentrated under reduced pressure, and then 100 mL of dichloromethane and 150 mL of water were added, and the reaction mixture was stirred for approximately 30 minutes. The organic layer was dried over anhydrous magnesium sulfate, and the solid was filtered. The filtrate was concentrated under reduced pressure to obtain the title compound (20.62 g, 93%). MS [M+H] = 245 (M+1) 1 H NMR (400 MHz, CD3OD) δ 4.27 (m, 1H), 3.77 (s, 1.8H), 3.76 (s, 1.2H), 3.75-3.67 (m, 1H), 3.50-3.42 (m, 1H), 3.22-3.17 (m, 1H), 2.58-2.47 (m, 1H), 1.82-1.71 (m, 1H), 1.48 (s, 4.5H), 1.42 (s, 4.5H)

[0064] Step C: Preparation of 1-(tert-butyl) 2-methyl(2S,4S)-4-(((1s,4R)-4-methylcyclohexyl)amino)pyrrolidine-1,2-dicarboxylate [ka]

[0065] 1-(tert-butyl) 2-methyl(2S,4S)-4-aminopyrrolidine-1,2-dicarboxylate (20.6 g, 84.4 mmol) obtained in Step B above was dissolved in 150 mL of 1,2-dichloroethane, and 4-methylcyclohexanone (9.50 mL, 101 mmol) was added. The mixture was cooled to 0°C, and sodium triacetoxyborohydride (26.8 g, 127 mmol) was added, followed by stirring at room temperature for 16 hours. The reaction mixture was concentrated under reduced pressure, followed by addition of water and extraction with ethyl acetate. The organic layer was washed with aqueous sodium chloride, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography to obtain the title compound (22.9 g, 80%). MS [M+H] = 341 (M+1) 1 H NMR (400 MHz, CD3OD) δ 4.26 (m, 1H), 3.76 (s, 1.8H), 3.75 (s, 1.2H), 3.78-3.71 (m, 1H), 3.49-3.40 (m, 1H), 3.22-3.16 (m, 1H), 2.69-2.60 (br, 1H), 2.58-2.46 (m, 1H), 1.87-1.77 (m, 1H), 1.73-1.63 (m, 1H), 1.62-1.35 (m, 8H), 1.48 (s, 4.5H), 1.42 (s, 4.5H), 0.96 (d, 3H)

[0066] Step D: Preparation of 1-(tert-butyl) 2-methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-1,2-dicarboxylate [ka]

[0067] 1-(tert-butyl) 2-methyl(2S,4S)-4-(((1s,4R)-4-methylcyclohexyl)amino)pyrrolidine-1,2-dicarboxylate (37.29 g, 109.5 mmol) obtained in Step C above was dissolved in 500 mL of dichloromethane, and triethylamine (61.1 mL, 438 mmol) was added. Then, isobutyl chloride (11.7 mL, 219 mmol) was slowly added at 0°C. After stirring at room temperature for 16 hours, the reaction solvent was concentrated under reduced pressure. Aqueous sodium bicarbonate solution and ethyl acetate were added to the concentrate, and the organic layer was separated. The organic layer was washed with aqueous sodium chloride solution and water, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography to obtain the title compound (38.79 g, 86%). MS [M+H] = 411 (M+1) 1 H NMR (400 MHz, CD3OD) δ 4.27 (m, 1H), 3.76 (s, 1.8H), 3.75 (s,1.2H), 3.78-3.72 (m, 1H), 3.50-3.41 (m, 1H), 3.33-3.14 (m, 1H), 2.69-2.60 (m, 2H), 2.57-2.43 (m, 1H), 1.87-1.79 (m, 1H), 1.70-1.61 (m, 1H), 1.60-1.32 (m, 8H), 1.47 (s, 4.5H), 1.41 (s, 4.5H), 1.10 (dd, 6H), 0.99 (d, 3H)

[0068] Step E: Preparation of (2S,4S)-1-(tert-butoxycarbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylic acid [ka]

[0069] 1-(tert-butyl) 2-methyl(2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-1,2-dicarboxylate (3.63 g, 8.85 mmol) obtained in Step D above was dissolved in 30 mL of ethanol, and a 1N aqueous solution of sodium hydroxide (26.5 mL, 26.5 mmol) was added, followed by stirring at room temperature for 2 hours. The reaction solution was diluted with water and then adjusted to pH 4 with a 1N aqueous solution of hydrochloric acid. The reaction solution was extracted with ethyl acetate, and the organic layer was separated and dried over anhydrous sodium sulfate. The solid was filtered, and the filtrate was concentrated under reduced pressure to obtain the title compound (2.40 g, 69%). MS [M+Na] = 419.4 (M+23) 1 H NMR (400 MHz, CD3OD) δ 4.27 (m, 1H), 4.00-3.85 (m, 2H), 3.66 (m, 1H), 3.44 (m, 1H), 2.86 (m, 1H), 1.95-1.80 (m, 3H), 1.76-1.53 ​​(m, 5H), 1.50-1.42 (m, 11H), 1.10-1.05 (m, 9H)

[0070] Step F: Preparation of tert-butyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)-2-(morpholine-4-carbonyl)pyrrolidine-1-carboxylate [ka]

[0071] (2S,4S)-1-(tert-butoxycarbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylic acid (2.40 g, 6.05 mmol) obtained in Step E above, 1H-benzo[d][1,2,3]triazol-1-ol monohydrate (1.11 g, 7.26 mmol), and 3-(((ethylimino)methylene)amino)-N,N-dimethylpropan-1-amine hydrochloride (1.39 g, 7.26 mmol) were dissolved in 30 mL of dimethylformamide. To the mixture were added morpholine (0.55 mL, 6.66 mmol) and N,N-diisopropylethylamine (3.10 mL, 18.2 mmol), and the mixture was stirred at room temperature for 16 hours. After the reaction was completed, the reaction mixture was concentrated under reduced pressure, and the filtrate was washed with aqueous sodium bicarbonate and extracted with ethyl acetate. The organic layer was collected, washed with water, dried over anhydrous magnesium sulfate, and the solid was filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography to give the title compound (1.87 g, 66%). MS [M+H] = 466.5 (M+1) 1 H NMR (400 MHz, CD3OD) δ 4.66 (m, 1H), 3.94 (m, 1H), 3.75-3.55 (m, 10H), 3.45 (m, 1H), 2.86 (m, 1H), 2.24 (m, 1H), 1.95-1.80 (m, 3H), 1.77-1.60 (m, 5H), 1.59-1.45 (m, 2H), 1.46-1.41 (m, 9H), 1.05 (m, 9H)

[0072] Step G: Preparation of N-((1s,4R)-4-methylcyclohexyl)-N-((3S,5S)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)isobutyramide hydrochloride [ka]

[0073] The tert-butyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)-2-(morpholine-4-carbonyl)pyrrolidine-1-carboxylate (1.87 g, 4.00 mmol) obtained in Step F above was dissolved in 30 mL of dichloromethane, cooled to 0° C., and 4 M hydrochloric acid in 1,4-dioxane (2.15 mL, 8.59 mmol) was added. The mixture was stirred at room temperature for 16 hours, and then the reaction mixture was concentrated under reduced pressure. The resulting solid was washed with ethyl ether and dried to obtain the title compound (1.22 g, 76%). MS [M+H] = 366.4 (M+1) 1 H NMR (400 MHz, DMSO-d6) δ 9.88 (brs, 1H), 8.12 (m, 1H), 4.51 (m, 1H), 4.20 (m, 1H), 3.63-3.35 (m, 10H), 3.30 (m, 1H), 2.85 (m, 1H), 2.51 (m, 1H), 2.01 (m, 1H), 1.91 (m, 1H), 1.76-1.60 (m, 4H), 1.53 (m, 2H), 1.40 (m, 2H), 0.99 (m, 9H)

[0074] Preparation Example 3: Preparation of methyl (2S,4S)-4-(N-(4,4-dimethylcyclohexyl)pivalamido)pyrrolidine-2-carboxylate hydrochloride [ka] The title compound was obtained via the following steps A and B.

[0075] Step A: Preparation of 1-(tert-butyl) 2-methyl(2S,4S)-4-(N-(4,4-dimethylcyclohexyl)pivalamido)pyrrolidine-1,2-dicarboxylate [ka]

[0076] The title compound was obtained by the method disclosed in International Publication No. WO 2008 / 007930. MS [M+Na] = 461.4 (M+23) 1 H NMR (400 MHz, CD3OD) δ 4.34 (t, 1H), 3.90-3.70 (m, 2H), 3.73 (m, 3H), 3.45 (m, 2H), 2.74-2.61 (m, 1H), 2.30 (m, 1H), 1.83 (m, 2H), 1.53 (m, 4H), 1.45-1.40 (m, 9H), 1.40-1.30 (m, 2H), 1.24 (s, 9H), 0.99 (s, 3H), 0.94 (s, 3H)

[0077] Step B: Preparation of methyl (2S,4S)-4-(N-(4,4-dimethylcyclohexyl)pivalamido)pyrrolidine-2-carboxylate hydrochloride [ka]

[0078] The 1-(tert-butyl) 2-methyl(2S,4S)-4-(N-(4,4-dimethylcyclohexyl)pivalamido)pyrrolidine-1,2-dicarboxylate (1.67 g, 3.81 mmol) obtained in Step A was dissolved in 4 mL of ethyl acetate, and a 4 M solution of hydrochloric acid in ethyl acetate (3.81 mL, 15.2 mmol) was added, followed by stirring at room temperature for 16 hours. The reaction mixture was concentrated under reduced pressure to give the title compound (1.43 g, 99%). MS [M+H] = 339.4 (M+1) 1 H NMR (400 MHz, CD3OD) δ 4.41 (t, 1H), 4.24 (m, 1H), 3.86 (m, 1H), 3.83 (s, 3H), 3.42 (m, 2H), 2.66 (m, 1H), 2.22 (m, 1H), 1.77 (m, 2H), 1.51 (m, 4H), 1.34 (m, 2H), 1.23 (s, 9H), 0.99 (s, 3H), 0.93 (s, 3H)

[0079] Preparation Example 4: Preparation of methyl (2S,4S)-4-(N-(4,4-dimethylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylate hydrochloride [ka] The title compound was obtained via the following steps A and B.

[0080] Step A: Preparation of 1-(tert-butyl) 2-methyl(2S,4S)-4-(N-(4,4-dimethylcyclohexyl)isobutyramido)pyrrolidine-1,2-dicarboxylate [ka]

[0081] The title compound was obtained by the method disclosed in International Publication No. WO 2008 / 007930. MS [M+Na] = 447.4 (M+23) 1 H NMR (400 MHz, CD3OD) δ 4.31 (t, 1H), 3.93 (m, 1H), 3.83 (m, 1H), 3.75-3.68 (m, 3H), 3.62 (m, 1H), 3.45 (m, 1H), 2.81 (m, 1H), 2.26 (m, 1H), 1.80 (m, 2H), 1.50-1.35 (m, 14H), 1.21 (m, 2H), 1.04 (m, 6H), 0.96 (s, 3H), 0.92 (s, 3H)

[0082] Step B: Preparation of methyl (2S,4S)-4-(N-(4,4-dimethylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylate hydrochloride [ka]

[0083] The 1-(tert-butyl) 2-methyl(2S,4S)-4-(N-(4,4-dimethylcyclohexyl)isobutyramido)pyrrolidine-1,2-dicarboxylate (1.1 g, 2.6 mmol) obtained in Step A above was dissolved in 2.5 mL of ethyl acetate, and a 4 M solution of hydrochloric acid in ethyl acetate (2.5 mL, 10 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours and then concentrated under reduced pressure to give the title compound (0.93 g, 99%). MS [M+H] = 325.4 (M+1) 1 H NMR (400 MHz, CD3OD) δ 4.41 (t, 1H), 4.30 (m, 1H), 3.84 (s, 3H), 3.66 (m, 1H), 3.47 (m, 3H), 2.85 (m, 1H), 2.72 (m, 1H), 2.24 (m, 1H), 1.75 (m, 2H), 1.51 (m, 4H), 1.38 (m, 2H), 1.06 (m, 6H), 0.98 (s, 3H), 0.93 (s, 3H)

[0084] Production Example 5: Production of (3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carboxylic acid [ka]

[0085] The title compound was obtained by the method disclosed in International Publication No. WO 2004 / 092126. MS [M+H] = 282 (M+1) 1 H NMR (400 MHz, CD3OD) δ 7.43-7.33 (m, 4H), 3.90-3.69 (m, 3H), 3.59 (m, 1H), 3.29 (m, 1H), 3.18-3.09 (m, 1H), 1.44 (s, 9H)

[0086] Production Example 6: Production of (3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carboxylic acid [ka]

[0087] The title compound was obtained by the method disclosed in International Publication No. WO 2004 / 092126. MS [M+H] = 284.2 (M+1) 1 H NMR (400 MHz, CD3OD) δ 7.50 (m, 1H), 6.97 (m, 2H), 3.93-3.75 (m, 3H), 3.60 (m, 1H), 3.26 (m, 2H), 1.43 (s, 9H)

[0088] Preparation Example 7: Preparation of (3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carboxylic acid [ka]

[0089] The title compound was obtained by the method disclosed in International Publication No. WO 2004 / 092126. MS [M+H] = 300.3 (M+1) 1 H NMR (400 MHz, CD3OD) δ 7.47 (t, 1H), 7.22 (m, 2H), 3.93-3.75 (m, 3H), 3.60 (m, 1H), 3.26 (m, 2H), 1.43 (s, 9H)

[0090] Example 1: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)pivalamide hydrochloride [ka]

[0091] The title compound was obtained through the following steps A and B. Step A: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)pivalamide [ka]

[0092] Dimethylformamide (12 mL), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.28 g, 1.5 mmol), 1-hydroxybenzotriazole hydrate (0.22 g, 1.5 mmol), and N,N-diisopropylethylamine (0.64 mL, 3.6 mmol) were added to N-((1s,4R)-4-methylcyclohexyl)-N-((3S,5S)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)pivalamide hydrochloride (0.50 g, 1.2 mmol) obtained in Production Example 1 and (3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carboxylic acid (0.34 g, 1.2 mmol) obtained in Production Example 6, and the mixture was stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure, and then ethyl acetate was added. The reaction mixture was washed with aqueous ammonium chloride and 1M aqueous sodium hydroxide, and dried over anhydrous sodium sulfate. The solid was filtered off, and the filtrate was concentrated under reduced pressure and purified by chromatography to give the title compound (0.32 g, 40%). MS [M+H] = 645.6 (M+1) 1H NMR (400 MHz, CD3OD) δ 7.58 (m, 1H), 6.95 (m, 2H), 4.76 (m, 1H), 4.18 (m, 1H), 3.84-3.40 (m, 15H), 3.11 (m, 1H), 2.89 (m, 1H), 2.12 (m, 1H), 1.95 (m, 1H), 1.79 (m, 1H), 1.65 (m, 4H), 1.44 (m, 2H), 1.28 (m, 2H), 1.19 (s, 9H), 1.17 (s, 9H), 1.03 (m, 3H)

[0093] Step B: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)pivalamide hydrochloride [ka]

[0094] To the N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)pivalamide (0.32 g, 0.50 mmol) obtained in Step A above, 5 mL of dichloromethane and a 4 M solution of hydrochloric acid in ethyl acetate (0.25 mL, 1.0 mmol) were added, and the mixture was stirred at room temperature for 20 minutes. The reaction mixture was concentrated under reduced pressure, and diethyl ether was added. The resulting solid was filtered and dried to obtain the title compound (0.23 g, 68%). MS [M+H] = 645.6 (M+1) 1H NMR (400 MHz, CD3OD) δ 7.63 (m, 1H), 7.07 (m, 2H), 4.82 (m, 1H), 4.19 (m, 1H), 4.00-3.50 (m, 15H), 3.03 (m, 1H), 2.69 (m, 1H), 2.17 (m, 1H), 1.95 (m, 1H), 1.79 (m, 1H), 1.64 (m, 4H), 1.47 (s, 9H), 1.45-1.25 (m, 4H), 1.19 (s, 9H), 1.00 (m, 3H)

[0095] Example 2 Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide hydrochloride [ka]

[0096] The title compound was obtained via the following steps A and B. Step A: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide [ka]

[0097] The title compound (0.10 g, 9%) was obtained in the same manner as in Step A of Example 1 using (3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carboxylic acid (0.50 g, 1.76 mmol) obtained in Production Example 6 and N-((1s,4R)-4-methylcyclohexyl)-N-((3S,5S)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)isobutyramide hydrochloride (0.71 g, 1.76 mmol) obtained in Production Example 2. MS [M+H] = 631.5 (M+1) 1 H NMR (400 MHz, CD3OD) δ 7.56 (m, 1H), 7.00 (m, 2H), 4.79 (m, 1H), 4.56 (m, 1H), 4.17 (m, 1H), 3.84 (m, 1H), 3.70-3.50 (m, 13H), 3.09 (m, 1H), 2.80 (m, 1H), 2.70 (m, 1H), 2.12 (m, 1H), 1.93 (m, 1H), 1.75-1.50 (m, 5H), 1.40 (m, 2H), 1.29 (s, 9H), 1.24 (m, 2H), 1.00 (m, 9H)

[0098] Step B: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide hydrochloride [ka]

[0099] Using N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide (0.10 g, 0.16 mmol) obtained in Step A, the title compound (0.083 g, 78%) was obtained in the same manner as in Step B of Example 1. MS [M+H] = 631.5 (M+1) 1 H NMR (400 MHz, CD3OD) δ 7.63 (m, 1H), 7.07 (m, 2H), 4.80 (t, 1H), 4.21 (m, 1H), 3.94 (m, 2H), 3.85-3.50 (m, 13H), 3.03 (m, 1H), 2.82 (m, 1H), 2.70 (m, 1H), 2.15 (m, 1H), 1.95 (m, 1H), 1.80-1.59 (m, 5H), 1.47 (s, 9H), 1.40-1.20 (m, 4H), 1.01 (m, 9H)

[0100] Example 3 Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide hydrochloride [ka]

[0101] The title compound was obtained through the following steps A and B. Step A: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide [ka]

[0102] The title compound (0.13 g, 12%) was obtained in the same manner as in Step A of Example 1 using (3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carboxylic acid (0.50 g, 1.67 mmol) obtained in Production Example 7 and N-((1s,4R)-4-methylcyclohexyl)-N-((3S,5S)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)isobutyramide hydrochloride (0.67 g, 1.67 mmol) obtained in Production Example 2. MS [M+H] = 647.5 (M+1) 1 H NMR (400 MHz, CD3OD) δ 7.54 (t, 1H), 7.20 (m, 2H), 4.79 (m, 1H), 4.18 (m, 1H), 3.85 (m, 1H), 3.50-3.50 (m, 13H), 3.40 (m, 1H), 3.02 (m, 1H), 2.78 (m, 1H), 2.68 (m, 1H), 2.14 (m, 1H), 1.92 (m, 1H), 1.80-1.39 (m, 7H), 1.29-1.17 (m, 11H), 1.01 (m, 9H)

[0103] Step B: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide hydrochloride [ka] Using N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide (0.13 g, 0.20 mmol) obtained in Step A, the title compound (0.1 g, 80%) was obtained in the same manner as in Step B of Example 1. MS [M+H] = 647.5 (M+1) 1 H NMR (400 MHz, CD3OD) δ 7.60 (m, 1H), 7.30 (m, 2H), 4.81 (m, 1H), 4.22 (m, 1H), 4.04-3.90 (m, 2H), 3.86-3.41 (m, 13H), 3.12 (m, 1H), 2.83 (m, 1H), 2.70 (m, 1H), 2.20 (m, 1H), 1.95 (m, 1H), 1.80-1.55 (m, 5H), 1.45-1.27 (m, 4H), 1.47 (s, 9H), 1.02 (m, 9H)

[0104] Example 4 Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)pivalamide hydrochloride [ka]

[0105] The title compound was obtained via the following steps A and B. Step A: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)pivalamide [ka]

[0106] The title compound (0.45 g, 56%) was obtained in the same manner as in Step A of Example 1 using (3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carboxylic acid (0.36 g, 1.21 mmol) obtained in Production Example 7 and N-((1s,4R)-4-methylcyclohexyl)-N-((3S,5S)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)pivalamide hydrochloride (0.50 g, 1.21 mmol) obtained in Production Example 1. MS [M+H] = 661.6 (M+1) 1 H NMR (400 MHz, CD3OD) δ 7.55 (m, 1H), 7.18 (m, 2H), 4.79 (m, 1H), 4.18 (m, 1H), 3.90-3.20 (m, 15H), 3.08 (m, 1H), 2.70 (m, 1H), 2.12 (m, 1H), 1.93 (m, 1H), 1.81-1.60 (m, 5H), 1.47 (m, 2H), 1.30-1.10 (m, 2H), 1.20 (s, 9H), 1.15 (s, 9H), 1.03 (m, 3H)

[0107] Step B: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)pivalamide hydrochloride [ka]

[0108] Using N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)pivalamide (0.25 g, 0.38 mmol) obtained in Step A, the title compound (0.18 g, 68%) was obtained in the same manner as in Step B of Example 1. MS [M+H] = 661.6 (M+1) 1 H NMR (400 MHz, CD3OD) δ 7.60 (t, 1H), 7.30 (m, 2H), 4.80 (m, 1H), 4.20 (m, 1H), 4.04-3.90 (m, 2H), 3.86-3.40 (m, 13H), 3.12 (m, 1H), 2.67 (m, 1H), 2.16 (m, 1H), 1.94 (m, 1H), 1.82-1.60 (m, 5H), 1.47 (s, 9H), 1.45-1.20 (m, 4H), 1.20 (s, 9H), 1.03 (m, 3H)

[0109] Example 5 Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-(4,4-dimethylcyclohexyl)isobutyramide hydrochloride [ka]

[0110] The title compound was obtained via the following steps A, B, C and D. Step A: Preparation of methyl (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-(4,4-dimethylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylate [ka]

[0111] The title compound (0.49 g, 32%) was obtained in the same manner as in Step A of Example 1 using (3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carboxylic acid (0.73 g, 2.58 mmol) obtained in Production Example 5 and methyl (2S,4S)-4-(N-(4,4-dimethylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylate hydrochloride (0.93 g, 2.58 mmol) obtained in Production Example 4. MS [M+H] = 588.5 (M+1) 1 H NMR (400 MHz, CD3OD) δ 7.30 (m, 4H), 4.42 (t, 1H), 4.01 (t, 1H), 3.69 (s, 3H), 3.65-3.50 (m, 3H), 3.34-3.20 (m, 2H), 3.13-3.04 (m, 2H), 2.87 (m, 1H), 2.79 (m, 1H), 2.67 (m, 1H), 2.15 (m, 1H), 1.69 (m, 1H), 1.56 (m, 1H), 1.50-1.26 (m, 7H), 1.16 (s, 9H), 1.00 (m, 6H), 0.93 (m, 6H)

[0112] Step B: Preparation of (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-(4,4-dimethylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylic acid [ka]

[0113] Methyl (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-(4,4-dimethylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylate (0.49 g, 0.83 mmol) obtained in Step A above was dissolved in methanol (2.8 mL), cooled to 0° C., and 6 M aqueous sodium hydroxide solution (0.7 mL, 4.2 mmol) was added. The mixture was stirred at room temperature for 16 hours and then concentrated under reduced pressure. Water was added thereto to adjust the pH to 4, and the mixture was concentrated under reduced pressure. The mixture was dissolved in dichloromethane, and the solid was removed by filtration. The filtrate was concentrated under reduced pressure to obtain the title compound (0.47 g, 98%). MS [M+H] = 574.4 (M+1) 1 H NMR (400 MHz, CD3OD) δ 7.40 (m, 4H), 4.41 (m, 1H), 4.13-3.65 (m, 5H), 3.60-3.35 (m, 3H), 2.96 (m, 1H), 2.82-2.69 (m, 2H), 2.18 (m, 1H), 1.73-1.55 (m, 2H), 1.45 (s, 9H), 1.42-1.20 (m, 7H), 1.00 (m, 6H), 0.94 (m, 6H)

[0114] Step C: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-(4,4-dimethylcyclohexyl)isobutyramide [ka]

[0115] The title compound (0.41 g, 78%) was obtained in the same manner as in Step A of Example 1 using (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-(4,4-dimethylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylic acid (0.47 g, 0.82 mmol) obtained in Step B above and morpholine (0.071 mL, 0.82 mmol). MS [M+H] = 643.4 (M+1) 1 H NMR (400 MHz, CD3OD) δ 7.34 (m, 2H), 7.30 (m, 2H), 4.77 (m, 1H), 4.12 (m, 1H), 3.80-3.30 (m, 15H), 3.05 (m, 1H), 2.77 (m, 1H), 2.64 (m, 1H), 2.09 (m, 1H), 1.80-1.58 (m, 2H), 1.50-1.24 (m, 7H), 1.14 (s, 9H), 0.99 (m, 6H), 0.95 (m, 6H)

[0116] Step D: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-(4,4-dimethylcyclohexyl)isobutyramide hydrochloride [ka]

[0117] Using N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-(4,4-dimethylcyclohexyl)isobutyramide (0.42 g, 0.65 mmol) obtained in Step C above, the title compound (0.37 g, 83%) was obtained in the same manner as in Step B of Example 1. MS [M+H] = 643.4 (M+1) 1 H NMR (400 MHz, CD3OD) δ 7.44 (m, 4H), 4.81 (m, 1H), 4.22 (m, 1H), 3.90 (m, 1H), 3.80-3.40 (m, 14H), 2.99 (m, 1H), 2.82 (m, 1H), 2.68 (m, 1H), 2.16 (m, 1H), 1.80-1.60 (m, 2H), 1.40-1.20 (m, 7H), 1.47 (s, 9H), 1.05 (m, 6H), 0.96 (m, 6H)

[0118] Example 6: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-(4,4-dimethylcyclohexyl)pivalamide hydrochloride [ka]

[0119] The title compound was obtained via the following steps A, B, C and D. Step A: Preparation of methyl (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-(4,4-dimethylcyclohexyl)pivalamido)pyrrolidine-2-carboxylate [ka]

[0120] The title compound (0.63 g, 49%) was obtained in the same manner as in Step A of Example 1 using (3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carboxylic acid (0.60 g, 2.13 mmol) obtained in Production Example 5 and methyl (2S,4S)-4-(N-(4,4-dimethylcyclohexyl)pivalamido)pyrrolidine-2-carboxylate hydrochloride (0.80 g, 2.13 mmol) obtained in Production Example 3. MS [M+H] = 602.5 (M+1) 1 H NMR (400 MHz, CD3OD) δ 7.34 (m, 4H), 4.47 (t, 1H), 3.98 (m, 1H), 3.80 (m, 1H), 3.71 (s, 3H), 3.60 (m, 2H), 3.48-3.34 (m, 3H), 3.17 (m, 1H), 3.10 (m, 1H), 2.63 (m, 1H), 2.16 (m, 1H), 1.73 (m, 1H), 1.58 (m, 1H), 1.52-1.38 (m, 3H), 1.36-1.20 (m, 4H), 1.23 (s, 9H), 1.17 (s, 9H), 0.97 (s, 3H), 0.94 (s, 3H)

[0121] Step B: Preparation of (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-(4,4-dimethylcyclohexyl)pivalamido)pyrrolidine-2-carboxylic acid [ka]

[0122] Using methyl (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-(4,4-dimethylcyclohexyl)pivalamido)pyrrolidine-2-carboxylate (0.63 g, 1.05 mmol) obtained in Step A, the title compound (0.51 g, 83%) was obtained in the same manner as in Step B of Example 5. MS [M+H] = 588.5 (M+1) 1H NMR (400 MHz, CD3OD) δ 7.42 (m, 4H), 4.41 (m, 1H), 4.06 (m, 1H), 3.90 (m, 2H), 3.80-3.60 (m, 3H), 3.47 (m, 2H), 2.94 (m, 1H), 2.72 (m, 1H), 2.18 (m, 1H), 1.71 (m, 1H), 1.61 (m, 1H), 1.50-1.20 (m, 7H), 1.47 (s, 9H), 1.19 (s, 9H), 0.98 (s, 3H), 0.94 (s, 3H)

[0123] Step C: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-(4,4-dimethylcyclohexyl)pivalamide [ka]

[0124] The title compound (0.44 g, 77%) was obtained in the same manner as in Step A of Example 1 using (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-(4,4-dimethylcyclohexyl)pivalamido)pyrrolidine-2-carboxylic acid (0.51 g, 0.87 mmol) obtained in Step B and morpholine (0.076 mL, 0.87 mmol). MS [M+H] = 657.6 (M+1) 1 H NMR (400 MHz, CD3OD) δ 7.36 (m, 2H), 7.30 (m, 2H), 4.77 (m, 1H), 4.14 (m, 1H), 3.80-3.20 (m, 15H), 3.05 (m, 1H), 2.68 (m, 1H), 2.09 (m, 1H), 1.75 (m, 1H), 1.61 (m, 1H), 1.51-1.24 (m, 7H), 1.15 (m, 18H), 0.97 (m, 6H)

[0125] Step D: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-(4,4-dimethylcyclohexyl)pivalamide hydrochloride [ka]

[0126] Using N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-(4,4-dimethylcyclohexyl)pivalamide (0.44 g, 0.67 mmol) obtained in Step C above, the title compound (0.39 g, 84%) was obtained in the same manner as in Step B of Example 1. MS [M+H] = 657.6 (M+1) 1 H NMR (400 MHz, CD3OD) δ 7.45 (m, 4H), 4.81 (m, 1H), 4.16 (m, 1H), 4.06-3.85 (m, 2H), 3.84-3.40 (m, 13H), 2.98 (m, 1H), 2.67 (m, 1H), 2.13 (m, 1H), 1.75 (m, 1H), 1.63 (m, 1H), 1.47 (s, 9H), 1.50-1.20 (m, 7H), 1.18 (s, 9H), 0.96 (m, 6H)

[0127] Comparative Example 1: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-(4,4-dimethylcyclohexyl)acetamide hydrochloride (A95) [ka]

[0128] The A95 compound of International Publication No. WO 2008 / 007930 was obtained by the same method as disclosed therein.

[0129] Comparative Example 2: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-(4,4-dimethylcyclohexyl)acetamide hydrochloride (A96) [ka]

[0130] Compound A96 of International Publication No. WO 2008 / 007930 was obtained by the same method as disclosed therein.

[0131] Experimental Example 1: Luciferase assay To measure MC4R (melanocortin-4 receptor) agonist activity, we established a cell line constitutively expressing the luciferase gene (CRE-LUC) under the control of MC4R and the cAMP response element (CRE). A mammalian cell expression vector (pCDNA3(Neo)) (Invitrogen) containing the MC4R gene was constructed, and then a human embryonic kidney (HEK) cell line was transfected with a vector (pCRE-Luc) (Stratagen) expressing the luciferase gene (CRE-LUC) under the control of the cAMP response element (CRE) using Lipofectamine 2000 (Invitrogen). The transformed cell line (HEK MC4R-Luc) was cultured for 24 hours in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (GIBCO / BRL) in a 37°C incubator with 5% CO2. The cell lines were cultured for 4 days in 10 mL of selective medium (Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal bovine serum (GIBCO / BRL), 100 units / mL penicillin (GIBCO / BRL), 100 units / mL streptomycin (GIBCO / BRL), and 800 μg / mL geneticin (G418) (GIBCO / BRL). The medium was replaced with 10 mL of fresh selective medium to remove cells killed by the selective medium, a process repeated three times every four days. Individual colonies formed by the finally selected and expanded clones were transferred under microscope observation to 24-well cell culture plates containing 1 mL of selective medium per well and cultured for 4 days. The cells were treated with forskolin (Sigma) to a final concentration of 10 μM and cultured in a 37°C incubator with 5% CO2 for 5 hours. Each well was treated with 50 μL of Bright-Glo luciferase reagent (Promega) and incubated at room temperature for 15 minutes. Luminescence was measured using a luminometer (Victor). Clones that showed luminescence levels 100-fold higher than the baseline level after forskolin treatment were selected and used to measure the MC4R agonist activity of each compound.

[0132] HEK MC4R-Luc cells were plated in each well of a 96-well luminometer cell culture plate (Costar) at 2.5 × 10 cells / well in 100 μL of medium. 4 The cells were then cultured in a 37°C incubator under 6% CO₂ for 18 hours. MCR agonists diluted to various concentrations using the medium were added so that the final DMSO concentration did not exceed 1%, and the cells were cultured in a 37°C incubator under 6% CO₂ for 5 hours. Each well was treated with 50 μL of Bright-Glo luciferase reagent (Promega) and left at room temperature for 5 minutes. Luminescence from each well was measured using a luminometer (Victor). The amount of luminescence induced by the agonist diluted at various concentrations was converted into a relative percentage value compared to the amount induced by 0 μM NDP-α-MSH treatment. EC 0.5 MSH was measured at the concentration that induces 50% of the maximum luminescence induced by NDP-α-MSH, EC 50 The values ​​were expressed as the concentration of each agonist that induced 50% of the maximum luminescence amount that could be induced by the agonist. The measured values ​​were calculated using statistical software (Prizm).

[0133] The MC4R agonist activity of each compound obtained in the above experiment was measured and the results were analyzed using EC 50 The values ​​are shown in Table 1 in nM units. [Table 1]

[0134] As shown in Table 1 above, among the well-known melanocortin receptors in vivo, it was confirmed that the compounds of the examples have superior MC4R agonist activity to the melanocortin-4 receptor (MC4R), which is involved in energy metabolism and body weight control in vivo, compared to the compounds of the comparative examples (A95 and A96).

[0135] Experimental Example 2: cAMP Assay Melanocortin receptors are a type of G protein-coupled receptor (GPCR), and the primary role of G proteins is to activate second messengers that control cellular responses to many physiological stimuli through signal transduction. MC4R is a Gs-coupled receptor, and its interaction with an agonist activates adenylate cyclase (AC), leading to an increase in the intracellular concentration of cyclic AMP (cAMP), a second messenger. Therefore, melanocortin receptor activity can be assessed by measuring the generation of cAMP signals.

[0136] To measure the increase in intracellular cAMP levels in response to agonists, cAMP-hunter Gs-coupled receptor cell lines (CHO-K1 cell line) were established by overexpressing MC1R, MC3R, MC4R, and MC5R. The cells were seeded into individual wells of a white cell culture plate and cultured for 24 hours at 37°C in a 5% CO2 incubator. After incubation, the medium was removed and 15 μL of 2:1 HBBS / 10 mM HEPES:cAMP XS+Ab reagent was added. Five μL of a sample diluted 4-fold with buffer was added, followed by the addition of MC4R agonist compounds diluted at various concentrations to a vehicle concentration of 1%. The mixture was incubated at 37°C for 30 minutes. The activity (%) of each agonist compound was expressed as 100% × (mean RLU value of sample − mean RLU value of vehicle control) / (mean RLU value of max control − mean RLU value of vehicle control), and the values ​​were analyzed using the CBIS data analysis suite (ChemInnovation, CA).

[0137] The melanocortin receptor agonist activity of each compound obtained in the above experiment was evaluated using EC 50 The results measured in nM are shown in Table 2 below. [Table 2]

[0138] As shown in Table 2 above, among the well-known melanocortin receptors in vivo, it was confirmed that the compounds of the examples have superior receptor agonist activity to the melanocortin-4 receptor (MC4R), which is involved in energy metabolism and body weight control in vivo, compared with the Kato compounds of the comparative examples (A95 and A96).

[0139] Experimental Example 3: β-Arrestin Assay Melanocortin receptors are a type of G protein-coupled receptor (GPCR) that transduce signals from many neurotransmitters, thereby regulating various physiological responses. When GPCRs are phosphorylated, β-arrestins bind to the phosphorylated portion of the receptor and play an important role in activating various intracellular signaling pathways through interactions with other proteins. When melanocortin receptors interact with agonists, β-arrestins are recruited and participate in β-arrestin-mediated signaling pathways. Therefore, measuring β-arrestin levels can be used to assess melanocortin receptor activity.

[0140] We established the Pathhunter eXpress β-arrestin cell line (U2OS cell line) by co-expressing Prolink (PK)-tagged MC1R, MC3R, MC4R, and MC5R with enzyme acceptor (EA)-tagged β-arrestin. When the MCR-PK portion of this cell line is activated, β-arrestin-EA is recruited, and the β-galactosidase enzyme fragment, oxygen acceptor (EA), interacts with Prolink (PK). The activated enzyme hydrolyzes the substrate via β-galactosidase activity, generating a chemiluminescent signal that can be measured. After culturing the Pathhunter eXpress β-arrestin cell line (U2OS cell line), the cells were seeded into individual wells of a cell culture plate and incubated at 37°C in a 5% CO2 incubator for 48 hours. After incubation, 5 μL of the sample diluted 5-fold with buffer was added to the cells, and the vehicle concentration was set to 1%. MC4R agonist compounds diluted at various concentrations were added and incubated for 90 minutes at 37° C. The activity (%) of each agonist compound was expressed as 100% × (mean RLU value of sample − mean RLU value of vehicle control) / (mean maximum value of control ligand − mean RLU value of vehicle control), and the values ​​were analyzed using the CBIS data analysis suite (ChemInnovation, CA).

[0141] The melanocortin receptor agonist activity of each compound obtained in the above experiment was evaluated using EC 50 The results measured in nM are shown in Table 3 below. [Table 3]

[0142] As shown in Table 3 above, it was confirmed that the compounds of the examples have superior receptor activity to the melanocortin-4 receptor (MC4R), which is involved in energy metabolism and body weight control in the body, among the well-known melanocortin receptors in the body, compared to the compounds of the comparative examples (A95 and A96).

[0143] Experimental Example 4: Binding Affinity There are five known subtypes of melanocortin receptors (MCRs) in vivo, and subtype 4, MC4R, is known to be involved in energy metabolism and weight control. Other MCR subtypes are involved in regulating various functions in the body, such as skin pigmentation, energy homeostasis, and exocrine function. Therefore, ensuring the selectivity of MC4R agonist compounds for MC4R is crucial to preventing potential side effects. Therefore, we measured the receptor binding ability of MC4R agonists for each MCR subtype.

[0144] After establishing the CHO-K1 cell line expressing human recombinant MC1R and the HEK-293 cell line expressing MC3R, MC4R, and MC5R, membranes were harvested from each cell line. 3 μg of MC1 membrane and 0.04 nM of MC1R were added per well of a 96-well cell culture plate. 125 I-NDP-α-MSH was added and reacted at 37°C for 2 hours. 125 I-NDP-α-MSH was reacted at 37°C for 1 hour and then incubated with 3.12 μg of MC4R membranes and 0.02 nM 125 I-NDP-α-MSH was reacted for 2 hours at 37°C. At this time, 25 mM HEPES-KOH adsorption buffer (pH 7.0) containing MCR agonist diluted at various concentrations was added to each well and allowed to react. The reacted solution was transferred to a filter, washed with adsorption buffer, and then radioactivity was measured. The values ​​obtained by subtracting the nonspecific binding amount in the presence of 1 μM (MC1R) and 3 μM (MC3R, MC4R, MC5R) NDP-α-MSH from the total binding amount were calculated. 125 The specific binding amount of I-NDP-α-MSH was determined by diluting the agonist at each concentration. 125 The degree to which I-NDP-α-MSH specific binding was inhibited was measured. 50 teeth, 125 The concentrations were expressed as the concentration of each agonist required to inhibit 50% of the specific binding of I-NDP-α-MSH.

[0145] The binding of each compound to the melanocortin receptor obtained in the above experiment was measured in Ki (nM) units, and the results are shown in Tables 4 and 5. [Table 4] [Table 5]

[0146] As shown in Tables 4 and 5, among the well-known melanocortin receptors in vivo, it was confirmed that the compounds of the Examples have superior receptor binding ability to the melanocortin-4 receptor (MC4R), which is involved in energy metabolism and body weight control in vivo, compared to the compounds of the Comparative Examples (A95 and A96).

Claims

[Claim 1] The invention described in the specification.

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