Melanocortin-4 receptor agonists

Abstract

The problem to be solved by the present invention is to provide a compound formula excellent in selective agonist activity for melanocortin receptors.SOLUTION: The present invention relates to novel compounds represented by formula (1), pharmaceutically acceptable salts or isomers thereof, which have excellent selective agonist activity against melanocortin receptors, particularly melanocortin-4 receptor (MC4R).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 present invention relates to a compound represented by the following formula (1): [ka] (wherein R1 is C2-C5 alkyl), 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 from fat cells (adipocytes), and its secretion increases as body fat mass increases. Leptin regulates the functions of various neuropeptides produced in the hypothalamus, thereby regulating various biological functions, including appetite, body fat mass, and energy metabolism (Non-Patent Document 1). Leptin signals regulating appetite and body weight are transmitted through the regulation of many downstream factors, the most representative of which are melanocortin, agouti-related peptide (AgRP), and neuropeptide Y (NPY).

[0003] Increased blood leptin levels resulting from excess calories in the body increase secretion of the proopiomelanocortin (POMC) protein hormone in the pituitary gland and decrease 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, decreased leptin levels due to calorie deficits increase expression of AgRP, an MC4R antagonist, and NPY, thereby effectively increasing appetite. Thus, in response to changes in leptin, α-MSH and AgRP hormones 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. Among 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 known to be involved in the production of glucocorticoid hormones, with ACTH (adrenocorticotropic hormone) derived from POMC as its ligand. MC3R and MC4R are primarily expressed in the central nervous system and are involved in regulating appetite, energy metabolism, and the efficiency of fat accumulation in the body. MC5R is expressed in various tissues and is known to regulate exocrine function (Non-Patent Document 2). In particular, activation of the MC4R receptor has been shown to effectively reduce body weight by inducing anorexia and increasing energy metabolism, and has been demonstrated to be a key target of action in the development of obesity treatments (see Non-Patent Documents 2, 3, 4, and 5).

[0005] The role of MC4R in appetite and body weight control was primarily established through experiments using an animal model (agouti mice) with abnormal expression of agouti protein. In agouti mice, agouti protein is expressed at high concentrations in the central nervous system due to a genetic mutation, and it was found to act as an MC4R antagonist in the hypothalamus, inducing obesity (Non-Patent Documents 6 and 7). Subsequent research has shown that Agouti-related peptide (AgRP), similar to the actual agouti protein, is expressed in hypothalamic neurons, and that AgRP is involved in appetite regulation as an MC4R antagonist (Non-Patent Documents 8 and 9).

[0006] In vivo, intracerebral administration of the MC4R agonist α-MSH to animals reduced appetite, and treatment with the MC4R antagonists SHU9119 (peptide) or HS014 (peptide) again increased appetite (Non-Patent Document 10). Furthermore, animal studies using melanotan II (MTII, Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2) and its analogous agonist HP228 demonstrated appetite suppression, weight loss, and increased energy metabolism after intracerebral, intraperitoneal, or subcutaneous administration (Non-Patent Documents 11, 12, 13). In contrast, administration of the representative MC4R agonist SHU9119 to animals resulted in significant and sustained increases in food intake and weight gain, providing pharmacological evidence that obesity can be treated using MCR agonists. The anorectic effect clearly observed during MTII administration was not observed in MC4RKO (knockout) mice, again demonstrating that the anorectic effect is primarily achieved through activation of MC4R (Non-Patent Document 14).

[0007] Appetite suppressants acting on the central nervous system are the predominant obesity treatments developed to date, and most inhibitors are drugs that modulate the action of neurotransmitters. Examples include the adrenergic agonists (phentermine and mazindol) and the serotonergic drugs 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 major drawbacks, such as a lack of selectivity for each subtype and various side effects when administered for a long period of time.

[0008] On the other hand, melanocortins are neuropeptides, not neurotransmitters, and all functions except energy metabolism are normal in MC4R gene knockout mice. 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 belongs to the most successful category of new drug targets developed to date. It is significantly different from conventional targets in that it is relatively easy to ensure subtype selectivity.

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

[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 preparing the compound represented by formula (1).

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

[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 for the prevention or treatment of obesity, diabetes, inflammation and erectile dysfunction.

[0016] It is yet another object of the present invention to provide a method for preventing or treating obesity, diabetes, inflammation and erectile dysfunction, which comprises administering a compound represented by formula (1) or a pharmaceutically acceptable salt or isomer thereof to a subject in need thereof. [Means for solving the problem]

[0017] 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, or a pharmaceutically acceptable salt or isomer thereof.

[0018] The compound of formula (1) according to the present invention may form a pharmaceutically acceptable salt.

[0019] 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 such isomers and mixtures are included within the scope of the present invention.

[0020] In this specification, unless otherwise specified, the compound of formula (1) is used to mean all compounds of formula (1), their pharmaceutically acceptable salts and isomers.

[0021] 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 straight-chain or branched C2-C4 alkyl, such as ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl.

[0022] In another embodiment according to the present invention, R1 in formula (1) is C2 or C3 alkyl. In another embodiment according to the present invention, R1 in formula (1) is straight or branched C2 or C3 alkyl, for example, ethyl, n-propyl or isopropyl.

[0023] In another embodiment according to the present invention, the compound of formula (1) is N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide of formula (2): [ka]

[0024] In another embodiment of the present invention, the compound of formula (1) is N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)propionamide of formula (3): [ka]

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

[0026] In another embodiment according to the present invention, examples of said pharmaceutically acceptable salts include, but are not limited to, acid addition salts 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.

[0027] In another embodiment according to the invention, the compound is a pharmaceutically acceptable salt of a compound of formula (1), wherein R1 is ethyl and the salt is an inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, etc.; an organic carboxylic acid 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.; or a sulfonic acid such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, etc.

[0028] In another embodiment according to the invention, the compound is a pharmaceutically acceptable salt of a compound of formula (1), wherein R1 is n-propyl and the salt is an inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, etc.; an organic carboxylic acid 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.; or a sulfonic acid such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, etc.

[0029] In another embodiment according to the present invention, the compound is a pharmaceutically acceptable salt of a compound of formula (1), wherein R1 is isopropyl and the salt is an inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, etc.; an organic carboxylic acid 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.; or a sulfonic acid such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, etc.

[0030] In another embodiment according to the invention, the compound is a pharmaceutically acceptable salt of a compound of formula (1), wherein R1 is n-butyl and the salt is an inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, etc.; an organic carboxylic acid 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.; or a sulfonic acid such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, etc.

[0031] In another embodiment according to the invention, the compound is a pharmaceutically acceptable salt of a compound of formula (1), wherein R1 is isobutyl and the salt is an inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, etc.; an organic carboxylic acid 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.; or a sulfonic acid such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, etc.

[0032] In another embodiment according to the invention, the compound is a pharmaceutically acceptable salt of a compound of formula (1), wherein R1 is sec-butyl and the salt is an inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, etc.; an organic carboxylic acid 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.; or a sulfonic acid such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, etc.

[0033] In another embodiment according to the invention, the compound is a pharmaceutically acceptable salt of a compound of formula (1), wherein R1 is tert-butyl and the salt is an inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, etc.; an organic carboxylic acid 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.; or a sulfonic acid such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, etc.

[0034] In another embodiment according to the present invention, said pharmaceutically acceptable salt is the hydrochloride salt.

[0035] In another embodiment according to the present invention, the compound of formula (1) is N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide hydrochloride of formula (5): [ka]

[0036] In another embodiment according to the present invention, the compound of formula (1) is N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)propionamide hydrochloride of formula (6): [ka]

[0037] In another embodiment according to the present invention, the compound of formula (1) is N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)pivalamide hydrochloride of formula (7): [ka]

[0038] In another embodiment of the present invention, N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide hydrochloride of the formula (5), N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide hydrochloride of the formula (6), (Morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)propionamide hydrochloride and N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)pivalamide hydrochloride of formula (7) can be prepared according to the following Reaction Scheme 1. (Reaction Scheme 1) [ka] wherein R2 is C1-C5 alkyl, R3 is C3-C8 cycloalkyl that is unsubstituted or substituted with one or two C1-C5 alkyl groups, and R4 and R5 are each independently hydrogen or halogen.

[0039] 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.

[0040] As used herein, "carrier" refers to a compound that facilitates the introduction of a compound into cells or tissues.

[0041] When the compounds of the present invention are administered for clinical purposes, the total daily dose administered to the host, either as a single dose or in separate doses, is preferably in the range of 0.01 to 10 mg per kg of body weight, although the specific dose level for an individual patient may vary depending on the particular compound used, the patient's weight, sex, health condition, diet, time of drug administration, method of administration, excretion rate, drug mixture, and severity of disease, etc.

[0042] 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.

[0043] Injectable preparations can be prepared according to known art by using suitable dispersing agents, wetting agents or suspending agents.

[0044] 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, disintegrating agents, binders, etc. [Effects of the Invention]

[0045] 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 usefully used in the prevention or treatment of obesity, diabetes, inflammation, and erectile dysfunction.

[0046] The compound of formula (1) according to the present invention exhibits a targeting effect on the melanocortin-4 receptor, exhibiting weight loss and food intake reduction effects, without affecting anxiety and depression, and can be administered without causing side effects such as hERG (human ether-a-go-go related gene) inhibition and safety issues such as 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

[0047] The present invention will be described in more detail below through Production Examples and Examples, but these examples are merely illustrative and should not be construed as limiting the scope of the present invention.

[0048] Preparation Example 1: Preparation of methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylate hydrochloride [ka]

[0049] The following steps A, B, C, D and E gave the title compound.

[0050] Step A: Preparation of 1-(tert-butyl) 2-methyl (2S,4S)-4-azidopyrrolidine-1,2-dicarboxylate Under nitrogen, 1-(tert-butyl) 2-methyl(2S,4R)-4-((methylsulfonyl)oxy)pyrrolidine-1,2-dicarboxylate (48.5 g, 150 mmol) was dissolved in N,N'-dimethylformamide (250 mL), and sodium azide (19.5 g, 300 mL) was added. After stirring at 80°C for 16 hours, the reaction solvent was concentrated under reduced pressure, water was added, and the mixture was extracted twice 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 crude product (39.59 g, 98%), which was used in the next step without further purification.

[0051] MS [M+H] = 271 (M+1) 1H 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)

[0052] Step B: Preparation of 1-(tert-butyl) 2-methyl (2S,4S)-4-aminopyrrolidine-1,2-dicarboxylate 1-(tert-butyl) 2-methyl(2S,4S)-4-azidopyrrolidine-1,2-dicarboxylate (24.59 g, 91.0 mmol) obtained in Step A was dissolved in tetrahydrofuran (180 mL), and 1 M trimethylphosphine tetrahydrofuran solution (109.2 mL, 109.2 mmol) was slowly added at 0°C. After stirring at the same temperature for 1 hour, the mixture was stirred at room temperature for 3 hours. The reaction solvent was concentrated under reduced pressure, and then dichloromethane (100 mL) and water (150 mL) were added and stirred for approximately 30 minutes. The layers were separated and extracted again with dichloromethane. The organic layer was dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the crude product (20.62 g, 93%), which was used in the next step without further purification.

[0053] 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)

[0054] Step C: Preparation of 1-(tert-butyl) 2-methyl(2S,4S)-4-(((1s,4R)-4-methylcyclohexyl)amino)pyrrolidine-1,2-dicarboxylate 1-(tert-butyl) 2-methyl(2S,4S)-4-aminopyrrolidine-1,2-dicarboxylate (20.62 g, 84.4 mmol) obtained in Step B above was dissolved in dichloroethane (150 mL) and 4-methylcyclohexanone (9.5 mL, 101.3 mmol) was added. Sodium triacetoxyborohydride (26.8 g, 126.6 mmol) was added at 0°C and the mixture was stirred at room temperature for 16 hours. The reaction solvent was concentrated under reduced pressure, water was added, and the mixture was extracted twice 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 give the title compound (22.9 g, 80%).

[0055] 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)

[0056] Step D: Preparation of 1-(tert-butyl) 2-methyl(2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-1,2-dicarboxylate 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 dichloromethane (500 mL), triethylamine (61.1 mL, 438.1 mmol) was added, and 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 was added, and the mixture was extracted twice 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 and purified by column chromatography to obtain the title compound (38.79 g, 86%).

[0057] 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)

[0058] Step E: Preparation of methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylate hydrochloride The 1-(tert-butyl) 2-methyl(2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-1,2-dicarboxylate (34.0 g, 82.8 mmol) obtained in Step D above was dissolved in dichloromethane (200 mL), and a 4 N solution of hydrochloric acid in 1,4-dioxane (82.8 mL, 331.3 mmol) was added at 0° C. After stirring at room temperature for 6 hours, the reaction solvent was concentrated under reduced pressure to obtain a crude product (28.7 g, 99%). This was used in the next step without purification.

[0059] MS[M+H] = 311 (M+1)

[0060] Preparation Example 2: Preparation of (3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carboxylic acid [ka] The title compound was obtained by the method described in International Publication No. WO2004 / 092126.

[0061] 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 (dd, J = 11.2, 10.0 Hz, 1H), 3.29 (dd, J = 11.2, 11.2 Hz, 1H), 3.18-3.09 (m, 1H), 1.44 (s, 9H)

[0062] Preparation Example 3: Preparation of methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)propionamido)pyrrolidine-2-carboxylate hydrochloride [ka] The title compound was obtained by the following steps A and B.

[0063] Step A: Preparation of 1-(tert-butyl) 2-methyl(2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)propionamido)pyrrolidine-1,2-dicarboxylate [ka] The title compound (0.98 g, 84%) was obtained in the same manner as in Step D of Production Example 1 using 1-(tert-butyl) 2-methyl(2S,4S)-4-(((1s,4R)-4-methylcyclohexyl)amino)pyrrolidine-1,2-dicarboxylate (1.0 g, 2.9 mmol) obtained in Step C of Production Example 1 and propionyl chloride (0.33 g, 3.5 mmol).

[0064] MS [M+Na] = 419.5 (M+23) 1 H NMR (400 MHz, CD3OD) δ 4.33 (m, 1H), 4.00-3.80 (m, 2H), 3.75 (m, 3H), 3.58 (m, 1H), 3.47 (m, 1H), 2.85-2.68 (m, 1H), 2.38 (q, 2H), 2.31 (m, 1H), 1.93 (m, 1H), 1.80 (m, 2H), 1.72-1.55 (m, 4H), 1.45 (m, 2H), 1.45-1.41 (m, 9H), 1.07 (m, 6H)

[0065] Step B: Preparation of methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)propionamido)pyrrolidine-2-carboxylate hydrochloride [ka] Using 1-(tert-butyl) 2-methyl(2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)propionamido)pyrrolidine-1,2-dicarboxylate (0.98 g, 2.4 mmol) obtained in Step A, the title compound (0.76 g, 93%) was obtained in the same manner as in Step E of Production Example 1.

[0066] MS [M+H] = 297.4 (M+1) 1 H NMR (400 MHz, DMSO-d6) δ 9.95 (brs, 1H), 8.63 (brs, 1H), 4.38 (m, 1H), 4.21 (m, 1H), 3.77 (s, 3H), 3.53 (m, 1H), 3.40 (m, 2H), 2.53 (m, 1H), 2.37 (q, 2H), 2.24 (m, 1H), 1.88 (m, 1H), 1.68-1.55 (m, 4H), 1.52 (m, 2H), 1.40 (m, 2H), 0.97 (m, 6H)

[0067] Preparation Example 4: Preparation of methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)pivalamido)pyrrolidine-2-carboxylate hydrochloride [ka] The title compound was obtained by the following steps A and B.

[0068] Step A: Preparation of 1-(tert-butyl) 2-methyl(2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)pivalamido)pyrrolidine-1,2-dicarboxylate [ka] The title compound was obtained by the method described in International Publication No. WO2008 / 007930.

[0069] MS [M+Na] = 447.5 (M+23) 1H NMR (400 MHz, CD3OD) δ 4.34 (m, 1H), 3.90-3.75 (m, 2H), 3.73 (m, 3H), 3.45 (m, 2H), 2.75-2.60 (m, 1H), 2.30 (m, 1H), 1.95 (m, 1H), 1.85 (m, 2H), 1.66 (m, 4H), 1.50 (m, 2H), 1.45-1.41 (m, 9H), 1.25-1.20 (m, 9H), 1.05 (d, 3H)

[0070] Step B: Preparation of methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)pivalamido)pyrrolidine-2-carboxylate hydrochloride [ka] The title compound (0.68 g, 99%) was obtained in the same manner as in Step E of Production Example 1 using 1-(tert-butyl) 2-methyl(2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)pivalamido)pyrrolidine-1,2-dicarboxylate (0.80 g, 1.88 mmol) obtained in Step A above.

[0071] MS [M+H] = 325.4 (M+1) 1 H NMR (400 MHz, DMSO-d6) δ 10.24 (brs, 1H), 8.60 (brs, 1H), 4.41 (m, 1H), 4.22 (m, 1H), 3.77 (m, 3H), 3.40-3.28 (m, 3H), 2.55 (m, 1H), 2.20 (m, 1H), 1.87 (m, 1H), 1.70-1.50 (m, 6H), 1.40 (m, 2H), 1.21-1.10 (m, 9H), 1.00 (m, 3H)

[0072] Example 1 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-((1s,4R)-4-methylcyclohexyl)isobutyramide hydrochloride [ka] The title compound was obtained by the following steps A, B, C and D.

[0073] Step A: Preparation of methyl (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylate Methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylate hydrochloride (28.7 g, 82.73 mmol) obtained in Production Example 1, (3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carboxylic acid (24.5 g, 86.87 mmol) obtained in Production Example 2, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (22.2 g, 115.83 mmol), and 1-hydroxybenzotriazole hydrate (15.7 g, 115.83 mmol) were dissolved in N,N′-dimethylformamide (400 mL), and N,N′-diisopropylethylamine (72.0 mL, 413.66 mmol) was slowly added. After stirring at room temperature for 16 hours, the reaction solvent was concentrated under reduced pressure, followed by the addition of 0.5N aqueous sodium hydroxide and extraction twice with ethyl acetate. The organic layer was washed twice with aqueous sodium chloride and water, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography to give the title compound (41.19 g, 87%).

[0074] MS [M+H] = 575 (M+1)

[0075] Step B: Preparation of (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylic acid Methyl (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylate (39.4 g, 68.62 mmol) obtained in Step A above was dissolved in methanol (450 mL), and 6N aqueous sodium hydroxide solution (57.2 mL, 343.09 mmol) was added. The mixture was stirred at room temperature for 16 hours, and the pH was adjusted to approximately 5 with 6N aqueous hydrochloric acid. The reaction solution was then concentrated under reduced pressure. The concentrate was dissolved in dichloromethane, and the insoluble solid was then filtered through filter paper. The filtrate was concentrated under reduced pressure to obtain the crude product (38.4 g, 99%). This was used in the next step without further purification.

[0076] MS [M+H] = 561 (M+1)

[0077] 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-((1s,4R)-4-methylcyclohexyl)isobutyramide (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylic acid (38.4 g, 68.60 mmol) obtained in Step B above, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (18.4 g, 96.04 mmol), and 1-hydroxybenzotriazole hydrate (13.0 g, 96.04 mmol) were dissolved in N,N′-dimethylformamide (200 mL), and morpholine (5.9 mL, 68.80 mmol) and N,N′-diisopropylethylamine (59.7 mL, 343.02 mmol) were added slowly and successively. After stirring at room temperature for 16 hours, the reaction solution was concentrated under reduced pressure, followed by the addition of 0.5N aqueous sodium hydroxide and extraction twice with ethyl acetate. The organic layer was washed twice with aqueous sodium chloride and water, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography to give the title compound (37.05 g, 86%).

[0078] MS [M+H] = 630 (M+1)

[0079] 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-((1s,4R)-4-methylcyclohexyl)isobutyramide hydrochloride N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide (5.0 g, 7.95 mmol) obtained in Step C above was dissolved in ethyl acetate (50 mL), and a 2N solution of hydrochloric acid in ethyl acetate (3.97 mL, 15.89 mmol) was slowly added. The mixture was stirred at room temperature for 30 minutes, and the reaction solvent was concentrated under reduced pressure. The resulting crude solid was purified by trituration with hexane and diethyl ether to give the title compound (5.23 g, 99%).

[0080] MS [M+H] = 630 (M+1) 1 H NMR (500 MHz, CD3OD) δ 7.49-7.44 (m, 4H), 4.83 (m, 1H), 4.23-4.20 (m, 1H), 3.95-3.91 (m, 2H), 3.79-3.47 (m, 14H), 3.03-3.00 (m, 1H), 2.86-2.82 (m, 1H), 2.73-2.67 (m, 1H), 2.20-2.14 (m, 1H), 1.97 (m, 1H), 1.80-1.62 (m, 5H), 1.50 (s, 9H), 1.44-1.27 (m, 3H), 1.06-1.04 (m, 9H)

[0081] 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-((1s,4R)-4-methylcyclohexyl)propionamide hydrochloride [ka] The title compound was obtained by the following steps A, B, C and D.

[0082] Step A: Preparation of methyl (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)propionamido)pyrrolidine-2-carboxylate [ka] The title compound (0.45 g, 35%) was obtained in the same manner as in Step A of Example 1 using methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)propionamido)pyrrolidine-2-carboxylate hydrochloride (0.76 g, 2.28 mmol) obtained in Production Example 3 and (3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carboxylic acid (0.64 g, 2.28 mmol) obtained in Production Example 2.

[0083] MS [M+H] = 560.4 (M+1) 1 H NMR (400 MHz, CD3OD) δ 7.39-7.30 (m, 4H), 4.45 (m, 1H), 4.04 (m, 1H), 3.71 (s, 3H), 3.65-3.35 (m, 6H), 3.13 (m, 2H), 2.99 (m, 1H), 2.71 (m, 1H), 2.34 (q, 2H), 2.20 (m, 1H), 1.92 (m, 1H), 1.75-1.55 (m, 6H), 1.42 (m, 2H), 1.22 (m, 9H), 1.03 (m, 6H)

[0084] Step B: Preparation of (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)propionamido)pyrrolidine-2-carboxylic acid [ka] Using methyl (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)propionamido)pyrrolidine-2-carboxylate (0.45 g, 0.80 mmol) obtained in Step A, the title compound (0.44 g, 99%) was obtained in the same manner as in Step B of Example 1.

[0085] MS [M+H] = 546.4 (M+1)

[0086] 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-((1s,4R)-4-methylcyclohexyl)propionamide [ka] Using (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)propionamido)pyrrolidine-2-carboxylic acid (0.44 g, 0.80 mmol) obtained in Step B above, the title compound (0.28 g, 53%) was obtained in the same manner as in Step C of Example 1.

[0087] MS [M+H] = 615.5 (M+1) 1 H NMR (400 MHz, CD3OD) δ 7.36 (m, 4H), 4.79 (m, 1H), 4.18 (m, 1H), 3.80-3.40 (m, 15H), 3.20 (m, 1H), 3.03 (m, 1H), 2.70 (m, 1H), 2.33 (q, 2H), 2.15 (m, 1H), 1.93 (m, 1H), 1.71-1.56 (m, 6H), 1.40-1.20 (m, 11H), 1.00 (m, 6H)

[0088] 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-((1s,4R)-4-methylcyclohexyl)propionamide hydrochloride [ka] The title compound (0.08 g, 94%) was obtained in the same manner as in Step D of Example 1 using N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)propionamide (0.08 g, 0.13 mmol) obtained in Step C above.

[0089] MS [M+H] = 615.5 (M+1) 1 H NMR (400 MHz, CD3OD) δ 7.43 (m, 4H), 4.82 (t, 1H), 4.20 (m, 1H), 4.06-3.40 (m, 15H), 2.97 (m, 1H), 2.69 (m, 1H), 2.33 (m, 2H), 2.15 (m, 1H), 1.93 (m, 1H), 1.80-1.53 ​​(m, 5H), 1.47 (s, 9H), 1.50-1.25 (m, 4H), 1.01 (m, 6H)

[0090] Example 3 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-((1s,4R)-4-methylcyclohexyl)pivalamide hydrochloride [ka] The title compound was obtained by the following steps A, B, C and D.

[0091] Step A: Preparation of methyl (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)pivalamido)pyrrolidine-2-carboxylate [ka] The title compound (0.70 g, 66%) was obtained in the same manner as in Step A of Example 1 using methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)pivalamido)pyrrolidine-2-carboxylate hydrochloride (0.65 g, 1.8 mmol) obtained in Production Example 4 and (3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carboxylic acid (0.50 g, 1.8 mmol) obtained in Production Example 2.

[0092] MS [M+H] = 588.5 (M+1) 1 H NMR (400 MHz, CD3OD) δ 7.40-7.30 (m, 4H), 4.49 (m, 1H), 4.00-3.50 (m, 4H), 3.71 (s, 3H), 3.40 (m, 3H), 3.20-3.05 (m, 2H), 3.00 (m, 1H), 2.70 (m, 1H), 2.27 (m, 1H), 1.90 (m, 1H), 1.73-1.60 (m, 6H), 1.60-1.35 (m, 2H), 1.25-1.17 (m, 18H), 1.01 (m, 3H)

[0093] Step B: Preparation of (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)pivalamido)pyrrolidine-2-carboxylic acid [ka] The title compound (0.10 g, 99%) was obtained in the same manner as in Step B of Example 1 using methyl (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)pivalamido)pyrrolidine-2-carboxylate (0.10 g, 0.18 mmol) obtained in Step A above.

[0094] MS [M+H] = 574.4 (M+1)

[0095] 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-((1s,4R)-4-methylcyclohexyl)pivalamide [ka] Using (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)pivalamido)pyrrolidine-2-carboxylic acid (0.10 g, 0.18 mmol) obtained in Step B above, the title compound (0.020 g, 17%) was obtained in the same manner as in Step C of Example 1.

[0096] MS [M+H] = 643.5 (M+1) 1 H NMR (400 MHz, CD3OD) δ 7.40-7.30 (m, 4H), 4.79 (m, 1H), 4.17 (m, 1H), 3.80-3.40 (m, 15H), 3.10 (m, 1H), 2.96 (m, 1H), 2.71 (m, 1H), 2.15 (m, 1H), 1.90 (m, 1H), 1.80-1.35 (m, 8H), 1.21-1.15 (m, 18H), 1.02 (m, 3H)

[0097] 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-((1s,4R)-4-methylcyclohexyl)pivalamide hydrochloride [ka] The title compound (0.29 g, 83%) was obtained in the same manner as in Step D of Example 1 using N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)pivalamide (0.33 g, 0.51 mmol) obtained in Step C above.

[0098] MS [M+H] = 643.5 (M+1) 1 H NMR (400 MHz, CD3OD) δ 7.41 (m, 4H), 4.80 (m, 1H), 4.13 (m, 1H), 3.90 (m, 2H), 3.80-3.40 (m, 13H), 2.94 (m, 1H), 2.63 (m, 1H), 2.11 (m, 1H), 1.93 (m, 1H), 1.75 (m, 2H), 1.60 (m, 4H), 1.46 (s, 9H), 1.15 (s, 9H), 1.45-1.30 (m, 3H), 1.01 (m, 3H)

[0099] 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] Compound A95 of International Publication No. WO2008 / 007930 was obtained by the same method as described therein.

[0100] 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] Compound A96 of International Publication No. WO2008 / 007930 was obtained by the same method as described therein.

[0101] Experimental Example 1: Luciferase assay To measure agonist activity against the melanocortin-4 receptor (MC4R), we established a cell line permanently expressing the luciferase gene (CRE-LUC) under the control of the MC4R and cAMP response element (CRE). After preparing a mammalian cell expression vector (pCDNA3(Neo)) (Invitrogen) containing the MC4R gene, human embryonic kidney (HEK) cell lines were 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 in Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal bovine serum (GIBCO / BRL) at 37°C in the presence of 5% CO2 for 24 hours. 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 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, 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. After treatment with forskolin (Sigma) to a final concentration of 10 μM, the cells were cultured for 5 hours in a 37°C incubator with 5% CO2. 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 the MC4R agonist activity of each compound was measured.

[0102] HEK MC4R-Luc cells were cultured at 2.5 × 10 in 100 μL of culture per well of a 96-well luminometer cell culture plate (Costar). 4 The cells were then cultured in an incubator at 37°C under 6% CO₂ for 18 hours. The cultures were treated with MCR agonists diluted at various concentrations so that the final DMSO concentration did not exceed 1%. The cultures were then cultured in an incubator at 37°C under 6% CO₂ for 5 hours. Each well was treated with 50 μL of Bright-Glo luciferase reagent (Promega) and incubated at room temperature for 5 minutes. Luminescence was measured using a luminometer (Victor). The luminescence induced by the agonists diluted at various concentrations was converted into a percentage value relative to the amount induced by 10 μM NDP-α-MSH treatment. EC 0.5 MSH was expressed as the concentration that induces 50% of the maximum luminescence induced by NDP-α-MSH, and EC 50 The α- and β-actin concentrations were expressed as the concentration that induced 50% of the maximum luminescence that could be induced by each agonist. The measurements were performed using statistical software (Prizm).

[0103] The MC4R agonist ability of each compound obtained in the above experiment was evaluated using EC 50 The results measured in nM are shown in Table 1. [Table 1]

[0104] 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 regulation in vivo, compared to the comparative compounds (A95 and A96).

[0105] Experimental Example 2: cAMP Assay Melanocortin receptors (MC4Rs) are a type of G-protein-coupled receptor (GPCR), and the primary role of G-proteins is to activate second-order transducers to regulate cellular responses to many physiological stimuli through signal transduction. MC4Rs are G-coupled receptors, and their interaction with agonists activates adenylate cyclase (AC), which increases the intracellular concentration of cyclic AMP (cAMP), one of the second-order transducers. Therefore, melanocortin receptor activity can be assessed by measuring the generation of cAMP signals.

[0106] To measure the increase in intracellular cAMP levels in response to agonists, we established cAMPH Hunter Gs-coupled cell lines (CHO-K1 cell line) overexpressing MC1R, MC3R, MC4R, and MC5R. The cells were seeded into individual wells of white cell culture plates and cultured for 24 hours in a 37°C incubator with 5% CO2. After incubation, the medium was removed and 15 μL of 2:1 HBBS / 10 mM HEPES:cAMPXS+Ab reagent was added. Five μL of a 4-fold diluted sample in buffer was added, followed by the addition of MC4R agonist compounds diluted at various concentrations to a vehicle concentration of 1%. The cells were then 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).

[0107] The MC4R agonist ability of each compound obtained in the above experiment was evaluated using EC 50 The results measured in nM are shown in Table 2. [Table 2]

[0108] 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 agonist activity to the melanocortin-4 receptor (MC4R), which is involved in energy metabolism and body weight regulation in vivo, compared to the comparative compounds (A95 and A96).

[0109] Experimental Example 3: β-Arrestin Assay Melanocortin receptors are a type of G-protein-coupled receptor (GPCR) that mediate various physiological responses by transmitting signals from numerous neurotransmitters. 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, melanocortin receptor activity can be assessed by measuring β-arrestin levels.

[0110] We established the Pathhuntere® Xpress β-arrestin cell line (U2OS cell line) in which Prolink (PK)-tagged MC1R, MC3R, MC4R, and MC5R and enzyme acceptor (EA)-tagged β-arrestin were co-expressed. When the MCR-PK portion of this cell line is activated, β-arrestin-EA is recruited, and the enzyme acceptor (EA), a β-galactosidase enzyme fragment, interacts with Prolink (PK). The activated enzyme hydrolyzes the substrate via β-galactosidase activity, generating a chemiluminescent signal, allowing for measurement of activity. The Pathhuntere® Xpress β-arrestin cell line (U2OS cell line) was cultured and seeded into each well of a cell culture plate. The cells were then incubated at 37°C in a 5% CO2 incubator for 48 hours. After incubation, 5 μL of a 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 the sample − mean RLU value of the vehicle control) / (mean maximum value of the control ligand − mean RLU value of the vehicle control), and the values ​​were analyzed using the CBIS data analysis suite (ChemInnovation, CA).

[0111] The MC4R agonist ability of each compound obtained in the above experiment was evaluated using EC 50 The results measured in nM are shown in Table 3. [Table 3]

[0112] As shown in Table 3 above, among the well-known melanocortin receptors in vivo, it was confirmed that the compounds of the examples have superior agonist activity to the melanocortin-4 receptor (MC4R), which is involved in energy metabolism and body weight regulation in vivo, compared to the comparative compounds (A95 and A96).

[0113] Experimental Example 4: Binding Affinity Five subtypes of melanocortin receptors (MCRs) are known in vivo, and subtype 4, MC4R, is known to be involved in energy metabolism and body weight regulation. Because other MCR subtypes are involved in regulating various biological functions, such as skin pigmentation, energy homeostasis, and exocrine function, 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.

[0114] 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. MC1R membranes were incubated with 3 μg of MC1R membranes and 0.04 nM of MC5R per well in a 96-well cell culture plate. 125 I-NDP-α-MSH was reacted for 2 hours at 37°C. 3 μg of MC3R and MC5 membranes and 0.035 nM 125 I-NDP-α-MSH was reacted at 37°C for 1 hour, and 3.12 μg of MC4R membranes and 0.02 nM 125 I-NDP-α-MSH was reacted at 37°C for 2 hours. At this time, 25 mM HEPES-KOH adsorption buffer (pH 7.0) containing MCR agonist diluted at each step concentration was added to each well and allowed to react. The reaction solution was transferred to a filter, washed with the adsorption buffer, and then radioactivity was measured. The value was calculated 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. 125 The specific binding amount of I-NDP-α-MSH was used as the specific binding amount of I-NDP-α-MSH. 125 The degree to which I-NDP-α-MSH specific binding was inhibited was measured. 50 is 50% 125 The concentrations were expressed as the concentration of each agonist that inhibited I-NDP-α-MSH specific binding.

[0115] The MC4R agonist potency of each compound obtained in the above experiment was measured in Ki (nM) units and the results are shown in Table 4. [Table 4]

[0116] As shown in Table 4, 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 regulation in vivo, compared to the comparative compounds (A95 and A96).

[0117] Experimental Example 5: Pharmacokinetics and Drug Metabolism Experimental Example 5-1: Pharmacokinetic profile To investigate the pharmacokinetic (PK) properties of the compound of Example 1 and the compound of the comparative example, the following experiment was carried out.

[0118] To conduct PK studies of the compound of Example 1 and the comparative compounds (A95 and A96), approximately 7-week-old C57BL6 mice were prepared and assigned to groups of 12 mice per substance. They were then starved for oral administration. On the day of administration, a drug solution was prepared at a concentration of 1 mg / mL using distilled water (DW) as a vehicle. Each mouse was orally administered 1 mL per kg of body weight for a final dose of 10 mg / kg. At 1, 3, 8, and 24 hours after administration, whole blood was collected from three mice in each group via cardiac puncture and placed in heparin tubes to prevent coagulation. The cerebrum of each mouse was then removed and placed in an EP tube. The tissue was weighed and stored frozen at -20°C.

[0119] On the day of analysis, DDW (four times the tissue weight) was added to each tissue tube and homogenized. The stored plasma was thawed at room temperature. Similarly to the plasma, 50 μL of tissue homogenate was collected and transferred to a separate tube. 200 μL of acetonitrile (AN), four times the total volume of the following samples, was added to each tube of plasma and tissue homogenate for deproteinization. At this time, AN contained an internal standard. To generate a calibration curve, AN solutions (containing the internal standard) with known concentrations of 0.1, 0.5, 5, 50, and 500 ng / mL were prepared, and plasma and blank brain plasma were deproteinized at four times the above dose. Therefore, final calibration curves were created ranging from 0.4 to 2,000 ng / mL for plasma and 1, 5, 50, 500, and 5,000 ng / mL for brain. After injecting 0.5 μL of the supernatant obtained after protein removal into the LC-MS / MS, the peak areas of the compounds of the examples and the comparative examples (A95 and A96) were corrected with the peak area of ​​IS to determine the peak response at each sample collection point, and concentration conversion was performed using a calibration curve.

[0120] Pharmacokinetic parameters (C max , AUC inf , t 1 / 2 The time course blood concentration values ​​for each dose group were calculated by non-compartmental analysis using WinNonlin 8.1.

[0121] The pharmacokinetic properties of each compound were compared by comparing the exposure level and half-life changes of each drug administration group, and the results are shown in Tables 5 and 6. In addition, the results based on the ratio of exposure to the brain and blood are shown in Table 7. [Table 5] [Table 6] [Table 7]

[0122] From the above results, the exposure of each compound was confirmed in the whole body and brain in the following order: compound of Example 1, compound of Comparative Example 2 (A96), and compound of Comparative Example 1 (A95). The half-life of brain elimination during the observation period was longer than that observed in blood, which is thought to indicate the effective persistence of the series of substances at the site of efficacy. Furthermore, when each compound was administered at the same dose, the compound of the Example was confirmed to have the best absolute exposure and persistence in the brain. Furthermore, compared to the comparative compounds (A95 and A96), the compound of Example 1 was also confirmed to have the best ratio of brain exposure to blood exposure.

[0123] Considering these results, as well as the in vitro activity and drug durability of each compound, the compound of Example 1 was expected to have the highest efficacy when administered in the same manner as the comparative compound. To achieve a certain level of efficacy, a lower dose can be administered, thus minimizing side effects caused by systemic exposure.

[0124] Experimental Example 5-2: CYP inhibition rate (%) To confirm drug interactions with CYP (cytochrome P450) isoenzymes, the following experiment was carried out.

[0125] Recombinant CYP1A2, 2C9, 2C19, 2D6, and 3A4 enzymes were prepared to measure and compare the inhibitory potency of the compound of Example 1 and the compound (A96) of Comparative Example 2. The probe substrates, positive control groups, and isozymes used to measure the inhibitory potency of each substance, as well as the measurement conditions, were determined with reference to Table 8 below. [Table 8]

[0126] The incubation was performed in a 96-well plate (Costar, 3792-black, round-bottom). The buffer system used for metabolism was 50 mM potassium phosphate buffer, pH 7.4, with a final reaction volume of 250 μL. The final concentrations of the compound of Example 1, the compound of Comparative Example 2 (A96), and the positive control group in the total buffer were 10 μM (2% methanol (v / v)). A negative control (methanol only, 2% (v / v)) was also included. The buffer spiked with the experimental compound was mixed with an equal volume of the CYP isozyme preparation solution to a final concentration of 10 μM and preheated in a fluorescence plate reader at 37 °C for 10 min. NRS solution (NADPH regeneration system: 0.22 mM β-NADP, 2.8 mM glucose-6-phosphate, and 0.6 units / mL glucose-6-phosphate dehydrogenase) was added to each well and preincubated for 30 min. The reaction was then initiated by adding substrate to the pre-incubated wells, and monitored at the measurement wavelength for each substrate at 1-minute intervals for 30 minutes. The fluorescence intensity measured using the positive control group and the compound of Example 1 was compared with that of the negative control (no inhibitor or compound) to confirm the inhibitory potency against the isozymes. The inhibitory potency (%) against each isozyme when each compound was treated at 10 μM is shown in Table 9. The inhibitory potency (%) was expressed based on the following criteria: A = <50%, B = >50%.

[0127] The therapeutic concentration of a compound is 10 μM, which is very high. Therefore, it is conservative to evaluate the inhibitory potential based on this therapeutic concentration and select as candidates drugs with an inhibition rate of less than 50%. In this case, the selected candidate drugs were considered to have a very low probability of inhibiting CYP isozymes. [Table 9]

[0128] These results confirmed that the inhibitory potency of the compound of Example 1 against major CYP isozymes was equal to or less than that of compound (A96) of Comparative Example 2 when treated at the same concentration. Compound (A96) of Comparative Example 2 has a high inhibitory potency against CYP2C9, raising concerns about drug interactions. CYP2C9 is involved in the metabolism of approximately 10% of all marketed drugs and is known to play a major role in the loss of efficacy of drugs with narrow therapeutic ranges. Furthermore, CYP2C9 is extremely important in both research and clinical settings due to the reported presence of individual polymorphisms. This is evidenced by the fact that CYP2C9 is listed as an essential isozyme whose inhibitory effects must be confirmed during drug development in the FDA DDI (Drug-Drug Interaction) Guidance.

[0129] Based on this, it is judged that when the compound of Example 1 is administered, drug interactions due to CYP inhibition are lower than those of the comparative compound (A96).

[0130] Experimental Example 6: Pharmacological effects The pharmacological effects of the compounds of the present invention as melanocortin-4 receptor agonists were evaluated in the following obesity model.

[0131] Experimental Example 6-1: High-fat diet-induced obesity mouse model The effects of melanocortin-4 receptor agonists on obesity were assessed using a mouse obesity model induced by a high-fat diet.

[0132] Obesity was induced in 5-week-old male C57BL / 6N Taconic mice by feeding them a 60 kcal% fat diet (D12492, Research Diet) for 15 weeks. The compound of Example 1, the comparative compounds (A95 and A96), and sibutramine as a positive control were prepared in distilled water and orally administered once daily from Day 1 to Day 16 to a 19-week-old mouse model of high-fat diet-induced obesity. Body weight was measured once daily, food intake five times a week, and drinking water intake twice a week from Day 1 to Day 16. Blood glucose and glycosylated hemoglobin were measured on Day 15, and all animals were sacrificed on Day 17. Blood was collected from the abdominal vena cava, and the liver and epididymal adipose tissue were excised and weighed. The collected blood was placed in a heparin tube and centrifuged to separate the plasma, followed by plasma biochemistry analysis.

[0133] Table 10 below shows the difference in the rate of change in body weight measured on day 12 for each dose of each compound relative to the vehicle. [Table 10]

[0134] In this mouse obesity model, significant weight loss was observed, particularly when only the compound of Example 1 was administered. When the compound of Example 1 was administered at doses of 10 mg / kg and 30 mg / kg, it caused a weight gain inhibition of -9.4% and -15.1%, respectively, compared to the solvent-administered vehicle control, demonstrating a statistically significant weight loss effect compared to the comparative compounds (A95 and A96).

[0135] This is believed to be due to the superior in vitro MC4R agonist potency, superior absolute brain exposure and durability, and superior brain exposure ratio compared to blood exposure of the compound of Example 1 compared to the compound of the comparative example, and is believed to show significant differences even at low doses.

[0136] Furthermore, the compound of Example 1 exhibits efficacy that is equal to or superior to that of sibutramine (Reductil), an obesity treatment agent in the related field, and is expected to exhibit significant pharmacological efficacy in actual clinical applications.

Claims

1. The following formula (1) 【Chemistry 1】 (Wherein R is C 2 -C 5 or a pharmaceutically acceptable salt or isomer thereof.

2. R1 is C 2 -C 4 2. The compound of claim 1, or a pharmaceutically acceptable salt or isomer thereof, wherein R is alkyl.

3. 3. The compound according to claim 2, or a pharmaceutically acceptable salt or isomer thereof, wherein R1 is ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl.

4. The compound according to claim 2, wherein the compound of formula (1) is selected from the following group: N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide; N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)propionamide; and N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)pivalamide.

5. 2. The compound of claim 1, or a pharmaceutically acceptable salt or isomer thereof, wherein the pharmaceutically acceptable salt is selected from the group consisting of hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, and hydroiodic acid.

6. 6. The compound according to claim 5, or a pharmaceutically acceptable salt or isomer thereof, wherein the pharmaceutically acceptable salt is a hydrochloride salt.

7. A melanocortin receptor agonist pharmaceutical composition comprising the compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt or isomer thereof, and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, which is for the prevention or treatment of obesity.

9. The pharmaceutical composition according to claim 7, which is for the prevention or treatment of diabetes.

10. The pharmaceutical composition according to claim 7, which is for the prevention or treatment of inflammation.

11. The pharmaceutical composition according to claim 7, which is for the prevention or treatment of erectile dysfunction.

12. Use of the melanocortin receptor agonist pharmaceutical composition according to claim 7 for the preparation of a medicament for the prevention or treatment of obesity.

13. Use of the melanocortin receptor agonist pharmaceutical composition according to claim 7 for the preparation of a medicament for the prevention or treatment of diabetes.

14. Use of the melanocortin receptor agonist pharmaceutical composition of claim 7 for the preparation of a medicament for the prevention or treatment of inflammation.

15. Use of the melanocortin receptor agonist pharmaceutical composition according to claim 7 for the preparation of a medicament for the prevention or treatment of erectile dysfunction.

16. A method for preventing or treating obesity, comprising administering the melanocortin receptor agonist pharmaceutical composition of claim 7 to a subject in need thereof.

17. A method for preventing or treating diabetes, comprising administering the melanocortin receptor agonist pharmaceutical composition of claim 7 to a subject in need thereof.

18. A method for preventing or treating inflammation, comprising administering the melanocortin receptor agonist pharmaceutical composition of claim 7 to a subject in need thereof.

19. A method for preventing or treating erectile dysfunction, comprising administering the melanocortin receptor agonist pharmaceutical composition of claim 7 to a subject in need thereof.

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