Melanocortin-4 receptor agonists

Abstract

The present invention relates to a compound showing excellent agonist activity on melanocortin receptors, more specifically, to a compound of Chemical Formula 1, a pharmaceutical composition containing the same as an active ingredient, and a use thereof. Specifically, the compound of the present invention exhibits excellent agonist activity on melanocortin-4 receptors, and thus can be particularly advantageously used for preventing or treating obesity, diabetes, inflammation, and erectile dysfunction.

Description

Technical Field

[0001] This invention relates to a compound that exhibits excellent agonist activity against the melanocortin receptor. More specifically, this invention relates to compounds of formula 1, pharmaceutical compositions comprising the compound as an active ingredient, and uses thereof, and the compounds of this invention exhibit excellent agonist activity against the melanocortin-4 receptor, and may be particularly useful in the prevention or treatment of obesity, diabetes, inflammation, and erectile dysfunction:

[0002] [Formula 1]

[0003]

[0004] R1, R2, R3, R4, and n are the same as those defined in this paper. Background Technology

[0005] Leptin is a hormone secreted by fat cells (adipocytes), and its secretion increases with increasing body fat content. Leptin regulates the function of various neuropeptides produced in the hypothalamus, thereby regulating appetite, body fat content, and various bodily functions, including energy metabolism (Schwartz et al., Nature 404, 661-671 (2000)). The signal transduction of leptin's control over appetite and weight is achieved by regulating many downstream factors, the most representative of which are melanocortin, muscarinic oleander-related peptide (AgRP), and neuropeptide Y (NPY) hormone.

[0006] When excess calories lead to increased leptin levels in the blood, the secretion of pro-pomelocortin (POMC) protein hormone from the pituitary gland increases, while the production of AgRP and NPY decreases. α-MSH (melanocyte-stimulating hormone), a small peptide hormone produced by POMC neurons, is an agonist of the melanocortin-4 receptor (MC4R) on the second neuron, ultimately causing a decrease in appetite. Conversely, when leptin levels decrease due to insufficient calorie intake, the expression of AgRP, an MC4R antagonist, increases, as does the expression of NPY, which ultimately enhances appetite. In other words, depending on changes in leptin levels, α-MSH and AgRP, as MC4R agonists and antagonists, participate in appetite regulation.

[0007] In addition to MC4R, α-MSH hormones also induce various physiological responses by binding to three MCR subtypes. To date, five MCR subtypes have been identified. Among these subtypes, MC1R is known to be mainly expressed in skin cells and involved in melanin deposition, MC2R is mainly expressed in the adrenal glands and involved in the production of glucocorticoids, and only ACTH (adrenocorticotropic hormone) derived from POMC is its ligand. MC3R and MC4R, mainly expressed in the central nervous system, are involved in regulating appetite, energy metabolism, and the efficiency of fat storage in the body, while MC5R, which is known to be expressed in various tissues, can regulate exocrine function (Wikberg et al., PharmRes 42(5)393-420(2000)). Specifically, activation of the MC4R receptor has been shown to be a key target for the development of obesity drugs because it has been shown to effectively reduce weight by causing decreased appetite and increased energy metabolism (Reviews: Wikberg, Eur. J. Pharmacol 375, 295-310 (1999); Wikberg et al., Pharm Res 42(5) 393-420 (2000); Douglas et al., Eur. J. Pharm 450, 93-109 (2002); and O'Rahilly et al., Nature Med 10, 351-352 (2004)).

[0008] The role of MC4R in appetite and weight control has been preliminarily demonstrated in experiments using an animal model of aberrant muskrat protein expression (agouti mice). In the case of agouti mice, muskrat protein was found to be expressed at high concentrations in the central nervous system and, due to gene mutations, to act as an antagonist of MC4R in the hypothalamus, thus causing obesity (Yen, TT et al., FASEBJ. 8, 479-488 (1994); and Lu D et al., Nature 371, 799-802 (1994)). In subsequent studies, AgRP (muskrat-associated peptide), which is similar to the actual muskrat protein, was observed to be expressed in hypothalamic neurons, and AgRP is known to participate in appetite regulation as an MC4R antagonist (Shutter et al., Genes Dev., 11, 593-602 (1997); and Ollman et al., Science 278, 135-138 (1997)).

[0009] In animal brain administration, α-MSH, as an in vivo MC4R agonist, showed an appetite-reducing effect, and when these animals were treated with SHU9119 (peptide) or HS014 (peptide), as MC4R antagonists, an appetite-increasing effect was again observed (Kask et al., Biochem. Biophys. Res. Comm. 245, 90-93 (1998)). Furthermore, in animal studies using melanin II (Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2) and HP228 (a similar agonist), appetite suppression, weight loss, and increased energy metabolism were confirmed after administration to the brain, intraperitoneally, or subcutaneously. (Thiele T.E. et al., Am J Physiol 274(1 Pt 2), R248-54(1998); Lee MD et al., FASEB J 12, A552(1998); Murphy B. et al., J Appl Physiol 89, 273-82(2000)). In contrast, when animals were administered the representative SHU9119, they showed a significant and sustained increase in food intake and body weight, providing pharmacological evidence that MCR agonists can be used to treat obesity. The appetite-reducing effect that was evident during MTII administration was not observed in MC4R KO (knockout) mice, which again demonstrates that the appetite-reducing effect is mainly achieved through MC4R activation (Marsh et al., Nat Genet 21119-122(1999)).

[0010] Appetite suppressants acting on the central nervous system are the main drugs developed to date for the treatment of obesity. Most suppressants are drugs that modulate neurotransmitter activity. Examples include norepinephrine agents (phentermine and masindole) and serotonergic agents such as fluoxetine and sibutramine. However, neurotransmitter modulators, in addition to suppressing appetite, have a wide range of effects on various physiological functions through many subtypes of receptors. Therefore, modulators lack selectivity for each subtype, and a major drawback is that long-term use can lead to various side effects.

[0011] On the other hand, melanocortin is a neuropeptide, not a neurotransmitter. Given that MC4R gene knockout mice function normally in all aspects except energy metabolism, the advantage of melanocortin agonists as a target is that they can induce weight loss solely through appetite suppression without affecting other physiological functions. Specifically, this receptor is a G protein-coupled receptor (GPCR), which belongs to the most successful class of drug targets developed to date and differs significantly from targets in related fields because it is relatively easy to ensure selectivity for subtype receptors.

[0012] As examples of compounds that utilize melanocortin receptors as sites of action, international publications WO 2008 / 007930 and WO2010 / 056022 disclose compounds that act as melanocortin receptor agonists. Summary of the Invention

[0013] Technical issues

[0014] The 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 melanocortin-4 receptor (MC4R).

[0015] Another object of the present invention is to provide a method for preparing a compound represented by Formula 1.

[0016] Another object of the present invention is to provide a melanocortin receptor agonist pharmaceutical composition comprising a compound represented by Formula 1 or a pharmaceutically acceptable salt or isomer thereof as an active ingredient.

[0017] Another object of the present invention is to provide the use of compounds represented by Formula 1 or pharmaceutically acceptable salts or isomers thereof in the prevention or treatment of obesity, diabetes, inflammation and erectile dysfunction.

[0018] Technical solutions

[0019] To achieve the above objectives, the present invention provides compounds of Formula 1 or pharmaceutically acceptable salts or isomers thereof:

[0020] [Formula 1]

[0021]

[0022] in

[0023] R1 is a C2-C5 alkyl group;

[0024] R2 is a halogen;

[0025] R3 is hydrogen or halogen;

[0026] R4 is a C1-C3 alkyl group; and

[0027] n is an integer of 1 or 2;

[0028] The condition is that n is 2 when R2 is chlorine and R3 is hydrogen.

[0029] The compounds of Formula 1 according to the present invention can form pharmaceutically acceptable salts.

[0030] Furthermore, since the compounds according to the invention may have asymmetric carbon centers and asymmetric axes or asymmetric planes, the compounds may exist as cis or trans isomers, R or S isomers, racemates, diastereomer mixtures and single diastereomers, all of which are included within the scope of the invention.

[0031] In this specification, unless otherwise stated, the use of compounds of Formula 1 shall include all compounds of Formula 1, their pharmaceutically acceptable salts and isomers.

[0032] The term "halogen" as used in this article refers to the free radical of fluorine (F), chlorine (Cl), bromine (Br), or iodine (I).

[0033] The term "alkyl" as used in this article refers to a straight-chain or branched hydrocarbon group.

[0034] In one embodiment of the invention, R1 in Formula 1 is a C2 to C4 alkyl group. In another embodiment of the invention, R1 in Formula 1 is a straight-chain or branched C2 to C4 alkyl group, such as ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl.

[0035] In another embodiment of the invention, R1 in Formula 1 is a C3 or C4 alkyl group. In another embodiment of the invention, R1 in Formula 1 is a branched C3 or C4 alkyl group, such as isopropyl or tert-butyl.

[0036] In another embodiment of the invention, the compound of formula 1 is N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholino-4-carbonyl)pyrrolidine-3-yl)-N-((1S,4R)-4-methylcyclohexyl)trimethylacetamide of formula 2:

[0037] [Equation 2]

[0038]

[0039] In another embodiment of the invention, the compound of formula 1 is N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholino-4-carbonyl)pyrrolidine-3-yl)-N-((1S,4R)-4-methylcyclohexyl)isobutyramide of formula 3:

[0040] [Formula 3]

[0041]

[0042] In another embodiment of the invention, the compound of formula 1 is N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholino-4-carbonyl)pyrrolidine-3-yl)-N-((1S,4R)-4-methylcyclohexyl)isobutyramide of formula 4:

[0043] [Formula 4]

[0044]

[0045] In another embodiment of the invention, the compound of formula 1 is N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholino-4-carbonyl)pyrrolidine-3-yl)-N-((1S,4R)-4-methylcyclohexyl)trimethylacetamide of formula 5:

[0046] [Formula 5]

[0047]

[0048] In another embodiment of the invention, the compound of formula 1 is N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholin-4-carbonyl)pyrrolidine-3-yl)-N-(4,4-dimethylcyclohexyl)isobutyramide of formula 6:

[0049] [Formula 6]

[0050]

[0051] In another embodiment of the invention, the compound of formula 1 is N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholino-4-carbonyl)pyrrolidine-3-yl)-N-(4,4-dimethylcyclohexyl)trimethylacetamide of formula 7:

[0052] [Formula 7]

[0053]

[0054] In another embodiment of the invention, examples of pharmaceutically acceptable salts include acid addition salts formed from acids such as inorganic acids, including hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, and hydroiodic acid; organic carboxylic acids, including tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, and maleic acid; and sulfonic acids, including, but not limited to, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and naphthalenesulfonic acid.

[0055] In another embodiment of the invention, the pharmaceutically acceptable salt is a hydrochloride salt.

[0056] In another embodiment of the invention, the compound of formula 1 is N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholino-4-carbonyl)pyrrolidine-3-yl)-N-((1S,4R)-4-methylcyclohexyl)trimethylacetamide hydrochloride of formula 8:

[0057] [Formula 8]

[0058]

[0059] In another embodiment of the invention, the compound of formula 1 is N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholino-4-carbonyl)pyrrolidine-3-yl)-N-((1S,4R)-4-methylcyclohexyl)isobutyramide hydrochloride of formula 9:

[0060] [Formula 9]

[0061]

[0062] In another embodiment of the invention, the compound of Formula 1 is N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholino-4-carbonyl)pyrrolidine-3-yl)-N-((1S,4R)-4-methylcyclohexyl)isobutyramide hydrochloride of Formula 10:

[0063] [Formula 10]

[0064]

[0065] In another embodiment of the invention, the compound of formula 1 is N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholino-4-carbonyl)pyrrolidine-3-yl)-N-((1S,4R)-4-methylcyclohexyl)trimethylacetamide hydrochloride of formula 11:

[0066] [Equation 11]

[0067]

[0068] In another embodiment of the invention, the compound of formula 1 is N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholino-4-carbonyl)pyrrolidine-3-yl)-N-(4,4-dimethylcyclohexyl)isobutyramide hydrochloride of formula 12:

[0069] [Equation 12]

[0070]

[0071] In another embodiment of the invention, the compound of formula 1 is N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholino-4-carbonyl)pyrrolidine-3-yl)-N-(4,4-dimethylcyclohexyl)trimethylacetamide hydrochloride of formula 13:

[0072] [Equation 13]

[0073]

[0074] In another embodiment of the present invention, the N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholin-4-carbonyl)pyrrolidine-3-yl)-N-((1s,4R)-4-methylcyclohexyl)trimethylacetamide hydrochloride of Formula 8, and the N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholin-4-carbonyl)pyrrolidine-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide hydrochloride of Formula 9, are... The N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholin-4-carbonyl)pyrrolidine-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide hydrochloride of Formula 10, and the N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholin-4-carbonyl)pyrrolidine-3-yl)-N-((1s,4R)-4-methylcyclohexyl)trimethylacetamide hydrochloride of Formula 11 can be prepared according to the following reaction scheme 1.

[0075] [Reaction Scheme 1]

[0076]

[0077] In reaction scheme 1,

[0078] R1, R2, and R3 are the same as those defined above.

[0079] In another embodiment of the present invention, the N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholin-4-carbonyl)pyrrolidine-3-yl)-N-(4,4-dimethylcyclohexyl)isobutyramide hydrochloride of Formula 12 and the N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholin-4-carbonyl)pyrrolidine-3-yl)-N-(4,4-dimethylcyclohexyl)trimethylacetamide hydrochloride of Formula 13 can be prepared according to the following reaction scheme 2.

[0080] [Reaction Scheme 2]

[0081]

[0082] In reaction scheme 2,

[0083] R1 is the same as defined above.

[0084] The compounds of Formula 1 according to the invention exhibit excellent agonist activity against melanocortin receptors, particularly melanocortin-4 receptor (MC4R). Therefore, the present invention also provides melanocortin receptor agonist pharmaceutical compositions comprising a compound of Formula 1 or a pharmaceutically acceptable salt or isomer thereof as an active ingredient, and a pharmaceutically acceptable carrier. Specifically, the compositions according to the invention exhibit excellent effects in the prevention or treatment of obesity, diabetes, inflammation, and erectile dysfunction, but are not limited thereto.

[0085] In this specification, "carrier" refers to a compound that facilitates the injection of the compound into cells or tissues.

[0086] When the compounds of the present invention are administered for clinical purposes, the total daily dose administered to the host as a single dose or individual dose is preferably in the range of 0.01 mg to 10 mg per kg of body weight, but the specific dose level for an individual patient may depend on the specific compound used, the patient's weight, sex, health status and diet, time of administration, method of administration, drug excretion rate, drug mixture and disease severity.

[0087] Depending on the intended use, the compounds of the present invention can be administered via any route. For example, the compounds of the present invention can be administered by injection or orally.

[0088] According to known techniques, injectable formulations can be prepared by using suitable dispersants, wetting agents, or suspending agents.

[0089] Examples of solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules, and solid dosage forms can be prepared by mixing an active compound of Formula 1 according to the invention with one or more carriers (e.g., inert diluents, lubricants, disintegrants, and binders).

[0090] Beneficial effects

[0091] The compounds of Formula 1 according to the present invention exhibit excellent agonist activity against melanocortin receptors, particularly melanocortin-4 receptor (MC4R), and are therefore effective for the prevention or treatment of obesity, diabetes, inflammation and erectile dysfunction.

[0092] The compounds of Formula 1 according to the invention exhibit a targeting effect on the melanocortin-4 receptor, showing weight loss and reduced food intake without affecting anxiety and depression. Furthermore, the compounds of Formula 1 can be administered without causing inhibitory side effects on the human ether-a-go-go-related gene (hERG) and without safety issues such as mutations. Moreover, the compounds of Formula 1 according to the invention can be safely administered due to the absence of cytotoxicity and hepatotoxicity. Detailed Implementation

[0093] The invention is described in more detail below through the following embodiments. However, it must be understood that the scope of protection of the invention is not limited to the embodiments.

[0094] Preparation Example 1: Preparation of N-((1s,4R)-4-methylcyclohexyl)-N-((3S,5S)-5-(morpholin-4-carbonyl)pyrrolidine-3-yl)trimethylacetamide hydrochloride

[0095]

[0096] The title compound was obtained through the following steps A, B, and C.

[0097] Step A: Preparation of (2S,4S)-1-(tert-butoxycarbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)trimethyl Acetamidopyrrolidine-2-carboxylic acid

[0098]

[0099] The title compound was obtained by the method disclosed in International Publication No. WO 2008 / 007930.

[0100] MS[M+Na]=433.4(M+23)

[0101] 1 H NMR(400MHz,CD3OD)δ4.25(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)

[0102] Step B: Preparation of (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)trimethylacetamyl)-2-(morpholine- 4-Carbonyl)pyrrolidine-1-carboxylic acid tert-butyl ester

[0103]

[0104] The (2S,4S)-1-(tert-butoxycarbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)trimethylacetamyl)pyrrolidine-2-carboxylic acid (0.81 g, 1.97 mmol) obtained in step A 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-dimethylpropyl-1-amine hydrochloride (0.45 g, 2.36 mmol) and N,N-diisopropylethylamine (1.0 mL, 5.92 mmol) were added to the solution, and the mixture was stirred at room temperature for 16 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure, the filtrate was washed with aqueous sodium bicarbonate solution, and extracted with ethyl acetate. The organic layer was collected, washed with water, dried over anhydrous magnesium sulfate, and the solid was filtered off. The filtrate was concentrated under reduced pressure and purified by column chromatography to obtain the title compound (0.62 g, 66%).

[0105] MS[M+H]=480.5(M+1)

[0106] 1 H NMR(400MHz,CD3OD)δ4.65(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)

[0107] Step C: Preparation of N-((1s,4R)-4-methylcyclohexyl)-N-((3S,5S)-5-(morpholin-4-carbonyl)pyrrolidine- 3-yl)trimethylacetamide hydrochloride

[0108]

[0109] The (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)trimethylacetamyl)-2-(morpholino-4-carbonyl)pyrrolidine-1-carboxylic acid tert-butyl ester (0.62 g, 1.3 mmol) obtained in step B was dissolved in 4 mL of dichloromethane, and a solution of 1,4-dioxane in 4 M hydrochloric acid (1.3 mL, 5.1 mmol) was added. The mixture was stirred at room temperature for 16 hours. After the reaction solution was concentrated under reduced pressure, the resulting solid was washed with diethyl ether and dried to obtain the title compound (0.55 g, 99%).

[0110] MS[M+H]=380.6(M+1)

[0111] 1H NMR(400MHz,DMSO-d6)δ10.00-8.00(brs,2H),4.46(m,1H),4.14(m,1H),3.76(m,1H),3.65-3.25(m,1 0H),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)

[0112] Preparation Example 2: Preparation of N-((1s,4R)-4-methylcyclohexyl)-N-((3S,5S)-5-(morpholin-4-carbonyl)pyrrolidine-3-yl)isobutyramide hydrochloride

[0113]

[0114] The title compound was obtained through the following steps A, B, C, D, E, F, and G.

[0115] Step A: Preparation of (2S,4S)-4-azidopyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl)2-methyl ester

[0116]

[0117] (2S,4R)-4-((methanesulfonyl)oxy)pyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl)2-methyl ester (48.5 g, 150 mmol) was dissolved in 250 mL of dimethylformamide, and sodium azide (19.5 g, 300 mmol) was added. After stirring the mixture at 80 °C for 16 hours, the solution was concentrated under reduced pressure. Water was added, followed by extraction with ethyl acetate. 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 to obtain the title compound (39.59 g, 98%), which was used in the next step without purification.

[0118] MS[M+H]=271(M+1)

[0119] 1 H NMR(400MHz,CD3OD)δ4.43-4.37(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)

[0120] Step B: Preparation ( 2S, 4S 1-(tert-butyl)-2-methyl 4-aminopyrrolidine-1,2-dicarboxylic acid

[0121]

[0122] The (2S,4S)-4-azidopyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl)2-methyl ester (24.6 g, 91.0 mmol) obtained in step A was dissolved in 180 mL of tetrahydrofuran, and a 1 M trimethylphosphine tetrahydrofuran solution (109 mL, 109 mmol) was slowly added to it at 0 °C. After stirring at the same temperature for 1 hour, the reaction mixture was stirred at room temperature for 3 hours. After concentrating the reaction solvent under reduced pressure, 100 mL of dichloromethane and 150 mL of water were added, and the reaction mixture was stirred for about 30 minutes. The organic layer was dried over anhydrous magnesium sulfate, and the solid was filtered off. The filtrate was concentrated under reduced pressure to obtain the title compound (20.62 g, 93%).

[0123] MS[M+H]=245(M+1)

[0124] 1 H NMR(400MHz,CD3OD)δ4.27(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)

[0125] Step C: Preparation of (2S,4S)-4-((1S,4R)-4-methylcyclohexyl)amino)pyrrolidine-1,2-dicarboxylic acid 1-(tert-) Butyl 2-methyl ester

[0126]

[0127] The (2S,4S)-4-aminopyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl)2-methyl ester (20.6 g, 84.4 mmol) obtained in step B was dissolved in 150 mL of 1,2-dichloroethane, and 4-methylcyclohexanone (9.50 mL, 101 mmol) was added. After cooling the mixture to 0 °C, sodium triacetoxyborohydride (26.8 g, 127 mmol) was added, and the reaction mixture was stirred at room temperature for 16 hours. After concentrating the reaction solution under reduced pressure, water was added and the reaction mixture was extracted with ethyl acetate. The organic layer was washed with aqueous sodium chloride solution, 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%).

[0128] MS[M+H]=341(M+1)

[0129] 1H NMR (400MHz, CD3OD) δ4.26(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)

[0130] Step D: Preparation of (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-1,2-di 1-(tert-butyl)-2-methyl formate

[0131]

[0132] After dissolving (2S,4S)-4-((1S,4R)-4-methylcyclohexyl)amino)pyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl)2-methyl ester (37.29 g, 109.5 mmol) in 500 mL of dichloromethane, triethylamine (61.1 mL, 438 mmol) was added, followed by the slow addition of isobutyryl chloride (11.7 mL, 219 mmol) at 0 °C. After stirring at room temperature for 16 hours, the reaction solvent was concentrated under reduced pressure. The organic layer was separated after adding aqueous sodium bicarbonate and ethyl acetate for concentration. 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%).

[0133] MS[M+H]=411(M+1)

[0134] 1 H NMR (400MHz, CD3OD) δ4.27(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)

[0135] Step E: Preparation of (2S,4S)-1-(tert-butoxycarbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyryl (amino)pyrrolidine-2-carboxylic acid

[0136]

[0137] The (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutamido)pyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl)2-methyl ester (3.63 g, 8.85 mmol) obtained in step D was dissolved in 30 mL of ethanol, and 26.5 mL of 1 N aqueous sodium hydroxide solution (26.5 mmol) was added to the solution, and the mixture was stirred at room temperature for 2 hours. After diluting the reaction solution with water, the pH was adjusted to 4 with 1 N aqueous hydrochloric acid solution. The reaction mixture 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%).

[0138] MS[M+Na]=419.4(M+23)

[0139] 1 H NMR(400MHz,CD3OD)δ4.27(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)

[0140] Step F: Preparation of (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)-2-(morpholino-4-carbonyl) tert-butyl pyrrolidine-1-carboxylate

[0141]

[0142] The (2S,4S)-1-(tert-butoxycarbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutamido)pyrrolidine-2-carboxylic acid (2.40 g, 6.05 mmol), 1H-benzo[d][1,2,3]triazol-1-ol monohydrate (1.11 g, 7.26 mmol), and 3-(((ethylimino)methylene)amino)-N,N-dimethylpropyl-1-amine hydrochloride (1.39 g, 7.26 mmol) obtained in step E were dissolved in 30 mL of dimethylformamide. Morpholine (0.55 mL, 6.66 mmol) and N,N-diisopropylethylamine (3.10 mL, 18.2 mmol) were added to the mixture, followed by stirring at room temperature for 16 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure, and the filtrate was washed with aqueous sodium bicarbonate solution and extracted with ethyl acetate. The organic layer was collected, washed with water, and dried over anhydrous magnesium sulfate. The solid was filtered off. The filtrate was concentrated under reduced pressure and purified by column chromatography to obtain the title compound (1.87 g, 66%).

[0143] MS[M+H]=466.5(M+1)

[0144] 1 H NMR(400MHz,CD3OD)δ4.66(m,1H),3.94(m,1H),3.75-3.55(m,10H),3.45(m,1H),2.86(m,1H),2.2 4(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)

[0145] Step G: Preparation of N-((1s,4R)-4-methylcyclohexyl)-N-((3S,5S)-5-(morpholin-4-carbonyl)pyrrolidine- 3-yl)isobutyramide hydrochloride

[0146]

[0147] The (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramidoyl)-2-(morpholino-4-carbonyl)pyrrolidine-1-carboxylic acid tert-butyl ester (1.87 g, 4.00 mmol) obtained in step F above was dissolved in 30 mL of dichloromethane and cooled to 0 °C, and 4 M 1,4-dioxane hydrochloride (2.15 mL, 8.59 mmol) was added to the solution. After stirring the mixture at room temperature for 16 hours, the reaction solution was concentrated under reduced pressure. The resulting solid was washed with diethyl ether and dried to obtain the title compound (1.22 g, 76%).

[0148] MS[M+H]=366.4(M+1)

[0149] 1 H NMR(400MHz,DMSO-d6)δ9.88(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)

[0150] Preparation Example 3: Preparation of (2S,4S)-4-(N-(4,4-dimethylcyclohexyl)trimethylacetamido)pyrrolidine-2-carboxylic acid methyl ester hydrochloride

[0151]

[0152] The title compound was obtained through the following steps A and B.

[0153] Step A: Preparation of (2S,4S)-4-(N-(4,4-dimethylcyclohexyl)trimethylacetamido)pyrrolidine-1,2-di 1-(tert-butyl)-2-methyl formate

[0154]

[0155] The title compound was obtained by the method disclosed in International Publication No. WO 2008 / 007930.

[0156] MS[M+Na]=461.4(M+23)

[0157] 1 H NMR (400MHz, CD3OD) δ4.34(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)

[0158] Step B: Preparation of (2S,4S)-4-(N-(4,4-dimethylcyclohexyl)trimethylacetamido)pyrrolidine-2-carboxylic acid Methyl ester hydrochloride

[0159]

[0160] The (2S,4S)-4-(N-(4,4-dimethylcyclohexyl)trimethylacetamyl)pyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl)2-methyl ester (1.67 g, 3.81 mmol) obtained in step A was dissolved in 4 mL of ethyl acetate, and 4 M ethyl acetate hydrochloride solution (3.81 mL, 15.2 mmol) was added to the solution. The mixture was stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure to obtain the title compound (1.43 g, 99%).

[0161] MS[M+H]=339.4(M+1)

[0162] 1 H NMR(400MHz,CD3OD)δ4.41(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)

[0163] Preparation Example 4: Preparation of (2S,4S)-4-(N-(4,4-dimethylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylic acid methyl ester hydrochloride

[0164]

[0165] The title compound was obtained through the following steps A and B.

[0166] Step A: Preparation of (2S,4S)-4-(N-(4,4-dimethylcyclohexyl)isobutyramido)pyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl)-2-methyl ester

[0167]

[0168] The title compound was obtained by the method disclosed in International Publication No. WO 2008 / 007930.

[0169] MS[M+Na]=447.4(M+23)

[0170] 1 H NMR (400MHz, CD3OD) δ4.31(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)

[0171] Step B: Preparation of methyl (2S,4S)-4-(N-(4,4-dimethylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylate hydrochloride

[0172]

[0173] The (2S,4S)-4-(N-(4,4-dimethylcyclohexyl)isobutamido)pyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl)2-methyl ester (1.1 g, 2.6 mmol) obtained in step A was dissolved in 2.5 mL of ethyl acetate, and 2.5 mL of 4M ethyl acetate hydrochloride solution (10 mmol) was added to the solution. The reaction solution was stirred at room temperature for 16 hours and concentrated under reduced pressure to obtain the title compound (0.93 g, 99%).

[0174] MS[M+H]=325.4(M+1)

[0175] 1H NMR(400MHz,CD3OD)δ4.41(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)

[0176] Preparation Example 5: Preparation of (3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carboxylic acid

[0177]

[0178] The title compound was obtained by the method disclosed in International Publication No. WO 2004 / 092126.

[0179] MS[M+H]=282(M+1)

[0180] 1 H NMR (400MHz, CD3OD) δ7.43-7.33(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)

[0181] Preparation Example 6: Preparation of (3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carboxylic acid

[0182]

[0183] The title compound was obtained by the method disclosed in International Publication No. WO 2004 / 092126.

[0184] MS[M+H]=284.2(M+1)

[0185] 1 H NMR (400MHz, CD3OD) δ7.50(m,1H),6.97(m,2H),3.93-3.75(m,3H),3.60(m,1H),3.26(m,2H),1.43(s,9H)

[0186] Preparation Example 7: Preparation of (3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carboxylic acid

[0187]

[0188] The title compound was obtained by the method disclosed in International Publication No. WO 2004 / 092126.

[0189] MS[M+H]=300.3(M+1)

[0190] 1 H NMR (400MHz, CD3OD) δ7.47(t,1H),7.22(m,2H),3.93-3.75(m,3H),3.60(m,1H),3.26(m,2H),1.43(s,9H)

[0191] Example 1: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholino-4-carbonyl)pyrrolidine-3-yl)-N-((1S,4R)-4-methylcyclohexyl)trimethylacetamide hydrochloride

[0192]

[0193] The title compound was obtained through the following steps A and B.

[0194] Step A: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3- (carbonyl)-5-(morpholino-4-carbonyl)pyrrolidine-3-yl)-N-((1s,4R)-4-methylcyclohexyl)trimethylacetamide

[0195]

[0196] 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-(morpholino-4-carbonyl)pyrrolidine-3-yl)carboxylic acid (0.34 g, 1.2 mmol) obtained in Preparation Example 1 and (3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carboxylic acid (0.34 g, 1.2 mmol) obtained in Preparation Example 6. The mixture was stirred at room temperature for 16 hours. After the reaction solution was concentrated under reduced pressure, ethyl acetate was added. The reaction mixture was washed with aqueous ammonium chloride and 1M sodium hydroxide solution, and then dried over anhydrous sodium sulfate. The solid was filtered, the filtrate was concentrated under reduced pressure and purified by chromatography to obtain the title compound (0.32 g, 40%).

[0197] MS[M+H]=645.6(M+1)

[0198] 1H NMR(400MHz,CD3OD)δ7.58(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)

[0199] Step B: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3- (carbonyl)-5-(morpholino-4-carbonyl)pyrrolidine-3-yl)-N-((1s,4R)-4-methylcyclohexyl)trimethylacetamide hydrochloride

[0200]

[0201] Add 5 mL of dichloromethane and a 4M ethyl acetate hydrochloride solution (0.25 mL, 1.0 mmol) to the N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholino-4-carbonyl)pyrrolidine-3-yl)-N-((1S,4R)-4-methylcyclohexyl)trimethylacetamide (0.32 g, 0.50 mmol) obtained in step A, and stir for 20 minutes at room temperature. Concentrate the reaction solution under reduced pressure and add diethyl ether. Filter the resulting solid and dry it to obtain the title compound (0.23 g, 68%).

[0202] MS[M+H]=645.6(M+1)

[0203] 1 H NMR (400MHz, CD3OD) δ7.63(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)

[0204] Example 2: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholin-4-carbonyl)pyrrolidine-3-yl)-N-((1S,4R)-4-methylcyclohexyl)isobutyramide hydrochloride

[0205]

[0206] The title compound was obtained through the following steps A and B.

[0207] Step A: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3- (carbonyl)-5-(morpholino-4-carbonyl)pyrrolidine-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide

[0208]

[0209] The title compound (0.10 g, 9%) was obtained using (3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carboxylic acid (0.50 g, 1.76 mmol) obtained in Preparation Example 6 and N-((1s,4R)-4-methylcyclohexyl)-N-((3S,5S)-5-(morpholino-4-carbonyl)pyrrolidine-3-yl)isobutyramide hydrochloride (0.71 g, 1.76 mmol) obtained in Preparation Example 2 in the same manner as in step A of Example 1.

[0210] MS[M+H]=631.5(M+1)

[0211] 1 H NMR (400MHz, CD3OD) δ7.56(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)

[0212] Step B: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3- (carbonyl)-5-(morpholino-4-carbonyl)pyrrolidine-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide hydrochloride

[0213]

[0214] The title compound (0.083 g, 78%) was obtained using N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholino-4-carbonyl)pyrrolidine-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide (0.10 g, 0.16 mmol) obtained in step A, in the same manner as in step B of Example 1.

[0215] MS[M+H]=631.5(M+1)

[0216] 1H NMR(400MHz,CD3OD)δ7.63(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)

[0217] Example 3: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholino-4-carbonyl)pyrrolidine-3-yl)-N-((1S,4R)-4-methylcyclohexyl)isobutyramide hydrochloride

[0218]

[0219] The title compound was obtained through the following steps A and B.

[0220] Step A: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3- (carbonyl)-5-(morpholino-4-carbonyl)pyrrolidine-3-yl)-N-((1s,4R)-4-methylcyclohexyl)

[0221] Isobutyramide

[0222]

[0223] The title compound (0.13 g, 12%) was obtained using (3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carboxylic acid (0.50 g, 1.67 mmol) obtained in Preparation Example 7 and N-((1s,4R)-4-methylcyclohexyl)-N-((3S,5S)-5-(morpholino-4-carbonyl)pyrrolidine-3-yl)isobutyramide hydrochloride (0.67 g, 1.67 mmol) obtained in Preparation Example 2 in the same manner as in step A of Example 1.

[0224] MS[M+H]=647.5(M+1)

[0225] 1H NMR (400MHz, CD3OD) δ7.54(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)

[0226] Step B: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3- (carbonyl)-5-(morpholino-4-carbonyl)pyrrolidine-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide hydrochloride

[0227]

[0228] The title compound (0.1 g, 80%) was obtained using N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholino-4-carbonyl)pyrrolidine-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide (0.13 g, 0.20 mmol) obtained in step A, in the same manner as in step B of Example 1.

[0229] MS[M+H]=647.5(M+1)

[0230] 1 H NMR (400MHz, CD3OD) δ7.60(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)

[0231] Example 4: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholino-4-carbonyl)pyrrolidine-3-yl)-N-((1S,4R)-4-methylcyclohexyl)trimethylacetamide hydrochloride

[0232]

[0233] The title compound was obtained through the following steps A and B.

[0234] Step A: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3- (carbonyl)-5-(morpholino-4-carbonyl)pyrrolidine-3-yl)-N-((1s,4R)-4-methylcyclohexyl)trimethylacetamide

[0235]

[0236] The title compound (0.45 g, 56%) was obtained using (3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carboxylic acid (0.36 g, 1.21 mmol) obtained in Preparation Example 7 and N-((1s,4R)-4-methylcyclohexyl)-N-((3S,5S)-5-(morpholino-4-carbonyl)pyrrolidine-3-yl)trimethylacetamide hydrochloride (0.50 g, 1.21 mmol) obtained in Preparation Example 1 in the same manner as in step A of Example 1.

[0237] MS[M+H]=661.6(M+1)

[0238] 1 H NMR (400MHz, CD3OD) δ7.55(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)

[0239] Step B: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3- (carbonyl)-5-(morpholino-4-carbonyl)pyrrolidine-3-yl)-N-((1s,4R)-4-methylcyclohexyl)trimethylacetamide hydrochloride

[0240]

[0241] The title compound (0.18 g, 68%) was obtained using N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholino-4-carbonyl)pyrrolidine-3-yl)-N-((1s,4R)-4-methylcyclohexyl)trimethylacetamide (0.25 g, 0.38 mmol) obtained in step A, in the same manner as in step B of Example 1.

[0242] MS[M+H]=661.6(M+1)

[0243] 1H NMR (400MHz, CD3OD) δ7.60(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)

[0244] Example 5: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholin-4-carbonyl)pyrrolidine-3-yl)-N-(4,4-dimethylcyclohexyl)isobutyramide hydrochloride

[0245]

[0246] The title compound was obtained through the following steps A, B, C, and D.

[0247] Step A: Preparation of (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4- Methyl (N-(4,4-dimethylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylate

[0248]

[0249] The title compound (0.49 g, 32%) was obtained using (3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carboxylic acid (0.73 g, 2.58 mmol) obtained in Preparation Example 5 and (2S,4S)-4-(N-(4,4-dimethylcyclohexyl)isobutamido)pyrrolidine-2-carboxylic acid methyl hydrochloride (0.93 g, 2.58 mmol) obtained in Preparation Example 4, in the same manner as in step A of Example 1.

[0250] MS[M+H]=588.5(M+1)

[0251] 1H NMR(400MHz,CD3OD)δ7.30(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)

[0252] 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

[0253]

[0254] The methyl (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-(4,4-dimethylcyclohexyl)isobutamido)pyrrolidine-2-carboxylate (0.49 g, 0.83 mmol) obtained in step A was dissolved in methanol (2.8 mL) and cooled to 0 °C. A 6 M sodium hydroxide aqueous solution (0.7 mL, 4.2 mmol) was added to the solution. The mixture was stirred at room temperature for 16 hours and then concentrated under reduced pressure. Water was added to adjust the pH to 4, and the reaction mixture was concentrated under reduced pressure. The mixture was dissolved in dichloromethane, and the solids were removed by filtration. The filtrate was concentrated under reduced pressure to obtain the title compound (0.47 g, 98%).

[0255] MS[M+H]=574.4(M+1)

[0256] 1 H NMR(400MHz,CD3OD)δ7.40(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)

[0257] Step C: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl) 5-(morpholino-4-carbonyl)pyrrolidine-3-yl)-N-(4,4-dimethylcyclohexyl)isobutyramide

[0258]

[0259] The title compound (0.41 g, 78%) was obtained using (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-(4,4-dimethylcyclohexyl)isobutamido)pyrrolidine-2-carboxylic acid (0.47 g, 0.82 mmol) and morpholine (0.071 mL, 0.82 mmol) obtained in step B, in the same manner as in step A of Example 1.

[0260] MS[M+H]=643.4(M+1)

[0261] 1 H NMR (400MHz, CD3OD) δ7.34(m,2H),7.30(m,2H),4.77(m,1H),4.12(m,1H),3.80-3.30(m,15H),3.05(m,1H),2.7 7(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)

[0262] Step D: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl) 3-(4,4-dimethylcyclohexyl)isobutyramide hydrochloride

[0263]

[0264] The title compound (0.37 g, 83%) was obtained using N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholino-4-carbonyl)pyrrolidine-3-yl)-N-(4,4-dimethylcyclohexyl)isobutyramide (0.42 g, 0.65 mmol) obtained in step C, in the same manner as in step B of Example 1.

[0265] MS[M+H]=643.4(M+1)

[0266] 1 H NMR (400MHz, CD3OD) δ7.44(m,4H),4.81(m,1H),4.22(m,1H),3.90(m,1H),3.80-3.40(m,14H),2.99(m,1H),2.8 2(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)

[0267] Example 6: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholin-4-carbonyl)pyrrolidine-3-yl)-N-(4,4-dimethylcyclohexyl)trimethylacetamide hydrochloride

[0268]

[0269] The title compound was obtained through the following steps A, B, C, and D.

[0270] Step A: Preparation of (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4- Methyl pyrrolidine-2-carboxylate (N-(4,4-dimethylcyclohexyl)trimethylacetamyl)pyrrolidine-2-carboxylate

[0271]

[0272] The title compound (0.63 g, 49%) was obtained using (3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carboxylic acid (0.60 g, 2.13 mmol) obtained in Preparation Example 5 and (2S,4S)-4-(N-(4,4-dimethylcyclohexyl)trimethylacetamyl)pyrrolidine-2-carboxylic acid methyl ester hydrochloride (0.80 g, 2.13 mmol) obtained in Preparation Example 3, in the same manner as in step A of Example 1.

[0273] MS[M+H]=602.5(M+1)

[0274] 1 H NMR(400MHz,CD3OD)δ7.34(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)

[0275] Step B: Preparation of (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4- (N-(4,4-dimethylcyclohexyl)trimethylacetamyl)pyrrolidine-2-carboxylic acid

[0276]

[0277] The title compound (0.51 g, 83%) was obtained using methyl pyrrolidine-2-carboxylate (0.63 g, 1.05 mmol) obtained in step A, in the same manner as in step B of Example 5.

[0278] MS[M+H]=588.5(M+1)

[0279] 1 H NMR(400MHz,CD3OD)δ7.42(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)

[0280] Step C: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl) 5-(morpholino-4-carbonyl)pyrrolidine-3-yl)-N-(4,4-dimethylcyclohexyl)trimethylacetamide

[0281]

[0282] The title compound (0.44 g, 77%) was obtained using (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-(4,4-dimethylcyclohexyl)trimethylacetamyl)pyrrolidine-2-carboxylic acid (0.51 g, 0.87 mmol) and morpholine (0.076 mL, 0.87 mmol) obtained in step B, in the same manner as in step A of Example 1.

[0283] MS[M+H]=657.6(M+1)

[0284] 1 H NMR(400MHz,CD3OD)δ7.36(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)

[0285] Step D: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl) 5-(morpholino-4-carbonyl)pyrrolidine-3-yl)-N-(4,4-dimethylcyclohexyl)trimethylacetamide hydrochloride

[0286]

[0287] The title compound (0.39 g, 84%) was obtained using N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholino-4-carbonyl)pyrrolidine-3-yl)-N-(4,4-dimethylcyclohexyl)trimethylacetamide (0.44 g, 0.67 mmol) obtained in step C, in the same manner as in step B of Example 1.

[0288] MS[M+H]=657.6(M+1)

[0289] 1 H NMR (400MHz, CD3OD) δ7.45(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)

[0290] 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)pyrrolidine-3-yl)-N-(4,4-dimethylcyclohexyl)acetamide hydrochloride (A95)

[0291]

[0292] Compound A95 of International Publication No. WO 2008 / 007930 was obtained by the same method disclosed therein.

[0293] Comparative Example 2: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl) 5-(morpholino-4-carbonyl)pyrrolidine-3-yl)-N-(4,4-dimethylcyclohexyl)acetamide hydrochloride (A96)

[0294]

[0295] Compound A96 in International Publication No. WO 2008 / 007930 was obtained by the same method disclosed therein.

[0296] Experimental Example 1: Luciferase Assay

[0297] To determine the agonist activity against MC4R (melanocortin-4 receptor), a cell line permanently expressing the luciferase gene under the control of MC4R and CRE (cAMP response element) was established (CRE-LUC). After preparing a mammalian cell expression vector containing the MC4R gene (pCDNA3(Neo)) (Invitrogen), human embryonic kidney (HEK) cell lines were transformed using Lipofectamine 2000 (Invitrogen) and a vector expressing the luciferase gene (CRE-LUC) under the control of the cAMP response element (CRE) (pCRE-Luc) (Stratagen). The transformed cell line (HEK MC4R-Luc) was cultured for 24 hours at 37°C in the presence of 5% CO2 in Dulbecco modified Eagles medium (DMEM) containing 10% heat-inactivated fetal bovine serum (GIBCO / BRL). Cell lines were cultured for four days in Dulbecco modified Eagles medium (DMEM) containing 10 ml of selective medium (10% heat-inactivated fetal bovine serum (GIBCO / BRL), 100 IU / ml penicillin (GIBCO / BRL), 100 IU / ml streptomycin (GIBCO / BRL), and 800 μg / ml genimycin (G418) (GIBCO / BRL). The process of replacing the selective medium with 10 ml of fresh selective medium to remove cells killed by the selective medium was repeated three times, every four days. Single colonies formed from the final selected and proliferated clones were transferred under a microscope to 24-well cell culture plates, each well containing 1 ml of selective medium, and cultured for 4 days. The cells were then treated with a final concentration of 10 μM Forskolin (SIGMA) and incubated at 37°C for 5% [the desired cell line concentration is missing from the original text]. Incubate for 5 hours in the presence of CO2. Each well is treated with 50 μl of Bright-Glo luciferase reagent (Promega) and incubated at room temperature for 15 minutes, after which the luminescence of each well is measured using a photometer (Victor). Clones that show luminescence levels 100 times or more than the baseline value after Forskolin treatment are selected for measuring the MC4R agonist activity of each compound.

[0298] HEK MC4R-Luc cells were added to each well of a 9-well 6-well spectrophotometer cell culture plate (Costar) at a scale of 2.5 × 10⁶ cells per 100 μl medium. 4Cells were cultured at 37°C for 18 hours in the presence of 6% CO2. The MCR agonist was diluted at each concentration level using the above medium to ensure a final DMSO concentration of no more than 1%, and then cultured at 37°C for 5 hours in the presence of 6% CO2. Each well was treated with 50 μl of Bright-Glo luciferase reagent (Promega) and incubated at room temperature for 5 minutes, after which luminescence was measured using a photometer (Victor). The amount of luminescence induced by each concentration dilution of the agonist was converted to a relative % value relative to the amount of luminescence shown by treatment with 10 μM NDP-α-MSH. EC 0.5 MSH is represented as the concentration that induces 50% of the maximum luminescence induced by NDP-α-MSH, and EC50 is represented as the concentration that induces 50% of the maximum luminescence induced by each agonist. Measurements were taken using statistical software (Prizm).

[0299] Table 1 shows the measured MC4R agonist activity of each compound obtained through the above experiments, in EC50. 50 (nM).

[0300] [Table 1]

[0301]

[0302] As shown in Table 1, it has been demonstrated that, among the well-known melanocorticoid receptors in vivo, the compounds in the examples have superior MC4R agonist activity compared to the comparative example compounds (A95 and A96) regarding the melanocorticoid-4 receptor (MC4R), which is involved in energy metabolism and weight control in vivo.

[0303] Experimental Example 2: cAMP Measurement

[0304] The melanocortin receptor is a G protein-coupled receptor (GPCR). The primary function of G proteins is to activate second messengers to regulate cellular responses to various physiological stimuli through signal transduction. MC4R is a Gs-coupled receptor, and it is well known that when MC4R interacts with an agonist, adenylate cyclase (AC) is activated to increase the concentration of cyclic adenosine monophosphate (cAMP), one of the cell's second messengers. Therefore, the activity of the melanocortin receptor can be assessed by measuring cAMP signaling.

[0305] After establishing cAMP-Hunter Gs-coupled cell lines (CHO-K1 cell lines) in which MC1R, MC3R, MC4R, and MC5R are each overexpressed, the increase in intracellular cAMP levels due to agonist response can be measured. Cells were seeded into each well of a white cell culture plate and cultured at 37°C in the presence of 5% CO2 for 24 hours. After culture, the medium was removed, and 15 μl of 2:1 HBBS / 10 mM HEPES:cAMP XS+Ab reagent was added. After adding 5 μl of the sample diluted four times with buffer, the solvent concentration was set to 1%, and the MC4R agonist compound diluted at each concentration level was added, followed by reaction at 37°C for 30 minutes. The activity (%) of each agonist compound is expressed as 100% × (mean RLU value of the sample - mean RLU value of the solvent control) / (mean RLU value of the maximum control - mean RLU value of the solvent control), which was analyzed using the CBIS data analysis suite (ChemInnovation, California).

[0306] Table 2 shows the measurement results of the melanocortin receptor agonist activity of the compounds obtained through the above experiments, in EC5. 50 (nM).

[0307] [Table 2]

[0308]

[0309] As shown in Table 2, it has been demonstrated that, among the well-known melanocortin receptors in vivo, the compounds in the examples have superior receptor agonist activity compared to the comparative example compounds (A95 and A96) for the melanocortin-4 receptor (MC4R), which is involved in energy metabolism and weight control in vivo.

[0310] Experimental Example 3: Measurement of β-repressor protein

[0311] The melanocortin receptor is a G protein-coupled receptor (GPCR) that regulates various physiological responses by transmitting signals from multiple neurotransmitters. When the GPCR is phosphorylated, β-repressor protein binds to the phosphorylated portion of the receptor and plays a crucial role in activating various signaling pathways in cells through interactions with other proteins. It is well known that when the melanocortin receptor interacts with agonists, β-repressor protein is mobilized and participates in β-repressor protein-mediated signaling pathways. Therefore, the activity of the melanocortin receptor can be assessed by measuring β-repressor protein levels.

[0312] A Pathhunter eXpress β-repressor protein cell line (U2OS cell line) was established, which simultaneously expresses Prolink (PK)-labeled MC1R, MC3R, MC4R, MC5R and enzyme receptor (EA)-labeled β-repressor protein. When the MCR-PK moiety of this cell line is activated, the β-repressor protein-EA is mobilized and interacts with Prolink (PK) as the enzyme receptor (EA) for the β-galactosidase fragment. The activated enzyme hydrolyzes the substrate through β-galactosidase activity, generating a chemiluminescent signal, which can be used to measure activity. After culturing the Pathhunter β-repressor protein cell line (U2OS cell line), cells were seeded into each well of a cell culture plate and cultured at 37°C in the presence of 5% CO2 for 48 h. After culture, 5 μl of sample diluted 5 times with buffer was added to set the solvent concentration to 1%, and MC4R agonist compound diluted at each concentration level was added, followed by reaction at 37°C for 90 min. The activity (%) of each agonist compound is expressed as 100% × (mean RLU value of the sample - mean RLU value of the solvent control) / (mean maximum value of the control ligand - mean RLU value of the solvent control), which was analyzed by the CBIS data analysis suite (ChemInnovation, California).

[0313] Table 3 shows the measurement results of melanocortin receptor activity of each compound obtained through the above experiments, in EC5. 50 (nM).

[0314] [Table 3]

[0315]

[0316] As shown in Table 3, it has been demonstrated that, among the well-known melanocortin receptors in vivo, the compounds in the examples have superior receptor agonist activity compared to the comparative example compounds (A95 and A96) for the melanocortin-4 receptor (MC4R), which is involved in energy metabolism and weight control in vivo.

[0317] Experiment Example 4: Binding Affinity

[0318] Five melanocortin receptor (MCR) subtypes are known in the body, with subtype 4 (MC4R) being involved in energy metabolism and weight control. Since other MCR subtypes are involved in the regulation of various functions in the body, such as skin pigmentation, energy homeostasis, and exocrine function, ensuring the selectivity of MC4R agonist compounds for MC4R is crucial for preventing potential future side effects. Therefore, the receptor binding affinity of MC4R agonists for each MCR subtype was determined.

[0319] After establishing the CHO-K1 cell line expressing recombinant human MC1R and the HEK-293 cell line expressing MC3R, MC4R, and MC5R, membranes were collected from each cell line. In 96-well cell culture plates, 3 μg of MC1R membrane and 0.04 nM [amount missing] were added to each well. 125 I-NDP-α-MSH was reacted at 37°C for 2 hours. 3 μg MC3R and MC5R membranes were reacted with 0.035 nM... 125 I-NDP-α-MSH was reacted at 37°C for 1 hour, and 3.12 μg MC4R membrane was reacted with 0.02 nM... 125 I-NDP-α-MSH was reacted at 37°C for 2 hours. During this time, 25 mM HEPES-KOH adsorption buffer (pH 7.0) containing MCR agonists diluted at each concentration level was added to each well and the reaction was carried out. The reaction solution was transferred to a filter and washed with the adsorption buffer, and then radioactivity was measured. The non-specific binding amounts of NDP-α-MSH in the presence of 1 μM (MC1R) and 3 μM (MC3R, MC4R, MC5R) were excluded from each total binding amount and used as the final value. 125 The specific binding amount of I-NDP-α-MSH was measured. The agonist's effect was assessed at each concentration level. 125 The degree of inhibition of I-NDP-α-MSH specific binding. IC50 represents 50% inhibition. 125 The concentration of each agonist that I-NDP-α-MSH specifically binds to.

[0320] Tables 4-1 and 4-2 show the measurement results of melanocortin receptor binding of the compounds obtained through the above experiments, in Ki (nM).

[0321] [Table 4-1]

[0322]

[0323] [Table 4-2]

[0324]

[0325] As shown in Tables 4-1 and 4-2, it has been confirmed that among known melanocortin receptors in vivo, the compounds in the examples have superior receptor binding ability for the melanocortin-4 receptor (MC4R), which is involved in energy metabolism and weight control in vivo.

Claims

1. A compound having a structural formula selected from the following or a pharmaceutically acceptable salt thereof: , , , and .

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of: N -((3 S 5 S )-1-((3 S 4 R )-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholin-4-carbonyl)pyrrolidine-3-yl)- N -((1 s 4 R )-4-methylcyclohexyl)isobutyramide; and N -((3 S 5 S )-1-((3 S 4 R )-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholin-4-carbonyl)pyrrolidine-3-yl)- N -((1 s 4 R 4-Methylcyclohexyl)isobutyramide.

3. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of: N -((3 S 5 S )-1-((3 S 4 R )-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholin-4-carbonyl)pyrrolidine-3-yl)- N -((1 s 4 R 4-Methylcyclohexyl)trimethylacetamide; N -((3 S 5 S )-1-((3 S 4 R )-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholin-4-carbonyl)pyrrolidine-3-yl)- N -((1 s 4 R 4-Methylcyclohexyl)trimethylacetamide; and N -((3 S 5 S )-1-((3 S 4 R )-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholin-4-carbonyl)pyrrolidine-3-yl)- N -(4,4-dimethylcyclohexyl)trimethylacetamide.

4. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is N -((3 S 5 S )-1-((3 S 4 R )-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholin-4-carbonyl)pyrrolidine-3-yl)- N -((1 s 4 R 4-methylcyclohexyl)trimethylacetamide.

5. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is N -((3 S 5 S )-1-((3 S 4 R )-1-(tert-butyl)-4-(2,4-difluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholin-4-carbonyl)pyrrolidine-3-yl)- N -((1 s 4 R 4-Methylcyclohexyl)isobutyramide.

6. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is N -((3 S 5 S )-1-((3 S 4 R )-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholin-4-carbonyl)pyrrolidine-3-yl)- N -((1 s 4 R 4-Methylcyclohexyl)isobutyramide.

7. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is N -((3 S 5 S )-1-((3 S 4 R )-1-(tert-butyl)-4-(4-chloro-2-fluorophenyl)pyrrolidine-3-carbonyl)-5-(morpholin-4-carbonyl)pyrrolidine-3-yl)- N -((1 s 4 R 4-methylcyclohexyl)trimethylacetamide.

8. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is N -((3 S 5 S )-1-((3 S 4 R )-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholin-4-carbonyl)pyrrolidine-3-yl)- N -(4,4-dimethylcyclohexyl)trimethylacetamide.

9. The compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt is an acid addition salt formed from an acid selected from hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid and hydroiodic acid.

10. The compound of claim 9 or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt is a hydrochloride salt.

11. A melanocortin-4 receptor agonist pharmaceutical composition comprising a compound as defined in any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

12. Use of a composition comprising any one of the compounds according to claims 1 to 10 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention or treatment of obesity, diabetes, inflammation, or erectile dysfunction.

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