Melanocortin-4 receptor agonists

Abstract

The present invention relates to melanocortin-4 receptor agonists. More specifically, the present invention relates to a compound of Formula 1, a pharmaceutical composition comprising the compound as an active ingredient, and a use thereof, and the compound of the present invention exhibits excellent agonist activity against the melanocortin-4 receptor, and can be particularly used for the prevention or treatment of obesity, diabetes, inflammation, and erectile dysfunction.

Description

[0001] This application is a divisional application of the application with international application date November 6, 2020, international application number PCT / KR2020 / 015462, entering the Chinese national phase on December 10, 2021, application number 202080042935.X, and invention name “Melanocortin-4 receptor agonist”. Technical Field

[0002] The present invention relates to a compound that exhibits excellent agonist activity against melanocortin receptors. More specifically, the present invention relates to a compound of the following formula 1, a pharmaceutical composition comprising the compound as an active ingredient, and use thereof, and the compound of the present invention exhibits excellent agonist activity against melanocortin-4 receptors and may be particularly useful in preventing or treating obesity, diabetes, inflammation, and erectile dysfunction:

[0003] [Formula 1]

[0004]

[0005] Wherein R1 is C2-C5 alkyl. Background Art

[0006] Leptin protein is a hormone secreted by body fat cells (adipocytes), and its secretion increases with the increase of body fat content. Leptin protein regulates the functions of various neuropeptides produced in the hypothalamus, thereby regulating appetite, body fat content and various body functions including energy metabolism, (Schwartz et al., Nature 404, 661-671 (2000)). The signal transduction of leptin protein to control appetite and body weight is achieved by regulating many downstream factors, among which the most representative are melanocortin, agouti-related peptide (AgRP) and neuropeptide Y (NPY) hormones.

[0007] When the leptin concentration in the blood increases due to excess calories in the body, the secretion of the pro-opiomelanocortin (POMC) protein hormone in the pituitary gland increases, and the production of AgRP and NPY decreases. α-MSH (melanocyte stimulating hormone) is a small peptide hormone produced by POMC neurons, which is an agonist of the melanocortin-4 receptor (MC4R) of the second neuron, ultimately causing a decrease in appetite. On the other hand, when the leptin concentration decreases due to insufficient calories, the expression of AgRP, which is an antagonist of MC4R, increases, and the expression of NPY also increases, which ultimately increases appetite. That is, according to the changes in leptin, the α-MSH hormone and the AgRP hormone participate in appetite regulation as agonists and antagonists of MC4R.

[0008] In addition to MC4R, α-MSH hormone also causes various physiological reactions by binding to 3 MCR subtypes. So far, 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, which are mainly expressed in the central nervous system, are involved in regulating appetite, energy metabolism and body fat storage efficiency, 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 an effective effect on reducing body weight by causing a decrease in appetite and an increase in energy metabolism, and has therefore been shown to be a major point of action for the development of obesity drugs (reviewed in: 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).

[0009] The role of MC4R in appetite and weight control has been preliminarily demonstrated by experiments on an animal model of abnormal expression of agouti protein (agouti mice). In the case of agouti mice, agouti protein was found to be expressed at high concentrations in the central nervous system and to act as an antagonist of MC4R in the hypothalamus due to gene mutation, thereby causing obesity (Yen, TT et al., FASEB J. 8, 479-488 (1994); and Lu D et al., Nature 371, 799-802 (1994)). In subsequent research results, it was observed that AgRP (agouti-related peptide) similar to actual agouti protein was expressed in hypothalamic nerves, and it is known that AgRP is involved 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)).

[0010] Intracerebral administration of α-MSH, which is an in vivo agonist of MC4R, to animals showed an effect of reducing appetite, and if the animals were treated with SHU9119 (peptide) or HS014 (peptide), which are MC4R antagonists, an effect of increasing appetite was observed again (Kask et al., Biochem. Biophys. Res. Comm. 245, 90-93 (1998)). In addition, in animal experiments using melanin II (MTII, Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2) and HP228 (which is a similar agonist), appetite suppression, weight loss, and energy metabolism increase effects were confirmed after brain, intraperitoneal or subcutaneous administration. (Thiele TE et al., Am J Physiol 274 (1Pt 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 the representative SHU9119 was administered to animals, significant and sustained increases in food intake and body weight were observed, providing pharmacological evidence that MCR agonists can be used to treat obesity. The appetite-reducing effect evident during MTII administration was not observed in MC4R KO (knockout) mice, and this experimental result once again demonstrated that the appetite-reducing effect was mainly achieved through activation of MC4R (Marsh et al., Nat Genet 21119-122 (1999)).

[0011] Appetite suppressants acting on the central nervous system are the main drugs for obesity treatment developed so far, and most inhibitors are drugs that regulate the action of neurotransmitters. Its embodiments include norepinephrine agents (phentermine and mazindol) and fluoxetine and positive controls, all of which are serotonergic agents. However, neurotransmitter regulators, in addition to suppressing appetite, also have a wide range of effects on various physiological effects through many subtype receptors. Therefore, regulators lack selectivity for each subtype, and there is a great disadvantage that long-term administration is accompanied by various side effects.

[0012] On the other hand, melanocortin is a neuropeptide, not a neurotransmitter, and considering that all other functions of MC4R gene KO mice are normal except for energy metabolism, the advantage of melanocortin agonists as a point of action is that they can only induce weight loss by suppressing appetite without affecting other physiological functions. Specifically, the receptor is a G protein-coupled receptor (GPCR), which belongs to the most successful category of new drug action points developed so far, and is very different from the action points in the related field because it is relatively easy to ensure selectivity for subtype receptors.

[0013] As examples of using the melanocortin receptor as an action point, International Publications WO 2008 / 007930 and WO 2010 / 056022 disclose compounds as melanocortin receptor agonists. Summary of the invention

[0014] Technical issues

[0015] 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).

[0016] Another object of the present invention is to provide a method for preparing the compound represented by Formula 1.

[0017] Another object of the present invention is to provide a melanocortin receptor agonist pharmaceutical composition comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt or isomer thereof as an active ingredient.

[0018] Another object of the present invention is to provide a compound represented by Formula 1 or a pharmaceutically acceptable salt or isomer thereof for use in preventing or treating obesity, diabetes, inflammation and erectile dysfunction.

[0019] Another object of the present invention is 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.

[0020] Technical Solutions

[0021] In order to achieve the above object, the present invention provides a compound of the following formula 1 or a pharmaceutically acceptable salt or isomer thereof:

[0022] [Formula 1]

[0023]

[0024] Wherein R1 is C2-C5 alkyl.

[0025] The compound of formula 1 according to the present invention can form a pharmaceutically acceptable salt.

[0026] In addition, since the compounds according to the present invention may have an asymmetric carbon center and an asymmetric axis or an asymmetric plane, the compounds may exist as cis or trans isomers, R or S isomers, racemates, diastereomeric mixtures and individual diastereomers, all of which are included within the scope of the present invention.

[0027] In the present specification, unless otherwise stated, the use of the compound of formula 1 shall include all compounds of formula 1, pharmaceutically acceptable salts and isomers thereof.

[0028] In one embodiment according to the present invention, R1 in Formula 1 is a C2 to C4 alkyl group. In another embodiment according to the present invention, R1 in Formula 1 is a linear or branched C2 to C4 alkyl group, such as ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl.

[0029] In another embodiment according to the present invention, R1 in Formula 1 is a C2 or C3 alkyl group. In another embodiment according to the present invention, R1 in Formula 1 is a linear or branched C2 or C3 alkyl group, such as ethyl, n-propyl or isopropyl.

[0030] 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 the following formula 2:

[0031] [Formula 2]

[0032]

[0033] 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 the following formula 3:

[0034] [Formula 3]

[0035]

[0036] 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)trimethylacetamide of the following formula 4:

[0037] [Formula 4]

[0038]

[0039] In another embodiment according to the present invention, examples of pharmaceutically acceptable salts include acid addition salts formed from inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid and hydroiodic acid; 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 and maleic acid; and sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and naphthalenesulfonic acid, but are not limited thereto.

[0040] In another embodiment of the present 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 and hydroiodic acid; 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 and maleic acid; and a sulfonic acid such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and naphthalenesulfonic acid.

[0041] In another embodiment of the present 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 and hydroiodic acid; 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 and maleic acid; and a sulfonic acid such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and naphthalenesulfonic acid.

[0042] In another embodiment of 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 and hydroiodic acid; 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 and maleic acid; and a sulfonic acid such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and naphthalenesulfonic acid.

[0043] In another embodiment of the present 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 and hydroiodic acid; 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 and maleic acid; and a sulfonic acid such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and naphthalenesulfonic acid.

[0044] In another embodiment of the present 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 and hydroiodic acid; 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 and maleic acid; and a sulfonic acid such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and naphthalenesulfonic acid.

[0045] In another embodiment of the present 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 and hydroiodic acid; 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 and maleic acid; and a sulfonic acid such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and naphthalenesulfonic acid.

[0046] In another embodiment of the present 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 and hydroiodic acid; 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 and maleic acid; and a sulfonic acid such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and naphthalenesulfonic acid.

[0047] In another embodiment according to the present invention, the pharmaceutically acceptable salt is hydrochloride.

[0048] 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 the following formula 5:

[0049] [Formula 5]

[0050]

[0051] 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 hydrochloride of the following formula 6:

[0052] [Formula 6]

[0053]

[0054] 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)trimethylacetamide hydrochloride of the following formula 7:

[0055] [Formula 7]

[0056]

[0057] In another embodiment of the present invention, the N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidine-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide hydrochloride of the above formula 5, the N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidine-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide hydrochloride of the above formula 6 -carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)propanamide 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)trimethylacetamide hydrochloride of the above formula 7 can be prepared according to the following reaction scheme 1.

[0058] [Reaction Scheme 1]

[0059]

[0060] In reaction scheme 1,

[0061] R2 is a C1-C5 alkyl group;

[0062] R3 is a C3-C8 cycloalkyl group which is unsubstituted or substituted with 1 or 2 C1-C5 alkyl groups; and

[0063] R4 and R5 are each independently hydrogen or halogen.

[0064] The compound of formula 1 according to the present invention exhibits excellent agonist activity against melanocortin receptors, particularly melanocortin-4 receptor (MC4R), and therefore the present invention also provides a melanocortin receptor agonist pharmaceutical composition, the pharmaceutical composition comprising the compound of formula 1 or a pharmaceutically acceptable salt or isomer thereof as an active ingredient, and a pharmaceutically acceptable carrier. Specifically, the composition according to the present invention exhibits excellent effects in preventing or treating obesity, diabetes, inflammation and erectile dysfunction, but is not limited thereto.

[0065] In the present specification, "carrier" refers to a compound that facilitates injection of a compound into cells or tissues.

[0066] When the compounds of the present invention are administered for clinical purposes, the total daily dosage administered to the host as a single dose or separate doses is preferably in the range of 0.01 mg to 10 mg per 1 kg body weight, but the specific dosage level for an individual patient may depend on the specific compound used, the patient's body weight, sex, health condition and diet, administration time, administration method, excretion rate of the drug, drug mixture and severity of the disease.

[0067] The compounds of the present invention can be administered by any route depending on the use. For example, the compounds of the present invention can be administered by injection or oral administration.

[0068] Injection preparations can be prepared according to known techniques by using appropriate dispersing agents, wetting agents or suspending agents.

[0069] Examples of solid dosage forms for oral administration include capsules, tablets, pills, powders and granules, and the solid dosage forms can be prepared by mixing the active compound of Formula 1 according to the present invention with one or more carriers such as inert diluents, lubricants, disintegrants and binders.

[0070] Beneficial effects

[0071] The compound of formula 1 according to the present invention exhibits excellent agonist activity against melanocortin receptors, particularly melanocortin-4 receptor (MC4R), and thus can be effectively used for preventing or treating obesity, diabetes, inflammation and erectile dysfunction.

[0072] The compound of formula 1 according to the present invention exhibits a targeting effect on the melanocortin-4 receptor, exhibits weight loss and diet reduction effects without affecting anxiety and depression, and can be administered without producing inhibitory side effects on human ether-a-go-go related genes (hERG) and without causing safety issues such as mutations. In addition, the compound of formula 1 according to the present invention can be safely administered due to the absence of cytotoxicity and liver toxicity. DETAILED DESCRIPTION

[0073] Hereinafter, the present invention will be described in more detail through preparation examples and examples. However, these examples are only illustrative, and the scope of the present invention is not limited thereto.

[0074] Preparation Example 1: Preparation of (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramide)pyrrolidine-2-carboxylate Methyl ester hydrochloride

[0075]

[0076] The title compound was obtained by following steps A, B, C, D and E.

[0077] Step A: Preparation of 1-(tert-butyl)2-methyl (2S,4S)-4-azidopyrrolidine-1,2-dicarboxylate

[0078] (2S, 4R) -4- ((methylsulfonyl) oxy) pyrrolidine -1,2- dicarboxylic acid 1- (tert-butyl) 2-methyl ester (48.5g, 150mmol) was dissolved in N, N'-dimethylformamide (250ml) under nitrogen, and sodium azide (19.5g, 300ml) was added thereto. After stirring at 80 ° C for 16 hours, the reaction solvent was concentrated under reduced pressure, water was added, and ethyl acetate was used for two extractions. The organic layer was washed with sodium chloride aqueous solution and water, dried over anhydrous magnesium sulfate, and then filtered. The filtrate was concentrated under reduced pressure to obtain a crude product (39.59g, 98%), which was used in the next step without purification.

[0079] MS[M+H]=271(M+1)

[0080] 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)

[0081] Step B: Preparation of (2S,4S)-4-aminopyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl) 2-methyl ester

[0082] The (2S, 4S)-4-azidopyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl) 2-methyl ester (24.59 g, 91.0 mmol) obtained in step A was dissolved in tetrahydrofuran (180 ml), and then a tetrahydrofuran solution of 1M-trimethylphosphine (109.2 ml, 109.2 mmol) was slowly added thereto at 0 ° C. After stirring for 1 hour at the same temperature, the mixture was stirred at room temperature for three hours. After the reaction solvent was concentrated under reduced pressure, dichloromethane (100 ml) and water (150 ml) were added and stirred for about 30 minutes. After the layers were separated and extracted with dichloromethane again, the organic layer was dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product (20.62 g, 93%), which was used in the next step without purification.

[0083] MS[M+H]=245(M+1)

[0084] 1H 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)

[0085] Step C: Preparation of (2S,4S)-4-((1s,4R)-4-methylcyclohexyl)amino)pyrrolidine-1,2-dicarboxylic acid 1-(tert- butyl) 2-methyl ester

[0086] The (2S, 4S)-4-aminopyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl) 2-methyl ester (20.62 g, 84.4 mmol) obtained in step B was dissolved in dichloroethane (150 ml), and 4-methylcyclohexanone (9.5 ml, 101.3 mmol) was added thereto. Sodium triacetoxyborohydride (26.8 g, 126.6 mmol) was added at 0 ° C, and stirred at room temperature for 16 hours. The reaction solvent was concentrated under reduced pressure, water was added, and extracted twice with ethyl acetate. The organic layer was washed with sodium chloride aqueous solution, dried over anhydrous magnesium sulfate, and then filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography to obtain the title compound (22.9 g, 80%).

[0087] MS[M+H]=341(M+1)

[0088] 1 H 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)

[0089] Step D: Preparation of (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-1,2-diol 1-(tert-Butyl) 2-methyl formate

[0090] The (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) obtained in step C was dissolved in dichloromethane (500 ml), triethylamine (61.1 ml, 438.1 mmol) was added, and isobutyryl 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, sodium bicarbonate aqueous solution was added, and extracted twice with ethyl acetate. The organic layer was washed with sodium chloride aqueous solution and water, dried over anhydrous magnesium sulfate, and then filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography to obtain the title compound (38.79 g, 86%).

[0091] MS[M+H]=411(M+1)

[0092] 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)

[0093] Step E: Preparation of (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylic acid Methyl ester hydrochloride

[0094] The (2S, 4S)-4-(N-((1s, 4R)-4-methylcyclohexyl)isobutylamino)pyrrolidine-1,2-dicarboxylic acid 1-(tert-butyl) 2-methyl ester (34.0 g, 82.8 mmol) obtained in step D was dissolved in dichloromethane (200 ml), and a 4N hydrochloric acid 1,4-dioxane solution (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%), which was used in the next step without purification.

[0095] MS[M+H]=311(M+1)

[0096] Preparation Example 2: Preparation of (3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carboxylic acid

[0097]

[0098] The title compound was obtained by the method described in International Publication No. WO 2004 / 092126.

[0099] MS[M+H]=282(M+1)

[0100] 1 H NMR (400MHz, CD3OD) δ7.43-7.33(m,4H),3.90-3.69(m,3H),3.59(dd,J=11.2 ,10.0Hz,1H),3.29(dd,J=11.2,11.2Hz,1H),3.18-3.09(m,1H),1.44(s,9H)

[0101] Preparation Example 3: Preparation of (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)propionamido)pyrrolidine-2-carboxylic acid Methyl ester hydrochloride

[0102]

[0103] The title compound was obtained via steps A and B.

[0104] Step A: (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)propionamido)pyrrolidine-1,2-dicarboxylic acid Prepare 1-(tert-butyl) 2-methyl ester

[0105]

[0106] The title compound (0.98 g, 84%) was obtained in the same manner as in Step D of Preparation Example 1 by using 1-(tert-butyl)2-methyl (2S,4S)-4-((1s,4R)-4-methylcyclohexyl)amino)pyrrolidine-1,2-dicarboxylate (1.0 g, 2.9 mmol) and propionyl chloride (0.33 g, 3.5 mmol).

[0107] MS [M+Na] = 419.5 (M+23)

[0108] 1 H NMR (400MHz, CD3OD) δ4.33(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)

[0109] Step B: Preparation of (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)propionamido)pyrrolidine-2-carboxylic acid Ester hydrochloride

[0110]

[0111] The title compound (0.76 g, 93%) was obtained in the same manner as in Step E of Preparation Example 1 by 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.

[0112] MS [M+H] = 297.4 (M+1)

[0113] 1 H NMR(400MHz,DMSO-d6)δ9.95(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)

[0114] Preparation Example 4: Preparation of (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)trimethylacetamido)pyrrolidine- 2-Methyl formate hydrochloride

[0115]

[0116] The title compound was obtained via steps A and B.

[0117] Step A: Preparation of (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)trimethylacetamido)pyrrolidine-1, 2-Dicarboxylic acid 1-(tert-butyl) 2-methyl ester

[0118]

[0119] The title compound was obtained by the method disclosed in International Publication No. WO 2008 / 007930.

[0120] MS [M+Na] = 447.5 (M+23)

[0121] 1 H NMR (400MHz, CD3OD) δ4.34(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)

[0122] Step B: Preparation of (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)trimethylacetamido)pyrrolidine-2-yl Methyl formate hydrochloride

[0123]

[0124] The title compound (0.68 g, 99%) was obtained in the same manner as in Step E of Preparation Example 1 by using 1-(tert-butyl)2-methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)trimethylacetamido)pyrrolidine-1,2-dicarboxylate (0.80 g, 1.88 mmol) obtained in Step A.

[0125] MS [M+H] = 325.4 (M+1)

[0126] 1 H NMR(400MHz,DMSO-d6)δ10.24(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)

[0127] Example 1: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl ((1s,4R)-4-methylcyclohexyl)isobutyramide hydrochloride

[0128]

[0129] The title compound was obtained via steps A, B, C and D.

[0130] Step A: 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 ester

[0131] The (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylic acid methyl ester hydrochloride (28.7 g, 82.73 mmol) obtained in Preparation Example 1, the (3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carboxylic acid (24.5 g, 86.87 mmol) obtained in Preparation 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, then 0.5N sodium hydroxide aqueous solution was added, and ethyl acetate was used for two extractions. The organic layer was washed twice with sodium chloride aqueous solution and water, dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography to obtain the title compound (41.19 g, 87%).

[0132] MS [M+H] = 575 (M+1)

[0133] 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

[0134] The (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 ester (39.4 g, 68.62 mmol) obtained in step A was dissolved in methanol (450 ml), and 6N aqueous sodium hydroxide solution (57.2 ml, 343.09 mmol) was added. After stirring at room temperature for 16 hours and adjusting the pH value to about 5 with 6N aqueous hydrochloric acid solution, the reaction solution was concentrated under reduced pressure. The concentrate was dissolved in dichloromethane, and the insoluble solid was filtered with a paper filter. The filtrate was concentrated under reduced pressure to give a crude product (38.4 g, 99%), which was used in the next step without purification.

[0135] MS[M+H]=561(M+1)

[0136] Step C: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl (1S,4R)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1S,4R)-4-methylcyclohexyl)isobutyramide

[0137] (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, 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 then morpholine (5.9 ml, 68.80 mmol) and N,N'-diisopropylethylamine (59.7 ml, 343.02 mmol) were slowly added in sequence. After stirring at room temperature for 16 hours, the reaction solution was concentrated under reduced pressure, 0.5 N sodium hydroxide aqueous solution was added, and extracted twice with ethyl acetate. The organic layer was washed twice with sodium chloride aqueous solution and water, dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography to obtain the title compound (37.05 g, 86%).

[0138] MS[M+H]=630(M+1)

[0139] Step D: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl ((1s,4R)-4-methylcyclohexyl)isobutyramide hydrochloride

[0140] 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 was dissolved in ethyl acetate (50 ml), and 2N hydrochloric acid ethyl acetate solution (3.97 ml, 15.89 mmol) was slowly added. After stirring at room temperature for 30 minutes, the reaction solvent was concentrated under reduced pressure. The obtained crude solid was purified by grinding with hexane and ether to obtain the title compound (5.23 g, 99%).

[0141] MS[M+H]=630(M+1)

[0142] 1H NMR(500 MHz, CD3OD) δ7.49-7.44(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)

[0143] Example 2: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl ((1s,4R)-4-methylcyclohexyl)propionamide hydrochloride

[0144]

[0145] The title compound was obtained via steps A, B, C and D.

[0146] Step A: Preparation of (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4- Methyl (N-((1s,4R)-4-methylcyclohexyl)propionamido)pyrrolidine-2-carboxylate

[0147]

[0148] The title compound (0.45 g, 35%) was obtained in the same manner as in Step A of Example 1 by using (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)propionamido)pyrrolidine-2-carboxylic acid methyl ester hydrochloride (0.76 g, 2.28 mmol) obtained in Preparation Example 3 and (3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carboxylic acid (0.64 g, 2.28 mmol) obtained in Preparation Example 2.

[0149] MS [M+H] = 560.4 (M+1)

[0150] 1 H NMR (400MHz, CD3OD) δ7.39-7.30(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)

[0151] 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

[0152]

[0153] The title compound (0.44 g, 99%) was obtained in the same manner as in step B of Example 1 by 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.

[0154] MS [M+H] = 546.4 (M+1)

[0155] Step C: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl (1s,4R)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)propionamide

[0156]

[0157] The title compound (0.28 g, 53%) was obtained in the same manner as in step C of Example 1 by 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.

[0158] MS [M+H] = 615.5 (M+1)

[0159] 1 H NMR(400MHz,CD3OD)δ7.36(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)

[0160] Step D: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl ((1s,4R)-4-methylcyclohexyl)propionamide hydrochloride

[0161]

[0162] The title compound (0.08 g, 94%) was obtained in the same manner as in step D of Example 1 by 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)propanamide (0.08 g, 0.13 mmol) obtained in step C.

[0163] MS [M+H] = 615.5 (M+1)

[0164] 1 H NMR(400MHz,CD3OD)δ7.43(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)

[0165] Example 3: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl ((1s,4R)-4-methylcyclohexyl)trimethylacetamide hydrochloride

[0166]

[0167] The title compound was obtained via steps A, B, C and D.

[0168] Step A: Preparation of (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4- (N-((1s,4R)-4-methylcyclohexyl)trimethylacetamido)pyrrolidine-2-carboxylic acid methyl ester

[0169]

[0170] Using (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)trimethylacetamido)pyrrolidine-2-carboxylic acid methyl ester hydrochloride (0.65 g, 1.8 mmol) obtained in Preparation Example 4 and (3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carboxylic acid (0.50 g, 1.8 mmol) obtained in Preparation Example 2, the title compound (0.70 g, 66%) was obtained in the same manner as in Step A of Example 1.

[0171] MS [M+H] = 588.5 (M+1)

[0172] 1H NMR (400MHz, CD3OD) δ7.40-7.30(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)

[0173] Step B: Preparation of (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4- (N-((1s,4R)-4-Methylcyclohexyl)trimethylacetamido)pyrrolidine-2-carboxylic acid

[0174]

[0175] The title compound (0.10 g, 99%) was obtained in the same manner as in step B of Example 1 by using (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)trimethylacetamido)pyrrolidine-2-carboxylic acid methyl ester (0.10 g, 0.18 mmol) obtained in step A.

[0176] MS [M+H] = 574.4 (M+1)

[0177] Step C: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl (1s,4R)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)trimethylacetamide

[0178]

[0179] The title compound (0.020 g, 17%) was obtained in the same manner as in step C of Example 1 by using (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)trimethylacetamido)pyrrolidine-2-carboxylic acid (0.10 g, 0.18 mmol) obtained in step B.

[0180] MS [M+H] = 643.5 (M+1)

[0181] 1H NMR(400MHz,CD3OD)δ7.40-7.30(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)

[0182] Step D: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl ((1s,4R)-4-methylcyclohexyl)trimethylacetamide hydrochloride

[0183]

[0184] The title compound (0.29 g, 83%) was obtained in the same manner as in step D of Example 1 by 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)trimethylacetamide (0.33 g, 0.51 mmol) obtained in step C.

[0185] MS [M+H] = 643.5 (M+1)

[0186] 1 H NMR (400MHz, CD3OD) δ7.41(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)

[0187] 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)

[0188]

[0189] The A95 compound of International Publication No. WO 2008 / 007930 was obtained by the same method as disclosed therein.

[0190] Comparative Example 2: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl 4-( ...

[0191]

[0192] The A96 compound in International Publication No. WO 2008 / 007930 was obtained by the same method as disclosed therein.

[0193] Experimental Example 1: Luciferase Assay

[0194] To determine the ability of agonists to target MC4R (melanocortin-4 receptor), a cell line permanently expressing the luciferase gene (CRE-LUC) under the control of MC4R and CRE (cAMP response element) was established. After preparing a mammalian cell expression vector (pCDNA3 (Neo)) (Invitrogen) containing the MC4R gene, a human embryonic kidney (HEK) cell line was transformed using Lipofectamine 2000 (Invitrogen) and a vector (pCRE-Luc) (Stratagen) expressing the luciferase gene (CRE-LUC) under the control of the cAMP response element (CRE). The transformed cell line (HEK MC4R-Luc) was cultured in a 37°C incubator in the presence of 5% CO2 for 24 hours using Dulbecco's modified Eagles medium (DMEM) containing 10% heat-inactivated fetal bovine serum (GIBCO / BRL). The cell line was cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10 ml of selection medium (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) for four days. The process of replacing the selection medium with 10 mL of new selection medium to remove cells killed by the selection medium was repeated three times every 4 days. Single colonies formed by the final selection and propagation of clones were transferred to 24-well cell culture plates under a microscope, each well containing 1 ml of selection medium, and cultured for 4 days. Forskolin (SIGMA) was treated with a final concentration of 10 μM and then incubated at 37°C in a 5% Cultivate for 5 hours in the presence of CO2. Each well is treated with 50 μl of Bright-Glo luciferase reagent (Promega) and placed at room temperature for 15 minutes, and then the luminescence of each well is measured using a luminometer (Victor). Select clones that show a luminescence amount of 100 times or more of the basic value by Forskolin treatment and use to measure the MC4R agonist ability of each compound.

[0195] HEK MC4R-Luc cells were added to each well of a 9-well luminometer cell culture plate (Costar) at a scale of 2.5 × 10 cells / well in 100 μl of culture medium. 4cells, and then cultured in a 37°C incubator in the presence of 6% CO2 for 18 hours. The MCR agonist was diluted at each step concentration using the above culture medium so that the final DMSO concentration did not exceed 1%, and then cultured in a 37°C incubator in the presence of 6% CO2 for 5 hours. Each well was treated with 50 μl of Bright-Glo luciferase reagent (Promega) and placed at room temperature for 5 minutes, and then the luminescence of each well was measured using a luminometer (Victor). The amount of luminescence induced by the diluted agonist at each step concentration was converted into a relative % value relative to the amount of luminescence displayed by 10 μM NDP-α-MSH treatment. EC 0.5 MSH is expressed as the concentration inducing 50% of the maximum luminescence inducible by NDP-α-MSH, and EC50 is expressed as the concentration inducing 50% of the maximum luminescence inducible by each agonist. The measured values ​​were measured using statistical software (Prizm).

[0196] Table 1 shows the measurement results of the MC4R agonist ability of each compound obtained by the above experiments, with the unit of EC 50 (nM).

[0197] [Table 1]

[0198]

[0199] As shown in Table 1, it was confirmed that, among well-known in vivo melanocortin receptors, regarding the melanocortin-4 receptor (MC4R) involved in in vivo energy metabolism and body weight control, the Example compounds had superior MC4R agonist ability than the Comparative Example compounds (A95 and A96).

[0200] Experimental Example 2: cAMP Assay

[0201] Melanocortin receptor is a G protein-coupled receptor (GPCR), and the main role of G protein is to activate secondary sensors through signal transduction, regulating the response of cells to various physiological stimuli. MC4R is a Gs-coupled receptor, and it is well known that if MC4R interacts with an agonist, adenylate cyclase (AC) is activated to increase the concentration of cyclic adenosine monophosphate (cAMP), one of the secondary sensors in the cell. Therefore, the activity of melanocortin receptor can be evaluated by measuring the generation of cAMP signal.

[0202] After establishing a cAMP-Hunter Gs coupled cell line (CHO-K1 cell line) in which MC1R, MC3R, MC4R, and MC5R are overexpressed, the increase in intracellular cAMP levels due to agonist response can be measured, and the cells are seeded into each well of a white cell culture plate and cultured in a 37°C incubator in the presence of 5% CO2 for 24 hours. After culture, the culture medium is removed and 15 μl of 2:1 HBBS / 10 mM HEPES:cAMP XS+Ab reagent is added. After adding 5 μl of a sample diluted 4 times with buffer, the solvent concentration is set to 1%, and the MC4R agonist compound diluted at each step concentration is added, and then reacted at 37°C for 30 minutes. The activity (%) of each agonist compound is expressed as 100% x (average RLU value of sample-average RLU value of solvent control) / (average RLU value of maximum control-average RLU value of solvent control), which is analyzed by the CBIS data analysis suite (ChemInnovation, California).

[0203] Table 2 shows the results of the measurement of the melanocortin receptor agonist ability of the compounds obtained by the above experiments, with the unit being EC 50 (nM).

[0204] [Table 2]

[0205]

[0206] As shown in Table 2, it was confirmed that, among well-known melanocortin receptors in vivo, regarding the melanocortin-4 receptor (MC4R) involved in energy metabolism and body weight control in vivo, the Example compounds had superior receptor agonist ability than the Comparative Example compounds (A95 and A96).

[0207] Experimental Example 3: β-arrestin assay

[0208] Melanocortin receptor is a G protein-coupled receptor (GPCR) that regulates various physiological responses by transmitting signals from a variety of neurotransmitters. When GPCR is phosphorylated, β-arrestin binds to the phosphorylated portion of the receptor and plays an important role in activating various signaling pathways in cells through interactions with other proteins. It is well known that when melanocortin receptor interacts with agonists, β-arrestin is mobilized and participates in β-arrestin-mediated signaling pathways. Therefore, the activity of melanocortin receptor can be evaluated by measuring β-arrestin.

[0209] A Pathhunter eXpress β-arrestin cell line (U2OS cell line) was established in which Prolink (PK)-tagged MC1R, MC3R, MC4R, MC5R and enzyme receptor (EA)-tagged β-arrestin were simultaneously expressed. When the MCR-PK portion of the cell line is activated, β-arrestin-EA is mobilized, and the enzyme receptor (EA) and Prolink (PK) as β-galactosidase fragments interact. The activated enzyme hydrolyzes the substrate through β-galactosidase activity to produce a chemiluminescent signal, so that the activity can be measured. After culturing the Pathhunter β-arrestin cell line (U2OS cell line), the cells were seeded into each well of a cell culture plate and cultured in a 37°C incubator in the presence of 5% CO2 for 48 hours. After culture, 5 μl of a sample diluted 5 times with a buffer was added, the solvent concentration was set to 1%, and the MC4R agonist compound diluted at each step concentration was added, and then reacted at 37°C for 90 minutes. The activity (%) of each agonist compound was expressed as 100% x (mean RLU value of sample - mean RLU value of vehicle control) / (mean maximum value of control ligand - mean RLU value of vehicle control) and analyzed by CBIS data analysis suite (ChemInnovation, CA).

[0210] Table 3 shows the results of the melanocortin receptor activity of each compound obtained by the above experiments, with the unit of EC 50 (nM).

[0211] [Table 3]

[0212]

[0213] As shown in Table 3, it was confirmed that, among well-known in vivo melanocortin receptors, regarding the melanocortin-4 receptor (MC4R) involved in in vivo energy metabolism and body weight control, the Example compounds had superior receptor agonist ability than the Comparative Example compounds (A95 and A96).

[0214] Experimental Example 4: Binding Affinity

[0215] There are five known subtypes of melanocortin receptors (MCRs) in vivo, and subtype 4 MC4R is known to be involved in energy metabolism and weight control. Since other MCR subtypes are involved in the regulation of various in vivo functions, such as skin pigmentation, energy homeostasis, and exocrine function, ensuring the selectivity of MC4R agonist compounds for MC4R is very important to prevent possible future side effects. Therefore, the receptor binding ability of MC4R agonists for each MCR subtype was determined.

[0216] After establishing a CHO-K1 cell line expressing human recombinant MC1R and a HEK-293 cell line expressing MC3R, MC4R, and MC5R, membranes were collected from each cell line. 3 μg of MC1R membrane and 0.04 nM 125 I-NDP-α-MSH was reacted at 37°C for 2 hours. 3 μg of 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 mixed with 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 the MCR agonist diluted at each step concentration was added to each well and reacted. The reaction solution was transferred to the filter and washed with the adsorption buffer, and then the radioactivity was measured. The value of the nonspecific binding amount in the presence of 1 μM (MC1R) and 3 μM (MC3R, MC4R, MC5R) NDP-α-MSH was excluded from each total binding amount and used as 125 The specific binding amount of I-NDP-α-MSH was measured at each concentration step. 125 IC50 is expressed as 50% inhibition of the specific binding of I-NDP-α-MSH. 125 The concentration of each agonist to which I-NDP-α-MSH specifically binds.

[0217] Table 4 shows the melanocortin receptor binding measurement results of the compounds obtained by the above experiments, with the unit being Ki (nM).

[0218] [Table 4]

[0219]

[0220] As shown in Table 4, it has been confirmed that, among known in vivo melanocortin receptors, the Example compounds have superior receptor binding ability than the Comparative Example (A95 and A96) compounds with respect to the melanocortin-4 receptor (MC4R) involved in in vivo energy metabolism and body weight control.

[0221] Experimental Example 5: Pharmacokinetics and Drug Metabolism

[0222] Experimental Example 5-1: Pharmacokinetic Curve

[0223] In order to study the pharmacokinetic (PK) properties of the compound of Example 1 and the compound of Comparative Example, the following experiments were performed.

[0224] In order to carry out PK test to the compound of Example 1 and comparative example compound (A95 and A96), C57BL6 mice of about 7 weeks of age were prepared, and 12 individuals were allocated to each administration substance, divided into groups, and starved for oral administration. On the day of administration, a drug solution with a concentration of 1 mg / mL was prepared with distilled water (DW) as a solvent, and was orally administered at a dose of 1 mL per kilogram of body weight, and the final dose was 10 mg / kg. At 1, 3, 8 and 24 hours after administration, whole blood was collected from 3 individuals in each group by heart blood sampling, and then placed in heparin tubes to prevent blood coagulation. Thereafter, the brain of each individual was collected and placed in an EP tube, the weight of the tissue was measured, and it was frozen and stored at -20 ° C.

[0225] On the day of analysis, DDW with a volume of 4 times the weight of the tissue was added to each tissue tube and homogenized, and the stored plasma was thawed at room temperature. As with plasma, 50 μL of tissue homogenate was taken and transferred to a separate tube, and then 200 μL of acetonitrile (AN) was added to each tube of plasma and tissue homogenate in a total amount of 4 times the sample volume for deproteinization. In this case, AN included an internal standard. To prepare the calibration curve, AN solutions (containing internal standards) with known concentrations of 0.1 ng / mL, 0.5 ng / mL, 5 ng / mL, 50 ng / mL, and 500 ng / mL were prepared and deproteinized in each plasma and blank plasma of the brain at the above 4 times volume. Therefore, final calibration curves were prepared for plasma 0.4 ng / mL to 2,000 ng / mL and for brain 1 ng / mL, 5 ng / mL, 50 ng / mL, 500 ng / mL, and 5,000 ng / mL. After 0.5 μL of the supernatant obtained after deproteinization was injected into LC-MS / MS, the peak area of ​​the example and comparative example compounds (A95 and A96) was corrected with the peak area of ​​IS to obtain the peak response of each sample collection point, and the concentration was converted using the calibration curve.

[0226] For the blood drug concentration values ​​of each dosing group that changed over time, the pharmacokinetic parameters (C 最大 , AUC inf ,t 1 / 2 wait).

[0227] The pharmacokinetic characteristics of each compound were compared by comparing the exposure, half-life changes, etc. of each dosing group, and the obtained values ​​are shown in Tables 5 and 6. In addition, the result values ​​obtained based on the ratio of brain exposure to blood exposure are shown in Table 7.

[0228] [Table 5]

[0229]

[0230] [Table 6]

[0231]

[0232] [Table 7]

[0233]

[0234] According to the above results, the exposure of each compound in the whole body and brain was confirmed in the order of Example 1 compound, Comparative Example 2 compound (A96) and Comparative Example 1 compound (A95). During the observation period, the half-life lost in the brain was higher than the value observed in the blood, assuming that this means the effective persistence of a series of substances in the site of efficacy expression. In addition, it has been confirmed that when each compound is administered at the same dose, the absolute exposure and persistence of the example compound in the brain are the most excellent. In addition, compared with the comparative example compounds (A95 and A96), it has been confirmed that the ratio of brain exposure to blood exposure of the example 1 compound is also the most excellent.

[0235] In addition to these results, when the in vitro activity of each compound and the persistence of the drug are considered comprehensively, the efficacy of the Example 1 compound can be expected to be the best when compared with the Comparative Example compound when the same dose is administered. In order to achieve a certain level of efficacy, a lower dose can be administered, and therefore, side effects caused by systemic exposure are expected to be minimized.

[0236] Experimental Example 5-2: CYP Inhibition (%)

[0237] To confirm the interaction between the drug and CYP (cytochrome P450) isozymes, the following experiments were performed.

[0238] In order to measure the inhibitory ability of the compound of Example 1 and the compound of Comparative Example 2 (A96), CYP1A2, 2C9, 2C19, 2D6 and 3A4 recombinant enzymes were prepared. For the use of probe substrates, positive controls, isozymes and the measurement conditions for measuring the inhibitory ability of each substance, refer to Table 8 below.

[0239] [Table 8]

[0240]

[0241] Cultivation was performed in a 96-well plate (Costar, 3792 black, round bottom), the buffer system for metabolism was 50mM potassium phosphate buffer, pH 7.4, and the final reaction volume was 250μL. The final concentration of the compound of Example 1, the compound of Comparative Example 2 (A96), and the positive control in the total buffer was 10μM (2% methanol (v / v)), and each included a negative control (methanol only, 2% (v / v)). The buffer to which the experimental compound was added was mixed with the same volume of the CYP isozyme preparation solution to a final concentration of 10μM, and then preheated in a 37°C fluorescent plate reader for 10 minutes. NRS solution (NADPH regeneration system: 0.22mMβ-NADP, 2.8mM glucose-6-phosphate and 0.6 units / mL glucose-6-phosphate dehydrogenase) was added to each well, followed by pre-incubation for 30 minutes. Thereafter, the reaction was initiated by adding the substrate to the pre-incubated wells and monitored at 1 minute intervals for 30 minutes at the measurement wavelength of each substrate. The measured values ​​obtained from the positive control and the compound of Example 1 were compared with the fluorescence intensity of the negative control (no inhibition or compound) to confirm the inhibitory ability of each compound to the isozyme. When each compound was treated at 10 μM, the inhibitory ability (%) to each isozyme was shown in Table 9. The expression of the inhibitory activity (%) was based on the following criteria: A = <50%, B = >50%.

[0242] The therapeutic concentration of 10 μM of the compound is very high, and it is a conservative standard to evaluate the inhibitory ability based on this therapeutic concentration and select drugs with an inhibition rate of less than 50% as candidate drugs. It can be considered that in this case, the selected candidate drug has the least possibility of inhibiting CYP isozymes.

[0243] [Table 9]

[0244]

[0245] According to the above results, when treated at the same concentration, it was confirmed that the inhibitory ability of Example 1 compound on the main CYP isozymes was lower than or similar to that of Comparative Example 2 compound (A96). It is worth noting that Comparative Example 2 compound (A96) has drug interactions due to its very high inhibitory ability on CYP2C9. It is known that CYP2C9 is involved in the metabolism of about 10% of all commercial drugs and plays a major role in the loss of efficacy of drugs with a narrow therapeutic window. In addition, CYP2C9 is of great significance in both research and clinical aspects because it is reported that polymorphism depends on each individual. This can also be known from the fact that CYP2C9 appears in the list of basic isozymes whose inhibition should be checked during drug development in the FDA Drug-Drug Interaction (DDI) Guide.

[0246] Based on this, it was determined that when the compound of Example 1 was administered, the drug interaction caused by CYP inhibition was lower than that of the comparative compound (A96).

[0247] Experimental Example 6: Pharmacology

[0248] The pharmacological effects of the compounds of the present invention as melanocortin-4 receptor agonists were evaluated in the following obesity model.

[0249] Experimental Example 6-1: High-fat diet-induced obesity model in mice

[0250] A high-fat diet-induced obesity model in mice was used to evaluate the effects of melanocortin-4 receptor agonists on obesity.

[0251] Five-week-old male C57BL / 6N taconic mice were fed with a 60kcal% fat diet (D12492, research diet) for 15 weeks to induce obesity. Example 1 compound, comparative example compounds (A95 and A96) and positive controls were prepared in distilled water and orally administered once a day from the 1st day to the 16th day to a 19-week-old mouse obesity model induced by a high-fat diet. From the 1st day to the 16th day, body weight was measured once a day, dietary intake was measured five times a week, and drinking water intake was measured twice a week. Blood glucose and glycosylated hemoglobin were measured on the 15th day, and all animals were killed on the 17th day. Blood was collected through the abdominal vena cava, and the liver and epididymal adipose tissue were removed and weighed. The collected blood was placed in a heparin tube, and the plasma was centrifuged and then subjected to plasma biochemical analysis.

[0252] Table 10 shows the difference in weight change rate measured on day 12 for each dose of each compound compared to vehicle.

[0253] [Table 10]

[0254] Day 12 10mg / kg 30mg / kg Example 1 -9.4％* -15.1％** Comparative Example 1 -4.8％ -4.0％ Comparative Example 2 -0.2％ -7.1％ Positive control -6.8％ NT(Not tested)

[0255] (Difference in weight change rate compared to the solvent)

[0256] Significant difference in body weight between vehicle control group and compound-treated groups: *p<0.05, **p<0.01 (mean ± SEM, Student's t-test, unpaired, two-tailed)

[0257] In this mouse obesity model, significant weight loss was shown, especially when only the compound of Example 1 was administered. When the compound of Example 1 was administered at a dose of 10 mg / kg and 30 mg / kg, it resulted in -9.4% and -15.1% suppression of weight gain, respectively, compared to the vehicle control of solvent administration, and showed a statistically significant weight loss effect compared to the comparative example compounds (A95 and A96).

[0258] This is because the Example 1 compound has superior in vitro MC4R agonist ability, superior absolute brain exposure and persistence, and excellent brain exposure to blood exposure ratio compared to the comparative example compound, and it is believed that significant differences can be shown even at low doses.

[0259] Furthermore, the compound of Example 1 showed equal or higher superior results in efficacy compared to the positive control, which is an obesity therapeutic agent in the related field, and thus the compound of Example 1 is expected to show significant drug efficacy in actual clinical applications.

Claims

1. A compound of the following formula 1, or a pharmaceutically acceptable salt or isomer thereof: [Formula 1] Wherein R1 is C2-C5 alkyl.

2. The compound according to claim 1 or a pharmaceutically acceptable salt or isomer thereof, wherein R1 is a C2-C4 alkyl group.

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 or a pharmaceutically acceptable salt or isomer thereof, wherein the compound is selected from the group consisting 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; 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)propanamide; 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)trimethylacetamide.

5. The compound according to claim 1 or a pharmaceutically acceptable salt or isomer thereof, wherein the pharmaceutically acceptable salt is selected from hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid and hydroiodic acid. 6 . The compound according to claim 5 , or a pharmaceutically acceptable salt or isomer thereof, wherein the pharmaceutically acceptable salt is a hydrochloride.

7. A melanocortin receptor agonist pharmaceutical composition, comprising a compound of formula 1 as defined in any one of claims 1 to 6 or a pharmaceutically acceptable salt or isomer thereof as an active ingredient, and a pharmaceutically acceptable carrier.

8. The melanocortin receptor agonist pharmaceutical composition according to claim 7, which is used for preventing or treating obesity. 9 . The melanocortin receptor agonist pharmaceutical composition according to claim 7 , which is used for preventing or treating diabetes. 10 . The melanocortin receptor agonist pharmaceutical composition according to claim 7 , which is used for preventing or treating inflammation.

11. The melanocortin receptor agonist pharmaceutical composition according to claim 7, which is used for preventing or treating erectile dysfunction.

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

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

14. Use of the melanocortin receptor agonist pharmaceutical composition according to claim 7 in the preparation of a medicament for preventing or treating inflammation.

15. Use of the melanocortin receptor agonist pharmaceutical composition according to claim 7 in the preparation of a medicament for preventing or treating erectile dysfunction. 16 . A method for preventing or treating obesity in a subject in need thereof, the method comprising administering to the subject the melanocortin receptor agonist pharmaceutical composition according to claim 7.

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

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

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

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