Amorphous melanocortin-4 receptor agonist

Abstract

The present invention relates to an amorphous compound represented by chemical formula 1, a preparation method therefor, and a pharmaceutical composition comprising same. The amorphous compound represented by chemical formula 1 according to the present invention may be characterized by an XRD pattern, a DSC profile, and / or a TGA profile.

Description

Amorphous melanocortin-4 receptor agonist

[0001] Cross-citation with related applications

[0002] This application claims the benefit of priority from Korean Patent Application No. 10-2020-0142399, filed October 29, 2020, the entire contents of which are incorporated herein by reference.

[0003] Technology field

[0004] The present invention relates to a novel amorphous compound exhibiting excellent agonistic activity against melanocortin receptors, a method for preparing the same, and a pharmaceutical composition comprising the same.

[0005] Leptin protein is a hormone secreted by body fat cells (adipocytes). The amount secreted increases as body fat content increases, and it regulates various body functions including appetite, body fat content, and energy metabolism by regulating the functions of various neuropeptides produced in the hypothalamus (Schwartz, et al., Nature 404, 661-671 (2000)). The signaling of appetite and body weight control by leptin protein is achieved through the regulation of many downstream factors, the most representative of which are melanocortin, AgRP (agoutirelated peptide), and neuropeptide Y (NPY) hormones.

[0006] When blood leptin levels increase due to calorie excess in the body, the secretion of the protein hormone proopiomelanocortin (POMC) from the pituitary gland increases and the production of AgRP and NPY decreases. The small peptide hormone alpha-MSH (melanocyte stimulating hormone) is produced from POMC neurons, which acts as an agonist of the melanocortin-4 receptor (MC4R) on second-order neurons and ultimately induces appetite reduction. Conversely, when leptin levels decrease due to calorie deprivation, the expression of AgRP, an MC4R antagonist, increases and the expression of NPY also increases, ultimately increasing appetite. In other words, depending on the changes in leptin, alpha-MSH and AgRP hormones participate in appetite regulation by acting as agonists and antagonists on MC4R.

[0007] Alpha-MSH hormone binds to three MCR subtypes in addition to MC4R, inducing various physiological responses. Five MCR subtypes have been identified to date, of which MC1R is mainly expressed in skin cells and is involved in melanin pigmentation regulation, MC2R is mainly expressed in the adrenal gland and is known to be involved in the production of glucocorticoid hormones, and its only ligand is adrenocorticotropic hormone (ACTH) derived from POMC. MC3R and MC4R, which are mainly expressed in the central nervous system, are involved in the regulation of appetite, energy metabolism, and body fat storage efficiency, and MC5R, which is expressed in various tissues, is known to regulate exocrine function (Wikberg, et al., Pharm Res 42 (5) 393-420 (2000)). In particular, activation of the MC4R receptor has been proven to be a key factor in the development of anti-obesity drugs because it effectively reduces body weight by inducing a decrease in appetite and an increase in energy metabolism (Review: 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); O'Rahilly et al., Nature Med 10, 351-352 (2004)).

[0008] The role of MC4R in appetite and body weight regulation was initially demonstrated through experiments with an animal model overexpressing agouti protein (agouti mouse). In the Agouti mouse, a genetic mutation resulted in high levels of agouti protein in the central nervous system, and it was found that agouti protein acts as an antagonist of MC4R in the hypothalamus, thereby inducing obesity (Yen, TT et al., FASEB J. 8, 479-488 (1994); Lu D., et al. Nature 371, 799-802 (1994)). Subsequent research results showed that AgRP (agouti-related peptide), which is similar to actual agouti protein, is expressed in hypothalamic neurons, and it is known that these are also involved in appetite control as antagonists of MC4R (Shutter, et al., Genes Dev., 11, 593-602 (1997); Ollman, et al. Science 278, 135-138 (1997)).

[0009] When alpha-MSH, an MC4R agonist in vivo, is administered intracerebral to animals, it has an appetite-reducing effect, and when treated with MC4R antagonists SHU9119 (peptide) or HS014 (peptide), it was observed that appetite was increased again (Kask et al., Biochem. Biophys. Res. Comm. 245, 90-93 (1998)). In addition, in animal tests using Melanotan II (MTII, Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2) and its similar agonist HP228, appetite suppression, body weight loss, and increased energy metabolism were confirmed after intracerebral, intraperitoneal, or subcutaneous administration. (Thiele TE, 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, administration of the representative SHU9119 to animals resulted in a significant and sustained increase in food intake and body weight, providing pharmacological evidence that MCR agonists can be anti-obesity agents. The anorexic effect that was evident with MTII administration was not observed in MC4R KO (knock-out) mice, further demonstrating that the anorexic effect was primarily mediated through MC4R activation (Marsh, et al., Nat Genet 21, 119-122 (1999)).

[0010] To date, the majority of obesity treatments developed are appetite suppressants that act on the central nervous system, most of which modulate the action of neurotransmitters. Examples include noradrenaline agents (phentermine and mazindol) and serotonergic agents such as fluoxetine and sibutramine. However, neurotransmitter modulators exert a wide range of physiological effects beyond appetite suppression through numerous subtypes of receptors. Therefore, these modulators lack selectivity for each subtype, leading to various side effects with long-term administration.

[0011] On the other hand, melanocortin agonists are neuropeptides, not neurotransmitters, and have the advantage of being able to induce weight loss through appetite suppression without affecting other physiological functions, given that all functions other than energy metabolism are normal in MC4R gene KO mice. In particular, the receptor is a G-protein coupled receptor (GPCR), which belongs to the most successful category of new drug agonists developed to date, and it is relatively easy to secure selectivity for the subtype receptor, which is a major difference from existing agonists.

[0012] As examples of utilizing these melanocortin receptors as a point of action, International Publication Nos. WO 2008 / 007930 and WO 2010 / 056022 disclose compounds as agonists of melanocortin receptors.

[0013] In addition, the inventors of the present invention have repeatedly conducted research and invented a novel compound of the following chemical formula 1 having excellent selective agonistic activity against melanocortin receptors, particularly melanocortin-4 receptor (MC4R), and a method for producing the same (Application No. 10-2019-0141649 (filed on November 7, 2019)).

[0014] [Chemical Formula 1]

[0015] (R1 is C2-C5 alkyl.)

[0016] Meanwhile, the salt form or crystal structure of a pharmaceutically active ingredient often affects the chemical stability of the drug. Different salt forms of a compound can alter its filterability, product stability, and storage stability. Furthermore, different crystallization and storage conditions can alter its crystal structure, sometimes resulting in the concurrent production of different crystalline forms. Therefore, it is necessary to study salt forms and / or crystal structures that exhibit high purity and good chemical stability for a given compound.

[0017] The object of the present invention is to provide a novel amorphous compound or salt thereof having excellent selective agonistic activity against melanocortin receptors, particularly melanocortin-4 receptor (MC4R).

[0018] Another object of the present invention is to provide a method for producing the amorphous compound or its salt.

[0019] Another object of the present invention is to provide a pharmaceutical composition comprising the amorphous compound or a salt thereof.

[0020] To achieve the above purpose,

[0021] In one aspect, the present invention provides an amorphous compound of the following chemical formula 1, or a salt thereof.

[0022] [Chemical Formula 1]

[0023]

[0024] In the above chemical formula 1, R1 is C2-C5 alkyl.

[0025]

[0026] The compounds according to the present invention may have an asymmetric carbon center and an asymmetric axis or asymmetric plane, and therefore may exist as cis or trans isomers, R or S isomers, racemates, diastereomeric mixtures and individual diastereoisomers, and all of these isomers and mixtures are included in the scope of the present invention.

[0027] Unless otherwise specified herein for convenience, the compound of formula 1 is used to mean the compound of formula 1, pharmaceutically acceptable salts thereof, and isomers thereof.

[0028] In one specific embodiment according to the present invention, R1 of the formula 1 is C2 to C5 alkyl. In another specific embodiment according to the present invention, R1 of the formula 1 is straight-chain or branched C2 to C5 alkyl, for example, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl or tert-butyl.

[0029] In another specific embodiment according to the present invention, R1 of the formula 1 is C2 or C3 alkyl. In another specific embodiment according to the present invention, R1 of the formula 1 is straight-chain or branched C2 or C3 alkyl, for example, ethyl, n-propyl or iso-propyl.

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

[0031] In another specific embodiment according to the present invention, the pharmaceutically acceptable salt of the amorphous compound of formula 1 may be a hydrochloride compound of formula 2 below.

[0032] [Chemical Formula 2]

[0033]

[0034] In the above chemical formula 2, R2 is C2-C5 alkyl.

[0035] In another specific example according to the present invention, the compound of formula 1 or a pharmaceutically acceptable salt thereof may be N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide hydrochloride of formula 3 below.

[0036] [Chemical Formula 3]

[0037]

[0038]

[0039] The amorphous compound according to the present invention or a pharmaceutically acceptable salt thereof may be confirmed by an X-ray diffraction (XRD) pattern in which no characteristic peak exists at a 2θ angle of 4° to 40°.

[0040] In one specific embodiment according to the present invention, the amorphous compound or a pharmaceutically acceptable salt thereof may have an XRD pattern as shown in FIG. 1.

[0041] The amorphous compound or a pharmaceutically acceptable salt thereof according to the present invention may not have an exothermic peak when heated to a temperature of 350°C or lower in a differential scanning calorimetry (DSC) profile. In addition, the amorphous compound or a pharmaceutically acceptable salt thereof may have an endothermic peak in at least one temperature range of 20 to 130°C and 210 to 350°C in a DSC profile. In addition, the amorphous compound or a pharmaceutically acceptable salt thereof may have a glass transition temperature (Tg) in a temperature range of 145°C to 165°C, for example, 150°C to 160°C, or at a temperature of 156°C in a DSC profile.

[0042] In one specific embodiment according to the present invention, the amorphous compound or a pharmaceutically acceptable salt thereof may have a DSC profile as shown in FIG. 2.

[0043] The amorphous compound according to the present invention or a pharmaceutically acceptable salt thereof may have a weight loss of 10% or less, for example, 1% to 10%, 5% to 9%, or 7%, when heated to a temperature of 180°C or less in a thermogravimetric analysis (TGA) profile.

[0044] In one specific embodiment according to the present invention, the amorphous compound or a pharmaceutically acceptable salt thereof may have a TGA profile as shown in FIG. 3.

[0045] In this specification,

[0046] X-ray diffraction (XRD) analysis was performed using a PANalytical X' Pert Pro MPD system, Malvern Panalytical Ltd.

[0047] Differential scanning calorimetry (DSC) analysis was performed using a DSC1, Mettler-Toledo AG.

[0048] Thermogravimetric analysis (TGA) was performed using a TGA / DSC 1, Mettler-Toledo AG.

[0049] Stability analysis was performed using HPLC, Agilent Technologies, Inc.

[0050]

[0051] The amorphous compound of chemical formula 1 or a pharmaceutically acceptable salt thereof may have a higher purity and may be more physically and chemically stable than the crude compound or a pharmaceutically acceptable salt thereof.

[0052] In addition, the compound of Chemical Formula 2 is a compound in the form of a hydrochloride salt of the compound of Chemical Formula 1, and the amorphous hydrochloride salt compound of Chemical Formula 2 may have higher purity than the compound of Chemical Formula 1 and may be more stable both physically and chemically. In addition, compared to known melanocortin-4 receptor agonists, it may have a more excellent anti-melanocortin-4 receptor activity and a more excellent preventive or therapeutic effect on diseases such as obesity, diabetes, inflammation, and erectile dysfunction, but the effects of the present invention are not limited thereto.

[0053]

[0054] In another aspect, the present invention provides a method for preparing an amorphous compound of formula 1 or a pharmaceutically acceptable salt thereof, comprising the steps of preparing a mixed solution by dissolving a crude compound represented by formula 1 in an organic solvent, cooling the mixed solution, and adding an acid dropwise to the cooled mixed solution.

[0055] First, the crude compound represented by the above chemical formula 1 is dissolved in an organic solvent.

[0056] For the preparation of the amorphous compound of formula 1 or a pharmaceutically acceptable salt thereof, the crude compound of formula 1 may be the compound of formula 1, a salt thereof, or an isomer thereof.

[0057] The compound of the above chemical formula 1 may be obtained by the manufacturing method described in the specification of application number 10-2019-0141649 (filed on November 7, 2019).

[0058] The solvent for dissolving the crude compound of the above chemical formula 1 may be used without particular limitation as long as it is a solvent capable of dissolving the crude compound of the above chemical formula 1. In one specific example, the solvent may be an organic solvent, and specifically may include an ether-based organic solvent.

[0059] The above ether organic solvents are not limited thereto, but include, for example, dialkyl ether solvents such as diethyl ether, dipropyl ether, dibutyl ether, diisoamyl ether, ethyl methyl ether, methyl propyl ether, methyl butyl ether, and ethyl propyl ether; cyclic ether solvents such as tetrahydrofuran and tetrahydropyran; and aromatic ring-containing ether solvents such as diphenyl ether and anisole. The above organic solvents may be used alone, or two or more of the above solvents may be mixed and used.

[0060] In one specific example according to the present invention, the organic solvent may include methyl tert-butyl ether (MTBE).

[0061] For 1 g of the crude compound of the above chemical formula 1, 10 to 30 mL or 15 to 25 mL of the organic solvent may be used.

[0062] The dissolution of the crude compound of the above chemical formula 1 into a solvent may be performed without or with stirring at a temperature of 15 to 30°C, specifically 23 to 28°C.

[0063] In one specific example according to the present invention, a mixed solution in which the crude compound of chemical formula 1 is dissolved can be obtained at 25°C using 19 mL of MTBE for 1 g of the crude compound of chemical formula 1.

[0064] Next, the mixed solution in which the crude compound of the above chemical formula 1 is dissolved is cooled.

[0065] The cooling may be performed so that the temperature of the mixed solution becomes 0°C or lower. Specifically, the cooling may be performed so that the temperature of the mixed solution becomes -10 to 0°C, specifically -5 to 0°C.

[0066] The manufacturing method of the present invention may further include a step of adding a non-polar organic solvent to the mixed solution before, after, or simultaneously with cooling of the mixed solution. By adding the non-polar organic solvent, the rate of formation of crystallized particles can be increased, thereby improving the yield or production stability of the obtained amorphous compound; however, the present invention is not limited thereto.

[0067] The above non-polar organic solvent may be used without particular limitation as long as it is an organic solvent having non-polar properties, but examples of non-polar organic solvents include hexane, heptane, cyclohexane, carbon tetrachloride, benzene, and chloroform.

[0068] In one specific example according to the present invention, after adding heptane to the mixed solution, it may be cooled to -5 to 0°C.

[0069] The step of adding the non-polar organic solvent may be any step in the method for producing the amorphous compound of the present invention.

[0070] Next, acid is added dropwise to the cooled mixed solution.

[0071] The above acid includes hydrochloric acid, and the dropwise addition of the acid may be performed in the presence of ethyl acetate.

[0072] In one specific example according to the present invention, the addition of the acid may be by adding a hydrochloric acid-ethyl acetate solution dropwise to the mixed solution.

[0073] The concentration of hydrochloric acid in the above hydrochloric acid-ethyl acetate solution may be 0.5 M to 6 M, specifically 1 M to 5 M, 3 M to 5 M, for example 4 M.

[0074] The precipitate formed by the addition of the above acid can be filtered and washed to obtain an amorphous compound of chemical formula 1, specifically an amorphous compound of chemical formula 2, or an amorphous pharmaceutically acceptable salt thereof.

[0075] The amorphous compound of chemical formula 1 obtained as described above may have a higher purity than the crude compound of chemical formula 1 and may be more physically and chemically stable, but the effects of the present invention are not limited thereto.

[0076]

[0077] In another aspect, the present invention provides a pharmaceutical composition comprising (i) the amorphous compound of formula 1, or a salt of the amorphous compound of formula 1; and (ii) a pharmaceutically acceptable carrier.

[0078] The amorphous compound of formula 1 according to the present invention, or a salt of the amorphous compound of formula 1, exhibits excellent agonistic action against melanocortin receptors, particularly melanocortin-4 receptors (MC4R). Therefore, the present invention can also provide a pharmaceutical composition for enhancing the function of a melanocortin receptor, which comprises the above-described amorphous compound or a salt thereof as an active ingredient. Specifically, the pharmaceutical composition can be a composition for enhancing the function of a melanocortin-4 receptor.

[0079] In addition, the pharmaceutical composition may exhibit excellent effects in the prevention or treatment of obesity, diabetes, inflammation, and erectile dysfunction, and thus may be a composition for the prevention or treatment of obesity, diabetes, inflammation, or erectile dysfunction, but the use of the present invention is not limited to these diseases.

[0080] As used herein, “carrier” means a compound that facilitates introduction of a compound into a cell or tissue.

[0081] When administering the amorphous compound of the present invention for clinical purposes, the total daily dose to be administered to the host as a single dose or separate doses is preferably in the range of 0.01 to 10 mg per 1 kg of body weight, but the specific dosage level for each patient may vary depending on the specific compound to be used, the patient's weight, sex, health condition, diet, administration time of the drug, administration method, excretion rate, drug mixture, and severity of the disease.

[0082] The amorphous compound of the present invention may be administered by any route, depending on the intended purpose. For example, the amorphous compound of the present invention may be administered by injection or orally.

[0083] The pharmaceutical composition of the present invention may be in various oral dosage forms such as tablets, pills, powders, capsules, granules, syrups or emulsions, or may be in parenteral dosage forms such as injectable preparations for intramuscular, intravenous or subcutaneous administration.

[0084] Injectable formulations can be prepared using suitable dispersing agents, wetting agents, suspending agents, or excipients according to known techniques.

[0085] Excipients that can be used in the pharmaceutical preparation of the present invention include, but are not limited to, sweeteners, binders, solubilizers, solubilizers, wetting agents, emulsifiers, isotonic agents, adsorbents, disintegrants, antioxidants, preservatives, lubricants, fillers, fragrances, etc. For example, excipients that can be used include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, silica, magnesium aluminum silicate, starch, gelatin, gum tragacanth, alginic acid, sodium alginate, methylcellulose, sodium carboxymethylcellulose, water, ethanol, polyethylene glycol, polyvinylpyrrolidone, sodium chloride, calcium chloride, orange essence, strawberry essence, vanilla flavor, etc.

[0086] When the pharmaceutical composition of the present invention is in an oral dosage form, examples of carriers used include, but are not limited to, cellulose, calcium silicate, corn starch, lactose, sucrose, dextrose, calcium phosphate, stearic acid, magnesium stearate, calcium stearate, gelatin, talc, and the like.

[0087] When the pharmaceutical composition of the present invention is in the form of an injectable preparation, the carrier may include, but is not limited to, water, saline solution, glucose aqueous solution, similar sugar aqueous solution, alcohol, glycol, ether, oil, fatty acid, fatty acid ester, glyceride, etc.

[0088]

[0089] In another aspect, the present invention provides an amorphous compound of formula 1 or an amorphous salt thereof for use in enhancing the function of melanocortin receptors, particularly melanocortin-4 receptor (MC4R).

[0090] In one embodiment, the present invention provides an amorphous compound of formula 1 or an amorphous salt thereof for use in the treatment or prevention of obesity, diabetes, inflammation or erectile dysfunction.

[0091] In another aspect, a method for enhancing the function of a melanocortin receptor, particularly a melanocortin-4 receptor (MC4R), is provided, comprising administering to a subject an amorphous compound of the above-described chemical formula 1 or an amorphous salt compound thereof.

[0092] In another aspect, a method for treating obesity, diabetes, inflammation or erectile dysfunction is provided, comprising administering to a subject an amorphous compound of the above-described chemical formula 1 or an amorphous salt compound thereof.

[0093] The amorphous compound of chemical formula 1 or a salt thereof according to the present invention exhibits excellent agonistic action against melanocortin receptors, particularly melanocortin-4 receptor (MC4R), and therefore can be usefully used for the prevention or treatment of obesity, diabetes, inflammation, and erectile dysfunction.

[0094] The amorphous compound of chemical formula 1 or a salt thereof according to the present invention exhibits an on-target effect on the melanocortin-4 receptor, thereby exhibiting weight loss and appetite reduction effects, while not affecting anxiety and depression, and can be administered without safety issues such as side effects of hERG (human ether-a-go-go related gene) inhibition or mutagenesis.

[0095] In addition, the amorphous compound of chemical formula 1 or its salt according to the present invention has superior purity, yield, and physical and chemical stability compared to the crude compound of chemical formula 1.

[0096] Specifically, the amorphous compound of the above chemical formula 1 or its salt may have superior solubility, storage stability, and production stability compared to the crude compound of the chemical formula 1.

[0097] Figure 1 is a graph of the XRD results of Example 1.

[0098] Figure 2 is a DSC result graph of Example 1.

[0099] Figure 3 is a TGA result graph of Example 1.

[0100] The present invention is described in more detail through the following manufacturing examples and examples. However, these examples are merely illustrative of the present invention and the scope of the present invention is not limited by them.

[0101]

[0102] Manufacturing Example 1: Manufacturing of methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylate hydrochloride

[0103]

[0104] The title compound was obtained through the following steps A, B, C, D and E.

[0105] Step A: Preparation of 1-(tert-butyl) 2-methyl (2S,4S)-4-azidopyrrolidine-1,2-dicarboxylate

[0106] 1-(tert-butyl) 2-methyl (2S,4R)-4-((methylsulfonyl)oxy)pyrrolidine-1,2-dicarboxylate (48.5 g, 150 mmol) was dissolved in N,N'-dimethylformamide (250 ml) under nitrogen, and sodium azide (19.5 g, 300 ml) was added. The mixture was stirred at 80°C for 16 h, and the reaction solvent was concentrated under reduced pressure. Water was added, and the mixture was extracted twice with ethyl acetate. The organic layer was washed with aqueous sodium chloride solution and water, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain crude 1-(tert-butyl) 2-methyl (2S,4S)-4-azidopyrrolidine-1,2-dicarboxylate (39.59 g, 98%), which was used in the next step without purification.

[0107] MS [M+H] = 271 (M+1)

[0108] 1 H NMR (400 MHz, CD3OD) δ 4.43-4.37 (m, 1H), 4.35-4.27 (br, 1H), 3.77 (s, 1.8H), 3.76 (s, 1.2H), 3.73-3.66 (m, 1H), 3.44-3.38 (m, 1H), 2.63-2.49 (m, 1H), 2.19-2.11 (m, 1H), 1.50 (s, 4.5H), 1.44 (s, 4.5H)

[0109]

[0110] Step B: Preparation of 1-(tert-butyl) 2-methyl (2S,4S)-4-aminopyrrolidine-1,2-dicarboxylate

[0111] 1-(tert-butyl) 2-methyl (2S,4S)-4-azidopyrrolidine-1,2-dicarboxylate (24.59 g, 91.0 mmol) obtained in Step A above was dissolved in tetrahydrofuran (180 ml), and then 1 M trimethylphosphine tetrahydrofuran solution (109.2 ml, 109.2 mmol) was slowly added at 0°C. The mixture was stirred at the same temperature for 1 hour and then at room temperature for 3 hours. After concentrating the reaction solvent under reduced pressure, dichloromethane (100 ml) and water (150 ml) were added, and the mixture was stirred for about 30 minutes. The layers were separated, extracted once more with dichloromethane, and the organic layer was dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain crude 1-(tert-butyl) 2-methyl (2S,4S)-4-aminopyrrolidine-1,2-dicarboxylate (20.62 g, 93%), which was used in the next step without purification.

[0112] MS [M+H] = 245 (M+1)

[0113] 1 H NMR (400 MHz, CD3OD) δ 4.27 (m, 1H), 3.77 (s, 1.8H), 3.76 (s, 1.2H), 3.75-3.67 (m, 1H), 3.50-3.42 (m, 1H), 3.22-3.17 (m, 1H), 2.58-2.47 (m,1H), 1.82-1.71 (m, 1H), 1.48 (s, 4.5H), 1.42 (s, 4.5H)

[0114]

[0115] Step C: Preparation of 1-(tert-butyl)2-methyl (2S,4S)-4-(((1s,4R)-4-methylcyclohexyl)amino)pyrrolidine-1,2-dicarboxylate

[0116] 1-(tert-butyl) 2-methyl (2S,4S)-4-aminopyrrolidine-1,2-dicarboxylate (20.62 g, 84.4 mmol) obtained in Step B above was dissolved in dichloroethane (150 ml), and 4-methylcyclohexanone (9.5 ml, 101.3 mmol) was added. Sodium triacetoxyborohydride (26.8 g, 126.6 mmol) was added at 0°C, and the mixture was stirred at room temperature for 16 hours. The reaction solvent was concentrated under reduced pressure, water was added, and the mixture was extracted twice with ethyl acetate. The organic layer was washed with aqueous sodium chloride solution, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography to obtain 1-(tert-butyl) 2-methyl (2S,4S)-4-(((1s,4R)-4-methylcyclohexyl)amino)pyrrolidine-1,2-dicarboxylate (22.9 g, 80%).

[0117] MS [M+H] = 341 (M+1)

[0118] 1 H NMR (400 MHz, CD3OD) δ 4.26 (m, 1H), 3.76 (s, 1.8H), 3.75 (s, 1.2H), 3.78-3.71 (m, 1H), 3.49-3.40 (m, 1H), 3.22-3.16 (m, 1H), 2.69-2.60 (br, 1H), 2.58-2.46 (m, 1H), 1.87-1.77 (m, 1H), 1.73-1.63 (m, 1H), 1.62-1.35 (m, 8H), 1.48 (s, 4.5H), 1.42 (s, 4.5H), 0.96 (d, 3H)

[0119]

[0120] Step D: Preparation of 1-(tert-butyl) 2-methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-1,2-dicarboxylate

[0121] 1-(tert-butyl) 2-methyl (2S,4S)-4-(((1s,4R)-4-methylcyclohexyl)amino)pyrrolidine-1,2-dicarboxylate (37.29 g, 109.5 mmol) obtained in the above 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, followed by addition of aqueous sodium hydrogen carbonate, and extraction was performed twice 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 and purified by column chromatography to obtain 1-(tert-butyl) 2-methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-1,2-dicarboxylate (38.79 g, 86%).

[0122] MS [M+H] = 411 (M+1)

[0123] 1 H NMR (400 MHz, CD3OD) δ 4.27 (m, 1H), 3.76 (s, 1.8H), 3.75 (s, 1.2H), 3.78-3.72 (m, 1H), 3.50-3.41 (m, 1H), 3.33-3.14 (m, 1H), 2.69-2.60 (m, 2H), 2.57-2.43 (m, 1H), 1.87-1.79 (m, 1H), 1.70-1.61 (m, 1H), 1.60-1.32 (m, 8H), 1.47 (s, 4.5H), 1.41 (s, 4.5H), 1.10 (dd, 6H), 0.99 (d, 3H)

[0124]

[0125] Step E: Preparation of methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylate hydrochloride

[0126] 1-(tert-butyl) 2-methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-1,2-dicarboxylate (34.0 g, 82.8 mmol) obtained in the above step D was dissolved in dichloromethane (200 ml), and then 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 (28.7 g, 99%), which was used in the next step without purification.

[0127] MS[M+H] = 311 (M+1)

[0128]

[0129] Manufacturing Example 2: Manufacturing of (3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carboxylic acid

[0130]

[0131] The title compound was obtained by the method described in International Publication No. WO 2004 / 092126.

[0132] MS[ M+H] = 282 (M+1)

[0133] 1 H NMR (400 MHz, CD3OD) δ 7.43-7.33 (m, 4H), 3.90-3.69 (m, 3H), 3.59 (dd, J = 11.2, 10.0 Hz, 1H), 3.29 (dd, J = 11.2, 11.2 Hz, 1H), 3.18-3.09 (m, 1H), 1.44 (s, 9H)

[0134]

[0135] Manufacturing Example 3: N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pi

[0136] Preparation of cyclohexyl-3-carbonyl-l)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide

[0137]

[0138]

[0139] Manufacturing Example 3 (MC70)

[0140] The title compound was obtained through the following steps A, B and C.

[0141] Step A: Preparation of methyl (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylate

[0142] Methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylate hydrochloride (28.7 g, 82.73 mmol) obtained in Preparation Example 1, (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. The mixture was stirred at room temperature for 16 hours, and the reaction solvent was concentrated under reduced pressure. 0.5 N aqueous sodium hydroxide solution was added, and extraction was performed twice with ethyl acetate. The organic layer was washed twice with aqueous sodium chloride solution and water, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography to obtain methyl (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylate (41.19 g, 87%).

[0143] MS [M+H] = 575 (M+1)

[0144]

[0145] 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

[0146] Methyl (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylate (39.4 g, 68.62 mmol) obtained in the above step A was dissolved in methanol (450 ml), and then 6N sodium hydroxide aqueous solution (57.2 ml, 343.09 mmol) was added. The mixture was stirred at room temperature for 16 hours, and the pH was adjusted to about 5 with 6N hydrochloric acid aqueous solution. The reaction solution was concentrated under reduced pressure. The concentrate was dissolved in dichloromethane, and the insoluble solid was filtered through a paper filter. The filtrate was concentrated under reduced pressure to obtain the crude title compound (38.4 g, 99%), which was used in the next step without purification.

[0147] MS [M+H] = 561 (M+1)

[0148]

[0149] Step C: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide

[0150] (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), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (18.4 g, 96.04 mmol) and 1-hydroxybenzotriazole hydrate (13.0 g, 96.04 mmol) obtained in the above step B were dissolved in N,N'-dimethylformamide (200 ml), and then morpholine (5.9 ml, 68.80 mmol) and N,N'-diisopropylethylamine were sequentially added. (59.7 ml, 343.02 mmol) was slowly added. The mixture was stirred at room temperature for 16 hours, and the reaction solution was concentrated under reduced pressure. 0.5 N aqueous sodium hydroxide solution was added, and extraction was performed twice with ethyl acetate. The organic layer was washed twice with aqueous sodium chloride solution and water, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography to obtain 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 (37.05 g, 86%, Preparation Example 3).

[0151] MS [M+H] = 630 (M+1)

[0152]

[0153] Example 1

[0154] Preparation of an amorphous compound of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide hydrochloride

[0155] Based on 1 g of the compound (MC70) finally manufactured in the above Manufacturing Example 3, the compound (MC70) was dissolved at 25°C using 19 mL of MTBE. After complete dissolution, 1 mL of heptane was added and cooled to -5 to 0°C. After reaching the set temperature, 1 equivalent of 4 M HCl / EtOAc was added dropwise, stirred for about 90 minutes, and filtered to obtain amorphous 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 (Example 1). (Yield: about 90%)

[0156] Amorphous properties

[0157] 1) X-ray diffraction (XRD)

[0158] Powder XRD diffraction patterns were obtained using a PANalytical X'Pert Pro MPD system equipped with a monochromatized radiation source and Ni filter as a solid-state detector, as follows.

[0159] Approximately 20 to 30 mg of sample was placed flat on a glass sample holder, and the generator of the device was set to 45 kV (acceleration voltage) and 40 mA (filament emission), and measurements were made in reflection mode (not-spin). Bragg angles (2θ) in the range of 4 to 40° were measured under the conditions of a step size of 0.026° and a time per step of 51 s.

[0160] The XRD results are shown in Fig. 1, and as shown in Fig. 1, the amorphous Example 1 showed no diffraction peak and a wide range of noise typical of an amorphous sample.

[0161] 2) Differential Scanning Calorimetry (DSC)

[0162] DSC measurements were performed using a Mettler Toledo DSC1 system. Approximately 2–5 mg of sample was weighed and placed in a 40 μL Al crucible (a flat-bottomed aluminum pan with a pin-hole lid), with a single pinhole created. The sample was then heated from 25 to 350°C at a rate of 10°C / min, and the DSC measurements were performed. During the measurement, nitrogen gas was supplied to the instrument at a rate of 70 mL / min to prevent the inflow of oxygen and other gases. Data collection and evaluation were performed using the STARe software.

[0163] The DSC results are shown in Figure 2. An endothermic peak was observed at approximately 33.2°C (Onset), and the glass transition temperature was observed at approximately 156.5°C (Midpoint ISO). An endothermic peak due to decomposition appeared after approximately 220°C. The temperature values ​​have an error of ±5°C.

[0164] 3) Thermogravimetric analysis (TGA)

[0165] TGA was measured using a Mettler Toledo TGA / DSC 1 module. Approximately 4–8 mg of sample was weighed and placed in a 100 μL Al crucible (flat-bottomed aluminum crucible). The sample was then heated from 30°C to 350°C at a rate of 10°C / min, and TGA measurements were taken. During the measurement, nitrogen gas was supplied to the instrument at a rate of 80 mL / min to prevent the inflow of oxygen and other gases. Data collection and evaluation were performed using the STARe software.

[0166] The TGA results are shown in Fig. 3, and as shown in Fig. 3, a weight loss of approximately 5.6% was observed at temperatures below 100°C. Thereafter, a weight loss of approximately 1.1% was observed at approximately 150°C to 180°C. Weight loss due to decomposition occurred after approximately 220°C. Temperature values ​​have an error of ±5°C.

[0167]

[0168] Experimental Example 1. Stability Evaluation

[0169] Approximately 10-30 mg of samples were stored in an open container under accelerated conditions (40°C, 75% RH) or in a sealed container in an oven at 80°C for 4 weeks under harsh conditions. HPLC analysis was performed using the method in Table 1 to compare these samples with samples stored at room temperature.

[0170] [Table 1]

[0171]

[0172] The stability results of Example 1 evaluated according to the above method are shown in Table 2 below.

[0173] [Table 2]

[0174]

Claims

1. An amorphous compound of the following chemical formula 1, or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] In the above chemical formula 1, R 1 Silver C 2 -C 5 It's alkyl.

2. In claim 1, A compound or a pharmaceutically acceptable salt thereof having an X-ray diffraction pattern as shown in FIG.

1.

3. In claim 1, A compound or a pharmaceutically acceptable salt thereof having a DSC profile as shown in FIG.

2.

4. In claim 1, A compound or a pharmaceutically acceptable salt thereof having a TGA profile as shown in FIG.

3.

5. In claim 1, A compound or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt of the compound of the above chemical formula 1 is selected from the group consisting of a hydrochloride salt, a sulfate salt, a nitrate salt, a phosphate salt, a hydrobromide salt and a hydroiodide salt of the compound.

6. In claim 1, A compound, which 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, or a pharmaceutically acceptable salt thereof.

7. A method for producing an amorphous compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 6, A step of preparing a mixed solution by dissolving a compound represented by the following chemical formula 1 in an organic solvent, A step of cooling the above mixed solution, and A manufacturing method comprising the step of adding acid dropwise to the cooled mixed solution: [Chemical Formula 1] In the above chemical formula 1, R 1 Silver C 2 -C 5 It's alkyl.

8. In claim 7, A manufacturing method, wherein the organic solvent comprises an ether-based organic solvent.

9. In claim 7, A manufacturing method further comprising a step of adding a non-polar organic solvent to the mixed solution before, after, or simultaneously with cooling of the mixed solution.

10. A pharmaceutical composition comprising a compound according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier.

11. A pharmaceutical composition for enhancing melanocortin-4 receptor function, comprising a compound according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier.

12. A pharmaceutical composition according to claim 11, wherein the composition is for preventing or treating obesity, diabetes, inflammation or erectile dysfunction.

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