Crystalline form iii of melanocortin receptor agonist compound and method for preparing same

Abstract

To provide a stable crystalline form of a novel compound having excellent selective agonistic activity for melanocortin receptors, particularly melanocortin-4 receptor (MC4R), and a method for producing the same.SOLUTION: The present invention relates to crystalline form III of a compound represented by chemical formula 1, a pharmaceutically acceptable salt thereof, or a solvate thereof, the crystalline form III having three or more characteristic peaks selected from the following diffraction angles (2θ values) in an X-ray powder diffraction (XRPD) pattern: 6.238±0.2°, 8.257±0.2°, 8.828±0.2°, 14.637±0.2°, 16.618±0.2°, 17.465±0.2°, 18.859±0.2°, 19.061±0.2°, 19.333±0.2°, 20.642±0.2°, 22.679±0.2°and 25.985±0.2°.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims the benefit of priority based on Korean Patent Application No. 10-2020-0180809, filed December 22, 2020, and all contents disclosed in the documents of this Korean patent application are incorporated herein by reference.

[0002] The present invention relates to crystalline form III of a novel compound that exhibits excellent enhancing activity against melanocortin receptors, a method for preparing the same, and a pharmaceutical composition containing the same. [Background technology]

[0003] Leptin is a hormone secreted by adipocytes. Its secretion level increases with increasing body fat content. It regulates various physiological functions, including appetite, body fat content, and energy metabolism, by regulating the function of various neuropeptides produced in the hypothalamus (Schwartz et al., Nature 404, 661-671 (2000)). Leptin signaling, which regulates appetite and body weight, is mediated by various downstream factors, most notably melanocortin, agouti-related peptide (AgRP), and neuropeptide Y (NPY).

[0004] When blood leptin levels increase as a result of a calorie surplus in the body, the pituitary gland increases secretion of the protein hormone proopiomelanocortin (POMC) and decreases production of AgRP and NPY. POMC neurons produce the small peptide hormone alpha-MSH (melanocyte stimulating hormone), which acts as an agonist to the melanocortin-4 receptor (MC4R) in second-order neurons, thereby inducing appetite reduction. On the other hand, when leptin levels decrease as a result of a calorie deficit, expression of AgRP, an MC4R antagonist, and NPY increase, resulting in increased appetite. Thus, in response to changes in leptin, alpha-MSH and AgRP hormones mediate appetite regulation by acting as agonists or antagonists to MC4R.

[0005] Alpha-MSH hormones bind to three MCR subtypes in addition to MC4R, inducing various physiological responses. Five MCR subtypes have been identified to date. MC1R is primarily expressed in skin cells and is involved in skin pigmentation. MC2R is primarily expressed in the adrenal gland and is known to be involved in the production of glucocorticoid hormones, with ACTH (adrenocorticotropic hormone) derived from POMC as its only ligand. MC3R and MC4R, primarily expressed in the central nervous system, are involved in regulating appetite, energy metabolism, and fat storage efficiency. MC5R, 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 major target for the development of anti-obesity drugs, as it effectively reduces body weight by reducing appetite and increasing 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)).

[0006] The role of MC4R in appetite and body weight regulation was first demonstrated by experiments using agouti mice, an animal model in which agouti protein is overexpressed. In agouti mice, a genetic mutation causes high levels of agouti protein to be expressed in the central nervous system, where it acts as an MC4R antagonist in the hypothalamus, inducing obesity (Yen, TT et al., FASEB J. 8, 479-488 (1994); Lu D., et al., Nature 371, 799-802 (1994)). Subsequent research has shown that the hypothalamic neurons do indeed express AgRP (agouti-related peptide), which is similar to agouti protein, and this is also known to be involved in appetite regulation as an antagonist of MC4R (Shutter, et al., Genes Dev., 11, 593-602 (1997); Ollman, et al., Science 278, 135-138 (1997)).

[0007] When alpha-MSH, an in vivo MC4R agonist, is administered intracerebrally to animals, it has been shown to have an appetite-reducing effect, whereas when treated with the MC4R antagonists SHU9119 (peptide) or HS014 (peptide), it has been observed to increase appetite again (Kask et al., Biochem. Biophys. Res. Comm. 245, 90-93 (1998)). Furthermore, animal studies using Melanotan II (MTII, Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2) and its analogous agonist HP228 have shown that cerebral, intraperitoneal, or subcutaneous administration suppresses appetite, reduces body weight, and increases energy metabolism (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)). Conversely, administration of the representative compound SHU9119 to animals resulted in significant and sustained increases in food intake and body weight, providing pharmacological evidence that MCR agonists can be used as therapeutic agents for obesity. The significant appetite-reducing effect observed upon MTII administration was not observed in MC4R KO (knock-out) mice, and these experimental results also demonstrate that the appetite-reducing effect is primarily mediated by activation of MC4R (Marsh, et al., Nat Genet 21, 119-122 (1999)).

[0008] The main type of obesity treatments developed to date are appetite suppressants that act on the central nervous system, most of which are drugs that modulate the activity of neurotransmitters. Examples include the noradrenaline agents phentermine and mazindol, and the serotonergic agents fluoxetine and sibutramine. However, these neurotransmitter modulators exert a wide range of effects on various physiological functions in addition to appetite suppression due to their numerous receptor subtypes. Therefore, these modulators have the major drawback of lacking selectivity for each subtype and causing various side effects when administered for a long period of time.

[0009] On the other hand, melanocortin agonists are neuropeptides, not neurotransmitters, and have the advantage of being able to induce weight loss by suppressing appetite without affecting other physiological functions, given that all functions except energy metabolism are normal in MC4R gene knockout mice. In particular, the receptor used is a G-protein coupled receptor (GPCR), which is one of the most successful receptors for new drugs developed to date, making it relatively easy to ensure selectivity for subtype receptors, which sets them apart from existing receptors.

[0010] As examples of utilizing such melanocortin receptors as a site of action, International Publication Nos. WO2008 / 007930 and WO2010 / 056022 disclose compounds that act as melanocortin receptor agonists.

[0011] Furthermore, the inventors of the present invention have conducted extensive research and invented a novel compound represented by the following Chemical Formula 1, which has excellent selective enhancing activity against melanocortin receptors, particularly against melanocortin-4 receptors (MC4R), and a method for producing the same (Korean Patent Application No. 10-2019-0141649 (filed November 7, 2019)).

[0012] [ka] (R1 is C2-C5 alkyl.)

[0013] On the other hand, the crystalline structure of a pharmaceutically active ingredient can sometimes affect the chemical stability of the drug. Different crystallization and storage conditions can change the crystalline structure of the compound, sometimes resulting in the production of different crystalline forms. Generally, amorphous drug products do not have a regular crystalline structure and sometimes have other defects such as poor product stability, small particle size, difficult filtration, tendency to agglomeration, and poor flowability. Therefore, it is necessary to improve various physical properties of the product. Thus, it is necessary to research a crystalline structure with high purity and good chemical stability for a compound. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] International Patent Application Publication No. WO2008 / 007930 [Patent Document 2] International Patent Application Publication No. WO2010 / 056022 Summary of the Invention [Problem to be solved by the invention]

[0015] An object of the present invention is to provide a stable crystalline form of a novel compound that has excellent selective stimulating activity for melanocortin receptors, particularly melanocortin-4 receptor (MC4R), and a method for producing the same.

[0016] Another object of the present invention is to provide a pharmaceutical composition containing a stable crystalline form of said novel compound. [Means for solving the problem]

[0017] To achieve the above objectives, In one aspect, the present invention provides crystalline Form III of the compound of the following Chemical Formula 1, a pharmaceutically acceptable salt thereof, or a solvate thereof, wherein the crystalline Form III has an X-ray powder diffraction (XRPD) pattern having three or more characteristic peaks selected from the following diffraction angles (2θ values): 6.238±0.2°, 8.257±0.2°, 8.828±0.2°, 14.637±0.2°, 16.618±0.2°, 17.465±0.2°, 18.859±0.2°, 19.061±0.2°, 19.333±0.2°, 20.642±0.2°, 22.679±0.2°, and 25.985±0.2°.

[0018] [ka]

[0019] In the above Chemical Formula 1, R1 is C2-C5 alkyl.

[0020] The compounds of Formula 1 may have asymmetric carbon centers and asymmetric axes or planes and may exist as cis or trans isomers, R or S isomers, racemates, partial stereoisomeric mixtures, and individual partial stereoisomers, all of which isomers and mixtures are included within the scope of the compounds of Formula 1.

[0021] For convenience, in this specification, unless otherwise specified, the compound of Chemical Formula 1 is used to mean the compound of Chemical Formula 1, its pharmaceutically acceptable salts, its isomers and solvates.

[0022] In one embodiment according to the present invention, R1 in Formula 1 is C2-C5 alkyl. In another embodiment according to the present invention, R1 in Formula 1 is linear or branched C2-C5 alkyl, for example, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl.

[0023] In another embodiment according to the present invention, R1 in Chemical Formula 1 is C2-C4 alkyl. In another embodiment according to the present invention, R1 in Chemical Formula 1 is linear or branched C2-C4 alkyl, for example, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl. Specifically, R1 may be isopropyl.

[0024] In one embodiment according to the present invention, the pharmaceutically acceptable salts include, but are not limited to, acid addition salts formed with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, etc.; organic carboxylic acids such as tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, etc.; sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, or naphthalenesulfonic acid, etc.

[0025] In one embodiment according to the present invention, the solvate may include hydrates; solvates with organic solvents such as methanol, ethanol, 2-propanol, 1,2-propanediol, 1,3-propanediol, n-butanol, 1,4-butanediol, tert-butanol, acetic acid, acetone, butyl acetate, methyl acetate, ethyl acetate, propyl acetate, t-butyl acetate, isobutyl acetate, methyl ethyl ketone, 2-pentanone, tetrahydrofuran, acetonitrile, formamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone, chloroform, toluene, and mixtures thereof.

[0026] In one embodiment according to the present invention, the crystalline form III may be a crystalline form of a solvate of the amide-based solvent of the compound of Chemical Formula 1.

[0027] In another embodiment according to the present invention, the crystalline form III may be a crystalline form of a formamide solvate of the compound of formula 1.

[0028] In one embodiment according to the present invention, crystalline Form III may have an X-ray powder diffraction pattern with three or more, five or more, seven or more, nine or more, ten or more, or eleven or more characteristic peaks selected from the following: 6.238±0.2°, 8.257±0.2°, 8.828±0.2°, 14.637±0.2°, 16.618±0.2°, 17.465±0.2°, 18.859±0.2°, 19.061±0.2°, 19.333±0.2°, 20.642±0.2°, 22.679±0.2°, and 25.985±0.2°.

[0029] In one embodiment according to the present invention, crystalline Form III may have characteristic peaks at 6.238±0.2°, 8.257±0.2°, 8.828±0.2°, 14.637±0.2°, 16.618±0.2°, 17.465±0.2°, 18.859±0.2°, 19.061±0.2°, 19.333±0.2°, 20.642±0.2°, 22.679±0.2°, and 25.985±0.2° in an X-ray powder diffraction pattern.

[0030] Crystalline Form III according to the present invention may be a crystalline form of a solvate of the compound of Chemical Formula 1, and therefore, a halo due to a residual solvent may be observed during XRPD measurement. For example, an XRPD peak observed at a 2θ angle of 20 to 30° may be a halo due to a residual solvent, such as formamide.

[0031] In one embodiment according to the present invention, the crystalline form III may have the X-ray powder diffraction (XRPD) pattern shown in FIG.

[0032] The crystalline form III according to the present invention may exhibit two endothermic peaks at 80 to 130°C and 200 to 260°C in a differential scanning calorimetry (DSC) profile.

[0033] In particular, the crystalline form III according to the present invention may contain an endothermic peak (200 to 260° C.) due to a solvent, such as formamide, in its differential scanning calorimetry (DSC) profile.

[0034] Furthermore, the crystalline form III according to the present invention may have a weight loss of 10% or less, for example, 5% or less, 4% or less, 3% or less, 2% or less, 1.5% or less, or 1.2% or less, in the range where an endothermic peak is observed at 80 to 130°C in a DSC profile.

[0035] Furthermore, the crystalline form III according to the present invention may have a weight loss of 20 to 40%, for example, 25 to 30%, or 27.9%, in the range where an endothermic peak is observed at 200 to 260°C in a DSC profile.

[0036] In one embodiment according to the present invention, the crystalline form III may have the TG / DTA propyl shown in FIG.

[0037] The NMR analysis of the crystalline form III according to the present invention may include peaks due to a solvent, such as formamide.

[0038] In one embodiment according to the present invention, the crystalline form III may have the NMR results shown in FIG.

[0039] In this specification, X-ray powder diffraction (XRPD) analysis was performed using a PANalytical X' Pert Pro MPD system, Malvern Panalytical Ltd.

[0040] Thermogravimetric analysis (TG / DTA) was performed using TGA / DSC1, Mettler-Toledo AG, and the results shown are shown below.

[0041] The nuclear magnetic resonance (NMR) results are shown as the results measured using a Bruker 500 MHz.

[0042] Crystalline Form III may have higher purity and be more physically and chemically stable than the crude compound of Formula 1, the amorphous compound of Formula 1, or other crystalline forms of the compound of Formula 1.

[0043] Furthermore, the crystalline form III of the compound of Chemical Formula 1 may have superior melanocortin-4 receptor activating ability and preventive or therapeutic effects against diseases such as obesity, diabetes, inflammation, and erectile dysfunction compared to known melanocortin-4 receptor agonists, but the effects of the present invention are not limited thereto.

[0044] In another aspect, the present invention provides a method for preparing crystalline Form III, comprising the steps of dissolving the compound of Formula 1 in a crystallization solvent to prepare a mixed solution, and obtaining crystals from the mixed solution.

[0045] First, the compound represented by the formula 1 is dissolved in a crystallization solvent.

[0046] The compound of Chemical Formula 1 for preparing the crystalline form III can be the compound of Chemical Formula 1, a salt thereof, an isomer thereof, or a solvate thereof.

[0047] The compound of Chemical Formula 1 can be obtained by the manufacturing method described in the specification of Korean Patent Application No. 10-2019-0141649 (filed on November 7, 2019).

[0048] The crystallization solvent is not particularly limited as long as it is a solvent suitable for crystallization of a compound. In one embodiment, the crystallization solvent includes a polar organic solvent.

[0049] The polar organic solvent may include, but is not limited to, an amide solvent.

[0050] The amide solvent may include, but is not limited to, formamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone, or a mixture thereof.

[0051] In one embodiment according to the present invention, the polar organic solvent may include formamide.

[0052] In one embodiment according to the present invention, the step of obtaining the crystals can be carried out by precipitation with anti-solvent.

[0053] In the antisolvent crystallization method, the crystallization solvent for dissolving the compound of Formula 1 can be the polar organic solvent described above, for example, formamide, and the antisolvent can be at least one of a nonpolar organic solvent and water. The nonpolar organic solvent can be any organic solvent having nonpolar properties, and examples thereof include hexane, heptane, cyclohexane, carbon tetrachloride, benzene, and chloroform.

[0054] In one embodiment of the present invention, the compound of Formula 1 is dissolved in formamide, and then heptane is added dropwise as an antisolvent to obtain crystalline Form III.

[0055] In another embodiment according to the present invention, the step of obtaining the crystals can be carried out by a slurry experiment.

[0056] The slurry method may be performed by dissolving the compound of Formula 1 in a sufficient amount of solvent at a desired temperature until undissolved solids remain, sealing the solution, stirring the solution for a predetermined period while maintaining the temperature, filtering, and drying the solution, but is not limited thereto.

[0057] In the slurry method, the crystallization solvent for dissolving the compound of Formula 1 may be the polar organic solvent described above, for example, formamide, but is not limited thereto.

[0058] In one embodiment according to the present invention, the compound of Chemical Formula 1 is added to a sufficient amount of a crystallization solvent at a constant temperature of 0°C to 80°C until a solid remains, and then the mixture is sealed, maintained at the same temperature, and stirred for 1 to 60 days to obtain Crystalline Form III.

[0059] In the slurry method, the certain temperature can be, for example, but not limited to, 0°C to 80°C, 5°C to 60°C, 5°C to 50°C, 5°C to 20°C, 20°C to 50°C, 15°C to 60°C, 5°C, 20°C, or 50°C.

[0060] In the slurry method, the period for maintaining the compound dissolved in the crystallization solvent is not limited thereto, but may be, for example, 1 to 60 days, 1 to 30 days, 1 to 15 days, 1 to 10 days, 5 to 7 days, or 6 days.

[0061] The thus obtained crystalline form III may have higher purity and be physically and chemically more stable than the crude compound of Chemical Formula 1, the amorphous compound of Chemical Formula 1, or any crystalline form of Chemical Formula 1, but the effects of the present invention are not limited thereto.

[0062] In yet another aspect, the present invention provides a pharmaceutical composition comprising: (i) said crystalline Form III; and (ii) a pharmaceutically acceptable carrier.

[0063] Since the crystalline form III of the present invention exhibits excellent enhancing activity on melanocortin receptors, particularly the melanocortin-4 receptor (MC4R), the present invention can provide a pharmaceutical composition for enhancing melanocortin receptor function, which contains the crystalline form III as an active ingredient. Specifically, the pharmaceutical composition can be a composition for enhancing melanocortin-4 receptor function.

[0064] Furthermore, the pharmaceutical composition may exhibit excellent effects in the prevention or treatment of obesity, diabetes, inflammation, and erectile dysfunction, and 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.

[0065] As used herein, the term "carrier" refers to a compound that facilitates the introduction of a compound into cells or tissues.

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

[0067] The crystalline Form III of the present invention can be administered by any route depending on the purpose. For example, the amorphous compound of the present invention can be administered by injection or orally.

[0068] The pharmaceutical compositions of the present invention can be in various oral administration forms such as tablets, pills, powders, capsules, granules, syrups or emulsions, or in parenteral administration forms such as injectable preparations for intramuscular, intravenous or subcutaneous administration.

[0069] Injectable preparations can be prepared using suitable dispersing agents, wetting agents, suspending agents, or excipients according to known techniques.

[0070] Excipients that can be used in the pharmaceutical formulation of the present invention may include, but are not limited to, sweeteners, binders, solubilizers, solubilizers, wetting agents, emulsifiers, isotonicity agents, adsorbents, disintegrants, antioxidants, preservatives, lubricants, fillers, flavorings, etc. For example, excipients that can be used include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, silica, magnesium aluminum silicate, starch, gelatin, tragacanth gum, alginic acid, sodium alginate, methylcellulose, sodium carboxymethylcellulose, water, ethanol, polyethylene glycol, polyvinylpyrrolidone, sodium chloride, calcium chloride, orange essence, strawberry essence, vanilla flavor, etc.

[0071] When the pharmaceutical composition of the present invention is in the form of oral administration, examples of the carrier to be 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, etc.

[0072] 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, aqueous glucose solution, aqueous saccharide solution, alcohol, glycol, ether, oil, fatty acid, fatty acid ester, glyceride, etc.

[0073] In yet another aspect, there is provided crystalline Form III as described above for use in enhancing the activity of melanocortin receptors, particularly melanocortin-4 receptors (MC4R).

[0074] In one embodiment, there is provided crystalline Form III as described above for use in the treatment or prophylaxis of obesity, diabetes, inflammation or erectile dysfunction.

[0075] In yet another aspect, there is provided a method for enhancing the activity of melanocortin receptors, particularly melanocortin-4 receptors (MC4R), comprising the step of administering to a subject crystalline Form III described above.

[0076] In yet another aspect, there is provided a method of treating obesity, diabetes, inflammation or erectile dysfunction comprising administering to a subject crystalline Form III as described above. [Effects of the Invention]

[0077] Crystalline Form III of the present invention exhibits excellent enhancing activity against melanocortin receptors, particularly melanocortin-4 receptor (MC4R), and can therefore be usefully used in the prevention or treatment of obesity, diabetes, inflammation, and erectile dysfunction.

[0078] Crystalline Form III of the present invention exhibits an on-target effect on the melanocortin-4 receptor, and therefore exhibits weight loss and food intake reduction effects, does not affect anxiety and depression, and can be administered without safety issues such as side effects and mutation induction due to hERG (human ether-a-go-go related gene) inhibition.

[0079] Furthermore, the crystalline form III of the present invention has superior purity, yield, physical and chemical stability compared to the crude compound of formula 1, the amorphous compound of formula 1, or any other crystalline form of formula 1.

[0080] Specifically, the crystalline form III may have superior solubility, storage stability, and production stability compared to the compound of Chemical Formula 1, the amorphous compound of Chemical Formula 1, or any other crystalline form of Chemical Formula 1. [Brief explanation of the drawings]

[0081] [Figure 1] 1 is a graph showing the XRPD results of Example 1. [Figure 2]1 is a graph showing the TG / DTA results of Example 1. [Figure 3] 1 is a graph showing the NMR results of Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0082] The present invention will be described in more detail below with reference to Production Examples and Examples, although these Examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention.

[0083] Preparation Example 1: Preparation of methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylate hydrochloride [ka]

[0084] The title compound was obtained through the following steps A, B, C, D and E.

[0085] Step A: Preparation of 1-(tert-butyl) 2-methyl (2S,4S)-4-azidopyrrolidine-1,2-dicarboxylate Under nitrogen, 1-(tert-butyl) 2-methyl(2S,4R)-4-((methylsulfonyl)oxy)pyrrolidine-1,2-dicarboxylate (48.5 g, 150 mmol) was dissolved in N,N'-dimethylformamide (250 mL) and sodium azide (19.5 g, 300 mL) was added. The mixture was stirred at 80°C for 16 hours, 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 and water, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give 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.

[0086] MS [M+H] = 271 (M+1) 1H NMR (400 MHz, CD3OD) δ 4.43-4.37 (m, 1H), 4.35-4.27 (br, 1H), 3.77 (s, 1.8H), 3.76 (s, 1.2H), 3.73-3.66 (m, 1H), 3.44-3.38 (m, 1H), 2.63-2.49 (m, 1H), 2.19-2.11 (m, 1H), 1.50 (s, 4.5H), 1.44 (s, 4.5H)

[0087] Step B: Preparation of 1-(tert-butyl) 2-methyl (2S,4S)-4-aminopyrrolidine-1,2-dicarboxylate 1-(tert-butyl) 2-methyl(2S,4S)-4-azidopyrrolidine-1,2-dicarboxylate (24.59 g, 91.0 mmol) obtained in Step A was dissolved in tetrahydrofuran (180 mL), and 1 M trimethylphosphine tetrahydrofuran solution (109.2 mL, 109.2 mmol) was slowly added at 0°C. The mixture was stirred at the same temperature for 1 hour and then at room temperature for 3 hours. The reaction solvent was concentrated under reduced pressure, and dichloromethane (100 mL) and water (150 mL) were added and stirred for approximately 30 minutes. The layers were separated and extracted again with dichloromethane. The organic layer was dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain 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.

[0088] MS [M+H] = 245 (M+1) 1H 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)

[0089] Step C: Preparation of 1-(tert-butyl) 2-methyl(2S,4S)-4-(((1s,4R)-4-methylcyclohexyl)amino)pyrrolidine-1,2-dicarboxylate 1-(tert-butyl) 2-methyl(2S,4S)-4-aminopyrrolidine-1,2-dicarboxylate (20.62 g, 84.4 mmol) obtained in Step B 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%).

[0090] MS [M+H] = 341 (M+1) 1H 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)

[0091] Step D: Preparation of 1-(tert-butyl) 2-methyl(2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-1,2-dicarboxylate 1-(tert-butyl) 2-methyl(2S,4S)-4-(((1s,4R)-4-methylcyclohexyl)amino)pyrrolidine-1,2-dicarboxylate (37.29 g, 109.5 mmol) obtained in Step C 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 the addition of aqueous sodium bicarbonate solution and extraction 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 give 1-(tert-butyl) 2-methyl(2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-1,2-dicarboxylate (38.79 g, 86%).

[0092] MS [M+H] = 411 (M+1) 1H 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)

[0093] Step E: Preparation of methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylate hydrochloride 1-(tert-butyl) 2-methyl(2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-1,2-dicarboxylate (34.0 g, 82.8 mmol) obtained in Step D was dissolved in dichloromethane (200 mL), and then 4N hydrochloric acid in 1,4-dioxane (82.8 mL, 331.3 mmol) was added at 0°C. After stirring at room temperature for 6 hours, the reaction solvent was concentrated under reduced pressure to obtain a crude product (28.7 g, 99%), which was used in the next step without purification.

[0094] MS[M+H] = 311 (M+1)

[0095] Production Example 2: Production of (3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carboxylic acid [ka]

[0096] The title compound was obtained by the method described in International Publication No. WO2004 / 092126.

[0097] MS[ M+H] = 282 (M+1) 1H 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)

[0098] Production Example 3: Production 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 (MC70) [ka]

[0099] The title compound was obtained through the following steps A, B and C.

[0100] Step A: Preparation of methyl (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylate Methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylate hydrochloride (28.7 g, 82.73 mmol) obtained in Production Example 1, (3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carboxylic acid (24.5 g, 86.87 mmol) obtained in Production Example 2, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (22.2 g, 115.83 mmol), and 1-hydroxybenzotriazole hydrate (15.7 g, 115.83 mmol) were dissolved in N,N'-dimethylformamide (400 ml), and N,N'-diisopropylethylamine (72.0 ml, 413.66 mmol) was slowly added. The mixture was stirred at room temperature for 16 hours, and the reaction solvent was concentrated under reduced pressure. Then, 0.5N aqueous sodium hydroxide was added and the mixture was extracted twice with ethyl acetate. The organic layer was washed twice with aqueous sodium chloride and water, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography to give 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%).

[0101] MS [M+H] = 575 (M+1)

[0102] Step B: Preparation of (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylic acid Methyl (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-((1S,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylate (39.4 g, 68.62 mmol) obtained in Step A was dissolved in methanol (450 mL), and 6N aqueous sodium hydroxide solution (57.2 mL, 343.09 mmol) was added. The mixture was stirred at room temperature for 16 hours, and the pH was adjusted to approximately 5 with 6N aqueous hydrochloric acid. The reaction solution was then concentrated under reduced pressure. The concentrated solution was dissolved in dichloromethane, and the undissolved 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.

[0103] MS [M+H] = 561 (M+1)

[0104] Step C: Preparation of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylic acid (38.4 g, 68.60 mmol) obtained in Step B, 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 thereto. The mixture was stirred at room temperature for 16 hours, concentrated under reduced pressure, and then 0.5N aqueous sodium hydroxide was added and extracted twice with ethyl acetate. The organic layer was washed twice with aqueous sodium chloride and water, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography to give 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%, MC70).

[0105] MS [M+H] = 630 (M+1)

[0106] [Example] Preparation of Crystalline Form III 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 [ka]

[0107] Example 1: Preparation by antisolvent method 25 mg of the compound (MC70) prepared in Preparation Example 3 was dissolved in formamide, and then 19 volumes of heptane as an antisolvent were added at room temperature. After storing at 4°C for 7 to 14 days, the solution was filtered and dried to obtain the title compound (crystalline form III of MC70).

[0108] Example 2: Production by the slurry method 25 mg of the compound (MC70) prepared in Preparation Example 3 was added to a sufficient amount of formamide at the target temperature (5°C) until an undissolved solid remained. The test tube was sealed and stirred for 6 days while maintaining the target temperature (5°C). The mixture was then filtered and dried to obtain the title compound (crystalline form III of MC70).

[0109] Example 3: Production by the slurry method The title compound (crystalline form III of MC70) was obtained in the same manner as in Example 2, except that the target temperature was set to 20°C.

[0110] Example 4: Production by the slurry method The title compound (crystalline form III of MC70) was obtained in the same manner as in Example 2, except that the target temperature was set to 50°C.

[0111] The compounds obtained in Examples 1 to 4 were subjected to XRPD, TG / DTA, NMR, and DVS analyses by the following methods, and the results confirmed that the compounds obtained in Examples 1 to 4 had the same crystalline form.

[0112] The analytical results of XRPD (FIG. 1), TG / DTA (FIG. 2), and NMR (FIG. 3) performed on Example 1, which represents the compounds of Examples 1 to 4, are shown in graphs in FIGS. 1 to 3. XRPD analysis was also performed after the DVS measurement, and it was confirmed that the crystalline form had not changed. The results are shown in FIG. 5.

[0113] Experimental Example 1. XRPD evaluation Powder XRPD diffraction patterns were obtained using a PANalytical X'Pert Pro MPD system equipped with a monochromatized radiation source and a Ni filter as a solid-state detector in the following manner.

[0114] Approximately 20-30 mg of sample was placed on a glass sample holder with a flat surface. The instrument generator was set to 45 kV (acceleration voltage) and 40 mA (filament emission), and measurements were performed in reflection mode (not-spin). Bragg angles (2θ) were measured in the range of 4-40° with a step size of 0.026° and a time per step of 51 seconds.

[0115] The obtained crystalline form III was measured by XRPD, and the results are shown in FIG.

[0116] As can be seen from the spectrum shown in Figure 1, the crystalline form III of the present invention is a crystalline material, and the specific XRPD values ​​are shown in Table 1 below. The amorphous pattern observed between 20 and 30° 2θ was interpreted as being due to unsolvated formamide remaining.

[0117] [Table 1]

[0118] Experimental Example 2: Thermogravimetric analysis (TG / DTA) TG / DTA measurements were performed using a Mettler Toledo DSC1 system. Approximately 2–5 mg of sample was weighed and placed in a 40 μL aluminum crucible (flat-bottomed aluminum pan with a pinhole lid), which was then placed in a TG furnace. The sample was then heated at a rate of 10°C / min up to 300°C, stabilizing at 30°C for 1 min before heating. Nitrogen gas was supplied to the inside of the equipment at a rate of 70 mL / min during the measurements to prevent the inflow of oxygen and other gases. Data collection and evaluation were performed using the software STARe.

[0119] The TG / DTA measurement results of the obtained crystalline form III are shown in FIG.

[0120] As can be seen from Figure 2, crystalline Form III exhibited two endothermic peaks at approximately 95.08°C (Onset) and approximately 216.53°C (Onset), with weight losses of 1.2% w / w (0.17 mol eq. formamide) and 27.9% w / w (5.4 mol eq. formamide), respectively. The temperature values ​​may have an error of ±5°C.

[0121] The temperature at which the endothermic peak appears at about 216.53°C (Onset) coincides with the boiling point of formamide, and formamide was also confirmed in the following NMR results, confirming that the crystalline form III is a solvate of formamide.

[0122] Experimental Example 3.1H Nuclear Magnetic Resonance Spectroscopy (NMR) The obtained crystalline form III was subjected to NMR analysis using a Bruker 500 MHz spectrometer with methanol-d4 solvent, and the results are shown in FIG.

[0123] NMR analysis confirmed that crystalline Form III was a formamide solvate of Formula 1.

Claims

1. Crystalline Form III of a compound of the following formula 1, a pharmaceutically acceptable salt thereof, or a solvate thereof: Crystalline Form III having an X-ray powder diffraction (XRPD) pattern with three or more characteristic peaks selected from the following diffraction angles (2θ values): 6.238±0.2°, 8.257±0.2°, 8.828±0.2°, 14.637±0.2°, 16.618±0.2°, 17.465±0.2°, 18.859±0.2°, 19.061±0.2°, 19.333±0.2°, 20.642±0.2°, 22.679±0.2°, and 25.985±0.2°. 【Chemical 1】 (In the above chemical formula 1, R １ is C ２ -C ５ alkyl)

2. The R １ is C ２ ~C ４ 2. The crystalline form III of claim 1, wherein the alkyl is alkyl.

3. 3. The crystalline form III of claim 2, wherein 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.

4. 2. The crystalline form III of claim 1, wherein the pharmaceutically acceptable salt is selected from the group consisting of hydrochloride, sulfate, nitrate, phosphate, hydrobromide, and hydroiodide.

5. 2. The crystalline form III of claim 1, which is a crystalline form of the formamide solvate of formula 1.

6. A method for preparing crystalline Form III according to any one of claims 1 to 5, comprising the steps of: dissolving the compound of Formula 1 in a crystallization solvent to prepare a mixed solution; and obtaining crystals from the mixed solution.

7. 7. The method for preparing crystalline Form III according to claim 6, wherein the crystals are obtained by antisolvent crystallization.

8. 7. The method for producing crystalline Form III according to claim 6, wherein the crystals are obtained by a slurry method.

9. 7. The method for producing crystalline Form III according to claim 6, wherein the crystallization solvent comprises a polar organic solvent.

10. 10. The method for preparing crystalline Form III according to claim 9, wherein the polar organic solvent comprises formamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone, or a mixture thereof.

11. 10. The method for producing crystalline Form III according to claim 9, wherein the crystallization solvent further comprises a non-polar organic solvent.

12. 12. The method for preparing crystalline Form III according to claim 11, wherein the non-polar organic solvent comprises hexane, heptane, cyclohexane, carbon tetrachloride, benzene, chloroform, or a mixture thereof.

13. A pharmaceutical composition comprising crystalline Form III of any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

14. A pharmaceutical composition for enhancing the activity of melanocortin-4 receptor, comprising crystalline Form III of any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

15. The pharmaceutical composition of claim 13, wherein the composition is for the prevention or treatment of obesity, diabetes, inflammation or erectile dysfunction.

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