Crystalline form ii of melanocortin receptor agonist compound and preparation method therefor

Abstract

To provide a crystalline form II of a compound represented by chemical formula 1, a method for preparing the same, and a pharmaceutical composition comprising the same.SOLUTION: The crystalline form II of a compound represented by chemical formula 1 of the present invention may be characterized by XRD patterns, DSC profiles, and / or TGA profiles.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] This application claims the benefit of priority based on Korean Patent Application No. 10-2020-0142397, filed on October 29, 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 II 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 and 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) being its only ligand. MC3R and MC4R, primarily expressed in the central nervous system, are involved in regulating appetite, energy metabolism, and the efficiency of fat storage in the body. 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 subtype receptors. 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 they use 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]

[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, smaller 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 novel amorphous compound or a salt thereof which has excellent selective enhancing activity against melanocortin receptors, particularly melanocortin-4 receptor (MC4R).

[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 II of a compound of Formula 1, a pharmaceutically acceptable salt thereof, or a solvate thereof: The X-ray powder diffraction pattern showed the following diffraction angles (2θ values): 7.77±0.2°, 9.82±0.2°, 10.50±0.2°, 11.37±0.2°, 12.36±0.2°, 15.17±0.2°, 15.46±0.2°, 15.88±0.2°, 16.75±0.2°, 17.59±0.2°, 17.93±0.2°, 18.33±0.2°, 19.64±0.2°, 20.19±0.2°, 21.19±0.2°, 22.19±0.2°, 23.19±0.2°, 24.19±0.2°, 25.19±0.2°, 26.19±0.2°, 27.19±0.2°, 28.19±0.2°, 29.19±0.2°, 30.19±0.2°, 31.19±0.2°, 32.19±0.2°, 33.19±0.2°, 34.19±0.2°, 35.19±0.2°, 36.19±0.2°, 37.19±0.2°, 38.19±0.2°, 39.19±0.2°, 40.19±0.2°, 41.19±0.2°, 42.19±0.2°, 43.19±0.2°, 44.19±0.2°, 45.19±0.2°, 46.19±0.2°, 47.19±0.2°, 48. The present invention provides crystalline Form II having 3 or more, 5 or more, 7 or more, 9 or more, or 10 or more characteristic peaks selected from 1.71±0.2°, 23.29±0.2°, 23.58±0.2°, 24.42±0.2°, 25.07±0.2°, 25.63±0.2°, 26.31±0.2°, 27.17±0.2°, 27.52±0.2°, and 28.96±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 a C2-C5 alkyl. In another embodiment according to the present invention, R1 in Formula 1 is a linear or branched C2-C5 alkyl, such as ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl.

[0023] In another embodiment according to the present invention, R1 in Formula 1 is C2 or C3 alkyl. In another embodiment according to the present invention, R1 in Formula 1 is linear or branched C2 or C3 alkyl, for example, ethyl, n-propyl, or iso-propyl.

[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, chloroform, toluene, and mixtures thereof.

[0026] In one embodiment according to the present invention, the crystalline form II may be a crystalline form of a pharmaceutically acceptable salt of the compound of formula 1.

[0027] The pharmaceutically acceptable salt of the compound of Formula 1 may be a hydrochloride compound of Formula 2 below.

[0028] [ka]

[0029] In the above formula 2, R2 is C2-C5 alkyl.

[0030] In still another embodiment according to the present invention, the pharmaceutically acceptable salt of the compound of Chemical Formula 1 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 salt of Chemical Formula 3 below.

[0031] [ka]

[0032] In another embodiment according to the present invention, the crystalline form II may be a crystalline form of a solvate, specifically a hydrate, of a pharmaceutically acceptable salt of the compound of formula 1.

[0033] More specifically, the crystalline form II may be a crystalline form of a hydrate of the hydrochloride salt of the compound of Chemical Formula 1.

[0034] In one embodiment according to the present invention, the crystalline form II may be a crystalline form of the compound of the following Chemical Formula 4:

[0035] [ka]

[0036] Crystalline Form II according to the present invention has the following properties when analyzed by X-ray powder diffraction (XRD): 7.77±0.2°, 9.82±0.2°, 10.50±0.2°, 11.37±0.2°, 12.36±0.2°, 15.17±0.2°, 15.46±0.2°, 15.88±0.2°, 16.75±0.2°, 17.59±0.2°, 17.93±0.2°, 18.33±0.2°, 19.64±0.2°, 20.1 and 28.96±0.2°.

[0037] In one embodiment according to the present invention, the crystalline form II may have the XRD pattern shown in FIG.

[0038] The crystalline form II according to the present invention exhibits two endothermic peaks at 30 to 200°C and an endothermic peak due to decomposition at 220°C or higher in a differential scanning calorimetry (DSC) profile.

[0039] In one embodiment according to the present invention, the crystalline form II may have a DSC profile as shown in FIG.

[0040] Crystalline Form II according to the present invention may have a thermogravimetric analysis (TGA) profile with a weight loss of 15% or less, e.g., 1% to 15%, 1% to 10%, 5% to 10%, or 6%, when heated at a temperature of 170°C or less.

[0041] In one embodiment according to the present invention, the crystalline form II may have a TGA profile as shown in FIG.

[0042] Crystalline Form II of the present invention was found to be stable to heat and humidity, as evidenced by the stability test results (HPLC) that it was chemically stable for 4 weeks under accelerated conditions (40°C, 75% RH) and harsh conditions (80°C).

[0043] In this specification, X-ray diffraction (XRD) analysis was performed using a PANalytical X' Pert Pro MPD system, Malvern Panalytical Ltd. The results shown are as follows.

[0044] Differential scanning calorimetry (DSC) analysis was performed using a DSC1, Mettler-Toledo AG, and the results are shown.

[0045] Thermogravimetric analysis (TGA) was performed using a TGA / DSC 1, Mettler-Toledo AG.

[0046] The stability analysis was performed using HPLC (Agilent Technologies, Inc.) and the results shown are shown below.

[0047] Crystalline Form II 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.

[0048] Furthermore, Crystal Form II 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.

[0049] In another aspect, the present invention provides a method for preparing crystalline Form II, comprising dissolving the compound of Formula 1 in a crystallization solvent to prepare a mixed solution, and obtaining crystals from the mixed solution.

[0050] First, the compound represented by the formula 1 is dissolved in a crystallization solvent.

[0051] The compound of Chemical Formula 1 for preparing Crystalline Form II can be the compound of Chemical Formula 1, a salt thereof, an isomer thereof, or a solvate thereof.

[0052] 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).

[0053] The crystallization solvent can be any solvent suitable for crystallization of the compound without particular limitation. In one embodiment, the crystallization solvent comprises a mixture of water and a polar aprotic organic solvent.

[0054] The polar aprotic organic solvent may include ethyl acetate, methyl isobutyl ketone, dimethylsulfoxide, tetrahydrofuran, acetone, dimethylformamide, acetonitrile, or a mixture thereof.

[0055] In one embodiment according to the present invention, the polar aprotic organic solvent may include ethyl acetate.

[0056] In one embodiment according to the present invention, the crystallization solvent may be a mixed solvent in which water and a polar aprotic organic solvent are mixed in a volume ratio of 20:1 to 1:20, specifically, a mixed solvent in which water and a polar aprotic organic solvent are mixed in a volume ratio of 15:1 to 1:15, 10:1 to 1:10, 1:1 to 1:10, 1:5 to 1:18, 1:7 to 1:16, 1:7.5 to 1:15, 1:8 to 1:13, 1:8.5 to 1:12, 1:9 to 1:11, 1:9.5 to 1:10.5, or 1:10.

[0057] The crystallization solvent can be used in an amount of 0.1 to 5 mL, 0.3 to 3 mL, 0.5 to 1.5 mL, 0.6 to 1 mL, 0.65 to 0.9 mL, 0.65 to 0.7 mL, or 0.66 mL per 1 g of the compound of Chemical Formula 1.

[0058] The compound of Formula 1 may be dissolved in a crystallization solvent at room temperature, for example, 20 to 30°C, specifically, 23 to 28°C or 25°C, without stirring or with stirring.

[0059] In one embodiment according to the present invention, a mixed solution in which the compound of Chemical Formula 1 is dissolved can be obtained by using 0.6 mL of EtOAc and 0.06 mL of distilled water per 1 g of the compound of Chemical Formula 1 at room temperature.

[0060] Next, a step of obtaining crystals from the mixed solution containing the compound of Formula 1 is included. The crystals can be obtained by, for example, cooling the solution, adding an acid dropwise to the solution to form a precipitate, evaporating the solvent, adding an anti-solvent to form a supersaturated solution, or using a slurry conversion method.

[0061] The crystallization step may include stirring the mixed solution, and may further include adding a non-polar organic solvent to the mixed solution before, after, or simultaneously with stirring the mixed solution.

[0062] The stirring can be carried out by known means, and the stirring time is not limited thereto, but can be, for example, 10 hours or more and 24 hours or less, specifically, 10 to 24 hours, 12 to 20 hours, 15 to 19 hours, 16 to 19 hours, or 18 hours.

[0063] In one embodiment according to the present invention, a non-polar organic solvent is dropwise added to the mixed solution, and the mixture is stirred. The precipitate formed is filtered and washed to obtain crystals.

[0064] The addition of the non-polar organic solvent can increase the rate of crystallization particle formation, thereby improving the yield or production stability of the obtained crystalline form II, but the present invention is not limited thereto.

[0065] The non-polar organic solvent can be used without any particular limitation as long as it is an organic solvent having non-polar properties, and examples thereof include hexane, heptane, cyclohexane, carbon tetrachloride, benzene, and chloroform.

[0066] In one embodiment according to the present invention, the method may include adding heptane to the solution during crystallization from the mixed solution.

[0067] The thus obtained Crystalline Form II 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 any crystalline form of Chemical Formula 1, but the effects of the present invention are not limited thereto.

[0068] In yet another aspect, the present invention provides a pharmaceutical composition comprising: (i) said crystalline Form II; and (ii) a pharmaceutically acceptable carrier.

[0069] Since the crystalline form II of the present invention exhibits excellent enhancing activity on melanocortin receptors, particularly the melanocortin-4 receptor (MC4R), the present invention also provides a pharmaceutical composition for enhancing melanocortin receptor function, which contains the crystalline form II as an active ingredient. Specifically, the pharmaceutical composition may be a composition for enhancing melanocortin-4 receptor function.

[0070] 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.

[0071] As used herein, the term "carrier" refers to a compound that facilitates the introduction of a compound into cells or tissues.

[0072] When crystalline Form II 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.

[0073] The crystalline Form II 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.

[0074] 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.

[0075] Injectable preparations can be prepared using suitable dispersing agents, wetting agents, suspending agents, or excipients according to known techniques.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] In yet another aspect, there is provided crystalline Form II as described above for use in enhancing the activity of melanocortin receptors, particularly melanocortin-4 receptors (MC4R).

[0080] In one embodiment, there is provided crystalline Form II as described above for use in the treatment or prevention of obesity, diabetes, inflammation or erectile dysfunction.

[0081] 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 II described above.

[0082] In yet another aspect, there is provided a method of treating obesity, diabetes, inflammation or erectile dysfunction comprising administering to a subject crystalline Form II as described above. [Effects of the Invention]

[0083] Crystalline Form II of the present invention exhibits excellent enhancing activity against melanocortin receptors, particularly melanocortin-4 receptor (MC4R), and can therefore be usefully used for the prevention or treatment of obesity, diabetes, inflammation, and erectile dysfunction.

[0084] Crystalline Form II of the present invention exhibits an on-target effect on the melanocortin-4 receptor, thereby exhibiting weight loss and food intake reduction effects, without affecting anxiety and depression, and can be administered without side effects due to hERG (human ether-a-go-go related gene) inhibition or stability issues such as induction of mutations.

[0085] Furthermore, the crystalline form II 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.

[0086] Specifically, the crystalline form II 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]

[0087] [Figure 1] 1 is a graph showing the results of XRD in Production Example 4. [Figure 2] 1 is a graph showing the results of DSC in Production Example 4. [Figure 3]1 is a graph showing the TGA results of Production Example 4. [Figure 4] 1 is a graph showing the XRD results of Example 1. [Figure 5] 1 is a graph showing the results of DSC in Example 1. [Figure 6] 1 is a graph showing the TGA results of Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0088] 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.

[0089] Preparation Example 1: Preparation of methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylate hydrochloride [ka]

[0090] The title compound was obtained through the following steps A, B, C, D and E.

[0091] 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.

[0092] MS [M+H] = 271 (M+1) 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)

[0093] 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.

[0094] MS [M+H] = 245 (M+1) 1 H NMR (400 MHz, CD3OD) δ 4.27 (m, 1H), 3.77 (s, 1.8H), 3.76 (s,1.2H), 3.75-3.67 (m, 1H), 3.50-3.42 (m, 1H), 3.22-3.17 (m, 1H), 2.58-2.47 (m,1H), 1.82-1.71 (m, 1H), 1.48 (s, 4.5H), 1.42 (s, 4.5H)

[0095] 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%).

[0096] MS [M+H] = 341 (M+1) 1 H NMR (400 MHz, CD3OD) δ 4.26 (m, 1H), 3.76 (s, 1.8H), 3.75 (s, 1.2H), 3.78-3.71 (m, 1H), 3.49-3.40 (m, 1H), 3.22-3.16 (m, 1H), 2.69-2.60 (br, 1H), 2.58-2.46 (m, 1H), 1.87-1.77 (m, 1H), 1.73-1.63 (m, 1H), 1.62-1.35 (m, 8H), 1.48 (s, 4.5H), 1.42 (s, 4.5H), 0.96 (d, 3H)

[0097] 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%).

[0098] MS [M+H] = 411 (M+1) 1 H NMR (400 MHz, CD3OD) δ 4.27 (m, 1H), 3.76 (s, 1.8H), 3.75 (s, 1.2H), 3.78-3.72 (m, 1H), 3.50-3.41 (m, 1H), 3.33-3.14 (m, 1H), 2.69-2.60 (m, 2H), 2.57-2.43 (m, 1H), 1.87-1.79 (m, 1H), 1.70-1.61 (m, 1H), 1.60-1.32 (m, 8H), 1.47 (s, 4.5H), 1.41 (s, 4.5H), 1.10 (dd, 6H), 0.99 (d, 3H)

[0099] 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.

[0100] MS[M+H] = 311 (M+1)

[0101] Production Example 2: Production of (3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carboxylic acid [ka]

[0102] The title compound was obtained by the method described in International Publication No. WO2004 / 092126.

[0103] MS[ M+H] = 282 (M+1) 1 H NMR (400 MHz, CD3OD) δ 7.43-7.33 (m, 4H), 3.90-3.69 (m, 3H), 3.59 (dd, J = 11.2, 10.0 Hz, 1H), 3.29 (dd, J = 11.2, 11.2 Hz, 1H), 3.18-3.09 (m, 1H), 1.44 (s, 9H)

[0104] 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 [ka]

[0105] The title compound was obtained through the following steps A, B and C.

[0106] 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%).

[0107] MS [M+H] = 575 (M+1)

[0108] 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.

[0109] MS [M+H] = 561 (M+1)

[0110] 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 reaction 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%).

[0111] MS [M+H] = 630 (M+1)

[0112] Preparation Example 4: Preparation of 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 [ka]

[0113] 1 g of compound (MC70) prepared in Preparation Example 3 was dissolved in 19 mL of MBTE at 25°C. After dissolution was complete, 1 mL of heptane was added and the mixture was cooled to -5 to 0°C. After the mixture reached the set temperature, 1 equivalent of 4M HCl / EtOAc was added dropwise, followed by stirring for approximately 90 minutes and filtration to obtain the title compound (MC71). (Yield: approximately 90%)

[0114] The results of XRD (FIG. 1), DSC (FIG. 2), and TGA (FIG. 3) analysis of the compound of Preparation Example 4 are attached in FIGS. 1 to 3, respectively, and the analysis results confirmed that the compound was amorphous. The analytical methods for XRD, DSC, and TGA are as described below in the experimental example for Example 1.

[0115] Example 1 Preparation of Crystalline Form II 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 Hydrate [ka]

[0116] 1 g of the compound (MC71) prepared in Preparation Example 4 was dissolved in 0.6 mL of EtOAc and 0.06 mL of distilled water at room temperature while stirring using a magnetic stirrer. After complete dissolution, 0.6 mL of heptane was added dropwise while stirring. After stirring for 18 hours, the mixture was filtered under nitrogen pressure to obtain the title crystalline form II (yield: approximately 60%).

[0117] Comparative Example 1 1 g of the compound (MC71) prepared in Preparation Example 4 was dissolved in 1 mL of EtOAc at room temperature. After dissolution was completed, the mixture was stirred with a magnetic stirrer for about 43 hours, but no crystals like those in Example 1 were obtained.

[0118] Experimental Example 1. XRD Evaluation Powder XRD 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.

[0119] 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.

[0120] The obtained crystalline form II was measured by XRD, and the results are shown in FIG.

[0121] As can be seen from the spectrum shown in FIG. 4, the crystalline form II according to the present invention exhibits characteristic peaks (2θ) at 7.77°, 9.82°, 10.50°, 11.37°, 12.36°, 15.17°, 15.46°, 15.88°, 16.75°, 17.59°, 17.93°, 18.33°, 19.64°, 20.19°, 21.19°, 21.71°, 23.29°, 23.58°, 24.42°, 25.07°, 25.63°, 26.31°, 27.17°, 27.52° and 28.96°, and the specific XRD values are shown in Table 1 below.

[0122] [Table 1]

[0123] Experimental Example 2. Differential Scanning Calorimetry (DSC) DSC 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) to form a pinhole. The sample was then heated from 25°C to 350°C at a rate of 10°C / min for DSC measurements. During the measurements, 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 software STARe.

[0124] The obtained crystalline form II was measured by DSC, and the results are shown in FIG.

[0125] As can be seen from Figure 5, crystalline Form II exhibited a first endothermic peak at about 28.8°C (Onset) and a second endothermic peak at about 172.6°C (Onset). After about 220°C, an endothermic peak due to decomposition was observed. The temperature values have an error of ±5°C.

[0126] Experimental Example 3: Thermogravimetric Analysis (TGA) TGA measurements were performed 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 subjected to TGA measurements. Nitrogen gas was supplied to the instrument at a rate of 80 mL / min during the measurements to prevent the inflow of oxygen and other gases. Data collection and evaluation were performed using the software STARe.

[0127] The obtained crystalline form II was subjected to TGA measurement, and the results are shown in FIG.

[0128] As can be seen from FIG. 6, crystalline form II exhibited a weight loss of about 5.9% at temperatures below 170°C. After about 220°C, weight loss due to decomposition occurred. The temperature values have an error of ±5°C. No weight loss was observed in the endothermic section of the DSC measurement. This confirmed that crystalline form II is a heat-stable crystalline form.

[0129] Experimental Example 4. Stability evaluation Approximately 10 to 30 mg of sample was stored for 4 weeks under accelerated conditions (40°C, 75% RH) in an open state, or under harsh conditions in a sealed state in an oven at 80°C. To compare the sample with a sample stored at room temperature, HPLC analysis was performed according to the method in Table 2 below.

[0130] [Table 2]

[0131] The stability of the obtained crystalline form II was evaluated, and the results are shown in Table 3 below.

[0132] [Table 3]

[0133] As can be seen from Table 3 above, the crystalline form II of the present invention exhibited excellent stability against heat and humidity, as it exhibited chemical stability for 4 weeks under accelerated conditions (40°C, 75% RH) and harsh conditions (80°C).

Claims

1. Crystalline Form II of a compound of the following formula 1, a pharmaceutically acceptable salt thereof, or a solvate thereof: X-ray powder diffraction pattern: the following diffraction angles (2θ values): 7.77±0.2°, 9.82±0.2°, 10.50±0.2°, 11.37±0.2°, 12.36±0.2°, 15.17±0.2°, 15.46±0.2°, 15.88±0.2°, 16.75±0.2°, 17.59±0.2°, 17.93±0.2°, 18.33±0.2°, 19.64±0.2° , 20.19±0.2°, 21.19±0.2°, 21.71±0.2°, 23.29±0.2°, 23.58±0.2°, 24.42±0.2°, 25.07±0.2°, 25.63±0.2°, 26.31±0.2°, 27.17±0.2°, 27.52±0.2° and 28.96±0.2°. 【Chemical 1】 In the above Chemical Formula 1, R １ is C ２ -C ５ It is alkyl.

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

3. The crystalline form II of claim 1, which is a crystalline form of a solvate of the hydrochloride salt of the compound of formula 1.

4. The crystalline form II of claim 3, wherein the solvate is a hydrate.

5. 5. The crystalline form II of claim 4, which is a crystalline form of the compound of formula 4: 【Chemistry 2】

6. A method for producing crystalline form II of 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 II according to claim 6, wherein the crystallization solvent comprises water, a polar aprotic organic solvent, or a mixture thereof.

8. 8. The method for preparing crystalline Form II according to claim 7, wherein the polar aprotic organic solvent comprises ethyl acetate, methyl isobutyl ketone, dimethyl sulfoxide, tetrahydrofuran, acetone, dimethylformamide, acetonitrile, or a mixture thereof.

9. 8. The method for producing crystalline form II according to claim 7, wherein the crystallization solvent is a mixed solvent of water and a polar aprotic organic solvent in a volume ratio of 20:1 to 1:

20.

10. 7. The method for producing crystalline form II according to claim 6, wherein the crystallization step further comprises adding a non-polar organic solvent to the mixed solution.

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

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

13. The pharmaceutical composition of claim 12, wherein the composition is for the prevention or treatment of obesity, diabetes, inflammation, or erectile dysfunction.

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