Amorphous melanocortin receptor agonist and method for preparing the same

Abstract

To provide an amorphous compound represented by a chemical formula 1, a method for preparing the same, and a pharmaceutical composition comprising the same.SOLUTION: The present invention provides an amorphous compound represented by a chemical formula 1, which may be characterized by the XPRD pattern, the DSC profile, and / or the NMR spectrum.SELECTED DRAWING: None

Description

Technical Field

[0001] This application claims the benefit of priority based on Korean Patent Application No. 10-2020-0180806, filed on Dec. 22, 2020, and all of the contents disclosed in the document of the Korean Patent Application are incorporated herein by reference in their entirety.

[0002] The present invention relates to an amorphous novel compound showing excellent enhancing activity against melanocortin receptors, a method for producing the same, and a pharmaceutical composition containing the same.

Background Art

[0003] Leptin protein is a hormone secreted by adipocytes, and its secretion amount increases as the content of body fat increases. By regulating the functions of various neuropeptides produced in the hypothalamus, it regulates various biological functions including appetite, body fat content, and energy metabolism (Schwartz, et al., Nature 404, 661-671 (2000)). The signal transduction of appetite and body weight regulation by leptin protein is carried out by regulating various factors downstream, and the most representative ones are melanocortin, AgRP (agouti-related peptide), and neuropeptide Y (NPY) hormones.

[0004] As a result of excessive calories in the body, when the concentration of leptin in the blood increases, the secretion of the proopiomelanocortin (POMC) protein hormone in 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, and this hormone ultimately induces a decrease in appetite as an agonist of the melanocortin-4 receptor (MC4R) in secondary neurons. On the other hand, as a result of calorie deficiency, when the concentration of leptin decreases, the expression of AgRP, which is an MC4R antagonist, increases, and the expression of NPY also increases, ultimately promoting appetite. That is, in response to changes in leptin, the alpha-MSH hormone and the AgRP hormone play roles of enhancement and antagonism against MC4R, respectively, and are involved in appetite regulation.

[0005] Alpha-MSH hormone binds to three MCR subtypes in addition to MC4R, inducing various physiological responses. To date, five MCR subtypes have been identified. Among them, MC1R is mainly expressed in skin cells and is involved in skin pigmentation regulation. MC2R is mainly expressed in the adrenal gland and is known to be involved in the production of glucocorticoid hormone. Only ACTH (adrenocorticotropic hormone) derived from POMC is its ligand. MC3R and MC4R, which are mainly expressed in the central nervous system, are involved in the regulation of appetite, energy metabolism, and body fat storage efficiency. 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 demonstrated as the main target for the development of anti-obesity agents because it shows the effect of efficiently reducing 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)).

[0006] In appetite and weight regulation, the role of MC4R was primarily demonstrated by experiments on animal models with abnormal expression of agouti protein (agouti mouse). In the case of Agouti mice, genetic mutations cause the agouti protein to be highly expressed in the central nervous system, where it acts as an antagonist of MC4R in the hypothalamus, leading to obesity (Yen, TT et al., FASEB J. 8, 479-488 (1994); Lu D., et al. Nature 371, 799-802 (1994)). Subsequent research findings have observed that AgRP (agouti-related peptide), which is similar to the agouti protein, is actually expressed in the hypothalamic neurons, and these are also known to be involved in appetite regulation as antagonists 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 shows an effect of reducing appetite. In contrast, when animals are treated with SHU9119 (peptide) or HS014 (peptide), which are MC4R antagonists, a phenomenon of increased appetite is observed (Kask et al., Biochem. Biophys. Res. Comm. 245, 90-93 (1998)). Moreover, in animal tests using Melanotan II (MTII, Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2) and its similar agonist HP228, after intracerebral, intraperitoneal or subcutaneous administration, effects such as appetite suppression, weight loss, and increased energy metabolism have been confirmed (Thiele T.E., et al. Am J Physiol 274(1 Pt 2), R248-54(1998); Lee M.D., et al. FASEB J 12, A552(1998); Murphy B., et al. J Appl Physiol 89, 273-82(2000)). Conversely, administration of the representative SHU9119 to animals shows significant and continuous feed intake and weight gain, providing pharmacological evidence that MCR agonists can be used as obesity therapeutics. The significant appetite-reducing effect that appears during MTII administration does not appear in MC4R KO (knock-out) mice, and this experimental result also proves that the appetite-reducing effect is mainly achieved by the activation of MC4R (Marsh, et al., Nat Genet 21, 119-122(1999)).

[0008] As obesity therapeutics developed to date, appetite suppressants that act on the central nervous system are the main type. Among them, most are drugs that regulate the action of neurotransmitters. Examples include phentermine and mazindol, which are noradrenalin agents, and fluoxetine and sibutramine, which are serotonergic agents. However, in the case of the neurotransmitter regulators, due to numerous subtypes of receptors, in addition to inhibiting appetite, they also have a wide range of effects on various physiological functions. Therefore, in the case of the regulators, there is a lack of selectivity for each subtype, and when administered for a long time, there are significant drawbacks accompanied by various side effects.

[0009] On the other hand, melanocortin agonists are neuropeptides rather than neurotransmitters. Considering that in MC4R gene KO mice, all functions other than energy metabolism are normal, they have the advantage as an action point in that they can induce only weight loss due to appetite inhibition without affecting other physiological functions. In particular, since the receptor belongs to the G-protein coupled receptor (GPCR), which is the most successful category among the new drug action points developed to date, ensuring selectivity for subtype receptors is relatively easy, which is greatly different from the existing action points.

[0010] As an example of utilizing such a melanocortin receptor as an action point, International Publication Nos. WO2008 / 007930 and WO2010 / 056022 disclose compounds as agonists of the melanocortin receptor.

[0011] In addition, the inventors of the present invention have intensively studied and invented a novel compound of the following Chemical Formula 1 having excellent selective enhancing activity against melanocortin receptors, particularly melanocortin-4 receptor (MC4R), and a method for producing the same (Korean Application No. 10-2019-0141649 (filed on November 7, 2019)).

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

[0013] On the other hand, the salt form and crystal structure of a pharmaceutically active ingredient sometimes affect the chemical stability of the drug. Different salt forms of a compound may change the filterability, product stability, storage stability, etc. of the compound, and different crystallization conditions and storage conditions of the compound may change the crystal structure of the compound, and sometimes cause production accompanied by different forms of crystal forms. Therefore, it is necessary to study the salt form and / or crystal structure having high purity and good chemical stability for one 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] [Problems to be Solved by the Invention]

[0015] An object of the present invention is to provide an amorphous novel compound or a salt thereof having excellent selective enhancing activity against melanocortin receptors, particularly melanocortin-4 receptor (MC4R).

[0016] Another object of the present invention is to provide a method for producing the amorphous compound or a salt thereof.

[0017] Another object of the present invention is to provide a pharmaceutical composition containing the amorphous compound or a salt thereof.

Means for Solving the Problems

[0018] To achieve the above object, In one aspect, the present invention provides an amorphous compound of the following Chemical Formula 1.

Chemical Formula

[0019] The compound of Chemical Formula 1 can have an asymmetric carbon center and an asymmetric axis or plane, and can exist as cis or trans isomers, R or S isomers, racemates, mixtures of diastereoisomers and individual diastereoisomers, and all these isomers and mixtures are included within the scope of the compound of Chemical Formula 1.

[0020] In the present specification, for convenience, unless otherwise specified, the compound of Chemical Formula 1 is used in the sense of including all of the compound of Chemical Formula 1, its pharmaceutically acceptable salts, its isomers and its solvates.

[0021] In one specific example according to the present invention, R1 of Chemical Formula 1 is C2-C5 alkyl. In another specific example according to the present invention, R1 of Chemical Formula 1 is linear or branched C2-C5 alkyl, for example, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl or tert-butyl.

[0022] In another specific example according to the present invention, R1 in the chemical formula 1 is C2-C4 alkyl. In another specific example according to the present invention, R1 in the chemical formula 1 is linear or branched C2-C4 alkyl, for example, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl. Specifically, the R1 can be iso-propyl.

[0023] In one specific example according to the present invention, the pharmaceutically acceptable salts include, but are not limited to, acid addition salts formed by inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, organic carboxylic acids such as tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, and sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or naphthalenesulfonic acid.

[0024] In one specific example according to the present invention, the solvate can 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.

[0025] The amorphous compound according to the present invention can have an x-ray powder diffraction (XRPD) pattern that does not have characteristic diffraction peaks and has broad noise.

[0026] In one specific example according to the present invention, the amorphous compound can have the XRPD pattern shown in Figure 1.

[0027] The amorphous compound according to the present invention can have no exothermic peak when heated at a temperature of 350 °C or lower in a Hyper DSC profile. Further, the amorphous compound is characterized by having a glass transition temperature (Tg) of about 98 °C in the Hyper DSC profile.

[0028] In one specific example according to the present invention, the amorphous compound can have the Hyper DSC profile shown in FIG. 2.

[0029] In this specification, The results of X-ray powder diffraction (XRPD) analysis were obtained using a PANalytical X’ Pert Pro MPD system, Malvern Panalytical Ltd.

[0030] The results of differential scanning calorimetry (DSC) analysis were obtained using a DSC8500, Perkin Elmer.

[0031] The results of stability analysis were obtained using HPLC, Agilent Technologies, Inc.

[0032] The results of nuclear magnetic resonance (NMR) spectra were obtained using a Bruker 500 MHz.

[0033] The compound of amorphous Chemical Formula 1 can have a higher purity compared to the crude compound and can be physically and chemically more stable.

[0034] The amorphous compound of Chemical Formula 1 according to the present invention can be formed from Crystal Form I of the compound of Chemical Formula 1. The Crystal Form I of the compound of Chemical Formula 1 has, in an X-ray powder diffraction (XRPD) pattern, the following diffraction angles (2θ values): 8.240 ± 0.2°, 9.363 ± 0.2°, 10.2693 ± 0.2°, 10.5969 ± 0.2°, 12.050 ± 0.2°, 12.841 ± 0.2°, 13.503 ± 0.2°, 15.5738 ± 0.2°, 16.6030 ± 0.2°, 17.009 ± 0.2°, 17.305 ± 0.2°, 18.364 ± 0.2°, 18.7390 ± 0.2°, 19.188 ± 0.2°, 19.476 ± 0.2°, 20.001 ± 0.2°, 20.477 ± 0.2°, 20.665 ± 0.2°, 21.348 ± 0.2°, 21.976 ± 0.2°, 22.580 ± 0.2°, 23.896 ± 0.2°, 4.334 ± 0.2°, 24.812 ± 0.2°, 25.243 ± 0.2°, 25.833 ± 0.2°, 26.646 ± 0.2°, 27.82 ± 0.2°, 28.316 ± 0.2°, 28.609 ± 0.2°, 29.692 ± 0.2°, 30.185 ± 0.2° and 30.875 ± 0.2°, and can have 3 or more, 5 or more, 7 or more, 9 or more, 10 or more, 13 or more, 15 or more, 17 or more, 20 or more, 23 or more, 25 or more, 27 or more or 30 or more characteristic peaks selected therefrom.

[0035] The amorphous compound of Chemical Formula 1 produced from Crystal Form 1 of the present invention can have a higher purity and can be physically and chemically more stable than the amorphous compound of Chemical Formula 1 formed from other crystal forms of the compound of Chemical Formula 1. Moreover, the amorphous compound of Chemical Formula 1 of the present invention can have a more excellent enhancing ability for the melanocortin-4 receptor and a preventive or therapeutic effect on diseases such as obesity, diabetes, inflammation, erectile dysfunction, etc., as compared with known melanocortin-4 receptor agonists, but the effects of the present invention are not limited thereto.

[0036] In another aspect, the present invention provides a method for producing an amorphous compound of Chemical Formula 1, which includes the step of rapidly cooling the compound of Chemical Formula 1 after melting it.

[0037] The amorphous compound of Chemical Formula 1 can be formed using the compound of Chemical Formula 1.

[0038] The compound of Chemical Formula 1 can be obtained by the production method described in the specification of Korean Application No. 10-2019-0141649 (filed on November 7, 2019).

[0039] The compound used for the production of the amorphous compound of Chemical Formula 1 of the present invention can include the crystalline form of the compound of Chemical Formula 1, and specifically, can include crystalline form I of the compound of Chemical Formula 1.

[0040] The crystalline form I of the compound has, in an X-ray powder diffraction (XPRD) pattern, three or more, five or more, seven or more, nine or more, ten or more, thirteen or more, fifteen or more, seventeen or more, twenty or more, twenty-three or more, twenty-five or more, twenty-seven or more, or thirty or more characteristic peaks selected from 8.240 ± 0.2°, 9.363 ± 0.2°, 10.2693 ± 0.2°, 10.5969 ± 0.2°, 12.050 ± 0.2°, 12.841 ± 0.2°, 13.503 ± 0.2°, 15.5738 ± 0.2°, 16.6030 ± 0.2°, 17.009 ± 0.2°, 17.305 ± 0.2°, 18.364 ± 0.2°, 18.7390 ± 0.2°, 19.188 ± 0.2°, 19.476 ± 0.2°, 20.001 ± 0.2°, 20.477 ± 0.2°, 20.665 ± 0.2°, 21.348 ± 0.2°, 21.976 ± 0.2°, 22.580 ± 0.2°, 23.896 ± 0.2°, 4.334 ± 0.2°, 24.812 ± 0.2°, 25.243 ± 0.2°, 25.833 ± 0.2°, 26.646 ± 0.2°, 27.82 ± 0.2°, 28.316 ± 0.2°, 28.609 ± 0.2°, 29.692 ± 0.2°, 30.185 ± 0.2°, and 30.875 ± 0.2°.

[0041] The crystalline form I can have the X-ray powder diffraction (XRPD) pattern shown in FIG. 4.

[0042] The amorphous compound of Chemical Formula 1 of the present invention can be formed by the melt quenching method using the compound of Chemical Formula 1.

[0043] First, the compound represented by Chemical Formula 1 is melted. The melting can be carried out under a nitrogen atmosphere.

[0044] The melting can be carried out, for example, at a temperature of 150°C to 200°C, 160°C to 200°C, 170°C to 190°C, 180°C to 190°C, 180°C or 190°C.

[0045] The melting can be carried out, for example, over a time of 30 seconds to 10 minutes, 30 seconds to 5 minutes, 30 seconds to 2 minutes, 1 minute to 2 minutes.

[0046] In one specific example according to the present invention, it can include melting the crystalline form I of the compound of Chemical Formula 1 at a temperature of 180°C to 190°C for 2 minutes.

[0047] Next, the melted compound is quenched.

[0048] The quenching can be carried out using liquid nitrogen.

[0049] The quenching can be carried out by bringing the melted compound of Chemical Formula 1 into contact with liquid nitrogen.

[0050] In one specific example according to the present invention, the melted compound of Chemical Formula 1 can be transferred to a liquid nitrogen tank to form an amorphous compound.

[0051] In another specific example according to the present invention, liquid nitrogen can be injected into a flask carrying the melted compound to form an amorphous compound.

[0052] The amorphous compound of Chemical Formula 1 obtained as described above can have a higher purity and can be physically and chemically more stable than the crude compound of Chemical Formula 1, but the effects of the present invention are not limited thereto.

[0053] In still another aspect, the present invention provides a pharmaceutical composition comprising (i) the amorphous compound of Chemical Formula 1 and (ii) a pharmaceutically acceptable carrier.

[0054] Since the amorphous compound of Chemical Formula 1 according to the present invention exhibits excellent enhancing effects on melanocortin receptors, particularly melanocortin-4 receptor (MC4R), the present invention can also provide a pharmaceutical composition for enhancing the function of melanocortin receptors containing the above-described amorphous compound as an active ingredient. Specifically, the pharmaceutical composition can be a composition for enhancing the function of melanocortin-4 receptor.

[0055] In addition, the pharmaceutical composition can exhibit excellent effects on the prevention or treatment of obesity, diabetes, inflammation, and erectile dysfunction, and can be a composition for preventing or treating obesity, preventing or treating diabetes, preventing or treating inflammation, or preventing or treating erectile dysfunction, but the uses of the present invention are not limited only to these diseases.

[0056] As used herein, the term "carrier" means a compound that facilitates the introduction of a compound into cells or tissues.

[0057] When the amorphous compound of the present invention is administered for clinical purposes, the total daily dose administered to the host in a single volume or divided volumes 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 can vary depending on the specific compound used, the patient's body weight, gender, health status, diet, time of drug administration, administration method, excretion rate, drug combination, and severity of the disease, etc.

[0058] The amorphous compound 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.

[0059] The pharmaceutical composition of the present invention can be in various oral dosage forms such as tablets, pills, powders, capsules, granules, syrups or emulsions, or in parenteral dosage forms such as injectable preparations for intramuscular, intravenous or subcutaneous administration.

[0060] Injectable preparations can be manufactured using suitable dispersants, wetting agents, suspending agents, or excipients according to known techniques.

[0061] Excipients that can be used in the pharmaceutical preparations of the present invention can include, but are not limited to, sweeteners, binders, solubilizers, solubilizing aids, wetting agents, emulsifiers, isotonic agents, adsorbents, disintegrants, antioxidants, preservatives, lubricants, fillers, fragrances, etc. For example, as excipients, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, silica, magnesium aluminum silicate, starch, gelatin, tragacanth gum, alginic acid, sodium alginate, methyl cellulose, sodium carboxymethyl cellulose, water, ethanol, polyethylene glycol, polyvinylpyrrolidone, sodium chloride, calcium chloride, orange essence, strawberry essence, vanilla flavor, etc. can be used.

[0062] When the pharmaceutical composition of the present invention is in an oral dosage form, examples of carriers that can 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.

[0063] When the pharmaceutical composition of the present invention is in the form of an injectable preparation, examples of the carrier include, but are not limited to, water, saline, glucose aqueous solution, pseudo-sugar aqueous solution, alcohol, glycol, ether, oil, fatty acid, fatty acid ester, glyceride, and the like.

[0064] In yet another aspect, there is provided the above-described amorphous compound for use in enhancing the function of melanocortin receptors, particularly melanocortin-4 receptor (MC4R).

[0065] In one embodiment, there is provided the above-described amorphous compound for use in the treatment or prevention of obesity, diabetes, inflammation or erectile dysfunction.

[0066] In yet another aspect, there is provided a method for enhancing the function of melanocortin receptors, particularly melanocortin-4 receptor (MC4R), comprising the step of administering the above-described amorphous compound to a subject.

[0067] In yet another aspect, there is provided a method for treating obesity, diabetes, inflammation or erectile dysfunction comprising the step of administering the above-described amorphous compound to a subject.

Advantages of the Invention

[0068] The amorphous compound of Chemical Formula 1 according to the present invention exhibits excellent enhancing effects on melanocortin receptors, particularly melanocortin-4 receptor (MC4R), and thus can be usefully used for the prevention or treatment of obesity, diabetes, inflammation and erectile dysfunction.

[0069] The amorphous compound of Chemical Formula 1 according to the present invention shows an on-target effect on the melanocortin-4 receptor, exhibits weight loss and diet reduction effects, does not affect anxiety and depression, and can be administered without safety problems such as side effects on hERG (human ether-a-go-go related gene) inhibition and induction of mutations.

[0070] In addition, the amorphous compound of Chemical Formula 1 according to the present invention is superior in purity, yield, physical and chemical stability compared to the crude compound of Chemical Formula 1.

[0071] Specifically, the amorphous compound of Chemical Formula 1 can be superior in solubility, storage stability, and production stability compared to the crude compound of Chemical Formula 1.

Brief Description of the Drawings

[0072]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Modes for Carrying Out the Invention

[0073] Hereinafter, the present invention will be described more specifically with reference to production examples and examples. However, these examples are illustrative of the present invention, and the scope of the present invention is not limited thereto.

[0074] Production Example 1: Production of Methyl (2S,4S)-4-((N-((1S,4R)-4-methylcyclohexyl)isobutyl)amide)pyrrolidine-2-carboxylate Hydrochloride

Chemical Formula

[0075] The title compound was obtained through the processes of the following Steps A, B, C, D, and E.

[0076] 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 mmol) was added. Stirring was carried out at 80 °C for 16 hours. After concentrating the reaction solvent under reduced pressure, water was added, and extraction was performed twice with ethyl acetate. The organic layer was washed with an aqueous sodium chloride solution and water, and then 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.

[0077] 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)

[0078] 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 the previous step A was dissolved in tetrahydrofuran (180 ml), and then a 1M tetrahydro solution of trimethylphosphine (109.2 ml, 109.2 mmol) was gradually added at 0 °C. After stirring for 1 hour at the same temperature, the mixture was stirred at room temperature for 3 hours. After concentrating the reaction solvent under reduced pressure, dichloromethane (100 ml) and water (150 ml) were added and stirred for about 30 minutes. After liquid separation and extraction with dichloromethane once again, 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.

[0079] 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)

[0080] Step C: Preparation of 1-(tert-butyl) 2-methyl (2S,4S)-4-(((1s,4R)-4-methylcyclohexyl)amino)pyrrolidine-1,2-dicarboxylate The 1-(tert-butyl) 2-methyl (2S,4S)-4-aminopyrrolidine-1,2-dicarboxylate (20.62 g, 84.4 mmol) obtained in the previous 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 an aqueous sodium chloride solution and then 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%).

[0081] 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)

[0082] Step D: Preparation of 1-(tert-butyl) 2-methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutylamide)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 the previous step C was dissolved in dichloromethane (500 ml), triethylamine (61.1 ml, 438.1 mmol) was added, and then isobutyryl chloride (11.7 ml, 219 mmol) was gradually added at 0 °C. After stirring at room temperature for 16 hours, the reaction solvent was concentrated under reduced pressure, an aqueous sodium bicarbonate solution was added, and extraction was performed twice with ethyl acetate. The organic layer was washed with an 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%).

[0083] 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)

[0084] Step E: Preparation of methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutylamide)pyrrolidine-2-carboxylate hydrochloride 1-(tert-Butyl) 2-methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutylamide)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 crude (28.7 g, 99%), which was used in the next step without purification.

[0085] MS[M+H] = 311 (M+1)

[0086] Production Example 2: Preparation of (3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carboxylic acid

Chemical formula

[0087] The title compound was obtained by the method described in International Publication No. WO2004 / 092126.

[0088] 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)

[0089] Production Example 3: 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)isobutylamide (MC70)

Chemical formula

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

[0091] 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)isobutylamide)pyrrolidine-2-carboxylate Methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutylamide)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 gradually added. Stirring was carried out at room temperature for 16 hours. After concentrating the reaction solvent under reduced pressure, 0.5N aqueous sodium hydroxide solution was added and extraction was performed twice with ethyl acetate. The organic layer was washed twice each with aqueous sodium chloride solution and water, and then 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)isobutylamide)pyrrolidine-2-carboxylate (41.19 g, 87%).

[0092] MS [M+H] = 575 (M+1)

[0093] Step B: Preparation of (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutylamide)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)isobutylamide)pyrrolidine-2-carboxylate (39.4 g, 68.62 mmol) obtained in the previous step A was dissolved in methanol (450 ml), and then 6N aqueous sodium hydroxide solution (57.2 ml, 343.09 mmol) was added. The mixture was stirred at room temperature for 16 hours, adjusted to about pH 5 with 6N aqueous hydrochloric acid solution, and then the reaction solution was 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.

[0094] MS [M+H] = 561 (M+1)

[0095] 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)isobutylamide (2S,4S)-1-((3S,4R)-1-(tert-Butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutylamide)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) 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 gradually added in turn. The mixture was stirred at room temperature for 16 hours, and the reaction solution was concentrated under reduced pressure. Then, 0.5 N aqueous sodium hydroxide solution was added, and the mixture was extracted twice with ethyl acetate. The organic layer was washed twice with aqueous sodium chloride solution and water, and then 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)pyrrolidine-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutylamide (37.05 g, 86%, MC70).

[0096] MS [M+H] = 630 (M+1)

[0097] Production Example 4: Production of Crystal Form I of N-((3S,5S)-1-((3S,4R)-1-(tert-Butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidine-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutylamide (Crystal Form I of MC70)

Chemical Structure

[0098] Using 0.975 mL of MTBE based on 0.6 g of the compound (MC70) produced in Production Example 3, the compound (MC70) was dissolved at room temperature for 30 minutes. After the dissolution was completed, it was cooled to 3°C and then stirred for about 21 hours, followed by filtration to obtain the title compound (Crystal Form I of MC70).

[0099] For the compound of Production Example 4, XRPD (Figure 4) and DSC (Figure 5) were analyzed according to the following method, and the resulting graphs are shown in Figures 4 and 5.

[0100] XRPD analysis of Production Example 4 The XRPD diffraction pattern was obtained by the following method using a PANalytical X’Pert Pro MPD system equipped with a monochromatized radiation source and a Ni filter as a solid-state detector.

[0101] After uniformly placing about 20 - 30 mg of the sample on a glass sample holder so as to have a flat surface, the generator of the instrument was set to 45 kV (acceleration voltage) and 40 mA (filament emission), and then measurement was carried out in reflection mode (not-spin). The Bragg angle (2θ) in the range of 4 - 40° was measured under the conditions of a step size of 0.026° and a time per step of 51 seconds.

[0102] DSC analysis of Production Example 4 DSC measurements were performed using a Mettler Toledo DSC1 system. A sample of approximately 2 - 5 mg was weighed and placed in a 40 μL Al crucible (flat-bottomed aluminum pan with one pin-hole lid) to form one pin hole. Next, the DSC was measured while heating the sample from 25 °C to 350 °C at a rate of 10 °C / min. During the measurement, nitrogen gas was supplied into the equipment 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.

[0103] Example 1 Preparation of an amorphous compound of N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidin-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide 25 mg of the crystalline form I compound prepared in Production Example 4 was placed in a vial and filled with nitrogen gas, and then heated at 180 °C or 190 °C for 2 minutes to melt. Next, the melted compound was quickly transferred to a liquid nitrogen bath to form an amorphous compound.

[0104] As a result of the analysis of the NMR spectrum (Figure 3) of Example 1, it was confirmed that the compound of Example 1 had the same structure as the compound of Production Example 4.

[0105] Amorphous characteristics 1) X-ray powder diffraction (XRPD) The XRPD pattern was obtained by the following method using a Panalytical Xpert Pro diffractometer equipped with a Cu-X-ray tube and a Pixcel detector system.

[0106] The sample was analyzed in transmission mode between low-density polyethylene films. The Bragg angle (2θ) in the range of 3 - 40° was measured under the conditions of a step size of 0.013° and a Time per step of 22 seconds. The analysis time of the sample was 5 minutes, and the sample was rotated at 60 rpm during data collection. The XRPD patterns were classified and processed using HighScorePlus 2.2c software.

[0107] The XRPD results are shown in Figure 1. As shown in Figure 1, it was confirmed that the amorphous Example 1 formed by melting at 180°C and 190°C showed the same pattern. These had no diffraction peaks and showed a broad range of noise typical of amorphous samples.

[0108] 2) Hyper differential scanning calorimetry (Hyper DSC) DSC was measured using a Perkin Elmer DSC8500. An accurately weighed sample was placed in an aluminum sample pan and sealed. Under nitrogen, after maintaining at -50°C for 1 minute, it was heated to 300°C at a rate of 30°C per minute, maintained for 1 minute, then cooled to -50°C and maintained for 2 minutes, and then heated to 300°C again at a rate of 30°C per minute.

[0109] The results of Hyper DSC are shown in Figure 2. As shown in Figure 2, it was confirmed that the substance of Example 1 had the characteristic of a glass transition temperature of about 98°C and showed characteristics typical of an amorphous sample.

[0110] Experimental Example 1. Stability Evaluation The following experiments were conducted for the stability evaluation of the amorphous compound under various solvent vapors and controlled humidity conditions.

[0111] The vapour stress experiment was carried out by putting about 25 mg of the amorphous compound of Example 1 into an unsealed vial, placing the vial in a large sealed container containing 1 mL of the selected solvent, and after 5 - 7 days, the results of analyzing the XRD pattern of the sample are shown in Table 1 below.

[0112] Also, in the humidity stress experiment, about 25 mg of the amorphous compound of Example 1 was placed in individual vials, stored for 5 to 7 days in various relative humidity chambers (23%RH, 59%RH, 7%RH, 98%RH, 40°C / 75%RH) without being sealed, and then the XRD patterns were analyzed. The results are shown in Table 2 below.

[0113] The experimental methods for the vapor stress and humidity stress are methods used as a way to generate stable solvates and hydrates because when a substance is exposed to vapor, the solid is plasticized and the molecular mobility is restricted. However, it was confirmed that the amorphous compound of Example 1 was maintained under various conditions by the above experimental methods and was stable under those conditions.

[0114] [Table 1]

[0115] [Table 2]

Claims

1. An amorphous compound of the following formula 1: 【Chemistry 1】 In the above Chemical Formula 1, R １ is C ２ -C ５ It is an alkyl.

2. 2. The amorphous compound of formula 1 according to claim 1, having an X-ray powder diffraction (XRPD) pattern with no characteristic diffraction peaks and with broad noise.

3. 2. The amorphous compound of formula 1 according to claim 1, having the X-ray powder diffraction pattern shown in FIG.

4. 2. The amorphous compound of formula 1 according to claim 1, having a DSC profile as shown in FIG.

5. The amorphous compound of formula 1 according to claim 1, 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.

6. A method for preparing an amorphous compound of formula 1 according to any one of claims 1 to 5, comprising the steps of: The method includes the steps of melting the compound of Formula 1 and then quenching the melted compound.

7. The compound of Formula 1 that is melted comprises Crystal Form I of the compound of Formula 1, The crystalline form I of the compound of Chemical Formula 1 has the following diffraction angles (2θ values) in an X-ray powder diffraction pattern (XRPD): 8.240±0.2°, 9.363±0.2°, 10.2693±0.2°, 10.5969±0.2°, 12.050±0.2°, 12.841±0.2°, 13.503±0.2°, 15.5738±0.2°, 16.6030±0.2°, 17.009±0.2°, 17.305±0.2°, 18.364±0.2°, 18.7390±0.2°, 19.188±0.2°, 19.476±0.2°, and 20.001±0.2°.

7. The method of claim 6, wherein the chromatogram has three or more characteristic peaks selected from the group consisting of 20.477±0.2°, 20.665±0.2°, 21.348±0.2°, 21.976±0.2°, 22.580±0.2°, 23.896±0.2°, 4.334±0.2°, 24.812±0.2°, 25.243±0.2°, 25.833±0.2°, 26.646±0.2°, 27.82±0.2°, 28.316±0.2°, 28.609±0.2°, 29.692±0.2°, 30.185±0.2° and 30.875±0.2°.

8. The method for preparing the amorphous compound of formula 1 according to claim 6, wherein the compound is melted at a temperature of 150°C to 200°C.

9. The method for preparing the amorphous compound of formula 1 according to claim 6, wherein the melting is carried out for a period of time ranging from 30 seconds to 10 minutes.

10. 7. The method for preparing an amorphous compound of formula 1 according to claim 6, wherein the quenching is carried out by contacting the molten compound of formula 1 with liquid nitrogen.

11. 10. A pharmaceutical composition comprising an amorphous compound of Formula 1 according to any one of claims 1 to 5 and a pharma- ceutically acceptable carrier.

12. A pharmaceutical composition for enhancing the activity of melanocortin-4 receptor, comprising an amorphous compound of formula 1 according to any one of claims 1 to 5 and a pharma- ceutically 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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