Amorphous melanocortin receptor agonists and methods for their preparation

Abstract

The present invention relates to an amorphous compound represented by formula 1, a method for preparing the same and a pharmaceutical composition containing the same. The amorphous compound represented by formula 1 of the present invention can be characterized by an XPRD pattern, a DSC profile and / or an NMR spectrum.

Description

[Technical field]

[0001] This application claims the benefit of priority based on Korean Patent Application No. 10-2020-0180806 filed on December 22, 2020, the entire contents of which are incorporated herein by reference.

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

[0003] Leptin is a hormone secreted by adipocytes, and its secretion increases with increasing body fat content. It regulates various functions in the body, including appetite, body fat content, and energy metabolism, by regulating the functions of various neuropeptides produced in the hypothalamus (Schwartz, et al., Nature 404, 661-671 (2000)). The signal transduction of appetite and body weight regulation by leptin is regulated by various downstream factors, the most representative of which are melanocortin, AgRP (agoutirelated peptide), and neuropeptide Y (NPY) hormone.

[0004] When the concentration of leptin in the blood increases as a result of excess calories in the body, the secretion of proopiomelanocortin (POMC) protein hormone in the pituitary gland increases, and the production of AgRP and NPY decreases. POMC neurons produce a small peptide hormone, melanocyte stimulating hormone (alpha-MSH), which acts as an agonist to the melanocortin-4 receptor (MC4R) of second-order neurons, thereby inducing a decrease in appetite. On the other hand, when the concentration of leptin decreases as a result of a lack of calories, the expression of AgRP, an MC4R antagonist, increases, and the expression of NPY also increases, resulting in an increase in appetite. In other words, in response to changes in leptin, the hormones alpha-MSH and AgRP play stimulatory and antagonistic roles on MC4R, thereby regulating appetite.

[0005] Alpha-MSH hormone binds to three MCR subtypes in addition to MC4R to induce various physiological responses. Five MCR subtypes have been identified to date, of which MC1R is mainly expressed in skin cells and is involved in the regulation of melanin pigmentation, MC2R is mainly expressed in the adrenal gland and is known to be involved in the production of glucocorticoid hormones, and its only ligand is ACTH (adrenocorticotropic hormone) derived from POMC. MC3R and MC4R, which are mainly expressed in the central nervous system, are involved in the regulation of appetite, energy metabolism, and the efficiency of fat storage in the body, and MC5R, which is expressed in various tissues, is known to regulate exocrine function (Wikberg, et al., Pharm Res 42(5)393-420(2000)). In particular, activation of the MC4R receptor has been proven to be a major target of action in the development of antiobesity drugs, as it effectively reduces body weight by inducing a decrease in appetite and an increase in energy metabolism (Review: Wikberg, Eur. J. Pharmacol 375, 295-310 (1999)); Wikberg, et al., Pharm Res 42(5) 393-420 (2000); Douglas et al., Eur J Pharm 450, 93-109 (2002); O'Rahilly et al., Nature Med 10, 351-352 (2004)).

[0006] The role of MC4R in appetite and body weight regulation was first demonstrated by agouti protein abnormal expression animal model (agouti mouse) experiments. In the case of agouti mouse, agouti protein is expressed at high concentrations in the central nervous system due to genetic mutation, and it has been revealed that it induces obesity by acting as an antagonist of MC4R in the hypothalamus (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 nerve actually expresses 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, was administered intracerebrally to animals, it was shown to have the effect of reducing appetite, whereas when the MC4R antagonists SHU9119 (peptide) or HS014 (peptide) were administered, appetite was increased again (Kask et al., Biochem. Biophys. Res. Comm. 245, 90-93 (1998)). Moreover, in animal studies using Melanotan II (MTII, Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2) and its similar agonist HP228, efficacy of appetite suppression, weight loss, and increased energy metabolism after cerebral, peritoneal or subcutaneous administration has been confirmed (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)). On the contrary, when a representative SHU9119 is administered to animals, it shows a significant and sustained increase in food intake and weight gain, providing pharmacological evidence that MCR agonists can be used as obesity treatment agents. The significant appetite-reducing effect observed upon administration of MTII is 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 obesity treatments developed to date are appetite suppressants that act on the central nervous system, and most of them are drugs that regulate the action of neurotransmitters. Examples include noradrenalin agents such as phentermine and mazindol, and serotonergic agents such as fluoxetine and sibutramine. However, the neurotransmitter regulators have a wide range of effects on various physiological functions in addition to appetite suppression due to the many subtype receptors. Therefore, the regulators have a major drawback in that they lack selectivity for each subtype and cause various side effects when administered for a long period of time.

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

[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] In addition, 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 the melanocortin-4 receptor (MC4R), and a method for producing the same (Korean Application No. 10-2019-0141649 (filed on November 7, 2019)).

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

[0013] On the other hand, the salt form and crystal structure of a pharma- ceutically active ingredient sometimes affect the chemical stability of the drug. Different salt forms of a compound may change the compound's filterability, product stability, storage stability, etc., and different crystallization and storage conditions of a compound may change the compound's crystal structure, sometimes resulting in the production of different crystal forms. Therefore, it is necessary to research salt forms and / or crystal structures 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 against melanocortin-4 receptor (MC4R).

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

[0017] Another object of the present invention is to provide a pharmaceutical composition comprising said amorphous compound or a salt thereof. [Means for solving the problem]

[0018] In order to achieve the above objectives, In one aspect, the present invention provides a compound of formula 1 in amorphous form: [ka] In the above formula 1, R1 is a C2-C5 alkyl.

[0019] 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.

[0020] 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 pharma- ceutically acceptable salts, its isomers and solvates thereof.

[0021] In one embodiment according to the present invention, R1 in the formula (1) is a C2-C5 alkyl. In another embodiment according to the present invention, R1 in the formula (1) is a linear or branched C2-C5 alkyl, such as ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, or tert-butyl.

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

[0023] In one embodiment according to the present invention, the pharma- ceutically 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.

[0024] 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.

[0025] Amorphous compounds according to the present invention may have x-ray powder diffraction (XRPD) patterns that are devoid of characteristic diffraction peaks and have broad noise.

[0026] In one embodiment according to the present invention, the amorphous compound may have an XRPD pattern as shown in FIG.

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

[0028] In one embodiment according to the present invention, the amorphous compound may have a Hyper DSC profile as shown in FIG.

[0029] In this specification, X-ray powder diffraction (XRPD) analysis was performed using a PANalytical X' Pert Pro MPD system, Malvern Panalytical Ltd.

[0030] Differential scanning calorimetry (DSC) analysis was performed using a DSC8500, Perkin Elmer.

[0031] The stability analysis was performed using HPLC (Agilent Technologies, Inc.) and the results shown are shown below.

[0032] Nuclear magnetic resonance (NMR) spectra were obtained using a Bruker 500 MHz.

[0033] The amorphous compound of Formula 1 may have higher purity and may be more physically and chemically stable than the crude compound.

[0034] The amorphous compound of Chemical Formula 1 according to the present invention may be formed from crystalline form I of the compound of Chemical Formula 1, and the crystalline form I of the compound of Chemical Formula 1 has the following diffraction angles (2θ values) in an X-ray powder diffraction (XRPD) pattern: 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°, 2 The molecule may 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 from 8.609±0.2°, 29.692±0.2°, 30.185±0.2°, and 30.875±0.2°.

[0035] The amorphous compound of Chemical Formula 1 prepared from Crystal Form 1 of the present invention may have higher purity and be more physically and chemically stable than the amorphous compound of Chemical Formula 1 formed from other crystalline forms of the compound of Chemical Formula 1. Furthermore, the amorphous compound of Chemical Formula 1 of the present invention may have better melanocortin-4 receptor enhancing ability and better preventive or therapeutic effects against diseases such as obesity, diabetes, inflammation, and erectile dysfunction than 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 preparing an amorphous compound of Formula 1, comprising melting the compound of Formula 1 and then quenching the melt.

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

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

[0039] The compound used to prepare the amorphous compound of formula 1 of the present invention may include a crystalline form of the compound of formula 1, specifically, crystalline form I of the compound of formula 1.

[0040] The crystalline form I of the compound has an X-ray powder diffraction (XPRD) pattern of 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°, and 16.60 30±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 may have the X-ray powder diffraction (XRPD) pattern shown in FIG.

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

[0043] First, the compound represented by Formula 1 is melted. The melting may be performed under a nitrogen atmosphere.

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

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

[0046] In one embodiment according to the present invention, the method may include melting the crystalline form I of the compound of Formula 1 at a temperature of 180° C. to 190° C. for 2 minutes.

[0047] The molten compound is then rapidly cooled.

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

[0049] The quenching can be carried out by contacting the molten compound of Formula 1 with liquefied nitrogen.

[0050] In one embodiment according to the present invention, the molten compound of Formula 1 can be transferred to a bath of liquid nitrogen to form an amorphous compound.

[0051] In another embodiment according to the present invention, liquid nitrogen can be injected into a flask containing the molten compound to form an amorphous compound.

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

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

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

[0055] In addition, 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 preventing or treating obesity, diabetes, inflammation, or erectile dysfunction, but the use of the present invention is not limited to these diseases.

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

[0057] When the amorphous compounds of the present invention are administered for clinical purposes, the total daily dose administered to the host in single or separate doses is preferably in the range of 0.01 to 10 mg per kg of body weight, although the specific dose level for an individual patient may vary depending on the particular compound used, the patient's weight, sex, health condition, diet, time of drug administration, method of administration, excretion rate, drug mixture, and severity of disease, etc.

[0058] The amorphous compounds of the present invention can be administered by any route depending on the purpose. For example, the amorphous compounds of the present invention can be administered by injection or orally.

[0059] 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.

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

[0061] The excipients that can be used in the pharmaceutical preparation 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, 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. may be used as excipients.

[0062] 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, and the like.

[0063] 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 sucrose solution, alcohol, glycol, ether, oil, fatty acid, fatty acid ester, glyceride, etc.

[0064] In yet another aspect, there is provided an amorphous compound as described above for use in enhancing the activity of a melanocortin receptor, in particular the melanocortin-4 receptor (MC4R).

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

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

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

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

[0069] The amorphous compound of formula 1 according to 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.

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

[0071] Specifically, the amorphous compound of Chemical Formula 1 may have superior solubility, storage stability, and production stability compared to the crude compound of Chemical Formula 1. [Brief description of the drawings]

[0072] [Figure 1] 1 is a graph showing the XRPD results of Example 1. [Diagram 2] 1 is a graph showing the results of DSC in Example 1. [Diagram 3] 1 is a graph showing the NMR results of Example 1. [Figure 4] 1 is a graph showing the XRPD results of Production Example 4. [Diagram 5] 1 is a graph showing the results of DSC in Production Example 4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0073] The present invention will be described in more detail below with reference to Production Examples and Examples, however, these Examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention.

[0074] Preparation Example 1: Preparation of methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylate hydrochloride [ka]

[0075] The title compound was obtained through 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 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 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 step A was dissolved in tetrahydrofuran (180 ml), and then 1M trimethylphosphine tetrahydrofuran solution (109.2 ml, 109.2 mmol) was gradually added at 0°C. The mixture was stirred at the same temperature for 1 hour, and then stirred 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 about 30 minutes. The layers were separated and extracted again with dichloromethane, and the organic layer was dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain crude 1-(tert-butyl) 2-methyl (2S, 4S)-4-aminopyrrolidine-1,2-dicarboxylate (20.62 g, 93%), which was used in the next step without purification.

[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 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 an 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%).

[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)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 gradually added at 0°C. After stirring at room temperature for 16 hours, the reaction solvent was concentrated under reduced pressure, and then an aqueous sodium bicarbonate solution was added, followed by extraction 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 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%).

[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)isobutyramido)pyrrolidine-2-carboxylate hydrochloride 1-(tert-butyl) 2-methyl (2S, 4S)-4-(N-((1s, 4R)-4-methylcyclohexyl) isobutyramide) 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 (28.7 g, 99%), which was used in the next step without purification.

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

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

[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: 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]

[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)isobutyramido)pyrrolidine-2-carboxylate Methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutylamido)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. 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 solution was added and 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 methyl (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramide)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)isobutyramido)pyrrolidine-2-carboxylic acid The methyl (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramide)pyrrolidine-2-carboxylate (39.4 g, 68.62 mmol) obtained in 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, and the pH was adjusted to about 5 with 6N aqueous hydrochloric acid solution, and 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)isobutyramide (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramide)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 solution 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 solution and water, 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)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide (37.05g, 86%, MC70).

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

[0097] Preparation Example 4: Preparation of Crystal Form I 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 (Crystal Form I of MC70) [ka]

[0098] 0.6 g of the compound (MC70) prepared in Preparation Example 3 was dissolved in 0.975 mL of MTBE at room temperature for 30 minutes. After dissolution was completed, the mixture was cooled to 3° C., stirred for about 21 hours, and filtered to obtain the title compound (crystalline form I of MC70).

[0099] The compound of Preparation Example 4 was analyzed by XRPD (FIG. 4) and DSC (FIG. 5) according to the following methods, and the resulting graphs are shown in FIGS.

[0100] XRPD analysis of Production Example 4 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.

[0101] Approximately 20-30 mg of sample was uniformly placed on a glass sample holder to ensure a flat surface, and the 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) in the range of 4-40° were 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 was measured using a Mettler Toledo DSC1 system. Approximately 2 to 5 mg of sample 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, 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 to the inside of 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 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 25 mg of the compound of crystalline form I prepared in Preparation Example 4 was placed in a vial, filled with nitrogen gas, and then melted by heating at 180° C. or 190° C. for 2 minutes. The molten compound was then quickly transferred to a liquid nitrogen bath to form an amorphous compound.

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

[0105] Amorphous properties 1) X-ray powder diffraction (XRPD) XRPD patterns were obtained using a Panalytical Xpert Pro diffractometer equipped with a Cu-X-ray tube and a Pixcel detector system in the following manner.

[0106] Samples were analyzed in transmission mode between low density polyethylene films. Bragg angles (2θ) ranging from 3 to 40° were measured with a step size of 0.013° and a time per step of 22 seconds. Sample analysis time was 5 minutes and the sample was rotated at 60 rpm during data collection. XRPD patterns were classified and processed using HighScorePlus 2.2c software.

[0107] The XRPD results are shown in Figure 1, which confirms that amorphous Example 1 formed by melting at 180°C and 190°C showed the same pattern, with no diffraction peaks and broad noise typical of amorphous samples.

[0108] 2) Hyper DSC DSC was measured using a Perkin Elmer DSC8500. Accurately weighed samples were placed in aluminum sample pans and sealed. Under nitrogen, samples were held at -50°C for 1 minute, then heated to 300°C at 300°C per minute, held for 1 minute, cooled back to -50°C, held for 2 minutes, and heated back to 300°C at 300°C per minute.

[0109] The results of the Hyper DSC are shown in FIG. 2. As shown in FIG. 2, the material of Example 1 exhibited characteristics having a glass transition temperature at about 98° C., and was confirmed to exhibit characteristics typical of an amorphous sample.

[0110] Experimental example 1. Stability evaluation To evaluate the stability of amorphous compounds under various solvent vapor and controlled humidity conditions, the following experiments were performed.

[0111] For the vapor stress experiment, approximately 25 mg of the amorphous compound of Example 1 was placed in an unsealed vial, and the vial was placed in a larger sealed container containing 1 mL of the selected solvent. After 5-7 days, the XRD patterns of the samples were analyzed, and the results are shown in Table 1 below.

[0112] In addition, a humidity stress experiment was performed by placing about 25 mg of the amorphous compound of Example 1 in individual vials and storing them unsealed in various relative humidity chambers (23% RH, 59% RH, 7% RH, 98% RH, 40°C / 75% RH) for 5 to 7 days, and then analyzing the XRD patterns. The results are shown in Table 2 below.

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

[0114] [Table 1]

[0115] [Table 2]

Claims

1. An amorphous compound represented by the following chemical formula 1: 【Chemistry 1】 (In the above Chemical Formula 1, R １ is C ２ -C ５ It is an alkyl group. An amorphous compound of formula 1 having the following differential scanning calorimetry (DSC) profile: 【Chemistry 2】 2. The amorphous compound of claim 1, having an X-ray powder diffraction (XRPD) pattern characterized by having no diffraction peaks at Bragg angles (2θ values) in the range of 3 to 40°.

3. An amorphous compound of chemical formula 1 described in claim 1, having an XRPD pattern as shown below. 【Chemistry 3】

4. 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.

5. A method for producing an amorphous compound of formula 1 according to any one of claims 1 to 4, comprising the steps of: The compound of Formula 1 is melted and then cooled; The cooling step comprises contacting the molten compound of Formula 1 with liquid nitrogen.

6. 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°.

6. The method of claim 5, 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°.

7. The method for preparing the amorphous compound of formula 1 according to claim 5, wherein the compound is melted at a temperature of 150°C to 200°C.

8. The method for preparing the amorphous compound of formula 1 according to claim 5, wherein the melting is carried out for a period of 30 seconds to 10 minutes.

9. A pharmaceutical composition comprising an amorphous compound of formula 1 according to any one of claims 1 to 4 and a pharma- ceutically acceptable carrier.

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

11. The pharmaceutical composition according to claim 10, wherein the composition is for the prevention or treatment of obesity, diabetes, inflammation or erectile dysfunction.

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