Amorphous melanocortin receptor agonist and preparation method thereof

Abstract

The present invention relates to an amorphous compound represented by Chemical Formula 1, a preparation method thereof, and a pharmaceutical composition comprising the same. The amorphous compound represented by Chemical Formula 1 of the present invention can be characterized by an XPRD pattern, a DSC curve, and / or an NMR spectrum.

Description

Technical Field

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to Korean Patent Application No. 10-2020-0180806, filed on December 22, 2020, and the entire disclosure of that Korean patent application is incorporated as a part of this specification. Technical Field

[0004] The present invention relates to a novel amorphous compound showing excellent agonistic activity on melanocortin receptors, a preparation method thereof and a pharmaceutical composition containing the same. Background Art

[0005] Leptin is a hormone secreted by adipocytes, and its secretion increases with increasing body fat content. It regulates the functions of various neuropeptides produced by the hypothalamus, thereby regulating various body functions, including appetite, body fat content, and energy metabolism (Schwartz et al., Nature 404, 661-671 (2000)). Leptin signaling, which controls appetite and body weight, is achieved by regulating many downstream factors, the most representative of which are melanocortin, agouti-related peptide (AgRP), and neuropeptide Y (NPY) hormones.

[0006] When the leptin concentration in the blood increases due to excess heat in the body, the secretion of proopiomelanocortin (POMC) in the pituitary gland increases, and the production of AgRP and NPY decreases. The small peptide hormone α-melanocyte stimulating hormone (MSH) is produced by POMC neurons. The hormone is an agonist of the melanocortin-4 receptor (MC4R) of secondary neurons, ultimately inducing a decrease in appetite. At the same time, when the leptin concentration decreases due to caloric deficiency, the expression of the MC4R antagonist AgRP increases, and the expression of NPY also increases, ultimately promoting appetite. That is, according to the changes in leptin, the α-MSH hormone and the AgRP hormone act as agonists and antagonists of MC4R, thereby participating in appetite control.

[0007] In addition to binding to MC4R, α-MSH hormone also binds to three MCR subtypes, thereby inducing a variety of physiological reactions. Five MCR subtypes have been identified so far. Among them, MC1R is mainly expressed in skin cells and is involved in regulating melanin deposition (skin pigmentation). MC2R is mainly expressed in the adrenal glands and is known to be involved in the production of glucocorticoids. Its ligand is only adrenocorticotropic hormone (ACTH) derived from POMC. MC3R and MC4R are mainly expressed in the central nervous system and are involved in regulating appetite, energy metabolism and body fat storage efficiency, while MC5R is expressed in a variety of tissues and is known to regulate exocrine function (Wikberg et al., Pharm Res 42 (5) 393-420 (2000)). In particular, activation of the MC4R receptor induces decreased appetite and increased energy metabolism, thereby effectively reducing weight. Therefore, it has been shown to be a major point of action for the development of anti-obesity drugs (reviewed in: Wikberg, Eur. J. Pharmacol 375, 295-310 (1999)); Wikberg et al., Pharm Res 42(5)393-420 (2000); Douglas et al., Eur J Pharm 450, 93-109 (2002); O'Rahilly et al., Nature Med 10, 351-352 (2004)).

[0008] The role of MC4R in controlling appetite and body weight has been primarily demonstrated through experiments in an animal model with abnormal expression of agouti protein (agouti mice). In agouti mice, it was found that due to a genetic mutation, agouti protein is expressed at high concentrations in the central nervous system and 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 results have shown that agouti-related peptides (AgRPs), similar to actual agouti protein, are expressed in hypothalamic nerves. These peptides are also known to be antagonists of MC4R and are involved in controlling appetite (Shutter et al., Genes Dev., 11, 593-602 (1997); Ollman et al., Science 278, 135-138 (1997)).

[0009] Intracerebral administration of the in vivo MC4R agonist α-MSH to animals produced anorexigenic effects. When animals were treated with the MC4R antagonists SHU9119 (peptide) or HS014 (peptide), appetite was again increased (Kask et al., Biochem. Biophys. Res. Comm. 245, 90-93 (1998)). In addition, in animal studies 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 suppressing appetite, reducing body weight, and increasing energy metabolism were found (Thiele TE et al., Am J Physiol 274(1Pt 2), R248-54(1998); Lee MD et al., FASEB J 12, A552(1998); Murphy B. et al., J Appl Physiol 89, 273-82(2000)). In contrast, administration of the representative SHU9119 to animals showed significant and sustained increases in food intake and body weight, which provides pharmacological evidence that MCR agonists may be anti-obesity agents. No obvious appetite-reducing effect was observed after administration of MTII in MC4R knockout (KO) mice. This experimental result once again demonstrated that the appetite-reducing effect is mainly achieved through activation of MC4R (Marsh et al., Nat Genet 21, 119-122 (1999)).

[0010] Anorexigenic agents acting on the central nervous system are the main types of anti-obesity drugs developed so far. Among them, most are drugs that regulate the action of neurotransmitters. Examples include noradrenergic agents (phentermine and mazindol), serotonergic agents, fluoxetine and sibutramine. However, in addition to suppressing appetite, neurotransmitter modulators also have a wide range of effects on various physiological activities through many subtype receptors. Therefore, the modulators lack selectivity for each subtype, so a major disadvantage is that they can be accompanied by various side effects when used for a long time.

[0011] At the same time, melanocortin agonists are neuropeptides, not neurotransmitters. Given that all functions except energy metabolism are normal in MC4R gene KO mice, their advantage as a site of action is that they can induce weight loss simply by suppressing appetite without affecting other physiological functions. In particular, the receptor is a G protein-coupled receptor (GPCR), which belongs to the most successful class of new drug sites developed so far. Therefore, the site of action is very different from existing sites of action, and it is relatively easy to ensure selectivity for subtype receptors.

[0012] As examples of using the melanocortin receptor as an action point, International Publication Nos. WO 2008 / 007930 and WO 2010 / 056022 disclose compounds that are melanocortin receptor agonists.

[0013] In addition, the inventors of the present invention have conducted extensive research and invented a novel compound of the following formula 1 having excellent agonist activity and selectivity for melanocortin receptors, particularly melanocortin-4 receptors (MC4R), and a preparation method thereof (application number KR 10-2019-0141649 (filed on November 7, 2019)):

[0014] [Formula 1]

[0015]

[0016] (R1 is a C2-C5 alkyl group.)

[0017] Furthermore, the salt form or crystal structure of a pharmaceutical active ingredient often affects the chemical stability of the drug. Different salt forms of a compound can alter its filterability, product stability, storage stability, and other aspects. Different crystallization and storage conditions can lead to changes in the compound's crystal structure, sometimes resulting in the formation of alternative crystalline forms. Therefore, for a single compound, it is necessary to develop salt forms and / or crystal structures with high purity and good chemical stability.

[0018] [Prior art literature]

[0019] [Patent Document]

[0020] (Patent Document 1) International Patent Application Publication No. WO 2008 / 007930

[0021] (Patent Document 2) International Patent Application Publication No. WO 2010 / 056022 Summary of the Invention

[0022] Technical issues

[0023] One object of the present invention is to provide a novel amorphous compound and a salt thereof having excellent agonist activity and selectivity for melanocortin receptors, particularly melanocortin-4 receptor (MC4R).

[0024] Another object of the present invention is to provide a method for preparing the amorphous compound or a salt thereof.

[0025] Another object of the present invention is to provide a pharmaceutical composition comprising the amorphous compound or a salt thereof.

[0026] Technical Solutions

[0027] In order to achieve the above goals,

[0028] According to one aspect of the present invention, there is provided an amorphous compound of the following formula 1:

[0029] [Formula 1]

[0030]

[0031] wherein R1 is a C2-C5 alkyl group.

[0032] Since the compound of Formula 1 may have an asymmetric carbon center and an asymmetric axis or plane, it may exist as cis or trans isomers, R or S isomers, racemates, diastereomeric mixtures and individual diastereomers, all of which are within the scope of the compound of Formula 1.

[0033] In this specification, unless otherwise specified, for convenience, the compound of Formula 1 is used to include all compounds of Formula 1, pharmaceutically acceptable salts, isomers and solvates thereof.

[0034] In one embodiment according to the present invention, in Formula 1, R1 is a C2 to C5 alkyl group. In another embodiment according to the present invention, in Formula 1, R1 is a linear or branched C2 to C5 alkyl group, such as ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl.

[0035] In another embodiment according to the present invention, in Formula 1, R1 is a C2 to C4 alkyl group. In another embodiment according to the present invention, in Formula 1, R1 is a linear or branched C2 to C4 alkyl group, such as ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl. Specifically, R1 can be an isopropyl group.

[0036] In one embodiment according to the present invention, pharmaceutically acceptable salts include, but are not limited to, acid addition salts formed by the following acids: inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid and hydroiodic acid; organic carbonic acids such as tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid and maleic acid; or sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or naphthalenesulfonic acid.

[0037] In one embodiment according to the present invention, the solvate may include a hydrate; and a solvate with an organic solvent, such as methanol, ethanol, 2-propanol, 1,2-propylene glycol, 1,3-propylene glycol, n-butanol, 1,4-butanediol, tert-butanol, acetic acid, acetone, butyl acetate, methyl acetate, ethyl acetate, propyl acetate, tert-butyl acetate, isobutyl acetate, methyl ethyl ketone, 2-pentanone, tetrahydrofuran, acetonitrile, chloroform, toluene, and a mixture thereof.

[0038] The amorphous compound according to the present invention may have an X-ray powder diffraction (XRPD) pattern in which a characteristic diffraction peak is absent and has broad noise.

[0039] In one embodiment according to the present invention, the amorphous compound may have a Figure 1 XRPD pattern shown.

[0040] In a high-speed differential scanning calorimetry (HSSC) curve, the amorphous compound according to the present invention may not have an exothermic peak when heated to a temperature below 350° C. In addition, in the high-speed DSC curve, the amorphous compound is characterized by a glass transition temperature (Tg) of about 98° C.

[0041] In one embodiment according to the present invention, the amorphous compound may have Figure 2 High-speed DSC curve shown.

[0042] In this manual,

[0043] X-ray powder diffraction (XRPD) analysis shows the results obtained using a PANalytical X'Pert Pro MPD system (Malvern Panalytical Ltd).

[0044] Differential Scanning Calorimetry (DSC) analysis shows the results obtained using DSC8500 (Perkin Elmer).

[0045] The stability analysis shows the results obtained using HPLC (Agilent Technologies).

[0046] Nuclear magnetic resonance (NMR) spectra show the results obtained using a Bruker 500 MHz.

[0047] The amorphous compound of Formula 1 has a higher purity than the crude compound and is more stable physically and chemically.

[0048] The amorphous compound of Formula 1 according to the present invention can be formed from the crystalline form I of the compound of Formula 1, which 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 in the X-ray powder diffraction (XRPD) pattern, wherein the characteristic peaks are selected from the peaks having 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°, 17.00 9±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°, 2 3.896±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°.

[0049] The amorphous compound of Formula 1 prepared from the crystalline Form I of the present invention may have a higher purity than the amorphous compound of Formula 1 formed from other crystalline forms of the compound of Formula 1 and may be more physically and chemically stable.

[0050] In addition, the amorphous compound of Formula 1 of the present invention may have a better ability to agonize the melanocortin-4 receptor and a better preventive or therapeutic effect on diseases such as obesity, diabetes, inflammation, erectile dysfunction, etc. than known melanocortin-4 receptor agonists. However, the effects of the present invention are not limited thereto.

[0051] In another aspect, the present invention provides a method for preparing an amorphous compound of Formula 1, comprising the steps of melting the compound of Formula 1 and then rapidly cooling it.

[0052] The amorphous compound of Formula 1 may be formed using the compound of Formula 1.

[0053] The compound of formula 1 can be obtained by the preparation method described in the specification of application number KR 10-2019-0141649 (filed on November 7, 2019).

[0054] 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.

[0055] The crystalline form I of the compound 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 in the X-ray powder diffraction (XPRD) pattern, wherein the characteristic peaks are selected from peaks having the following 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°, 1 8.364±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°.

[0056] The crystalline form I may have Figure 4 X-ray powder diffraction (XRPD) pattern shown.

[0057] The amorphous compound of Formula 1 of the present invention can be formed from the compound of Formula 1 by a melt quenching method.

[0058] First, the compound represented by Formula 1 is melted. The melting may be performed under a nitrogen atmosphere.

[0059] 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, 180°C or 190°C.

[0060] Melting can be performed for, for example, 30 seconds to 10 minutes, 30 seconds to 5 minutes, 30 seconds to 2 minutes, or 1 minute to 2 minutes.

[0061] In one embodiment according to the present invention, the method may comprise melting the crystalline Form I of the compound of Formula 1 at a temperature of 180°C to 190°C for 2 minutes.

[0062] Next, the molten compound is rapidly cooled.

[0063] Rapid cooling can be performed using liquid nitrogen.

[0064] Rapid cooling can be performed by contacting the molten compound of Formula 1 with liquid nitrogen.

[0065] In one embodiment according to the present invention, the molten compound of Formula 1 may be transferred into a liquid nitrogen bath to form an amorphous compound.

[0066] In another embodiment according to the present invention, liquid nitrogen may be introduced into the flask containing the molten compound to form the amorphous compound.

[0067] The amorphous compound of Formula 1 obtained as above may have a higher purity than the crude compound of Formula 1 and may be more physically and chemically stable. However, the effects of the present invention are not limited thereto.

[0068] In another aspect, the present invention provides a pharmaceutical composition comprising: (i) an amorphous compound of Formula 1 and (ii) a pharmaceutically acceptable carrier.

[0069] The amorphous compound of Formula 1 according to the present invention exhibits excellent agonist effects on melanocortin receptors, particularly the melanocortin-4 receptor (MC4R). Therefore, the present invention can provide a pharmaceutical composition for agonizing melanocortin receptors, the composition comprising the amorphous compound as an active ingredient. Specifically, the pharmaceutical composition can be a composition that agonizes the function of the melanocortin-4 receptor.

[0070] In addition, since the pharmaceutical composition can show excellent effects in preventing or treating obesity, diabetes, inflammation and erectile dysfunction, it may be a composition for preventing or treating obesity, diabetes, inflammation or erectile dysfunction. However, the use of the present invention is not limited to the above diseases.

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

[0072] When the amorphous compound of the present invention is administered for clinical purposes, the total daily dose administered to the host in a single dose or divided doses may preferably be in the range of 0.01 to 10 mg / kg body weight. However, the specific dosage level for an individual patient may vary depending on the specific compound to be used, the patient's weight, sex, health status, diet, time of drug administration, method of administration, excretion rate, drug combination, severity of disease, etc.

[0073] The amorphous compound of the present invention can be administered by any route as desired. For example, the amorphous compound of the present invention can be administered by injection or orally.

[0074] The pharmaceutical composition of the present invention may be in various oral dosage forms, such as tablets, pills, powders, capsules, granules, syrups or emulsions, or parenteral dosage forms, such as injection preparations for intramuscular, intravenous or subcutaneous administration.

[0075] Injection preparations can be prepared according to known techniques using appropriate dispersants, wetting agents, suspending agents or excipients.

[0076] Excipients that can be used in the pharmaceutical preparations of the present invention include, but are not limited to, sweeteners, binders, solubilizers, solubilizing agents, wetting agents, emulsifiers, isotonic agents, adsorbents, decomposing agents, antioxidants, preservatives, lubricants, fillers, aromatics, etc. For example, as excipients, lactose, glucose, sucrose, mannitol, sorbitol, cellulose, glycine, silicon dioxide, magnesium aluminum silicate, starch, gelatin, tragacanth gum, arginine, sodium alginate, methylcellulose, sodium carboxymethylcellulose, water, ethanol, polyethylene glycol, polyvinyl pyrrolidone, sodium chloride, calcium chloride, orange flavor, strawberry flavor, vanilla flavor, etc. can be used.

[0077] When the pharmaceutical composition of the present invention is in an oral dosage form, examples of carriers to be used may include, but are not limited to, cellulose, calcium silicate, corn starch, lactose, sucrose, glucose, calcium phosphate, stearic acid, magnesium stearate, calcium stearate, gelatin, talc, and the like.

[0078] When the pharmaceutical composition of the present invention is in the form of an injectable preparation, examples of carriers may include, but are not limited to, water, saline, aqueous glucose solution, sugar-like aqueous solution, alcohol, glycol, ether, oil, fatty acid, fatty acid ester, glyceride, and the like.

[0079] In another aspect, an amorphous compound as described above is provided for use in agonizing the function of a melanocortin receptor, particularly a melanocortin-4 receptor (MC4R).

[0080] In one embodiment, an amorphous compound as described above is provided for use in treating or preventing obesity, diabetes, inflammation or erectile dysfunction.

[0081] In another aspect, a method for agonizing the function of melanocortin receptors, particularly melanocortin-4 receptor (MC4R), is provided, comprising the step of administering the above-mentioned amorphous compound to a subject.

[0082] In another aspect, provided is a method for treating obesity, diabetes, inflammation or erectile dysfunction, comprising the step of administering the above-mentioned amorphous compound to a subject.

[0083] Beneficial effects

[0084] The amorphous compound of Formula 1 according to the present invention exhibits excellent agonistic effects on melanocortin receptors, particularly melanocortin-4 receptor (MC4R), and thus can be effectively used for preventing or treating obesity, diabetes, inflammation and erectile dysfunction.

[0085] The amorphous compound of Formula 1 according to the present invention exhibits an on-target effect on the melanocortin-4 receptor, thereby exhibiting weight loss and reduced appetite without affecting anxiety and depression. Furthermore, it can be administered without any safety concerns, such as side effects such as human ether-a-go-go related gene (hERG) inhibition or mutagenesis.

[0086] Furthermore, the amorphous compound of Formula 1 according to the present invention has more excellent purity, yield, physical and chemical stability than the crude compound of Formula 1.

[0087] Specifically, the amorphous compound of Formula 1 may have better solubility, storage stability, and production stability than the crude compound of Formula 1. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 This is a graph showing the XRPD results of Example 1.

[0089] Figure 2 This is a graph showing the DSC results of Example 1.

[0090] Figure 3 This is a graph showing the NMR results of Example 1.

[0091] Figure 4 This is a graph showing the XRPD results of Preparation Example 4.

[0092] Figure 5 This is a graph showing the DSC results of Preparation Example 4. DETAILED DESCRIPTION

[0093] The present invention will be described in more detail below through preparation examples and examples. However, these examples are merely illustrative of the present invention, and the scope of the present invention is not limited thereto.

[0094] Preparation Example 1: Preparation of methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylate hydrochloride

[0095]

[0096] The title compound was obtained by following steps A, B, C, D and E.

[0097] Step A: Preparation of 2-methyl (2S,4S)-4-azidopyrrolidine-1,2-dicarboxylate

[0098] 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. After stirring at 80°C 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 and water, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure to yield 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.

[0099] MS[M+H]=271(M+1)

[0100] 1 H NMR(400MHz,CD3OD)δ4.43-4.37(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)

[0101] Step B: Preparation of 2-methyl (2S,4S)-4-aminopyrrolidine-1,2-dicarboxylate

[0102] 2-Methyl (2S,4S)-4-azidopyrrolidine-1,2-dicarboxylate (24.59 g, 91.0 mmol) was dissolved in tetrahydrofuran (180 ml), and a 1 M trimethylphosphine tetrahydrofuran solution (109.2 ml, 109.2 mmol) was slowly added at 0°C. After stirring at the same temperature for 1 hour, the mixture was 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 the mixture was 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 yield crude 2-methyl (2S,4S)-4-aminopyrrolidine-1,2-dicarboxylate (20.62 g, 93%), which was used in the next step without purification.

[0103] MS[M+H]=245(M+1)

[0104] 1 H NMR(400MHz,CD3OD)δ4.27(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)

[0105] Step C: 1-(tert-Butyl)-(2S,4S)-4-(((1s,4R)-4-methylcyclohexyl)amino)pyrrolidine-1,2-dicarboxylate Preparation of 2-methyl ester

[0106] 2-Methyl (2S,4S)-4-aminopyrrolidine-1,2-dicarboxylate (20.62 g, 84.4 mmol) 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 2-methyl (2S,4S)-4-(((1S,4R)-4-methylcyclohexyl)amino)pyrrolidine-1,2-dicarboxylate (22.9 g, 80%).

[0107] MS[M+H]=341(M+1)

[0108] 1H NMR (400MHz, CD3OD) δ4.26(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)

[0109] Step D: (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-1,2-dicarboxylic acid Preparation of 1-(tert-butyl) 2-methyl ester

[0110] 2-Methyl (2S,4S)-4-(((1s,4R)-4-methylcyclohexyl)amino)pyrrolidine-1,2-dicarboxylate (37.29 g, 109.5 mmol) obtained in step C above 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 slowly added at 0°C. After stirring at room temperature for 16 hours, the reaction solvent was concentrated under reduced pressure, and aqueous sodium bicarbonate was added, followed by 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 obtain 2-methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-1,2-dicarboxylate (38.79 g, 86%).

[0111] MS[M+H]=411(M+1)

[0112] 1 H NMR (400MHz, CD3OD) δ4.27(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)

[0113] Step E: Methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylate Preparation of hydrochloride

[0114] The 1-(tert-butyl)-2-methyl (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-1,2-dicarboxylate (34.0 g, 82.8 mmol) obtained in the above step D was dissolved in dichloromethane (200 ml), and 1,4-dichloromethane containing 4N hydrochloric acid solution 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.

[0115] MS[M+H]=311(M+1)

[0116] Preparation Example 2: Preparation of (3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carboxylic acid

[0117]

[0118] The title compound was obtained according to the method described in International Patent Publication No. WO 2004 / 092126.

[0119] MS[M+H]=282(M+1)

[0120] 1 H NMR (400MHz, CD3OD) δ7.43-7.33(m,4H),3.90-3.69(m,3H),3.59(dd,J=11.2 ,10.0Hz,1H),3.29(dd,J=11.2,11.2Hz,1H),3.18-3.09(m,1H),1.44(s,9H)

[0121] Preparation 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)isobutyramide (MC70)

[0122]

[0123] The title compound was obtained by following steps A, B and C.

[0124] Step A: (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N- Preparation of methyl ((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylate

[0125] The (2S,4S)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylic acid methyl ester hydrochloride (28.7 g, 82.73 mmol) obtained in Preparation Example 1, the (3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carboxylic acid (24.5 g, 86.87 mmol) obtained in Preparation Example 2, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (22.2 g, 115.83 mmol) and 1-hydroxybenzotriazole hydrate (15.7 g, 115.83 mmol) were dissolved in N,N'-dimethylformamide (400 ml), and N,N'-diisopropylethylamine (72.0 ml, 413.66 mmol) was slowly added. After stirring at room temperature for 16 hours, the reaction solvent was concentrated under reduced pressure, 0.5N 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, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography to obtain (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 ester (41.19 g, 87%).

[0126] MS[M+H]=575(M+1)

[0127] Step B: (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N- Preparation of ((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylic acid

[0128] 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 above was dissolved in methanol (450 ml), followed by the addition of 6N aqueous sodium hydroxide solution (57.2 ml, 343.09 mmol). After stirring at room temperature for 16 hours, the pH was adjusted to approximately 5 with 6N aqueous hydrochloric acid, and the reaction solution was concentrated under reduced pressure. The concentrate was dissolved in dichloromethane, and the insoluble solid was filtered through filter paper. 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.

[0129] MS[M+H]=561(M+1)

[0130] Step C: N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5- Preparation of (morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide

[0131] The (2S,4S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-4-(N-((1s,4R)-4-methylcyclohexyl)isobutyramido)pyrrolidine-2-carboxylic acid (38.4 g, 68.60 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (18.4 g, 96.04 mmol) and 1-hydroxybenzotriazole hydrate (13.0 g, 96.04 mmol) obtained in the above step B were dissolved in N,N'-dimethylformamide (200 ml), and then morpholine (5.9 ml, 68.80 mmol) and N,N'-diisopropylethylamine (59.7 ml, 343.02 mmol) were slowly added in sequence. After stirring at room temperature for 16 hours, the reaction solution was concentrated under reduced pressure, 0.5N sodium hydroxide aqueous solution was added, and the mixture was extracted twice with ethyl acetate. The organic layer was washed twice with sodium chloride aqueous solution and water, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography to obtain N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide (37.05 g, 86%, MC70).

[0132] MS[M+H]=630(M+1)

[0133] Preparation Example 4: Preparation of Crystalline 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 (Crystalline Form I of MC70)

[0134]

[0135] Based on 0.6g of the compound (MC70) prepared in Preparation Example 3 above, 0.975mL of MTBE was used to dissolve the compound (MC70) at room temperature for 30 minutes. After dissolution was complete, the mixture was cooled to 3°C and stirred for approximately 21 hours, then filtered to obtain the title compound (Crystallized Form I of MC70).

[0136] The compound of Preparation Example 4 was subjected to XRPD ( Figure 4 ) and DSC( Figure 5 ) analysis. The resulting diagram is shown in Figure 4 and Figure 5 middle.

[0137] XRPD analysis of Preparation Example 4

[0138] XRPD diffractograms were obtained using a PANalytical X'PertPro MPD system equipped with a monochromatized radiation source and a Ni filter as a solid-state detector by the following method.

[0139] Approximately 20 to 30 mg of sample was compressed in a glass sample holder so that the sample had a flat surface, and the instrument's generator was set to 45 kV (accelerating voltage) and 40 mA (filament emission), and then the measurement was performed in reflection mode (no spinning). The Bragg angle (2θ) was measured in the range of 4° to 40° with a step size of 0.026° and a step time of 51 seconds.

[0140] DSC analysis of Preparation Example 4

[0141] DSC measurements were performed using a Mettler Toledo DSC1 system. Approximately 2 to 5 mg of sample was weighed and placed in a 40 μL Al crucible (a flat-bottomed aluminum pan with a pinhole lid), and a pinhole was punched. DSC measurements were then performed while the sample was heated from 25°C to 350°C at a rate of 10°C / min. During the measurement, nitrogen was supplied to the instrument at a rate of 70 mL / min to prevent the influx of oxygen and other gases. Data collection and evaluation were performed using the STARe software.

[0142] Example 1

[0143] 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

[0144] 25 mg of the crystalline Form I compound prepared in Preparation Example 4 was placed in a vial. The vial was filled with nitrogen, and then the compound was 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.

[0145] NMR spectrum of Example 1 ( Figure 3 ) showed that the compound of Example 1 had the same structure as the compound of Preparation Example 4.

[0146] Amorphous characteristics

[0147] 1) X-ray powder diffraction (XRPD)

[0148] XRPD patterns were obtained using a Panalytical Xpert Pro diffractometer equipped with a Cu-X-ray tube and a Pixcel detector system.

[0149] The samples were analyzed in transmission mode between low-density polyethylene films. The Bragg angle (2θ) in the range of 3° to 40° was measured under the condition that the step length was 0.013° and the time per step was 22 seconds. The analysis time of the sample 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.

[0150] The XRPD results are shown in Figure 1 In. Figure 1 As shown, it was confirmed that the amorphous Example 1 formed by melting at 180° C. and 190° C. showed the same pattern. They had no diffraction peaks and showed broad noise, which is unique to amorphous samples.

[0151] 2) High-speed differential scanning calorimetry (HSDSC)

[0152] DSC measurements were performed using a Perkin Elmer DSC8500. Accurately weighed samples were placed in sealed aluminum sample pans. The samples were held at -50°C under nitrogen for 1 minute, then heated to 300°C at a rate of 300°C / min, held for 1 minute, cooled back to -50°C, held for 2 minutes, and then heated again to 300°C at a rate of 300°C / min.

[0153] High-speed DSC results are shown in Figure 2 In. Figure 2 As shown, the material of Example 1 is characterized by a glass transition temperature of about 98° C., confirming that it exhibits typical characteristics of an amorphous sample.

[0154] Experimental Example 1. Stability Evaluation

[0155] The following experiments were performed to evaluate the stability of amorphous compounds under various solvent vapor and controlled humidity conditions.

[0156] A steam stress experiment was performed by placing approximately 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 analyzing the XRD pattern of the sample after 5 to 7 days. The results of the analysis are shown in Table 1 below.

[0157] In addition, a humidity stress experiment was conducted by placing approximately 25 mg of the amorphous compound of Example 1 in individual vials, storing the vials in an unsealed state in different relative humidity chambers (23% RH, 59% RH, 7% RH, 98% RH, 40°C / 75% RH) for 5 to 7 days, and analyzing the XRD patterns. The analysis results are shown in Table 2 below.

[0158] Steam stress and humidity stress tests are methods used to generate stable solvates or hydrates because, when a substance is exposed to steam, the solid plasticizes and limits molecular mobility. However, the amorphous compound of Example 1 was maintained under various conditions using the above-described test methods. Therefore, the amorphous compound was confirmed to be stable under the corresponding conditions.

[0159] [Table 1]

[0160] Input solvent result XRD Amorphous acetone solid Amorphous Amorphous ACS solid Amorphous Amorphous Benzyl alcohol solid Amorphous Amorphous chlorobenzene solid Amorphous Amorphous Chloroform solid Amorphous Amorphous DCM solid Amorphous Amorphous DME solid Amorphous Amorphous DMSO solid Amorphous Amorphous ethanol solid Amorphous Amorphous Formamide solid Amorphous Amorphous heptane solid Amorphous Amorphous HFIPA solid Amorphous Amorphous MeOH solid Amorphous Amorphous MEK solid Amorphous Amorphous 2-Me-THF solid Amorphous Amorphous Nitromethane solid Amorphous Amorphous NMP solid Amorphous Amorphous IPA solid Amorphous Amorphous THF solid Amorphous Amorphous water solid Amorphous Amorphous Pyridine solid Amorphous

[0161] [Table 2]

[0162] Input Screening methods result XRD Amorphous 23% RH stress solid Amorphous Amorphous 59% RH stress solid Amorphous Amorphous 75% RH stress solid Amorphous Amorphous 98% RH stress solid Amorphous Amorphous 40℃ / 75%RH stress solid Amorphous

Claims

1. An amorphous compound of the following formula 1: [Formula 1] wherein R1 is a C2-C5 alkyl group, and wherein the amorphous compound of Formula 1 has a differential scanning calorimetry (DSC) curve as shown in FIG2 . 2 . The amorphous compound of Formula 1 according to claim 1 , which has an X-ray powder diffraction (XRPD) pattern having no characteristic diffraction peak and broad noise. 3 . The amorphous compound of Formula 1 according to claim 1 , having an X-ray powder diffraction pattern as shown in FIG. 1 .

4. The amorphous compound of Formula 1 according to claim 1, which is N-((3S,5S)-1-((3S,4R)-1-(tert-butyl)-4-(4-chlorophenyl)pyrrolidine-3-carbonyl)-5-(morpholine-4-carbonyl)pyrrolidin-3-yl)-N-((1s,4R)-4-methylcyclohexyl)isobutyramide.

5. A method for preparing the amorphous compound of formula 1 according to any one of claims 1 to 4, comprising the steps of melting the compound of formula 1 and then rapidly cooling it.

6. The preparation method according to claim 5, wherein the compound of formula 1 to be melted comprises crystalline form I of the compound of formula 1, and The X-ray powder diffraction (XRPD) pattern of the crystalline form I of the compound of formula 1 has more than three characteristic peaks, and the characteristic peaks are selected from the peaks having 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°. 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°. 7 . The method for preparing the amorphous compound of Formula 1 according to claim 5 , wherein the melting is performed 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 performed for 30 seconds to 10 minutes.

9. The method for preparing the amorphous compound of Formula 1 according to claim 5, wherein the rapid cooling is performed by contacting the melted compound of Formula 1 with liquid nitrogen.

10. A pharmaceutical composition comprising the amorphous compound of Formula 1 according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

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

12. The pharmaceutical composition according to claim 11, which is used for preventing or treating obesity, diabetes, inflammation or erectile dysfunction.

Images