A new crystalline form of 5-[(1,1-dioxido-4-thiomorpholinyl)methyl]-2-phenyl-N-(tetrahydro-2H-pyran-4-yl)-1H-indol-7-amine

Abstract

The present invention relates to crystalline form A of the compound of Chemical Formula 1, which has superior physicochemical properties compared to amorphous and other crystalline forms of the compound of Chemical Formula 1. The crystalline form A of the compound of Chemical Formula 1 according to the present invention does not denature even under harsh conditions for a long period of time compared to amorphous and other crystalline forms, has low sorption, and does not change in crystal form even when subjected to pressure or pulverization, which is advantageous for formulation, and the stability of the crystalline form itself is also very excellent, making it useful for long-term storage.

Description

[Technical field]

[0001] The present invention relates to a novel crystalline form of the compound of formula 1 below, 5-[(1,1-dioxido-4-thiomorpholinyl)methyl]-2-phenyl-N-(tetrahydro-2H-pyran-4-yl)-1H-indol-7-amine.

[0002] [ka] [Background technology]

[0003] Different crystalline or amorphous forms can exhibit different solid-state physical properties, such as sorption properties, behavior toward compression, stability during storage, and flowability of milled solids. Such properties also affect the suitability of a particular solid state as an active pharmaceutical substance for commercial production. For example, flowability affects the ease with which a substance can be handled during processing into a pharmaceutical product. If the particles of a powdered compound do not flow easily, the formulation specialist must take this fact into account when developing a tablet or capsule formulation, which may require the use of lubricants such as colloidal silicon dioxide, talc, starch, or tribasic calcium phosphate.

[0004] Also, different crystalline or amorphous forms of the same drug can have substantial differences in pharma- ceutical important properties such as dissolution rate and bioavailability. Dissolution rate is not only a consideration in preparing syrups, elixirs, and other liquid medicaments, but can also alter therapeutic outcomes. For example, differences in dissolution rate of an active ingredient in a patient's gastric juices can affect the time it takes for an orally administered active ingredient to reach the patient's bloodstream, altering therapeutic outcomes.

[0005] The compound of Chemical Formula 1, 5-[(1,1-dioxide-4-thiomorpholinyl)methyl]-2-phenyl-N-(tetrahydro-2H-pyran-4-yl)-1H-indol-7-amine, is a compound disclosed in International Patent Publication WO2009-025478 and is known to have preventive, therapeutic and ameliorative effects on cell necrosis and related diseases.

[0006] The present inventors have continuously studied crystalline forms of the compound of Chemical Formula 1, which are easy to prepare and have excellent stability, during the product development process of the compound of Chemical Formula 1. As a result, the present inventors have confirmed that, among the crystalline forms of the compound of Chemical Formula 1, crystalline form A has the most excellent physicochemical properties compared with amorphous and other crystalline forms. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Patent Publication WO2009-025478 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide crystalline form A of compound of formula 1, which has superior physicochemical properties compared to the amorphous form and other crystalline forms of compound of formula 1.

[0009] The present invention also provides a method for preparing crystalline form A of the compound of formula 1.

[0010] The present invention also provides a pharmaceutical composition comprising crystalline form A of the compound of formula 1 as an active ingredient. [Means for solving the problem]

[0011] The present invention provides crystalline form A of the compound of Chemical Formula 1, which has superior physicochemical properties compared to the amorphous form and other crystalline forms of the compound of Chemical Formula 1. "Crystal form A" is a substance named crystalline form XII in the process of screening the crystalline forms of the compound of Chemical Formula 1, and is hereafter renamed as "pattern A" or "crystalline form A". In this specification, "crystalline form A", "pattern A" or "crystalline form XII" are used interchangeably. "Crystal form" and "pattern" are also used interchangeably. According to X-ray powder diffraction (XRPD) analysis, crystalline form A has a crystal structure different from other crystalline forms.

[0012] According to one embodiment of the present invention, crystalline form A of compound of formula 1 is characterized by an X-ray powder diffraction pattern having 4 or more, e.g., 4, 5, 6, 7, 8, 9, 10 or more, diffraction peaks at 2[θ] values ​​selected from 7.64±0.2, 12.32±0.2, 12.62±0.2, 15.26±0.2, 17.32±0.2, 19.18±0.2, 19.61±0.2, 19.95±0.2, 20.60±0.2, 21.12±0.2, 22.94±0.2, 24.11±0.2 and 28.15±0.2.

[0013] In particular, the X-ray powder diffraction pattern is characterized by having diffraction peaks at 2[θ] values ​​selected from 7.64±0.2, 15.26±0.2, 17.32±0.2, 19.18±0.2, 21.12±0.2, and 22.94±0.2.

[0014] More specifically, crystalline Form A of the compound of Formula 1 is characterized by an X-ray powder diffraction pattern having peak positions consistent with the peak positions listed in Table 1 below.

[0015] [Table 1]

[0016] In another embodiment, the crystalline form A of the compound of formula 1 provided by the present invention is characterized by an onset of melting point at 272.7° C. (±2.5) and a maximum of 273.4° C. (±2.5), as measured by differential scanning calorimetry.

[0017] The present invention provides a method for preparing crystalline form A of the compound of chemical formula 1, comprising: stirring a solution containing the compound of chemical formula 1 and a solvent selected from DMSO, DMF, and NMP under heating for 1 to 24 hours; and cooling the solution to 0 to 25° C. for 1 to 72 hours while stirring to grow crystals.

[0018] Here, the compound of formula 1 used as the starting material may be amorphous or in any crystalline form.

[0019] The heating can be carried out at a temperature elevated to a range of 50 to 120°C.

[0020] The cooling can be carried out at a rate of 5 to 20° C. / h.

[0021] Another embodiment of the present invention provides a method for preparing crystalline form A of compound of formula 1, comprising: adding a poor solvent to a solution obtained by dissolving compound of formula 1 in a solvent to cause crystallization; and cooling the solution to 0-25° C. for 6-48 hours while stirring to grow crystals.

[0022] Similarly, the compound of formula 1 used as the starting material herein may be amorphous or in any form of crystalline form.

[0023] In one embodiment of the present invention, the method for preparing crystalline form A of compound of Formula 1 may further include adding crystalline form A of compound of Formula 1 as a seed crystal during the addition of the antisolvent.

[0024] The solvent can be used without limitation as long as it can dissolve the compound of Chemical Formula 1. Without being limited thereto, the solvent may be selected from, for example, DMSO, DMF, or NMP.

[0025] The "antisolvent" is a solvent that shows low solubility or is insoluble in the target compound, and can be used to precipitate the target compound by adding the antisolvent to a solution in which the compound is dissolved. Therefore, in the method for preparing crystalline form A according to the present invention, the compound can be crystallized by selecting and adding an appropriate antisolvent in consideration of the type of solvent in which the compound of Chemical Formula 1 is dissolved and the solubility of the compound therein, and the crystals produced can have an X-ray powder diffraction pattern and an endothermic peak.

[0026] In one embodiment of the present invention, the anti-solvent may be, but is not limited to, ethanol or MTBE.

[0027] In one embodiment of the present invention, the weight of the solvent may be 2 or more times the weight of the compound of Chemical Formula 1. Since it is sufficient to dissolve the compound of Chemical Formula 1, the maximum amount of the solvent used is not limited, but in consideration of reaction economics, the weight ratio of the solvent to the compound of Chemical Formula 1 is preferably in the range of 2 to 10 times.

[0028] The amount of the poor solvent used for crystallization is determined depending on the amount of the solvent used. In one specific example of the present invention, the weight of the poor solvent may be two or more times the weight of the solvent. There is no upper limit to the amount of the poor solvent used, but the weight ratio of the poor solvent to the solvent is preferably in the range of 2 to 15 times.

[0029] In the preparation method of crystalline form A of the compound of Formula 1, a filtration or drying step may be further carried out to obtain a product with high purity by removing foreign matter, excess solvent, etc.

[0030] The present invention also provides a pharmaceutical composition comprising crystalline form A of the compound of formula 1 and a pharma- ceutically acceptable carrier.

[0031] In relation to the pharmaceutical composition containing crystalline form A of the compound of Chemical Formula 1 according to the present invention, the following uses of the compound of Chemical Formula 1 have been revealed:

[0032] According to WO2009-025478, the compound of Chemical Formula 1 is known to have preventive or therapeutic effects on cell necrosis and related diseases. According to WO2009-025478, the cell necrosis and related diseases include acute and chronic liver diseases (e.g., hepatitis, liver fibrosis, liver cirrhosis), neurodegenerative diseases (e.g., dementia, Parkinson's disease, Huntington's disease), ischemic heart disease, reperfusion injury (Korean Patent Registration No. 10-1325272), ischemic stroke or ischemic injury, pancreatitis, bacterial / viral sepsis, diabetes or diabetic complications, diabetic vascular disease (these diabetes are particularly caused by pancreatic cell-destroying substances and are mediated by viruses, hyperglycemia, fatty acids, diet, toxins, streptozotocin, etc.), necrotizing proctitis, and the like. proctitis), cystic fibrosis, rheumatoid arthritis, degenerative arthritis, nephropathy, bacterial infections, viral infections (e.g., HIV), multiple sclerosis, leukemia, lymphoma, neonatal respiratory distress syndrome, asphyxiation, tuberculosis, endometriosis, vascular weakness, psoriasis, frostbite, complications of steroid treatment, gangrene, tenderness, hemoglobinuria, burns, hyperpyrexia, Crohn's disease, celiac disease, compartment syndrome, spinal cord injury, glomerulonephritis, muscular dystrophy, inherited metabolic disorders, mycoplasmosis, anthrax, Andersen's disease, congenital mitochondrial diseases, phenylketonuria, placental infarction, syphilis, and aseptic necrosis. In addition, the cell necrosis and associated diseases caused by drugs and toxic substances are selected from the group consisting of necrosis associated with exposure to and / or administration and / or self-administration of alcoholism and cocaine, drugs (e.g., paracetamol, antibiotics, anticancer drugs, adriamycin, puromycin, bleomycin, NSAIDs, cyclosporine, chemical toxins (e.g., carbon tetrachloride, cyanide, methanol, ethylene glycol), poison gases, pesticides, heavy metals (e.g., lead, mercury, cadmium), and radiation / UV exposure damage and associated cell necrosis.

[0033] In addition, the compound of Formula 1 is also expected to have preventive, therapeutic and ameliorative effects on cell necrosis and related diseases, including acute and chronic kidney disease, traumatic brain injury, neurodegenerative disease Lugelig's disease, necrotizing colitis, viral infection (e.g., SARS-CoV), skin diseases including psoriasis and allergic dermatitis, and organ preservation / organ transplantation (see Korean Patent Registration Nos. 10-1098583 and 10-1941004).

[0034] In addition, the pharmaceutical composition containing the compound of Chemical Formula 1 has the function of regulating intracellular calcium and can improve endoplasmic reticulum (ER) stress and mitochondrial dysfunction caused by abnormal intracellular calcium levels. Therefore, the pharmaceutical composition containing the compound of Chemical Formula 1 is expected to show preventive or therapeutic effects and improve related diseases. The related diseases are as follows.

[0035] Acute lung injury syndrome / acute lung disease, pneumonia, tuberculosis, asthma, pulmonary arterial hypertension, chronic obstruction pulmonary disease, idiopathic pulmonary fibrosis, and chronic inflammatory pulmonary disease, including cystic fibrosis (see Mitochondrial dysfunction in fibrotic diseases. Cell Death Discov. 2020 Sep 5;6:80. Mitochondrial dysfunction in lung aging and diseases. Eur Respir Rev. 2020 Oct 15;29(157):200165. See Korean Patent No. 10-1636563)

[0036] Demyelination and demyelinating diseases including amyotrophic lateral sclerosis (ALS), hypertension including pulmonary arterial hypertension, stroke, prion disease, epilepsy, ataxia, migraine, cognitive decline, seizures, tremors, psychiatric disorders (e.g., depression) (see Neuronal and glial calcium signaling in Alzheimer's disease. Cell Calcium. Oct-Nov 2003;34(4-5):385-97. Mitochondrial disorders: challenges in diagnosis & treatment. Indian J Med Res. 2015 Jan;141(1):13-26.)

[0037] Insulin resistance, hyperlipidemia, atherosclerosis, inflammatory bowel disease (IBD) including Crohn's disease and ulcerative colitis, various cancers and cancer metastasis (see Reticulum stress and oxidative stress in cell fate decision and human disease. Antioxid Redox Signal. 2014 Jul 20;21(3):396-413.)

[0038] Vision-related diseases (e.g., retinitis pigmentosa, optic neuropathy, cataract, glaucoma), anemia, cholestasis, hypoparathyroidism, pancytopenia, pancreatic disorders, lactic acidosis, lactacidemia, hearing impairment, short stature, intestinal obstruction, cardiac conduction defect, cardiomyopathy, endometriosis, infertility, premature menopause.Mitochondrial diseases: the contribution of organelle stress responses to pathology.Nat Rev Mol Cell Biol.2018 Feb;19(2):77-92.Seminars in medicine of the Beth Israel Hospital,Boston.Mitochondrial DNA and disease.N Engl J Med.1995 Sep 7;333(10):638-44.Mitochondrial injury and dysfunction in hypertension-induced cardiac damage.Eur Heart J.2014 Dec 7;35(46):3258-3266.)

[0039] Muscular dystrophies, including limb girdle / Becker muscular dystrophy (LGMD / BMD) and Duchenne muscular dystrophy (DMD) (Duchenne muscular dystrophy is associated with the inhibition of calcium uniport in mitochondria and an increased sensitivity of the organelles to the calcium-induced permeability transition. Biochim Biophys Acta Mol Basis Dis. 2020 May 1; 1866(5): 165674.)

[0040] Aging and age-related diseases (Interrelation between ROS and Ca2+ in aging and age-related diseases. Redox Biology. 2020;6:101678.)

[0041] According to WO2009-025478, the pharmaceutical composition containing the compound of formula 1 not only exhibits liver protection and liver function improving effects, but also exhibits preventive or therapeutic effects on acute and chronic liver diseases such as chronic liver diseases such as fatty liver, liver fibrosis and liver cirrhosis, and hepatitis caused by viruses or drugs. As a result, complications of liver diseases such as portal hypertension can be prevented or treated, but are not limited to these. In particular, the pharmaceutical composition according to the present invention is also effective in treating or preventing liver diseases selected from liver transplantation, alcoholic or non-alcoholic fatty liver (see Korean Patent Registration No. 10-2006247), liver fibrosis, liver cirrhosis, and hepatitis caused by viruses or drugs, and is effective for alcoholic acute and chronic liver diseases. The composition according to the present invention is also effective in treating or preventing fatty liver caused by fatty liver or acute and chronic liver diseases caused by fatty liver.

[0042] According to Korean Patent Registration No. 10-1852304, the compound of Chemical Formula 1 can improve the differentiation efficiency and maturity of stem cell-derived cardiomyocytes by culturing stem cells.

[0043] Therefore, the present invention also provides a composition for inducing differentiation of stem cells into cardiomyocytes, comprising crystalline form A of the compound of Chemical Formula 1.

[0044] In addition, according to WO2016-072692, the compound of Chemical Formula 1 can also be used for the prevention and treatment of mucositis.

[0045] Thus, the present invention provides a use of a pharmaceutical composition comprising crystalline form A of the compound of formula 1 and a pharma- ceutical acceptable carrier for the treatment of the recited diseases, and a method for preventing or treating the recited diseases, comprising administering the pharmaceutical composition to a subject in need thereof. As used herein, "treatment" means stopping or delaying the progression of a disease when used on an object showing symptoms of disease, and "prevention" means stopping or delaying the onset of disease when used on an object not showing symptoms of disease but at high risk.

[0046] In the present invention, the "pharmaceutical composition" may contain the compound of the present invention together with a pharma- ceutically acceptable carrier, if necessary.

[0047] The compound of formula 1 according to the present invention can be administered in various oral and parenteral dosage forms in clinical administration, and when formulated, it is prepared using a diluent or excipient such as a commonly used filler, extender, binder, wetting agent, disintegrant, surfactant, etc.

[0048] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, lozenges, etc., and such solid preparations are prepared by mixing one or more compounds of the present invention with at least one or more excipients, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, liquids, emulsions, or syrups, and may contain various excipients, such as wetting agents, sweeteners, flavorings, preservatives, etc., in addition to water and liquid paraffin, which are frequently used simple diluents.

[0049] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, etc. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. can be used. As bases for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, gelatin, etc. can be used.

[0050] In addition, the effective dosage of the compound of formula 1 of the present invention to the human body may vary depending on the age, weight, sex, administration form, health condition and disease level of the patient, and is generally about 0.001 to 100 mg / kg / day, preferably 0.01 to 35 mg / kg / day. Based on an adult patient weighing 70 kg, the dosage is generally 0.07 to 7000 mg / day, preferably 0.7 to 2500 mg / day, and may be administered once or several times a day at regular intervals according to the judgment of a doctor or pharmacist. Effect of the Invention

[0051] The crystalline form A of the compound of Chemical Formula 1 according to the present invention is advantageous for formulation since it does not denature even under harsh conditions for a long period of time, has low sorption, and does not change in crystal form even when subjected to pressure or pulverization, compared with amorphous forms and other crystalline forms. The crystalline form itself is also very stable, making it useful for long-term storage. [Brief description of the drawings]

[0052] [Figure 1] FIG. 1 shows the results of X-ray powder diffraction pattern (XRPD) analysis of unpurified P9. [Diagram 2] FIG. 2 shows the results of differential scanning calorimetry (DSC) of P9 before purification. [Diagram 3] FIG. 3 shows the results of thermal gravimetric analysis (TGA) of unpurified P9. [Figure 4]FIG. 4 shows the X-ray powder diffraction pattern (XRPD) analysis of crystalline Form IV. [Diagram 5] FIG. 5 shows the TGA / DSC overlay results for crystalline Form IV. [Figure 6] FIG. 6 shows the XRPD results measured after TGA testing of crystalline form IV. [Figure 7] FIG. 7 shows the TGA / DSC overlay results for crystalline Form IV. [Figure 8] FIG. 8 shows the X-ray powder diffraction pattern (XRPD) analysis of crystalline form V. [Figure 9] FIG. 9 shows the TGA / DSC overlay results of crystalline form V. [Figure 10] FIG. 10 shows the XRPD results measured after TGA testing of crystalline form V. [Figure 11] FIG. 11 shows the X-ray powder diffraction pattern (XRPD) analysis of crystalline Form VIII. [Figure 12] FIG. 12 shows the TGA / DSC overlay results for crystalline Form VIII. [Figure 13] FIG. 13 shows the XRPD results measured after TGA testing of crystalline form VIII. [Figure 14] FIG. 14 shows the X-ray powder diffraction pattern (XRPD) analysis of crystalline form XII. [Figure 15] FIG. 15 shows the TGA / DSC overlay results of crystalline Form XII. [Figure 16] FIG. 16 shows the X-ray powder diffraction pattern (XRPD) analysis of crystalline form XI. [Figure 17] FIG. 17 shows the TGA / DSC overlay results for crystalline form XI. [Figure 18] FIG. 18 shows the XRPD results measured after TGA testing of crystalline form XI. [Figure 19] FIG. 19 shows a schematic diagram of small-scale recrystallization tests (Tests 1 to 3) of the compound of Chemical Formula 1. [Figure 20]FIG. 20 shows the X-ray powder diffraction pattern (XRPD) analysis result of intermediate form I, which corresponds to the intermediate stage of the conversion process from crystalline form V to crystalline form A (test 4). [Figure 21] FIG. 21 shows the X-ray powder diffraction pattern (XRPD) analysis result of intermediate form II, which corresponds to the intermediate conversion process from crystalline form V to crystalline form A (test 5). [Figure 22] FIG. 22 shows the X-ray powder diffraction pattern (XRPD) analysis of the compound of Formula 1 recrystallized from different DMSO / EtOH ratios. [Figure 23] FIG. 23 shows the X-ray powder diffraction pattern (XRPD) analysis of compound of Formula 1 recrystallized in different DMSO / EtOH ratios (1:2, 1:3, 1:4, 1:5, 1:7) at 50° C. [Figure 24] FIG. 24 shows the XRPD analysis results of the compound of Formula 1 slurried in a DMSO / EtOH mixed solvent at various temperatures. [Diagram 25] FIG. 25 shows XRPD results for each recrystallization step of the compound of Chemical Formula 1; [Figure 26] FIG. 26 shows the HPLC analysis results of the recrystallized product of the compound of Chemical Formula 1. [Figure 27] FIG. 27 shows photographs of the compound of formula 1 before and after recrystallization. [Figure 28] FIG. 28 shows a photograph of crystalline form A produced by a seedless method; [Figure 29] FIG. 29 shows the XRPD analysis of Form A produced by the seedless method; [Diagram 30] FIG. 30 shows the HPLC chromatogram of crystalline form A produced using a seedless method. [Diagram 31] FIG. 31 shows a polarized microscope image of crystalline form A produced by the seedless method; [Diagram 32] FIG. 32 shows the TGA / DSC results of crystalline form A produced by the seedless method. [Diagram 33] FIG. 33 shows the particle size distribution of Form A produced by the seedless method. [Diagram 34] FIG. 34 shows the XRPD analysis of crystalline form A produced by the final process; [Diagram 35] FIG. 35 shows a polarized light microscope (PLM) image of crystalline form A produced by the final production method; [Diagram 36] FIG. 36 shows the TGA / DSC results of crystalline form A produced by the final production method. [Figure 37] FIG. 37 shows the particle size distribution (PSD) of crystalline form A produced by the final manufacturing method. [Figure 38] FIG. 38 shows the results of isothermal moisture sorption / desorption analysis for crystalline form A. [Figure 39] FIG. 39 shows the calcium concentration regulating effect of the compound of Chemical Formula 1 under tBHP treatment conditions. [Diagram 40] FIG. 40 shows imaging results related to the regulating effect of the compound of Chemical Formula 1 on intramitochondrial calcium concentration in the presence of additional treatment with tBHP, thapsigargin, and CaCl 2 . [Diagram 41] FIG. 41 shows the intramitochondrial reactive oxygen scavenging effect of the compound of Chemical Formula 1 under tBHP treatment conditions. [Diagram 42] FIG. 42 shows imaging results related to the intramitochondrial superoxide scavenging effect of the compound of Chemical Formula 1 under tBHP treatment conditions. [Diagram 43] FIG. 43 shows the intracellular nitric oxide scavenging effect of the compound of Chemical Formula 1 under SIN-1 treatment conditions. [Diagram 44] FIG. 44 shows the improving effect of total time when the compound of Chemical Formula 1 was treated in a MPTP-induced Parkinson's disease mouse model. [Diagram 45] FIG. 45 shows the effect of improving T-turn when the compound of Chemical Formula 1 is treated in a MPTP-induced Parkinson's disease mouse model. [Figure 46] FIG. 46 shows the grip strength improving effect of the compound of Chemical Formula 1 in a mouse model of MPTP-induced Parkinson's disease. [Figure 47]FIG. 47 shows the effect of restoring TH levels in the striatum upon treatment with the compound of Chemical Formula 1 in a mouse model of MPTP-induced Parkinson's disease. [Figure 48] FIG. 48 shows the effect of restoring TH levels in the substantia nigra upon treatment with the compound of formula 1 in a mouse model of MPTP-induced Parkinson's disease. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] The advantages and features of the present invention, and the methods for achieving them, will become clearer with reference to the following detailed examples. However, the present invention is not limited to the following examples, and may be embodied in various different forms. The present examples are provided to fully explain the scope of the present invention and to fully inform those skilled in the art of the present invention, and the present invention is only defined by the scope of the claims. EXAMPLES

[0054] Abbreviation The following abbreviations are used in this application: TIFF2024515630000004.tif82153

[0055] Equipment, standards and methodologies used 1. X-ray Powder Diffraction (XRPD) X-ray diffraction patterns were recorded using a Bruker D8 Advance powder diffractometer with Cu K-alpha X-ray (l=1.54179 Å) radiation (40 kV / 40 mA) while rotating the sample from 3° to 40°. Sample rotation speed = 15 RPM Scanning rate=18.5° / min 2.Differential scanning calorimetry (DSC) Using a TA DSC Q2000 and a Discovery DSC-2500, approximately 1 mg of a sample was placed in a hermetic aluminum pan with a pinhole and heated from 25°C to 300°C at a rate of 10°C / min. 3.Thermal gravimetric analysis (TGA) Using a TGA Q50 or TA Q5000, approximately 4 mg of a sample was placed in an open platinum pan and measured by heating from 30°C to 300°C at a rate of 10°C / min. 4. Particle size distribution analyzer (PSD) The particles were measured using a Sympatec HELOS particles size analyzer in a RODOS dispersing system at a pressure of 0.5 bar. 5. Polarized Light Microscopy (PLM) A 5 megapixel CCD Nikon LV100POL microscope with a 20x physical lens was used. 6. Isothermal water sorption / desorption study (DVS cycle)

[0056] 10 mg of sample was placed in a mesh stainless steel basket using the SMS DVS Advantage 1. The entire experimental cycle consisted of two scans (sorption and desorption) at constant temperature (25 °C and 10% RH intervals ranging from 40 to 90% (60 to 360 min for each humidity level).

[0057] Example 1: Preparation of 5-[(1,1-dioxido-4-thiomorpholinyl)methyl]-2-phenyl-N-(tetrahydro-2H-pyran-4-yl)-1H-indol-7-amine

[0058] [Reaction Scheme 1] [ka]

[0059] The method for producing the compound of Chemical Formula 1 disclosed in WO2009-025478 is shown in Reaction Scheme 1.

[0060] P9 produced by reaction 1 contains a large amount of impurities and foreign matter. The compound of formula 1 has very low solubility, so purification is a very important process. In order to remove foreign matter and color from P9 obtained for early research, it was dissolved in DMSO, filtered, and the purified compound obtained by adding EtOH as a poor solvent was called the compound of formula 1.

[0061] Figure 1 and Table 2 show the X-ray powder diffraction pattern (XRPD) analysis results of P9 before purification. Figure 2 shows the differential scanning calorimetry (DSC) results of P9 before purification, and Figure 3 shows the thermal gravimetric analysis (TGA) results of P9 before purification.

[0062] Subsequent research has confirmed that P9 is crystalline form V. It showed a mass loss of 11.4% below 160°C and a melting point of 271°C.

[0063] [Table 2]

[0064] In order to reduce or remove the residual DMSO content, color, impurities, etc. of the primarily purified material (P9), the solid obtained in the secondary purification step of heating / slurrying with EtOH was named the compound of formula 1, and depending on the temperature, concentration, and time during this process, IV, V, VIII, XI, XII, or other crystal forms were also identified.

[0065] In order to mass-produce high-purity, stable clinical substances in a GMP facility, it is very important to establish standardized, optimal purification and most stable crystal form production conditions. Therefore, we decided to start purification and polycrystalline form research using the compound of formula 1 produced for research purposes.

[0066] Example 2: Solvent Screening for Purification In order to find a suitable solvent for removing foreign matter, decolorizing and obtaining a high-purity compound of formula 1 with the most stable crystal form, the solubility of the compound of formula 1 was screened. Specifically, the solubility and purity of the compound of formula 1 in various solvents were analyzed using the supernatant at 25°C and 50°C.

[0067] [Table 3]

[0068] As can be seen from Table 3, the solubility of the compound of formula 1 is very low in many solvents, and the solvents that showed a solubility of 40 mg / mL or more were DMF, NMP, and DMSO. However, DMF, NMP, and DMSO also have high solubility for impurities, and we thought that recrystallization studies using poor solvents would be suitable for purification studies. As poor solvents, MeOH, EtOH, IPA, and EtOAc, which have low amounts of total related substances (TRS), were considered as priority.

[0069] Based on the above results, the purification and polycrystalline form research was carried out, and it was found that the compound of Formula 1 has various crystal structures. This process is described in Example 3.

[0070] Example 3: Preparation of crystalline forms 3-1: Crystal Form IV - Slurry Method (DMF Solvate) 600 mg of the compound of formula 1 was added to 4 mL of DMF and stirred at 25°C for 1 day. The supernatant was removed using an ultracentrifuge, and the remaining solid was dried in vacuum to obtain crystals. This was named crystalline form IV of the compound of formula 1, and was analyzed by XRPD, DSC / TGA, and1 H NMR was measured.

[0071] [Table 4]

[0072] FIG. 4 and Table 4 show the X-ray powder diffraction pattern (XRPD) analysis of crystalline Form IV.

[0073] The crystalline form IV is characterized by an X-ray powder diffraction pattern having four or more diffraction peaks at 2[θ] values ​​selected from 8.52±0.2, 17.06±0.2, 21.12±0.2, 21.76±0.2, 24.18±0.2, 24.35±0.2 and 28.27±0.2.

[0074] FIG. 5 shows the TGA / DSC overlay results for crystalline Form IV.

[0075] In addition, 1 The H NMR results are as follows: 1 H NMR (400MHz, DMSO-d6)δppm 1.41-1.54(m, 1H), 2.06(brd, J=11.29Hz, 2H), 2.74(s, 3H, DMF), 2.89(s, 7H), 3.09(brd, J=4.77Hz, 4H), 3.46-3.56(m, 2H), 3.59-3.71(m, 3H), 3.90-4.00(m , 2H), 5.35(d, J=7.78Hz, 1H), 6.32(s, 1H), 6.76(s, 2H), 7.27-7.35(m, 1H), 7. 48(t, J=7.65Hz, 2H), 7.80(d, J=7.28Hz, 2H), 7.96(s, 1H, DMF), 10.94(s, 1H).

[0076] XRPD and DSC / TGA, 1 Considering the H NMR results, the crystalline form IV is a DMF solvate (compound of formula 1:DMSO=1:1).

[0077] In addition, Figure 6 shows the XRPD results measured after the TGA test of crystalline form IV (heated to 200°C and then cooled). As can be seen from the XRPD results in Figure 6, when crystalline form IV was heated to 200°C and cooled, DMF was removed and another crystalline form was observed, which was named crystalline form XII (hereinafter, renamed as "Pattern A" or "crystalline form A").

[0078] 3-2: Crystal form IV (DMF solvate) - Recrystallization 200 mg of the compound of formula 1 was added to 0.4 mL of DMF and heated at 50° C. The resulting suspension was filtered at 50° C. The resulting solution was cooled to 0° C. at a rate of 0.5° C. / min, and the resulting solid was vacuum dried to obtain a crystalline form. As a result of TGA and DSC analysis of the resulting crystalline form, as shown in FIG. 7, the TGA / DSC overlay results of crystalline form IV, which is the same as that of FIG. 5, were observed, and it was confirmed that the crystalline form was crystalline form IV (compound of formula 1:DMSO=1:1).

[0079] 3-3: Crystal form V (metastable) - Solvent / antisolvent 600 mg of the compound of formula 1 was added to 2 mL of DMSO, and the resulting suspension was centrifuged to obtain a supernatant (1.5 mL). EtOH (10.5 mL) was added slowly at room temperature for 1 hour (stirred at 700 RPM) in an amount of 7 times that of DMSO. The solid obtained by centrifuging was dried in vacuum to obtain crystals. This was named crystalline form V of the compound of formula 1, and was analyzed by XRPD, DSC / TGA, and 1 H NMR was measured.

[0080] [Table 5]

[0081] FIG. 8 and Table 5 show the X-ray powder diffraction pattern (XRPD) analysis of crystalline form V.

[0082] The crystalline form V is characterized by an X-ray powder diffraction pattern having four or more diffraction peaks at 2[θ] values ​​selected from 6.82±0.2, 13.84±0.2, 19.08±0.2, 19.66±0.2, 20.51±0.2, 22.84±0.2, 23.78±0.2, and 38.69±0.2.

[0083] FIG. 9 shows the TGA / DSC overlay results of crystalline form V.

[0084] In addition, 1 The H NMR results are as follows: 1 H NMR (400MHz, CHLOROFORM-d)δppm 1.22-1.34(m, 1H), 1.56-1.72(m, 4H), 2.16(brd, J=12.88Hz, 2H), 2.64(s, 3H), 3.05(brd, J=9.26Hz, 8H), 3.55-3.81(m, 6H), 4.10(brd, J=11.63Hz, 2H ), 6.49(s, 1H), 6.77(d, J=1.50Hz, 1H), 7.02(s, 1H), 7.28(s, 1H), 7.32-7. 39(m, 1H), 7.47(t, J=7.63Hz, 2H), 7.73(brd, J=7.38Hz, 2H), 8.68(brs, 1H)

[0085] Crystal form V is 1 The H NMR and TGA / DSC data showed the presence of DMSO, but after heating to 200°C, removing the DMSO, and cooling, the crystalline form V continued to be maintained, indicating that it is not a DMSO solvate (Figure 10). Figure 10 shows the XRPD results of crystalline form V after TGA testing (heating to 200°C and cooling).

[0086] 3-4: Crystalline Form VIII (DMSO solvate) - Slurry method 50 mg of the compound of formula 1 was stirred in 1 mL of DMSO at room temperature for 4 weeks in a slurry state. The solid obtained by using a centrifuge was dried in vacuum to obtain crystals. This was named crystalline form VIII of the compound of formula 1 and was characterized by XRPD, DSC / TGA and 1H NMR was measured.

[0087] [Table 6-1] [Table 6-2]

[0088] FIG. 11 and Table 6 show the X-ray powder diffraction pattern (XRPD) analysis of crystalline Form VIII.

[0089] The crystalline form VIII is characterized by an X-ray powder diffraction pattern having four or more diffraction peaks at 2[θ] values ​​selected from 6.88±0.2, 13.75±0.2, 16.17±0.2, 17.93±0.2, 20.18±0.2, 22.82±0.2, 25.84±0.2, 26.41±0.2 and 29.68±0.2.

[0090] FIG. 12 shows the TGA / DSC overlay results for crystalline Form VIII.

[0091] In addition, 1 The H NMR results are as follows: 1 H NMR (400MHz, CHLOROFORM-d) δppm 1.58-1.77(m, 4H), 2.16(brd, J=11.54Hz, 2H), 2.58-2.70(m, 7H, DMSO peak), 2.93-3.14(m, 8H), 3.55-3.78(m, 5H), 4.10(dt, J=11.80,3.26Hz, 2H), 6.47(s, 1H), 6.76(d, J=2.01Hz) , 1H), 7.00(s, 1H), 7.28(s, 1H), 7.31-7.38(m, 1H), 7.46(t, J=7.65Hz, 2H), 7.70-7.82(m, 2H), 8.99(brs, 1H).

[0092] Crystalline Form VIII is 1From the H NMR and TGA / DSC data, it was found to be a DMSO solvate (compound of formula 1:DMSO=1:1), and from the TGA / XRPD, it was found that DMSO was removed from the DMSO solvate upon cooling after heating to 200°C, and another crystalline form was observed, which was confirmed to be crystalline form XII (Figure 13). Figure 13 shows the XRPD result measured after TGA test (heating to 200°C and cooling) of crystalline form VIII.

[0093] 3-5: Crystal Form XII - Slurry Method 100 mg of the compound of formula 1 having crystalline form V obtained in Example 3-3 was added to 1 mL of EtOH and stirred at room temperature for 3 days. The solid was filtered using a 0.45 μm PTFE (Polytetrafluoroethylene) filter and dried at 50° C. to obtain crystals. This was named as crystalline form XII of the compound of formula 1 (hereinafter, re-named as "Pattern A" or "crystalline form A"), and XRPD and DSC were measured.

[0094] [Table 7-1] [Table 7-2]

[0095] FIG. 14 and Table 7 show the X-ray powder diffraction pattern (XRPD) analysis of crystalline Form XII.

[0096] The crystalline form XII is characterized by an X-ray powder diffraction pattern having four or more diffraction peaks at 2[θ] values ​​selected from 7.64±0.2, 12.32±0.2, 12.62±0.2, 15.26±0.2, 17.32±0.2, 19.18±0.2, 19.61±0.2, 19.95±0.2, 20.60±0.2, 21.12±0.2, 22.94±0.2, 24.11±0.2 and 28.15±0.2.

[0097] FIG. 15 shows the TGA / DSC overlay results of crystalline Form XII.

[0098] From the above results, it was found that the quasicrystalline form V was transformed into the more stable crystalline form XII by slurrying.

[0099] 3-6: Crystal Form XI (NMP solvate) - Slurry method 500 mg of the compound of formula 1 was added to 1 mL of NMP and stirred at 25°C for 3 hours. The supernatant was removed using an ultracentrifuge, and the remaining solid was dried in vacuum to obtain crystals. This was named crystalline form XI of the compound of formula 1, and was analyzed by XRPD, DSC / TGA, and 1 H NMR was measured.

[0100] [Table 8-1] [Table 8-2]

[0101] FIG. 16 and Table 8 show the X-ray powder diffraction pattern (XRPD) analysis of crystalline form XI.

[0102] The crystalline form XI is characterized by an X-ray powder diffraction pattern having four or more diffraction peaks at 2[θ] values ​​selected from 16.89±0.2, 17.30±0.2, 20.37±0.2, 20.95±0.2, 21.18±0.2, 21.34±0.2, 23.16±0.2, 24.04±0.2 and 27.85±0.2.

[0103] FIG. 17 shows the TGA / DSC overlay results for crystalline form XI.

[0104] In addition, 1 The H NMR results are as follows: 1H NMR (400MHz, DMSO-d6)δppm 1.40-1.54(m, 2H), 1.84-1.96(m, 2H), 2.06(brd, J=11.54Hz, 2H), 2.13-2.23(m, 2H), 2.70(s, 3H, NMP- Me ), 2.89(brs, 4H), 3.08(brd, J=4.52Hz, 4H), 3.46-3.56(m, 2H), 3.59-3.72(m, 3H), 3.89-3.99(m, 2H), 5.35(d, J=7.5 3Hz, 1H), 6.32(s, 1H), 6.76(s, 2H), 7.28-7.35(m, 1H), 7.48(t, J=7.78Hz, 2H), 7.80(d, J=7.53Hz, 2H), 10.94(s, 1H).

[0105] XRPD, DSC / TGA and 1 Considering the H NMR results, the crystalline form XI was an NMP solvate (the compound of formula 1:NMP=1:1).

[0106] The TGA / XRPD results confirmed that the NMP was removed during re-cooling after heating to 200°C, resulting in the conversion to Form XII (Figure 18). Figure 18 shows the XRPD results after TGA testing of Form XI (heated to 200°C and then cooled).

[0107] Summary of Example 3

[0108] [Table 9]

[0109] Example 4: Stability study of five crystalline forms The five crystalline forms obtained in Example 3 were subjected to a stability test for 8 weeks under harsh conditions (40° C., relative humidity 75%).

[0110] Table 10 below shows the stability test results for the five crystalline forms.

[0111] [Table 10]

[0112] As can be seen from Table 10, all five crystal forms showed little change in purity under harsh conditions. Crystal forms IV, VIII and XI were 1 H NMR spectroscopy and DSC / TGA measurements confirmed that the solvates were DMF, DMSO and NMP, respectively, and after 8 weeks of follow-up observation under harsh conditions (40°C, 75% relative humidity), it was found that the crystal form was converted to crystal form XII, and crystal form V was found to be converted to crystal form XII in a metastable form, although not a solvate. Therefore, crystal form XII was determined to be the most stable crystal structure, and was named crystal form A or pattern A (crystal form XII = crystal form A or pattern A).

[0113] Example 5: Optimization Process I 5-1. Determining the initial concentration of the compound of formula 1 FIG. 19 shows a schematic diagram of small-scale recrystallization tests of the compound of formula 1 (Tests 1-3). As shown in FIG. 19, in order to determine the initial concentration of the compound of formula 1 in DMSO solvent, it was dissolved at concentrations of 100, 150, and 200 mg / mL, and then 7 times the amount of EtOH was added at 50° C. for 2 hours, followed by stirring at room temperature for 19 hours. The XRPDs of the solids (XRPD-1) produced immediately after the addition of EtOH, the solids (XRPD-2) produced after stirring at room temperature for 1 day, and the solids (XRPD-3) vacuum-dried at 50° C. were measured and the yields were calculated.

[0114] [Table 11]

[0115] As can be seen from Table 11, initial crystal form V was found to be produced at concentrations of 150 and 200 mg / mL, and it was observed that over time it was converted to crystal form A. Based on the test results, 200 mg / mL, which gave the highest yield, was selected as the initial crystallization concentration.

[0116] 5-2.Determining the ratio of DMSO / EtOH antisolvent To determine the ratio of EtOH, a poor solvent, to a 200 mg / mL DMSO solution of the compound of formula 1, 5-fold and 7-fold EtOH were added at 50°C for 2 hours, and then stirred at room temperature for 16 hours. The solid obtained by filtration was dried under vacuum at 50°C. XRPD was measured for each sample before and after drying, and the changes during the drying process were also confirmed, and the yield was calculated (Table 12). The solid obtained with 7-fold EtOH showed crystalline form V before drying with a yield of 84.15%, and an XRPD spectrum (test 4, intermediate form I) with a characteristic peak corresponding to the intermediate conversion process from crystalline form V to crystalline form A after drying (Figure 20). In contrast, the solid obtained with 5-fold EtOH showed crystalline form V before drying with a yield of 76.55%, and a new characteristic intermediate crystalline form (test 5, intermediate form II) different from the conversion process pattern from crystalline form V to crystalline form A after drying (Figure 21). Based on these results, DMSO:EtOH=1:7 was considered to be a better yield and recrystallization condition for crystalline form A than DMSO:EtOH=1:5.

[0117] [Table 12]

[0118] FIG. 20 and Table 13 show the XRPD analysis of intermediate Form I.

[0119] [Table 13]

[0120] FIG. 21 and Table 14 show the XRPD analysis of intermediate Form II.

[0121] [Table 14]

[0122] 100 mg of intermediate form I obtained above was placed in 1 mL of EtOH and stirred at room temperature for 3 days. The solid formed was dried at 50°C under vacuum using a 0.45 μm PTFE filter. XRPD analysis of the dried sample confirmed that it was crystalline form A (Table 15). From this result, it was found that crystalline form V is a metastable crystalline form, and when the time and conditions are sufficient, it will be converted to the more stable crystalline form A.

[0123] [Table 15]

[0124] In addition, when using a DMSO / EtOH mixed solvent, other patterns of crystal forms that are undergoing intermediate conversion during the crystallization process can appear depending on the conditions, making it necessary to establish a production process that would produce the most stable crystal form from the time recrystallization first begins.

[0125] 5-3. Optimization of dropwise addition time of antisolvent (EtOH) to DMSO solution Using the crystallization method used in Example 5-2, the dropwise addition time of EtOH, which is 7 times that of DMSO, was 24 and 35 hours (in test 7, 6.7% EtOH was added dropwise, and then 20% crystalline form A was added to the seed crystals) and the addition was slower, and the XRPD of the crystalline form obtained at each step was analyzed. Considering the results including the results of Example 5-1 (dropwise addition time 19 hours), it was found that the change in dropwise addition time and the use of seed crystals made it difficult to avoid the formation of initial crystalline form V, but it was possible to obtain pure crystalline form A without other crystalline forms after drying was completed.

[0126] [Table 16]

[0127] Example 6: Recrystallization of the compound of formula 1 in DMSO / MTBE solvent To a 150mg / mL DMSO solution of the compound of formula 1 at 50℃, 5x and 7x MTBE, which are poor solvents, were added over a period of 3 hours, and then stirred at room temperature for 18 hours. The solid obtained after filtration was vacuum dried at 50℃ for 18 hours. Here, the XRPD of each was measured before and after vacuum drying to confirm the changes during the drying process. The solid obtained with 7x MTBE had a yield of 64.73% and showed the XRPD of crystalline form V both before and after drying, and even with 5x, it showed the same crystalline form V both before and after drying with a yield of 59.27%.

[0128] [Table 17]

[0129] Example 7: Recrystallization of the compound of Formula 1 in NMP / EtOH solvent Seven times the amount of EtOH, a poor solvent for a 150 mg / mL NMP solution of the compound of formula 1, was added at 25°C for 24-35 hours, and then stirred at room temperature for 22-33 hours. The solid obtained by filtration was vacuum dried at 50°C for 2 hours. Here, the XRPD of each was measured before and after vacuum drying, and changes during the drying process were also confirmed. It was obtained with a yield of 65-74%, and the formation of crystalline form XI was observed early even with the combination of NMP / EtOH.

[0130] [Table 18]

[0131] Analysis of the results of Examples 5 to 7 Looking at the results of Examples 5, 6, and 7, it was confirmed that colored crystals appeared under various possible mixed solvent conditions, and that there were also intermediate crystal forms that were not stable. Considering the shape, yield, and residual solvent toxicity, the combination of DMSO / EtOH was found to be the best candidate, and further optimization was carried out.

[0132] [Table 19]

[0133] Example 8: Optimization of DMSO / EtOH Solvent Ratio 8-1: Optimization experiment of DMSO / EtOH antisolvent ratio 200 mg of the compound of formula 1 was added to 1 mL of DMSO, heated to 60°C, and filtered through a 0.45 μm PTFE membrane. The resulting solution was slowly added with 10 mL of EtOH at 50°C for 35 hours. The crystal forms of the initial solids obtained when EtOH was added at 1.5, 2, 3, 5, 7, and 10 times the amount of DMSO were measured and analyzed. Figure 22 shows the X-ray powder diffraction pattern (XRPD) analysis results of the compound of formula 1 recrystallized at different DMSO / EtOH ratios. As can be seen from Figure 22, it was confirmed that when the ratio was 1:3 or less, the solvate crystalline form VIII was formed, and when the ratio was 1:5 or more, the crystalline form A was formed. In order to find a more accurate ratio, the optimum slurry conditions were carried out in the next step.

[0134] 8-2: Optimization experiment of DMSO / EtOH mixed solvent ratio at 50℃ Five containers were filled with 1 mL of DMSO / EtOH mixed solvent (1:2, 1:3, 1:4, 1:5, 1:7) and 200 mg of the compound of formula 1, and stirred at 50°C for one day. The crystalline forms of the solids obtained using a centrifuge were measured and analyzed by XRPD. Figure 23 shows the X-ray powder diffraction pattern (XRPD) analysis results of the compound of formula 1 slurried with different DMSO / EtOH ratios (1:2, 1:3, 1:4, 1:5, 1:7) at 50°C. As can be seen from Figure 23, the crystal form A was stably produced at 50°C when the DMSO / EtOH ratios were 1:4, 1:5, and 1:7, and the optimal ratio was determined to be DMSO:EtOH=1:4.

[0135] Example 9: Equilibrium solubility and supersaturation at different temperatures in DMSO / EtOH mixed solvents 200 mg of the compound of formula 1 was added to 1 mL of two kinds of DMSO / EtOH (1:4, 1:5) mixed solutions to prepare 200 mg / mL turbid solutions, which were then heated and maintained at 50°C, 60°C, and 75°C, respectively, and stirred for 6 hours. After 6 hours, the supernatant of the turbid solution was analyzed by HPLC to determine the equilibrium solubility, and the XRPD analysis results of the solid were also shown in Table 20 and Figure 24. Figure 24 shows the XRPD analysis results of the compound of formula 1 slurried in DMSO / EtOH mixed solvent at various temperatures. It was confirmed that the solid present under all conditions of various temperatures (50°C, 60°C, 75°C) and mixed solvents (1:4, 1:5) had crystalline form A. The solubility in a 1:4 DMSO / EtOH mixed solution was 31 mg / mL at 75°C, and since a recrystallization method using a 1:4 DMSO / EtOH mixed solvent from the beginning was not appropriate, we decided to consider the supersaturation method.

[0136] [Table 20]

[0137] 200mg of the compound of formula 1 was dissolved in 1mL of DMSO, heated at 75°C, and 4 times EtOH was added to obtain a transparent supersaturated solution, with a concentration of 40mg / mL and a degree of supersaturation of 1.3 times (40 / 31) with respect to the mixed solvent. Based on the results of Example 5-1, it was determined that a high yield could be maintained if the solubility of the compound of formula 1 was at least 40mg / mL, aiming for 200mg of the compound of formula 1 / 1mL of DMSO, and therefore the supersaturation method was used.

[0138] Example 10: Method for preparing crystalline form A by the seeded supersaturation / recrystallization method 3g of the compound of formula 1 was dissolved in 15mL of DMSO at 75℃, and foreign matter was removed using a 0.45μm PTFE membrane filter. While maintaining the temperature at 75℃, 60mL of EtOH was added (this confirmed that a clear solution was maintained), and 2% seed crystals (crystal form A) were added, and suspension began to be observed. While maintaining the temperature at 75℃, an additional 45mL of EtOH was slowly added for 15 hours. All crystallization processes were carried out under stirring at 250RPM. After adding all the EtOH, the mixture was cooled to 5℃ at a rate of 10℃ / h, and the resulting solid was collected using a 0.45μm PTFE membrane filter and then vacuum dried at 50℃ for 21 hours. As shown in Table 21, the recrystallization of the compound of formula 1 using seed crystals resulted in a yield of 74.58% and a purity of 99.95%.

[0139] [Table 21]

[0140] Figure 25 shows XRPD results for each recrystallization step of the compound of Chemical Formula 1, Figure 26 shows HPLC analysis results for the recrystallized product of the compound of Chemical Formula 1, and Figure 27 shows photographs of the compound of Chemical Formula 1 before and after recrystallization. Referring to Figure 27, it was found that the off-white to white impurities were removed from the compound of Chemical Formula 1 after recrystallization.

[0141] Example 11: Method for preparing crystalline form A by a supersaturation / recrystallization method without using seed crystals 2g of the compound of formula 1 was dissolved in 10mL of DMSO at 60℃, and foreign matter was removed using a 0.45μm PTFE membrane filter. While maintaining the temperature at 60℃, 40mL of EtOH was added and stirred for 30 minutes (turned into a suspension). [The XRPD of the solid obtained by immediately cooling a part of the suspension to room temperature was measured and confirmed to be crystal form V - Figure 29 (rapid cooling) solid separated out at room temperature] The temperature was then slowly increased to 75℃ for 12 minutes and maintained for 10 minutes (continued suspension). The temperature was slowly decreased to 50℃ for 1.5 hours, and XRPD-1 of the solid formed at 60℃ was measured. While maintaining the temperature at 50℃, 30mL of EtOH was slowly added for 15 hours. All crystallization processes were carried out under stirring at 250RPM. The solid formed by cooling to 5°C at a rate of 10°C / hr was separated using a 0.45μm PTFE membrane filter and measured for XRPD-2, and then dried under vacuum at 50°C for 17 hours and measured for XRPD-3 (yield 70.35%, purity 99.95%). From the results shown in Figure 29, the XRPDs for each stage were all crystalline form A.

[0142] [Table 22]

[0143] Table 22 summarizes the recrystallization results of compound of formula 1 (2g). As a result of analyzing the XRPD of the crystal form at each stage, when seed crystals were not used, the yield was about 5% lower than that of Example 10, and a laminated type crystal form A with a slightly greenish color was obtained. Figure 28 shows a photo of crystal form A produced by a method without using seed crystals, Figure 29 shows the XRPD analysis result of crystal form A produced by a method without using seed crystals, Figure 30 shows the HPLC chromatogram result of crystal form A produced by a method without using seed crystals, Figure 31 shows a polarizing microscope image of crystal form A produced by a method without using seed crystals, Figure 32 shows the TGA / DSC result of crystal form A produced by a method without using seed crystals, and Figure 33 shows the particle size distribution of crystal form A produced by a method without using seed crystals.

[0144] In addition, if the temperature is rapidly decreased during the recrystallization process, the formed crystalline form V may act as a nucleus during the crystallization process, resulting in the formation of undesired crystalline forms. Therefore, it was confirmed that monitoring of temperature control is also very important (Figure 29 - rapid cooling).

[0145] Comparing Examples 10 and 11, it was found that the use of seeds showed a higher yield, and it was decided to adjust the total amount of EtOH used as the poor solvent.

[0146] Example 12: Adjustment of the amount of antisolvent used in Example 10 (total DMSO:EtOH = 1:5) 1g of the compound of formula 1 was dissolved in 5mL of DMSO at 60℃, and foreign matter was removed using a 0.45μm PTFE membrane filter. After raising the temperature to 75℃ and maintaining it for 30 minutes, 20mL of EtOH was added, and 2% seed crystals (crystal form A) were added, and suspension began to be observed. The temperature was slowly lowered to 50℃ for 1.5 hours, and then 5mL of EtOH was slowly added while maintaining the temperature for 5 hours, and stirred at the same temperature for another 5 hours. All crystallization processes were carried out under 300RPM stirring. The mixture was cooled to 5℃ at a rate of 10℃ / hr, and further stirred at the same temperature for another 5 hours. The resulting solid was collected using a 0.45μm PTFE membrane filter, and then vacuum dried at 50℃ for 20 hours to obtain crystal form A with a yield of 72.12%.

[0147] Example 13: Adjustment of the amount of antisolvent used in Example 10 (total DMSO:EtOH = 1:6) 1g of the compound of formula 1 was dissolved in 5mL of DMSO at 60℃, and foreign matter was removed using a 0.45μm PTFE membrane filter. After raising the temperature to 75℃ and maintaining it for 30 minutes, 20mL of EtOH was added, and 2% seed crystals (crystal form A) were added, and suspension began to be observed. The temperature was slowly lowered at 50℃ for 1.5 hours, and then 10mL of EtOH was slowly added for 5 hours while maintaining the temperature, and stirred at the same temperature for another 5 hours. All crystallization processes were carried out under 300RPM stirring. The mixture was cooled to 5℃ at a rate of 10℃ / hr, and further stirred at the same temperature for another 5 hours. The resulting solid was collected using a 0.45μm PTFE membrane filter, and then vacuum dried at 50℃ for 20 hours, obtaining crystal form A with a yield of 73.28%.

[0148] Considering the results of Examples 10, 12 and 13, Example 10 using a total DMSO:EtOH=1:7 was found to be the best, and in order to confirm reproducibility, the final step was carried out using 30 g of the compound of Formula 1 according to the method of Example 10 using seed crystals.

[0149] Example 14: Final preparation method of crystalline form A (30 g scale) 30g of the compound of formula 1 was dissolved in 150mL of DMSO at 75℃, and foreign matter was removed using a 0.45μm PTFE membrane filter. While maintaining the temperature at 75℃, 600mL of EtOH was added (this confirmed that the solution was maintained at this state), and 2% seed crystals (crystal form A) were added, and suspension began to be observed. While maintaining the temperature at 75℃, an additional 450mL of EtOH was slowly added for 15 hours. All crystallization processes were carried out under 250RPM stirring. After adding all the EtOH, the mixture was cooled to 5℃ at a rate of 10℃ / h, and the resulting solid was obtained using a 0.45μm PTFE membrane filter, and then vacuum dried at 50℃ for 15 hours. As shown in Table 22, the recrystallization of the compound of formula 1 using the seed crystals gave a yield of 70.35% and a purity of 99.96%.

[0150] Using the obtained crystalline form A, various analyses such as XRPD, TSA / DSC, PLM, HPLC, PSM and residual solvent were performed, and the results are shown in Tables 23 to 24 and Figures 34 to 38. Figure 34 shows the XRPD analysis result of crystalline form A produced by the final production method, Figure 35 shows a polarizing microscope (PLM) image of crystalline form A produced by the final production method, Figure 36 shows the TGA / DSC result of crystalline form A produced by the final production method, and Figure 37 shows the particle size distribution (PSD) of crystalline form A produced by the final production method.

[0151] [Table 23]

[0152] The crystalline form A is characterized by an X-ray powder diffraction pattern containing four or more 2θ values ​​(±0.2) selected from 7.64, 12.32, 12.62, 15.26, 17.32, 19.18, 19.61, 19.95, 20.60, 21.12, 22.94, 24.11, 28.15 at a temperature of 25±5° C.

[0153] [Table 24]

[0154] NMR data of pattern A 1 H NMR (400MHz, DMSO-d6) δppm 1.40-1.54(m, 2H), 2.06(brd, J=11.04Hz, 2H), 2.88(brs, 4H), 3.09(brd, J=4.52Hz, 4H), 3.44-3.55(m, 2H), 3.60-3.70(m, 3H), 3.95(brd, J=11 .29Hz, 2H), 5.35(d, J=7.78Hz, 1H), 6.32(s, 1H)6.76(s, 2H), 7.24-7.36(m, 1H), 7.48(t, J=7.78Hz, 2H), 7.80(d, J=7.28Hz, 2H), 10.93(s, 1H).

[0155] Isothermal water sorption / desorption study (DVS cycle) Using the SMS DVS Advantage 1, 10 mg of form A was placed in a mesh stainless steel basket. The entire experimental cycle consisted of two scans (sorption and desorption) at a constant temperature (25°C and 40-90% RH intervals (60-360 min for each humidity level). The weight gain of about 0.7% and the absorption and loss relative to the humidity level indicate a non-sorbing sample. Figure 38 shows the isothermal curve (% weight change vs. RH%) and the kinetic curve of % weight change vs. time and % relative humidity for form A. After the DVS procedure, the XRPD analysis showed no change in the crystal form, which appears to be the most stable form.

[0156] Example 15: Evaluation of Formulation Suitability of Crystalline Form A 15-1. Pressure test of crystal form A 100 mg of Form A powder was filled into the die cavity and compressed with the upper punch at pressures of 2, 3, and 4 MPa for 3 minutes each. After crushing each tablet, XRPD analysis showed no change, indicating that this is the most stable form.

[0157] 15-2.Grinding test of crystal form A 1) Dry grinding 30 mg of Form A was manually ground in a mortar for 5 minutes. XRPD analysis showed no change. 2) Wet grinding with EtOH 30 mg of Form A and 40 mL of EtOH were placed in a mortar and manually ground for 5 minutes. XRPD analysis showed no change. 3) Wet grinding with H2O 30 mg of Form A and 40 mL of H2O were placed in a mortar and manually ground for 5 minutes. XRPD analysis showed no change.

[0158] Example 16: Homeostatic effect of cytosolic and mitochondrial calcium concentration in cardiomyocytes To confirm the effect of maintaining calcium concentration in cardiomyocytes, H9C2 cells were plated in a 96-well plate at 1.5 × 10 4The cells were then dispensed at 1000x1000 cells / well and cultured for 24 hours. Cytoplasmic calcium concentration was measured using FLIPR Calcium 6 assay kit (Molecular devices; #R8190). The cells were treated with probenecid and calcium specific dye according to the manufacturer's experimental method, and after 1.75 hours, the compound of formula 1 was treated with the final concentrations of 0.1, 0.03, 0.01, 0.003, and 0.001 μM for 0.25 hours. To stimulate the cells, tBHP was treated with the final concentration of 150 μM, and the calcium concentration in the cytoplasm was measured in real time every 30 seconds. Figure 39 shows the calcium concentration regulation effect of the compound of formula 1 under tBHP treatment conditions. The ΔF / F(Max-Min) value on the vertical axis is the maximum minus the minimum fluorescence value of the calcium specific dye generated after tBHP treatment, and the greater the increase in cytoplasmic calcium concentration, the greater the value. The horizontal axis indicates the concentration of the compound of formula 1, and VC is the control group treated with only the carrier. The calcium concentration of cells increased most significantly upon tBHP treatment. When treated with 0.01 μM of the compound of formula 1, the abnormally increased cytoplasmic calcium concentration caused by tBHP treatment was reduced, and when treated with 0.1 μM, the cytoplasmic calcium concentration was at a normal level.

[0159] To confirm the effect of suppressing the increase in mitochondrial calcium concentration in cardiomyocytes by cell imaging, H9C2 cells were cultured in a 35-mm culture dish at 1.5 × 10 4The cells were then cultured for 24 hours. Rhod-2 was used for imaging of calcium concentration in mitochondria. Rhod-2 was treated with MitoTracker Green (200nM) and Hoechst33342 (2drops / ml) for 50 minutes, washed twice with KRB buffer solution, and then the buffer solution was added and cultured for another 30 minutes. The cells were treated with the compound of formula 1 at a final concentration of 10μM for 0.25 hours, treated with tBHP at a final concentration of 150μM for 30 minutes, treated with thapsigargin 1.2μM for 10 minutes, and then treated with CaCl2 2mM, and the fluorescence emitted by calcium present in the mitochondria of living cells was observed through imaging. Figure 40 shows the imaging results related to the effect of the compound of formula 1 in suppressing the increase in calcium in mitochondria under the condition of further treatment with tBHP, thapsigargin, and CaCl2. As a result of the experiment, it was observed through the decrease in Rhod-2 fluorescence that the compound of Chemical Formula 1 effectively suppressed the increase in intramitochondrial calcium concentration that occurred under the condition of further treatment with tBHP, thapsigargin and CaCl2, and it was confirmed through this that the compound of Chemical Formula 1 strongly suppresses the increase in intramitochondrial calcium.

[0160] The experimental results are expected to show preventive or therapeutic effects and ameliorative effects on related diseases associated with the mechanism of improving ER stress and mitochondrial dysfunction through the function of intracellular calcium regulation.

[0161] Example 17: Effect of removing reactive oxygen species from mitochondria of cardiomyocytes To confirm the effect of removing reactive oxygen species generated in mitochondria in cardiomyocytes, H9C2 cells were plated in a 96-well plate at 1.5 × 10 4The cells were dispensed at 100x100x10 cells / well and cultured for 24 hours. Measurement of mitochondrial specific reactive oxygen species was performed using a fluorescence measurement method using dihydrorhodamine-123 (DHR-123). After treating the cells with DHR-123 for 0.5 hours, the cells were then treated with the compound of formula 1 at final concentrations of 0.0001, 0.001, 0.003, 0.03, 0.1, 0.3, 1, 10, and 30 μM for 0.25 hours. After adding tBHP to the cells at a final concentration of 400 μM, intracellular reactive oxygen species were measured in real time every 3 minutes using fluorescence intensity. Figure 41 shows the intramitochondrial reactive oxygen scavenging effect of the compound of formula 1 under tBHP treatment conditions. The horizontal axis shows the concentration of the compound of formula 1 on a log scale, and the vertical axis shows the total amount of intramitochondrial reactive oxygen species generated 2 hours after tBHP treatment. tBHP caused a rapid increase in intramitochondrial reactive oxygen species, but treatment with the compound of formula 1 showed a concentration-dependent suppression effect on the generation of reactive oxygen species.

[0162] To confirm the reactive oxygen scavenging effect of the compound of formula 1 by cell imaging, H9C2 cells were cultured in a 35 mm confocal microscope culture dish at 1.5 × 10 4 The cells were then cultured for 24 hours. MitoSOX was used to measure the amount of superoxide produced in mitochondria, a type of reactive oxygen species. TMIntracellular fluorescence imaging was performed using 1μM Mitochondrial Superoxide Indicator (Red Mitochondrial Superoxide Indicator). The cells were pretreated with the compound of formula 1 at a concentration of 1μM for 0.25 hours, and tBHP was added to the cells at a final concentration of 50μM and treated for 1 hour. The cells were replaced with a medium without phenol red, and treated with HOECHST (0.5 drop / ml), MitoTracker Green (150nM), and MitoSOX (2μM) for 30 minutes, and intracellular fluorescence was observed using imaging. Figure 42 shows the imaging results related to the mitochondrial superoxide scavenging effect of the compound of formula 1 under tBHP treatment conditions. As a result of the experiment, it was observed by the decrease in MitoSOX fluorescence that the compound of formula 1 effectively scavenges reactive oxygen species including intramitochondrial superoxide, and it was confirmed that the compound of formula 1 acts as an antioxidant that strongly scavenges reactive oxygen species generated in mitochondria.

[0163] The effect of removing intramitochondrial reactive oxygen species as seen from these experimental results is associated with cell necrosis, and is therefore expected to have preventive, therapeutic and ameliorative effects on diseases associated with cell damage and necrosis.

[0164] Example 18: Scavenging effect of nitric oxide produced by macrophages and astrocytes To confirm the effect of nitric oxide scavenging in macrophages, RAW264.7 cells were plated in a 96-well plate at 1 × 10 5 The cells were dispensed at 1000 cells / well and cultured for 24 hours. Nitric oxide was measured using a DAX-J2 TM Fluorescence measurement was performed using PON Green dye. TMAfter treatment with PON Green dye for 1 hour, the compound of formula 1 and the positive control ascorbic acid were treated for 5 minutes at final concentrations of 10 and 30 μM, respectively. SIN-1 was then treated at a final concentration of 200 μM, and the amount of intracellularly generated nitric oxide was measured in real time every 2 minutes using fluorescence intensity. To confirm the nitric oxide scavenging effect in astrocytes, C8-D1A cells were cultured at 6 × 10 in a 96-well plate. 4 The cells were dispensed at 100x the cell / well and cultured for 24 hours, and the experiment was carried out under the same conditions. Figure 43 shows the intracellular nitric oxide scavenging effect of the compound of formula 1 under SIN-1 treatment conditions. The left side shows the results in macrophages, and the right side shows the results in astrocytes. The horizontal axis shows the concentration of the compound of formula 1 and ascorbic acid, and VC is the control group treated with only the carrier, and the intracellular nitric oxide concentration increases the most when treated with SIN-1. The vertical axis shows the total amount of nitric oxide generated 1 hour after SIN-1 treatment. Intracellular nitric oxide increased rapidly with SIN-1, but the generation of nitric oxide was inhibited concentration-dependently by treatment with the compound of formula 1, showing a more excellent inhibitory effect than the positive control group, ascorbic acid.

[0165] By confirming the nitric oxide removal effect of macrophages and astrocytes involved in inflammation, the results of this experiment are expected to show preventive, therapeutic and ameliorative effects on various inflammation-based diseases such as pancreatitis, rheumatoid arthritis, degenerative arthritis, bacterial and viral infections, glomerulonephritis, acute and chronic kidney disease, necrotizing colitis, pneumonia, hepatitis and mucositis, as well as aging and aging-related brain diseases such as Alzheimer's disease, Parkinson's disease, Lu Gehrig's disease, limb-girdle / Becker muscular dystrophy and Duchenne muscular dystrophy.

[0166] Example 19: Improved behavioral performance in MPTP-induced Parkinson's disease mouse model MPTP (1-Methy-4-phenyl-1,2,3,6-tetrahydropyridine) is a neurotoxin. +MPTP is a pro-drug of (1-methy-4-phenylpyridium), which destroys dopaminergic neurons when administered and induces symptoms of Parkinson's disease. C57BL / 6 mice were administered 20mg / kg MPTP for a total of four times at two-hour intervals to induce behavioral disorders. Starting two days after MPTP administration, the compound of formula 1 was intraperitoneally administered once a day at low, medium and high doses for six days. A pole test was performed to confirm the behavioral ability improving effect of the compound of formula 1. Figure 44 shows the improving effect of total time when the compound of formula 1 is treated in an MPTP-induced Parkinson's disease mouse model, and Figure 45 shows the improving effect of T-turn when the compound of formula 1 is treated in an MPTP-induced Parkinson's disease mouse model. The total time and T-turn values ​​of the MPTP-administered group were significantly increased compared to the control group. It was confirmed that the total time and T-turn values ​​of the group administered with the compound of formula 1 were recovered in a concentration-dependent manner.

[0167] In addition, a grip strength test was conducted to confirm the behavioral ability improving effect of the compound of Chemical Formula 1. Figure 46 shows the grip strength improving effect of the compound of Chemical Formula 1 on the MPTP-induced Parkinson's disease mouse model. The grip strength value of the MPTP-administered group was significantly decreased compared to the control group. It was confirmed that the grip strength value of the group administered with the compound of Chemical Formula 1 was recovered in a concentration-dependent manner.

[0168] Example 20: Protective effect on dopaminergic neurons in MPTP-induced Parkinson's disease mouse model C57BL / 6 mice were administered 20 mg / kg MPTP at 2-hour intervals for a total of four times to induce behavioral disorders. Starting two days after MPTP administration, the compound of formula 1 was intraperitoneally administered once a day for six days at low, medium and high doses. On the sixth day after administration of the compound of formula 1, the mice were sacrificed, and frozen sections of brain tissue were prepared and stained with TH (Tyrosine hydroxylase). Figure 47 shows the effect of recovering TH levels in the striatum when the compound of formula 1 is treated in an MPTP-induced Parkinson's disease mouse model. The TH level in the striatum of the MPTP-administered group was significantly reduced compared to the control group. In this case, the TH level in the group administered with the compound of formula 1 was concentration-dependently restored, and was significantly increased at medium and high doses. Figure 48 shows the effect of recovering TH levels in the substantia nigra when the compound of formula 1 is treated in an MPTP-induced Parkinson's disease mouse model. The TH level in the substantia nigra of the MPTP-administered group was significantly reduced compared to the control group. In this case, in the group administered with the compound of Chemical Formula 1, the TH level was restored in a concentration-dependent manner, and a significant difference was observed at high doses.

Claims

1. Crystal A of a compound represented by the following formula 1: 【Chemistry 1】 The crystal is characterized by being identified by an X-ray powder diffraction pattern having four or more diffraction peaks at 2[θ] values ​​selected from 7.64±0.2, 12.32±0.2, 12.62±0.2, 15.26±0.2, 17.32±0.2, 19.18±0.2, 19.61±0.2, 19.95±0.2, 20.60±0.2, 21.12±0.2, 22.94±0.2, 24.11±0.2 and 28.15±0.

2.

2. The crystal according to claim 1, characterized in that the X-ray powder diffraction pattern is characterized by an X-ray powder diffraction pattern having four or more diffraction peaks at 2[θ] values ​​selected from 7.64±0.2, 15.26±0.2, 17.32±0.2, 19.18±0.2, 21.12±0.2 and 22.94±0.

2.

3. The crystal of claim 1, characterized in that it has an X-ray powder diffraction pattern with peak positions matching those listed in the table below: 【Table 1】

4. Stirring a solution containing a compound of Formula 1 below and a solvent selected from DMSO, DMF, and NMP under heating for 1 to 24 hours; The method for producing crystalline A of the compound of formula 1 according to claim 1, comprising: cooling the solution to 0-25° C. for 1-72 hours while stirring to grow crystals. 【Chemistry 2】

5. The method for producing crystalline A of the compound of formula 1 according to claim 4, wherein the heating is carried out at an elevated temperature of 50 to 120°C.

6. The method for producing crystalline A of compound of formula 1 according to claim 4, wherein the cooling is carried out at a rate of 5 to 20° C. / h.

7. a step of adding a poor solvent to a solution obtained by dissolving a compound of the following formula 1 in a solvent, and crystallizing the solution; The method for producing crystalline A of the compound of formula 1 according to claim 1, comprising: cooling the solution to 0-25°C for 6-48 hours while stirring to grow crystals. 【Chemistry 3】

8. The method for preparing the crystalline A of the compound of formula 1 according to claim 7, further comprising adding the crystalline A of the compound of formula 1 as a seed crystal during the addition of the poor solvent.

9. The method for preparing crystalline A of the compound of formula 1 according to claim 7, wherein the solvent is selected from DMSO, DMF, and NMP.

10. The method for preparing crystalline A of the compound of formula 1 according to claim 7, wherein the anti-solvent is ethanol or MTBE.

11. The method for preparing Crystal A of the compound of formula 1 according to claim 7, wherein the weight of the solvent is at least twice as much as the weight of the compound of formula 1.

12. The method for preparing the crystalline A of the compound of formula 1 according to claim 7, wherein the weight of the anti-solvent is at least twice as much as the weight of the solvent.

13. A pharmaceutical composition comprising a crystalline form of the compound of formula 1 according to any one of claims 1 to 3, and a pharma- ceutically acceptable carrier.

14. A pharmaceutical composition for preventing or treating cell necrosis and associated diseases selected from the following group, comprising a crystalline form of the compound of formula 1 according to any one of claims 1 to 3 and a pharma- ceutical acceptable carrier: acute or chronic liver disease, dementia, Parkinson's disease, Huntington's disease, ischemic disease, diabetes, pancreatitis, bacterial or viral sepsis, necrotizing proctitis, and the like. proctitis), cystic fibrosis, rheumatoid arthritis, degenerative arthritis, nephropathy, bacterial infection, viral infection, multiple sclerosis, leukemia, lymphoma, neonatal respiratory distress syndrome, asphyxiation, tuberculosis, endometriosis, vascular weakness, psoriasis, frostbite, steroid treatment complications, gangrene, tenderness, hemoglobinuria, burns, hyperthermia, Crohn's disease, celiac disease, compartment syndrome, spinal cord injury, glomerulonephritis, muscular dystrophy, mycoplasmosis, anthrax, Andersen's disease, congenital mitochondrial disease, phenytoin, ketonuria, placental infarction, syphilis and avascular necrosis; and necrosis associated with alcoholism and exposure to or administration or self-administration of cocaine, antibiotics, anti-cancer drugs, nonsteroidal anti-inflammatory drugs (NSAIDs), cyclosporine, chemical toxins, toxic gases, pesticides, heavy metals, injury from radiation or UV exposure and associated cellular necrosis, acute and chronic kidney disease, traumatic brain injury, Lou Gehrig's disease, necrotizing colitis, colis), viral infections, skin diseases including psoriasis and allergic dermatitis, organ preservation / transplantation, acute lung injury syndrome / acute lung disease, pneumonia, tuberculosis, asthma, pulmonary arterial hypertension, chronic obstructive pulmonary disease, inflammatory lung diseases including idiopathic pulmonary fibrosis and cystic pulmonary fibrosis, demyelination and amyotrophic lateral sclerosis (ALS;Demyelinating diseases including amyotrophic lateral sclerosis, hypertension including pulmonary arterial hypertension, stroke, prion diseases, epilepsy, ataxia, migraine, cognitive decline, seizures, tremors, psychiatric disorders, insulin resistance, hyperlipidemia, atherosclerosis, inflammatory bowel disease (IBD) including Crohn's disease and ulcerative colitis, cancer and cancer metastasis, vision-related diseases including retinitis pigmentosa, optic neuropathy, cataracts and glaucoma, anemia, cholestasis, hypoparathyroidism, pancytopenia, pancreatic disorders, lactic acidosis, acidosis, lactacidemia, hearing loss, short stature, intestinal obstruction, cardiac conduction defect, cardiomyopathy, endometriosis, infertility, premature menopause, muscular dystrophic diseases including limb girdle / Becker muscular dystrophy (LGMD / BMD) and Duchenne muscular dystrophy (DMD), aging and aging-related diseases, and mucositis.

15. A composition for inducing differentiation of stem cells into cardiomyocytes, comprising crystalline A of the compound of chemical formula 1 according to any one of claims 1 to 3.

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