New crystal form of magnesium serine and preparation method thereof

Abstract

The invention relates to a novel crystal form of serine magnesium represented by a chemical formula 1 and a preparation method thereof. The crystal form of the present invention has excellent thermal stability and moisture stability, can be stably stored without decomposition or crystal form transition even during long-term storage, and thus can be advantageously used as a pharmaceutical raw material for pharmaceutical products. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a new crystal form of magnesium serine and a preparation method thereof. Background Art

[0002] Serine (L-serine), traditionally considered a non-essential amino acid, is used to synthesize glutathione, a precursor to glycine and cysteine. Serine is known to mediate neuroprotection in the mammalian central nervous system and, when consumed at appropriate concentrations over a long period of time, reduces age-related oxidative stress and inflammation in the hypothalamus. Furthermore, functional foods containing high concentrations of serine have recently been reported to exhibit antioxidant effects.

[0003] Magnesium (Mg) is an essential mineral used as a cofactor in the human body, participating in over 300 biochemical reactions required to maintain homeostasis. Furthermore, it not only induces the production of nucleic acids and participates in ATP reactions, but also has a wide range of different physiological functions, such as insulin secretion, muscle contraction, and regulation of intracellular calcium concentrations.

[0004] Magnesium intake is reportedly inadequate in Western populations, and inadequate magnesium intake is known to be associated with the development of hypertension, cardiovascular disease, and type 2 diabetes. Furthermore, magnesium intake is known to be effective for mild and moderate depression in adults. Clinical results have been reported: taking four 500mg magnesium chloride tablets daily, resulting in an intake of 248mg of elemental magnesium, significantly reduced symptoms of depression and anxiety, regardless of age, depression severity, or whether or not taking antidepressants.

[0005] Meanwhile, Korean Patent No. 10-1952443 (Patent Document 1) reports a compound represented by the following Formula 2, in which a magnesium atom is chelated with L-serine.

[0006] [Formula 2]

[0007]

[0008] The compound of Formula 2 has a structure in which two serine molecules are bound to a single magnesium atom via ionic bonds and coordination bonds, and each carboxylic acid group (COO - ) forms an ionic bond with the magnesium atom, and two amine groups (-NH2) form a coordination bond. The compound of the above formula 2 can be obtained by reacting L-serine with magnesium oxide (MgO) or magnesium hydride (MgH2), and water (H2O) or hydrogen (H2) is produced as a by-product, respectively.

[0009] Due to very high water absorption, the compound of Formula 2 exhibits decreased stability when stored at room temperature, including discoloration and denaturation. These issues require strict storage and formulation requirements, such as refrigeration, which makes its use in large-scale pharmaceutical production unfavorable.

[0010] [Prior art literature]

[0011] [Patent Document]

[0012] (Patent Document 0001) Korean Patent No. 10-1952443

[0013] [Non-patent literature]

[0014] (Non-patent document 0001) Zhou X, Zhang H, He L, Wu X and Yin Y (2018) Long-Term L-Serine Administration Reduces Food Intake and Improves Oxidative Stress andSirt1 / NFkB Signaling in the Hypothalamus of Aging Mice. Front. Endocrinol. 9:476.

[0015] (Non-patent document 0002) Zhou X, He L, Zuo S, Zhang Y, Wan D, Long C et al., Serineprevented high-fat diet-induced oxidative stress by activating AMPK and epigenetically modulating the expression of glutathione synthesis-relatedgenes. Biochim Biophys Acta (2018) 1864:488-98.

[0016] (Non-patent document 0003) Metcalf JS, Dunlop RA, Powell JT, Banack SA, Cox PA. L-Serine: a naturally-occurring amino acid with therapeutic potential. Neurotox Res. (2018) 33: 213-21.

[0017] (Non-patent document 0004) Dunlop RA, Powell JT, Metcalf JS, Guillemin GJ, Cox PA. L-serine-mediated neuroprotection includes the upregulation of the ER stresschaperone protein disulfide isomerase (PDI). Neurotox Res. (2018) 33:113-22.

[0018] (Non-patent document 0005) Tarleton EK, Littenberg B, MacLean CD, Kennedy AG, Daley C (2017) Role of magnesium supplementation in the treatment of depression: Arandomized clinical trial. PLoS ONE 12(6):e0180067.

[0019] (Non-patent document 0006) Swaminathan R. Magnesium metabolism and its disorders. Clin. Biochem. Rev. 2003; 242: 47-66.

[0020] (Non-patent document 0007) Topf JM, Murray PT Hypermagnesemia and hypermagnesemia. Rev. Endocr. Metab. Disord. 2003; 42: 195-206.

[0021] (Non-patent document 0008) King DE, Mainous AG, 3rd, Geesey ME, Woolson R.F. Dietary magnesium and C-reactive protein levels. J. Am. Coll. Nutr. 2005; 243: 166-171.

[0022] (Non-patent document 0009) Song Y., Sesso HD, Manson JE, Cook NR, Buring JE, Liu S. Dietary magnesium intake and risk of incident hypertension among middle-aged and older US women in a 10-year follow-up study. Am.J. Cardiol. 2006; 9812:1616-1621.

[0023] (Non-patent document 0010) Song YQ, Manson JE, Cook NR, Albert CM, Buring J.E., Liu S. Dietary magnesium intake and risk of cardiovascular disease among women. Am. J. Cardiol. 2005; 968: 1135-1141.

[0024] (Non-patent document 0011) Song YQ, Manson JE, Buring JE, Liu S. Dietary magnesiumintake in relation to plasma insulin levels and risk of type 2 diabetes in women. Diabetes Care. 2004; 271: 59-65. Summary of the Invention

[0025] Technical issues

[0026] The purpose of the present invention is to provide a new crystal form of a magnesium serine compound and a preparation method thereof.

[0027] The object of the present invention is to provide a pharmaceutical composition for preventing or treating developmental disorders and / or neurodegenerative diseases, which comprises a crystalline form of a magnesium serine compound.

[0028] The object of the present invention is to provide a health functional food for preventing or improving developmental disorders and / or neurodegenerative diseases, which comprises a crystal form of a magnesium serine compound.

[0029] The object of the present invention is to provide a crystal form of a magnesium serine compound for use in preventing and / or treating developmental disorders and / or neurodegenerative diseases.

[0030] An object of the present invention is to provide a method for preventing or treating developmental disorders and / or neurodegenerative diseases, which comprises administering a crystalline form of a magnesium serine compound to an individual.

[0031] The present invention aims to provide a crystal form of a magnesium serine compound for use in preparing a medicament for preventing or treating developmental disorders and / or degenerative neurological diseases.

[0032] Solution to the problem

[0033] The present inventors have discovered that a new crystalline form of a magnesium serine compound not only exhibits excellent physical properties, stability, and low hygroscopicity, but also has thermodynamic stability. Therefore, it is suitable for use as a pharmaceutical raw material because it does not undergo discoloration, denaturation, or polymorphic transition caused by aging during storage, thereby completing the present invention.

[0034] Hereinafter, the present invention will be described in more detail.In the present specification, when a part is referred to as "comprising" a certain component, it means that it can further include other components, rather than excluding other components, unless otherwise specified.

[0035] Crystal form

[0036] The present invention provides a crystalline form of a magnesium serine compound represented by the following formula 1:

[0037] [Formula 1]

[0038]

[0039] The chemical name of the compound of Formula 1 is magnesium di-(2S)-2-amino-3-hydroxypropionate, and in the present invention, it is named "magnesium L-serine" or "magnesium serine".

[0040] The magnesium serine compound represented by Formula 1 of the present invention has a structure in which two L-serine molecules form an ionic bond and a coordinate bond with one magnesium (Mg) atom. Specifically, in the magnesium serine compound represented by Formula 1, the carboxyl groups of the two L-serine molecules are ionically bonded to the magnesium atom, while the hydroxyl group of one L-serine molecule and the amine group of the other L-serine molecule form a coordinate bond with the magnesium atom.

[0041] That is, the crystal form of the compound represented by Formula 1 is a crystalline solid compound composed of a structure in which two L-serine molecules form an ionic bond and a coordinate bond with one magnesium atom, as shown in the following structural formula.

[0042]

[0043] The crystalline form of the magnesium serine compound represented by Formula 1 of the present invention is a novel crystalline compound, which is a sufficiently robust crystalline form that is stable to heat and moisture. Moreover, the active ingredient does not decompose or undergo polymorphic transformation during production, formulation, storage, and distribution, and thus has stability, thereby facilitating drug production.

[0044] In an embodiment of the present invention, the crystalline form of the magnesium serine compound represented by Formula 1 may include diffraction peaks at diffraction angles (2θ±0.2°) of 13.53°, 16.00°, and 22.05° in an X-ray powder diffraction pattern; and three or more diffraction peaks at diffraction angles (2θ±0.2°) selected from 18.36°, 19.75°, 20.00°, 20.95°, 24.39°, and 28.37°.

[0045] In addition, in an embodiment of the present invention, in addition to the above-mentioned diffraction peaks, the crystalline form of the magnesium serine compound represented by Formula 1 may further include one or more diffraction peaks at diffraction angles (2θ±0.2°) selected from 18.89°, 25.73°, 26.39°, 29.55°, 30.07°, 32.39°, 33.51°, 33.99°, 34.42° and 40.36° in the X-ray powder diffraction pattern.

[0046] In addition, the crystal form of the magnesium serine compound represented by Formula 1 of the present invention may further include in the X-ray powder diffraction pattern Figure 4 The diffraction peaks are shown.

[0047] In an embodiment of the present invention, the crystal form of the magnesium serine compound represented by Formula 1 may have a maximum endothermic peak at 283±3° C. when analyzed by differential scanning calorimetry (DSC).

[0048] That is, the crystalline form of the present invention has excellent thermal stability with a thermal decomposition temperature of 283±3°C, and can be stably preserved without decomposition of the active ingredient even during long-term storage.

[0049] The present invention provides a magnesium serine compound, which is a crystal form of a magnesium serine compound in which two serine molecules are bound to one magnesium atom and includes diffraction peaks at diffraction angles (2θ±0.2°) of 13.53°, 16.00°, and 22.05° in an X-ray powder diffraction pattern.

[0050] In addition, in an embodiment of the present invention, the crystalline form of the magnesium serine compound in which two serine molecules are bound to one magnesium atom may further include three or more diffraction peaks at diffraction angles (2θ±0.2°) selected from 18.36°, 19.75°, 20.00°, 20.95°, 24.39° and 28.37° in the X-ray powder diffraction pattern.

[0051] In addition, in an embodiment of the present invention, the crystalline form of the magnesium serine compound in which two serine molecules are bound to one magnesium atom may further include one or more diffraction peaks at diffraction angles (2θ±0.2°) selected from 18.89°, 25.73°, 26.39°, 29.55°, 30.07°, 32.39°, 33.51°, 33.99°, 34.42° and 40.36° in the X-ray powder diffraction pattern.

[0052] In this case, the crystalline form of the magnesium serine compound according to the present invention may have a maximum endothermic peak at 283±3° C. when analyzed by differential scanning calorimetry (DSC).

[0053] In the present invention, the magnesium serine compound in the following crystal form may refer to the same crystal form as the magnesium serine compound represented by Formula 1: a crystal form of the magnesium serine compound in which two serine molecules are bound to one magnesium atom and including diffraction peaks at diffraction angles (2θ±0.2°) of 13.53°, 16.00°, and 22.05° in an X-ray powder diffraction pattern.

[0054] Method for preparing a crystalline form of a compound represented by Formula 1

[0055] As a method for preparing a crystalline form of a compound represented by Formula 1, the present invention provides a method for preparing a crystalline form of a magnesium serine compound, comprising:

[0056] a) mixing L-serine, magnesium (Mg) or a magnesium compound with a solvent and stirring the mixture; and

[0057] b) Filter the crystals produced after stirring.

[0058] In step a), magnesium (Mg) may be in the form of powder or particles. The magnesium compound may be a divalent compound of a stable form of magnesium, specifically one or more selected from magnesium methoxide (Mg(OCH3)2), magnesium ethoxide (Mg(OC2H5)2), magnesium chloride (MgCl2), magnesium bromide (MgBr2), magnesium carbonate (MgCO3), and magnesium citrate (C6H6O7Mg). More specifically, the magnesium compound may be magnesium methoxide or magnesium ethoxide. Commercially available products may be used as the magnesium or magnesium compound.

[0059] The solvent may be a polar solvent, specifically, an alcohol solvent having 1 to 9 carbon atoms, which may be at least one selected from methanol, ethanol, isopropanol, and tert-butanol. More specifically, it may be methanol or ethanol.

[0060] The stirring in step a) can be carried out at room temperature (1° C. to 30° C.), or can be carried out under reflux stirring in a temperature range of ±20° C. of the boiling point of the solvent used. In addition, the stirring in step a) can be vigorously carried out until L-serine and magnesium or the magnesium compound are completely dissolved in the solvent, and can be continued for 1 to 120 hours.

[0061] In step b), filtration may be performed at room temperature using a Buchner funnel, but is not limited thereto.

[0062] The preparation method of the present invention may further include the steps of washing and drying the crystals produced in step b) after the step of filtering the crystals.

[0063] The washing can be performed using a reaction solvent cooled to room temperature. In particular, as the reaction solvent, at least one selected from methanol, ethanol, isopropanol, and tert-butanol can be used, specifically methanol or ethanol can be used.

[0064] Drying can be performed at a temperature ranging from room temperature (1°C to 30°C) to 120°C.

[0065] Pharmaceutical composition or health functional food containing the crystal form of the compound represented by formula 1

[0066] In another embodiment, the present invention provides a pharmaceutical composition comprising a crystalline form of the compound represented by Formula 1 above.

[0067] In another embodiment, the present invention provides a pharmaceutical composition for preventing or treating a developmental disorder or a neurodegenerative disease, comprising a crystalline form of the compound represented by Formula 1 as an active ingredient.

[0068] In the present invention, the developmental disorder may be one or more diseases selected from autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD), intellectual disability, language disorder, movement disorder including motor developmental delay, tic disorder and Tourette syndrome.

[0069] In the present invention, the neurodegenerative disease may be one or more diseases selected from Alzheimer's disease, vascular dementia, mild cognitive impairment, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis and cerebellar degenerative diseases.

[0070] In the present invention, the pharmaceutical composition can be formulated into one or more preparations selected from powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, suppositories, eye drops and injections.

[0071] The content of the crystalline form as an active ingredient in the pharmaceutical composition of the present invention can be calculated taking into account the patient's age, weight, etc. Generally, based on the total weight of the pharmaceutical composition, 0.1 wt% to 80.0 wt% of the crystalline form can be included.

[0072] In addition, the pharmaceutical composition of the present invention can include pharmaceutically acceptable additives such as excipients, diluents, binders, disintegrants, glidants, pH regulators, antioxidants and solubilizers within the scope of not damaging the effect of the active ingredient. Examples of pharmaceutically acceptable additives that can be used to prepare the pharmaceutical composition of the present invention include microcrystalline cellulose, xylitol, erythritol, methylcellulose, polyvinyl pyrrolidone, starch, gum arabic, alginate, gelatin, lactose, glucose, sucrose, propyl hydroxybenzoate, cellulose, methyl hydroxybenzoate, magnesium stearate, talc, sorbitol, mannitol, maltitol, calcium phosphate, calcium silicate, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethylcellulose, hypromellose, mineral oil, etc., but are not limited thereto.

[0073] In addition, the pharmaceutical composition of the present invention can be formulated into one or more formulations selected from powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, suppositories, eye drops, and injections according to conventional formulation methods, but is not limited thereto. The present invention does not have any particular limitations on the above formulations.

[0074] In addition, when eye drops or injections are prepared using the pharmaceutical composition according to the present invention, water for injection can be used for preparation. The eye drops or injections may optionally contain isotonic agents, buffers, osmotic agents, etc. commonly used in the art.

[0075] In an embodiment, the present invention provides a health functional food for preventing or improving developmental disorders or neurodegenerative diseases, comprising a crystalline form of a magnesium serine compound represented by Formula 1. The functional health food can be effectively used to improve energy utilization, maintain nerve and muscle function, improve memory, and / or prevent or improve cognitive decline due to aging.

[0076] The terms "developmental disorder" and "degenerative neurological disease" may have the same meaning as described above.

[0077] The present invention provides a method for preventing or treating a developmental disorder or a neurodegenerative disease, comprising administering to an individual a crystalline form of a magnesium serine compound represented by Formula 1 or a pharmaceutical composition comprising the crystalline form.

[0078] The method for preventing or treating a developmental disorder or a neurodegenerative disease of the present invention may comprise administering the crystalline form of the present invention in a therapeutically effective amount.

[0079] In the present invention, "administering" means introducing a predetermined substance into an individual by an appropriate method.

[0080] In the present invention, "individual" means all animals including humans, such as rats, mice, livestock, etc., and specifically can be mammals including humans, but is not limited thereto.

[0081] In the present invention, "prevention" refers to all actions to inhibit or delay the onset of a disease by administering the crystalline form of the present invention.

[0082] In the present invention, "treatment" refers to all actions to improve or beneficially change the symptoms of a suspected or sick individual by administering the crystalline form of the present invention.

[0083] In the present invention, "therapeutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment without causing side effects, which can be determined by those skilled in the art based on the patient's sex, age, weight, health status, disease type, severity, drug activity, sensitivity to the drug, administration method, administration time, administration route, excretion rate, duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field. It is preferred to administer a specific therapeutically effective amount to a specific patient based on various factors including the type and extent of the response to be achieved, whether the specific composition contains other agents used as appropriate, the patient's age, weight, overall health status, sex and diet, administration time, administration route, excretion rate of the composition, duration of treatment, drugs used in combination or concurrently with the specific composition, and similar factors well known in the medical field.

[0084] The pharmaceutical composition and health functional food of the present invention can be prepared by conventional pharmaceutical compositions and conventional methods for preparing health functional foods in the art.

[0085] The present invention provides use of a crystal form of a magnesium serine compound for preventing or treating developmental disorders or neurodegenerative diseases.

[0086] The present invention provides use of a crystal form of a magnesium serine compound in preparing a medicament for preventing or treating developmental disorders or neurodegenerative diseases.

[0087] Matters mentioned in each of the present invention, namely the crystal form, preparation method, pharmaceutical composition, health functional food, treatment method and use, are equally applicable unless they conflict with each other.

[0088] Advantageous Effects of the Invention

[0089] The crystal form of the present invention does not decompose even when stored for a long time due to its excellent stability against heat and moisture, and the crystal form does not undergo polymorphic transformation, so it can be stably stored; therefore, its large-scale production is easy and economical, and it can be effectively used as a pharmaceutical raw material for drugs formulated into oral preparations, injectable preparations, eye drops, etc.

[0090] The crystalline form of the present invention not only has properties that are beneficial for drug production, but also exhibits a bioavailability comparable to that of water-soluble L-serine despite its low solubility; therefore, it can be effectively used to prevent, improve or treat developmental disorders or neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 This is a graph showing the crystal form of magnesium serine of Example 1. 13 C NMR peak (a) and theory based on three structures of the compound in which magnesium and serine are bound 13 C NMR peaks (b), (c), and (d).

[0092] Figure 2 1 is a diagram showing six structures of compounds in which six theoretically possible combinations of magnesium and serine are present.

[0093] Figure 3 This is the powder crystal structure of the crystallographic information file (CIF) of the magnesium serine crystal form of Example 1 analyzed using the CIF / PLATON program according to Experimental Example 1.

[0094] Figure 4 This is a diagram showing the X-ray powder diffraction pattern of the magnesium serine crystal form of Example 1.

[0095] Figure 5 This is a graph showing the TG / DSC-MS thermal analysis of crystalline magnesium serine of Example 1.

[0096] Figure 6 The results are shown by dissolving the magnesium serine crystal form of Example 1 in D2O solvent. 1 H-NMR spectrum (a) and 13 C-NMR spectrum (b).

[0097] Figure 7 Images showing properties of the compound of Formula 2 according to Experimental Example 7 (a) and properties of the crystal form of the present application (b).

[0098] Figure 8 This is a diagram for evaluating the pharmacological effects of Comparative Examples 1 and 2 according to Experimental Example 8 and the pharmacological effects of the crystal form of the present application. DETAILED DESCRIPTION

[0099] Hereinafter, the embodiments of the present invention will be described in more detail. However, it will be apparent to those skilled in the art that the following embodiments are intended to illustrate the present invention and the scope of the present invention is not limited to these embodiments.

[0100] <Example>

[0101] Example 1. Preparation of Crystalline Magnesium Serine

[0102] L-serine (10.53g) and methanol (50mL) are added to a round flask, and stirred at room temperature for 10 minutes under a nitrogen atmosphere.While the reaction solution is stirred at room temperature, magnesium (shavings / fragments) (1.21g) is added thereto, and the reaction solution is vigorously stirred for 90 hours.Filtered through a Buchner funnel, washed with methanol (10mL), and after being dried at 110 DEG C for 8 hours, the crystal formation (11.5g, 99% yield) of the serine magnesium compound represented by Formula 1 as a white crystalline solid is obtained.The analytical results of the crystal formation prepared in Example 1 are as follows.

[0103] [Table 1]

[0104] Test items result heavy metal 20ppm or less content 98.6％

[0105] Heavy metal test was carried out according to method 1 of the heavy metal test method as a general test method in the 12th edition of the Korean Pharmacopoeia. In the content test, the sample prepared in Example 1 of about 200 mg was accurately quantified and dissolved in 50 mL of water, to which was added about 2 mL of ammonia / ammonium chloride buffer (pH 10.7) and 3 to 4 drops of chrome black T indicator, followed by titration with 0.05 M-EDTA 2Na. The endpoint of the titration was determined to be the time when red turned blue and lasted for at least 30 seconds. In the same way, a blank test was performed and corrected, and the content was then calculated.

[0106] Example 2. Preparation of Crystalline Magnesium Serine

[0107] 85g of magnesium methoxide solution (Mg content is 8 weight % to 9 weight %) is added to a round flask, L-serine (10g) is added thereto, and the mixture is then refluxed and stirred for 4 hours.Then, the reaction solution is cooled to room temperature, filtered through a Buchner funnel, washed with the methanol of about 30g, and dried in vacuo at 60 DEG C for 12 hours or longer, to obtain the crystal formation (10.92g, 99% yield) of the magnesium serine compound represented by Formula 1 as a white crystalline solid. The obtained crystal formation is identified by PXRD, DSC and NMR, confirming substantially the same result as Example 1. The analytical results of the embodiment 2 prepared are as follows.

[0108] [Table 2]

[0109] Test items result pH 10.3 heavy metal 20ppm or less Moisture 0.1％ content 100.0％

[0110] The moisture and pH evaluations were performed in the same manner as in Experimental Example 7, and the heavy metal test and content test were performed in the same manner as in Example 1.

[0111] Experimental Example 3. Preparation of Crystalline Magnesium Serine

[0112] After adding methanol (150mL) in a round flask, L-serine (10g) and magnesium ethoxide (5.44g) were added thereto, and the mixture was refluxed and stirred for about 5 hours. Then, the reaction solution was cooled to room temperature, filtered through a Buchner funnel, washed with about 30g of methanol, and vacuum-dried at 60°C for 12 hours or longer to obtain a crystal form (11.03g, 100% yield) of the magnesium serine compound represented by Formula 1 as a white crystalline solid. The obtained crystal form was identified by PXRD, DSC and NMR, confirming substantially the same result as Example 1.

[0113] <Experimental Example>

[0114] Hereinafter, AST-011 (Comparative Example 2) was prepared in the same manner as described in Example 1.1 of Korean Patent No. 10-1952443.

[0115] Experimental Example 1. Analysis of crystal form

[0116] The crystal structure of the magnesium serine compound represented by Formula 1 of the present invention is identified using the structure determination of the powder diffraction (SDPD) method. The SDPD method requires high-quality powder XRD data. The initial structure candidate is identified by several operating steps such as baseline correction, peak search and data indexing, and the structure is then refined by Rietveld refinement, and finally the crystal structure is confirmed by verifying the refined structure. The SDPD method includes three methods: direct method, real space method and charge reversal method. In the present invention, the real space method is used for structural analysis, which utilizes actually measured PXRD data. The real space method involves confirming whether the obtained structure is consistent with the physicochemical theoretical value by matching the measured value with the theoretically derived PXRD simulation value using the least squares method, and comparing the actually measured PXRD data with the theoretical PXRD data of the structure with respect to the base value.

[0117] To obtain the structure of the ground state candidate, solid-state NMR was used to analyze how the Mg atom and two L-serine molecules are coordinated according to the crystal structure. Figure 1 As shown, the theory of three types of compounds in which magnesium and serine bind according to the binding structure 13The C NMR peaks are different. This model looks similar to solid-state NMR, as it is the expected spectrum for nitrogen or oxygen coordination. Generally speaking, simulations of liquid samples do not differ, as they cannot be used unless they are strongly coordinated models due to their high degrees of freedom.

[0118] When measuring the SS of the magnesium serine crystal form of the present invention 13 C-NMR (solid-state 13 C-NMR) Figure 1 a), it was confirmed that the pentagonal-hexagonal coordination compound ( Figure 1 b) Similar results were obtained. Considering the presence of the same amount of hexagonal-hexagonal compounds ( Figure 1 c) and pentagon-pentagon compounds ( Figure 1 d), three peaks will appear around 60ppm.

[0119] According to these experimental results, pentagon-hexagon compounds can be selected as ground state candidates. The number of such compounds can be six structures, e.g. Figure 2 shown.

[0120] In order to confirm the magnesium serine of the present invention among the six candidate crystal structures, the structures were subsequently constructed by the SDPD method and analyzed accordingly.

[0121] The PXRD data were processed using the EXPO2014 program by background setting and peak searching, followed by indexing and space group determination using the built-in N-TREOR90 algorithm. The Le Bail method was then used to obtain the integrated intensity, followed by the reciprocal space method (simulated annealing) to deduce the approximate crystal structure, and finally, Rietveld refinement was performed to obtain the confirmed crystal structure represented by Formula 1.

[0122] Figure 3 The confirmation of the crystalline structure of the magnesium serine crystalline form represented by Formula 1 of the present invention is shown, which is achieved by uploading to the CheckCIF website (http: / / checkcif.iucr.org) provided by IUCr using the PLATON program, using the Crystallographic Information File (CIF), which is a standardized data format for crystallographic information developed under the leadership of the Working Group on Crystallographic Information (WPCI) of the International Union of Crystallography (IUCr).

[0123] Experimental Example 2. Analysis of X-ray Powder Diffraction (PXRD)

[0124] According to the analysis conditions and measuring instrument specifications shown in Table 3 below, X-ray powder diffraction (PXRD) analysis was performed on the magnesium serine crystalline form prepared in Example 1 of the present invention. The main X-ray diffraction pattern diffraction angles (2θ±0.2°) and relative intensities are shown in Tables 4 and Figure 4 In. Figure 4 In the figure, the x-axis is 2θ (Bragg angle, unit: °).

[0125] [Table 3]

[0126]

[0127]

[0128] [Table 4]

[0129]

[0130]

[0131] Refer to Table 4 and Figure 4 , it can be confirmed that the crystal form of the present invention is a novel crystalline compound and is different from the amorphous AST-011 (Comparative Example 2).

[0132] Experimental Example 3. Differential Scanning Calorimetry (DSC) Analysis

[0133] In order to analyze the crystal form of the present application (Example 1) by differential scanning calorimetry, DSC-N650 (SCINCO, Korea) was used as a DSC analyzer, and measurement was performed while increasing the temperature from 0°C to 500°C at a heating rate of 10°C / min. The maximum endothermic peak temperature of the crystal form obtained by differential scanning calorimetry (DSC) analysis is shown in FIG. Figure 5 middle.

[0134] refer to Figure 5 , it can be seen that the crystal form of the present application exhibits a maximum endothermic peak at 283±3°C. That is, it can be confirmed that the crystal form of the present invention exhibits new thermodynamic properties different from AST-011 (Comparative Example 2) (which does not show a main endothermic peak), and is thus a new thermodynamically stable crystal.

[0135] Experimental Example 4. Nuclear Magnetic Resonance (NMR) Analysis

[0136] The crystal form of the present application prepared in Example 1 was subjected to 1 H NMR and 13 C NMR analysis. 20 mg of the crystalline sample of the present application was dissolved in 0.7 mL of D2O, and the result was subjected to nuclear magnetic resonance analysis at 25°C using JEOL / 400YH (JEOL, USA). The results are shown in Figure 6 middle.

[0137] Experimental Example 5. Stability test under harsh conditions

[0138] In order to confirm the stability of the crystalline form of the present invention as conditions change, a stability test was performed under harsh conditions.

[0139] The magnesium serine crystalline form prepared in Example 2 was stored in a solid state at 70° C. for 21 days and in an aqueous solution at a concentration of 0.5 mg / mL at 25° C. (60.0% relative humidity) for 21 days, respectively. Maleic acid was then used as a standard substance, and stability (measurement of content and related substances) was confirmed by qNMR analysis.

[0140] For the qNMR analysis method, the analysis conditions were established by measuring the nuclide of magnesium serine on the sample obtained by the preliminary stress test. 1 The spin-lattice relaxation time T1 of H. The T1 value is set to 4.708 seconds, and the measurement wait time (d1: delay time) and measurement time (at: acquisition time) are set based on this value. The measurement wait time (d1) is set to 30 seconds, and the measurement time (at) is set to be equal to or greater than the T1 value.

[0141] For qNMR analysis 1 For H-NMR measurements, a JEOL JNM-ECX400I (400 MHz) NMR instrument was used, and the spectral width (sw) was set to 6000 Hz to fully reflect the frequency range of 0 ppm to 10 ppm.

[0142] The analysis results are shown in Table 5 below.

[0143] [Table 5]

[0144]

[0145]

[0146] As can be seen from the results in Table 5 above, it is confirmed that the magnesium serine crystalline form of the present invention is very stable in the solid state and in aqueous solution even under harsh conditions, and does not decompose to produce products and related substances. Therefore, it is confirmed that the crystalline form of the present invention can be used very effectively as a pharmaceutical raw material.

[0147] Experimental Example 6. Evaluation of polymorphic transition

[0148] In order to confirm whether the magnesium serine crystalline form of the present invention undergoes polymorphic transformation over time, PXRD and DSC analyses were performed in the initial state and after 4 weeks to confirm whether polymorphic transformation occurred.

[0149] Specifically, the crystalline form prepared in Example 2 was placed in a polyethylene bag, placed in a stabilization chamber at 40°C and 75% relative humidity, and then stored for 4 weeks. The PXRD and DSC results measured at the beginning and after 4 weeks are shown in Table 6 below. PXRD and DSC analysis were performed in substantially the same manner as in Experimental Examples 2 and 3.

[0150] [Table 6]

[0151]

[0152] As can be seen from the results of PXRD and DSC analysis in Table 6 above, the magnesium serine crystalline form of the present invention did not undergo polymorphic transformation even after storage for 4 weeks. That is, it was confirmed that the magnesium serine crystalline form of the present invention is a very stable crystalline form in which polymorphic transformation does not occur due to aging, and thus can be used very effectively as a pharmaceutical raw material.

[0153] Experimental Example 7. Comparison of the Crystalline Form of the Present Application with Amorphous AST-011 (Comparative Example 2)

[0154] The crystalline form and amorphous form of AST-011 (Comparative Example 2) of the present application prepared in Example 1 were comparatively evaluated for the following test items by the following methods. The results are shown in Table 7 below.

[0155] -Appearance evaluation: visual observation

[0156] - Moisture measurement: According to the moisture measurement method (Karl Fischer method) of the general test method in the 12th edition of the Korean Pharmacopoeia, 0.5 g of the sample was added to a moisture meter (Mettler Toledo, V10-S, Switzerland).

[0157] - Infrared spectrum measurement method (IR): Measurement was performed using an FT-IR device (PerkinElmer, Spectrum 2, USA) according to the ATR measurement method of the general test method in the 12th edition of the Korean Pharmacopoeia.

[0158] - pH measurement: 0.5 g of the sample was added to a 10 mL volumetric flask, water was added to a final volume of 10 mL, and then the pH was measured using a pH meter (Mettler Toledo, FP-20, Switzerland).

[0159] - XRD and DSC analysis: The analysis was performed in the same manner as in Experimental Examples 2 and 3.

[0160] [Table 7]

[0161]

[0162] As can be seen from Table 7 above, AST-011 (Comparative Example 2) was confirmed to be an amorphous compound with a high moisture content and no main endothermic peak. It was confirmed that it was an unstable compound, and its appearance showed a change after 7 days of storage. In contrast, the crystal form of the present application was confirmed to be a stable crystalline compound with a low moisture content, an endothermic peak at 283±3°C, and a stable compound that did not change in properties even after 3 months of storage under the same conditions as AST-011.

[0163] That is, it was confirmed that the crystal form of the present invention can be used very effectively as a pharmaceutical raw material in a large-scale production process or the like.

[0164] Experimental Example 8. Testing of pharmacological effects

[0165] Take out the mouse brain of 1 day old, take out the olfactory bulb, and then peel off the meninges. After the obtained product is collected in a 50mL tube containing HBSS (Hank's balanced salt solution) and centrifuged at 250 × g for 1 minute, remove the supernatant. 2mL of trypsin / EDTA (0.5%), 8mL of HBSS buffer and 0.2mL of deoxyribonuclease are mixed, and then reacted in a 37°C constant temperature water bath for 30 minutes. After adding 18mL of culture medium and 2mL of trypsin / EDTA and pipetting 10 times, the obtained product is filtered using a 40μm cell strainer. Then, after centrifugation at 800 × g for 2 minutes, remove the supernatant. After adding 5mL of culture fluid thereto, the cell number is confirmed using a hemocytometer, and the cells are cultured to obtain primary mouse astrocytes.

[0166] The protective effects of L-serine (AST-001, Comparative Example 1), AST-011 (Comparative Example 2), and the crystalline form of the present application (Example 1) against oxidative stress induced by DMNQ (1,4-naphthoquinone) in these cells were confirmed. Cells (primary astrocytes, 1×10 5cells) and cultured at 5% CO2 and 37°C for 24 hours to obtain a cell monolayer, the composition was diluted with a cell seeding medium, and the diluted composition was inoculated at concentrations of 0mM, 1mM, 2mM and 5mM at 100 μL / well, followed by incubation at 5% CO2 and 37°C for 6 hours. After removing the culture medium, 0mM, 1mM, 2mM and 5mM of DMNQ (30 μM) causing cytotoxicity were mixed with each of the compounds of Comparative Examples 1 and 2 and the crystalline form of Example 1 of the present application and inoculated at 100 μL / well, followed by incubation at 5% CO2 and 37°C for 16 hours. Then, after removing the culture medium, 0.5 mg / mL of MTT solution was added to each well in an amount of 100 μL, followed by reaction in a 5% CO2 and 37°C incubator for 2 hours. After completely removing the MTT solution and drying the culture plate, the formazan crystals formed in the cells were dissolved with DMSO, and the absorbance was measured at 590 nm using an ELISA plate reader to evaluate cell viability and drug efficacy. Figure 8 middle.

[0167] If through Figure 8 It can be seen that about 50% of the cells were killed by the oxidative stress induced by DMNQ. In contrast, in the group pretreated with the compound of Comparative Example 1 (AST-001) or Comparative Example 2 (AST-011) or the crystal form of the present application and then post-treated with DMNQ, the cell viability was excellent due to the cytoprotective effect of the drug, thereby confirming their pharmacological effects. It was confirmed that the cytotoxic protective effect of the magnesium serine crystal form of the present invention against DMNQ was similar to that of L-serine and AST-011, thereby confirming that the pharmacological effect of the crystal form of the present invention is within a range substantially equivalent to that of L-serine and AST-011.

[0168] According to the above experimental results, it is confirmed that the magnesium serine crystal form of the present invention has excellent stability, does not undergo polymorphic transformation over time, and has excellent pharmacological effects, and is therefore optimized as a pharmaceutical raw material.

[0169] Although the specific parts of the present invention have been described in detail above, it is obvious to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereto. Therefore, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims

1. A crystalline form of a magnesium serine compound represented by the following formula 1: [Formula 1] 2. The crystal form of the magnesium serine compound according to claim 1, wherein X-ray powder diffraction pattern: including diffraction peaks at diffraction angles (2θ±0.2°) of 13.53°, 16.00°, and 22.05°; and Three or more diffraction peaks are included at diffraction angles (2θ±0.2°) selected from the group consisting of 18.36°, 19.75°, 20.00°, 20.95°, 24.39°, and 28.37°.

3. The crystal form of the magnesium serine compound according to claim 2, wherein The X-ray powder diffraction pattern further includes one or more diffraction peaks at diffraction angles (2θ±0.2°) selected from 18.89°, 25.73°, 26.39°, 29.55°, 30.07°, 32.39°, 33.51°, 33.99°, 34.42° and 40.36°. The crystalline form of the magnesium serine compound according to claim 1 , which has an endothermic peak at 283±3° C. when analyzed by differential scanning calorimetry (DSC).

5. A crystal form of a magnesium serine compound, wherein two serine molecules are bound to one magnesium atom, The X-ray powder diffraction pattern includes diffraction peaks at diffraction angles (2θ±0.2°) of 13.53°, 16.00° and 22.05°. The crystalline form of the magnesium serine compound according to claim 5, which has an endothermic peak at 283±3°C when analyzed by differential scanning calorimetry (DSC).

7. A method for preparing a crystalline form of the magnesium serine compound according to any one of claims 1 to 6, comprising: a) mixing L-serine, magnesium (Mg) or a magnesium compound with a solvent and stirring the mixture; as well as b) Filter the crystals produced after stirring.

8. The method according to claim 7, wherein: Magnesium (Mg) is in the form of powder or granules.

9. The method according to claim 7, wherein: The magnesium compound is one or more selected from magnesium methoxide, magnesium ethoxide, magnesium chloride, magnesium bromide, magnesium carbonate and magnesium citrate.

10. The method according to claim 7, wherein: The solvent is a polar solvent.

11. The method according to claim 7, wherein: The solvent is one or more selected from methanol, ethanol, isopropanol and tert-butanol.

12. A pharmaceutical composition for preventing or treating developmental disorders or neurodegenerative diseases, comprising the crystal form of the magnesium serine compound according to any one of claims 1 to 6 as an active ingredient.

13. The pharmaceutical composition according to claim 12, wherein The developmental disorder is one or more diseases selected from autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD), intellectual disability, language disorder, movement disorder, tic disorder and Tourette syndrome.

14. The pharmaceutical composition according to claim 12, wherein The neurodegenerative disease is one or more diseases selected from Alzheimer's disease, vascular dementia, mild cognitive impairment, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis and cerebellar degenerative disease.

15. The pharmaceutical composition according to claim 12, wherein The pharmaceutical composition is formulated into one or more preparations selected from powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, suppositories, eye drops and injections.

16. A health functional food for preventing or improving developmental disorders or neurodegenerative diseases, comprising the crystal form according to any one of claims 1 to 6.

17. A method for preventing or treating a developmental disorder or a neurodegenerative disease, comprising administering to an individual the crystalline form of the magnesium serine compound according to any one of claims 1 to 6.

18. Use of the crystal form of the magnesium serine compound according to any one of claims 1 to 6 for preventing or treating developmental disorders or neurodegenerative diseases.

19. Use of the crystal form of the magnesium serine compound according to any one of claims 1 to 6 in the preparation of a medicament for preventing or treating developmental disorders or neurodegenerative diseases.

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