Crystalline form of oral dnmt inhibitor

Abstract

The present disclosure relates to a crystalline form of a hemihydrate of 5'- O-triethylsilyl -2'- deoxy-5-azacytidine, a method for preparing the same, and a pharmaceutical composition containing the crystalline form.

Description

Crystalline forms of oral DNMT inhibitors

[0001] The present disclosure relates to crystalline forms of orally available DNMT inhibitors that have high stability against the hydrolytic metabolic enzyme cytidine deaminase and can replace 2'-deoxy-5-azacytidine (decitabine), and methods for preparing the same.

[0002] DNMTs is an abbreviation for DNA methyltransferases, which methylate the amino group at position 6 of the adenine ring in DNA (Adenine N 6 -specific DNA-methyltransferase: EC 2.1.1.72) or methylation of the amino group at position 4 of the cytosine ring (Cytosine N 4 -specific DNA-methyltransferase: EC 2.1.1.113) or methylation of the 5-position of the cytosine ring (Cytosine C 5 DNMTs are a group of enzymes that catalyze the methyltransferase (EC 2.1.1.37) and play important roles in various biological phenomena, such as cell differentiation and ontogeny, through the regulation of gene expression (Non-Patent Documents 1 and 2). 5-Azacytidine and decitabine are known as selective enzyme inhibitors (DNA methyltransferase inhibitors) of DNMTs (Non-Patent Document 3).

[0003] Dacogen®, an intravenous formulation of decitabine indicated for the treatment of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), has two dosage regimens: (1) 3-day regimen: 15 mg / m²; (2) 3-day regimen: 15 mg / m²; (3) 3-day regimen: 15 mg / m²; (4) 3-day regimen: 15 mg / m²; (5) 3-day regimen: 15 mg / m²; (6) 3-day regimen: 15 mg / m²; (7) 3-day regimen: 15 mg / m²; (8) 3-day regimen: 15 mg / m²; (9) 3-day regimen: 15 mg / m²; (10) 3-day regimen: 15 mg / m²; (11) 3-day regimen: 15 mg / m²; (12) 3 2 Administered as a continuous intravenous infusion over at least 3 hours every 8 hours, this cycle is repeated for 3 days. This cycle is repeated every 6 weeks. (2) 5-Day Regimen: 20 mg / m 2(The drug is administered by continuous intravenous infusion over at least one hour per day for five days. This cycle is repeated every four weeks.) Regardless of the dosage regimen, four or more cycles are required to achieve complete or partial remission. Because treatment with Dacogen® is long-term and the initially considered 3-day regimen required hospitalization, a new 5-day regimen was devised that allows outpatient treatment. However, these regimens result in a very low quality of life for patients requiring this treatment, and frequent hospital visits are required. Therefore, the development of an oral formulation is needed for convenience.

[0004] US Patent No. 2006014949 (A1) (Compositions and formulations of decitabine polymorphs and methods of use Japanese Patent No. 5159307 (Compositions and Formulations of Decitabine Polymorphs and Methods for Using the Same) (Patent Documents 1 and 2) discloses that decitabine exists in the crystalline forms of anhydrous, hemihydrate, and monohydrate, and discloses methods for producing the anhydrous crystalline form using solvents such as methanol, acetone, 2-butanone, chloroform, dichloromethane, ethyl ether, hexane, methyl sulfide, 2-propanol, and 1,1,1-trichloroethane; methods for producing the hemihydrate crystalline form using 2,2,2-trifluoroethanol and water; and methods for producing the monohydrate crystalline form using solvents such as dichloromethane and methanol (1:1), 1,2-dimethoxyethane, 1,1,1,3,3,3-hexafluoro-2-propanol, methanol, methanol and 2,2,2-trifluoroethanol (1:1), 2,2,2-trifluoroethanol, 2,2,2-trifluoroethanol and water (9:1), and water. It has also been disclosed that the hemihydrate crystal is highly unstable and readily transforms into the monohydrate in the presence of atmospheric moisture, while the anhydrous and monohydrate crystals are thermally stable. The high stability of the anhydrous and monohydrate crystal forms is due to the ease with which the free hydroxyl groups at the 3' and 5' positions of decitabine are hydrogen-bonded within each crystal lattice. Furthermore, the monoclinic crystal coordinates of decitabine monohydrate prepared from dimethyl sulfoxide have been published, confirming the existence of intermolecular interactions via hydrogen bonds between the 5' hydroxyl group, a water molecule, and the 2-carbonyl group of the 5-azacytosine ring, as well as between the 5' hydroxyl group, a water molecule, and the 3' hydroxyl group (Non-Patent Document 4).

[0005] In recent years, in order to solve the above problems, (1) guadecitabine (compound number: SGI-110), a prodrug of decitabine that has high stability against cytidine deaminase (Non-Patent Documents 5 and 6), and (2) INQOVI (registered trademark) (oral C-DEC, ASTX727) (Non-Patent Document 7), an oral combination drug consisting of decitabine and cedazuridine, an enzyme inhibitor of cytidine deaminase, have been developed and are currently undergoing clinical development.

[0006] The present inventors have reported a decitabine prodrug (5'-O-triethylsilyl-2'-deoxy-5-azacytidine) (compound represented by formula (1), hereinafter referred to as compound (1)) that has high stability against cytidine deaminase (Patent Documents 3 and 4) (Non-Patent Documents 8, 9, and 10), and are currently conducting clinical development of its oral formulation. However, the preparation method of the crystalline form of the drug substance and its chemical and physicochemical stability have not been clearly stated.

[0007] US Patent Publication No. 2006014949 Japanese Patent No. 5159307 Japanese Patent No. 6162349 Japanese Patent No. 6956937

[0008] Science Advances, 2024, 10(9), eadl3188.Chemico-Biological Interactions, 2024, 392, 110907.Frontiers in Oncology, 2022, 12, 849895.Acta Crystallographica, 1991 , C47, 1418-1420.Expert Opinion on Investigational Drugs, 2019, 28(10), 835-849.Journal of Geriatric Oncology, 2023, 14(3), 101406.Therapeutic Advances in Hematology, 2023, 14, 20406207231205429.Clinical Epigenetics, 2019, 11(1), 111.Blood,2020, 136(7), 871-884.Molecular Cancer Therapeutics, 2021, 20(8), 1412-1421.

[0009] 5'-O-triethylsilyl-2'-deoxy-5-azacytidine: Compound (1) can be easily prepared by triethylsilylation of the 5'-hydroxyl group of decitabine and isolated as anhydrous crystals by crystallization in an organic solvent. However, it has been found that anhydrous crystals with different crystalline forms (anhydrous Form I crystals and anhydrous Form II crystals) are obtained when an organic solvent with a boiling point below 100°C is used as the crystallization solvent, compared to when an organic solvent with a boiling point above 100°C is used. Furthermore, both crystalline forms are unstable even under constant temperature and humidity conditions, and the crystalline form gradually changes. Based on the above background, the objective of this study is to clarify the newly formed crystalline form of compound (1) and to provide a crystalline form suitable for the production of oral formulations.

[0010] In order to solve the above-mentioned problems, the present inventors have conducted a detailed analysis of a new crystalline form that precipitates when anhydrous Form I crystals and anhydrous Form II crystals of 5'-O-triethylsilyl-2'-deoxy-5-azacytidine (Compound (1)) represented by formula (I) are aerated under high humidity conditions, and have also investigated a simple method for preparing this crystalline form, thereby completing the present invention. The present disclosure includes the following features: [1] A crystal of 5'-O-triethylsilyl-2'-deoxy-5-azacytidine hemihydrate (Compound (1)). [2] The crystal according to [1], wherein the crystalline form of compound (1) exhibits a powder X-ray diffraction pattern containing characteristic diffraction peaks at angles of 2θ=12.5°±0.2°, 15.0°±0.2°, 19.1°±0.2°, 20.4°±0.2°, 25.1°±0.2°, and 25.9°±0.2° with CuKα radiation of a wavelength of 1.5419 angstroms. [3] The crystal according to [1], characterized in that the crystalline form of compound (1) exhibits a powder X-ray diffraction pattern containing characteristic diffraction peaks at angles of 2θ = 15.4° ± 0.2°, 15.8° ± 0.2°, 16.1° ± 0.2°, 17.1° ± 0.2°, 22.4° ± 0.2°, 23.4° ± 0.2°, and 28.1° ± 0.2° with CuKα radiation having a wavelength of 1.5419 angstroms. [4] The crystal according to [1], characterized in that the crystalline form of compound (1) exhibits a powder X-ray diffraction pattern containing characteristic diffraction peaks at angles of 2θ = 5.6° ± 0.2°, 12.9° ± 0.2°, and 13.2° ± 0.2° with CuKα radiation having a wavelength of 1.5419 angstroms. [5] The crystal according to [1], wherein the crystalline form of compound (1) exhibits a powder X-ray diffraction pattern containing characteristic diffraction peaks at angles of 2θ=5.6°±0.2°, 12.5°±0.2°, 12.9°±0.2°, 13.2°±0.2°, 15.0°±0.2°, 15.4°±0.2°, 15.8°±0.2°, 16.1°±0.2°, 17.1°±0.2°, 19.1°±0.2°, 20.4°±0.2°, 22.4°±0.2°, 23.4°±0.2°, 25.1°±0.2°, 25.9°±0.2°, and 28.1°±0.2° with CuKα radiation of a wavelength of 1.5419 angstroms.[6] The crystal according to any one of [1] to [5], further characterized in that when measured using a differential scanning calorimeter at a rate of 15°C / min, the crystal exhibits onset temperatures (extrapolated peak onset temperatures) of 86°C±5°C, 136°C±5°C, and 185°C±5°C. [7] A method for producing the crystal according to any one of [2] to [6], characterized in that compound (1) is crystallized from an aqueous organic solvent. [8] The production method according to [7], wherein the organic solvent is one or more solvents selected from the group consisting of C1-C4 acetic acid alkyl ester solvents, linear or branched C1-C6 alcohol solvents, C3-C6 ketone solvents, and C2-C5 saturated aliphatic nitrile solvents. [9] A method for producing the crystal according to any one of [2] to [6], characterized in that compound (1) is dissolved in aqueous acetone with heating and crystallized by adding methyl tert-butyl ether.

[10] A method for producing the crystal according to any one of [2] to [6], which comprises exposing anhydrous crystals of compound (1) to water.

[11] A method for producing the crystal according to any one of [2] to [6], which comprises exposing anhydrous crystals of compound (1) to air and humidity.

[12] A method for producing the crystal according to any one of [2] to [6], which comprises exposing anhydrous crystals of compound (1) to water in an organic solvent.

[13] A method for producing the crystal according to any one of [2] to [6], which comprises suspending and stirring anhydrous crystals of compound (1) in water.

[14] The method according to [8], wherein the organic solvent that can be added to promote the precipitation of hemihydrate crystals of compound (1) is one or more solvents selected from linear or branched ether solvents having C4 to C8 carbon atoms, C4 to C8 saturated hydrocarbon solvents, etc.

[15] A pharmaceutical composition comprising the crystal according to any one of [1] to [6] and a pharmaceutically acceptable carrier.

[16] A pharmaceutical composition for preventing or treating myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), acute or chronic myelogenous leukemia (AML or CML), or adult T-cell leukemia (ATL), comprising the crystal according to any one of [1] to [6] and a pharmaceutically acceptable carrier.

[0011] According to the present disclosure, when 5'-O-triethylsilyl-2'-deoxy-5-azacytidine (compound (1)), a prodrug of decitabine, is crystallized from organic solvents with different boiling points, two types of anhydrous crystals (anhydrous Form I crystals and anhydrous Form II crystals) are precipitated. However, both crystalline forms are unstable under constant temperature and humidity conditions and gradually transition to a hemihydrate crystal form. Because this hemihydrate crystal is stable for long periods under constant temperature and humidity conditions, oral formulations manufactured using this crystalline form as a drug substance are expected to be highly stable under constant temperature and humidity storage conditions. Furthermore, unlike the two anhydrous crystals (anhydrous Form I crystals and anhydrous Form II crystals), this crystalline form of compound (1) is less likely to be electrostatically charged. Therefore, this crystalline form of compound (1) is easy to handle not only in the drug substance manufacturing process but also in the formulation manufacturing process. This oral formulation (oral DNA methyltransferase inhibitor) can be effectively used as a tumor prevention and treatment agent.

[0012]

[0033] Figure 1 shows a PXRD pattern for the anhydrous Type I crystalline form of compound (1). Figure 2 shows a PXRD pattern for the anhydrous Type II crystalline form of compound (1). Figure 3 shows a PXRD pattern for the hemihydrate crystalline form of compound (1). Figure 4 shows a thermal analysis pattern by DSC for the anhydrous Type I crystalline form of compound (1). Figure 5 shows a thermal analysis pattern by DSC for the anhydrous Type II crystalline form of compound (1). Figure 6 shows a thermal analysis pattern by DSC for the hemihydrate crystalline form of compound (1). Figure 7 shows the crystal coordinates for the hemihydrate single crystalline form of compound (1). Figure 8 shows the state of intermolecular interactions via hydrogen bonds formed between the carbonyl group at position 2 of each 5-azacytosine ring, a water molecule, and the hydroxyl group at position 3' of each sugar moiety. The diagram shows the state of intermolecular interactions via hydrogen bonds formed between the amino group at position 4 and the nitrogen atom at position 3 of each 5-azacytosine ring, which contribute to the formation of the crystal lattice of the hemihydrate of compound (1), and the state of intermolecular interactions via hydrogen bonds formed between the carbonyl group at position 2 of each 5-azacytosine ring, a water molecule, and the hydroxyl group at position 3' of each sugar moiety. The diagram shows the state of intermolecular stacking between 5-azacytosine rings due to π-π electron interactions, which contribute to the formation of the crystal lattice of the hemihydrate of compound (1), and the state of intermolecular interactions via hydrogen bonds formed between the carbonyl group at position 2 of each 5-azacytosine ring, a water molecule, and the hydroxyl group at position 3' of each sugar moiety. The diagram shows the state of intermolecular interactions via intermolecular hydrophobic bonds involving the O-triethylsilyl group at position 5' of the sugar moiety, which contribute to the formation of the crystal lattice of the hemihydrate of compound (1). The Raman absorption spectrum data for the anhydrous crystalline forms (two types) and the hemihydrate crystalline form of compound (1) are shown in a table.

[0013] The crystalline form of the oral DNA methyltransferase inhibitor of the present disclosure, Compound (1), will be described in detail below. However, the following description is merely an example for explaining the present disclosure and is not intended to limit the present disclosure to the scope of the description.

[0014] In the 5'-O-triethylsilyl-2'-deoxy-5-azacytidine (compound (1)) of the present disclosure, the hydroxyl group at the 5'-position is protected with a triethylsilyl group, which is a hydrophobic functional group, making it difficult for intermolecular hydrogen bonds to form at this position. Therefore, it is not possible to expect the formation of a crystal lattice via intermolecular hydrogen bonds at the 5'-position of the sugar moiety, as in the crystals of decitabine described in prior art documents (Patent Documents 1 and 2, Non-Patent Document 4), and therefore the anhydrous crystals of compound (1) cannot maintain a stable crystalline form under conditions of water or humidity. On the other hand, the hemihydrate crystal of compound (1) was interpreted to form a crystal lattice through intermolecular interactions via hydrogen bonds formed between the carbonyl group at position 2 of each 5-azacytosine ring, a water molecule, and the hydroxyl group at position 3' of each sugar moiety (see Figure 7), intermolecular interactions via hydrogen bonds formed between the amino group at position 4 of each 5-azacytosine ring and the nitrogen atom at position 3 (see Figure 8), intermolecular stacking between 5-azacytosine rings due to π-π electron interactions (see Figure 9), and intermolecular interactions via intermolecular hydrophobic bonds involving the O-triethylsilyl group at position 5' of the sugar moiety (see Figure 10).

[0015] Compound (1) (5'-O-triethylsilyl-2'-deoxy-5-azacytidine) of the present disclosure can be prepared by any method known to those skilled in the art. For example, it can be prepared according to the method disclosed in Japanese Patent No. 6162349, the disclosure of which is incorporated herein by reference in its entirety. Compound (1) of the present disclosure is a prodrug of decitabine that has high stability against cytidine deaminase, the hydrolytic metabolic enzyme of decitabine, and can gradually release decitabine under physiological conditions.

[0016] To facilitate understanding of this disclosure, a number of terms and phrases are defined below. Unless otherwise stated, the terms and phrases used in the specification and claims have the meanings set forth below.

[0017] As used herein, the term "anhydrate" refers to a compound whose structural formula does not include water. As used herein, the term "hemihydrate" refers to a hydrate in which one molecule of water is combined with two molecules of 5'-O-triethylsilyl-2'-deoxy-5-azacytidine.

[0018] The term "crystallization" as used herein refers to dissolving an individual compound having a sufficient content for crystallization in a single or mixed anhydrous or aqueous organic solvent at room temperature or under heating (or concentrating to a required amount), and then leaving the solution at room temperature or cooled to about -10°C or by slow stirring to precipitate crystals.

[0019] The term "aqueous organic solvent" as used herein refers to an organic solvent that is water-soluble either alone or as a mixed solvent, and examples thereof include, but are not limited to, one or a mixed solvent of two or more selected from the group consisting of C1-C5 acetic acid alkyl ester solvents, linear or branched C1-C6 alcohol solvents, C3-C6 ketone solvents, and C2-C5 nitrile solvents. From the viewpoint of smooth progress of crystal precipitation, the organic solvent required for the crystallization of the present invention may be any organic solvent as long as crystal precipitation proceeds. Examples of C1-C5 alkyl ester solvents include methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, and pentyl acetate; examples of linear or branched C1-C6 alcohol solvents include linear or branched C1-C6 alcohols, such as methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, pentanol, and hexanol; examples of C3-C6 ketone solvents include acetone, methyl ethyl ketone, diethyl ketone, methyl isopropyl ketone, methyl isobutyl ketone, and cyclohexanone; examples of C2-C5 nitrile solvents include acetonitrile, propionitrile, and butyronitrile. These solvents may be used alone or in combination as a mixed organic solvent of two or more. The water content of the aqueous organic solvent is not particularly limited as long as it is an amount that allows the crystallization of hemihydrate crystals to proceed, but is preferably in the range of 1% to 50%, more preferably 2% to 25%, and even more preferably 3% to 10%.

[0020] The amount of organic solvent used in the crystallization of the present invention may be any amount as long as crystal precipitation proceeds. For example, the total amount of solvent used may be in the range of 5 to 30 parts by volume (V / W) relative to compound (1), with a range of 7 to 15 parts by volume (V / W) being preferred. The amount of organic solvent used necessary for the crystallization of the present invention can be appropriately adjusted by one skilled in the art.

[0021] The heating temperature required for the crystallization of the present invention is not particularly limited. In one embodiment, the heating temperature is, for example, in the range of room temperature to the boiling point of each solvent from the viewpoints of improving the recovery rate, suppressing by-products, and economic efficiency.

[0022] The time required for the crystallization of the present invention is not particularly limited. In one embodiment, from the viewpoints of improving the recovery rate, suppressing by-products, and economic efficiency, the time can be exemplified as being in the range of 0.5 to 120 hours, preferably 1 to 72 hours, more preferably 1 to 48 hours, and even more preferably 1 to 24 hours. However, the time required for the recrystallization of the present invention can be appropriately adjusted by those skilled in the art.

[0023] In the process of crystallizing hemihydrate crystals from an aqueous organic solvent, an organic solvent miscible with the aqueous organic solvent can be added to promote the precipitation of the hemihydrate crystals. Examples of such organic solvents include, but are not limited to, the organic solvents used as the aqueous organic solvents described above, as well as organic solvents selected from linear or branched chain C4 to C8 ether solvents and C4 to C8 saturated hydrocarbon solvents. Examples of linear or branched chain C4 to C8 ether solvents include diethyl ether, diisopropyl ether, and methyl tert-butyl ether. Examples of linear or branched chain C5 to C8 saturated hydrocarbon solvents include n-pentane, n-hexane, n-heptane, and n-octane.

[0024] The hemihydrate crystals of the present invention can also be produced by exposing anhydrous crystals to water. Examples of methods for exposing anhydrous crystals to water include exposing the anhydrous crystals to air and humidity, exposing the anhydrous crystals to water in an organic solvent, and suspending the anhydrous crystals in water. The humidity for the method of exposing the anhydrous crystals to air and humidity is not particularly limited, but a relative humidity of 50% or higher is preferred. In addition to the hydrous organic solvents used in the above-mentioned crystallization, water-immiscible organic solvents can also be used as organic solvents for the method of exposing the anhydrous crystals to water in an organic solvent. Examples of water-immiscible organic solvents include aromatic hydrocarbon solvents, ether solvents, and ester solvents. Examples of aromatic hydrocarbon solvents include toluene and xylene, and examples of ether solvents include diethyl ether, diisopropyl ether, and methyl tert-butyl ether.

[0025] Oral formulations are dosage forms of pharmaceutical compositions that can be safely administered orally, and examples thereof include, but are not limited to, tablets, capsules (including soft capsules and microcapsules), granules, powders, syrups, emulsions, suspensions, and sustained-release formulations.

[0026] Oral preparations can be produced by methods commonly used in the field of pharmaceutical technology, such as those described in the Japanese Pharmacopoeia.

[0027] Static electricity is the electrical charge that accumulates within a substance. All substances possess positive and negative charges, which are normally balanced. However, friction and other stimuli can disrupt the electrical balance, resulting in the accumulation of static electricity. Static electricity is one of the most troublesome issues in the production of pharmaceutical active ingredients and powder formulations. Organic powders with high electrical resistance tend to accumulate large amounts of static electricity during the handling process. In the pharmaceutical active ingredient and powder formulation manufacturing processes, static electricity can cause abnormal adhesion of powders to pipes and containers, resulting in reduced yields, inability to transport and package fixed quantities, and even fires during the active ingredient and formulation manufacturing processes. Therefore, when manufacturing oral powder formulations, it is desirable to select crystalline forms of active ingredients that are less susceptible to static charge, as well as formulation additives.

[0028] Compound (1) of the present disclosure has many therapeutic and prophylactic uses. In a preferred embodiment, compound (1) is a prodrug of decitabine and is therefore used to treat and prevent a wide variety of diseases susceptible to treatment with decitabine. Preferred indications that can be treated with compound (1) of the present disclosure include those involving undesired or uncontrolled cell division. Such indications include, but are not limited to, hematological disorders, benign tumors, various types of cancer (e.g., primary tumors and metastatic tumors), restenosis (e.g., coronary, carotid, and cerebral arterial lesions), abnormal endothelial cell stimulation (atherosclerosis), surgical injury to body tissues, abnormal wound healing, abnormal angiogenesis, diseases resulting in tissue fibrosis, repetitive movement disorders, disorders of highly non-vascularized tissues, and proliferative responses associated with organ transplantation.

[0029] Hematological disorders include abnormal proliferation of blood cells and hematological malignancies (e.g., various leukemias) that can result in dysplastic changes of blood cells. Examples of hematological disorders include, but are not limited to, acute myeloid leukemia, acute promyelocytic leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, myeloaplastic syndrome, and sickle cell anemia.

[0030] In some embodiments, compounds (1) of the present disclosure are used to treat blood disorders, including inherited blood disorders and / or hemoglobin deficiency disorders (e.g., sickle cell anemia). In some embodiments, compounds (1) of the present disclosure can be used to treat cancer. These cancers include leukemia, preleukemia, and other bone marrow-related cancers, such as myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML), acute or chronic myeloid leukemia (AML or CML), or adult T-cell leukemia (ATL), as well as lung cancer, such as non-small cell lung cancer (NSCL). NSCL can include epidermoid or squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. MDS can include, but are not limited to, refractory anemia, refractory anemia with excess blasts undergoing transformation, and myelomonocytic leukemia.

[0031] Powder X-ray diffraction (PXRD) is a rapid analytical technique used for the phase identification of crystalline materials, primarily for the identification of their phases. The technique involves irradiating a sample with X-rays and measuring the intensity of the diffracted X-rays while varying the irradiation angle θ. This technique can be used to determine lattice spacing and lattice constants. When X-rays with wavelengths comparable to the atomic spacing (0.5 Å to 3 Å) are incident on a material with a regularly arranged structure of atoms, the X-rays are scattered by the electrons belonging to each atom. The scattered X-rays interfere with each other and reinforce each other in specific directions, resulting in X-ray diffraction. PXRD is also utilized in pharmaceutical development, where it is useful for evaluating the amorphous or crystalline nature of active pharmaceutical ingredients and for assessing properties such as physical stability and manufacturability. Throughout this specification and the claims, when a crystalline form of Compound (1) is identified using one or more characteristic PXRD peaks given as angles 2θ, each 2θ value is understood to mean the given value ±0.2°. It is noted that PXRD values, particularly those at lower angles (large d values), may shift slightly depending on the milling conditions of the sample.

[0032] A differential scanning calorimetry (DSC) is an instrument that measures the temperature difference between a sample and a reference material as a function of temperature or time while changing the temperature according to a certain program, and measures the phase transition temperature and changes in heat (enthalpy) of solids and liquids. Thermal property measurements using DSC make it possible to understand not only simple heat-induced state changes such as melting, but also structural phase transitions and crystallization, and are widely used to evaluate the physical properties of polymeric materials, organic materials, metals, ceramics, etc.

[0033] Thermogravimetry (TGA) is a technique for measuring the change in weight of a sample when it is heated or cooled at a constant rate, or when it is held at a constant temperature, and is applied to measuring chemical and physical changes that accompany weight changes such as evaporation, decomposition, oxidation, reduction, adsorption, etc. By measuring these, it is possible to quantify the moisture, solvent, or components contained in a sample, analyze the thermal decomposition mechanism, and evaluate thermal stability, reactivity, etc.

[0034] Hereinafter, the present disclosure will be described with reference to examples, but the present disclosure is not limited to the following examples. Comparative Example 1

[0035] According to the method described in Japanese Patent No. 6,162,349, 5'-O-triethylsilyl-2'-deoxy-5-azacytidine (compound (1) [Method A (DMF-Imidazole system; reaction time: approximately 1 hour, column elution solvent: ethyl acetate-methanol system, isolated yield: 81%)] was prepared as a white solid. 1 H-NMR (400MHz, CDCl3) δ : 8.62 (s, 1H), 6.26 (t, J= 6Hz, 1H), 6.25 (br, 1H), 5.58 (br, 1H), 4.47-4.51 (m, 1H), 4.09-4.11 (m, 1H), 3.93 (dd, J= 11 and 2Hz, 1H), 3.82 (dd, J= 12 and 2Hz, 1H), 2.64-2.70 (m, 1H), 2.66 (br, 1H), 2.23 (dt, J= 12 and 6Hz, 1H), 0.96 (t, J= 8Hz, 9H), and 0.63 (t, J= 8Hz, 6H) ppm. 13 C-NMR (CDCl3) δ : 166.3, 156.0, 154.1, 87.6, 86.8, 71.6, 62.3, 42.6, 6.7, and 4.1 ppm. Mass: 343.3 (M + +1) (calcd. for C 14 H 26 N4O4Si, MW= 342.47). Comparative Example 2

[0036] 1.0 g of compound (1), prepared and column-purified by the method described in Comparative Example 1, was dissolved in 20 mL of acetonitrile by heating (65°C) and then allowed to stand overnight at room temperature to crystallize, yielding 770 mg of white crystals (plates) (HPLC purity = >99.5%). PXRD (Cu Kα radiation, 1.5406 Å) showed characteristic peaks at 2θ angles of 5.74°, 11.53°, 12.67°, 13.56°, 14.26°, 15.92°, 19.27°, 19.89°, 20.37°, 21.56°, 25.09°, and 27.41° (see Figure 1). In addition, when measured by DSC at a rate of 15°C / min, this crystal showed a slight endothermic peak at 137°C and a sharp endothermic peak at 188°C, and in TGA, it showed a weight change of about 0.4% between 25 and 170°C (see Figure 4). 1 The H-NMR spectrum matched the chemical shift data of the white solid shown in Comparative Example 1. From these findings, it was determined that this crystal was an anhydrous form of compound (1), and this crystal was designated as an anhydrous form I crystal of 5'-O-triethylsilyl-2'-deoxy-5-azacytidine. Note that this crystal was prone to static electricity, making it difficult to handle during isolation and weighing. Comparative Example 3

[0037] 1.0 g of compound (1), prepared and column-purified using the method described in Comparative Example 1, was dissolved in 100 mL of toluene at 140°C and allowed to stand overnight at room temperature to crystallize, yielding 778 mg of white crystals (columnar crystals) (HPLC purity = >99.5%). PXRD analysis of the crystals showed characteristic peaks at 2θ = 9.69°, 12.90°, 13.41°, 14.76°, 16.07°, 16.73°, 18.53°, 19.18°, 19.46°, 20.39°, 22.45°, 23.14°, 24.22°, and 27.44° (see Figure 2). In addition, when this crystal was measured by DSC at a rate of 15°C / min, a sharp endothermic peak was observed at 188°C, and in TGA, a weight change of about 0.1% was observed between 25 and 170°C (see Figure 5). 1The H-NMR spectrum matched the chemical shift data of the white solid shown in Comparative Example 1. From these findings, it was determined that this crystal was an anhydrous form of compound (1), and this crystal was designated as an anhydrous form II crystal of 5'-O-triethylsilyl-2'-deoxy-5-azacytidine. Note that this crystal was prone to static electricity, making it difficult to handle during isolation and weighing.

[0038] The crystals (100 mg each) obtained in Comparative Example 1 (anhydrous Form I crystals) and Comparative Example 2 (anhydrous Form II crystals) were exposed to saturated saline (a saturated aqueous solution of sodium chloride at 22.2°C±0.2°C and 75.3±0.5% RH) at room temperature for 10 days. In both cases, they transformed into crystals showing the same PXRD chart. The obtained crystals showed characteristic peaks at 2θ=5.55°, 12.51°, 12.88°, 13.18°, 15.03°, 15.38°, 15.79°, 16.14°, 17.10°, 19.06°, 20.42°, 22.43°, 23.40°, 25.09°, and 25.94° (see Figure 3). In addition, this crystal showed endothermic peaks at 85.9°C, 136.2°C, and 184.8°C when measured by DSC at a rate of 15°C / min, and in TGA, it showed a weight change of 2.5% wt (a loss of 0.5% of water) between 25 and 170°C (see Figure 6). 1 The H-NMR spectrum matched the chemical shift data of the white solid shown in Comparative Example 1, except for the integrated intensity of the peak derived from water molecules observed at a δ value of approximately 3.3 ppm, which was 1 H higher. Based on these findings, this crystal was determined to be a hemihydrate of compound (1), and was therefore designated as a hemihydrate crystal of 5'-O-triethylsilyl-2'-deoxy-5-azacytidine. Furthermore, this crystal was not easily charged with static electricity, making it easy to handle during isolation and weighing.

[0039] 1.71 g (5 mmol) of the white solid compound (1), prepared and column-purified using the method described in Comparative Example 1, was dissolved in 15 mL of acetone with heating. Subsequently, while heating, 180 μL (10 mmol) of purified water and 75 mL of MTBE (methyl tert-butyl ether) were added and the mixture was stirred overnight at room temperature. The resulting white solid was collected by filtration, yielding 1.07 g (63% recovery) of the desired crystalline powder of 5'-O-triethylsilyl-2'-deoxy-5-azacytidine (fine columnar crystals). The HPLC purity of the resulting crystalline powder was >99%. The PXRD pattern of this crystalline powder was consistent with that of 5'-O-triethylsilyl-2'-deoxy-5-azacytidine hemihydrate crystals.

[0040] 330 mg (1 mmol) of the hemihydrate of compound (1) was dissolved in 3 mL of acetone with heating. Then, while heating, 18 μL (1.0 mmol) of purified water (1), 27 μL (1.5 mmol) of (2), and 36 μL (2.0 mmol) of (3) were added. 15 mL of MTBE was added to each solution, and the mixture was stirred overnight at room temperature. The resulting solids were collected by filtration, yielding 167 mg (51% recovery) of a white solid in the case of (1), 178 mg (54% recovery) of a white solid in the case of (2), and 254 mg (77% recovery) of a white solid in the case of (3). PXRD data of these solids indicated that (1) and (2) were anhydrous Form I crystals of 5'-O-triethylsilyl-2'-deoxy-5-azacytidine, and (3) was a hemihydrate crystal of 5'-O-triethylsilyl-2'-deoxy-5-azacytidine. Test Example 1

[0041] First, saturated solutions of compound (1) were prepared at 20°C using the following solutions: (1) acetonitrile (MeCN) only, (2) 3% aqueous MeCN, (3) 10% aqueous MeCN, and (4) purified water only. To each of these saturated solutions, a mixed powder of 10 mg of hemihydrate crystals, 10 mg of anhydrous Form I crystals, and 10 mg of anhydrous Form II crystals of compound (1) was added under stirring, and each suspension mixture was stirred at 20°C for 3 days. The solids in each suspension were filtered and analyzed by PXRD. It was determined that each solid obtained was the crystalline form of compound (1) shown in the right column of Table 1. Table 1 The results of this test show that both anhydrous crystals of compound (1) (anhydrous Form I and anhydrous Form II) readily transform into hemihydrate crystals in the presence of water. These results suggest that the hemihydrate crystals of compound (1) can maintain a stable crystalline form even when stored under constant temperature and humidity conditions, including a certain level of humidity, and are therefore effective in ensuring quality as a drug substance. As shown in the following Test Examples 2 and 3, hemihydrate crystals lose water of crystallization and transform into anhydrous crystals under extremely high temperature conditions, so storage at room temperature is recommended. Test Example 2

[0042] 5'-O-triethylsilyl-2'-deoxy-5-azacytidine: When measured by DSC at a rate of 15°C / min, the hemihydrate crystal of Compound (1) exhibited endothermic peaks at 85.9°C, 136.2°C, and 184.8°C. Based on this finding, 30 mg of the hemihydrate crystal of Compound (1) was placed on a cover glass and heated on a hot plate at 100°C ± 5°C for 30 minutes, followed by cooling to room temperature. PXRD analysis data of the resulting solid indicated that the hemihydrate crystal form of Compound (1) was converted to the anhydrous Form I crystal form by heat treatment under these conditions. Test Example 3

[0043] 5'-O-triethylsilyl-2'-deoxy-5-azacytidine: When measured by DSC at a rate of 15°C / min, the hemihydrate crystal of Compound (1) exhibited endothermic peaks at 85.9°C, 136.2°C, and 184.8°C. Based on this finding, 30 mg of the hemihydrate crystal of Compound (1) was placed on a cover glass and heated on a hot plate at 150°C ± 5°C for 10 minutes, followed by cooling to room temperature. PXRD analysis data of the resulting solid indicated that the hemihydrate crystal form of Compound (1) was converted to anhydrous Form II crystal form by heat treatment under these conditions.

[0044] While the foregoing disclosure has been described in some detail for purposes of clarity and understanding, these specific examples should be considered illustrative rather than restrictive. It will be readily apparent to those skilled in the art from a reading of this disclosure that various changes in form and detail may be made without departing from the true scope of the present disclosure, which is to be defined not by the specific examples but by the claims. Furthermore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In the event of any conflict, the present disclosure, including definitions, will control.

Claims

1. A crystal of 5'-O-triethylsilyl-2'-deoxy-5-azacytidine hemihydrate (compound (1)).

2. The crystal of claim 1, wherein the crystalline form of compound (1) exhibits a powder X-ray diffraction pattern containing characteristic diffraction peaks at angles of 2θ = 12.5° ± 0.2°, 15.0° ± 0.2°, 19.1° ± 0.2°, 20.4° ± 0.2°, 25.1° ± 0.2°, and 25.9° ± 0.2° with CuKα radiation of a wavelength of 1.5419 angstroms.

3. The crystal of claim 1, wherein the crystalline form of compound (1) exhibits a powder X-ray diffraction pattern containing characteristic diffraction peaks at angles of 2θ = 15.4° ± 0.2°, 15.8° ± 0.2°, 16.1° ± 0.2°, 17.1° ± 0.2°, 22.4° ± 0.2°, 23.4° ± 0.2°, and 28.1° ± 0.2° with CuKα radiation of a wavelength of 1.5419 angstroms.

4. The crystal of claim 1, wherein the crystalline form of compound (1) exhibits a powder X-ray diffraction pattern containing characteristic diffraction peaks at angles of 2θ = 5.6° ± 0.2°, 12.9° ± 0.2°, and 13.2° ± 0.2° with CuKα radiation of a wavelength of 1.5419 angstroms.

5. The crystal of claim 1, wherein the crystalline form of compound (1) exhibits a powder X-ray diffraction pattern containing characteristic diffraction peaks at angles of 2θ=5.6°±0.2°, 12.5°±0.2°, 12.9°±0.2°, 13.2°±0.2°, 15.0°±0.2°, 15.4°±0.2°, 15.8°±0.2°, 16.1°±0.2°, 17.1°±0.2°, 19.1°±0.2°, 20.4°±0.2°, 22.4°±0.2°, 23.4°±0.2°, 25.1°±0.2°, 25.9°±0.2°, and 28.1°±0.2° with CuKα radiation of a wavelength of 1.5419 angstroms.

6. The crystal according to any one of claims 1 to 5, further characterized in that when measured using a differential scanning calorimeter at a rate of 15°C / min, it exhibits onset temperatures (extrapolated peak onset temperatures) of 86°C±5°C, 136°C±5°C, and 185°C±5°C.

7. A method for producing the crystal according to any one of claims 2 to 6, characterized in that compound (1) is crystallized from a water-containing organic solvent.

8. The production method according to claim 7, wherein the organic solvent is one or more solvents selected from the group consisting of acetic acid C1 to C4 alkyl ester solvents, linear or branched C1 to C6 alcohol solvents, C3 to C6 ketone solvents, and C2 to C5 saturated aliphatic nitrile solvents.

9. A method for producing the crystal according to any one of claims 2 to 6, characterized in that compound (1) is dissolved in aqueous acetone by heating and crystallized by adding methyl tert-butyl ether.

10. A method for producing the crystals according to any one of claims 2 to 6, which comprises exposing anhydrous crystals of compound (1) to water.

11. A method for producing the crystals according to any one of claims 2 to 6, characterized in that the anhydrous crystals of compound (1) are exposed to air and humidity.

12. A method for producing the crystals according to any one of claims 2 to 6, characterized in that anhydrous crystals of compound (1) are exposed to water in an organic solvent.

13. A method for producing the crystals according to any one of claims 2 to 6, characterized in that anhydrous crystals of compound (1) are suspended in water and stirred.

14. The production method according to claim 8, wherein the organic solvent that can be added to promote the precipitation of hemihydrate crystals of compound (1) is one or more solvents selected from linear or branched C4-C8 ether solvents and C4-C8 saturated hydrocarbon solvents.

15. A pharmaceutical composition comprising the crystal according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier.

16. A pharmaceutical composition for preventing or treating myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), acute or chronic myelogenous leukemia (AML or CML), or adult T-cell leukemia (ATL), comprising the crystal according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier.

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