Crystal Forms of Compounds Inhibiting Prolyl Hydroxylase Activity and Their Applications

Abstract

The present invention relates to crystal forms of the compound shown in Structural Formula I, a preparation method thereof including a preparation method of an intermediate, a pharmaceutical composition thereof, and the use of any one of the above crystal forms in treating a disease, disorder or condition or in preparing a pharmaceutical preparation for treating a disease, disorder or condition. #imgabs0#

Description

Technical Field

[0001] This invention relates to various crystal forms of a novel compound and their use in inhibiting the activity of prolyl hydroxylase. The invention also relates to methods for treating diseases, conditions, or symptoms associated with increased or decreased HIF levels or activity in vivo by using crystal forms of at least one of the aforementioned compounds. Background Technology

[0002] The cellular transcription factor HIF (Hypoxia-inducible factor) plays a central role in oxygen homeostasis in many organisms and is also a key regulator of the hypoxia response. Genes regulated by HIF transcriptional activity play crucial roles in angiogenesis, erythropoiesis, hemoglobin F production, energy metabolism, inflammation, vasomotor function, apoptosis, and cell proliferation. HIF also plays a role in cancer (typically with elevated expression in cancer cells) and in the physiological response to ischemia and hypoxia.

[0003] The HIF transcriptional complex includes the αβ heterodimer (HIFαβ): HIF-β is a basic nuclear protein that forms a dimer with the oxygen-regulated HIF-α subunit. Oxygen regulation occurs through the hydroxylation of the HIF-α subunit, which is subsequently rapidly destroyed by the proteasome. In oxygen-rich cells, the pVHL protein (von Hippel-Lindautumorsuppressor protein) binds to the hydroxylated HIF-α subunit to promote its ubiquitin-dependent proteolysis. This process is inhibited under hypoxic conditions, HIF-α becomes more stable, and the transcription and activation of the HIFαβ dimer are promoted.

[0004] Hydroxylation of the HIF-α subunit can occur on proline and aspartic acid residues and can be catalyzed by a series of 2-ketoglutarate-dependent enzymes. This series includes HIF prolyl hydroxylase isozymes (PHDs) of Pro402 and Pro564 that hydroxylate human HIF1α, and the HIF factor inhibiting HIF (FIH) of Asn803 that hydroxylates human HIF1. Inhibition of PHDs or FIH increases HIF stability and favors its transcription and activation.

[0005] Inhibition of PHDs in the HIF complex also increases HIF stability and promotes its transcription and activation, which in turn could offer potential treatments for ischemia and anemia. Several patents mention potential PHDs. sFor the chemical structure design of the inhibitor, see WO2004108681, WO2007070359 and WO2011006355. Summary of the Invention

[0006] This invention relates to various substantially pure crystalline forms, which are various crystalline forms of compounds represented by structural formula I below, and / or their hydrates, and / or their solvates.

[0007]

[0008] The compounds of structural formula I described in this invention exist in one or more crystal forms. The inventors have designated these crystal forms as crystal form I, crystal form II, crystal form III, crystal form IV, crystal form V, crystal form VI, and crystal form VII, respectively.

[0009] The present invention provides a crystal form of the compound shown in structural formula I, the X-ray powder diffraction pattern of which has characteristic peaks with diffraction angles 2θ of approximately 5.9°, 11.0° and 25.9°.

[0010] The present invention further provides preferred embodiments of the above-described crystal form:

[0011] Preferably, the interplanar spacings corresponding to the diffraction angle 2θ of the above crystal form are respectively and

[0012] Preferably, the X-ray powder diffraction pattern of this crystal form has characteristic peaks with diffraction angles 2θ of approximately 5.9°, 11.0°, 17.6°, 22.6°, 25.9° and 26.9°.

[0013] Preferably, the interplanar spacings corresponding to the diffraction angle 2θ of the above crystal form are respectively and

[0014] Preferably, the X-ray powder diffraction pattern of this crystal form has characteristic peaks with diffraction angles 2θ of approximately 5.9°, 11.0°, 14.8°, 17.6°, 22.6°, 24.0°, 25.9°, and 26.9°.

[0015] Preferably, the interplanar spacings corresponding to the diffraction angle 2θ of the above crystal form are respectively and

[0016] Preferably, the above-mentioned crystal form has the following characteristics: Figure 1 The X-ray powder diffraction pattern shown is shown.

[0017] Table 1 summarizes... Figure 1The X-ray powder diffraction pattern shown is shown.

[0018] Table 1:

[0019]

[0020]

[0021] Preferably, the melting point of this crystal form is 174-177℃.

[0022] Preferably, the purity of this crystal form is ≥85%.

[0023] Preferably, the purity of this crystal form is ≥95%.

[0024] Preferably, the purity of this crystal form is ≥99%.

[0025] The present invention also provides a method for preparing the above-mentioned crystal form, comprising the following steps: dissolving the compound of structural formula I prepared in Example 1 in a methanol / methyl tert-butyl ether (MTBE) mixed solvent at room temperature, and then evaporating and precipitating to obtain the crystal form.

[0026] The present invention also provides another crystal form of the compound shown in structural formula I, which has characteristic peaks with diffraction angles of approximately 8.2°, 14.5° and 26.6° in its X-ray powder diffraction pattern.

[0027] Preferably, the interplanar spacings corresponding to the diffraction angle 2θ of the above crystal form are respectively and

[0028] Preferably, the X-ray powder diffraction pattern of this crystal form has characteristic peaks with diffraction angles 2θ of approximately 8.2°, 13.3°, 14.5°, 21.2° and 26.6°.

[0029] Preferably, the interplanar spacings corresponding to the diffraction angle 2θ of the above crystal form are respectively and

[0030] Preferably, the X-ray powder diffraction pattern of this crystal form has characteristic peaks with diffraction angles 2θ of approximately 8.2°, 9.6°, 13.3°, 14.5°, 21.2°, 22.8°, 25.4° and 26.6°.

[0031] Preferably, the interplanar spacings corresponding to the diffraction angle 2θ of the above crystal form are respectively and

[0032] Preferably, the above-mentioned crystal form has the following characteristics: Figure 2 The X-ray powder diffraction pattern shown is shown.

[0033] Table 2 summarizes... Figure 2 The X-ray powder diffraction pattern shown is shown.

[0034] Table 2:

[0035]

[0036]

[0037] Preferably, the melting point of this crystal form is 209-212℃.

[0038] Preferably, the purity of this crystal form is ≥85%.

[0039] Preferably, the purity of this crystal form is ≥95%.

[0040] Preferably, the purity of this crystal form is ≥99%.

[0041] The present invention also provides a method for preparing the above-mentioned crystal form, comprising the following steps: an excess of the compound of structural formula I prepared in Example 1 is suspended and stirred at room temperature or 50°C for at least 48 hours in a mixed solvent of water / acetonitrile (3:1) or water / ethanol; or, in a mixed solvent of methanol / water, it is suspended and stirred at room temperature for at least 48 hours to obtain the crystal form.

[0042] The present invention further provides a crystal form of the compound shown in structural formula I, which has characteristic peaks with diffraction angles 2θ of approximately 6.2°, 17.8° and 26.2° in its X-ray powder diffraction pattern.

[0043] Preferably, the interplanar spacings corresponding to the diffraction angle 2θ of the above crystal form are respectively and

[0044] Preferably, the X-ray powder diffraction pattern of this crystal form has characteristic peaks with diffraction angles 2θ of approximately 6.2°, 17.8°, 22.0°, 26.2° and 26.9°.

[0045] Preferably, the interplanar spacings corresponding to the diffraction angle 2θ of the above crystal form are respectively and

[0046] Preferably, the X-ray powder diffraction pattern of this crystal form has characteristic peaks with diffraction angles 2θ of approximately 6.2°, 12.1°, 15.6°, 17.8°, 22.0°, 26.2°, 26.9° and 28.9°.

[0047] Preferably, the interplanar spacings corresponding to the diffraction angle 2θ of the above crystal form are respectively and

[0048] Preferably, the above-mentioned crystal form has the following characteristics: Figure 3 The X-ray powder diffraction pattern shown.

[0049] Table 3 summarizes... Figure 3 The X-ray powder diffraction pattern shown.

[0050] Table 3:

[0051]

[0052]

[0053] Preferably, the melting point of this crystal form is 198-200℃.

[0054] Preferably, the purity of this crystal form is ≥85%.

[0055] Preferably, the purity of this crystal form is ≥95%.

[0056] Preferably, the purity of this crystal form is ≥99%.

[0057] The present invention also provides a method for preparing the above-mentioned crystal form, comprising the following steps: dissolving the compound of structural formula I prepared in Example 1 in a methanol / acetonitrile mixed solvent at room temperature, followed by evaporation and precipitation to obtain the crystal form. Alternatively, the method comprises the following steps: suspending and stirring an excess of the compound of structural formula I prepared in Example 1 in water, dichloromethane, isopropyl acetate, ethyl acetate, or an isopropyl acetate / heptane mixture at 50°C for at least 48 hours to obtain the crystal form.

[0058] The present invention further provides another crystal form of the compound shown in structural formula I, which has characteristic peaks with diffraction angles 2θ of approximately 12.4°, 20.3° and 26.6° in its X-ray powder diffraction pattern.

[0059] Preferably, the interplanar spacings corresponding to the diffraction angle 2θ of the above crystal form are respectively and

[0060] Preferably, the X-ray powder diffraction pattern of this crystal form has characteristic peaks with diffraction angles 2θ of approximately 11.3°, 12.4°, 20.3°, 21.4° and 26.6°.

[0061] Preferably, the interplanar spacings corresponding to the diffraction angle 2θ of the above crystal form are respectively and

[0062] Preferably, the X-ray powder diffraction pattern of this crystal form has characteristic peaks with diffraction angles 2θ of approximately 11.3°, 12.4°, 15.0°, 17.9°, 20.3°, 21.4°, 24.8° and 26.6°.

[0063] Preferably, the interplanar spacings corresponding to the diffraction angle 2θ of the above crystal form are respectively and

[0064] Preferably, the above-mentioned crystal form has the following characteristics: Figure 4 The X-ray powder diffraction pattern shown.

[0065] Table 4 summarizes... Figure 4 The X-ray powder diffraction pattern shown.

[0066] Table 4:

[0067]

[0068]

[0069] Preferably, the melting point of this crystal form is 204-207℃.

[0070] Preferably, the purity of this crystal form is ≥85%.

[0071] Preferably, the purity of this crystal form is ≥95%.

[0072] Preferably, the purity of this crystal form is ≥99%.

[0073] The present invention also provides a method for preparing the above-mentioned crystal form, comprising the following steps: an excess of the compound of structural formula I prepared in Example 1 is suspended and stirred at room temperature for at least 48 hours in a mixed solvent of methyl tert-butyl ether (MTBE), isopropyl acetate / heptane, or ethyl acetate / heptane to obtain the crystal form. Alternatively, the method comprises the following steps: an excess of the compound of structural formula I prepared in Example 1 is suspended and stirred at 50°C for at least 48 hours in a mixed solvent of ethyl acetate / heptane to obtain the crystal form.

[0074] The following steps are included: the crystal form III of the compound with structural formula I prepared in Example 4 is suspended and stirred at 50°C for 12-14 days in a mixed solvent of water / acetone to obtain the final crystal form.

[0075] The present invention further provides a crystal form of the compound shown in structural formula I, which has characteristic peaks with diffraction angles 2θ of approximately 6.0°, 11.1° and 24.1° in its X-ray powder diffraction pattern.

[0076] Preferably, the interplanar spacings corresponding to the diffraction angle 2θ of the above crystal form are respectively and

[0077] Preferably, the X-ray powder diffraction pattern of this crystal form has characteristic peaks with diffraction angles 2θ of approximately 6.0°, 11.1°, 17.7°, 24.1° and 26.9°.

[0078] Preferably, the interplanar spacings corresponding to the diffraction angle 2θ of the above crystal form are respectively and

[0079] Preferably, the X-ray powder diffraction pattern of this crystal form has characteristic peaks with diffraction angles 2θ of approximately 6.0°, 8.8°, 11.1°, 11.9°, 14.9°, 17.7°, 24.1° and 26.9°.

[0080] Preferably, the interplanar spacings corresponding to the diffraction angle 2θ of the crystal form V are respectively and

[0081] Preferably, the above-mentioned crystal form has the following characteristics: Figure 5 The X-ray powder diffraction pattern shown.

[0082] Table 5 summarizes... Figure 5 The X-ray powder diffraction pattern shown.

[0083] Table 5:

[0084]

[0085]

[0086] Preferably, the melting point of this crystal form is 190-193℃.

[0087] Preferably, the purity of this crystal form is ≥85%.

[0088] Preferably, the purity of this crystal form is ≥95%.

[0089] Preferably, the purity of this crystal form is ≥99%.

[0090] The present invention also provides a method for preparing the above-mentioned crystal form, comprising the following steps: suspending and stirring an excess of the compound of structural formula I prepared in Example 1 in a mixed solvent of methyl tert-butyl ether (MTBE) / heptane at 50°C for at least 48 hours to obtain the crystal form; or, adding water as an antisolvent to a methanol solution of the compound of structural formula I prepared in Example 1 to precipitate it to obtain the crystal form.

[0091] The present invention further provides a crystal form of the compound shown in structural formula I, which has characteristic peaks with diffraction angles 2θ of approximately 7.1°, 22.2° and 26.9° in its X-ray powder diffraction pattern.

[0092] Preferably, the interplanar spacings corresponding to the diffraction angle 2θ of the above crystal form are respectively and

[0093] Preferably, the X-ray powder diffraction pattern of this crystal form has characteristic peaks with diffraction angles 2θ of approximately 7.1°, 10.6°, 18.8°, 22.2° and 26.9°.

[0094] Preferably, the interplanar spacings corresponding to the diffraction angle 2θ of the above crystal form are respectively and

[0095] Preferably, the X-ray powder diffraction pattern of this crystal form has characteristic peaks with diffraction angles 2θ of approximately 7.1°, 9.4°, 10.6°, 16.5°, 18.8°, 21.3°, 22.2° and 26.9°.

[0096] Preferably, the interplanar spacings corresponding to the diffraction angle 2θ of the above crystal form are respectively and

[0097] Preferably, the above-mentioned crystal form has the following characteristics: Figure 6 The X-ray powder diffraction pattern shown.

[0098] Table 6 summarizes... Figure 6 The X-ray powder diffraction pattern shown.

[0099] Table 6:

[0100]

[0101]

[0102] Preferably, the melting point of this crystal form is 200-203℃.

[0103] Preferably, the purity of this crystal form is ≥85%.

[0104] Preferably, the purity of this crystal form is ≥95%.

[0105] Preferably, the purity of this crystal form is ≥99%.

[0106] The present invention also provides a method for preparing the above-mentioned crystal form, comprising the following steps: an excess of the compound of structural formula I prepared in Example 1 is suspended and stirred at room temperature in a mixed solvent of acetonitrile / water (volume ratio 1:1) or tetrahydrofuran / water for at least 48 hours to obtain the crystal form. Alternatively, the method comprises the following steps: the compound of structural formula I prepared in Example 1 is added to a mixed solvent of methanol / ethyl acetate, and the crystal form prepared in Example 5 is added as a seed crystal, followed by evaporation and precipitation to obtain the crystal form.

[0107] The present invention further provides a crystal form of the compound shown in structural formula I, which has characteristic peaks with diffraction angles 2θ of approximately 6.9°, 11.7° and 21.1° in its X-ray powder diffraction pattern.

[0108] Preferably, the interplanar spacings corresponding to the diffraction angles of the above-mentioned crystal forms are respectively and

[0109] Preferably, the X-ray powder diffraction pattern of this crystal form has characteristic peaks with diffraction angles 2θ of approximately 6.9°, 11.7°, 15.1°, 21.1° and 25.8°.

[0110] Preferably, the interplanar spacings corresponding to the diffraction angles of the above-mentioned crystal forms are respectively and

[0111] Preferably, the X-ray powder diffraction pattern of this crystal form has characteristic peaks with diffraction angles 2θ of approximately 6.9°, 7.5°, 11.7°, 15.1°, 19.3°, 21.1°, 22.6° and 25.8°.

[0112] Preferably, the interplanar spacings corresponding to the diffraction angle 2θ of the above crystal form are respectively and

[0113] Preferably, the above-mentioned crystal form has the following characteristics: Figure 7 The X-ray powder diffraction pattern shown.

[0114] Table 7 summarizes... Figure 7 The X-ray powder diffraction pattern shown.

[0115] Table 7:

[0116]

[0117]

[0118] Preferably, the purity of this crystal form is ≥85%.

[0119] Preferably, the purity of this crystal form is ≥95%.

[0120] Preferably, the purity of this crystal form is ≥99%.

[0121] The present invention also provides a method for preparing the above-mentioned crystal form, comprising the following steps: heating the crystal form VI prepared in Example 7 to 180°C to obtain the crystal form.

[0122] The present invention further provides applications of the above-mentioned crystal form.

[0123] A pharmaceutical composition comprising a therapeutically effective amount of the crystal form of the present invention, and pharmaceutically acceptable excipients, auxiliaries, or carriers.

[0124] The present invention also provides preferred embodiments of the above-described pharmaceutical composition:

[0125] Preferably, the above-mentioned pharmaceutical composition contains a therapeutically effective amount of the crystal form of the present invention, in combination with at least one other active ingredient.

[0126] Preferably, the pharmaceutical composition is for oral administration.

[0127] Preferably, the pharmaceutical composition is used in tablets or capsules.

[0128] Preferably, the pharmaceutical composition contains 1% to 99% by weight of the crystal form of the present invention.

[0129] Preferably, the pharmaceutical composition contains 1% to 70% by weight of the crystal form of the present invention.

[0130] Preferably, the pharmaceutical composition contains 10% to 30% by weight of the crystal form of the present invention.

[0131] The application of the crystal form of the present invention in the preparation of drugs for regulating HIF levels or HIF activity in vivo.

[0132] The present invention also provides a preferred embodiment of the application of the crystal form:

[0133] Preferably, the crystal form of the present invention is used in the preparation of medicaments for treating diseases, conditions or symptoms related to HIF levels or HIF activity.

[0134] Preferably, the crystal form of the present invention is used in the preparation of medicaments for treating anemia, ischemia, or diseases, symptoms, or conditions related to anemia or ischemia.

[0135] Preferably, the crystal form of the present invention is used in the preparation of a medicament for treating a patient’s disease, condition or symptom, wherein the patient’s disease, condition or symptom is selected from ischemia, anemia, wound healing, orthotopic transplantation, heterotopic transplantation, allogeneic transplantation, systemic hypertension, thalassemia, diabetes, cancer or inflammation, or a combination of two or more of these.

[0136] The present invention also provides a method for regulating HIF levels or activity in a patient by administering one of the crystal forms of the present invention to the patient.

[0137] The present invention further provides a method for treating a patient with a disease, condition, or symptom related to HIF levels or HIF activity by administering one of the crystal forms of the present invention to the patient.

[0138] The present invention also provides a method for treating patients with anemia or ischemia, or diseases, symptoms or conditions related to anemia or ischemia, by applying one of the crystal forms of the present invention to a patient.

[0139] The present invention also provides a method for treating a patient’s disease, condition or symptom by applying one of the crystal forms of the present invention to the patient, wherein the disease, condition or symptom is selected from ischemia, anemia, wound healing, orthotopic transplantation, heterotopic transplantation, allogeneic transplantation, systemic hypertension, thalassemia, diabetes, cancer or inflammation, or a combination of two or more of them.

[0140] All crystal forms of this invention are substantially pure.

[0141] As used herein, the term "substantially pure" means that at least 85% by weight, preferably at least 95% by weight, and more preferably at least 99% by weight, of the compound represented by structural formula I is present in the crystal forms of the present invention, particularly in crystal forms I, II, III, IV, V, VI and VII.

[0142] The crystal forms described above only summarize the main peaks. These main peaks are reproducible and within the error limit (value ±0.2).

[0143] In this invention, "having as Figure 1 The X-ray powder diffraction pattern shown refers to the main peaks displayed in the X-ray powder diffraction pattern, such as... Figure 1 As shown, the main peak refers to the peak that is related to... Figure 1 Compared to the highest peak (whose relative intensity is specified as 100%), peaks with a relative intensity exceeding 10%, preferably exceeding 30%. Similarly, in this invention, as... Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 or Figure 7The X-ray powder diffraction pattern shown refers to the main peaks displayed in the X-ray powder diffraction pattern, such as... Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 or Figure 7 As shown, the main peaks refer to those that are respectively related to... Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 or Figure 7 Compared to the highest peak (whose relative intensity is specified as 100%), peaks with a relative intensity exceeding 10%, preferably exceeding 30%, are preferred.

[0144] The present invention also provides a method for preparing the compound shown in structural formula I, as follows:

[0145]

[0146] The present invention also provides a method for preparing crystal forms I, II, III, IV, V, VI or VII of the compound shown in structural formula I.

[0147] The crystallization of the compounds of the present invention from a suitable solvent system containing at least one solvent can be achieved by natural precipitation (solvent evaporation), cooling and / or adding an antisolvent (in which the compounds of the present invention have relatively low solubility) to achieve supersaturation in the solvent system.

[0148] Crystallization can also be achieved by using seed crystals of compounds suitable for crystallizing the present invention, with or without the use of such seed crystals.

[0149] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of one or more crystal forms of a compound of structural formula I, including crystal form I, crystal form II, crystal form III, crystal form IV, crystal form V, crystal form VI, and crystal form VII, and a pharmaceutically acceptable excipient or adjuvant or carrier. The pharmaceutical composition contains 1% to 99% by weight, preferably 1% to 70% by weight, more preferably 10% to 30% by weight of any one of crystal forms I, II, III, IV, V, VI, and VII of a compound of structural formula I.

[0150] The present invention also provides the use of a compound of structural formula I or a crystal form selected from crystal forms I, II, III, IV, V, VI and VII in the preparation of a medicament for regulating HIF levels or HIF activity.

[0151] The present invention also provides the use of a compound of structural formula I or a crystal form selected from crystal forms I, II, III, IV, V, VI and VII in the preparation of a medicament for treating anemia or ischemia, or for diseases, conditions or symptoms associated with anemia or ischemia.

[0152] Furthermore, the present invention also provides the use of a compound of structural formula I or a crystal form selected from crystal forms I, II, III, IV, V, VI and VII in the preparation of a medicament for treating a combination of ischemia, anemia, wound healing, orthotopic transplantation, heterotopic transplantation, allogeneic transplantation, systemic hypertension, thalassemia, diabetes, cancer or inflammation, or a combination of two or more of these diseases, conditions or symptoms.

[0153] As used herein, the term "therapeutic effective amount" refers to the amount of a compound, when administered to a subject, sufficient to affect the treatment of a disease, condition, or symptom. "Therapeutic effective amount" can vary with the compound, the disease, condition, and / or the symptoms of the disease or condition, the severity of the disease, condition, and / or the symptoms of the disease or condition, the age of the patient being treated, and / or the weight of the patient being treated. In any given case, an appropriate amount may be obvious to those skilled in the art or may be determined by routine experiments. In the case of combination therapy, "therapeutic effective amount" refers to the total amount of the combined treatment effective in treating the disease, condition, or symptom.

[0154] Pharmaceutical compositions containing the compounds of the present invention can be administered to patients requiring treatment via oral, nasal inhalation, rectal, parenteral, or topical administration. For oral administration, the pharmaceutical composition can be formulated into conventional solid dosage forms such as tablets, powders, granules, capsules, etc., liquid dosage forms such as aqueous or oil suspensions, or other liquid dosage forms such as syrups, solutions, suspensions, etc. For parenteral administration, the pharmaceutical composition can be formulated into solutions, aqueous solutions, oily suspensions, lyophilized powders for injection, etc. Preferably, the dosage form of the pharmaceutical composition is selected from tablets, coated tablets, capsules, suppositories, nasal sprays, or injections, more preferably tablets or capsules. The pharmaceutical composition can be administered as a single unit dose with a precise dosage. Furthermore, the pharmaceutical composition may also include other active ingredients.

[0155] All dosage forms of the pharmaceutical compositions of the present invention can be prepared using conventional methods in the pharmaceutical field. For example, the active ingredient is mixed with one or more excipients and then formulated into the desired dosage form.

[0156] "Pharmaceutically acceptable carriers" refer to conventional pharmaceutical carriers suitable for the desired drug formulation, such as: diluents and excipients such as water and various organic solvents; fillers such as starch and sucrose; binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone (PVP); wetting agents such as glycerin; disintegrants such as agar, calcium carbonate, and sodium bicarbonate; absorption enhancers such as quaternary ammonium compounds; surfactants such as hexadecyl alcohol; absorption carriers such as kaolin and bentonite; and lubricants such as talc, calcium stearate, magnesium stearate, and polyethylene glycol. Other pharmaceutically acceptable excipients may also be added to the drug composition, such as dispersants, stabilizers, thickeners, complexing agents, buffers, penetration enhancers, polymers, flavorings, sweeteners, and dyes. Excipients suitable for the desired dosage form and route of administration are preferred.

[0157] The term “disease” or “symptom” or “condition” refers to any disease, discomfort, illness, symptom or indication. Attached Figure Description

[0158] Figure 1 X-ray powder diffraction pattern of crystal form I of the compound shown in structural formula I;

[0159] Figure 2 X-ray powder diffraction pattern of crystal form II of the compound shown in structural formula I;

[0160] Figure 3 X-ray powder diffraction pattern of crystal form III of the compound shown in structural formula I;

[0161] Figure 4 X-ray powder diffraction pattern of crystal form IV of the compound shown in structural formula I;

[0162] Figure 5 X-ray powder diffraction pattern of crystal form V of the compound shown in structural formula I;

[0163] Figure 6 X-ray powder diffraction pattern of crystal form VI of the compound shown in structural formula I;

[0164] Figure 7 X-ray powder diffraction pattern of crystal form VII of the compound shown in structural formula I.

[0165] Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 and Figure 7The X-ray powder diffraction pattern shown was obtained on a PANalytical X-ray diffraction system equipped with an Empyrean console. The diffraction peak positions are indicated by a value at 28.443. ° The 2θ value of the single-crystal silicon was corrected. The X-ray source was an Empyrean copper LEFX-X-ray tube emitting K-Alpha rays. Detailed Implementation

[0166] The present invention will be further illustrated below by giving examples, but these examples do not constitute any limitation on the scope of protection claimed by the present invention. In the specific embodiments of the present invention, unless otherwise specified, the techniques or methods described are conventional techniques or methods in the art.

[0167] Example 1

[0168] Synthesis of the compound shown in structural formula I:

[0169]

[0170] Synthesis of Compound 1

[0171] Under an inert gas (such as nitrogen) atmosphere, 9.2 g of 4-nitrophthalonitrile, 5.0 g of phenol, 7.3 g of potassium carbonate, and 40 mL of DMSO were added to a flask. The mixture was stirred at room temperature for 48 hours, and then heated to 60 °C for 2 hours. After cooling, the reaction mixture was filtered and the resulting yellow solid was dried to give 11.6 g of compound 1.

[0172] Synthesis of Compound 2

[0173] 11.3 g of compound 1 was dissolved in methanol, and 25 mL of 50% sodium hydroxide solution was added. The solution was heated under reflux for 48 hours until the reaction was complete. The pH was adjusted to 3 with concentrated hydrochloric acid. The precipitate was filtered and dried to give 10.5 g of compound 2.

[0174] Synthesis of Compound 3

[0175] 6.0 g of compound 2 was dissolved in 60 mL of glacial acetic acid and 60 mL of acetic anhydride, and the mixture was heated under reflux for 3 hours. After removing the solvent using a rotary evaporator, compound 3 was obtained.

[0176] Synthesis of Compound 4

[0177] 6.0 g of compound 3 and 2.65 g of methyl isocyanate were dissolved in 60 mL of tetrahydrofuran. 3.54 g of DBU (CAS registration number: 6674-22-2) was added dropwise at room temperature, followed by stirring at room temperature for 1 hour after the addition was complete. Impurities were removed by extraction with ethyl acetate under alkaline conditions, and the pH of the aqueous phase was adjusted to 3 with dilute hydrochloric acid. The mixture was then extracted with ethyl acetate, washed with water, dried over anhydrous Na₂SO₄, filtered, and the final organic phase was distilled using a rotary evaporator to obtain 9.0 g of compound 4.

[0178] Synthesis of Compound 5

[0179] 9.0 g of compound 4 was dissolved in methanol, concentrated hydrochloric acid was added, and the mixture was heated to 60 °C and reacted for 4 hours. The final precipitate was filtered to give 5.8 g of crude product. The product was further purified by column chromatography to give 1.85 g of compound 5.

[0180] Synthesis of Compound 6

[0181] 1.77 g of compound 5 was heated to 70 °C and reacted with 10 mL of phosphorus oxychloride for 3 hours. After cooling, the mixture was poured into ice. After complete decomposition of phosphorus oxychloride, the final precipitate was filtered and washed with water to obtain 1.45 g of compound 6.

[0182] Synthesis of Compound 7

[0183] Under nitrogen protection, 1.41 g of compound 6, 20 mL of dioxane, 0.49 g of tetra(triphenylphosphine)palladium, 1.78 g of potassium carbonate, and 0.54 g of trimethylborane were added, stirred, and refluxed for 3 hours, followed by stirring at room temperature for 48 hours. After concentration, the resulting mixture was extracted with ethyl acetate, washed with water, dried, and filtered. The mixture was then distilled on a rotary evaporator and further purified by column chromatography to give 0.42 g of compound 7.

[0184] Synthesis of Compound 8

[0185] 1.02 g of compound 7 was added to a mixture of 10 mL ethanol and 10 mL 2N sodium hydroxide, and refluxed for 1.5 hours. After filtering to remove impurities, the resulting mixture was distilled off the ethanol using a rotary evaporator. The resulting pale yellow solid was washed with water and dried to give 0.5 g of compound 8.

[0186] Synthesis of Compound 9

[0187] 0.37 g of compound 8, 0.44 g of glycine methyl ester hydrochloride, and 1.00 g of PyBOP (CAS registration number: 128625-52-5) were added to 15 mL of dichloromethane, followed by 0.74 mL of triethylamine and 1.0 mL of di(isopropyl)ethylamine. The mixture was stirred at room temperature for 3 hours. After filtration, the organic phase was washed with water, dried, and filtered again. The mixture was then subjected to rotary evaporation and further purified by column chromatography to obtain 0.29 g of compound 9.

[0188] Synthesis of compound 10, i.e., the compound shown in structural formula I

[0189] 0.28 g of compound 9 was dissolved in tetrahydrofuran, 5 mL of 1N sodium hydroxide was added, and the mixture was stirred at room temperature for 1 hour. After removing the tetrahydrofuran using a rotary evaporator, the pH of the residue was adjusted to 3 with dilute hydrochloric acid. The residue was then washed with ethyl acetate, filtered, and dried to obtain 0.21 g of compound 10, which is the compound shown in structural formula I.

[0190] Example 2

[0191] Preparation of crystal form I of the compound shown in structural formula I

[0192] The compound of structural formula I prepared by the method disclosed in Example 1 above was first dissolved in a methanol / MTBE (methyl tert-butyl ether) mixed solvent at room temperature, and then volatilized and precipitated to obtain crystal form I, which has a melting point of 174-177℃.

[0193] Example 3

[0194] Preparation of crystal form II of the compound shown in structural formula I

[0195] An excess of the compound of structural formula I prepared by the method disclosed in Example 1 above is suspended and stirred at room temperature or 50°C for at least 48 hours in a mixed solvent of water / acetonitrile (volume ratio of 3:1) or water / ethanol. Alternatively, an excess of the compound of structural formula I prepared by the method disclosed in Example 1 above is suspended and stirred at room temperature for at least 48 hours in a mixed solvent of methanol / water to obtain crystal form II, which has a melting point of 209-212°C.

[0196] Example 4

[0197] Preparation of crystal form III of the compound shown in structural formula I

[0198] At room temperature, the compound of structural formula I prepared by the method disclosed in Example 1 above was first dissolved in a methanol / acetonitrile mixed solvent, and then volatilized and precipitated to obtain crystal form III.

[0199] Alternatively, an excess of the compound of structural formula I prepared by the method disclosed in Example 1 above is suspended and stirred at 50°C for at least 48 hours in water, dichloromethane, isopropyl acetate, ethyl acetate (EtOAc), or a mixed solvent of isopropyl acetate / heptane or water / acetone to obtain crystal form III, which has a melting point of 198-200°C.

[0200] Example 5

[0201] Preparation of crystal form IV of the compound shown in structural formula I

[0202] An excess of the compound of structural formula I prepared by the method disclosed in Example 1 above was suspended and stirred at room temperature for at least 48 hours in methyl tert-butyl ether, or a mixed solvent of methyl tert-butyl ether / heptane, isopropyl acetate / heptane, ethyl acetate / heptane, or water / acetone to obtain crystal form IV.

[0203] Alternatively, an excess of the compound of structural formula I prepared by the method disclosed in Example 1 above is suspended and stirred at 50°C for at least 48 hours in a mixed solvent of ethyl acetate / heptane to obtain crystal form IV.

[0204] Alternatively, excess crystal form III prepared in Example 4 above can be suspended and stirred at 50°C for 12-14 days to obtain crystal form IV, which has a melting point of 204-207°C.

[0205] Example 6

[0206] Preparation of crystal form V of the compound shown in structural formula I

[0207] The excess of the compound of structural formula I prepared in Example 1 above was suspended and stirred at 50°C for at least 48 hours to obtain crystal form V; or, water was added to a methanol solution of the compound of structural formula I as an antisolvent to precipitate it to obtain crystal form V, which has a melting point of 190-193°C.

[0208] Example 7

[0209] Preparation of crystal form VI of the compound shown in structural formula I

[0210] An excess of the compound of structural formula I prepared by the method disclosed in Example 1 above was suspended and stirred at room temperature for at least 48 hours in a mixed solvent of acetonitrile / water (1:1) or tetrahydrofuran / water to obtain crystal form VI.

[0211] Alternatively, at room temperature, the compound of structural formula I prepared by the method disclosed in Example 1 is dissolved in a mixed solvent of methanol / ethyl acetate, and then the crystal form IV prepared in Example 5 is added as a seed crystal. After evaporation and precipitation, crystal form VI is obtained, with a melting point of 200-203°C.

[0212] Example 8

[0213] Preparation of crystal form VII of the compound shown in structural formula I

[0214] The crystal form V prepared by the method in Example 6 was heated to 180°C to obtain crystal form VII.

[0215] Example 9

[0216] Determination of HIF-PHD2 enzyme activity

[0217] HIF-PHD2 activity was determined using homogeneous TR-FRET technology (see US2008 / 004817; Dao JH et al., Anal Biochem. 2009, 384: 213-23). ​​In a 96-well plate, 2 μL of DMSO solution of the test compound and 40 μL of assay buffer (50 mM Tris pH 7.4 / 0.01% Tween-20 / 0.1 mg / mL BSA / 1 mM sodium ascorbate / 20 μg / mL catalase / 10 μM ferrous sulfate) containing 600 nM of full-chain PHD2 were added to each well. After pre-incubation at room temperature for 30 min, 8 μL of substrate (final concentration of 0.2 μM 2-ketoglutarate and 0.5 μM HIF-1α-peptide biotinyl dLDLEMLAPYIPMDDDFQL) was added to initiate the enzyme reaction. Two hours later, at room temperature, 50 μL of a quencher / detector mixture (final concentrations of 1 mM o-phenanthroline, 0.1 mM EDTA, 0.5 nM anti-(His)6LANCE reagent, 100 nM MAF647-labeled antibiotic streptavidin, and 30 nM (His)6-VHL-elonginB-elonginC) was added to terminate the reaction and generate a signal. The time-resolved fluorescence signal intensity ratio at 665 and 620 nm was measured, and the percentage of inhibition relative to the parallel uninhibited control sample was calculated. The IC50 of the compound of formula I prepared according to the method disclosed in Example 1 was approximately 2 μM.

[0218] Example 10

[0219] Assay of erythropoietin (EPO) induction in normal mice

[0220] Eight-week-old male C57BL / 6 rats were orally administered a suspension containing one crystalline form of compound I at doses of 20, 60, and 100 mg / kg in 0.5% CMC. Serum samples were obtained from the orbital vein six hours after administration (see Robinson A, et al., Gastroenterology. 2008, 134:145-55; Hsieh MM, et al., Blood. 2007, 110:2140-7). Erythropoietin in the samples was analyzed using an electrochemiluminescence immunoassay (MSD) analyzer as directed by the manufacturer. When administered the suspension of crystalline form VI of the present invention, the induced erythropoietin level in the sample was approximately 6297, which was at least 300 times higher than that in the uninduced blank control sample.

[0221] Example 11

[0222] Determination of crystal form stability

[0223] 8.3 mg of compound of formula I, prepared according to the method disclosed in Example 1, was added to 1 mL of isopropyl acetate, stirred, and filtered. Then, 9.6 mg of crystal form IV and 1.97 mg of crystal form VI, as disclosed in this invention, were added to the solution and stirred at room temperature for 36 hours. After centrifugation and drying, the resulting crystal form was determined to be pure crystal form VI. This indicates that crystal form VI is the most thermodynamically stable crystal form in this study.

Claims

1. A crystal form VI of the compound shown in structural formula I, characterized in that: Its X-ray powder diffraction pattern has characteristic peaks with diffraction angles 2θ of 7.1°, 10.6°, 18.8°, 22.2° and 26.9°.

2. The crystal form VI according to claim 1, characterized in that: The interplanar spacings corresponding to the diffraction angle 2θ are respectively and 3. The crystal form VI according to claim 1, characterized in that: Its X-ray powder diffraction pattern has characteristic peaks with diffraction angles 2θ of 7.1°, 9.4°, 10.6°, 16.5°, 18.8°, 21.3°, 22.2° and 26.9°.

4. The crystal form VI according to claim 3, characterized in that: The interplanar spacings corresponding to the diffraction angle 2θ are respectively and 5. The crystal form VI of the compound of structural formula I according to claim 1, characterized in that: It has the X-ray powder diffraction pattern shown in Figure 6.

6. The crystal form VI according to any one of claims 1 to 5, characterized in that, The melting point of crystal form VI is 200-203℃.

7. The crystal form VI according to claim 6, characterized in that, The purity of crystal form VI is ≥85%.

8. The crystal form VI according to claim 7, characterized in that, The purity of crystal form VI is ≥95%.

9. The crystal form VI according to claim 8, characterized in that, The purity of crystal form VI is ≥99%.

10. A method for preparing a compound of structural formula I, comprising the following steps: Synthesis of Compound 1 Under inert gas protection, 4-nitrophthalonitrile, phenol, potassium carbonate and DMSO were added to a flask and stirred at room temperature for 45-50 hours. Then the temperature was raised to 55-65℃ and the reaction was carried out for 1.5-2.5 hours. After cooling, the precipitate was collected and filtered to obtain compound 1. Synthesis of Compound 2 Compound 1 was dissolved in methanol, added to a 40-60% sodium hydroxide solution, heated under reflux until the reaction was complete, the pH was adjusted to 2.5-3.5 with concentrated hydrochloric acid, a precipitate was formed, filtered, and dried to obtain compound 2; Synthesis of Compound 3 Compound 2 was dissolved in glacial acetic acid and acetic anhydride, heated under reflux until the reaction was complete, and the solvent was removed by rotary evaporation to obtain compound 3. Synthesis of Compound 4 Compound 3 and methyl isocyanate were dissolved in tetrahydrofuran, and DBU (1,8-diazacyclo[5,4,0]undec-7-ene) was added dropwise at room temperature. The mixture was stirred at room temperature for 0.5-1.5 hours. Impurities were removed by washing with ethyl acetate under alkaline conditions. The pH of the aqueous phase was adjusted to 2.5-3.5 with dilute hydrochloric acid. The mixture was extracted with ethyl acetate, washed with water, dried over Na2SO4, filtered, and the resulting extract was distilled on a rotary evaporator to obtain compound 4. Synthesis of Compound 5 Compound 4 was dissolved in methanol, concentrated hydrochloric acid was added, and the mixture was heated to 55-65°C for 3.5-4.5 hours. The precipitate was filtered and purified by column chromatography to obtain compound 5. Synthesis of Compound 6 Compound 5 was heated with phosphorus oxychloride to 65-75℃ and reacted for 2.5-3.5 hours. After cooling, the mixture was poured into ice. After the phosphorus oxychloride was completely decomposed, the precipitate was filtered and washed with water to obtain compound 6. Synthesis of Compound 7 Under inert gas protection, compound 6, dioxane, tetra(triphenylphosphine)palladium, potassium carbonate and trimethylborane were added and stirred. The mixture was heated under reflux for 2.5-3.5 hours, then stirred at room temperature for 45-50 hours. The mixture was concentrated, extracted with ethyl acetate, washed with water, dried and filtered. The mixture was then distilled on a rotary evaporator and further purified by column chromatography to obtain compound 7. Synthesis of Compound 8 Compound 7 was added to ethanol and 1.5-2.5N sodium hydroxide, refluxed for 1-2 hours, and impurities were removed by filtration. The resulting mixture was then distilled on a rotary evaporator to remove ethanol, and then filtered to obtain a light yellow solid. The solid was washed with water and dried to obtain compound 8. Synthesis of Compound 9 Compound 8, glycine methyl ester hydrochloride, and PyBOP were added to dichloromethane, followed by the addition of triethylamine and di(isopropyl)ethylamine. The mixture was stirred at room temperature for 2.5-3.5 hours, filtered, washed with water, dried, filtered, rotary evaporated, and further purified by silica gel column chromatography to obtain compound 9. Synthesis of compound 10, i.e., the compound shown in structural formula I Compound 9 was dissolved in tetrahydrofuran, and 0.5-1.5N sodium hydroxide was added. The mixture was stirred at room temperature for 0.5-1.5 hours. After removing the tetrahydrofuran using a rotary evaporator, the pH of the residue was adjusted to 3 with dilute hydrochloric acid. The residue was further washed with ethyl acetate, filtered, and dried to obtain compound 10, which is the compound shown in structural formula I.

11. The method for preparing crystal form VI according to any one of claims 1 to 9, characterized in that: The method includes the following steps: an excess of the compound of structural formula I prepared according to the method of claim 10 is suspended and stirred at room temperature for at least 48 hours in a tetrahydrofuran / water or a mixed solvent of acetonitrile / water in a volume ratio of 1:1 to obtain the final crystal form.

12. The method for preparing crystal form VI according to any one of claims 1 to 9, characterized in that: The process includes the following steps: at room temperature, the compound of structural formula I prepared according to the method of claim 10 is dissolved in a mixed solvent of methanol / ethyl acetate, crystal form IV is added as a seed crystal, and then volatilized and precipitated to obtain the final crystal form; wherein the X-ray powder diffraction pattern of crystal form IV has characteristic peaks with diffraction angles 2θ of 11.3°, 12.4°, 20.3°, 21.4° and 26.6°.

13. The method for preparing crystal form VI according to claim 12, wherein the X-ray powder diffraction pattern of crystal form IV has characteristic peaks with diffraction angles 2θ of 11.3°, 12.4°, 15.0°, 17.9°, 20.3°, 21.4°, 24.8° and 26.6°.

14. A pharmaceutical composition, characterized in that: Contains a therapeutically effective amount of crystal form VI as described in any one of claims 1 to 9, and a pharmaceutically acceptable excipient, adjuvant, or carrier.

15. The pharmaceutical composition according to claim 14, characterized in that: The composition is for oral administration.

16. The pharmaceutical composition according to claim 15, characterized in that... The composition is in the form of tablets or capsules.

17. A pharmaceutical composition, characterized in that: Containing a therapeutically effective amount of crystal form VI as described in any one of claims 1 to 9, in combination with at least one other active ingredient.

18. The pharmaceutical composition according to claim 17, characterized in that: The composition is for oral administration.

19. The pharmaceutical composition according to claim 18, characterized in that... The composition is in the form of tablets or capsules.

20. The pharmaceutical composition according to any one of claims 14 to 19, characterized in that: The composition contains 1% to 99% by weight of crystal form VI as described in any one of claims 1 to 9.

21. The pharmaceutical composition according to claim 20, characterized in that: The composition contains 1% to 70% by weight of crystal form VI as described in any one of claims 1 to 9.

22. The pharmaceutical composition according to claim 21, characterized in that: The pharmaceutical composition contains 10% to 30% by weight of crystal form VI as described in any one of claims 1 to 9.

23. The use of crystal form VI according to any one of claims 1 to 9 in the preparation of a medicament for regulating HIF levels or HIF activity.

24. The use of crystal form VI according to any one of claims 1 to 9 in the preparation of a medicament for treating diseases, conditions or symptoms related to HIF levels or HIF activity.

25. The use of crystal form VI according to any one of claims 1 to 9 in the preparation of a medicament for treating anemia, ischemia, or diseases, conditions, or symptoms related to HIF levels or HIF activity.

26. The use of crystal form VI according to any one of claims 1 to 9 in the preparation of a medicament for treating a patient's disease, condition, or symptom, wherein, The patient’s disease, condition or symptom is selected from ischemia, anemia, wound healing, orthotopic transplantation, heterotopic transplantation, allogeneic transplantation, systemic hypertension, diabetes, cancer or inflammation, or a combination of two or more of these diseases that are related to HIF levels or HIF activity.

27. The application according to claim 26, characterized in that... The anemia mentioned is thalassemia.

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