TWI582078B - Crystal types of compounds inhibiting activity of prolyl hydroxylase and use thereof - Google Patents

Description

Crystal form of compound inhibiting proline hydroxylase activity and application thereof

The present invention relates to various crystalline forms of a novel compound and their inhibitory activity against prolyl hydroxylase. The invention also relates to methods of using at least one of the above compounds and a plurality of crystalline forms to increase the level or activity of HIF in vivo, or to treat conditions associated with an increase or decrease in HIF levels or activity in vivo.

The cellular transcription factor HIF (Hypoxia Inducible Factor) plays a central role in regulating the homeostasis of intracellular oxygen in many organisms, and is also a key regulatory factor for cells to respond to hypoxia. A series of genes regulated by HIF transcription play a pivotal role in the processes of neovascularization, erythropoiesis, hemoglobin F production, energy metabolism, inflammation, vasomotor regulation, apoptosis and cell proliferation. HIF is generally elevated in cancer cells and plays a role in the pathological response of ischemia and hypoxia in vivo.

The HIF transcription complex is an αβ heterodimer (HIFαβ), a nuclear protein composed of an intrinsic structure that forms a dimer with HIF-α, which is regulated by oxygen levels in the receptor. The body's oxygen level regulation of HIF-α is rapidly decomposed by the proteasome by hydroxylation of HIF-α. In cells with sufficient oxygen, the pVHL protein (von Hippel-Lindau tumor suppressor protein) and the hydroxyl group HIF-binding to promote its ubiquitin-dependent proteolysis. This process is inhibited under hypoxic conditions, and HIF-α becomes more stable, which is more conducive to the transcriptional formation and activation of HIFαβ dimer.

Hydroxylation of HIF-α is primarily at the proline and aspartate residues and can be catalyzed by a series of 2-ketoglutarate-dependent enzymes. The series of enzymes include PHDs-HIF prolyl hydroxylase isozymes which can hydroxylate Pro402 and Pro564 on human HIF1α, and FIH (Factor Inhibiting HIF) which can hydroxylate Asn803 on human HIF1. Inhibition of PHDs or FIH increases the stability of HIF and is more conducive to its transcriptional formation and activation. See: Schofield and Ratcliffe, Nature Rev. Mol. Cell Biol., Vol 5, 343-354 (2004). Inhibition of PHDs on the HIF complex also increases the stability of HIF and facilitates its transcriptional formation and activation, thereby providing the potential for treatment of ischemia and anemia. A number of patents mention the chemical structural design of potential PHDs inhibitors, see WO2004108681, WO2007070359 and WO2011006355.

The present invention relates to a plurality of substantially pure crystalline forms which are various crystalline forms of the compounds of formula I below, and/or hydrates, and/or solvates,

The compounds of structural formula I of the present invention may exist in one or more crystalline forms. The inventors have abbreviated these crystal forms as Form I, Form II, Form III, Form IV, Form V, Form VI, and Form VII.

The present invention provides a crystalline form of the compound of formula I having an X-ray powder diffraction pattern having diffraction peaks having diffraction angles 2θ of 5.9°, 11.0°, 17.6°, 22.6°, 25.9°, and 26.9°. The inventors defined it as Form I.

The present invention further provides a preferred embodiment of the above crystal form I. Preferably, the diffraction angle 2θ corresponding to the crystal form I corresponds to a plane spacing of 14.9 Å, 8.0 Å, 5.1 Å, 3.9 Å, 3.4 Å, and 3.3, respectively. Å.

Preferably, the X-ray powder diffraction pattern has diffraction peaks having diffraction angles 2θ of 5.9°, 11.0°, 14.8°, 17.6°, 22.6°, 24.0°, 25.9°, and 26.9°.

Preferably, the diffraction angles corresponding to the diffraction angle 2θ of the above crystal form I are 14.9 Å, 8.0 Å, 6.0 Å, 5.1 Å, 3.9 Å, 3.7 Å, 3.4 Å, and 3.3 Å, respectively.

Preferably, the above Form I has an X-ray powder diffraction pattern as shown in FIG.

Table 1 summarizes the X-ray powder diffraction pattern shown in Figure 1.

Preferably, the melting point is 174-177 °C.

Preferably, the crystal form purity 85%.

Preferably, the crystal form purity 95%.

Preferably, the crystal form purity 99%.

The present invention also provides a method for preparing the above crystal form, comprising the steps of dissolving the compound of the formula I prepared in the first embodiment in a mixed solvent of methanol/methyl tert-butyl ether (MTBE) at room temperature. The crystal form I is obtained by further volatilization and precipitation.

The present invention further provides a crystal form of the compound of the formula I having a diffraction peak of diffraction angles 2θ of 8.2°, 13.3°, 14.5°, 21.2° and 26.6° in the X-ray powder diffraction pattern, the inventors It is defined as Form II.

The present invention further provides a preferred embodiment of the above crystal form II. Preferably, the interplanar spacing of the diffraction angle 2θ of the above crystal form II is 10.8 Å, 6.7 Å, 6.1 Å, 4.2 Å, and 3.4 Å, respectively.

Preferably, the X-ray powder diffraction pattern has diffraction peaks having diffraction angles 2θ of 8.2°, 9.6°, 13.3°, 14.5°, 21.2°, 22.8°, 25.4°, and 26.6°.

Preferably, the interplanar spacing of the diffraction angle 2θ of the above crystal form II is 10.8 Å, 9.3 Å, 6.7 Å, 6.1 Å, 4.2 Å, 3.9 Å, 3.5 Å, and 3.4 Å, respectively.

Preferably, the above Form II has an X-ray powder diffraction pattern as shown in FIG.

Table 2 summarizes the X-ray powder diffraction pattern shown in Figure 2.

Preferably, the melting point is 209-212 °C.

Preferably, the crystal form purity 85%.

Preferably, the crystal form purity 95%.

Preferably, the crystal form purity 99%.

The present invention also provides a method for preparing the above crystal, comprising the following steps: the compound of the formula I prepared in the embodiment 1 is in a mixed solvent of water/acetonitrile (3:1) or water/ethanol, at room temperature or 50. The suspension was stirred at ° C for more than 48 hours; or, in a mixed solvent of methanol/water, the suspension was stirred at room temperature for more than 48 hours, and finally, Form II was obtained.

The invention further provides a crystalline form of the compound of formula I having a diffraction peak in the X-ray powder diffraction pattern having diffraction angles 2θ of 6.2°, 17.8°, 22.0°, 26.2° and 26.9°. The inventors defined it as Form III.

The present invention further provides a preferred embodiment of the above crystal form III. Preferably, the interplanar spacing of the diffraction angle 2θ of the above crystal form III is 14.3 Å, 5.0 Å, 4.0 Å, 3.4 Å, and 3.3 Å, respectively.

Preferably, the X-ray powder diffraction pattern has diffraction peaks at diffraction angles 2θ of 6.2°, 12.1°, 15.6°, 17.8°, 22.0°, 26.2°, 26.9° and 28.9°.

Preferably, the interplanar spacing of the diffraction angle 2θ of the above crystal form III is 14.3 Å, 7.3 Å, 5.7 Å, 5.0 Å, 4.0 Å, 3.4 Å, 3.3 Å, and 3.1 Å, respectively.

Preferably, the crystal form purity 85%.

Preferably, the crystal form purity 95%.

Preferably, the crystal form purity 99%.

Preferably, the above Form III has an X-ray powder diffraction pattern as shown in FIG.

Table 3 summarizes the X-ray powder diffraction pattern shown in Figure 3.

Preferably, the melting point is from 198 to 200 °C.

The present invention also provides a method for preparing the above crystal, comprising the steps of dissolving the compound of the formula I prepared in the first embodiment in a methanol/acetonitrile mixed solvent at room temperature, and then volatilizing and precipitating. Alternatively, comprising the following steps, the compound of the formula I prepared in Example 1 is suspended in a solvent of water, dichloromethane, isopropanol, ethyl acetate or isopropanol / heptane at 50 ° C Stirring for more than 48 hours, finally obtaining Form III.

The present invention still further provides a crystal form of the compound of the formula I, which has a diffraction peak having a diffraction angle 2θ of 11.3°, 12.4°, 20.3°, 21.4° and 26.6° in the X-ray powder diffraction pattern. People define it as Form IV.

The present invention further provides a preferred technical solution of the above Form IV: Preferably, the interplanar spacing of the diffraction angle 2θ of the above crystal form IV is 7.9 Å, 7.1 Å, 4.4 Å, 4.1 Å, and 3.4 Å, respectively.

Preferably, the X-ray powder diffraction pattern has diffraction peaks having diffraction angles 2θ of 11.3°, 12.4°, 15.0°, 17.9°, 20.3°, 21.4°, 24.8°, and 26.6°.

Preferably, the diffraction angles corresponding to the diffraction angle 2θ of the above crystal form IV are 7.9 Å, 7.1 Å, 5.9 Å, 5.0 Å, 4.4 Å, 4.1 Å, 3.6 Å, and 3.4 Å, respectively.

Preferably, the above Form IV has an X-ray powder diffraction pattern as shown in FIG.

Table 4 summarizes the X-ray powder diffraction pattern shown in Figure 4.

Preferably, the melting point is from 204 to 207 °C.

Preferably, the crystal form purity 85%.

Preferably, the crystal form purity 95%.

Preferably, the crystal form purity 99%.

The present invention also provides a process for the preparation of the above Form IV, comprising the steps of the compound of the formula I prepared in Example 1 in methyl tert-butyl ether (MTBE), isopropyl acetate / heptane Or a mixed solvent of ethyl acetate / heptane, suspension stirring at room temperature for more than 48 hours, and finally crystal form IV is obtained. Alternatively, the compound of the formula I prepared in Example 1 is suspended and stirred at 50 ° C for more than 48 hours in a mixed solvent of ethyl acetate / heptane, and finally Form IV is obtained. Alternatively, the crystals prepared in Example 4 were suspended and stirred in a mixed solvent of water/acetone at 50 ° C for 12-14 days, and finally Form IV was obtained.

The present invention still further provides a crystal form of the compound of the formula I, which has a diffraction peak having a diffraction angle 2θ of 6.0°, 11.1°, 17.7°, 24.1° and 26.9° in the X-ray powder diffraction pattern. People define it as Form V.

The present invention further provides a preferred embodiment of the above-mentioned crystal form V. Preferably, the diffraction angles corresponding to the diffraction angle 2θ of the above-mentioned crystal form V are 14.8 Å, 8.0 Å, 5.0 Å, 3.7 Å, and 3.3 Å, respectively.

Preferably, the X-ray powder diffraction pattern has diffraction peaks having diffraction angles 2θ of 6.0°, 8.8°, 11.1°, 11.9°, 14.9°, 17.7°, 24.1° and 26.9°.

Preferably, the crystal plane spacing corresponding to the diffraction angle 2θ of the above-mentioned crystal form V is 14.8 Å, 10.0 Å, 8.0 Å, 7.4 Å, 6.0 Å, 5.0 Å, 3.7 Å, and 3.3 Å, respectively.

Preferably, the above crystal form V has an X-ray powder diffraction pattern as shown in FIG.

Table 5 summarizes the X-ray powder diffraction pattern shown in Figure 5.

table 5:

Preferably, the melting point is from 190 to 193 °C.

Preferably, the crystal form purity 85%.

Preferably, the crystal form purity 95%.

Preferably, the crystal form purity 99%.

The present invention also provides a method for preparing the above Form V, comprising the steps of: the compound of the formula I prepared in Example 1 in a mixed solvent of methyl tert-butyl ether (MTBE) / heptane at 50 ° C Suspension stirring for more than 48 hours, finally obtaining Form V; or, in a methanol solution of the compound of Structural Formula I prepared in Example 1, water is added as a reverse solution to precipitate, thereby obtaining Form V.

The present invention still further provides a crystal form of the compound of the formula I, which has a diffraction peak having a diffraction angle 2θ of 7.1°, 10.6°, 18.8°, 22.2° and 26.9° in the X-ray powder diffraction pattern. People define it as a crystal form VI.

The present invention further provides a preferred embodiment of the above-mentioned Form VI: Preferably, the interplanar spacing of the diffraction angle 2θ of the above Form VI is 12.4 Å, 8.4 Å, 4.7 Å, 4.0 Å, and 3.3 Å, respectively.

Preferably, the X-ray powder diffraction pattern has diffraction peaks having diffraction angles 2θ of 7.1°, 9.4°, 10.6°, 16.5°, 18.8°, 21.3°, 22.2°, and 26.9°.

Preferably, the interplanar spacing corresponding to the diffraction angle 2θ of the above-mentioned Form VI is 12.4 Å, 9.4 Å, 8.4 Å, 5.4 Å, 4.7 Å, 4.2 Å, 4.0 Å, and 3.3 Å, respectively.

Preferably, the above Form VI has an X-ray powder diffraction pattern as shown in FIG.

Table 6 summarizes the X-ray powder diffraction pattern shown in Figure 6.

Preferably, the melting point is from 200 to 203 °C.

Preferably, the crystal form purity 85%.

Preferably, the crystal form purity 95%.

Preferably, the crystal form purity 99%.

The invention also provides a preparation method of the above crystalline form VI, comprising the following steps: the compound of the structural formula I prepared in the embodiment 1 is dissolved in an acetonitrile/water (1:1 by volume) or tetrahydrofuran/water solvent. The suspension was stirred at room temperature for more than 48 hours, and finally the crystal form VI was obtained. or, Including the following steps, the compound of the structural formula I prepared in Example 1 was added to the crystal prepared in Example 5 as a seed crystal in a mixed solvent of methanol/ethyl acetate, and then volatilized and precipitated to obtain a crystal form VI.

The present invention still further provides a crystal form of the compound of the formula I, which has a diffraction peak having a diffraction angle 2θ of 6.9°, 11.7°, 15.1°, 21.1° and 25.8° in an X-ray powder diffraction pattern. It is defined by humans as Form VII.

The present invention further provides a preferred embodiment of the above Form VII: Preferably, the diffraction angle corresponding to the diffraction angle of the above Form VII is 12.8 Å, 7.5 Å, 5.9 Å, 4.2 Å, and 3.5 Å, respectively.

Preferably, the X-ray powder diffraction pattern has diffraction peaks having diffraction angles 2θ of 6.9°, 7.5°, 11.7°, 15.1°, 19.3°, 21.1°, 22.6° and 25.8°.

Preferably, the interplanar spacing of the diffraction angle 2θ of the above Form VII is 12.8 Å, 11.8 Å, 7.5 Å, 5.9 Å, 4.6 Å, 4.2 Å, 3.9 Å, and 3.5 Å, respectively.

Preferably, the above Form VII has an X-ray powder diffraction pattern as shown in FIG.

Table 7 summarizes the X-ray powder diffraction pattern shown in Figure 7.

Preferably, the crystal form purity 85%.

Preferably, the crystal form purity 95%.

Preferably, the crystal form purity 99%.

The present invention also provides a process for the preparation of the above Form VII, comprising the steps of heating the Form VI prepared in Example 7 to 180 ° C.

The invention further provides for the use of the above crystalline forms.

A pharmaceutical composition comprising a therapeutically effective amount of any of the crystalline forms provided herein, and a pharmaceutically acceptable excipient, adjuvant or carrier.

The invention further provides a preferred embodiment of the above pharmaceutical composition: preferably, a therapeutically effective amount of the above crystalline form; and at least one additional active ingredient.

Preferably, the pharmaceutical composition is suitable for oral administration.

Preferably, the pharmaceutical composition is a tablet or capsule.

Preferably, the above crystalline form is contained in the pharmaceutical composition in an amount of from 1 to 99% by weight.

Preferably, the above crystalline form is contained in the pharmaceutical composition in an amount of from 1 to 70% by weight.

Preferably, the above crystalline form is contained in the pharmaceutical composition in an amount of from 10 to 30% by weight.

The use of the above crystalline form for the preparation of a medicament for regulating the level of HIF in vivo or its activity.

The invention also provides a preferred technical solution for the above application: Preferably, the crystalline form is used in the manufacture of a medicament for the treatment of a disease associated with HIF level or HIF activity.

Preferably, the crystalline form is used in the manufacture of a medicament for the treatment of anemia, ischemia or a disease associated with anemia or ischemia.

Preferably, the crystalline form is used in the preparation of a medicament for treating a disease of a patient, wherein the disease of the patient is selected from the group consisting of ischemia, anemia, wound healing, orthotopic transplantation, ectopic transplantation, allogeneic transplantation, and high Blood pressure, thalassemia, diabetes, cancer or various inflammations, or a combination of two or more of these diseases.

All of the above crystalline forms may be present in substantially pure form.

The term "substantially pure" as used herein is understood in the context of the present invention to mean, in particular, at least 85%, preferably at least 95%, optimally at least 99% by weight of the compound of the formula I. The crystalline forms described are, in particular, crystalline Form I, Form II, Form III, Form IV, Form V, Form VI and Form VII. The above crystal forms only summarize the main peaks (i.e., the most characteristic, significant, unique, and/or reproducible peaks). The main peak described above can be reproduced and within the margin of error (±2 at the given decimal point position in the present invention, i.e., the value ± 0.2).

In the present invention, "having an X-ray powder diffraction pattern as shown in Fig. 1" means that the X-ray diffraction pattern is substantially as shown in Fig. 1, that is, the relative intensity is the largest in the map shown in Fig. 1. The relative intensity of the line exceeds 10%, especially those above 30%, compared to the line (the relative intensity of the line is set to 100). In other words, in the present invention, having an X-ray powder diffraction pattern as shown in FIG. 2, FIG. 3, FIG. 4, FIG. 5, FIG. 6, or FIG. 7 means that the X-ray diffraction pattern is substantially as As shown in FIG. 2, FIG. 3, FIG. 4, FIG. 5, FIG. 6, or FIG. 7, at least FIG. 2, FIG. 3, FIG. 4, FIG. 5, FIG. The main line in the spectrum, that is, the line with the highest relative intensity (the relative intensity of the line is set to 100) in the map shown in the figure, the relative intensity of the line exceeds 10%, especially over 30%. Those lines.

The invention also provides a process for the preparation of a compound of formula I, as follows:

The invention further provides a process for the preparation of Form I, Form II, Form III, Form IV, Form V, Form VI or Form VII of the compound of Formula I.

Crystallization of a compound of the invention from a suitable solvent system containing at least one solvent may be by natural crystallization (solvent evaporation), temperature drop and/or by the addition of an antisolvent (i.e., a solvent in which the compound of the invention is less soluble). Supersaturation is achieved in the solvent system.

Crystallization can be achieved with or without the use of seed crystals suitable for crystallization of the compounds of the invention.

The present invention still further provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of the formula I or a crystalline form I, a crystalline form II, a crystalline form III, a crystalline form IV, a crystalline form V, a crystalline form VI and a crystalline form thereof. One or more of VII, and a pharmaceutically acceptable excipient or adjuvant or carrier. Wherein the active compound of the formula I, or any of the above crystal form I, form II, form III, form IV, form V, form VI and form VII, in the drug The content in the composition is from 1 to 99% by weight, preferably from 1 to 70% by weight, more preferably from 10 to 30% by weight.

Further, the present invention also provides a compound of the formula I or any one of the above crystal forms I, Form II, Form III, Form IV, Form V, Form VI and Form VII in preparation for conditioning Application in HIF level or active drugs.

Still further, the present invention also provides a compound of the formula I or any one of the above crystal forms I, Form II, Form III, Form IV, Form V, Form VI and Form VII in preparation for use in the preparation of The use of drugs for the treatment of diseases associated with anemia or ischemia.

Still further, the present invention also provides a compound of the formula I or any one of the above crystal forms I, Form II, Form III, Form IV, Form V, Form VI and Form VII in preparation for use in the preparation. Use in the treatment of drugs for ischemia, anemia, wound healing, orthotopic transplantation, ectopic transplantation, allogeneic transplantation, hypertension, thalassemia, diabetes, cancer or various inflammations, or a combination of two or more of these diseases.

As used herein, "therapeutically effective amount" means a compound which, when used in the treatment of an article, treats and prevents and/or inhibits at least one clinical condition of a disease, condition, symptom, indication, and/or discomfort, is sufficient for the disease An effective dose that produces a certain effect in the treatment of the condition, indications, discomfort or symptoms. The specific "therapeutically effective amount" may vary depending on the compound, the route of administration, the age of the patient, the weight of the patient, the type, symptom and severity of the disease or condition being treated. Wherever practicable, a suitable dosage will be apparent to those skilled in the art and may be determined by routine experimentation. In the case of combination therapy, "therapeutically effective amount" refers to the total amount of a combination of articles effective to treat the disease.

The compounds of the invention may be administered to a patient in need of treatment by oral, nasal, rectal, parenteral or topical administration in the form of a composition. When used for oral administration, it can be made often Solid dosage forms such as tablets, powders, granules, capsules, etc., in liquid dosage forms such as water or oil suspensions or other liquid dosage forms such as syrups, solutions, suspensions, etc.; for parenteral administration, It is prepared as a solution for injection, an aqueous solution, an oily suspension, a lyophilized powder, and the like. Preferred dosage forms are tablets, coated tablets, capsules, suppositories, nasal sprays or injections, more preferably tablets or capsules. The pharmaceutical composition may be in unit dosage form suitable for single administration of precise dosages. In addition, the pharmaceutical composition may also include other active ingredients.

The various dosage forms of the pharmaceutical compositions of the present invention can be prepared according to conventional methods of manufacture in the pharmaceutical arts. For example, the active ingredient is mixed with one or more excipients and then formulated into the desired dosage form.

As used herein, "pharmaceutically acceptable carrier" refers to any pharmaceutically acceptable carrier suitable for the desired pharmaceutical formulation in the pharmaceutical art, such as diluents such as water, various organic solvents and the like; fillers such as starch, sucrose, etc.; binders Such as cellulose derivatives, alginates, gelatin and PVP (polyvinylpyrrolidone); wetting agents such as glycerin; disintegrants such as agar, calcium carbonate and sodium hydrogencarbonate; absorption enhancers such as quaternary ammonium compounds; surfactants such as ten Hexadecanol; absorption carriers such as kaolin and soap clay; lubricants such as talc, calcium stearate, magnesium stearate and polyethylene glycol. It is also possible to add other pharmaceutically acceptable excipients such as dispersing agents, stabilizers, thickeners, complexing agents, buffers, penetration enhancers, polymers, fragrances, sweeteners and dyes to the pharmaceutical compositions. . It is preferred to use an excipient suitable for the desired dosage form and the desired mode of administration.

As used herein, "disease" means that the term "disease" refers to any disease, discomfort, condition, symptom or indication.

Figure 1: X-ray powder diffraction diffraction pattern of the crystalline form I of the compound of the formula I; Figure 2: X-ray powder diffraction diffraction pattern of the crystalline form II of the compound of the formula I; Figure 3: X-ray powder diffraction diffraction pattern of the crystalline form III of the compound of the formula I; Figure 4: Structure X-ray powder diffraction-diffraction pattern of Form IV of the compound of Formula I; Figure 5: X-ray powder diffraction-diffraction pattern of Form V of the compound of Formula I; Figure 6: Structural Formula I X-ray powder diffraction-diffraction pattern of Form VI of the compound shown; Figure 7: X-ray powder diffraction-diffraction pattern of Form VII of the compound of Formula I.

The X-ray powder diffraction pattern shown in Figures 1, 2, 3, 4, 5, 6, and 7 was measured on a PANalytical X-ray diffraction system with an Empyrean console. Single crystal germanium was corrected. The 2θ diffraction peak of single crystal germanium is 28.443°, and the X-ray source is a K-alpha beam of an Empyrean copper X-ray tube.

The invention is further illustrated by the following examples and test examples, but the examples and test examples do not constitute any limitation to the scope of the invention. In the specific embodiments of the present invention, unless otherwise stated, it is a conventional technical means or method of the art.

Example 1

Synthesis of the compound of formula I:

Synthesis of Compound 1

Under the protection of an inert gas (such as nitrogen), add 9.2 g of 4-nitrophthalonitrile, 5.0 g of phenol, 7.3 g of potassium carbonate, 40 ml of DMSO, and stir at room temperature for 48 hours, then raise the temperature to 60 ° C. After the lapse of time, the heating was stopped, and after cooling, it was precipitated and filtered to give a yellow solid.

Synthesis of Compound 2

11.3 g of the compound 1 was dissolved in 30 ml of methanol, and added to 25 ml of a 50% sodium hydroxide solution, and the mixture was heated under reflux for 48 hours. After completion of the reaction, pH was adjusted to about 3 with concentrated hydrochloric acid, and the precipitate was precipitated, filtered, and dried to give 10.5 g of Compound 2.

Synthesis of Compound 3

6.0 g of the 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 to remove the solvent to obtain the compound 3.

Synthesis of Compound 4

Compound 3 (6.0 g) and 2.65 g of methyl isocyanoacetate were dissolved in 60 ml of tetrahydrofuran, and 3.54 g of DBU (CAS registration number: 6674-22-2) was added dropwise at room temperature, and the mixture was stirred at room temperature after completion of the dropwise addition. After 1 hour, the impurities were extracted with ethyl acetate under basic conditions to remove impurities. The aqueous phase was adjusted to pH 3 with dilute hydrochloric acid, and ethyl acetate was evaporated, washed with water and dried over anhydrous Na ₂ SO ₄ to give 9.0 g of Compound 4.

Synthesis of Compound 5

9.0 g of the compound 4 was dissolved in methanol, and concentrated hydrochloric acid was added thereto, and the mixture was heated to 60 degrees for 4 hours. A solid was produced during the reaction. After the reaction was completed, 5.8 g of the crude product of the compound 5 was obtained by filtration, and the column chromatography was purified to obtain 1.85. g compound 5.

Synthesis of Compound 6

1.77 g of the compound 5 and 10 ml of phosphorus oxychloride were heated to 70 ° C for 3 hours, cooled, poured into ice, and the phosphorus oxychloride was completely decomposed, and the precipitate was eluted, filtered, and washed with water to obtain 1.45 g of the compound 6.

Synthesis of Compound 7

Under a nitrogen atmosphere, 1.41 g of compound 6, 20 ml of dioxane, 0.49 g of tetrakis(triphenylphosphine)palladium, 1.78 g of potassium carbonate, 0.54 g of trimethylborane were added, and the mixture was stirred and heated under reflux for 3 hours. After stirring for 48 hours, it was concentrated and the mixture was extracted with ethyl acetate. After column chromatography purification, 0.42 g of Compound 7 was obtained.

Synthesis of Compound 8

1.02 g of the compound 7 was added to a mixture of 10 ml of ethanol and 10 ml of 2 mol/L of sodium hydroxide, and the mixture was refluxed for 1.5 hours. After a small amount of impurities were filtered off, ethanol was removed to obtain a pale yellow solid, which was filtered and washed with water to give 0.5 g of Compound 8.

Synthesis of Compound 9

0.37 g of compound 8, 0.44 g of glycine methyl ester hydrochloride, 1.00 g of PyBOP (CAS registration number: 128625-52-5) was added to 15 ml of dichloromethane, and 0.74 ml of triethylamine, 1.0 ml of di(isopropyl) was added. Ethylamine was stirred at room temperature for 3 hours, filtered, and the organic phase was washed with water, dried, and purified by column chromatography to give the compound.

Synthesis of Compound 10, a compound of formula I

0.28 g of the compound 9 was dissolved in 10 ml of tetrahydrofuran, 5 ml of 1 mol/L sodium hydroxide was added, and the reaction was stirred at room temperature for 1 hour, the tetrahydrofuran was removed, the pH was adjusted to 3 with dilute hydrochloric acid, and the mixture was filtered and dried to remove solvent. 0.21 g of compound 10, i.e., 0.21 g of the compound of formula I, [M+H] ⁺ 353.4.

Example 2

Preparation of Form I of Compound of Formula I

The compound of the above formula I prepared is dissolved in a methanol/MTBE (methyl tert-butyl ether) mixed solvent at room temperature, and then volatilized and precipitated to obtain crystal form I.

Melting point: 174-177 ° C.

Example 3

Preparation of Form II of Compound of Formula I

The compound of the formula I prepared as described above is suspended and stirred at room temperature or at 50 ° C for more than 48 hours in a mixed solvent of water / acetonitrile (3:1 by volume) or water / ethanol; or The compound of the formula I prepared as described above was suspended and stirred at room temperature for more than 48 hours in a mixed solvent of methanol/water; finally, Form II was obtained.

Melting point: 209-212 ° C.

Example 4

Preparation of Form III of Compound of Formula I

In the greenhouse, the compound of the formula I prepared above is first dissolved in a methanol/acetonitrile mixed solvent, and then volatilized and precipitated to obtain crystal form III. Alternatively, the compound of the formula I prepared as described above is dissolved in water, dichloromethane, isopropyl acetate (IPAc), ethyl acetate, isopropyl acetate/heptane or water/acetone at 50 ° C. The suspension was stirred for more than 48 hours, and finally Form III was obtained.

Melting point: 198-200 ° C.

Example 5

Preparation of Form IV of Compound of Formula I

The compound of the formula I prepared as described above is in a mixed solvent of methyl tert-butyl ether, methyl tert-butyl ether / heptane, isopropyl acetate / heptane, ethyl acetate / heptane or water / acetone. The suspension was stirred at room temperature for more than 48 hours, and finally, Form IV was obtained. Alternatively, the compound of the formula I prepared as described above is suspended and stirred at 50 ° C for more than 48 hours in a mixed solvent of ethyl acetate / heptane, and finally crystal form IV is obtained. Alternatively, the crystals prepared in Example 4 were suspended and stirred in a mixed solvent of water/acetone at 50 ° C for 12-14 days, and finally, Form IV was obtained.

Melting point: 204-207 ° C.

Example 6

Preparation of Form V of Compound of Formula I

The compound of the formula I prepared as described above is suspended and stirred in a mixed solvent of methyl tert-butyl ether/heptane at 50 ° C for more than 48 hours, and finally the form V is obtained; or the methanol of the compound of the formula I is obtained. Water is added to the solution as a reverse solution to precipitate it, and finally, Form V is obtained.

Melting point: 190-193 ° C.

Example 7

Preparation of Form VI of Compound of Formula I

The compound of the formula I prepared as described above is suspended and stirred at room temperature for more than 48 hours in an acetonitrile/water (1:1 by volume) or tetrahydrofuran/water solvent, and finally the form VI is obtained. Alternatively, the compound of the formula I prepared as described above is added to the crystal prepared in Example 5 as a seed crystal in a mixed solvent of methanol/ethyl acetate at room temperature, and then volatilized and precipitated to obtain a crystal form VI.

Melting point: 200-203 ° C.

Example 8

Preparation of Form VII of Compound of Formula I

The crystal prepared in Example 6 was heated to 180 ° C to obtain Form VII.

Example 9

Determination of HIF-PHD2 enzyme activity

The HIF-PHD2 enzyme activity was determined using a similar TR-FRET technique (see US 2008/004817; Dao JH et al., Anal Biochem. 2009, 384: 213-23). In the upper half of the 96-well plate, 2 μL of test compound in DMSO and 40 μL of assay buffer (50 mM Tris PH7.4/0.01% Tween-20/0.1 mg/ml BSA/1 mM sodium ascorbate/) were added dropwise to each well. 20 μg/ml catalase/10 μM ferrous sulfate), which also contained 600 nM of the full-chain PHD2 enzyme. Pre-incubation was carried out for 30 minutes at room temperature, and 8 μL of the substrate (final concentration of 0.2 μM 2-ketovaleric acid and 0.5 μM biotin-labeled short peptide DLDLEMLAPYIPMDDDFQL of HIF-1α) was added to initiate the enzymatic reaction. After 2 hours of reaction, 50 μL of a quencher/detection reagent mixture was added at room temperature (final concentration of 1 mM phenanthroline, 0.1 mM EDTA, 0.5 nM anti-(His) ₆ LANCE reagent, 100 nM AF647-labeled Streptavidin and 30nM(His) ₆ -VHL-elonginB-elonginC). The time-resolved fluorescence signal intensity ratio at 665 and 620 nm was measured while measuring the fluorescence signal of the uninhibited control, and the percent inhibition of the sample compound was calculated. The IC50 measurement of the compound of formula I prepared according to the procedure of Example 1 was about 2 [mu]M.

Example 10

Determination of erythropoietin (EPO) in normal mice

Eight-week old C57BL/6 male rats were orally administered with a suspension containing one of the crystal forms of Compound I at doses of 20, 60, 100 mg/kg, respectively, and the solvent was 0.5% CMC. Serum samples were obtained from the orbital veins 6 hours after administration (see Robinson A, et al., Gastroenterology. 2008, 134: 145-55; Hsieh MM, et al., Blood. 2007, 110: 2140-7). The erythropoietin in the sample was analyzed using an electrochemiluminescence immunoassay (MSD) as directed. When given a blend of Form VI In the suspension, the amount of erythropoietin produced by the stimulation in the sample was higher than that of the erythropoietin in the blank control sample without stimulation.

Example 11

Determination of crystal form stability

8.3 mg of the compound of formula I prepared according to Example 1 was added to 1 mL of isopropyl acetate, stirred and filtered to remove insolubles. 9.6 mg of Form IV and 1.97 mg of Form VI were added to the solution and stirred at room temperature for 36 hours. After centrifugation and drying, the resulting crystals were determined to be pure Form VI, indicating that Form VI is the most thermodynamically stable crystalline form in the study.

no

Claims (38)

A crystalline form III of a compound of the formula I, wherein the X-ray powder diffraction pattern of the crystalline form III has diffraction at a diffraction angle 2θ of 6.2°, 17.8°, 22.0°, 26.2° and 26.9° peak According to the crystal form III described in claim 1, wherein the diffraction angle 2θ of the crystal form III corresponds to a pitch of 14.3 Å, 5.0 Å, 4.0 Å, 3.4 Å, and 3.3 Å, respectively. The crystal form III according to claim 1, wherein the X-ray powder diffraction pattern of the crystal form III has a diffraction angle 2θ of 6.2°, 12.1°, 15.6°, 17.8°, 22.0°, 26.2°, 26.9. ° and a diffraction peak of 28.9 °. According to the crystal form III described in claim 3, wherein the diffraction angle 2θ of the crystal form III corresponds to a plane spacing of 14.3 Å, 7.3 Å, 5.7 Å, 5.0 Å, 4.0 Å, 3.4 Å, 3.3 Å, and 3.1Å. The crystal form III according to any one of claims 1 to 4, wherein the crystal form III has a melting point of 198 to 200 °C. Form III according to claim 5, wherein the crystal form III purity 85%. Form III according to claim 6, wherein the crystal form III purity 95%. Form III according to claim 7, wherein the crystal form III purity 99%. Form VI of a compound of formula I, wherein the X-ray powder diffraction pattern of the form VI has a diffraction peak with diffraction angles 2θ of 7.1°, 10.6°, 18.8°, 22.2°, and 26.9° The crystal form VI according to claim 9, wherein the diffraction angle 2θ of the crystal form VI corresponds to a pitch of 12.4 Å, 8.4 Å, 4.7 Å, 4.0 Å, and 3.3 Å, respectively. The crystal form VI according to claim 9, wherein the X-ray powder diffraction pattern of the form VI has a diffraction angle 2θ of 7.1°, 9.4°, 10.6°, 16.5°, 18.8°, 21.3°, 22.2. ° and a diffraction peak of 26.9 °. According to the crystal form VI described in claim 11, wherein the diffraction angle 2θ of the crystal form VI corresponds to a pitch of 12.4 Å, 9.4 Å, 8.4 Å, 5.4 Å, 4.7 Å, 4.2 Å, 4.0 Å, and 3.3Å. The crystalline form VI according to any one of claims 9 to 12, wherein the crystalline form VI has a melting point of from 200 to 203 °C. Form VI according to claim 13, wherein the crystal form VI is pure 85%. Form VI according to claim 14, wherein the crystal form VI is pure 95%. Form VI according to claim 15 wherein the crystal form VI is pure 99%. a method for preparing a compound of the formula I, Synthesis of Compound 1 Under the protection of an inert gas, 4-nitrophthalonitrile, phenol, potassium carbonate and DMSO were added, and the reaction was stirred at room temperature for 45-50 hours, and then the temperature was raised to 55-65 ° C for 1.5-2.5 hours. The reaction solution is removed to obtain Compound 1 ; Compound 2 is synthesized. Compound 1 is dissolved in methanol, added to 40-60% sodium hydroxide solution, and heated to reflux. After completion of the reaction, pH is adjusted to 2.5-3.5 with concentrated hydrochloric acid. The reaction solution is removed to obtain the compound 2 ; the synthesis of the compound 3 is carried out by dissolving the compound 2 in glacial acetic acid and acetic anhydride, heating to reflux until the reaction is completed, and removing the solvent to obtain the compound 3 ; the synthesis of the compound 4 is the compound 3 and the isocyano group. The methyl acetate was dissolved in tetrahydrofuran, DBU was added dropwise at room temperature, and the mixture was stirred at room temperature for 0.5-1.5 hours. The impurities were extracted with ethyl acetate under basic conditions to remove impurities. The aqueous phase was adjusted to pH=2.5 with dilute hydrochloric acid. 3.5, the reaction solution was removed, to obtain compound 4; synthesis of compound 5 compound 4 was dissolved in methanol, and concentrated hydrochloric acid was heated to 55-65 deg.] C 3.5-4.5 hours the reaction, the reaction solution was removed, to obtain compound 5; compound 6 compound 5 Phosphorus oxychloride the reaction was heated to 65-75 deg.] C 2.5-3.5 hours, cooled and poured into ice, phosphorus oxychloride was completely decomposed after the liquid phase was removed, to obtain compound 6; Synthesis of Compound 7 under the protection of inert gas, Adding compound 6 , dioxane, tetrakis(triphenylphosphine)palladium, potassium carbonate and trimethylborane, stirring and mixing, heating under reflux for 2.5-3.5 hours, then stirring at room temperature for 45-50 hours, removing the reaction solution. compound 7; after 8 synthesis of compound 7 compound of ethanol was added and 1.5-2.5mol / L sodium hydroxide was refluxed for 1-2 hours, a small amount of impurity filtered off, ethanol was removed to give a pale yellow solid, i.e. compound 8; compound Synthesis of 9 Compound 8 , glycine methyl ester hydrochloride, PyBOP was added to dichloromethane, triethylamine, di(isopropyl)ethylamine was added, and the reaction was stirred at room temperature for 2.5-3.5 hours to remove the reaction solution. Compound 9 ; Compound 10, that is, the compound of the formula I, the compound 9 is dissolved in tetrahydrofuran, 0.5-1.5 mol/L sodium hydroxide is added, and the reaction is stirred at room temperature for 0.5-1.5 hours to remove tetrahydrofuran, and diluted with Hydrochloric acid adjusted pH=3, washed with ethyl acetate, over Filtration and removal of the solvent gave compound 10 , a compound of formula I. A process for the preparation of the crystalline form I of the compound of the formula I of claim 1, wherein the preparation comprises the step of preparing the structure prepared according to the preparation method of claim 17 at room temperature. The compound of the formula I is first dissolved in a mixed solvent of methanol/methyl tert-butyl ether, and then volatilized and precipitated, wherein the X-ray powder diffraction pattern of the crystal form I has a diffraction angle 2θ of 5.9° and 11.0°. , 16.6°, 22.6°, 25.9°, and 26.9°; or diffraction peaks of 5.9°, 11.0°, 14.8°, 17.6°, 22.6°, 24.0°, 25.9°, and 26.9°. A process for the preparation of the crystalline form II of the compound of the formula I of claim 1, wherein the process comprises the step of preparing the compound of the formula I according to the preparation method of claim 17 in water/acetonitrile ( 3:1) or water/ethanol mixed solvent, suspension stirring at room temperature or 50 ° C for more than 48 hours; or, in a mixed solvent of methanol / water, stirring at room temperature for more than 48 hours; Wherein the diffraction pattern of the X-ray powder of the Form II has diffraction angles 2 θ of 8.2°, 13.3°, 14.5°, 21.2° and 26.6°; or 8.2°, 9.6°, 13.3°, 14.5°, 21.2 Diffraction peaks of °, 22.8°, 25.4° and 26.6°. A process for the preparation of the crystalline form III of the compound of the formula I of claim 1, wherein the process comprises the steps of: the compound of the formula I prepared according to the preparation method of claim 17 at room temperature Dissolving in a methanol/acetonitrile mixed solvent, and then volatilizing and precipitating, wherein the X-ray powder diffraction pattern of the crystal form III has a diffraction angle 2θ of 6.2°, 17.8°, 22.0°, 26.2° and 26.9°; Or diffraction peaks of 6.2°, 12.1°, 15.6°, 17.8°, 22.0°, 26.2°, 26.9° and 28.9°. A process for the preparation of the crystalline form III of the compound of the formula I of claim 1, wherein the preparation comprises the step of preparing the compound of the formula I according to the preparation method of claim 17 in water, dichloro In a mixed solvent of methane, isopropyl acetate, ethyl acetate or isopropyl acetate/heptane, the suspension is stirred at 50 ° C for more than 48 hours, and finally crystals are obtained, wherein the X-ray powder diffraction pattern of the crystal form III is There are diffraction peaks having diffraction angles 2θ of 6.2°, 17.8°, 22.0°, 26.2°, and 26.9°; or 6.2°, 12.1°, 15.6°, 17.8°, 22.0°, 26.2°, 26.9°, and 28.9°. A process for the preparation of the crystalline form IV of the compound of the formula I of claim 1, wherein the preparation comprises the step of preparing the compound of the formula I according to the preparation method of claim 17 in methyl tert-butyl The mixture is stirred and stirred at room temperature for more than 48 hours in a mixed solvent of isopropyl ether, isopropyl acetate/heptane or ethyl acetate/heptane, and finally crystals are obtained, wherein the crystal form IV has an X-ray powder diffraction pattern. The diffraction angles 2θ are 11.3°, 12.4°, 20.3°, 21.4°, and 26.6°; or diffraction peaks of 11.3°, 12.4°, 15.0°, 17.9°, 20.3°, 21.4°, 24.8°, and 26.6°. A process for the preparation of the crystalline form IV of the compound of the formula I of claim 1, wherein the preparation comprises the step of preparing the compound of the formula I according to the preparation method of claim 17 in ethyl acetate/ In a mixed solvent of heptane, the suspension is stirred at 50 ° C for more than 48 hours, wherein the diffraction pattern of the X-ray powder of the crystalline form IV has a diffraction angle 2θ of 11.3°, 12.4°, 20.3°, 21.4° and 26.6°; or diffraction peaks of 11.3°, 12.4°, 15.0°, 17.9°, 20.3°, 21.4°, 24.8° and 26.6°. A process for the preparation of the crystalline form V of the compound of the formula I of claim 1, wherein the preparation comprises the step of preparing the compound of the formula I according to the preparation method of claim 17 in methyl tert-butyl In a mixed solvent of ether/heptane, suspension and stirring at 50 ° C for more than 48 hours, and finally crystals are obtained; or, in a methanol solution of the compound of the formula I prepared according to the method of claim 17, water is added as a reverse solution. Precipitating, the crystal obtained, wherein the diffraction pattern of the X-ray powder of the Form V has a diffraction angle 2 θ of 6.0°, 11.1°, 17.7°, 24.1° and 26.9°; or 6.0°, 8.8°, 11.1 Diffraction peaks of °, 11.9°, 14.9°, 17.7°, 24.1° and 26.9°. A process for the preparation of a crystalline form VI of the compound of the formula I of claim 9 to 16, wherein the process comprises the step of preparing a compound of the formula I prepared in accordance with the preparation method of claim 17 in acetonitrile / In a solvent of water (1:1) or tetrahydrofuran/water, the suspension is stirred at room temperature for more than 48 hours, and finally crystals are obtained, wherein the crystal form VI has a diffraction angle 2θ in the diffraction pattern of the X-ray powder. They are diffraction peaks of 7.1, 10.6, 18.8, 22.2 and 26.9; or 7.1, 9.4, 10.6, 16.5, 18.8, 21.3, 22.2 and 26.9. A process for the preparation of a crystalline form VI of the compound of the formula I of claim 9 to 16 wherein the process comprises the steps of the formula I prepared according to the preparation method of claim 17 at room temperature. The compound is dissolved in a mixed solvent of methanol/ethyl acetate, and the crystal form prepared by the preparation method according to claim 22 or 23 is added as a seed crystal, and then volatilized, and the precipitate is obtained, wherein the X-ray powder diffraction pattern of the crystal form VI is obtained. a diffraction peak having a diffraction angle 2θ of 7.1°, 10.6°, 18.8°, 22.2°, and 26.9°; or 7.1°, 9.4°, 10.6°, 16.5°, 18.8°, 21.3°, 22.2°, and 26.9° . A process for the preparation of the form VII of the compound of the formula I of claim 1, wherein the preparation comprises the step of heating the crystal prepared in claim 24 to 180 ° C, wherein the form VII The X-ray powder diffraction pattern has diffraction angles 2θ of 6.9°, 11.7°, 15.1°, 21.1°, and 25.8°; or 6.9°, 7.5°, 11.7°, 15.1°, 19.3°, 21.1°, 22.6°, and A diffraction peak of 25.8°. A pharmaceutical composition comprising a therapeutically effective amount of the crystalline form of any one of claims 1 to 16, and a pharmaceutically acceptable excipient, adjuvant or carrier. A pharmaceutical composition comprising at least a therapeutically effective amount of the crystalline form of any one of claims 1 to 16. The pharmaceutical composition according to claim 28 or 29, wherein the pharmaceutical composition is suitable for oral administration. The pharmaceutical composition according to claim 30, wherein the pharmaceutical composition is a tablet or a capsule. The pharmaceutical composition according to claim 31, wherein the content of the crystalline form according to any one of claims 1 to 16 is from 1 to 99% by weight. The pharmaceutical composition according to claim 32, wherein the content of the crystalline form according to any one of claims 1 to 16 is from 1 to 70% by weight. The pharmaceutical composition according to claim 33, wherein the content of the crystalline form according to any one of claims 1 to 16 is from 10 to 30% by weight. Use of the crystalline form of any one of claims 1 to 16 for the manufacture of a medicament for modulating HIF levels or activity in vivo. Use of the crystalline form of any one of claims 1 to 16 for the manufacture of a medicament for the treatment of a disease associated with HIF level or HIF activity. Use of the crystalline form of any one of claims 1 to 16 for the manufacture of a medicament for the treatment of an anemia or ischemia-related disease. The use of the crystalline form of any one of claims 1 to 16 for the preparation of a medicament for treating a disease in a patient, wherein the patient's disease is selected from the group consisting of ischemia, anemia, ischemia, wound healing, and in situ Transplantation, ectopic transplantation, allogeneic transplantation, hypertension, thalassemia, diabetes, cancer or various inflammations, or a combination of two or more of them.