WO2025167361A1 - Crystal form a of 6-((5,6-diphenyl-1,2,4-triazine-3-yl)(isopropyl)amino)-n-(methylsulfonyl)hexanamide, and use thereof and preparation method therefor - Google Patents

Abstract

The present application discloses a crystal form A of compound I 6-((5,6-diphenyl-1,2,4-triazine-3-yl)(isopropyl)amino)-N-(methylsulfonyl)hexanamide, a pharmaceutical composition comprising same, a use thereof and a preparation method therefor. The crystal form A of compound I of the present application has diffraction peaks in an X-ray powder diffraction pattern at least at the following diffraction angles 2θ: 9.50±0.2°, 11.05±0.2°, 15.54±0.2°, 16.23±0.2°, 18.23±0.2° and 22.13±0.2°. The crystal form A of compound I of the present application has the advantages of low hygroscopicity, good stability and the like, and has great significance for drug development.

Description

6-((5,6-diphenyl-1,2,4-triazin-3-yl)(isopropyl)amino)-N-(methylsulfonyl)hexanamide crystalline form A and its use and preparation method

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Chinese patent application CN202410169645.1, filed on February 6, 2024. This application claims priority to Chinese patent application CN202410898621.X, filed on July 5, 2024. This application cites the full text of the above-mentioned Chinese patent application.

Technical Field

The present application belongs to the field of pharmacy, and specifically relates to a crystalline form of the compound 6-((5,6-diphenyl-1,2,4-triazin-3-yl)(isopropyl)amino)-N-(methylsulfonyl)hexanamide, its pharmaceutical composition, use and preparation method.

Background Art

Pulmonary arterial hypertension (PAH) is a rare, incurable pulmonary vascular disease that can gradually lead to right heart failure and ultimately death. PAH is characterized by pulmonary microvascular remodeling, which leads to a progressive increase in pulmonary artery resistance (PVR), which in turn causes right heart failure. This makes PAH a progressive and fatal disease. 75% of PAH patients die within 5 years of diagnosis, with an average survival of 1.9 years after symptom onset. It is often referred to as the "malignant tumor of the cardiovascular and pulmonary vascular system."

Currently, treatment options for PAH worldwide include conventional therapy and targeted therapies. Conventional therapy often only improves symptoms but cannot effectively halt disease progression. Regarding targeted therapy, there are three pathways for PAH: the nitric oxide pathway, the endothelin pathway, and the prostacyclin (PGI2) pathway. PGI2 is an important endothelial relaxing factor that stimulates cyclic adenosine monophosphate (cAMP) production, causing pulmonary vascular smooth muscle relaxation and inhibiting smooth muscle growth. PGI2 deficiency can cause pulmonary hypertension, making PGI2 drugs the most active treatment for PAH. PGI2 drugs include PGI2 analogs and PGI2 receptor agonists. PGI2 analogs, which share a native PGI2 backbone, are rapidly metabolized and have a very short biological half-life. They require frequent dosing or intravenous infusion, leading to poor patient compliance. Furthermore, PGI2 analogs have poor target selectivity, making it difficult to separate their therapeutic effects from other effects and prone to adverse reactions.

The compound of formula I, also known as Compound I, has the chemical name 6-((5,6-diphenyl-1,2,4-triazin-3-yl)(isopropyl)amino)-N-(methylsulfonyl)hexanamide, and its structural formula is shown in Formula I. It is a PGI2 receptor agonist with a novel structure and good drugability. Compound I has strong target selectivity, and its agonist activity at the prostacyclin IP receptor is more than 1,000 times that of the other seven prostacyclin receptor targets. It mainly activates the IP receptor to promote the production of cAMP in pulmonary artery smooth muscle cells, thereby inhibiting abnormal contraction of the pulmonary artery, inhibiting the proliferation of pulmonary artery smooth muscle cells, and reducing pulmonary artery pressure, thereby achieving the treatment of pulmonary hypertension. Compared with similar drugs already on the market, it has higher efficacy and safety.

At present, the crystal form of Compound I has not been disclosed. This application focuses on the crystal form research of Compound I during the drug development process, and provides a drug crystal form with good stability, as well as its pharmaceutical composition, use and preparation method.

Technical issues

After extensive research, it was discovered that Compound I exists in various crystalline forms. Extensive research has been conducted on the crystalline forms of Compound I to identify and prepare crystalline forms that meet pharmaceutical requirements. Based on these studies, this application provides Compound I Form A, which is non-hygroscopic and exhibits excellent storage stability, making it suitable for formulation development. This application also provides pharmaceutical compositions and uses of Compound I Form A, as well as a method for preparing Compound I Form A, which operates under mild process conditions and is suitable for large-scale production.

Technical Solutions

To achieve the purpose of this application, the following technical solutions are adopted:

One object of the present application is to provide a crystalline form A of compound I, whose X-ray powder diffraction pattern expressed in 2θ angles has diffraction peaks at 9.50±0.2°, 11.05±0.2°, 15.54±0.2°, 16.23±0.2°, 18.23±0.2° and 22.13±0.2°.

In some embodiments, the crystalline form A of compound 1 has an X-ray powder diffraction pattern expressed in 2θ angles at 6.38±0.2°, 9.50±0.2°, 11.05±0.2°, 13.11±0.2°, 15.54±0.2°, 16.23±0.2°, 18.23±0.2°, 18.96±0.2°, 19.42±0.2°, 20.52±0.2°, 21.43±0.2°, 22.13±0.2°, and 23.07±0.2°.

In some embodiments, the crystalline form A of Compound I has an X-ray powder diffraction pattern expressed in 2θ angles as shown in FIG1 .

In some embodiments, when Form A of Compound 1 is characterized by TGA/DSC, its TGA graph can confirm that Form A does not contain crystalline water or solvate.

In some embodiments, when Form A of Compound I is characterized by TGA/DSC, its DSC chart shows that the melting point of Form A is 130°C-135°C.

In some embodiments, the TGA/DSC graph of Form A of Compound 1 is shown in FIG2 .

The second object of the present application is to provide a preparation method for Compound I Form A, which comprises the following steps: heating and dissolving Compound I in a solvent, cooling to 25°C to 45°C, slowly cooling to crystallize or keeping warm to crystallize, continuing to cool to 0°C to 10°C to keep warm to crystallize, separating, and drying to obtain Form A.

In some embodiments, the solvent is one or a mixed solvent of alcohols, ethers, esters, alkanes, ketones, acetonitrile, and water.

In some embodiments, the solvent is one or a mixed solvent of methanol, ethanol, isopropanol, acetonitrile, tetrahydrofuran, methyl tert-butyl ether, ethyl acetate, dichloromethane, n-hexane, acetone, and water.

In certain preferred embodiments, the solvent is one or a mixed solvent of isopropyl alcohol, ethanol or water.

In certain preferred embodiments, the solvent is isopropyl alcohol.

In some embodiments, the mass volume ratio (g/mL) of the compound I to the solvent is 1:4-15.

In some embodiments, the heating and dissolving can be performed by heating and dissolving in a single solvent or a mixed solvent, or by first heating and dissolving in one solvent and then adding another one or more solvents.

In some embodiments, the heating condition is heating under reflux.

In some embodiments, the cooling to 25°C to 45°C can be performed in a variety of conventional ways, such as cooling in a water bath or a cold water bath, cooling by turning off the heating, or cooling at room temperature.

In some embodiments, the temperature is lowered to 25°C to 45°C, and the cooling time is 0-50 minutes.

In some embodiments, the temperature is lowered to 25°C to 45°C, preferably to 35°C to 45°C.

In some embodiments, the crystallization is carried out by slowly cooling or keeping the temperature at room temperature after cooling to 25°C to 45°C, which can be natural slow cooling and crystallization at room temperature, or keeping the temperature at room temperature.

In some embodiments, the crystallization by slow cooling or heat preservation can be performed by standing still or by stirring, such as paddle stirring, suspension stirring, etc.

In some embodiments, the crystallization is carried out by slowly cooling or keeping warm, and the crystallization time is 1 to 24 hours, preferably 1 to 3 hours.

In some embodiments, the continued cooling is performed in an ice bath or an ice salt bath.

In some embodiments, the temperature is continuously lowered to 0°C to 10°C for crystallization, and the crystallization can be performed by standing still or under stirring, such as paddle stirring, suspension stirring, etc.

In some embodiments, the temperature is continued to be lowered to 0°C to 10°C for crystallization, and the crystallization time is 1 to 24 hours, preferably 1 to 2 hours.

In some embodiments, the separation can be performed by conventional methods, such as centrifugation or filtration.

In some embodiments, the drying is a conventional drying method, such as vacuum drying.

In some embodiments, the drying entails drying to constant weight.

The third object of the present application is to provide a second method for preparing Form A of Compound I, which comprises the following steps: dissolving Compound I in a solvent by heating, cooling to 0°C to 10°C, preserving the temperature for crystallization, separating, and drying to obtain Form A.

In some embodiments, the solvent is one or a mixed solvent of alcohols, ethers, esters, alkanes, ketones, acetonitrile, and water.

In some embodiments, the solvent is one or a mixed solvent of methanol, ethanol, isopropanol, acetonitrile, tetrahydrofuran, methyl tert-butyl ether, ethyl acetate, dichloromethane, n-hexane, acetone, and water.

In certain preferred embodiments, the solvent is one or a mixed solvent of isopropyl alcohol, ethanol or water.

In certain preferred embodiments, the solvent is isopropyl alcohol.

In some embodiments, the mass volume ratio (g/mL) of the compound I to the solvent is 1:3.5-15.

In some embodiments, the heating condition is heating under reflux.

In some embodiments, the heating and dissolving can be performed by heating and dissolving in a single solvent or a mixed solvent, or by first heating and dissolving in one solvent and then adding another one or more solvents.

In some embodiments, the cooling to 0°C to 10°C is performed in a cold water bath, an ice bath, or an ice salt bath.

In some embodiments, the temperature is lowered to 0°C to 10°C, and the cooling time is 5-15 minutes.

In some embodiments, in the heat preservation crystallization, the crystallization method can be static crystallization or crystallization under stirring, such as paddle stirring, suspension stirring, etc.

In some embodiments, during the heat preservation crystallization, the crystallization time is 2 to 24 hours, preferably 2 to 5 hours.

In some embodiments, the separation can be performed by conventional methods, such as centrifugation or filtration.

In some embodiments, the drying is a conventional drying method, such as vacuum drying.

In some embodiments, the drying entails drying to constant weight.

The fourth object of the present application is to provide a third method for preparing Compound I Form A, which comprises the following steps: heating and dissolving Compound I in a solvent, cooling to 10°C to 25°C, keeping warm for crystallization, continuing to cool to 0°C to 10°C, keeping warm for crystallization, separating, and drying to obtain Form A.

In some embodiments, the solvent is one or a mixed solvent of alcohols, ethers, esters, alkanes, ketones, acetonitrile, and water.

In some embodiments, the solvent is one or a mixed solvent of methanol, ethanol, isopropanol, acetonitrile, tetrahydrofuran, methyl tert-butyl ether, ethyl acetate, dichloromethane, n-hexane, acetone, and water.

In certain preferred embodiments, the solvent is one or a mixed solvent of isopropyl alcohol, ethanol or water.

In certain preferred embodiments, the solvent is isopropyl alcohol.

In some embodiments, the mass volume ratio (g/mL) of the compound I to the solvent is 1:4-15.

In some embodiments, the heating condition is heating under reflux.

In some embodiments, the heating and dissolving can be performed by heating and dissolving in a single solvent or a mixed solvent, or by first heating and dissolving in one solvent and then adding another one or more solvents.

In some embodiments, the cooling to 10°C to 25°C is performed in a cold water bath or a water bath.

In some embodiments, the temperature is lowered to 10°C to 25°C, and the cooling time is 10 to 40 minutes.

In some embodiments, the temperature is lowered to 10° C. to 25° C. and then kept warm for crystallization. The crystallization can be performed by standing still or under stirring, such as paddle stirring, suspension stirring, etc.

In some embodiments, the temperature is lowered to 10° C. to 25° C. and then kept warm for crystallization, and the crystallization time is 0 to 24 hours, preferably 1 to 3 hours.

In some embodiments, the further cooling to 0°C to 10°C is performed in an ice bath or an ice salt bath.

In some embodiments, the temperature is continuously lowered to 0°C to 10°C for crystallization, and the crystallization can be performed by standing still or under stirring, such as paddle stirring, suspension stirring, etc.

In some embodiments, the temperature is continued to be lowered to 0°C to 10°C for crystallization, and the crystallization time is 1 to 24 hours, preferably 1 to 2 hours.

In some embodiments, the separation can be performed by conventional methods, such as centrifugation or filtration.

In some embodiments, the drying is a conventional drying method, such as vacuum drying.

In some embodiments, the drying entails drying to constant weight.

The fifth object of the present application is to provide a pharmaceutical composition comprising a therapeutically effective dose of Compound I Form A and a pharmaceutically acceptable carrier and excipient.

Furthermore, the pharmaceutical composition can be formulated into a variety of dosage forms for easy administration, for example, oral preparations (such as tablets, capsules, granules, powders, solutions or suspensions, etc.); injectable preparations (such as injectable solutions or suspensions, or injectable dry powders that can be used immediately after adding a drug solvent before injection).

The sixth object of the present application is to provide a therapeutically effective dose of Compound I Form A or the pharmaceutical composition for use in the preparation of a drug for preventing and/or treating a disease or condition.

In certain preferred embodiments, the disease or condition is associated with PGI2 receptor agonism.

In certain preferred embodiments, the disease or condition is selected from: pulmonary hypertension, cardiovascular and cerebrovascular diseases related to platelet aggregation, and diabetic nephropathy.

The seventh object of the present application is to provide a therapeutically effective dose of Compound I Form A or the pharmaceutical composition thereof for use in the preparation of a PGI2 receptor agonist drug.

Definitions and Explanations of Terms

As used herein, the term "room temperature" or "RT" refers to an ambient temperature of 20 to 25°C (68-77°F).

On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present application.

Beneficial effects

The positive advances of this application are: Form A of Compound I of this application exhibits low hygroscopicity and good stability in accelerated and long-term stability studies, making it suitable for formulation development. Furthermore, experiments have shown that Form A has high bioavailability, indicating good drug absorption and enhancing efficacy. Furthermore, its preparation process is mild and suitable for large-scale production. Therefore, Form A offers significant advantages in terms of drugability and ease of industrial production, and is of great significance for drug development.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the XRPD spectrum of Compound 1 Form A prepared in Example 1;

FIG2 shows the TGA/DSC spectrum of Compound 1 Form A prepared in Example 1;

FIG3 shows the ¹ H-NMR spectrum of Compound I Form A prepared in Example 1;

FIG4 shows the IR spectrum of Compound 1 Form A prepared in Example 1;

FIG5 shows the XRPD superposition of Compound I Form A prepared in Example 1 at the end of the 6th month of the accelerated experiment, the end of the 6th month of the long-term experiment, and day 0;

FIG6 shows the average concentration-time curve of Compound I in plasma of Group 1 animals after intravenous administration in the bioavailability study of Experimental Example 7;

FIG7 shows the average concentration-time curve of Compound I in plasma after oral administration to the second group of animals in the bioavailability test of Experimental Example 7.

Modes for Carrying Out the Invention

The present application is further described in detail below through specific implementation methods, but this is only intended to help understand the present application so that professionals in the field can implement or use the present application, and does not constitute any limitation to the present application.

Example 1

10 g of the Compound I drug substance was dissolved in 150 mL of isopropanol by heating under reflux, then placed in a water bath and rapidly cooled to 45°C after 10 minutes. The water bath was removed, and the solution was naturally cooled slowly at room temperature and stirred for crystallization for 2 hours. A large amount of solid precipitated during the slow cooling process. The solution was then cooled to 10°C in an ice bath, stirred at this temperature for 1.5 hours, filtered, and vacuum dried to constant weight to obtain a sample of Compound I Form A with a yield of 91%.

Example 2

10 g of the Compound I drug substance was dissolved in 100 mL of anhydrous ethanol by heating under reflux. The mixture was then placed in a cold water bath and rapidly cooled to 35°C over 5 minutes. The cold water bath was removed and the mixture was stirred at 35°C for 1.5 hours to allow crystallization. A large amount of solid precipitated. The mixture was then cooled to 5°C in an ice bath, stirred at this temperature for 2.0 hours, filtered, and vacuum dried to constant weight to obtain a sample of Compound I Form A with a yield of 82%. The X-ray powder diffraction pattern of the sample obtained by this method was consistent with that of the Compound I Form A sample obtained in Example 1.

Example 3

10 g of the Compound I drug substance was dissolved in 80 mL of methanol under reflux. The heat was turned off, and the temperature was cooled to 40°C over 35 minutes. The mixture was then stirred and crystallized at 40°C for 1.0 hour, resulting in the precipitation of a large amount of solid. The mixture was then cooled to 0°C in an ice-salt bath, stirred at this temperature for 2.0 hours, filtered, and vacuum-dried to constant weight to obtain a sample of Compound I Form A in a yield of 68%. The X-ray powder diffraction pattern of the sample obtained by this method was consistent with that of the Compound I Form A sample obtained in Example 1.

Example 4

10 g of the Compound I drug substance was dissolved in 40 mL of acetonitrile under reflux. The heat was turned off and the temperature was cooled to 45°C over 25 minutes. The solution was then allowed to slowly cool naturally at room temperature while stirring for 1 hour to crystallize. A large amount of solid precipitated during the slow cooling process. The solution was then cooled to 0°C in an ice-salt bath, stirred at this temperature for 10 hours, filtered, and vacuum-dried to constant weight to obtain a sample of Compound I Form A in a yield of 61%. The X-ray powder diffraction pattern of the sample obtained by this method was consistent with that of the Compound I Form A sample obtained in Example 1.

Example 5

10 g of the Compound I drug substance was dissolved in 45 mL of ethyl acetate under reflux. The mixture was then allowed to stand at room temperature, cooled to 35°C over 15 minutes, and stirred at 35°C for 1.5 hours to allow crystallization. A large amount of solid precipitated. The mixture was then cooled to 5°C in an ice bath, stirred at this temperature for 20 hours, filtered, and vacuum-dried to constant weight to obtain a sample of Compound I Form A in a 64% yield. The X-ray powder diffraction pattern of the sample obtained by this method was consistent with that of the Compound I Form A sample obtained in Example 1.

Example 6

10 g of the Compound I drug substance was dissolved in 40 mL of acetone under reflux. The mixture was then allowed to stand at room temperature for 25 minutes, cooled to 26°C, and allowed to crystallize at 26°C for 20 hours. A large amount of solid precipitated. The mixture was then cooled to 10°C in an ice bath, allowed to crystallize at this temperature for 24 hours, filtered, and vacuum-dried to constant weight to obtain a sample of Compound I Form A with a yield of 60%. The X-ray powder diffraction pattern of the sample obtained by this method was consistent with that of the Compound I Form A sample obtained in Example 1.

Example 7

10 g of the Compound I drug substance was dissolved in 50 mL of tetrahydrofuran under reflux. 100 mL of n-hexane was added, the heat was turned off, and the temperature was cooled to 35°C over 45 minutes. The temperature was then slowly cooled naturally at room temperature while stirring for 3.0 hours to allow crystallization. A large amount of solid precipitated during the slow cooling process. The solution was then cooled to 8°C in an ice bath, stirred at this temperature for 12.0 hours, filtered, and vacuum dried to constant weight to obtain a sample of Compound I Form A with a yield of 78%. The X-ray powder diffraction pattern of the sample obtained by this method was consistent with that of the Compound I Form A sample obtained in Example 1.

Example 8

10 g of the Compound I drug substance was dissolved in 30 mL of dichloromethane under reflux. 60 mL of methyl tert-butyl ether was added, and the mixture was allowed to cool slowly at room temperature while stirring for 3.0 hours to allow crystallization. A large amount of solid precipitated. The mixture was then cooled to 2°C in an ice-salt bath, stirred at this temperature for 5.0 hours, filtered, and vacuum-dried to constant weight to obtain a sample of Compound I Form A in a yield of 73%. The X-ray powder diffraction pattern of the sample obtained by this method was consistent with that of the Compound I Form A sample obtained in Example 1.

Example 9

10 g of the Compound I drug substance was dissolved in 150 mL of a mixed solvent of isopropanol and ethanol (volume ratio 1:1) under reflux. The heat was turned off, and the temperature was cooled to 35°C over 50 minutes. The mixture was then stirred and crystallized at 35°C for 2.0 hours. A large amount of solid precipitated. The temperature was then further cooled to 6°C in an ice bath, stirred at this temperature for 2.0 hours, filtered, and vacuum-dried to constant weight to obtain a sample of Compound I Form A in an 87% yield. The X-ray powder diffraction pattern of the sample obtained by this method was consistent with that of the Compound I Form A sample obtained in Example 1.

Example 10

10 g of the Compound I drug substance was dissolved in 100 mL of 95% ethanol under reflux. The solution was then placed in a water bath and rapidly cooled to 35°C over 15 minutes. The water bath was removed and the solution was allowed to slowly cool naturally at room temperature while stirring for 2 hours to crystallize. A large amount of solid precipitated during the slow cooling process. The solution was then cooled to 10°C in an ice bath, stirred at this temperature for 1 hour, filtered, and vacuum dried to constant weight to obtain a sample of Compound I Form A with a yield of 85%. The X-ray powder diffraction pattern of the sample obtained by this method was consistent with that of the Compound I Form A sample obtained in Example 1.

Example 11

10 g of the Compound I drug substance was dissolved in 60 mL of a mixed solvent of acetone and water (5:3 by volume) under reflux. The mixture was then placed in a water bath and rapidly cooled to 28°C over 15 minutes. The water bath was removed and the mixture was allowed to slowly cool naturally at room temperature while stirring for 3.0 hours to allow crystallization. A large amount of solid precipitated during the slow cooling process. The mixture was then cooled to 3°C in an ice bath, stirred at this temperature for 1.6 hours, filtered, and vacuum dried to constant weight to obtain a sample of Compound I Form A with a yield of 75%. The X-ray powder diffraction pattern of the sample obtained by this method was consistent with that of the Compound I Form A sample obtained in Example 1.

Example 12

10 g of Compound I API was dissolved in 100 mL of isopropanol under reflux, then placed in an ice-salt bath. The temperature was rapidly cooled to 0°C over 15 minutes, stirred and crystallized for 3.0 hours, filtered, and vacuum-dried to constant weight to obtain a sample of Compound I Form A in a 94% yield. The X-ray powder diffraction pattern of the sample obtained by this method was consistent with that of the Compound I Form A sample obtained in Example 1.

Example 13

10 g of Compound I API was dissolved in 90 mL of anhydrous ethanol under reflux. The mixture was then placed in an ice bath and rapidly cooled to 5°C over 15 minutes. The mixture was stirred and crystallized for 5 hours. The mixture was filtered and dried under vacuum to a constant weight to obtain a sample of Compound I Form A in an 84% yield. The X-ray powder diffraction pattern of the sample obtained by this method was consistent with that of the Compound I Form A sample obtained in Example 1.

Example 14

10 g of Compound I API was dissolved in 50 mL of methanol under reflux, then placed in an ice bath. The temperature was rapidly cooled to 10°C over 5 minutes, maintained at this temperature for 24 hours to allow crystallization, filtered, and vacuum-dried to constant weight to obtain a sample of Compound I Form A in a 75% yield. The X-ray powder diffraction pattern of the sample obtained by this method was consistent with that of the Compound I Form A sample obtained in Example 1.

Example 15

10 g of Compound I API was dissolved in 35 mL of acetonitrile under reflux, then placed in an ice bath. The temperature was rapidly cooled to 8°C over 10 minutes, stirred and maintained for 10 hours to allow crystallization. The mixture was filtered and dried under vacuum to a constant weight to obtain a sample of Compound I Form A in a 62% yield. The X-ray powder diffraction pattern of the sample obtained by this method was consistent with that of the Compound I Form A sample obtained in Example 1.

Example 16

10 g of Compound I API was dissolved in 45 mL of ethyl acetate under reflux. The mixture was then placed in an ice bath and rapidly cooled to 2°C over 5 minutes. The mixture was stirred and crystallized for 6.0 hours. The mixture was filtered and dried under vacuum to a constant weight to obtain a sample of Compound I Form A in a 64% yield. The X-ray powder diffraction pattern of the sample obtained by this method was consistent with that of the Compound I Form A sample obtained in Example 1.

Example 17

10 g of Compound I API was dissolved in 35 mL of acetone under reflux, then placed in an ice-salt bath. The temperature was rapidly cooled to 0°C over 10 minutes, stirred and maintained for 5 hours to allow crystallization. The mixture was filtered and vacuum-dried to constant weight to obtain a sample of Compound I Form A in a 63% yield. The X-ray powder diffraction pattern of the sample obtained by this method was consistent with that of the Compound I Form A sample obtained in Example 1.

Example 18

10 g of Compound I API was dissolved in 50 mL of tetrahydrofuran under reflux, 100 mL of n-hexane was added, and the mixture was then placed in an ice bath. The temperature was rapidly cooled to 5°C over 10 minutes, and the mixture was stirred and crystallized for 7.0 hours. The mixture was filtered and dried under vacuum to constant weight to obtain a sample of Compound I Form A in a yield of 77%. The X-ray powder diffraction pattern of the sample obtained by this method was consistent with that of the Compound I Form A sample obtained in Example 1.

Example 19

10 g of Compound I API was dissolved in 30 mL of dichloromethane under reflux, and 60 mL of methyl tert-butyl ether was added. The mixture was then placed in an ice-salt bath and rapidly cooled to 0°C over 8 minutes. Crystallization was allowed to proceed by stirring at this temperature for 2.0 hours, followed by filtration and vacuum drying to constant weight to obtain a sample of Compound I Form A in a yield of 74%. The X-ray powder diffraction pattern of the sample obtained by this method was consistent with that of the Compound I Form A sample obtained in Example 1.

Example 20

10 g of the Compound I drug substance was dissolved in 100 mL of a mixed solvent of isopropanol and ethanol (volume ratio 1:1) by heating under reflux. The mixture was then placed in an ice bath and rapidly cooled to 2°C over 15 minutes. The mixture was then allowed to stand for 20 hours to crystallize. The mixture was then filtered and dried under vacuum to a constant weight to obtain a sample of Compound I Form A in a 90% yield. The X-ray powder diffraction pattern of the sample obtained by this method was consistent with that of the Compound I Form A sample obtained in Example 1.

Example 21

10 g of Compound I API was dissolved in 100 mL of 95% ethanol under reflux. The mixture was then placed in an ice-salt bath and rapidly cooled to 0°C over 10 minutes. The mixture was stirred and crystallized for 3.0 hours. The mixture was filtered and dried under vacuum to constant weight to obtain a sample of Compound I Form A with a yield of 86%. The X-ray powder diffraction pattern of the sample obtained by this method was consistent with that of the Compound I Form A sample obtained in Example 1.

Example 22

10 g of Compound I API was dissolved in 55 mL of a mixed solvent of acetone and water (volume ratio 5:3) under reflux. The mixture was then placed in a cold water bath and rapidly cooled to 10°C over 15 minutes. The mixture was stirred and crystallized for 15 hours, filtered, and dried under vacuum to constant weight to obtain a sample of Compound I Form A in a yield of 76%. The X-ray powder diffraction pattern of the sample obtained by this method was consistent with that of the Compound I Form A sample obtained in Example 1.

Example 23

10 g of the Compound I drug substance was dissolved in 100 mL of isopropanol by heating under reflux. The solution was then placed in a cold water bath and cooled to 20°C over 40 minutes. The solution was stirred and crystallized for 1.0 hour. The solution was then cooled to 10°C in an ice bath, stirred and crystallized for 2.0 hours. The solution was filtered and dried under vacuum to constant weight to obtain a sample of Compound I Form A in a yield of 92%. The X-ray powder diffraction pattern of the sample obtained by this method was consistent with that of the Compound I Form A sample obtained in Example 1.

Example 24

10 g of the Compound I drug substance was dissolved in 100 mL of anhydrous ethanol under reflux. The solution was then placed in a cold water bath and cooled to 15°C over 20 minutes. The solution was stirred and crystallized for 2.0 hours. The solution was then cooled to 0°C in an ice bath, stirred and crystallized for 1.0 hours. The solution was filtered and dried under vacuum to constant weight to obtain a sample of Compound I Form A with a yield of 84%. The X-ray powder diffraction pattern of the sample obtained by this method was consistent with that of the Compound I Form A sample obtained in Example 1.

Example 25

10 g of the Compound I drug substance was dissolved in 50 mL of methanol under reflux. The solution was then placed in a water bath and cooled to 25°C over 15 minutes. The solution was then stirred and crystallized for 3 hours. The solution was then cooled to 5°C in an ice bath, allowed to stand for 24 hours, and filtered. The solution was then dried under vacuum to a constant weight to obtain a Compound I Form A sample with a yield of 74%. The X-ray powder diffraction pattern of the sample obtained by this method was consistent with that of the Compound I Form A sample obtained in Example 1.

Example 26

10 g of the Compound I drug substance was dissolved in 40 mL of acetonitrile by heating under reflux. The solution was then placed in a cold water bath and cooled to 10°C over 25 minutes. The solution was then stirred and crystallized for 8 hours. The solution was then cooled to 0°C in an ice-salt bath, held for 20 hours, and allowed to crystallize. The solution was filtered and vacuum-dried to constant weight to obtain a sample of Compound I Form A in a 60% yield. The X-ray powder diffraction pattern of the sample obtained by this method was consistent with that of the Compound I Form A sample obtained in Example 1.

Example 27

10 g of the Compound I drug substance was dissolved in 45 mL of ethyl acetate under reflux. The mixture was then placed in a cold water bath, cooled to 20°C over 10 minutes, and allowed to stand for crystallization for 24 hours. The mixture was then further cooled to 2°C in an ice bath, stirred and allowed to crystallize for 4 hours. The mixture was filtered and dried under vacuum to constant weight to obtain a sample of Compound I Form A in a 65% yield. The X-ray powder diffraction pattern of the sample obtained by this method was consistent with that of the Compound I Form A sample obtained in Example 1.

Example 28

10 g of the Compound I drug substance was dissolved in 40 mL of acetone under reflux. The mixture was then placed in a cold water bath, cooled to 10°C over 10 minutes, and allowed to stand for crystallization for 15 hours. The mixture was then further cooled to 3°C in an ice bath, stirred and allowed to crystallize for 8 hours. The mixture was filtered and vacuum dried to constant weight to obtain a Compound I Form A sample with a yield of 60%. The X-ray powder diffraction pattern of the sample obtained by this method was consistent with that of the Compound I Form A sample obtained in Example 1.

Example 29

10 g of the Compound I drug substance was dissolved in 50 mL of tetrahydrofuran under reflux. 100 mL of n-hexane was added, and the mixture was then placed in a water bath. The temperature was cooled to 25°C over 20 minutes. Without incubation for crystallization, the mixture was directly cooled to 5°C in an ice bath. Crystallization was allowed to proceed by incubation with stirring for 6.0 hours, followed by filtration and vacuum drying to constant weight to obtain a sample of Compound I Form A with a yield of 76%. The X-ray powder diffraction pattern of the sample obtained by this method was consistent with that of the Compound I Form A sample obtained in Example 1.

Example 30

10 g of the Compound I drug substance was dissolved in 30 mL of dichloromethane under reflux. 60 mL of methyl tert-butyl ether was added, and the mixture was placed in a cold water bath. The temperature was cooled to 15°C over 15 minutes, and the mixture was stirred and crystallized for 5.0 hours. The mixture was then cooled to 10°C in an ice bath, stirred and crystallized for 2.0 hours, filtered, and vacuum dried to constant weight to obtain a sample of Compound I Form A with a yield of 72%. The X-ray powder diffraction pattern of the sample obtained by this method was consistent with that of the Compound I Form A sample obtained in Example 1.

Example 31

10 g of the Compound I drug substance was dissolved in 100 mL of a mixed solvent of isopropanol and ethanol (volume ratio 1:1) under reflux. The mixture was then placed in a cold water bath and cooled to 20°C over 20 minutes. Crystallization was allowed to proceed with stirring for 1.0 hour. The mixture was then cooled to 0°C in an ice-salt bath, crystallized with stirring for 1.0 hour, filtered, and vacuum-dried to constant weight to obtain a sample of Compound I Form A with a yield of 88%. The X-ray powder diffraction pattern of the sample obtained by this method was consistent with that of the Compound I Form A sample obtained in Example 1.

Example 32

10 g of the Compound I drug substance was dissolved in 100 mL of 95% ethanol under reflux. The solution was then placed in a water bath, cooled to 25°C over 25 minutes, and stirred and crystallized for 10 hours. The solution was then cooled to 1°C in an ice-salt bath, stirred and crystallized for 3 hours, filtered, and vacuum-dried to constant weight to obtain a Compound I Form A sample with a yield of 87%. The X-ray powder diffraction pattern of the sample obtained by this method was consistent with that of the Compound I Form A sample obtained in Example 1.

Example 33

10 g of the Compound I drug substance was dissolved in 55 mL of a mixed solvent of acetone and water (volume ratio of 5:1) by heating under reflux. The mixture was then placed in a cold water bath and cooled to 10°C over 20 minutes. Crystallization was allowed to proceed with stirring at this temperature for 3.0 hours. The mixture was then cooled to 5°C in an ice bath, crystallized with stirring at this temperature for 2.0 hours, filtered, and vacuum dried to constant weight to obtain a sample of Compound I Form A with a yield of 77%. The X-ray powder diffraction pattern of the sample obtained by this method was consistent with that of the Compound I Form A sample obtained in Example 1.

Experimental Example 1: Determination of the diffraction angle (2θ) of the X-ray powder diffraction peak

Regarding the Compound I Form A sample prepared in Example 1, its X-ray powder diffraction (XRPD) spectrum is shown in FIG1 , and the diffraction angle (2θ) values of its X-ray powder diffraction peaks are shown in Table 1.

In addition, the physical property data of each crystal described in Experimental Example 1 were measured under the following conditions.

Testing instrument: Japan Rigaku SmartLab SE fully automatic multi-functional X-ray diffractometer.

Operating conditions: X-ray tube: cathode: copper; tube voltage: 40 kV; tube current: 30 mA; scanning mode: one-dimensional scanning; scanning rate: 10°/min; scanning axis: θ/2θ; scanning range: 3-35°; step interval: 0.01°.

Test results: Form A has characteristic peaks at diffraction angles 2θ of 6.38±0.2°, 9.50±0.2°, 11.05±0.2°, 13.11±0.2°, 15.54±0.2°, 16.23±0.2°, 18.23±0.2°, 18.96±0.2°, 19.42±0.2°, 20.52±0.2°, 21.43±0.2°, 22.13±0.2°, and 23.07±0.2° as determined by X-ray powder diffraction.

Table 1 Diffraction angle (2θ) values of X-ray powder diffraction peaks of Compound I Form A sample prepared in Example 1

Experimental Example 2: Thermogravimetric-Differential Scanning Calorimetry Analysis

Regarding the Compound I Form A sample prepared in Example 1, its thermogravimetric-differential scanning calorimetry (TGA/DSC) spectrum is shown in FIG2 .

Testing instrument model: Synchronous thermal analyzer STA449F3

Test conditions: Temperature: 25°C, Humidity: 35% RH

Crucible: DSC/TG pan Al ₂ O ₃

Atmosphere: AIR(80/20)--/NITROGEN/50/NITROGEN/20

Test results: The TGA graph can confirm that Form A does not contain crystalline water or solvate; the DSC graph can confirm that the melting point (extrapolated onset temperature) of Form A is 132.6±2°C.

Experimental Example 3: Nuclear Magnetic Resonance Spectroscopy ( ¹ H-NMR)

Regarding the Form A sample of Compound I prepared in Example 1, its nuclear magnetic resonance ( ¹ H-NMR) spectrum is shown in FIG3 .

Measurement conditions: The data were collected on a Bruker 500 MHz NMR spectrometer with CDCl ₃ as the solvent.

Test results: ¹ H-NMR: δ: 10.144 (br, 1H), 7.480~7.497 (m, 2H), 7.447~7.466 (m, 2H), 7.379~7.414 (m, 1H), 7.293~7.328 (m, 5H), 5.075 (m, 1H), 3.622 (m, 2H), 3.236 (s, 3H), 2.370~2.399 (t, 2H), 1.854~1.746 (m, 4H), 1.460~1.487 (m, 2H), 1.292~1.306 (m, 6H), ppm.

Experimental Example 4: Infrared Spectroscopy (IR)

Regarding the Form A sample of Compound I prepared in Example 1, its infrared (IR) spectrum is shown in FIG4 .

Instrument model: Nicolet.

Detection method: Take an appropriate amount of this product (about 1-2 mg) and an appropriate amount of ground and dried potassium bromide, place them in an agate mortar, mix the sample and potassium bromide and grind them evenly, take an appropriate amount of the ground mixture and put it into a tablet pressing mold to press the tablet, and measure the infrared spectrum.

Test results: The infrared spectrum of the sample has characteristic peaks at 3243±5cm ^-1 , 2931±5cm ^-1 , 1706±5cm ^-1 , 1530±5cm ^-1 , 1488±5cm ^-1 , 1446±5cm ^-1 , 1430±5cm ^-1 , 1226±5cm ^-1 , 1125±5cm ^-1 , 1110±5cm ^-1 , 706±5cm ^{- 1} , and 692±5cm ^-1 .

Experimental Example 5: Moisture-absorbing properties

Regarding the sample of Compound I Form A prepared in Example 1, its hygroscopicity was determined with reference to the 2020 edition of the Chinese Pharmacopoeia, Part IV, General Chapter 9103, Guidelines for Hygroscopicity Tests of Drugs. The specific test method is as follows:

Take a dry glass weighing bottle with a stopper (outer diameter of 50 mm, height of 15 mm), place it in a suitable constant temperature and humidity desiccator at 25℃±1℃ one day before the test, and accurately weigh the weight (m ₁ ).

Take an appropriate amount of the test sample and spread it evenly in the above-mentioned weighing bottle. The thickness of the test sample is generally about 1 mm. Accurately weigh the weight (m ₂ ).

Open the weighing bottle and place it with the bottle cap under the above constant temperature and humidity conditions for 24 hours. Cover the weighing bottle cap and accurately weigh the weight (m ₃ ).

Test results: The weight gain due to moisture absorption is less than 0.2%, indicating that the crystal form A obtained in this application is not hygroscopic.

Experimental Example 6: Stability Test

A stability study was conducted on the Compound I Form A sample prepared in Example 1. The sample was placed at 40°C ± 2°C/75% RH ± 5% RH for 6 months for an accelerated stability experiment, and at 30°C ± 2°C/65% RH ± 5% RH for 6 months for a long-term stability experiment. The XRPD spectra of the sample were measured on the 0th day, 1 month, 2 months, 3 months, and at the end of 6 months to determine the change in the crystal form. At the same time, its purity was determined by high performance liquid chromatography to examine the changes in related substances.

The stability test results of the 6-month accelerated test and the 6-month long-term test of the Form A sample are shown in Tables 2 and 3. The XRPD overlay images at the end of the 6th month in the accelerated test and at the end of the 6th month and day 0 in the long-term test are shown in Figure 5.

Results showed that Form A maintained its form unchanged compared to Day 0 during both a six-month accelerated stability test (40°C ± 2°C/75% ± 5% RH) and a six-month long-term stability test (30°C ± 2°C/65% ± 5% RH). Related substances also showed no significant changes. After six months, Form A maintained a purity exceeding 99.9%, demonstrating high purity and stability. These stability studies demonstrate that this form is suitable for formulation development.

Table 2 Results of 6-month accelerated stability test of Form A samples

Table 3 Results of 6-month long-term stability test of Form A samples

Experimental Example 7: Bioavailability Test

A bioavailability test was conducted on the Compound I Form A sample prepared in Example 1. The Compound I Form A sample was first prepared into an injection and a suspension using conventional methods.

This study involved 12 Beagle dogs (half male and half female) randomly divided into two groups, each containing three male and three female animals. Group 1 animals were not fasting and received a single intravenous injection of 0.5 mg/kg of Compound I (injection). Blood samples were collected until 24 hours after administration, specifically at pre-dose, 0.083, 0.25, 0.5, 1, 2, 3, 4, 5, 6, 8, 12, and 24 hours after administration. Group 2 animals were fasting and received a single oral gavage of 0.5 mg/kg of Compound I (suspension). Blood samples were collected until 24 hours after administration, specifically at pre-dose, 0.25, 0.5, 1, 2, 3, 4, 5, 6, 8, 12, and 24 hours after administration. The homogeneity and concentration of the drug formulations were analyzed using a validated HPLC-UV analytical method. The concentration of Compound I in plasma samples was determined using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) analytical method. Using blood drug concentration data, Phoenix 7.0 software uses a non-compartmental model to calculate its bioavailability.

The average concentration-time curves of Compound I in the plasma of Group 1 animals after intravenous administration and the average concentration-time curves of Compound I in the plasma of Group 2 animals after oral administration in the bioavailability study are shown in Figures 6 and 7, respectively.

The results showed that the bioavailability of Form A was 95.81%, which was relatively high, indicating that the drug was well absorbed and more helpful in improving the efficacy.

The Compound I Form A samples prepared in Examples 2 to 33 have comparable properties to the Compound I Form A sample prepared in Example 1, including hygroscopicity, stability, and bioavailability.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent replacements or improvements made within the spirit and principles of the present application should be included in the scope of protection of the present application.

Claims (20)

A crystalline form A of a compound of formula I, characterized in that the free base crystalline form A of the compound of formula I has an X-ray powder diffraction pattern expressed in 2θ angles at 9.50±0.2°, 11.05±0.2°, 15.54±0.2°, 16.23±0.2°, 18.23±0.2° and 22.13±0.2°,
The crystalline form A of the compound of formula I according to claim 1, characterized in that the free base crystalline form A of the compound of formula I has an X-ray powder diffraction pattern expressed in 2θ angles at 6.38±0.2°, 9.50±0.2°, 11.05±0.2°, 13.11±0.2°, 15.54±0.2°, 16.23±0.2°, 18.23±0.2°, 18.96±0.2°, 19.42±0.2°, 20.52±0.2°, 21.43±0.2°, 22.13±0.2° and 23.07±0.2°. There are diffraction peaks at. The crystalline form A of the compound of formula I according to claim 2, characterized in that the free base crystalline form A of the compound of formula I has an X-ray powder diffraction pattern expressed in 2θ angles having the pattern shown in Figure 1. The crystalline form A of the compound of formula I according to claim 1, characterized in that the free base crystalline form A of the compound of formula I has a melting point of 130°C-135°C as measured by differential scanning calorimetry. The crystalline form A of the compound of formula I according to claim 1, characterized in that the free base crystalline form A of the compound of formula I has a thermogravimetric-differential scanning calorimetry analysis spectrum as shown in Figure 2. The method for preparing Form A according to any one of claims 1 to 5, characterized in that it comprises: heating and dissolving the compound of formula I in a solvent, cooling to 25° C. to 45° C., slowly cooling or holding for crystallization, further cooling to 0° C. to 10° C. for crystallization, separating, and drying to obtain Form A. The method for preparing Form A according to any one of claims 1 to 5, comprising: heating and dissolving Compound I in a solvent, cooling to 0°C to 10°C, maintaining the temperature for crystallization, separating, and drying to obtain Form A. The method for preparing Form A according to any one of claims 1 to 5, comprising: heating and dissolving Compound I in a solvent, cooling to 10°C to 25°C, maintaining the temperature for crystallization, further cooling to 0°C to 10°C, maintaining the temperature for crystallization, separating, and drying to obtain Form A. The method for preparing Form A according to any one of claims 6 to 8, wherein the solvent is one or a mixed solvent of alcohols, ethers, esters, alkanes, ketones, acetonitrile or water. The method for preparing Form A according to claim 9, wherein the solvent is one or a mixed solvent of methanol, ethanol, isopropanol, acetonitrile, tetrahydrofuran, methyl tert-butyl ether, ethyl acetate, dichloromethane, n-hexane, acetone or water. The method for preparing Form A according to claim 10, wherein the solvent is one or a mixed solvent of isopropanol, ethanol or water. The method for preparing Form A according to claim 11, wherein the solvent is isopropanol. The method for preparing Form A according to any one of claims 6 to 8, characterized in that the mass volume ratio (g/mL) of the compound of formula I to the solvent is 1:3.5 to 15. The method for preparing Form A according to any one of claims 6 to 8, characterized in that the heating and dissolving is: heating and dissolving in a single solvent or a mixed solvent, or first heating and dissolving in one solvent and then adding one or more other solvents. The preparation method of Form A according to claim 6, characterized in that after cooling to 25°C to 45°C, the slow cooling and crystallization or the heat preservation and crystallization step comprises: cooling to 25°C to 45°C and then naturally slowly cooling and crystallizing at room temperature for 1 to 24 hours or cooling to 25°C to 45°C and then heat preservation and crystallization for 1 to 24 hours; the further cooling to 0°C to 10°C and heat preservation and crystallization step comprises: continuing to cool to 0°C to 10°C and heat preservation and crystallization for 1 to 24 hours. The method for preparing Form A according to claim 7, wherein after cooling to 0°C to 10°C, the crystallization is maintained for 2 to 24 hours. The method for preparing Form A according to claim 8, wherein the crystallization time during the crystallization process after cooling to 10°C to 25°C and holding is 0 to 24 hours; and the crystallization time during the crystallization process of continuing to cool to 0°C to 10°C and holding is 1 to 24 hours. A pharmaceutical composition comprising a pharmaceutically acceptable carrier, an excipient, and a therapeutically effective dose of Form A of the compound of Formula I according to any one of claims 1 to 5. A therapeutically effective dose of the crystalline form A of the compound of formula I according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 18 in the preparation of a medicament for preventing and/or treating pulmonary hypertension, platelet aggregation-related cardiovascular and cerebrovascular diseases, or diabetic nephropathy. Use of a therapeutically effective dose of the crystalline form A of the compound of formula I according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 18 in the preparation of a PGI2 receptor agonist drug.