WO2024125591A1 - Crystal of benzoxazinone compound and method for preparing same - Google Patents

Abstract

The present application discloses a crystal of a benzoxazinone compound and a method for preparing same, and particularly, discloses a compound of formula (I), a method for preparing a crystal thereof, and use thereof.

Description

Crystallization and preparation of benzoxazinone compounds

This application claims the following priority:

Application number: CN202211615935.1, application date: December 15, 2022.

Technical Field

The present application relates to a crystal of a benzoxazinone compound and a preparation method thereof, and specifically discloses a preparation method and application of a compound of formula (I) and its crystal.

Background technique

Aldosterone is a mineralocorticoid hormone mainly secreted by the zona glomerulosa of the adrenal cortex. It is an important substance for regulating water-sodium balance and maintaining homeostasis in the body. The latest research has found that excessive aldosterone can promote inflammation, cause myocardial remodeling and fibrosis, and can also directly lead to renal tissue damage, increase proteinuria, and participate in the occurrence and development of various diseases such as chronic kidney disease, hypertension, and heart failure. Mineralocorticoid receptor antagonists antagonize the binding of aldosterone and mineralocorticoid receptors (MR) to prevent excessive activation of the aldosterone-MR complex, thereby delaying disease progression. Although early clinical trials have shown that MR antagonists can improve the prognosis of patients with heart failure, increase survival rates, and have kidney protection effects in addition to lowering blood pressure, the first-generation drug spironolactone has poor nuclear receptor selectivity, and long-term use can cause adverse reactions such as male breast development, impotence, and female menstrual disorders; although the second-generation drug eplerenone improves nuclear receptor selectivity, its activity is weak and difficult to synthesize. Both spironolactone and eplerenone have the risk of hyperkalemia in clinical use, which limits the application of MR antagonists.

Summary of the invention

In one aspect, the present application provides a crystal of a compound of formula (I),

In some embodiments of the present application, the crystal of the compound of formula (I) of the present application is A crystal, and its X-ray powder diffraction pattern has diffraction peaks at the following 2θ angles: 10.52±0.20°, 11.45±0.20°, 13.88±0.20° and 15.59±0.20°.

In some embodiments of the present application, the above-mentioned A crystal has an X-ray powder diffraction pattern having diffraction peaks at the following 2θ angles: 10.52±0.20°, 11.45±0.20°, 13.21±0.20°, 13.88±0.20°, 15.59±0.20°, 16.59±0.20°, 19.52±0.20° and 25.92±0.20°.

In some embodiments of the present application, the above-mentioned A crystal has an X-ray powder diffraction pattern having diffraction peaks at the following 2θ angles: 10.52±0.20°, 11.45±0.20°, 12.38±0.20°, 13.21±0.20°, 13.88±0.20°, 15.59±0.20°, 16.59±0.20°, 19.22±0.20°, 19.52±0.20°, 23.82±0.20°, 25.05±0.20° and 25.92±0.20°.

In some schemes of the present application, the above-mentioned A crystal has an X-ray powder diffraction pattern having diffraction peaks at the following 2θ angles: 10.52±0.20°, 11.45±0.20°, 12.38±0.20°, 13.21±0.20°, 13.88±0.20°, 15.59±0.20°, 15.99±0.20°, 16.59±0.20°, 18.35±0.20°, 18.60±0.20°, 19.22±0.20°, 19.52±0.20°, 23.82±0.20°, 25.05±0.20°, 25.92±0.20° and 27.10±0.20°.

In some embodiments of the present application, the X-ray powder diffraction pattern of the above-mentioned A crystal, expressed by 2θ value, comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15 or 16 diffraction peaks selected from the following: 10.52±0.20°, 11.45±0.20°, 12.38±0.20°, 13.21±0.20°, 13.88±0. 20°, 15.59±0.20°, 15.99±0.20°, 16.59±0.20°, 18.35±0.20°, 18.60±0.20°, 19.22±0.20°, 19.52±0.20°, 23.82±0.20°, 25.05±0.20°, 25.92±0.20° and 27.10±0.20°.

In some embodiments of the present application, the above-mentioned A crystal, in its X-ray powder diffraction pattern, expressed by 2θ values, comprises 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 diffraction peaks selected from the following: 10.52±0.20°, 11.45±0.20°, 12.38±0.20°, 13.21±0.20°, 13.88±0.20°, 15.59±0.20°, 16.59±0.20°, 19.22±0.20°, 19.52±0.20°, 23.82±0.20°, 25.05±0.20° and 25.92±0.20°.

In some embodiments of the present application, the above-mentioned A crystal, in its X-ray powder diffraction pattern, expressed by 2θ values, contains 3, 4, 5, 6, 7 or 8 diffraction peaks selected from the following: 10.52±0.20°, 11.45±0.20°, 13.21±0.20°, 13.88±0.20°, 15.59±0.20°, 16.59±0.20°, 19.52±0.20° and 25.92±0.20°.

In some embodiments of the present application, the above-mentioned A crystal has an X-ray powder diffraction pattern with diffraction peaks at the following 2θ angles: 6.20±0.20°, 9.04±0.20°, 9.60±0.20°, 10.52±0.20°, 11.45±0.20°, 12.38±0.20°, 12.82±0.20°, 13.21±0.20°, 13.88±0.20°, 15.59±0.20°, 15.99 ±0.20°, 16.59±0.20°, 17.18±0.20°, 17.54±0.20°, 18.35±0.20°, 18.60±0.20°, 19.22±0.20°, 19.52±0.20°, 20.03±0.20°, 20.29±0.20°, 20.96±0.20°, 21.11±0.20°, 21.57±0.20°, 22.23±0.20° , 23.08±0.20°, 23.33±0.20°, 23.82±0.20°, 24.22±0.20°, 24.86±0.20°, 25.05±0.20°, 25.26±0.20°, 25.52±0.20°, 25.92±0.20°, 27.10±0.20°, 27.33±0.20°, 27.64±0.20°, 28.82±0.20°, 29.20 ±0.20°, 29.68±0.20°, 30.21±0.20°, 30.59±0.20°, 31.19±0.20°, 31.75±0.20°, 32.52±0.20°, 33.71±0.20°, 34.51±0.20°, 35.88±0.20°, 36.54±0.20°, 37.45±0.20°, 38.23±0.20° and 38.60±0.20°.

In some embodiments of the present application, the above-mentioned A crystal has an X-ray powder diffraction pattern having diffraction peaks at the following 2θ angles: 6.20°, 9.04°, 9.60°, 10.52°, 11.45°, 12.38°, 12.82°, 13.21°, 13.88°, 15.59°, 15.99°, 16.59°, 17.18°, 17.54°, 18.35°, 18.60°, 19.22°, 19.52°, 20.03°, 20.29°, 20.96°, 21.11°, 21.57° , 22.23°, 23.08°, 23.33°, 23.82°, 24.22°, 24.86°, 25.05°, 25.26°, 25.52°, 25.92°, 27.10°, 27.33°, 27.64°, 28.82°, 29.20°, 29.68°, 30.21°, 30.59°, 31.19°, 31.75°, 32.52°, 33.71°, 34.51°, 35.88°, 36.54°, 37.45°, 38.23° and 38.60°.

In some embodiments of the present application, the above-mentioned A crystal has an X-ray powder diffraction pattern having diffraction peaks at the following 2θ angles: 10.52±0.20°, 11.45±0.20°, 13.88±0.20°, and/or 15.59±0.20°, and/or 6.20±0.20°, and/or 9.04±0.20°, and/or 9.60±0.20°, and/or 12.38±0.20°, and/or 12.82±0.20°, and/or 13.21±0.20°, and/or 15.99±0.20°, and and/or 16.59±0.20°, and/or 17.18±0.20°, and/or 17.54±0.20°, and/or 18.35±0.20°, and/or 18.60±0.20°, and/or 19.22±0.20°, and/or 19.52±0.20°, and/or 20.03±0.20°, and/or 20.29±0.20°, and/or 20.96±0.20°, and/or 21.11±0.20°, and/or 21.57±0.20°, and/or 22.23±0.20°, and/or 23.08±0.20°, and/or 23.33±0.20°, and/or 23.82±0.20°, and/or 24.22±0.20°, and/or 24.86±0.20°, and/or 25.05±0.20°, or 25.26±0.20°, and/or 25.52±0.20°, and/or 25.92±0.20°, and/or 27.10±0.20°, and/or 27.33±0.20°, and/or 27.64±0.20°, and/or 28.82±0.20°, and/or 29.20 ±0.20°, and/or 29.68±0.20°, and/or 30.21±0.20°, and/or 30.59±0.20°, and/or 31.19±0.20°, and/or 31.75±0.20°, and/or 32.52±0.20°, and/or 33.71±0.20°, and/or 34.51±0.20°, and/or 35.88±0.20°, and/or 36.54±0.20°, and/or 37.45±0.20°, and/or 38.23±0.20°, and/or 38.60±0.20°.

In some embodiments of the present application, the peak position, intensity, interplanar spacing and relative intensity of the diffraction peak of the above-mentioned A crystal in its X-ray powder diffraction pattern are shown in Table 1 below:

Table 1 XRPD spectrum analysis data of compound A of formula (I)

In some schemes of the present application, the XRPD pattern of the above-mentioned A crystal is basically as shown in Figure 1.

In some embodiments of the present application, the above-mentioned A crystal has a differential scanning calorimetry curve (DSC graph) having an onset of an endothermic peak at 215.9°C±5°C.

In some schemes of the present application, the DSC spectrum of the above-mentioned A crystal is basically as shown in Figure 2.

In some embodiments of the present application, the thermogravimetric analysis curve (TGA) of the above-mentioned A crystal shows a weight loss of 0.90% at 200°C±3°C.

In some schemes of the present application, the TGA spectrum of the above-mentioned A crystal is basically as shown in Figure 3.

In some embodiments of the present application, the above-mentioned A crystal has a moisture absorption weight increase at 25° C. and 80% relative humidity greater than 0.2% and less than 2%.

In some embodiments of the present application, the water adsorption isotherm (DVS) spectrum of the above-mentioned A crystal is basically as shown in Figure 4.

On the other hand, the present application also provides a method for preparing the above-mentioned A crystal, which comprises the following steps:

(1) dissolving the compound of formula (I) in a crystallization solvent;

(2) Crystallization, and then separation of the solid to obtain crystal A of the compound of formula (I).

In some embodiments of the present application, the crystallization solvent described in step (1) above is selected from dichloromethane, tetrahydrofuran, dioxane, acetone, acetonitrile, ethanol, methanol, water or a mixed solvent of any two of these solvents.

In some embodiments of the present application, the crystallization solvent described in step (1) above is selected from tetrahydrofuran, acetone, acetonitrile, water or a mixed solvent of any two of these solvents.

In some embodiments of the present application, the crystallization solvent described in the above step (1) is selected from tetrahydrofuran, acetone, a mixed solvent of acetonitrile and water, and a mixed solvent of acetone and water.

On the other hand, the present application also provides a single crystal of a compound of formula (I).

In some embodiments of the present application, the compound of formula (I) is a single crystal, and the single crystal has the following characteristics: it belongs to the orthorhombic system, and the unit cell parameters are α＝90°，β＝90°，γ＝90°， Z=8, crystal density is 1.439 mg/m ³ .

In some embodiments of the present application, the compound of formula (I) is a single crystal, and the absolute configuration of the single crystal of the compound of formula (I) is S configuration.

In some embodiments of the present application, the compound of formula (I) is a single crystal, and the ellipsoid diagram of the single crystal molecular structure is shown in FIG5 .

On the other hand, the present application also provides a crystalline composition, wherein the A crystal of the compound of formula (I) accounts for more than 50% by weight of the crystalline composition, preferably more than 75%, more preferably more than 90%, and most preferably more than 95%. The crystalline composition may also contain a small amount of other crystalline or non-crystalline forms of the compound of formula (I). The crystalline composition may also contain a small amount of other crystalline or amorphous forms of the compound of formula (I), or impurities other than these substances.

On the other hand, the present application provides a pharmaceutical composition, which comprises a therapeutically effective amount of crystal A of the compound of formula (I) above, or a crystalline composition of the compound of formula (I) above; the pharmaceutical composition may comprise at least one pharmaceutically acceptable carrier or other excipient.

In some embodiments of the present application, the above-mentioned crystal A of the compound of formula (I), the above-mentioned crystalline composition of the compound of formula (I) or the above-mentioned pharmaceutical composition is selected from a pharmaceutical composition for oral administration, subcutaneous administration, intramuscular administration or intravenous administration.

In some embodiments of the present application, the above-mentioned crystal A of the compound of formula (I), the above-mentioned crystalline composition of the compound of formula (I) or the above-mentioned pharmaceutical composition is selected from a pharmaceutical composition for oral administration, intramuscular administration or intravenous administration.

In some embodiments of the present application, the above-mentioned crystal A of the compound of formula (I), the above-mentioned crystalline composition of the compound of formula (I) or the above-mentioned pharmaceutical composition is administered via a solid pharmaceutical composition.

In some embodiments of the present application, the solid pharmaceutical composition is selected from tablets or capsules.

In some embodiments of the present application, the dosage of the above-mentioned crystal A of the compound of formula (I), the above-mentioned crystalline composition of the compound of formula (I), or the above-mentioned pharmaceutical composition is 0.0001 mg/kg to 100 mg/kg.

In some embodiments of the present application, the dosage of the above-mentioned crystal A of the compound of formula (I), the above-mentioned crystalline composition of the compound of formula (I), or the above-mentioned pharmaceutical composition is 0.001 mg/kg to 80 mg/kg.

In some embodiments of the present application, the dosage of the above-mentioned crystal A of the compound of formula (I), the above-mentioned crystalline composition of the compound of formula (I), or the above-mentioned pharmaceutical composition is 0.01 mg/kg to 60 mg/kg.

On the other hand, the present application also provides the use of the A crystal of the compound of formula (I), the crystalline composition of the compound of formula (I) or the pharmaceutical composition in the preparation of drugs for treating diseases related to mineralocorticoid receptor antagonists.

On the other hand, the present application also provides the use of the A crystal of the compound of formula (I), the crystalline composition of the compound of formula (I) or the pharmaceutical composition in the preparation of a drug for treating diabetic nephropathy.

On the other hand, the present application also provides a method for treating a disease associated with a mineralocorticoid receptor antagonist in a subject in need thereof, comprising providing the subject with an effective dose of crystal A of the compound of formula (I), a crystalline composition of the compound of formula (I) or the pharmaceutical composition.

On the other hand, the present application also provides the use of the A crystal of the compound of formula (I), the crystalline composition of the compound of formula (I) or the pharmaceutical composition in the treatment of diseases associated with mineralocorticoid receptor antagonists.

On the other hand, the present application also provides crystal A of the compound of formula (I), a crystalline composition of the compound of formula (I) or the pharmaceutical composition for treating diseases associated with mineralocorticoid receptor antagonists.

In some embodiments of the present application, the above-mentioned mineralocorticoid receptor antagonist-related diseases are selected from diabetic nephropathy.

Technical Effects

The compounds of the present application have good antagonistic activity against mineralocorticoid receptors. In addition, the compounds exhibit excellent pharmacokinetic and pharmacodynamic properties, and have good membrane permeability and solubility. The crystals of the compounds of the present application have the advantages of being stable, having good hygroscopicity, and being less affected by light and heat, and have broad prospects for drug development.

Definition and Description

Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular phrase or term should not be considered to be uncertain or unclear in the absence of a special definition, but should be understood according to its ordinary meaning. When a trade name appears in this article, it is intended to refer to its corresponding commercial product or its active ingredient.

Unless otherwise stated, X-ray powder diffraction (XRPD) can detect information such as changes in crystallinity, crystallinity, and crystal structure state, and is a common means of identifying crystallinity. The peak position of the XRPD spectrum depends mainly on the structure of the crystal, is relatively insensitive to experimental details, and its relative peak height depends on many factors related to sample preparation and instrument geometry. Therefore, for any given crystalline form, the relative intensity of the diffraction peak can change due to preferred orientation caused by factors such as crystal morphology, which is well known in the field of crystallography. Where there is a preferred orientation effect, the peak intensity is changed, but the diffraction peak position of the crystal cannot be changed. In addition, for any given crystal, there may be a slight error in the position of the peak, which is also well known in the field of crystallography. For example, due to changes in temperature, movement of the sample, or calibration of the instrument when analyzing the sample, the position of the peak can be moved, and the measurement error of the 2θ value is sometimes about ± 0.2 degrees. The measurement of 2θ of the XRPD spectrum may be slightly different between different instruments and different samples, so the numerical value of the 2θ cannot be regarded as absolute. Therefore, it is well known to those skilled in the art that this error should be taken into account when determining each crystalline structure. Therefore, in some embodiments, the crystals of the present application are characterized by an XRPD pattern with certain peak positions, which is substantially as shown in the XRPD pattern provided in the accompanying drawings of the present application.

DSC measures the transition temperature when a crystal absorbs or releases heat due to changes in its crystalline structure or melting of the crystal. For the same crystal of the same compound, the error of thermal transition temperature and melting point is typically within about 5°C in consecutive analyses. When we say that a compound has a given DSC peak or melting point, this refers to the DSC peak or melting point ±5°C. DSC provides an auxiliary method to distinguish different crystals. Different crystalline forms can be identified based on their different transition temperature characteristics. It should be pointed out that for mixtures, their DSC peaks or melting points may vary over a larger range. In addition, since decomposition is accompanied by the melting of the substance, the melting temperature is related to the heating rate.

The term "treatment" means administering the compound or formulation described herein to improve or eliminate a disease or one or more symptoms associated with the disease, and includes:

(i) inhibiting a disease or disease state, i.e. arresting its development;

(ii) ameliorating a disease or condition, even if causing regression of the disease or condition.

The term "prevention" means administering the compounds or formulations described herein to prevent one or more symptoms associated with the disease, and includes preventing the disease or disease state from occurring in a mammal, particularly when such mammal is susceptible to the disease state but has not yet been diagnosed as having the disease state.

References throughout this specification to "one embodiment" or "an embodiment" or "in another embodiment" or "in certain embodiments" mean that at least one embodiment includes the specific referenced elements, structures, or features described in connection with that embodiment. Thus, the phrases "in one embodiment" or "in an embodiment" or "in another embodiment" or "in certain embodiments" appearing in different places throughout the specification do not necessarily all refer to the same embodiment. Furthermore, the specific elements, structures, or features may be combined in any appropriate manner in one or more embodiments.

It should be understood that the singular article "a", "an" and "the" used in the specification and the appended claims includes plural objects unless the context clearly dictates otherwise. Thus, for example, a reaction mentioned including "a catalyst" includes one catalyst, or two or more catalysts. It should also be understood that the term "or" is generally used in its sense including "and/or" unless the context clearly dictates otherwise.

The intermediate compounds of the present application can be prepared by a variety of synthesis methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitution methods well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present application.

The chemical reactions of the specific embodiments of the present application are carried out in a suitable solvent, which must be suitable for the chemical changes of the present application and the reagents and materials required. In order to obtain the compounds of the present application, it is sometimes necessary for those skilled in the art to modify or select the synthesis steps or reaction processes based on the existing embodiments.

The present application will be described in detail below through examples, which do not imply any limitation to the present application.

All solvents used in this application were commercially available and used without further purification.

The solvents used in this application are commercially available.

This application uses the following abbreviations:

N2: nitrogen; RH: relative humidity; mL: milliliter; L: liter; min: minute; ℃: degree Celsius; μm: micrometer; mm: millimeter; μL: microliter; moL/L: mole per liter; mg: milligram; s: second; nm: nanometer; MPa: megapascal; lux: lux; μw/cm2: microwatt per square centimeter; h: hour; Kg: kilogram; nM: nanomole; RT: retention time; RRT: relative retention time; rpm: rotational speed; Kv: kilovolt; mA: milliampere; DMF stands for N,N-dimethylformamide.

Instruments and analytical methods

1. The X-ray powder diffractometer (XRPD) method of this application, the test parameters are shown in Table 2.

Table 2 XRPD test parameters

2. The differential scanning calorimeter (DSC) method of this application, the test parameters are shown in Table 3.

Table 3 DSC test parameters

3. Thermogravimetric analysis (TGA) method of this application, test parameters are shown in Table 4.

Table 4 TGA test parameters

4. This application applies the dynamic vapor sorption analysis (DVS) method, and the test parameters are shown in Table 5.

Table 5 DVS test parameters

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a Cu-Kα radiation XRPD spectrum of a crystal of compound A of formula (I);

FIG2 is a DSC spectrum of the crystals of compound A of formula (I);

FIG3 is a TGA spectrum of the crystals of compound A of formula (I);

FIG4 is a DVS spectrum of a crystal of compound A of formula (I);

FIG5 is an ellipsoid diagram of the three-dimensional structure of a single crystal of the compound of formula (I).

Detailed ways

The present application is described in detail below by examples, but it is not meant to limit the present application in any adverse way. The compounds of the present application can be prepared by a variety of synthesis methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by the combination thereof with other chemical synthesis methods, and equivalent substitution methods well known to those skilled in the art, and preferred embodiments include but are not limited to the embodiments of the present application. It will be apparent to those skilled in the art that various changes and improvements are made to the specific embodiments of the present application without departing from the spirit and scope of the present application.

Example 1: Preparation of Intermediate 1

Synthesis line:

first step

Compound 1-1 (25 g, 203 mmol) and potassium carbonate (84.2 g, 609 mmol) were dissolved in N, N-dimethylformamide (200 mL), chloroacetyl chloride (17.0 mL, 213 mmol) was added at 0°C, and the mixture was reacted at 20°C for 2 hours. Water (1.5 L) was added to the reaction solution, and the mixture was filtered after stirring for 20 minutes. The filter cake was dried to obtain compound 1-2. MS-ESI calculated value [M+H] ⁺ 164, found value 164.

Step 2

Compound 1-2 (25.6 g, 157 mmol) was dissolved in N, N-dimethylformamide (250 mL), N-bromosuccinimide (30.7 g, 173 mmol) was added, and the mixture was stirred at 20°C for 0.5 hours. Water (1500 mL) was added to the reaction solution and stirred for 30 minutes, filtered, and the filter cake was dried under reduced pressure to obtain compound 1-3. MS-ESI calculated value [M+H] ⁺ 242, 244, found value 242, 244.

third step

Compound 1-3 (170 g, 702.28 mmol,), compound 1-(tributyltin)-methanol (315.69 g, 983.19 mmol), chloro(2-dicyclohexylphosphino-2,4,6-triisopropyl-1,1-biphenyl)[2-(2-amino-1,1-biphenyl)]palladium(II) (16.58 g, 21.07 mmol), 2-di-tert-butylphosphino-2',4',6'-triisopropylbiphenyl (20.09 g, 42.14 mmol) were dissolved in dioxane (1360 mL), the reaction solution was replaced with nitrogen three times, and the reaction solution was stirred at 80° C. for 16 hours under a nitrogen atmosphere. The reaction solution was cooled to 25° C., 6.2 L of n-heptane was added to the reaction solution, filtered, and the filter cake was slurried with 1 L of n-heptane, filtered and dried to obtain intermediate 1. MS-ESI calculated value [M+H] ⁺ 194 found 194. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ =10.17 (s, 1H), 6.93 (d, J=8.0 Hz, 1H), 6.77 (d, J=8.4 Hz, 1H), 4.97 (t, J=5.4 Hz, 1H), 4.46 (s, 2H), 4.41 (d, J=5.6 Hz, 2H), 2.16 (s, 3H).

Example 2: Preparation of Intermediate 2

Synthesis line:

first step

Compound 2-1 (1.00 g, 5.71 mmol) was dissolved in ethanol (5 mL), palladium/carbon (100 mg, 10% purity) was added, and the reaction mixture was stirred for 4 hours at 20° C. under a hydrogen atmosphere (15 psi). The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain compound 2-2. MS-ESI calculated value [M+H] ⁺ 146, found value 146.

Step 2

Compound 2-2 (820 mg, 5.65 mmol) and ethyl 4-chloroacetoacetate (1.12 g, 6.78 mmol) were dissolved in tetrahydrofuran (10 mL), heated to 50 ° C, triethylamine (787 μL, 5.65 mmol) was added to the reaction solution, and stirred at 50 ° C for 4 hours. After the reaction was completed, ethyl acetate (50 mL) was added to the reaction solution for extraction, the organic phase was washed with water (50 mL), washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was separated and purified by column chromatography (eluent: petroleum ether/ethyl acetate, 100/1~10/1, V/V) to obtain compound 2-3. MS-ESI calculated value [M+H] ⁺ 256, measured value 256.

third step

Compound 2-3 (500 mg, 1.96 mmol) was dissolved in ethanol (5 mL), palladium/carbon (100 mg, 10% purity) was added to the reaction solution, and the reaction mixture was stirred for 14 hours at 20°C under a hydrogen atmosphere (15 psi). The reaction solution was filtered, and the crude product obtained after concentration under reduced pressure was dissolved in ethanol (10 mL), palladium/carbon (200 mg, 10% purity) was added, and the reaction mixture was stirred for 14 hours under a hydrogen atmosphere (15 psi) and 20°C, and the reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain compound 2-4. MS-ESI calculated value [M+H] ⁺ 258, found value 258.

the fourth step

Compound 2-4 (5.5 g, 21.38 mmol) was dissolved in methanol (60 mL), and a solution of lithium hydroxide monohydrate (1.35 g, 32.07 mmol) dissolved in water (15 mL) was added to the reaction solution. The reaction mixture was stirred at 20 ° C for 12 hours. After the reaction was completed, it was concentrated under reduced pressure to remove methanol, and water (50 mL) was added to dilute it. The pH was adjusted to 4 with aqueous hydrochloric acid solution (2M), and extracted with ethyl acetate (50 mL×2). The combined organic phases were washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 2-5. MS-ESI calculated value [M+H] ⁺ 230, found value 230.

the fifth step

Compound 2-5 (64 g, 279.26 mmol) was dissolved in acetonitrile (960 mL), and compound 2-6 (59.01 g, 279.26 mmol) was added, and the reaction mixture was stirred at 25 ° C for 0.5 hours. The reaction mixture was heated to 70 ° C and stirred for 1 hour. The temperature was slowly lowered to 25 ° C and stirred at 25 ° C for 12 hours. The reaction solution was filtered, the filter cake was washed with acetonitrile (32 mL * 2), and the filter cake was collected. The filter cake was added to the reaction bottle, 320 mL of water was added, the pH was adjusted to 10-11 with 1M sodium hydroxide aqueous solution, and extracted 3 times with ethyl acetate (320 mL * 3). The aqueous phase was adjusted to pH 2-3 with 12M hydrochloric acid solution, and extracted twice with ethyl acetate (320 mL * 2). The combined organic phase was washed with saturated brine (640 mL * 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 2-7. MS-ESI calculated value [M+H] ⁺ 230, found value 230.

Step 6

Compound 2-7 (48 g, 209.44 mmol) was dissolved in N, N-dimethylformamide (240 mL), 1, 1'-carbonyldiimidazole (44.15 g, 272.28 mmol) was added to the reaction solution, and the reaction mixture was stirred at 25°C for 2 hours. Then, ammonium chloride (33.61 g, 628.33 mmol) and N, N-diisopropylethylamine (40.60 g, 314.16 mmol, 54.72 mL) were added to the reaction solution, and the reaction mixture was stirred at 25°C for 2 hours. The reaction solution was diluted with water (480 mL), extracted with ethyl acetate (480 mL×4), the combined organic phase was washed once with 1M hydrochloric acid (0.96 L), the organic phase was washed with saturated brine (0.96 L*3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was dissolved in ethyl acetate (90 mL), and n-heptane (450 mL) was added and stirred at 25°C for 0.5 hours, filtered, and the filter cake was washed with n-heptane (90 mL*3), and dried under reduced pressure to obtain intermediate 2. MS-ESI calculated value [M+H] ⁺ 229, found value 229.

Example 3: Preparation of the compound of formula (I)

synthetic route:

first step

Intermediate 1 (52 g, 269.15 mol) and trimethylsilyl chloride (58.48 g, 538.31 mol) were dissolved in dichloromethane (234 mL), and DMSO (5.26 g, 67.29 mmol) was slowly added dropwise. The reaction solution was stirred at 25°C for 2 hours. The reaction solution was filtered, and the filter cake was rinsed with dichloromethane (52 mL) and dried to obtain a crude compound 3. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ＝10.28 (s, 1H), 7.02 (d, J＝8.2 Hz, 1H), 6.81 (d, J＝8.2 Hz, 1H), 4.76 (s, 2H), 4.51 (s, 2H), 2.26 (s, 3H).

Step 2

Compound 3 (37 g, 174.82 mmol) and intermediate 2 (38.70 g, 169.58 mmol) were dissolved in acetone (222 mL), sodium iodide (5.24 g, 34.96 mmol) and sodium bicarbonate (15.42 g, 183.56 mmol) were added to the reaction solution, and the reaction mixture was stirred at 50°C for 16 hours. Water (666 mL) was added to the reaction solution in 3 batches, and stirred at 20 to 30°C for 0.5 to 1 hour. The reaction solution was filtered, and the filter cake was washed with water (74 mL*3) until the pH value of the aqueous phase reaches 7. At 20-30°C, add water (370mL) to the crude product and stir for 0.5-1 hour. Filter, wash the filter cake with water (74mL*3), and dry the filter cake under vacuum. Dry the filter cake under reduced pressure to obtain the crude product. Dissolve the crude product (62.5g, 154.94mmol) in tetrahydrofuran (437.5mL), heat to 65°C, and stir for 2 hours. Filter while hot, cool the filtrate to 50-60°C, and slowly add methyl tert-butyl ether (647.85g, 7.35mol, 875.00mL) at 50-60°C. Stir for 1 hour at 50-60°C. Slowly cool to 20°C and stir at 20°C for 12 hours. Filter, wash the filter cake with methyl tert-butyl ether (125mL*3), and dry the filter cake under reduced pressure to obtain the compound of formula (I). The compound was tested for ee value by SFC (chromatographic column: Chiralpak IG-3 50×4.6 mm ID, 3 μm; mobile phase: supercritical CO ₂ -0.05% ethylene glycol amine in ethanol; gradient: 0.05% ethylene glycol amine in ethanol: 5%-40%). MS-ESI calculated value [M+H] ⁺ 404, found value 404. ee value = 100%, RT = 1.196 min. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ=10.19 (s, 1H), 7.23 (s, 1H), 7.06 (d, J=8.4 Hz, 1H), 6.90-6.75 (m, 3H), 6.67 (m, 1H), 4.50 (s, 2H), 4.19 (d, J=14.8 Hz, 1H), 4.13-3.89 (m, 3H), 3.34-3.29 (m, 1H), 2.24 (s, 3H), 2.17-2.05 (m, 1H), 2.04-1.93 (m, 1H).

Example 4: Preparation of crystals of compound A of formula (I)

50 mg of the compound of formula (I) was weighed and added to a glass vial, and an appropriate amount of tetrahydrofuran was added. After adding a magnetic bar, the sample was placed at room temperature (25°C) and magnetically stirred overnight, filtered, and the solid sample on the filter cake was placed in a vacuum drying oven (45°C) and dried overnight to obtain a crystal of the compound of formula (I). The XRPD, DSC and TGA test results of the crystal of compound (I) A are shown in Figures 1, 2 and 3 respectively.

The above tetrahydrofuran was replaced with the solvents listed in Table 6, and the operation was carried out in the same manner as above. The results are shown in Table 6.

Table 6 Room temperature stirring test

Experimental Example 1: Hygroscopicity study of crystal A of compound of formula (I)

Experimental Materials:

SMS DVS intrinsic dynamic water vapor adsorption instrument

experimental method:

10-30 mg of crystal A of the compound of formula (I) was placed in a DVS sample tray for testing.

The classification of moisture absorption evaluation is shown in Table 7.

Table 7 Moisture absorption evaluation classification table

Note: ΔW% indicates the weight gain of the test sample at 25±1℃ and 80±2%RH.

Experimental results:

The DVS spectrum of the crystal A of the compound of formula (I) is shown in Figure 4. The sample was tested according to the procedure of 0-95-0% RH/25°C, and the sample was pre-equilibrated at 0% RH. Compared with the initial humidity of 0%, when the humidity rose to 80%, the sample absorbed moisture and gained weight by 0.2976% (cyclic adsorption), and the sample was slightly hygroscopic. The XRPD test results showed that the crystallization of the sample did not change before and after the DVS test.

Experimental Example 2: Solid Stability Test of Compound A of Formula (I)

According to the Guiding Principles for Stability Testing of APIs and Drug Products (Chinese Pharmacopoeia 2015 Edition, Part IV, General Rules 9001), the stability of crystal A of compound of formula (I) under high temperature (60°C, open), high humidity (room temperature/relative humidity 92.5%, open) and light (total illumination = 1.2×10 ⁶ Lux·hr/near ultraviolet = 200 w·hr/m ² , open) conditions was investigated.

Weigh 10 mg of crystal A of the compound of formula (I), place it at the bottom of a glass sample bottle, and spread it into a thin layer. For samples placed under high temperature (60°C) and high humidity (relative humidity 92.5% RH), seal the bottle mouth with aluminum foil, and punch some small holes in the aluminum foil to ensure that the sample can fully contact the ambient air, and place it in the corresponding constant temperature and humidity chamber; the illuminated sample (open, not covered with aluminum foil) and the illuminated control (the entire sample bottle is covered with aluminum foil) are placed in the illumination box. Weigh 2 portions at each time point as formal test samples. In addition, weigh about 50 mg of crystal A of the compound of formula (I) for XRPD testing, wrap the sample bottle with aluminum foil and punch small holes, and also place it in the corresponding constant temperature and humidity chamber. Samples were sampled and tested (XRPD) on the 5th and 10th days, and the test results were compared with the initial test results on day 0. The results of the solid pre-stability test of crystal A of the compound of formula (I) are shown in Table 8.

Table 8 Solid pre-stability test results of A crystal of compound of formula (I)

Experimental conclusion: Crystal A of the compound of formula (I) has good stability under high temperature, high humidity and strong light conditions.

Experimental Example 3: Single crystal X-ray diffraction analysis of the compound of formula (I)

Instrument parameters

Instrument: Bruker D8 VENTURE single crystal X-ray diffractometer; Cryogenic system: Oxford Cryostream 800; Light source: Cu rotating target, 2.5kW; distance from sample to detector: d=45mm; tube voltage: 50kV; tube current: 45mA.

Crystal cultivation

Take 10 mg of the compound of formula (I) and add it to 4 mL of methanol and stir until the sample is partially dissolved. Then add 1 mL of DMF and stir until the sample is completely dissolved. Place the sample clear solution in a 4 mL semi-sealed sample bottle and evaporate slowly at room temperature. After two weeks, colorless block crystals are obtained.

Data collection and tabulation

The single crystal is a compound of formula (I). The crystals were collected and the diffraction intensity data were collected using a Bruker D8 VENTURE single crystal X-ray diffractometer. The crystal structure data of the compound of formula (I) are shown in Tables 9 to 14.

Table 9 Single crystal structure data of compound of formula (I)

Table 10 Atomic coordinates (×10 ⁴ ) and equivalent isotropic shift parameters of single crystal of compound of formula (I)

Table 11 Bond lengths of single crystals of compounds of formula (I)

Table 12 Bond angles of single crystals of compound of formula (I)

Table 13 Twist angle of single crystal of compound of formula (I)

Table 14 Hydrogen atomic coordinates (×10 ⁴ ) and isotropic displacement parameters of single crystal of compound of formula (I)

Experimental results

The single crystal data show that the single crystal is the compound of formula (I), and it can be determined that the absolute configuration of the compound of formula (I) is S configuration. The stereoscopic structure ellipsoid diagram of the single crystal of the compound of formula (I) is shown in FIG5 .

Experimental Example 4: In vitro evaluation of MR antagonist activity

Experimental purpose: To test the MR antagonist activity of the compound

Experimental Materials:

DMEM medium was purchased from BI; fetal bovine serum was purchased from Biosera; HEK293/Gal4/MR cell line was provided by Wuhan Heyan Biopharmaceutical Technology Co., Ltd. Bright Glo was purchased from Promega; EnVision multi-label analyzer (PerkinElmer).

experimental method:

MR cells were seeded in a white 96-well plate, with 40,000 cells per well in 80 ml of cell suspension. The cell plate was placed in a carbon dioxide incubator for overnight culture.

The compound to be tested was diluted with a gun to 8 concentration points, 5-fold gradient dilution, and the compound concentration was 2 millimolar to 0.026 micromolar. 38 microliters of culture medium was added to the middle plate, and then 2 microliters of compound per well were added to the middle plate according to the corresponding position. After mixing, 10 microliters of compound solution per well were transferred to the cell plate, and the cell plate was placed in a carbon dioxide incubator for incubation for 1 hour. Aldosterone was diluted to 10 nanomolar using culture medium, that is, 20 microliters of aldosterone with a concentration of 1 micromolar was added to 1980 microliters of culture medium, and after mixing, 10 microliters of aldosterone solution was added to each well of the cell plate except the positive control well. The cell plate was placed in a carbon dioxide incubator and incubated for 24 hours. The final concentration of the compound was 10 micromolar to 0.128 nanomolar, and the final concentration of aldosterone was 1 nanomolar.

After the incubation, discard the cell supernatant, add 100 μl of BrightGlo reagent per well to the cell plate, and read immediately on the EnVision Multi-label Analyzer.

data analysis:

The raw data were converted to inhibition rate using the equation %Inhibition=((RFU _Cmpd -AVER(RFU _Neg.Ctrl ))/((AVER(RFU _Pos.Ctrl )-AVER(RFU _Neg.Ctrl ))×100%, and the compound curves were fitted by log(inhibitor) vs.response-Variable slope of Graphpad Prism 5. The IC ₅₀ value was calculated by the software using the formula Y=Bottom+(Top-Bottom)/(1+10^((LogIC50-X)*HillSlope)).

Experimental results: see Table 15.

Table 15 MR antagonist activity test results

Experimental conclusion: The compound of the present application has a good antagonistic activity to the mineralocorticoid receptor.

Experimental Example 5: Pharmacokinetic Evaluation

Experimental Materials:

SD rats (male, 7-9 weeks old, Beijing Weitonglihua)

Experimental operation:

The pharmacokinetic characteristics of the compound after intravenous (IV) and oral administration (PO) in rodents were tested using standard protocols. Rats were given a single intravenous injection and oral administration. The intravenous injection vehicle was water. The intravenous injection vehicle was a clear solution of 80% polyethylene glycol 400/20% water, and the oral vehicle was A homogeneous suspension of 99.9% (0.5% hydroxypropyl methylcellulose: polyethylene glycol stearate 15 = 95:5)/0.1% Tween 80. Four male SD rats were used in this project. Two rats were intravenously injected and plasma samples were collected at 0h (before administration) and 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24h after administration. The other two rats were orally gavaged and plasma samples were collected at 0h (before administration) and 0.25, 0.5, 1, 2, 4, 8, and 24h after administration. Whole blood samples were collected within 24 hours. All blood samples were immediately transferred to commercial centrifuge tubes containing K2-EDTA with labels. After blood sample collection, centrifuge at 4°C, 3200g for 10 minutes to absorb the supernatant plasma, quickly put it in dry ice, and then stored at -60°C or lower for LC-MS/MS analysis. The non-compartmental model was used to analyze the plasma drug concentration-time data and calculate the pharmacokinetic parameters, such as peak concentration (C _max ), clearance (CL), tissue distribution (Vdss), area under the concentration-time curve (AUC _0-last ), bioavailability (F), etc. using the WinNonlin software package (Version 6.3 and above).

The experimental results are shown in Table 16.

Table 16 Pharmacokinetic evaluation results of the compounds of the present application in rats

Experimental conclusion: The compound of the present application has good pharmacokinetic properties.

Experimental Example 6: In vivo efficacy test in unilateral nephrectomized rats

Experimental Materials:

SD rats (male, Beijing Weitonglihua Experimental Animal Technology Co., Ltd.)

Experimental operation:

SD male rats were adaptively fed for 2-3 days and then had their right kidney removed. At this time, they were fed with ordinary feed and drinking water. After one week of recovery, they were grouped according to their body weight. After grouping, an osmotic pump was implanted subcutaneously and aldosterone was injected at a concentration of 3 mg/mL (dissolved in 0.15% DMSO/prepared with sterile water) (Source Leaf Biology S30644-5mg), with a flow rate of 0.75 μg/hr (Alzet model 2004). At the same time, 6% NaCl high-salt feed (customized by Beijing Keao Xieli Feed Co., Ltd.) and 0.3% KCl drinking water (Source Leaf Biology S24120) were fed. While the osmotic pump was implanted subcutaneously, oral administration was performed. The dosage was detailed in the grouping. Oral solvent (0.5% hydroxypropyl methylcellulose, polyethylene glycol stearate 15=95:5 (v/v, containing 0.1% Tween 80)) was administered once a day for 4 consecutive weeks. Body weight was measured and recorded every day. After 4 weeks of administration, urine was collected for 24 hours to detect urine albumin and urine creatinine, and the urine albumin:urine creatinine ratio (UACR) was calculated.

Experimental conclusion: The compound of the present application has a significant protective effect on the kidneys and can effectively reduce the ratio of albumin to creatinine in rat urine.

Claims (14)

The crystals of the compound of formula (I) have an X-ray powder diffraction pattern with diffraction peaks at the following 2θ angles: 10.52±0.20°, 11.45±0.20°, 13.88±0.20° and 15.59±0.20°;
According to the crystal of claim 1, its X-ray powder diffraction pattern has diffraction peaks at the following 2θ angles: 10.52±0.20°, 11.45±0.20°, 13.21±0.20°, 13.88±0.20°, 15.59±0.20°, 16.59±0.20°, 19.52±0.20° and 25.92±0.20°. According to the crystal of claim 2, its X-ray powder diffraction pattern has diffraction peaks at the following 2θ angles: 10.52±0.20°, 11.45±0.20°, 12.38±0.20°, 13.21±0.20°, 13.88±0.20°, 15.59±0.20°, 16.59±0.20°, 19.22±0.20°, 19.52±0.20°, 23.82±0.20°, 25.05±0.20° and 25.92±0.20°. The crystal according to claim 3, has an X-ray powder diffraction pattern having diffraction peaks at the following 2θ angles: 10.52±0.20°, 11.45±0.20°, 12.38±0.20°, 13.21±0.20°, 13.88±0.20°, 15.59±0.20°, 15.99±0.20°, 16.59±0.20°, 18.35±0.20°, 18.60±0.20°, 19.22±0.20°, 19.52±0.20°, 23.82±0.20°, 25.05±0.20°, 25.92±0.20° and 27.10±0.20°. The crystal according to claim 4, wherein the X-ray powder diffraction pattern has diffraction peaks at the following 2θ angles: 6.20±0.20°, 9.04±0.20°, 9.60±0.20°, 10.52±0.20°, 11.45±0.20°, 12.38±0.20°, 12.82±0.20°, 13.21±0.20°, 13.88±0.20°, 15.59±0.20°, 15.99±0.2 0°、16.59±0.20°、17.18±0.20°、17.54±0.20°、18.35±0.20°、18.60±0.20°、19.22±0.20°、19.52±0.20°、20.03±0.20°、20.29±0.20°、20.96±0.20°、21.11±0.20°、21.57±0.20°、22.23±0.20°、23 .08±0.20°, 23.33±0.20°, 23.82±0.20°, 24.22±0.20°, 24.86±0.20°, 25.05±0.20°, 25.26±0.20°, 25.52±0.20°, 25.92±0.20°, 27.10±0.20°, 27.33±0.20°, 27.64±0.20°, 28.82±0.20°, 29.20±0. .20°, 29.68±0.20°, 30.21±0.20°, 30.59±0.20°, 31.19±0.20°, 31.75±0.20°, 32.52±0.20°, 33.71±0.20°, 34.51±0.20°, 35.88±0.20°, 36.54±0.20°, 37.45±0.20°, 38.23±0.20° and 38.60±0.20°. The crystal according to claim 5, wherein the X-ray powder diffraction pattern has diffraction peaks at the following 2θ angles: 6.20°, 9.04°, 9.60°, 10.52°, 11.45°, 12.38°, 12.82°, 13.21°, 13.88°, 15.59°, 15.99, 16.59°, 17.18°, 17.54°, 18.35°, 18.60°, 19.22°, 19.52°, 20.03°, 20.29°, 20.96°, 21.11°, 21.57°, 22. .23°, 23.08°, 23.33°, 23.82°, 24.22°, 24.86°, 25.05°, 25.26°, 25.52°, 25.92°, 27.10°, 27.33°, 27.64°, 28.82°, 29.20°, 29.68°, 30.21°, 30.59°, 31.19°, 31.75°, 32.52°, 33.71°, 34.51°, 35.88°, 36.54°, 37.45°, 38.23° and 38.60°. The XRPD pattern of the crystal of the compound of formula (I) is basically as shown in Figure 1. The crystal according to any one of claims 1 to 7, wherein the differential scanning calorimetry curve has a starting point of an endothermic peak at 215.9°C±5°C. The crystal according to claim 8, whose DSC spectrum is substantially as shown in Figure 2. The crystal according to any one of claims 1 to 7, wherein the weight loss of the crystal at 200°C±3°C is 0.90%. The crystal according to claim 10, has a TGA spectrum substantially as shown in Figure 3. A crystalline composition comprising any one of claims 1 to 11, wherein the crystals of the compound of formula (I) account for more than 50% by weight of the crystalline composition, preferably more than 80%, more preferably more than 90%, and most preferably more than 95%. A pharmaceutical composition comprising a therapeutically or prophylactically effective amount of the crystal according to any one of claims 1 to 11 or the crystalline composition according to claim 12. Use of the crystal according to any one of claims 1 to 11, the crystalline composition according to claim 12, or the pharmaceutical composition according to claim 13 in the preparation of a drug for a disease associated with a mineralocorticoid receptor antagonist; preferably, the disease associated with a mineralocorticoid receptor antagonist is selected from diabetic nephropathy.