WO2020228745A1

WO2020228745A1 - Crystal form of tetrahydroisoxazolo[4,3-c]pyridine compound against hbv

Info

Publication number: WO2020228745A1
Application number: PCT/CN2020/090059
Authority: WO
Inventors: 吴立方; 孙飞; 杜金华; 丁照中; 徐宏江; 杨玲; 张喜全
Original assignee: 正大天晴药业集团股份有限公司; 南京明德新药研发有限公司
Priority date: 2019-05-14
Filing date: 2020-05-13
Publication date: 2020-11-19
Also published as: CN113825758B; CN113825758A

Abstract

Disclosed is a new crystal form of a tetrahydroisoxazolo[4,3-c]pyridine compound against HBV, and in particular, disclosed are a crystal form of a compound of formula (I), a hydrate thereof, a crystal form of the hydrate thereof, and the use of the crystal form in the preparation of an anti-HBV drug.

Description

The crystal form of tetrahydroisoxazolo[4,3-c]pyridine compound against HBV

Cross references to related applications

This application claims the following priority: Chinese patent application: CN201910399200.1, application date: May 14, 2019, the full text of which is incorporated herein by reference.

Technical field

This application relates to a new crystal form of tetrahydroisoxazolo[4,3-c]pyridine compound against HBV, and specifically to the crystal form of the compound of formula (I), its hydrate and the crystal form of its hydrate The form also includes the application of the crystal form in the preparation of anti-HBV drugs.

Background technique

In patients infected with HBV, a stable covalently closed circular DNA, namely cccDNA, is formed in the nucleus of the host's liver cells, which serves as a template for HBV to continuously replicate. All subgenomic RNA (sgRNA) and pregenomic RNA (pgRNA) are formed by cccDNA transcription. After exiting the nucleus, sgRNA is translated into X protein and three other envelope proteins, and pgRNA is translated into core protein and viral polymerase. pgRNA and core protein self-assemble under the action of polymerase to form RNA that wraps the nucleocapsid. In the nucleocapsid, pgRNA is reverse transcribed into negative-strand DNA, and the positive strand of DNA is further synthesized from this, forming rcDNA. On the one hand, the rcDNA wrapped by nucleocapsid re-uncoated into the nucleus to further amplify the cccDNA; on the other hand, it recombines with the envelope protein and releases the cell through the endoplasmic reticulum to form a new HBV. In the replication cycle of HBV, the synthesis of nucleocapsid is a key step in the HBV genome replication process. The synthesis of viral DNA can only occur specifically inside the nucleocapsid. The assembly of nucleocapsid is an evolutionary constraint process that limits the diversity of HBV, and it is very sensitive to even subtle molecular interference. For the development of new therapies for different hepatitis B virus genotypes and drug-resistant strains, targets that act on the synthesis and degradation of nucleocapsid are extremely promising. Some anti-HBV virus compounds related to nucleocapsid have been reported. Several related compounds such as NVR 3-778 (WO 2015109130A1), JNJ-56136379, and GLS-4JHS are in the clinical research stage.

Summary of the invention

In one aspect, this application provides a hydrate of the compound of formula (I), the structure of which is shown in formula (I-1),

Here, x is 0.8 to 1.2.

In some aspects of the application, x of the hydrate is 0.9 to 1.1, preferably 1.0.

On the other hand, this application provides a crystal form of the compound of formula (I-1),

Here, x is 0.8 to 1.2.

In some solutions of the present application, x of the crystal form is 0.9 to 1.1, preferably 1.0.

In another aspect, the present application provides the crystalline form A of the compound of formula (I-1), and its Cu Kα radiation X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 8.54±0.20°, 12.35±0.20°, 19.31±0.20°, 24.50±0.20° and 28.01±0.20°,

Here, x is 0.8 to 1.2.

In some solutions of the present application, x of the crystal form A is 0.9 to 1.1, preferably 1.0.

In some solutions of this application, the X-ray powder diffraction pattern of Cu Kα radiation of the crystal form A has characteristic diffraction peaks at the following 2θ angles: 8.54±0.20°, 12.35±0.20°, 15.27±0.20°, 19.31±0.20° , 22.78±0.20°, 24.50±0.20°, 28.01±0.20° and 29.43±0.20°.

In some solutions of this application, the X-ray powder diffraction pattern of Cu Kα radiation of the above crystal form A has characteristic diffraction peaks at the following 2θ angles: 8.54±0.20°, 12.35±0.20°, 13.43±0.20°, 15.27±0.20° , 16.57±0.20°, 17.83±0.20°, 19.31±0.20°, 22.78±0.20°, 24.50±0.20°, 28.01±0.20° and 29.43±0.20°.

In some solutions of this application, the X-ray powder diffraction pattern of Cu Kα radiation of the above crystal form A has characteristic diffraction peaks at the following 2θ angles: 8.54±0.20°, 12.35±0.20°, 12.68±0.20°, 13.43±0.20° , 15.27±0.20°, 16.57±0.20°, 17.83±0.20°, 19.31±0.20°, 19.79±0.20°, 20.93±0.20°, 22.05±0.20°, 22.78±0.20°, 24.50±0.20°, 25.41±0.20° , 25.74±0.20°, 27.56±0.20°, 28.01±0.20°, 29.07±0.20°, 29.43±0.20°, 30.17±0.20°, 31.53±0.20°, 32.27±0.20°, 33.77±0.20° and 35.88±0.20° .

In some solutions of the present application, in the XRPD pattern of Cu Kα radiation of the above crystal form A, the peak positions and relative intensities of the diffraction peaks are shown in Table 1 below:

Table 1 XRPD data of Form A

In some solutions of this application, the XRPD pattern of the above-mentioned crystal form A is shown in FIG. 1.

In some solutions of this application, the differential scanning calorimetry (DSC) curve of the above-mentioned crystal form A has endothermic peaks at 89.41°C and 131.81°C.

In some solutions of this application, the DSC spectrum of the above-mentioned crystal form A is shown in FIG. 2.

In some solutions of the present application, the thermogravimetric analysis (TGA) curve of the above crystal form A has a weight loss of 4.229% at 78.45±3°C. According to the results of TGA, the above-mentioned crystal form A is a water-containing crystal form.

In some solutions of this application, the TGA pattern of the above-mentioned crystal form A is shown in FIG. 3.

In some solutions of this application, the above-mentioned crystal form A is prepared in a mixed solvent of ethanol and water.

In another aspect, the present application provides a method for preparing crystal form A, the method comprising the following steps:

The compound of the above formula (I) is added to the solvent, and then the solid is separated.

In some aspects of the application, in the preparation method of the above-mentioned crystal form A, the solvent is a mixed solvent of ethanol and water.

In some schemes of the present application, in the preparation method of the above-mentioned crystal form A, after the compound of formula (I) is added to the solvent, it can be selectively heated, and the heating temperature is selected from 30 to 60°C; in some embodiments, the heating temperature It is 40°C. In some specific embodiments, the heating is performed under stirring or shaking conditions for 60 hours.

In some aspects of the application, in the preparation method of the above-mentioned crystal form A, the method of separating the solid is selected from centrifugation or filtration.

In another aspect, this application also provides the crystalline form C of the compound of formula (I-1), and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 10.53±0.20°, 13.36±0.20°, 19.39±0.20 ° and 21.08±0.20°,

Here, x is 0.8 to 1.2.

In some solutions of the present application, x of the crystal form C is 0.9 to 1.1, preferably 1.0.

In some solutions of this application, the X-ray powder diffraction pattern of the above crystal form C has characteristic diffraction peaks at the following 2θ angles: 9.53±0.20°, 10.53±0.20°, 13.36±0.20°, 19.39±0.20°, 21.09±0.20 °, 21.58±0.20°, 22.08±0.20° and 28.96±0.20°.

In some solutions of this application, the X-ray powder diffraction pattern of the above crystal form C has characteristic diffraction peaks at the following 2θ angles: 9.53±0.20°, 10.53±0.20°, 13.36±0.20°, 17.06±0.20°, 19.39±0.20 °, 21.09±0.20°, 21.58±0.20°, 22.08±0.20°, 23.65±0.20°, 28.53±0.20°, 28.96±0.20° and 30.51±0.20°.

In some solutions of this application, the X-ray powder diffraction pattern of the above crystal form C has characteristic diffraction peaks at the following 2θ angles: 9.53±0.20°, 10.53±0.20°, 13.36±0.20°, 17.06±0.20°, 18.80±0.20 °、19.39±0.20°、19.69±0.20、21.09±0.20°、21.58±0.20°、22.08±0.20°、23.65±0.20°、24.26±0.20°、25.90±0.20°、26.85±0.20°、28.53±0.20° , 28.96±0.20°, 29.91±0.20°, 30.51±0.20°, 31.82±0.200°, 34.52±0.20°, 34.93±0.20°, 37.30±0.20° and 38.17±0.20°.

In some solutions of the present application, in the XRPD pattern of Cu Kα radiation of the crystal form C, the peak positions and relative intensities of the diffraction peaks are shown in Table 2 below:

Table 2 XRPD data of Form C

In some solutions of this application, the XRPD pattern of the above-mentioned crystal form C is shown in FIG. 4.

In some solutions of this application, the differential scanning calorimetry (DSC) curve of the above-mentioned crystal form C has an endothermic peak at 134.93°C.

In some solutions of this application, the DSC spectrum of the above-mentioned crystal form C is shown in FIG. 5.

In some solutions of the present application, the thermogravimetric analysis curve of the above-mentioned crystal form C has a weight loss of 4.741% at 132.15±3°C.

In some solutions of this application, the TGA pattern of the above-mentioned crystal form C is shown in FIG. 6.

In some solutions of this application, the above-mentioned crystal form C is prepared in a mixed solvent of acetone and water.

In another aspect, this application provides a method for preparing the above-mentioned crystal form C, the method comprising the following steps: adding a compound of formula (I) to a solvent, and then depositing a solid.

In some embodiments, the above-mentioned preparation method of Type C, wherein the solvent is a mixed solvent of acetone and water.

In some embodiments, in the preparation method of the above-mentioned crystal form C, after the compound of formula (I) is added to the solvent, it can be selectively heated under reflux and stirred. In some specific embodiments, the heating and stirring are cooled to room temperature.

In some embodiments, in the preparation method of the above crystal form C, after the solid is precipitated, the solid can be separated by centrifugation or filtration.

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

In another aspect, this application also provides the crystalline form B of the compound of formula (I) above, and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 8.91±0.20°, 11.80±0.20°, 15.46±0.20° and 16.15±0.20°.

In some solutions of this application, the X-ray powder diffraction pattern of the above-mentioned crystalline form B has characteristic diffraction peaks at the following 2θ angles: 8.91±0.20°, 11.80±0.20°, 15.46±0.20°, 16.15±0.20°, 17.63±0.20 °, 24.72±0.20° and 26.39±0.20°.

In some solutions of this application, the X-ray powder diffraction pattern of the above-mentioned crystalline form B has characteristic diffraction peaks at the following 2θ angles: 8.91±0.20°, 11.80±0.20°, 15.46±0.20°, 16.15±0.20°, 17.63±0.20 °, 19.28±0.20°, 23.46±0.20°, 24.72±0.20°, 25.94±0.20°, and 26.39±0.20°.

In some solutions of this application, the X-ray powder diffraction pattern of the above crystal form B has characteristic diffraction peaks at the following 2θ angles: 8.91±0.20°, 11.80±0.20°, 13.83±0.20°, 15.46±0.20°, 16.15±0.20 °、16.49±0.20°、17.63±0.20°、19.28±0.20°、20.53±0.20°、22.66±0.20°、23.46±0.20°、24.72±0.20°、25.94±0.20°、26.39±0.20°、26.95±0.20 °, 27.99±0.20°, 29.35±0.20°, 30.32±0.20°, 30.99±0.20°, 31.20±0.20°, 34.54±0.20°, 36.10±0.20°, 38.13±0.20°, 38.83±0.20° and 39.47±0.20 °.

In some solutions of this application, in the XRPD pattern of Cu Kα radiation of the above-mentioned crystal form B, the peak positions and relative intensities of the diffraction peaks are shown in Table 3 below:

Table 3 XRPD data of Form B

In some solutions of the present application, the XRPD pattern of the above crystal form B is shown in FIG. 8.

In some solutions of the present application, the differential scanning calorimetry (DSC) curve of the above-mentioned crystal form B has an endothermic peak at 178.62°C.

In some solutions of this application, the DSC spectrum of the above-mentioned crystal form B is shown in FIG. 9.

In some schemes of this application, the DVS pattern of the crystal form B of the compound of formula (I) is shown in FIG. 10.

In some schemes of this application, the above-mentioned crystalline form B is prepared in ethyl acetate. In another aspect, the present application provides a method for preparing the above-mentioned crystal form B, the method comprising the following steps: adding the compound of formula (I) to a solvent, and then separating the solid.

In some aspects of the present application, in the preparation method of the above-mentioned crystal form B, the solvent is ethyl acetate.

In some schemes of the application, in the preparation method of the above-mentioned crystal form B, after the compound of formula (I) is added to the solvent, it can be selectively heated, and the heating temperature is selected from 30 to 60°C; in some embodiments, the heating temperature It is 40°C. In some specific embodiments, the heating is performed under stirring or shaking conditions for 60 hours.

In some aspects of the application, in the preparation method of the above-mentioned crystal form B, the method of separating the solid is selected from centrifugation or filtration.

In some specific embodiments, the preparation method of the above-mentioned crystal form A, crystal form B, and crystal form C further includes drying the separated solid, for example, drying in a vacuum drying oven.

In another aspect, the present application provides a crystal form composition comprising the above-mentioned crystal form A, crystal form B, and crystal form C, wherein the crystal form A, crystal form B, and crystal form C account for the weight of the crystal form composition 50% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more.

In another aspect, the application provides a pharmaceutical composition, which contains a therapeutically effective amount of the crystal form A, crystal form B, crystal form C, or crystal form composition thereof. The pharmaceutical composition of the present application may or may not contain pharmaceutically acceptable excipients. In addition, the pharmaceutical composition of the present application may further include one or more other therapeutic agents.

On the other hand, this application also provides the application of the above-mentioned crystal form A, crystal form B, crystal form C, crystal form composition, or pharmaceutical composition thereof in the preparation of anti-hepatitis B drugs.

On the other hand, this application also provides the application of the above-mentioned crystal form A, crystal form B, crystal form C, crystal form composition thereof, or pharmaceutical composition thereof in the prevention or treatment of hepatitis B.

On the other hand, this application also provides a method for the treatment or prevention of hepatitis B, comprising administering a therapeutically effective amount of the above-mentioned crystal form A, crystal form B, and crystal form C to a mammal in need of such treatment or prevention, preferably a human. , Its crystal composition, or its pharmaceutical composition.

On the other hand, this application also provides the above-mentioned crystal form A, crystal form B, crystal form C, crystal form composition thereof, or pharmaceutical composition thereof for treating or preventing hepatitis B.

The preparation process of the crystal form of the present application is simple, and the crystal form is stable and less affected by heat, humidity, and light, which is convenient for preparation. The crystal form of the present application has good pharmacokinetic properties and is suitable for use as drugs. The pharmacokinetic properties can be measured in preclinical animal experiments such as SD rats and beagle dogs, or in Measured in clinical human trials.

Definition and description

Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings. A specific phrase or term should not be considered uncertain or unclear without a special definition, but should be understood in its ordinary meaning. When a trade name appears herein, it is intended to refer to its corresponding commodity or its active ingredient.

It should be noted that in the powder X-ray diffraction spectrum, the position of the peak or the relative intensity of the peak may be different due to factors such as the measuring instrument and the measuring method/condition. For any particular crystal form, there may be an error in the position of the peak, and the measurement error of the 2θ value may be ±0.2°. Therefore, when determining each crystal type, this error should be taken into consideration, and the error also belongs to the scope of this application.

It should be noted that for the same crystal form, the position of the endothermic peak of DSC may be different due to factors such as measuring instrument, measuring method/condition and so on. For any specific crystal form, there may be an error in the position of the endothermic peak, which can be ±5°C. Therefore, when determining each crystal type, this error should be taken into consideration, and the error also belongs to the scope of this application.

It should be noted that, for the same crystal type, the location of the TGA weight loss temperature may be different due to factors such as the measuring instrument, measuring method/condition and other factors. For any particular crystal type, there may be an error in the position of the weight loss temperature, which can be ±5°C. Therefore, when determining each crystal type, this error should be taken into consideration, and the error also belongs to the scope of this application.

The "pharmaceutically acceptable excipients" refer to inert substances that are administered together with the active ingredients to facilitate the administration of the active ingredients, including but not limited to those acceptable for use in humans or animals approved by the State Food and Drug Administration. (Such as livestock) any glidant, sweetener, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersant, disintegrant, suspending agent, stabilizer Agent, isotonic agent, solvent or emulsifier. Non-limiting examples of the auxiliary materials include calcium carbonate, calcium phosphate, various sugars and various starches, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

The pharmaceutical composition of this application can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, solutions, suppositories, injections, inhalants, Gels, microspheres and aerosols, etc.

Typical routes of administration of the pharmaceutical composition of the present application include, but are not limited to, oral, rectal, transmucosal, enteral administration, or topical, transdermal, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, and peritoneal administration. Intramuscular, subcutaneous, and intravenous administration. The preferred route of administration is oral administration.

The application will be specifically described below through examples, and these examples do not imply any limitation on the application.

All solvents used in this application are commercially available and can be used without further purification.

The following acronyms are used in this application: TFA stands for trifluoroacetic acid; Boc ₂ O stands for di-tert-butyl dicarbonate; Boc stands for tert-butoxycarbonyl, which is a protecting group for amino groups; LCMS stands for liquid mass chromatography; HPLC Represents liquid chromatography; QD represents once a day; BID represents twice a day.

Instruments and analysis methods

1.1 X-ray powder diffraction (X-ray powder diffractometer, XRPD)

Instrument model: Bruker D8 advanced X-ray diffractometer

Test method: Approximately 10-20mg sample is used for XRPD detection.

The detailed XRPD parameters are as follows:

Light pipe: Cu, kα,

Light tube voltage: 40kV, light tube current: 40mA

Divergence slit: 0.60mm

Detector slit: 10.50mm

Anti-scatter slit: 7.10mm

Scanning range: 3 or 4-40deg

Step diameter: 0.02deg

Step length: 0.12 seconds

Sample plate speed: 15rpm

1.2 Differential Scanning Calorimeter (DSC)

Instrument model: TA DSC Q2000 Differential Scanning Calorimeter

Test method: Take a sample (0.5～1mg) and place it in a DSC aluminum pan for testing. Under the condition of 50mL/min N ₂ and at a heating rate of 10℃/min, heat the sample from room temperature (25℃) to 300℃ or 350 ℃.

1.3 Thermogravimetric Analysis (Thermal Gravimetric Analyzer, TGA)

Instrument model: TA Q5000 thermogravimetric analyzer

Test method: Take a sample (2～5mg) and place it in a TGA platinum pot for testing. Under the condition of 25mL/min N ₂ and at a heating rate of 10℃/min, heat the sample from room temperature (25℃) to 300℃, 350 ℃ or 20% weight loss.

1.4 Dynamic Gas Sorption System (DVS)

Instrument model: DVS Advantage-1 (SMS)

Test conditions: Approximately 10-15 mg of sample is used for DVS detection.

Balance dm/dt: 0.01%/min: (time: 10min maximum 180min)

Drying: 0%RH, 120min

RH (%) measurement gradient: 10%

RH(%) measuring gradient range: 0%～90%～0%

1.5 High Performance Liquid Chromatography (High Performance Liquid Chromatograph, HPLC)

Instrument model: Shimadzu high performance liquid chromatograph

The analysis method is as follows:

Table 4 HPLC analysis method of related substance content test

设备equipment	岛津Shimadzu高效液相色谱仪Shimadzu High Performance Liquid Chromatograph
色谱柱Column	Zorbax SB-C-18，4.6mm×150mm，5μm(PDS-HPLC-007)Zorbax SB-C-18, 4.6mm×150mm, 5μm (PDS-HPLC-007)
流动相AMobile phase A	0.1％TFA 水溶液0.1%TFA aqueous solution
流动相BMobile phase B	乙腈溶液Acetonitrile solution
流速Flow rate	1mL/min1mL/min
检测波长Detection wavelength	220nm 220nm

柱温Column temperature	40℃40℃
稀释剂Thinner	1/1(v/v)乙腈：纯水1/1(v/v) Acetonitrile: pure water

Description of the drawings

Figure 1 is an XRPD spectrum of the crystal form A of the compound of formula (I-1).

Figure 2 is a DSC chart of the crystal form A of the compound of formula (I-1).

Figure 3 is a TGA spectrum of the crystal form A of the compound of formula (I-1).

Figure 4 is an XRPD spectrum of the crystal form C of the compound of formula (I-1).

Figure 5 is a DSC spectrum of the crystal form C of the compound of formula (I-1).

Figure 6 is a TGA spectrum of the crystal form C of the compound of formula (I-1).

Figure 7 is a DVS spectrum of the crystal form C of the compound of formula (I-1).

Figure 8 is an XRPD spectrum of the crystalline form B of the compound of formula (I).

Figure 9 is a DSC spectrum of the crystal form B of the compound of formula (I).

Figure 10 is a DVS spectrum of the crystal form B of the compound of formula (I).

Detailed ways

The following examples describe the application in detail, but they do not mean any disadvantageous restriction on the application. The application has been described in detail herein, and its specific embodiments are also disclosed. For those skilled in the art, various changes and improvements can be made to the specific embodiments of the application without departing from the spirit and scope of the application. It will be obvious.

Preparation examples

Example 1 Preparation of compound of formula (I)

Step A: Synthesis of compound 1-2

At 0 degrees Celsius, to a solution of 1-1 (50.0 g, 560.94 mmol) and potassium carbonate (77.53 g, 560.94 mmol) in acetonitrile (500 mL) was added 3-bromopropyne (66.73 g, 560.94 mmol) dropwise . The reaction mixture was naturally warmed to room temperature, and after stirring was continued for 12 hours, it was concentrated under reduced pressure to obtain a residue. The residue was diluted with water and extracted with ethyl acetate. The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product of 1-2. MS (ESI) m/z: 128 [M+H ⁺ ].

Step B: Synthesis of compound 1-3

At room temperature, potassium carbonate (26.56 g, 192.16 mmol) and Boc ₂ O (41.94 g, 192.16 mmol) were added to a solution of 1-2 (52.0 g, 192.16 mmol) in tetrahydrofuran (500 mL) at room temperature. After the reaction solution was stirred at 18 degrees Celsius for 12 hours, the solid was removed by filtration, and the filtrate was collected and concentrated under reduced pressure. The obtained residue was dissolved in ethyl acetate (800 ml), the organic phase was washed successively with water, saturated citric acid solution and saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 1 -3 crude product. MS (ESI) m/z: 228 [M+H ⁺ ].

Step C: Synthesis of Compound 1-4

At 0 degrees Celsius, to a solution of 1-3 (40.0 g, 175.98 mmol) in dichloromethane (400 mL) was added Dess-Martin reagent (82.10 g, 193.58 mmol) in batches. The reaction mixture was naturally warmed to room temperature and stirred for 2 hours, then washed twice with saturated sodium bicarbonate solution and saturated sodium thiosulfate solution with a volume ratio of 1:1, and then washed with saturated sodium bicarbonate solution and saturated brine in turn After the organic phase was dried with anhydrous sodium sulfate and filtered, the filtrate was concentrated under reduced pressure to obtain a crude product of 1-4. MS (ESI) m/z: 226 [M+H ⁺ ].

Step D: Synthesis of compound 1-5

At room temperature, to a mixed solution of 1-4 (42.0 g, 186.43 mmol) in ethanol (400 ml) and water (40 ml) was added sodium acetate (22.94 g, 279.65 mmol) and hydroxylamine hydrochloride (16.84 g, 242.36). Millimoles). After the reaction mixture was stirred at room temperature for 12 hours, it was concentrated under reduced pressure to remove ethanol to obtain a residue. After the residue was dissolved in ethyl acetate, washed with water and saturated brine in turn, dried over anhydrous sodium sulfate and filtered, the filtrate was concentrated under reduced pressure to obtain a crude product of 1-5. MS (ESI) m/z: 241 [M+H ⁺ ].

Step E: Synthesis of compound 1-6

At room temperature, to a solution of 1-5 (300 mg, 1.25 mmol) and 2-iodopyrimidine (257.16 mg, 1.25 mmol) in N,N-dimethylformamide (8 mL) was added cuprous iodide ( 11.89 mg, 62.42 micromoles), triethylamine (252.66 mg, 2.50 mmoles) and bistriphenylphosphine palladium dichloride (43.81 mg, 62.42 micromoles). Under the protection of nitrogen, the reaction mixture was stirred at 12 degrees Celsius for 12 hours. The reaction solution was diluted with ethyl acetate and filtered, the filtrate was concentrated under reduced pressure, and the residue was separated on a preparative TLC plate to obtain 1-6.

MS(ESI) m/z: 319 [M+H ⁺ ].

Step F: Synthesis of compounds 1-7

At room temperature, to a mixed solution of 1-6 (180 mg, 565.38 micromoles) of methanol (4 mL) and water (0.8 mL), bis(trifluoroacetoxy)iodobenzene (291.76 mg, 678.46 micromolar). After the reaction mixture was stirred at 10-20 degrees Celsius for 0.5 hours, it was diluted with 20 ml of water and then extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was separated by preparative TLC plate to obtain 1-7. MS(ESI) m/z: 317 [M+H ⁺ ].

Step G: Synthesis of compound 1-8

1-7 (90 mg, 284.49 micromoles) was dissolved in a dioxane solution (4 mol/L, 4 ml) of hydrochloric acid. After stirring the reaction mixture at 20 degrees Celsius for 0.5 hours, it was concentrated under reduced pressure to obtain 1- 8's crude product. MS (ESI) m/z: 217 [M+H ⁺ ].

Step H: Synthesis of compound A-1

Phenyl chloroformate (58.54 g, 373.90 mmol, 46.83 ml) was slowly added dropwise to dissolve 3-cyano-4-fluoroaniline (46.23 g, 339.91 mmol) and pyridine (29.58 g, 373.90 mmol) at 0 degrees Celsius. Mol, 30.18 mL) in dichloromethane (300 mL). After the reaction mixture was stirred at 25 degrees Celsius for 3 hours, it was quenched by adding 250 ml of water, and a white precipitate precipitated. After filtering the precipitate, it was dried under reduced pressure to obtain Intermediate A-1.

¹ H NMR (400MHz, CDCl ₃ ) δ: 7.85-7.74 (m, 1H), 7.67 (dd, J=3.8, 8.3 Hz, 1H), 7.46-7.41 (m, 2H), 7.31-7.18 (m, 5H) ); MS (ESI) m/z: 257 [M+H ⁺ ].

Step I: Synthesis of compound of formula (I)

At room temperature, add N,N-diisopropylethylamine (110.31 mg, 853.48 micromoles) to a solution of 1-8 (61.52 mg, 243.45 micromoles) in N,N-dimethylformamide (2 mL) ) And intermediate A-1 (72.90 mg, 284.51 micromole). The reaction mixture was stirred at 70 degrees Celsius for 1 hour. After the reaction solution was diluted with 20 ml of water, it was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and filtered. After the filtrate was concentrated under reduced pressure, the residue was purified by preparative high performance liquid chromatography (separation column: Phenomenex Synergi C18, 150×30mm×4μm, mobile phase: [water (0.225% trifluoroacetic acid)-acetonitrile]; acetonitrile%: 35% -65%, 10 min) to obtain the compound of formula (I).

¹ H NMR (400MHz, CD ₃ OD) δ: 8.97 (d, J = 5.0 Hz, 2H), 7.83 (dd, J = 2.8, 5.6 Hz, 1H), 7.75-7.71 (m, 1H), 7.52 (t , J=5.0Hz, 1H), 7.30(t, J=9.0Hz, 1H), 5.42(d, J=17.9Hz, 1H), 5.01(quin, J=6.4Hz, 1H), 4.59(d, J = 17.7 Hz, 1H), 3.14 (dd, J=5.7, 16.4 Hz, 1H), 2.95 (dd, J=1.2, 16.4 Hz, 1H), 1.27 (d, J=6.8 Hz, 3H). MS(ESI) m/z: 379 [M+H ⁺ ].

Example 2 Preparation of Compound Form A of Formula (I-1)

Weigh approximately 50 mg of the compound of formula (I) prepared according to Example 1 into a sample bottle, and add a certain volume of a mixed solvent of ethanol and water (V/V=3/1) to form a suspension. The suspension was continuously shaken at 40 degrees Celsius for 60 hours, and after centrifugation, the residual solid was placed in a vacuum drying oven to dry, to obtain the crystal form A of the compound of formula (I-1). The samples were tested by XRPD, as shown in Figure 1, DSC testing was shown in Figure 2, and TGA was shown in Figure 3.

Example 3 Preparation of Compound Form B of Formula (I)

Weigh about 2 g of the compound of formula (I) prepared according to Example 1 into a 40 mL sample bottle, and add 20 mL of ethyl acetate to form a suspension. The suspension was continuously stirred at 40 degrees Celsius for 60 hours and then naturally cooled to room temperature. After the white solid was filtered, it was placed in a vacuum drying oven to dry, to obtain the crystal form B of the compound of formula (I). The samples were tested by XRPD, as shown in Figure 8, DSC testing was shown in Figure 9, and DVS was shown in Figure 10.

Example 4 Preparation of Compound Form C of Formula (I-1)

220 g of the compound of formula (I) prepared according to Example 1 was added to a mixed solvent of acetone and water (420 mL, V/V=2/1), heated to reflux and naturally cooled to room temperature under stirring. The precipitated white solid was filtered and put into a vacuum drying oven to dry to obtain the crystal form C of the compound of formula (I-1). The samples are tested by XRPD, as shown in Figure 4, DSC testing is shown in Figure 5, TGA is shown in Figure 6, and DVS is shown in Figure 7.

Characterization data

1. Stability experiment of crystal form A, B, C

1.1 Preparation of samples for solid stability test of raw materials

Weigh 5 mg each of crystal form A, crystal form B, and crystal form C, put them in a dry and clean glass bottle, spread them into a thin layer, and use them as test samples. Each sample is weighed in two in parallel and labeled as sample 1 and sample 2. Place the sample under test conditions of influencing factors (60°C, 92.5%RH) and accelerated conditions (40°C, 75%RH and 60°C, 75%RH). The sample is fully exposed and covered with aluminum foil. Make small holes. Sampling analysis in 5 days, 10 days, 1 month, 2 months, and 3 months. The samples placed under the conditions of light (visible light 1200000 Lux, ultraviolet 200W) are fully exposed at room temperature.

1.2 Preparation of mobile phase and diluent

Diluent: acetonitrile/water=50/50 (V/V).

Mobile phase A: 0.1% TFA aqueous solution.

Mobile phase B: 100% pure acetonitrile solution.

1.3 Preparation of sample solution (0.5mg/mL)

Remove the sample from the refrigerator, place it at room temperature, and add 10 mL of diluent to dissolve it. As for the detection of the concentration of related substances, the results are shown in Table 5, and each crystal form did not undergo crystal transformation or other changes under various conditions.

Table 5 Stability test results of crystal forms A, B and C

Assay (%): indicates the relative content of the main peak; TRS: total impurity content; RH: relative humidity.

Activity test

1. In vitro anti-HBV activity test of compound of formula (I)

1) 100 microliters of HepG2.2.15 cells are seeded in a 96-well cell culture plate at 1.2×10 ⁵ cells per well, and the cells are cultured overnight in an incubator containing 5% carbon dioxide (CO ₂ ) at 37°C. On the second day, the test compound and the reference compound entecavir (ETV) were diluted three-fold with DMSO to a total of 8 concentrations, then the compound was diluted 100-fold with the medium, and 100 μl of the diluted compound was added to the cell-containing The final volume of the culture plate is 200 microliters, the final concentration of DMSO is 0.5%, double wells. The cells were cultured in an incubator containing 5% CO ₂ at 37°C for 3 days. On the fifth day, the cell culture plate was replaced with fresh medium containing the same concentration of compound. When the culture reaches the eighth day, collect the cell culture plate supernatant for extraction of HBV DNA.

2) Real-time quantitative PCR to detect HBV DNA: Extract the total DNA in the supernatant with QIAamp 96DNA Blood Kit, and use HBV-specific primers and probes to detect the HBV DNA content by quantitative PCR. Add 20 microliters of PCR master mix and 5 microliters of HBV DNA sample or HBV plasmid standard to the quantitative PCR plate for reaction. The HBV plasmid standard was diluted 7 points per microliter in a 10-fold gradient from 10 ⁷ to 10 copies. The quantitative PCR reaction program is as follows: 95°C pre-denaturation for 10 minutes; 95°C denaturation for 15 seconds, 60°C for 1 minute, and repeat the cycle 40 times. Calculate the inhibition rate of each well against HBV DNA according to the following formula, and use GraphPad Prism software to perform a nonlinear fitting analysis on the inhibition rate data of the compound to obtain the EC ₅₀ value of the compound.

HBV DNA inhibition rate% = (1-sample HBV DNA copy number/DMSO control HBV DNA copy number) × 100%

The HepG2.2.15EC ₅₀ value of the compound of formula (I) is shown in Table 6 below.

Table 6 In vitro anti-HBV activity results of compounds of formula (I)

化合物Compound	HepG2.2.15EC ₅₀(nM) HepG2.2.15EC ₅₀ (nM)
式(I)化合物Compound of formula (I)	25.3525.35

2. Mouse pharmacokinetic study

This experiment aims to evaluate the pharmacokinetic behavior of the compound of formula (I) after a single intravenous injection or intragastric administration in mice. For intravenous injection, the compound is formulated as a clear solution of 0.5 mg/mL, and the solvent: 5% DMSO/5% dodecyl hydroxystearate (solutol)/90% water; for intragastric administration, the compound is formulated as 2 mg/mL The suspension, solvent: 0.5% sodium carboxymethyl cellulose/0.2% Tween 80/99.3% water.

The concentration of the compound in plasma was determined by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). The retention time of the compound and the internal standard (diclofenac), chromatogram acquisition and chromatogram integration are processed by the software Analyst (Applied Biosystems), and the data statistics are processed by the software Watson LIMS (Thermo Fisher Scientific) or Analyst (Applied Biosystems). The unit of the analyte concentration in the sample is ng/mL, with 3 significant digits reserved, and all values expressed as percentages (such as% deviation and% coefficient of variation, etc.) are kept to one decimal place. Each calibration curve contains at least 6 concentration levels. The preparation of calibration standards requires stock solutions from different sources from the quality control samples. The deviation between the calculated concentration of the calibration standard and the labeled value exceeds ±15.0% (the lower limit of quantification exceeds ±20.0%). The standard should be rejected in regression analysis. The rejected calibration standards should be less than 25%, and each calibration curve contains at least 6 calibration standards that meet the acceptance criteria. If the lower limit of quantification and upper limit of quantification need to be rejected, the upper and lower limit of quantification of the analysis batch will be increased and decreased accordingly.

The non-compartmental model of WinNonlin ^TM Version 6.3 (Pharsight, Mountain View, CA) pharmacokinetic software was used to process the plasma concentration, and the linear logarithmic ladder method was used to calculate the pharmacokinetic parameters. The pharmacokinetic parameters to be calculated include but are not limited to (data allowed) T _1/2 , Vdss, CL, AUC _{0-24h in the} intravenous injection group; C _max , T _max , AUC _0-24h , and oral gavage group Bioavailability (F%).

The pharmacokinetic parameters of the compound of formula (I) in mice are shown in Table 7 below.

Table 7

3. In vivo activity test of mouse tail vein high-pressure water injection of HBV DNA plasmid model

The purpose of this study is to detect the inhibitory effect of compounds (compounds of formula (I)) on HBV in mice through a mouse model of high-pressure tail vein injection. This experiment uses female BABL/c mice, 6-7 weeks old. The HBV plasmid DNA was extracted with pAAV2-HBV 1.3mer and Qiagen EndoFree Plasmid Giga kit at a concentration of 1000ng/μL. Dilute with normal saline before use and store at 4°C until use.

3.1 Animal grouping

The grouping of experimental animals is shown in Table 8 below:

Table 8

*: Only one dose is given on the 7th day.

3.2 Drug preparation

The drug preparation situation is shown in Table 9 below:

Table 9

3.3 Dosing schedule

Table 10

组别Group	待测化合物Compound to be tested	给药方案(剂量/给药方式/频率/总时长)Dosing schedule (dose/dosing method/frequency/total duration)
11	溶剂对照组Solvent control group	10mL/Kg，灌胃，一天1次，从第1天到第7天 ^* 10mL/Kg, gavage, once a day, from day 1 to day 7 ^*
22	式(I)化合物Compound of formula (I)	60mg/Kg，灌胃，一天1次，从第1天到第7天 ^* 60mg/Kg, gavage, once a day, from day 1 to day 7 ^*
33	式(I)化合物Compound of formula (I)	60mg/Kg，灌胃，一天2次(8hr/16hr)，从第1天到第7天 ^* 60mg/Kg, gavage, 2 times a day (8hr/16hr), from day 1 to day 7 ^*

*: The medicine is given only once on the 7th day.

3.4 Experimental results

1) The detection values of HBV DNA concentration (Log HBV DNA) in blood samples collected on the 1, 3, 5, and 7 days after the administration of the mice in each test group are shown in Table 11 below:

Table 11

2) After 7 days of administration, the detection value of HBV DNA concentration (Log HBV DNA) in the liver tissue of each test group animal is shown in Table 12:

Table 12

Claims

The hydrate of the compound of formula (I) has the structure shown in formula (I-1),

Among them, x is 0.8 to 1.2, preferably 0.9 to 1.1, and more preferably 1.0.
The crystal form of the compound of formula (I-1),

Among them, x is 0.8 to 1.2, preferably 0.9 to 1.1, and more preferably 1.0.
The crystalline form A of the compound of formula (I-1), the X-ray powder diffraction pattern of Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 8.54±0.20°, 12.35±0.20°, 19.31±0.20°, 24.50±0.20 ° and 28.01±0.20°,

Among them, x is 0.8 to 1.2, preferably 0.9 to 1.1, and more preferably 1.0.
The crystal form A of claim 3, the X-ray powder diffraction pattern of Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 8.54±0.20°, 12.35±0.20°, 15.27±0.20°, 19.31±0.20° , 22.78±0.20°, 24.50±0.20°, 28.01±0.20° and 29.43±0.20°.
The crystal form A according to claim 4, the X-ray powder diffraction pattern of Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 8.54±0.20°, 12.35±0.20°, 13.43±0.20°, 15.27±0.20° , 16.57±0.20°, 17.83±0.20°, 19.31±0.20°, 22.78±0.20°, 24.50±0.20°, 28.01±0.20° and 29.43±0.20°.
The crystal form A according to claim 5, the X-ray powder diffraction pattern of Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 8.54±0.200°, 12.35±0.20°, 12.68±0.20°, 13.43±0.20° , 15.27±0.20°, 16.57±0.20°, 17.83±0.20°, 19.31±0.20°, 19.79±0.20°, 20.93±0.20°, 22.05±0.20°, 22.78±0.20°, 24.50±0.20°, 25.41±0.20° , 25.74±0.20°, 27.56±0.20°, 28.01±0.20°, 29.07±0.20°, 29.43±0.20°, 30.17±0.20°, 31.53±0.20°, 32.27±0.20°, 33.77±0.20° and 35.88±0.20° .
The crystal form A according to any one of claims 3 to 6, whose XRPD pattern is shown in FIG. 1.
The crystalline form A of the compound of formula (I-1) has endothermic peaks at 89.41°C and 131.81°C in its differential scanning calorimetry curve,

Among them, x is 0.8 to 1.2, preferably 0.9 to 1.1, and more preferably 1.0.
The crystal form A according to claim 8, and its DSC spectrum is shown in Fig. 2.
The crystal form A according to any one of claims 3-9, which is prepared in a mixed solvent of ethanol and water.
The preparation method of crystal form A according to any one of claims 3 to 9 comprises the following steps:

1) The compound of formula (I)
Add to the solvent;

2) Separate solids;

The solvent is a mixed solvent of ethanol and water.
Form C of the compound of formula (I-1), its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 10.53±0.20°, 13.36±0.20°, 19.39±0.20° and 21.08±0.20°,

Among them, x is 0.8 to 1.2, preferably 0.9 to 1.1, and more preferably 1.0.
The crystal form C according to claim 12, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 9.53±0.20°, 10.53±0.20°, 13.36±0.20°, 19.39±0.20°, 21.09±0.20 °, 21.58±0.20°, 22.08±0.20° and 28.96±0.20°.
The crystalline form C according to claim 13, whose X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 9.53±0.20°, 10.53±0.20°, 13.36±0.20°, 17.06±0.20°, 19.39±0.20 °, 21.09±0.20°, 21.58±0.20°, 22.08±0.20°, 23.65±0.20°, 28.53±0.20°, 28.96±0.20° and 30.51±0.20°.
The crystalline form C according to claim 14, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 9.53±0.20°, 10.53±0.20°, 13.36±0.20°, 17.06±0.20°, 18.80±0.20 °、19.39±0.20°、19.69±0.20、21.09±0.20°、21.58±0.20°、22.08±0.20°、23.65±0.20°、24.26±0.20°、25.90±0.20°、26.85±0.20°、28.53±0.20° , 28.96±0.20°, 29.91±0.20°, 30.51±0.20°, 31.82±0.200°, 34.52±0.20°, 34.93±0.20°, 37.30±0.20° and 38.17±0.20°.
The crystal form C according to any one of claims 12-15, and its XRPD pattern is shown in FIG. 4.
The crystalline form C of the compound of formula (I-1) has an endothermic peak at 134.93°C in its differential scanning calorimetry curve,

Among them, x is 0.8 to 1.2, preferably 0.9 to 1.1, and more preferably 1.0.
The crystal form C according to claim 17, whose DSC spectrum is shown in FIG. 5.
The crystal form C according to any one of claims 12 to 18, its preparation method comprises the following steps:

1) The compound of formula (I)
Add to the solvent;

2) Separated solid;

The solvent is a mixed solvent of acetone and water.
Form B of the compound of formula (I), its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 8.91±0.20°, 11.80±0.20°, 15.46±0.20° and 16.15±0.20°,
The crystal form B according to claim 20, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 8.91±0.20°, 11.80±0.20°, 15.46±0.20°, 16.15±0.20°, 17.63±0.20 °, 24.72±0.20° and 26.39±0.20°.
The crystal form B according to claim 21, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 8.91±0.20°, 11.80±0.20°, 15.46±0.20°, 16.15±0.20°, 17.63±0.20 °, 19.28±0.20°, 23.46±0.20°, 24.72±0.20°, 25.94±0.20°, and 26.39±0.20°.
The crystal form B according to claim 22, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 8.91±0.20°, 11.80±0.20°, 13.83±0.20°, 15.46±0.20°, 16.15±0.20 °、16.49±0.20°、17.63±0.20°、19.28±0.20°、20.53±0.20°、22.66±0.20°、23.46±0.20°、24.72±0.20°、25.94±0.20°、26.39±0.20°、26.95±0.20 °, 27.99±0.20°, 29.35±0.20°, 30.32±0.20°, 30.99±0.20°, 31.20±0.20°, 34.54±0.20°, 36.10±0.20°, 38.13±0.20°, 38.83±0.20° and 39.47±0.20 °.
According to the crystal form B according to any one of claims 20 to 23, the XRPD pattern is shown in FIG. 8.
The crystalline form B of the compound of formula (I) has an endothermic peak at 178.62°C in its differential scanning calorimetry curve,
According to the crystalline form B of claim 25, the DSC spectrum is shown in FIG. 9.
The crystal form B according to any one of claims 20 to 26, its preparation method comprises the following steps:

1) Add the compound of formula (I) to the solvent,

2) Separate solids;

Wherein the solvent is ethyl acetate.
A crystal composition comprising the crystal form A according to any one of claims 3 to 10, crystal form C according to any one of 12 to 18, and crystal form according to any one of 20 to 26. Form B, the crystal form A, crystal form B, and crystal form C account for more than 50% of the weight of the crystal composition, preferably more than 80%, more preferably more than 90%, and most preferably more than 95%.
A pharmaceutical composition comprising a therapeutically effective amount of the crystal form A according to any one of claims 3 to 10, the crystal form C according to any one of 12 to 18, and the crystal form according to any one of 20 to 26. Form B or the crystalline composition of claim 28.
The crystal form A according to any one of claims 3 to 10, the crystal form C according to any one of 12 to 18, the crystal form B according to any one of 20 to 26, or the crystal form according to claim 28 Use of the composition or the pharmaceutical composition according to claim 29 in the preparation of a medicine for treating and preventing hepatitis B.