HK40060045B - Crystal form of wee1 inhibitor compound and use thereof - Google Patents

Description

Crystal forms and applications of Wee1 inhibitor compounds

Cross-references to related applications

CN201910364694.X, application date: April 30, 2019.

Technical Field

This invention discloses the crystal form of the compound of formula (Ⅰ) and its application in the preparation of drugs for treating Wee1-related diseases.

Background Technology

The cell cycle is a complex process controlled by a series of cell cycle regulatory systems. The core component of the cell cycle regulatory system is the CDKs/Cyclins complex, which is formed by the binding of cyclin-dependent kinases (CDKs) and cyclins. These complexes can promote the cell to enter the proliferation cycle. Among them, the CDK1 (human homolog is also called CDC2)/Cyclin B complex plays a key role in controlling the cell to enter the M phase.

Before cells enter the M phase, DNA replication must be completed. Due to interference from various endogenous and exogenous factors, DNA often mutates or is damaged. This abnormal DNA must be repaired; otherwise, it can cause mitotic catastrophe and cell death. The main function of cell cycle checkpoints is to pause the cell cycle, allowing cells to complete DNA repair before entering the M phase. The G1/S checkpoint at the end of G1 phase and the G2/M checkpoint in G2 phase are two main cell cycle checkpoints, jointly responsible for recognizing and repairing DNA damage. Normal cells can complete DNA repair using the G1/S checkpoint in the G1 phase, but nearly 50% of cancer cells have a deficiency in the tumor suppressor gene p53, which also causes them to lack the function of the G1/S checkpoint. They need to rely more heavily on the G2/M checkpoint for DNA repair. The G2/M checkpoint rarely mutates; it is precisely because of this that cancer cells can evade DNA-damaging agents and radiation therapy.

Wee1 protein kinase is a cell cycle regulator, belonging to the nuclear serine and threonine protein kinase family, and is a key kinase at the G2/M checkpoint. The human "Wee" protein kinase family mainly includes Wee1 and Myt1, both of which phosphorylate the Tyr15 site on CDC2, inhibiting the activation of the CDC2/Cyclin B complex and arresting cells from entering the M phase until DNA repair is completed. Myt1 can also phosphorylate the Thr14 site on CDC2, which is also a negative regulation of CDC2 activity. Wee1 kinase is highly expressed in many types of cancerous cells. By inhibiting Wee1 kinase, tumor cells can skip the G2 phase DNA repair, prematurely enter mitosis, and lead to tumor cell death, thus achieving the goal of cancer treatment.

AstraZeneca's Wee1 inhibitor AZD1775 is currently in Phase II clinical trials, with over 30 clinical trials under development and showing promising therapeutic effects. AZD1775 was originally developed by Merck and is therefore also known as MK-1775. In September 2013, Merck transferred the compound globally to AstraZeneca, with related patents including US20070254892, WO2007126122, EP2213673, WO2008133866, and WO2011034743. Abbott and Abbvie have also conducted research on Wee1 inhibitors, with related patents including US2012220572, WO2013126656, WO2013012681, WO2013059485, WO2013013031, and WO2013126656. Almac's patents related to Wee1 inhibitors include WO2014167347, WO2015019037, and WO2015092431.

WO2008133866 discloses compound AZD1775, with the following structure:

Summary of the Invention

The present invention provides the A crystal form of the compound of formula (Ⅰ), whose X-ray powder diffraction (XRPD) pattern has characteristic diffraction peaks at the following 2θ angles: 5.71±0.2°, 12.68±0.2° and 15.32±0.2°.

In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned A-type crystal has characteristic diffraction peaks at the following 2θ angles: 5.71±0.2°, 12.68±0.2°, 15.32±0.2°, 19.72±0.2°, 21.44±0.2°, 23.61±0.2°, and 25.68±0.2°.

In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned A-type crystal has characteristic diffraction peaks at the following 2θ angles: 5.71±0.2°, 12.68±0.2°, 15.32±0.2°, 18.04±0.2°, 19.72±0.2°, 21.44±0.2°, 23.61±0.2°, and 25.68±0.2°.

In some embodiments of the present invention, the XRPD pattern of the above-mentioned A crystal form is shown in Figure 1.

In some embodiments of the present invention, the XRPD spectra analysis data of the above-mentioned A-type crystal form are shown in Table 1:

Table 1: XRPD pattern analysis data of crystal form A

In some embodiments of the present invention, the differential scanning calorimetry (DSC) curves of the above-mentioned A crystal form have an endothermic peak starting point at 34.95±3℃, 174.75±3℃ and 219.12±3℃ respectively.

In some embodiments of the present invention, the DSC spectrum of the above-mentioned A crystal form is shown in Figure 2.

In some embodiments of the present invention, the thermogravimetric analysis (TGA) curve of the above-mentioned A crystal form shows a weight loss of 0.7367% at 70.33±3℃ and a weight loss of 3.123% at 209.42±3℃.

In some embodiments of the present invention, the TGA spectrum of the above-mentioned A crystal form is shown in Figure 3.

The present invention also provides the B crystal form of the compound of formula (I), whose X-ray powder diffraction (XRPD) pattern has characteristic diffraction peaks at the following 2θ angles: 5.58±0.2°, 12.44±0.2° and 22.16±0.2°.

In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned B crystal form has characteristic diffraction peaks at the following 2θ angles: 5.58±0.2°, 11.71±0.2°, 12.44±0.2°, 14.48±0.2°, 15.13±0.2°, 18.64±0.2°, 22.16±0.2°, and 26.33±0.2°.

In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned B crystal form has characteristic diffraction peaks at the following 2θ angles: 5.58±0.2°, 11.71±0.2°, 12.44±0.2°, 14.48±0.2°, 15.13±0.2°, 17.57±0.2°, 18.64±0.2°, 22.16±0.2°, and 26.33±0.2°.

In some embodiments of the present invention, the XRPD pattern of the B crystal form is shown in Figure 4.

In some embodiments of the present invention, the XRPD spectra analysis data of the above-mentioned B crystal form are shown in Table 2:

Table 2: XRPD pattern analysis data of B crystal form

In some embodiments of the present invention, the differential scanning calorimetry (DSC) curves of the B crystal form have an endothermic peak starting point at 42.88±3℃, 198.79±3℃, and 222.36±3℃, respectively.

In some embodiments of the present invention, the DSC spectrum of the B crystal form is shown in Figure 5.

In some embodiments of the present invention, the thermogravimetric analysis (TGA) curve of the above-mentioned B crystal form shows a weight loss of 3.265% at 64.21±3℃ and a further weight loss of 1.516% at 243.05±3℃.

In some embodiments of the present invention, the TGA spectrum of the B crystal form is shown in Figure 6.

The present invention also provides the C crystal form of the compound of formula (I), whose X-ray powder diffraction (XRPD) pattern has characteristic diffraction peaks at the following 2θ angles: 5.05±0.2°, 5.58±0.2° and 12.44±0.2°.

In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned C-type crystal has characteristic diffraction peaks at the following 2θ angles: 5.05±0.2°, 5.58±0.2°, 12.44±0.2°, 15.91±0.2°, 16.68±0.2°, 17.61±0.2°, 22.19±0.2°, and 26.37±0.2°.

In some embodiments of the present invention, the XRPD pattern of the C crystal form is shown in Figure 7.

In some embodiments of the present invention, the XRPD spectra analysis data of the above-mentioned C-type crystal are shown in Table 3:

Table 3: XRPD pattern analysis data of C-type crystals

In some embodiments of the present invention, the differential scanning calorimetry (DSC) curves of the above-mentioned C crystal form have an endothermic peak starting point at 37.06±3℃, 189.16±3℃ and 218.61±3℃ respectively.

In some embodiments of the present invention, the DSC spectrum of the above-mentioned C crystal form is shown in Figure 8.

In some embodiments of the present invention, the thermogravimetric analysis (TGA) curve of the above-mentioned C-type crystal shows a weight loss of 2.211% at 64.98±3℃ and a further weight loss of 1.127% at 224.71±3℃.

In some embodiments of the present invention, the TGA spectrum of the C crystal form is shown in Figure 9.

The present invention also provides the D crystal form of the compound of formula (I), whose X-ray powder diffraction (XRPD) pattern has characteristic diffraction peaks at the following 2θ angles: 5.22±0.2°, 15.99±0.2° and 16.57±0.2°.

In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned D crystal form has characteristic diffraction peaks at the following 2θ angles: 5.22±0.2°, 15.99±0.2°, 16.57±0.2° and 21.22±0.2°.

In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned D-type crystal has characteristic diffraction peaks at the following 2θ angles: 5.22±0.2°, 15.18±0.2°, 15.99±0.2°, 16.57±0.2°, 17.08±0.2°, 18.60±0.2°, 21.22±0.2°, and 21.89±0.2°.

In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned D-type crystal has characteristic diffraction peaks at the following 2θ angles: 5.22±0.2°, 15.18±0.2°, 15.99±0.2°, 16.57±0.2°, 17.08±0.2°, 17.90±0.2°, 18.60±0.2°, 21.22±0.2°, 21.89±0.2°, 25.24±0.2°, and 27.00±0.2°.

In some embodiments of the present invention, the XRPD pattern of the above-mentioned D crystal form is shown in Figure 10.

In some embodiments of the present invention, the XRPD spectra analysis data of the above-mentioned D crystal form are shown in Table 4:

Table 4: XRPD pattern analysis data of D crystal form

In some embodiments of the present invention, the differential scanning calorimetry (DSC) curves of the above-mentioned D crystal form have an endothermic peak starting point at 56.07±3℃, 193.93±3℃ and 216.54±3℃ respectively.

In some embodiments of the present invention, the differential scanning calorimetry (DSC) curves of the above-mentioned D crystal form have an endothermic peak starting point at 56.07±3℃, 193.93±3℃ and 216.54±3℃ respectively, and an exothermic peak at 206.82±3℃.

In some embodiments of the present invention, the DSC pattern of the above-mentioned D crystal form is shown in Figure 11.

In some embodiments of the present invention, the thermogravimetric analysis (TGA) curve of the above-mentioned D crystal form shows a weight loss of 1.977% at 79.35±3℃ and a weight loss of 1.589% at 223.66±3℃.

In some embodiments of the present invention, the TGA spectrum of the above-mentioned D crystal form is shown in Figure 12.

The present invention also provides the E crystal form of the compound of formula (I), whose X-ray powder diffraction (XRPD) pattern has characteristic diffraction peaks at the following 2θ angles: 8.65±0.2°, 14.22±0.2° and 24.58±0.2°.

In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned E-type crystal has characteristic diffraction peaks at the following 2θ angles: 8.65±0.2°, 11.41±0.2°, 13.13±0.2°, 14.22±0.2°, 17.35±0.2°, 18.34±0.2°, 20.39±0.2°, 20.94±0.2°, and 24.58±0.2°.

In some embodiments of the present invention, the XRPD pattern of the E crystal form is shown in Figure 13.

In some embodiments of the present invention, the XRPD spectra analysis data of the above-mentioned E crystal form are shown in Table 5:

Table 5: XRPD pattern analysis data of E-crystal form

In some embodiments of the present invention, the differential scanning calorimetry (DSC) curves of the E-type crystal form have an endothermic peak starting point at 121.57±3℃, 197.26±3℃ and 217.23±3℃ respectively; and an exothermic peak at 168.31±3℃ and 212.95±3℃ respectively.

In some embodiments of the present invention, the DSC spectrum of the above-mentioned E crystal form is shown in Figure 14.

In some embodiments of the present invention, the thermogravimetric analysis (TGA) curve of the above-mentioned E-type crystal shows a weight loss of 6.775% at 143.31±3℃ and a weight loss of 0.3184% at 213.62±3℃.

In some embodiments of the present invention, the TGA spectrum of the above-mentioned E crystal form is shown in Figure 15.

The present invention also provides the F crystal form of the compound of formula (I), whose X-ray powder diffraction (XRPD) pattern has characteristic diffraction peaks at the following 2θ angles: 5.06±0.2°, 15.91±0.2° and 16.68±0.2°.

In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned F crystal form has characteristic diffraction peaks at the following 2θ angles: 5.06±0.2°, 8.34±0.2°, 10.98±0.2°, 15.13±0.2°, 15.91±0.2°, 16.68±0.2°, 17.63±0.2°, and 18.87±0.2°.

In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned F crystal form has characteristic diffraction peaks at the following 2θ angles: 5.06±0.2°, 8.34±0.2°, 10.98±0.2°, 15.13±0.2°, 15.91±0.2°, 16.68±0.2°, 17.63±0.2°, 18.87±0.2°, 20.33±0.2°, 21.44±0.2°, 22.01±0.2°, 24.04±0.2°, 25.32±0.2°, and 25.66±0.2°.

In some embodiments of the present invention, the XRPD pattern of the above-mentioned F crystal form is shown in Figure 16.

In some embodiments of the present invention, the XRPD spectra analysis data of the above-mentioned F crystal form are shown in Table 6:

Table 6: XRPD pattern analysis data of F crystal form

In some embodiments of the present invention, the differential scanning calorimetry (DSC) curves of the above-mentioned F crystal form have an endothermic peak starting point at 48.69±3℃ and 225.26±3℃, respectively.

In some embodiments of the present invention, the DSC spectrum of the above-mentioned F crystal form is shown in Figure 17.

In some embodiments of the present invention, the thermogravimetric analysis (TGA) curve of the above-mentioned F crystal form shows a weight loss of 3.404% at 100±3℃.

In some embodiments of the present invention, the TGA spectrum of the above-mentioned F crystal form is shown in Figure 18.

This invention also provides a method for preparing crystal form F of compound (I), comprising:

(a) Add the compound of formula (I) to an alcohol solvent, stir and heat to an oil bath at 55-65°C;

(b) Stir at 47℃-53℃ for 72 hours;

(c) Turn off the heating and let it cool naturally for 1 hour until it reaches 27°C while stirring.

(d) Let stand for 18 hours, filter, and then rinse the filter cake with methanol;

(e) Vacuum drying at 60℃ for 48 hours.

In some embodiments of the present invention, the alcohol solvent is methanol.

The present invention also provides the use of the above-described crystal form A, crystal form B, crystal form C, crystal form D, crystal form E, or crystal form F in the preparation of a medicament for treating Wee1-related diseases.

Technical effect

The compounds of this invention have stable crystal forms A, B, C, D, E, and F, are minimally affected by light, heat, and humidity, and exhibit very high solubility, thus showing promising prospects for drug development.

Definitions and Explanations

Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings. A particular phrase or term should not be considered uncertain or unclear unless specifically defined, but should be understood in its ordinary sense. When trade names appear herein, they are intended to refer to the corresponding product or its active ingredient.

The intermediate compounds of the present invention can be prepared by various synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments of the present invention.

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

The present invention will be described in detail below through embodiments, which are not intended to limit the present invention in any way.

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

The solvents used in this invention are commercially available. The following abbreviations are used in this invention: DCM represents dichloromethane; DMF represents N,N-dimethylformamide; DMSO represents dimethyl sulfoxide; EtOH represents ethanol; MeOH represents methanol; TFA represents trifluoroacetic acid; TsOH represents p-toluenesulfonic acid; mp represents melting point; _EtSO₃H represents ethanesulfonic acid; _MeSO₃H represents methanesulfonic acid; ATP represents adenosine triphosphate; HEPES represents 4-hydroxyethylpiperazine ethanesulfonic acid; EGTA represents ethylene glycol bis(2-aminoethyl ether)tetraacetic acid; _MgCl₂ represents magnesium dichloride; _MnCl₂ represents manganese dichloride; DTT represents dithiothreitol; DCC represents dicyclohexylcarbodiimide; DMAP represents 4-dimethylaminopyridine; DIEA represents N,N-diisopropylethylamine; wt%: mass percentage; THF represents tetrahydrofuran.

Instruments and Analytical Methods

1.1 X-ray powder diffraction (XRPD)

Instrument Model: Bruker D8 Advance X-ray Diffractometer

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

The detailed XRPD parameters are as follows:

Fluorescent tube:

Phototube voltage: 40kV, Phototube current: 40mA

Diverging slit: 0.60mm

Detector slit: 10.50mm

Anti-scattering slit: 7.10mm

Scan range: 4-40 degrees

Step size: 0.02deg

Step length: 0.12 seconds

Sample tray rotation speed: 15 rpm

1.2 Differential Scanning Calorimeter (DSC)

Instrument Model: TA Q2000 Differential Scanning Calorimeter Test Method: Take a sample (~1mg) and place it in a DSC aluminum pot for testing. Under N ₂ conditions of 50mL/min, heat the sample from 30℃ to 300℃ at a heating rate of 10℃/min.

1.3 Thermogravimetric Analysis (TGA)

Instrument Model: TA Q5000 Thermogravimetric Analyzer Test Method: Take a sample (2-5 mg) and place it in a TGA platinum pot for testing. Under N ₂ conditions at a heating rate of 10℃/min, heat the sample from 30℃ (room temperature) to 300℃ or until it loses 20% of its weight.

Attached Figure Description

Figure 1 shows the Cu-Kα radiation XRPD spectrum of the A crystal form of compound (I);

Figure 2 shows the DSC spectrum of crystal form A of compound (I);

Figure 3 shows the TGA spectrum of crystal form A of compound (I);

Figure 4 shows the Cu-Kα radiation XRPD spectrum of the B crystal form of compound (I);

Figure 5 shows the DSC spectrum of crystal form B of compound (I);

Figure 6 shows the TGA spectrum of the B crystal form of compound (I);

Figure 7 shows the Cu-Kα radiation XRPD spectrum of the C crystal form of compound (I);

Figure 8 shows the DSC spectrum of the C-crystal form of compound (I);

Figure 9 shows the TGA spectrum of the C-crystal form of compound (I);

Figure 10 shows the Cu-Kα radiation XRPD spectrum of the D crystal form of compound (I);

Figure 11 shows the DSC spectrum of the D crystal form of compound (I);

Figure 12 shows the TGA spectrum of the D crystal form of compound (I);

Figure 13 shows the Cu-Kα radiation XRPD spectrum of the E crystal form of compound (I);

Figure 14 shows the DSC spectrum of the E crystal form of compound (I);

Figure 15 shows the TGA spectrum of the E crystal form of compound (I);

Figure 16 shows the Cu-Kα radiation XRPD spectrum of the F crystal form of compound (I);

Figure 17 shows the DSC spectrum of the F crystal form of compound (I);

Figure 18 shows the TGA spectrum of the F crystal form of compound (I).

Detailed Implementation

The present invention will be described in detail below with reference to embodiments, but this does not imply any adverse limitation on the invention. The present invention has been described in detail, and specific embodiments thereof have also been disclosed. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope thereof.

Intermediate 1

Prepared using the synthesis method described in patent WO2007126122.

Example 1: Compound of Formula (I)

Synthesis route:

Step 1: Synthesis of compound 1-A.

Under nitrogen protection, 3-butenylmagnesium bromide (1M, 55.12 mL) was added dropwise to a THF (150 mL) solution of 2-acetyl-6-bromopyridine (7.35 g, 36.74 mmol). The reaction mixture was then stirred at 10–20 °C for 3 hours. The reaction was quenched by adding 100 mL of saturated ammonium chloride solution. The organic layer was separated, washed with 50 mL of saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated to dryness to obtain a brown oil. This brown oil was then purified by silica gel column chromatography (PE/EA = 7/1) to obtain 1-A. ¹ H NMR (400MHz, DMSO-d ₆ )δ7.73(t,J＝8.0Hz,1H),7.64(d,J＝7.2Hz,1H),7.46(d,J＝7.2Hz,1H),5.78～5. 7(m,1H),4.94～4.85(m,2H),2.07～2.01(m,1H),1.90～1.71(m,3H),1.41(s,3H).

Step 2: Synthesis of compound 1-B.

To a mixture of 1-A (3.47 g, 13.55 mmol) and 1-C (3.01 g, 13.55 mmol) in 150 mL of dioxane, N,N''-dimethylethylenediamine (1.31 g, 14.90 mmol, 1.60 mL), cuprous iodide (2.58 g, 13.55 mmol), and potassium carbonate (2.62 g, 18.97 mmol) were added. The mixture was purged with nitrogen three times, and then stirred at 95 °C for 1.5 h under nitrogen protection. 200 mL of ammonia (28%) was added, followed by extraction with ethyl acetate (300 mL × 2). The organic layers were combined, washed with 200 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure. The mixture was purified by silica gel column chromatography (PE/EA = 3/1) to give 1-B. ¹ H NMR (400MHz, DMSO-d ₆ )δ9.02(s,1H),8.04(t,J＝8.0Hz,1H),7.76(d,J＝7.2Hz,1H),7.64(d,J＝7.6Hz,1H),5.77～5.67(m,2H), 5.01～4.79(m,6H),2.56(s,3H),2.15～2.11(m,1H),1.85～1.75(m,2H),1.70～1.60(m,1H),1.46(s,3H).

Step 3: Synthesis of compound 1-D.

Grubbs second-generation catalyst (1.32 g, 1.55 mmol) was added to a toluene (700 mL) solution of 1-B (2.06 g, 5.18 mmol), and the mixture was stirred at 80 °C under a nitrogen atmosphere for 6 hours. Then, Grubbs second-generation catalyst (0.65 g, 0.775 mmol) was added, and the mixture was stirred at 80 °C under a nitrogen atmosphere for 3 hours. The mixture was cooled to room temperature, filtered, and the filtrate was concentrated to dryness to give a brown residue. Purification by silica gel column chromatography (PE/EA = 1/1) yielded 1-D. ¹ H NMR (400MHz, DMSO-d ₆ )δ9.07(s,1H),8.06(t,J＝8.0Hz,1H),7.79(d,J＝8.2Hz,1H),7.70(d,J＝7.6Hz,1H),5.39～5.25 (m,3H),4.66(d,J＝5.2Hz,2H),,2.62(s,3H),2.40～1.95(m,3H),1.85～1.65(m,1H)1.64(s,3H).

Step 4: Synthesis of compound 1-E.

To a toluene (35 mL) solution of compound 1-D (360 mg, 974.45 μmol), m-chloroperoxybenzoic acid (265.09 mg, 1.31 mmol, 85% purity) was added, and the reaction was stirred at 25 °C for 2 hours. 4-(4-methylpiperazine)aniline (242.30 mg, 1.27 mmol) and N,N-diisopropylethylamine (503.76 mg, 3.90 mmol) were added to the reaction solution. The reaction solution was stirred at 25 °C for 12 hours. 25 mL of water was added to the reaction solution, and the mixture was stirred. The aqueous phase was extracted with ethyl acetate (30 mL × 3). The organic phases were combined, washed once each with saturated sodium bicarbonate solution (30 mL) and saturated brine solution (30 mL), and dried over anhydrous sodium sulfate. The mixture was filtered, concentrated under vacuum to obtain a crude product, which was then separated by preparative liquid chromatography (neutral) to obtain 1-E. ¹ HNMR (400MHz, CDCl ₃ ) δppm 1.70 (s, 3H) 1.78 (br d, J = 13.54Hz, 2H) 2.04 (br d,J＝6.54Hz,1H)2.08-2.23(m,2H)2.39(s,3H)2.62-2.64(m,4H)3.21-3.24(m,4H)4.24(br s,1H)4.51(br MS m/z:513.1[M+H] ⁺ .

Step 5: Synthesis of compound (I).

Compound 1-E (100 mg, 195.08 μmol) was chirally separated by SFC (column: AD 250×50 mm ID, 10 μm; mobile phase: A: supercritical _CO2 , B: EtOH (0.1% _{NH3 H2O} ), A:B = 55:45 at 200 mL/min), retention time: 21 min, yielding compound (Ⅰ). ¹ H NMR (400MHz, CDCl ₃ ) δppm 1.60 (s, 3H) 1.68 (br s,2H)1.94(brd,J＝7.04Hz,1H)2.00-2.20(m,2H)2.30(s,3H)2.52-2.55(m,4H)3.12-3.15(m,4H)4.24(brs,1H)4.42(br d,J＝9.54Hz,1H)5.65(br s,2H)6.85(d,J＝9.04Hz,2H)7.17(s,1H)7.40(d,J＝9.04Hz,2H)7.69-7.81(m,2H)8.77(s,1H). MS m/z:513.1[M+H] ⁺ .

Example 2: Preparation of crystal form A of compound (I)

Approximately 50 mg of compound (I) was added to different glass vials, and 0.5 mL of acetone was added to each vial to form a suspension. The suspension samples were then tested in a constant-temperature shaker (40°C). After shaking at 40°C for two days, the suspension samples were centrifuged, the supernatant was discarded, and the remaining samples were dried overnight in a vacuum drying oven (40°C) to obtain crystal form A of compound (I).

Example 3: Preparation of crystal form B of compound (I)

Approximately 50 mg of compound (I) was added to different glass vials, and 0.3 mL of ethanol was added to each vial to form a suspension. The suspension samples were then tested in a constant-temperature shaker (40 °C). After shaking at 40 °C for two days, the suspension samples were centrifuged, the supernatant was discarded, and the remaining samples were then dried overnight in a vacuum drying oven (40 °C) to obtain the B crystal form of compound (I).

Example 4: Preparation of the C crystal form of compound (I)

Approximately 50 mg of compound (I) was added to different glass vials, and 0.2 mL of methanol was added to each vial to form a suspension. The suspension samples were then tested in a constant-temperature shaker (40 °C). After shaking at 40 °C for two days, the suspension samples were centrifuged, the supernatant was discarded, and the remaining samples were then dried overnight in a vacuum drying oven (40 °C) to obtain the C crystal form of compound (I).

Example 5: Preparation of crystal form D of compound (I)

Approximately 50 mg of compound (I) was added to different glass vials, and 0.4 mL of methanol/water (1/1) was added to each to form a suspension. The suspension samples were then tested in a constant-temperature shaker (40°C). After shaking at 40°C for two days, the suspension samples were centrifuged, the supernatant was discarded, and the remaining samples were dried overnight in a vacuum drying oven (40°C) to obtain the D crystal form of compound (I).

Example 6: Preparation of crystal form E of compound (I)

Approximately 50 mg of compound (I) was added to different glass vials, and 0.5 mL of acetonitrile was added to each vial to form a suspension. The suspension samples were then tested in a constant-temperature shaker (40 °C). After shaking at 40 °C for two days, the suspension samples were centrifuged, the supernatant was discarded, and the remaining samples were then dried overnight in a vacuum drying oven (40 °C) to obtain the E crystal form of compound (I).

Example 7: Preparation of crystal form F of compound (I)

At 25–26°C, 134.19 g of compound (I) was added to a 1 L three-necked flask containing 560 mL of MeOH. The mixture was stirred and heated to an oil bath temperature of 55–65°C. After 20 minutes, the internal temperature was maintained at 47°C–53°C. The mixture was kept at this temperature and stirred for 72 hours. The heating was then turned off, and the mixture was allowed to cool naturally for 1 hour until it reached 27°C. After standing for 18 hours, the mixture was filtered, and the filter cake was washed with 30 mL of MeOH. The filter cake was then vacuum-dried at 60°C for 48 hours. The F crystal form of compound (I) was obtained.

Experimental Example 1: Solid stability test of compound F of formula (I) under high temperature, high humidity and light conditions.

Based on the influencing factors and accelerated testing conditions, approximately 5 mg of compound F (I) crystal form was accurately weighed and placed in a dry, clean glass bottle, in duplicate. Each copy was spread into a thin layer and used as the official test sample. These samples were placed under the influencing factor testing conditions (60℃, 92.5% RH) and accelerated testing conditions (40℃/75% RH and 60℃/75% RH), with complete exposure. The samples were covered with aluminum foil and small holes were punched in the foil. Small amounts of the samples were also placed in 40 mL glass sample bottles under the same conditions to determine the crystal form. Samples were taken and analyzed at 5 days, 10 days, and 1 month. The analytical methods are shown in Table 7, and the results are shown in Table 8. Samples placed under light conditions (1200000 Lux visible light, 200W ultraviolet light) were placed in complete exposure at room temperature.

Table 7 High Performance Liquid Chromatography (HPLC) Methods for Related Substances

Table 8 shows the solid stability of compound F (Formula I) samples, including content and related substance analysis results (5 days, 10 days, and 1 month).

Conditions and timing Crystal form Total impurities % content% 0 days F crystal form 0.63 99.38 60℃_5 days F crystal form 0.74 99.39 60℃_10 days F crystal form 0.84 99.98 92.5% humidity - 5 days F crystal form 0.61 98.72 92.5% humidity - 10 days F crystal form 0.64 98.38 Avoid light F crystal form 0.61 97.77 illumination F crystal form 0.67 98.58 40℃-75% humidity-10 days F crystal form 0.63 98.01 40℃-75% humidity-1 month F crystal form 0.62 97.94 60℃-75% humidity-10 days F crystal form 0.69 100.43 60℃-75% humidity-1 month F crystal form 0.75 99.46

Conclusion: During the one-month solid-state influencing factor and accelerated testing, the crystal form of compound F of formula (I) remained unchanged, demonstrating good physical stability. Related substances analysis showed a slight increase in impurities at high temperature (60℃) and under accelerated conditions (60℃/75%RH), while the total amount of impurities remained almost unchanged under other conditions, indicating a slight sensitivity to temperature.

Experimental conclusion: The crystal form of this invention has good stability and is easy to process into a drug.

Experimental Example 2: In vitro enzymatic inhibitory activity of compound (I)

The experimental tests were conducted at Eurofins, and the results were provided by the company.

In the test system, 20 mM Tris-HCl, pH 8.5, 0.2 mM EDTA, 500 μM peptide substrate (LSNLYHQGKFLQTFCGSPLYRRR), 10 mM magnesium acetate, and 10 μM [γ- ^33P ]-ATP (at a strength of approximately 500 cpm/pmol) were added. After adding the ^Mg²⁺ and ATP mixture, the reaction was initiated and incubated at room temperature for 40 min. The reaction was terminated by adding 3% phosphate buffer. 10 μL of the reaction solution was filtered through a P30 continuous filter, washed three times with 75 mM phosphate buffer and once with methanol, each wash lasting 5 min. The solution was dried and read using a scintillation counting method. The experimental results are shown in Table 9.

Table 9: Results of in vitro enzymatic activity assays ( _IC50 ) of the compounds of this invention

Compound numbering <![CDATA[Wee1(IC₅₀ nM)]]> AZD1775 47 Compound of formula (I) 29

Experimental Example 3: In vitro permeability test of the compound of the present invention

This study used MDR1-MDCK II cells, licensed from the Piet Borst laboratory at the Netherlands Cancer Institute. These are Madin-Darby canine kidney cells transfected with the human multidrug resistance gene (MDR1). These cells stably express the efflux transporter P-glycoprotein (P-gp), making them suitable for screening P-gp substrates or inhibitors and predicting the permeability of compounds through highly efflux-inducing barriers such as the duodenum, blood-brain barrier, hepatocyte nucleus, and nephrons. We used passages 5-35 of MDR1-MDCK II cells for permeability studies.

MDR1-MDCK II cells were cultured in α-MEM (α-Minimum Essential Media) at 37±1℃, 5% _CO2 , and saturated relative humidity. Cells were then seeded in BD Transwell-96-well plates at a density of 2.3 × ^10⁵ cells/ ^cm² , and cultured in a CO2 incubator for 4–7 days before being used for transport experiments. The α-MEM medium was prepared as follows: liquid medium was prepared by dissolving powder (α-MEM powder from Gibco, Cat#: 11900) in pure water, with the addition of L-(L-glutamine) and _NaHCO₃ . Before use, 10% FBS (fetal bovine serum), 1% PS (antibiotics), and 1% NEAA were added to make it a complete medium. The α-MEM medium composition is shown in Table 10.

Table 10. Ingredients list for αMEM (1L×)

AZD1775 (or the compound of this invention) and digoxin were administered at a concentration of 2 μM, in both directions (A-B and B-A), with two replicates for each. Fenoterol and propranolol were tested at a concentration of 2 μM, in both directions (A-B), with two replicates for each.

Pre-incubate the solution to be used in a water bath at 37±1℃ for 30 minutes. Add the drug-injection solution and the receiving solution to the corresponding wells of the cell plate (75 μL and 250 μL to each top and base well, respectively), and start the bidirectional transport experiment. After adding the samples, incubate the cell plate in an incubator at 37±1℃, 5% _CO2 , and saturated relative humidity for 150 minutes. Sample collection information is shown in Table 11.

Table 11. Sample Collection Information

Note: _T0 represents the initial drug solution sample.

All samples were vortexed and centrifuged at 3220g for 10 minutes. An appropriate volume of the supernatant was transferred to a sample analysis plate. After sealing the plate, if the samples were not to be analyzed immediately, they were stored at 2-8℃ and analyzed using LC-MS/MS.

After the transport experiment, the integrity of MDR1-MDCK II cells was tested using the Lucifer Yellow Rejection Assay. After incubation with Lucifer Yellow solution for 30 minutes, the Lucifer Yellow samples were collected, and the relative fluorescence unit (RFU) of Lucifer Yellow in the samples was detected using a 2e plate reader at 425/528 nm (excitation/emission) wavelengths.

Semi-quantitative analysis was performed on the test sample AZD1775 (or the compound of this invention), reference standards fenoterol, propranolol, and digoxin. The peak area ratio of the analyte to the internal standard was used as the concentration of the reference standard.

The experimental results are shown in Table 12.

Table 12. Permeation rate ( ^10⁻⁶ cm/s)

AZD1775 Compound of formula (I) A to B 2.83 4.55 B to A 29.3 17.38 External discharge ratio 10.37 3.82

Experimental conclusion:

The permeability of compound (I) is greatly improved compared to AZD1775, which is beneficial for the body's utilization of the drug.

Experimental Example 4: Pharmacokinetic Evaluation of Compounds

This experiment aims to investigate the pharmacokinetics of AZD1775 (or the compound of this invention) in the plasma of female Balb/c Nude mice after a single intravenous or single oral administration.

Twelve mice (provided by Lingchang) were randomly divided into two groups of six females each, with blood samples collected using a cross-sample method. In the intravenous group, all animals were intravenously injected with 1 mg/kg of AZD1775 (or the compound of this invention), prepared as a clear solution of 5% HP-betaCD (Kunshan Ruisike Chemical Raw Materials Co., Ltd.) containing 0.2 mg/mL AZD1775 (or the compound of this invention). In the oral group, animals were administered 10 mg/kg of AZD1775 (or the compound of this invention) by gavage, prepared as a homogeneous suspension of 0.5% methylcellulose containing 1 mg/mL AZD1775 (or the compound of this invention).

Plasma samples were collected from animals in the intravenous group at nine time points: 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, and 24 hours after drug administration. Plasma samples were also collected from the oral group at eight time points: 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, and 24 hours after drug administration. The plasma concentration data of AZD1775 (or the compound of this invention) were obtained by LC-MS/MS analysis, and pharmacokinetic parameters such as peak concentration, time to peak concentration, clearance rate, half-life, area under the curve, and bioavailability were calculated. The experimental results are shown in Table 13.

Table 13. Pharmacokinetic Test Results

Experimental conclusion: Compared with AZD1775, compound (I) can significantly improve multiple pharmacokinetic parameters in mice, including clearing rate, half-life, in vivo concentration integral and bioavailability.

Experimental Example 5: In vivo study

(1) In vivo pharmacodynamic study of the compound on a BALB/c nude mouse model of human colon cancer LoVo cell subcutaneous xenograft tumor.

Experimental methods: The experimental animals used (provided by Shanghai Xipu-Bikai Experimental Animal Co., Ltd.) were BALB/c nude mice, 6-8 weeks old, weighing 18-22 grams.

Human colon cancer LoVo cells were cultured in vitro in a monolayer at 37°C in Ham's F-12 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM glutamine, under 5% _CO2 conditions. Cells were passaged twice weekly using trypsin-EDTA digestion. Cells were harvested, counted, and seeded when confluence reached 80%-90%. 0.1 mL (10 × ^10⁶ cells) of LoVo cells were subcutaneously seeded into the right posterior dorsal region of each nude mouse. Drug administration began when the average tumor volume reached 213 ^mm³ . The oral gavage dose was 40 mg/kg twice daily. The experimental endpoint was to assess whether tumor growth was inhibited, delayed, or cured. Tumor diameter was measured twice weekly using calipers. The tumor volume was calculated using the formula: V = 0.5a × ^b² , where a and b represent the major and minor diameters of the tumor, respectively.

The antitumor efficacy of the compound was evaluated using TGI (%) or relative tumor proliferation rate (T/C) (%). TGI (%) reflects the tumor growth inhibition rate. The calculation of TGI (%) is: TGI (%) = [(1 - (mean tumor volume at the end of treatment in a certain treatment group - mean tumor volume at the beginning of treatment in that treatment group)) / (mean tumor volume at the end of treatment in the solvent control group - mean tumor volume at the beginning of treatment in the solvent control group)] × 100%.

The final results of the experiment after 16 days of drug administration are shown in Table 14.

Table 14. Results of efficacy trials in a mouse model of human colon cancer LoVo cell xenograft tumor.

compound TGI (%) AZD1775 26.73 Compound of formula (I) 84.74

Conclusion: Compared with AZD1775, the compound of this invention can significantly improve the inhibitory effect on tumors in mice, and the chirality of the compound has an unexpected effect on the in vivo efficacy.

(2) In vivo pharmacodynamic study of the compound on a human pancreatic cancer BxPC-3BALB/c subcutaneous xenograft model in nude mice

Experimental methods: The experimental animals used were BALB/c nude mice, 6-8 weeks old, weighing 18-22 grams.

BxPC-3 cells, passage 10, were cultured in vitro in monolayers under the following conditions: RPMI 1640 medium (manufacturer: Gibco, catalog number: 22400-089) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin, at 37°C with 5% _CO2 , and passaged four times. Passage was performed twice weekly using trypsin-EDTA digestion. When cell saturation reached 80%-90%, cells were digested with trypsin-EDTA, counted, and resuspended in PBS at a density of 5 x ^10⁷ cells/mL. 0.1 mL (5 x ^10⁶ cells) of BxPC-3 cells were inoculated into the right posterior dorsal region of each animal. When the average tumor volume reached 153 ^mm³ , animals were randomly assigned to groups based on tumor volume, and drug administration began. The oral gavage dose was 25 mg/kg, once daily.

The experimental endpoint was to examine whether tumor growth was inhibited, delayed, or cured. Tumor diameter was measured twice weekly using calipers. The antitumor efficacy of the compound was evaluated using TGI (%) or relative tumor proliferation rate (T/C%). TGI (%) reflects the tumor growth inhibition rate. The calculation of TGI (%) is: TGI (%) = [(1 - (mean tumor volume at the end of treatment in a certain treatment group - mean tumor volume at the beginning of treatment in that treatment group)) / (mean tumor volume at the end of treatment in the solvent control group - mean tumor volume at the beginning of treatment in the solvent control group)] × 100%.

The final results of the 27-day drug administration experiment are shown in Table 15.

Table 15. Results of efficacy trials in a mouse model of human pancreatic cancer BxPC-3 cell xenograft tumor.

compound TGI (%) AZD1775 24.3 Compound of formula (I) 73.3

Conclusion: As shown in Table 15, compound (I) significantly enhances the inhibitory effect on mouse tumors compared to AZD1775. (3) In vivo antitumor efficacy study of the compound against CT26 mouse colon cancer cell xenograft model

Experimental methods: The experimental animals used were BALB/c nude mice, 7 weeks old, weighing 16-20 grams, and female.

Cells: Mouse colon cancer CT26 cells (cell bank of the Chinese Academy of Sciences Type Culture Collection) were cultured in vitro in a monolayer using RPMI-1640 medium containing 10% fetal bovine serum in a 37°C, 5% _CO2 incubator. Cells were passaged using trypsin-EDTA digestion. When cells were in the exponential growth phase and saturation reached 80%-90%, cells were harvested, counted, and seeded. 0.1 mL of DPBS (containing 3 × ^10⁵ CT26 cells) was subcutaneously injected into the right posterior dorsal region of each mouse. When the average tumor volume reached 50–70 ^mm³ , mice were randomly assigned to receive the appropriate treatment based on tumor volume. Oral administration dose: 30 mg/kg, twice daily.

The experimental endpoint was to assess whether tumor growth was inhibited, delayed, or cured. Tumor diameter was measured twice weekly using calipers. The antitumor efficacy of the compound was evaluated using TGI (%) or relative tumor proliferation rate (T/C%). TGI (%) reflects the tumor growth inhibition rate. The calculation of TGI (%) is as follows:

TGI (%) = [(1 - (mean tumor volume at the end of treatment - mean tumor volume at the start of treatment)) / (mean tumor volume at the end of treatment in solvent control group - mean tumor volume at the start of treatment in solvent control group)] × 100%.

The final results of the 18-day drug administration experiment are shown in Table 16.

Table 16. In vivo efficacy test results of CT26 cell allograft tumor model of colon cancer in mice

compound TGI (%) AZD1775 66.43 Compound of formula (I) 93.38

Conclusion: As shown in Table 16, compound (I) significantly enhances the inhibitory effect on tumors in mice compared to AZD1775.

Claims (45)

1. The A crystal form of compound (Ⅰ) has characteristic diffraction peaks at the following 2θ angles in its X-ray powder diffraction (XRPD) pattern: 5.71±0.2°, 12.68±0.2°, and 15.32±0.2°. 2. The A-type crystal according to claim 1, wherein its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 5.71±0.2°, 12.68±0.2°, 15.32±0.2°, 18.04±0.2°, 19.72±0.2°, 21.44±0.2°, 23.61±0.2° and 25.68±0.2°. 3. The XRPD pattern of the A crystal form according to claim 2 is shown in Figure 1. 4. The A crystal form according to any one of claims 1 to 3, wherein the differential scanning calorimetry (DSC) curve has an endothermic peak starting point at 34.95±3℃, 174.75±3℃ and 219.12±3℃ respectively. 5. The A-type crystal according to claim 4, its DSC spectrum is shown in Figure 2. 6. The A crystal form according to any one of claims 1 to 3, wherein the thermogravimetric analysis (TGA) curve shows a weight loss of 0.7367% at 70.33±3℃ and a further weight loss of 3.123% at 209.42±3℃. 7. The TGA spectrum of the A crystal form according to claim 6 is shown in Figure 3. 8. The B crystal form of compound (I) has characteristic diffraction peaks at the following 2θ angles in its X-ray powder diffraction (XRPD) pattern: 5.58±0.2°, 12.44±0.2°, and 22.16±0.2°. 9. The B-type crystal according to claim 8, wherein its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 5.58±0.2°, 11.71±0.2°, 12.44±0.2°, 14.48±0.2°, 15.13±0.2°, 18.64±0.2°, 22.16±0.2°, and 26.33±0.2°. 10. The B crystal form according to claim 9, its XRPD pattern is shown in Figure 4. 11. The B crystal form according to any one of claims 8 to 10, wherein the differential scanning calorimetry (DSC) curve has an endothermic peak starting point at 42.88±3℃, 198.79±3℃ and 222.36±3℃ respectively. 12. The B crystal form according to claim 11, its DSC spectrum is shown in Figure 5. 13. The B-type crystal according to any one of claims 8 to 10, wherein the thermogravimetric analysis (TGA) curve shows a weight loss of 3.265% at 64.21±3℃ and a further weight loss of 1.516% at 243.05±3℃. 14. The B crystal form according to claim 13, its TGA spectrum is shown in Figure 6. 15. The C-crystal form of compound (I) has characteristic diffraction peaks at the following 2θ angles in its X-ray powder diffraction (XRPD) pattern: 5.05±0.2°, 5.58±0.2°, and 12.44±0.2°. 16. The C-type according to claim 15, wherein its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 5.05±0.2°, 5.58±0.2°, 12.44±0.2°, 15.91±0.2°, 16.68±0.2°, 17.61±0.2°, 22.19±0.2°, and 26.37±0.2°. 17. The C-type crystal according to claim 16, its XRPD pattern is shown in Figure 7. 18. The C-type according to any one of claims 15 to 17, wherein the differential scanning calorimetry (DSC) curve has an endothermic peak starting point at 37.06±3℃, 189.16±3℃ and 218.61±3℃ respectively. 19. The C-type crystal according to claim 18, its DSC spectrum is shown in Figure 8. 20. The C-type according to any one of claims 15 to 17, wherein the thermogravimetric analysis (TGA) curve shows a weight loss of 2.211% at 64.98±3℃ and a further weight loss of 1.127% at 224.71±3℃. 21. The C-type crystal according to claim 20, its TGA spectrum is shown in Figure 9. 22. The D crystal form of compound (I) has characteristic diffraction peaks at the following 2θ angles in its X-ray powder diffraction (XRPD) pattern: 5.22±0.2°, 15.99±0.2°, and 16.57±0.2°. 23. The D-type according to claim 22, wherein its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 5.22±0.2°, 15.18±0.2°, 15.99±0.2°, 16.57±0.2°, 17.08±0.2°, 18.60±0.2°, 21.22±0.2°, and 21.89±0.2°. 24. The D-crystal form according to claim 23, wherein the XRPD pattern of the D-crystal form is shown in Figure 10. 25. The D-type according to any one of claims 22 to 24, wherein the differential scanning calorimetry (DSC) curve has an endothermic peak starting point at 56.07±3℃, 193.93±3℃ and 216.54±3℃ respectively. 26. The D-type according to claim 25, its DSC spectrum is shown in Figure 11. 27. The D-type according to any one of claims 22 to 24, wherein the thermogravimetric analysis (TGA) curve shows a weight loss of 1.977% at 79.35±3℃ and a further weight loss of 1.589% at 223.66±3℃. 28. The D-type crystal according to claim 27, its TGA spectrum is shown in Figure 12. 29. The E crystal form of compound (I) has characteristic diffraction peaks at the following 2θ angles in its X-ray powder diffraction (XRPD) pattern: 8.65±0.2°, 14.22±0.2°, and 24.58±0.2°. 30. The E-type according to claim 29, wherein its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 8.65±0.2°, 11.41±0.2°, 13.13±0.2°, 14.22±0.2°, 17.35±0.2°, 18.34±0.2°, 20.39±0.2°, 20.94±0.2°, and 24.58±0.2°. 31. The E-crystal form according to claim 30, the XRPD pattern of which is shown in Figure 13. 32. The E-type according to any one of claims 29 to 31, wherein the differential scanning calorimetry (DSC) curve has an endothermic peak at 121.57±3℃, 197.26±3℃ and 217.23±3℃ respectively; and an exothermic peak at 168.31±3℃ and 212.95±3℃ respectively. 33. The E-type crystal according to claim 32, its DSC spectrum is shown in Figure 14. 34. The E-type according to any one of claims 29 to 31, wherein the thermogravimetric analysis (TGA) curve shows a weight loss of 6.775% at 143.31±3℃ and a further weight loss of 0.3184% at 213.62±3℃. 35. The E-type crystal according to claim 34, its TGA spectrum is shown in Figure 15. 36. The F crystal form of compound (I) has characteristic diffraction peaks at the following 2θ angles in its X-ray powder diffraction (XRPD) pattern: 5.06±0.2°, 15.91±0.2°, and 16.68±0.2°. 37. The F-type according to claim 36, wherein its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 5.06±0.2°, 8.34±0.2°, 10.98±0.2°, 15.13±0.2°, 15.91±0.2°, 16.68±0.2°, 17.63±0.2° and 18.87±0.2°. 38. The F-type crystal according to claim 37, its XRPD pattern is shown in Figure 16. 39. The F-type according to any one of claims 36 to 38, wherein the differential scanning calorimetry (DSC) curve has an endothermic peak starting point at 48.69±3℃ and 225.26±3℃ respectively. 40. The F-type crystal according to claim 39, its DSC spectrum is shown in Figure 17. 41. The F-type according to any one of claims 36 to 38, wherein the thermogravimetric analysis (TGA) curve shows a weight loss of 3.404% at 100±3℃. 42. The F-type crystal according to claim 41, its TGA spectrum is shown in Figure 18. 43. The use of crystal form A according to any one of claims 1 to 7, crystal form B according to any one of claims 8 to 14, crystal form C according to any one of claims 15 to 21, crystal form D according to any one of claims 22 to 28, crystal form E according to any one of claims 29 to 35, or crystal form F according to any one of claims 36 to 42 in the preparation of a medicament for treating Wee1-related diseases. 44. The method for preparing the F-crystal form according to any one of claims 36 to 42, comprising: (a) Add the compound of formula (I) to an alcohol solvent, stir and heat to an oil bath at 55-65°C; (b) Stir at 47℃-53℃ for 72 hours; (c) Turn off the heating and let it cool naturally for 1 hour until it reaches 27°C while stirring. (d) Let stand for 18 hours, filter, and then rinse the filter cake with methanol; (e) Vacuum drying at 60℃ for 48 hours. 45. The preparation method according to claim 44, wherein the alcohol solvent is methanol.