RU2842334C2 - Crystalline form of inhibiting wee1 compound and use thereof - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a crystalline form A of a compound of formula (I), wherein crystalline form A has an X-ray powder diffraction pattern containing characteristic diffraction peaks at following angles 2θ: 5.71 ± 0.2°, 12.68 ± 0.2°, 15.32 ± 0.2°, 21.44 ± 0.2°, 23.61 ± 0.2° and 25.68 ± 0.2°.

EFFECT: crystalline form A of the compound of formula (I) according to the invention is used to treat Wee1-related disease.

6 cl, 16 tbl, 18 dwg, 12 ex

Description

Cross reference to related application

Application CN201910364694.X, filed April 30, 2019.

Field of the invention

Crystalline forms of the compound of formula (I) and the use of the crystalline forms in the manufacture of a medicinal product for the treatment of a Wee1-associated disease are disclosed.

Prior art of the present invention

The cell cycle process is a complex process that is controlled by a number of cell cycle regulatory systems. The major cell cycle regulatory system is the CDK-cyclin complex, which is formed by the combination of cyclin-dependent kinases (CDKs) and cyclins. This complex can ensure the entry of cells into the proliferation cycle, in which the CDK1 (human autoploid is also known as CDC2) and cyclin B complex plays a major role in directing cells into the M phase.

DNA replication must be completed before a cell enters M phase. Mutations or damage to DNA frequently occur due to a variety of endogenous and exogenous factors. Abnormal DNA must be repaired or its damage will cause mitotic failure and result in cell death. The cell cycle checkpoint terminates the cell cycle and ensures DNA repair before entry into M phase. The G1/S checkpoint at the end of G1 phase and the G2/M checkpoint at the end of G2 phase are the two major cell cycle checkpoints that are jointly responsible for recognizing and repairing DNA damage. In normal cells, the G1/S checkpoint is used to complete DNA repair in G1 phase, whereas approximately 50% of cancer cells contain defects in the tumor suppressor gene p53 that abolish G1/S checkpoint function. These cells are forced to rely more heavily on the G2/M checkpoint to complete DNA repair. The G2/M checkpoint is rarely mutated, allowing cancer cells to evade DNA-damaging agents and radiation.

Wee1 protein kinase is a cell cycle regulator, a member of the nuclear serine-threonine protein kinase family, and a major kinase for the G2/M checkpoint. The human Wee protein kinase family includes Wee1 and Myt1, both of which can phosphorylate Tyr15 of CDC2, inhibit the activation of the CDC2 and cyclin B complex, and block cell entry into M phase until DNA repair is completed. Myt1 can also phosphorylate Tyr14 of CDC2, which also negatively regulates CDC2 activity. Wee1 is highly expressed in many cancer cells. By inhibiting Wee1, cancer cells can be directly induced to bypass DNA repair at G2 and enter mitosis early, resulting in cancer cell death and achieving the goal of cancer therapy.

At present, AstraZeneca's Wee1 inhibitor AZD1775 has entered Phase II clinical trials, and more than thirty clinical trials are ongoing, showing good therapeutic effects. AZD1775 was first developed by Merck, and thus it is also known as MK-1775. In September 2013, Merck transferred this compound to AstraZeneca worldwide, and the related patents are mainly US20070254892, WO2007126122, EP2213673, WO2008133866, WO2011034743, etc. Abbott and Abbvie have also conducted studies on Wee1 inhibitors, and the relevant patents are mainly US2012220572, WO2013126656, WO2013012681, WO2013059485, WO2013013031, WO2013126656, etc. Almac's patents on Wee1 inhibitors are WO2014167347, WO2015019037, WO2015092431.

Document WO2008133866 discloses the compound AZD1775, which has the following structure:

Brief summary of the present invention

A crystalline form A of the compound of formula (I) is proposed, wherein the crystalline form A has an X-ray powder diffraction pattern containing characteristic diffraction peaks at the following 2θ angles: 5.71±0.2°, 12.68±0.2(, and 15.32±0.2°.

Formula (I)

According to some embodiments of the present invention, crystalline form A has an X-ray powder diffraction pattern comprising 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°.

According to some embodiments of the present invention, crystalline form A has an X-ray powder diffraction pattern comprising 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°.

According to some embodiments of the present invention, crystalline form A has an X-ray powder diffraction pattern that is shown in Fig. 1.

According to some embodiments of the present invention, crystalline form A has an X-ray powder diffraction pattern, the analysis results of which are presented in Table 1:

Table 1. Results of the analysis of the X-ray powder diffraction pattern of crystalline form A

No. Angle 2(°) Interplanar distance ( ) Relative intensity (%) No. Angle 2(°) Interplanar distance ( ) Relative intensity (%) 1 5,046 17,4994 0.7 17 20.08 4,4184 0.4 2 5,046 17,4994 0.7 18 20,413 4,3471 0.3 3 5,711 15,4612 100 19 21.2 4,1874 0.6 4 7,904 11,176 0.6 20 21,435 4,142 2.1 5 11,218 7,881 0.5 21 21,669 4,0979 0.5 6 12,677 6,9773 27.4 22 22,268 3,989 0.9 7 14.71 6,0172 1.4 23 23,234 3,8252 0.8 8 14,948 5,9216 0.8 24 23,607 3,7656 4.2 9 15,318 5,7794 1.5 25 25,517 3,488 1.9 10 16,051 5,5173 0.5 26 25,678 3,4665 2,2 11 17,159 5,1634 0.9 27 26,043 3,4187 0.5 12 17,768 4,9877 0.5 28 27,765 3,2104 0.4 13 18,043 4,9125 1 29 28,007 3,1832 1 14 18,597 4,7672 0.4 30 29,289 3,0467 0.4 15 18,906 4.69 0.9 31 30,426 2,9354 0.4 16 19,719 4,4984 1.7 -

According to some embodiments of the present invention, crystalline form A has a differential scanning calorimetry curve containing a single endothermic peak onset at 34.95±3°C, 174.75±3°C, and 219.12±3°C, respectively.

According to some embodiments of the present invention, crystalline form A has a differential scanning calorimetry curve as shown in Fig. 2.

According to some embodiments of the present invention, crystalline form A has a thermogravimetric analysis curve in which the mass loss at 70.33±3°C is 0.7367%; and the mass loss at 209.42±3°C is 3.123%.

According to some embodiments of the present invention, crystalline form A has a thermogravimetric analysis curve as shown in Fig. 3.

Furthermore, a crystalline form B of the compound of formula (I) is provided, wherein the crystalline form B has an X-ray powder diffraction pattern containing characteristic diffraction peaks at the following 2θ angles: 5.58±0.2°, 12.44±0.2(, and 22.16±0.2°.

According to some embodiments of the present invention, crystalline form B has an X-ray powder diffraction pattern comprising 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°.

According to some embodiments of the present invention, crystalline form B has an X-ray powder diffraction pattern comprising 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°.

According to some embodiments of the present invention, crystalline form B has an X-ray powder diffraction pattern that is shown in Fig. 4.

According to some embodiments of the present invention, crystalline form B has an X-ray powder diffraction pattern, the analysis results of which are presented in Table 2:

Table 2. Results of the X-ray powder diffraction pattern analysis of crystalline form B

No. Angle 2θ (°) Interplanar distance Relative intensity (%) No. Angle 2θ (°) Interplanar distance Relative intensity (%) 1 5,577 15,8346 100 17 19,582 4,5295 4.3 2 7,838 11,2705 1,1 18 20,016 4,4325 5.3 3 9,807 9,0113 1.5 19 21,831 4,0677 3.2 4 11,028 8,0162 2.4 20 22,164 4,0075 18.7 5 11,713 7,549 6.4 21 23,018 3,8607 2.8 6 12,056 7,3351 1.3 22 23,626 3,7626 2.1 7 12,439 7,1098 30.4 23 25,007 3,5579 2,3 8 13,706 6,4552 4.6 24 25,261 3,5227 1.4 9 14,233 6,2177 2.9 25 25,808 3,4493 1.3 10 14,476 6,1138 5.7 26 26,327 3,3824 8.5 11 15,127 5,8522 5.8 27 28,535 3,1255 1 12 16,717 5,2989 1,1 28 30,939 2,8879 0.9 13 17,146 5,1674 1,2 29 31,076 2,8755 0.9 14 17,567 5,0443 7.7 30 32,083 2,7875 0.7 15 18,241 4,8596 2 31 34,201 2,6196 0.7 16 18,637 4,757 6.5 -

According to some embodiments of the present invention, crystalline form B has a differential scanning calorimetry curve containing a single endothermic peak onset at 42.88±3°C, 198.79±3°C, and 222.36±3°C, respectively.

According to some embodiments of the present invention, crystalline form B has a differential scanning calorimetry curve as shown in Fig. 5.

According to some embodiments of the present invention, crystalline form B has a thermogravimetric analysis curve in which the mass loss at 64.21±3°C is 3.265%; and the mass loss at 243.05±3°C is 1.516%.

According to some embodiments of the present invention, crystalline form B has a thermogravimetric analysis curve as shown in Fig. 6.

In addition, a crystalline form C of the compound of formula (I) is provided, wherein the crystalline form C has an X-ray powder diffraction pattern containing characteristic diffraction peaks at the following 2θ angles: 5.05±0.2°, 5.58±0.2° and 12.44±0.2°.

According to some embodiments of the present invention, crystalline form C has an X-ray powder diffraction pattern comprising 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°.

According to some embodiments of the present invention, crystalline form C has an X-ray powder diffraction pattern that is shown in Fig. 7.

According to some embodiments of the present invention, crystalline form C has an X-ray powder diffraction pattern, the analysis results of which are presented in Table 3:

Table 3. Results of the analysis of the X-ray powder diffraction pattern of the crystalline form C

No. Angle 2θ (°) Interplanar distance Relative intensity (%) No. Angle 2θ (°) Interplanar distance Relative intensity (%) 1 5,047 17,4944 12.4 17 19.6 4,5254 0.8 2 5,577 15,8334 100 18 20,032 4,4288 0.7 3 10,945 8,077 0.6 19 21,275 4,1728 0.5 4 11,137 7,9381 1.4 20 21,436 4,1418 0.7 5 11,712 7,5495 1,2 21 21,869 4,0608 0.8 6 12,438 7,1105 12.8 22 22,186 4,0036 6.1 7 13,726 6,4461 0.6 23 23,035 3,8578 1.8 8 14,221 6,2226 0.8 24 23,627 3,7626 0.9 9 14,496 6,1055 0.9 25 24,832 3,5826 0.3 10 15,126 5,8527 1,1 26 25,002 3,5586 0.6 11 15,911 5,5654 6.4 27 25,278 3,5203 0.5 12 16,108 5,4978 2.8 28 26,367 3,3773 3 13 16,685 5,309 3.3 29 26.98 3,302 0.4 14 17.61 5,0322 2.6 30 28,633 3,1151 0.2 15 17,921 4,9455 0.4 31 28,939 3,0828 0.4 16 18,694 4,7427 2,2 32 34,293 2,6127 0.2

According to some embodiments of the present invention, crystalline form C has a differential scanning calorimetry curve containing a single endothermic peak onset at 37.06±3°C, 189.16±3°C, and 218.61±3°C, respectively.

According to some embodiments of the present invention, crystalline form C has a differential scanning calorimetry curve as shown in Fig. 8.

According to some embodiments of the present invention, crystalline form C has a thermogravimetric analysis curve in which the mass loss at 64.98±3°C is 2.211%; and the mass loss at 224.71±3°C is 1.127%.

According to some embodiments of the present invention, crystalline form C has a thermogravimetric analysis curve as shown in Fig. 9.

In addition, a crystalline form D of the compound of formula (I) is proposed, wherein the crystalline form D has an X-ray powder diffraction pattern containing characteristic diffraction peaks at the following 2θ angles: 5.22±0.2°, 15.99±0.2° and 16.57±0.2°.

According to some embodiments of the present invention, crystalline form D has an X-ray powder diffraction pattern comprising 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°.

According to some embodiments of the present invention, crystalline form D has an X-ray powder diffraction pattern comprising 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°.

According to some embodiments of the present invention, crystalline form D has an X-ray powder diffraction pattern comprising 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°.

According to some embodiments of the present invention, crystalline form D has an X-ray powder diffraction pattern that is shown in Fig. 10.

According to some embodiments of the present invention, crystalline form D has an X-ray powder diffraction pattern, the analysis results of which are presented in Table 4:

Table 4. Results of the analysis of the X-ray powder diffraction pattern of the crystalline form D

No. Angle 2θ (°) Interplanar distance Relative intensity (%) No. Angle 2θ (°) Interplanar distance Relative intensity (%) 1 5,224 16,9027 100 16 25,242 3,5253 8.2 2 10,424 8,4794 4 17 26,048 3,418 3.5 3 12,774 6,9241 1.9 18 26,609 3,3472 2 4 15,18 5,8316 7.9 19 26,997 3.3 7.6 5 15,595 5,6774 22.3 20 27,492 3,2417 2.9 6 15,992 5,5373 58.7 21 28,517 3,1274 1.6 7 16,567 5,3466 18.1 22 29,898 2,986 3.5 8 17,075 5,1886 15.3 23 30.55 2,9238 3.8 9 17,904 4,9503 4.9 24 31,333 2,8525 2 10 18,201 4.87 5.1 25 32,348 2,7653 1.7 11 18,599 4,7668 12.1 26 32.95 2,7161 1.3 12 19,506 4,5472 4.1 27 34,522 2,596 1.9 13 21,22 4,1834 25.5 28 34,545 2,5943 2 14 21,888 4,0573 4.8 29 36,655 2,4496 2.1 15 22,936 3,8743 1.6 30 39,522 2,2783 1.7

According to some embodiments of the present invention, crystalline form D has a differential scanning calorimetry curve containing a single endothermic peak onset at 56.07±3°C, 193.93±3°C, and 216.54±3°C, respectively.

According to some embodiments of the present invention, crystalline form D has a differential scanning calorimetry curve comprising one endothermic peak onset at 56.07±3°C, 193.93±3°C, and 216.54±3°C, respectively; and one exothermic peak value at 206.82±3°C.

According to some embodiments of the present invention, crystalline form D has a differential scanning calorimetry curve as shown in Fig. 11.

According to some embodiments of the present invention, crystalline form D has a thermogravimetric analysis curve in which the mass loss at 79.35±3°C is 1.977%; and the mass loss at 223.66±3°C is 1.589%.

According to some embodiments of the present invention, crystalline form D has a thermogravimetric analysis curve, which is shown in Fig. 12.

In addition, a crystalline form E of the compound of formula (I) is proposed, wherein the crystalline form E has an X-ray powder diffraction pattern containing characteristic diffraction peaks at the following 2θ angles: 8.65±0.2°, 14.22±0.2° and 24.58±0.2°.

According to some embodiments of the present invention, crystalline form E has an X-ray powder diffraction pattern comprising 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°.

According to some embodiments of the present invention, crystalline form E has an X-ray powder diffraction pattern that is shown in Fig. 13.

According to some embodiments of the present invention, crystalline form E has an X-ray powder diffraction pattern, the analysis results of which are presented in Table 5:

Table 5. Results of the analysis of the X-ray powder diffraction pattern of the crystalline form E

No. Angle 2θ (°) Interplanar distance Relative intensity (%) No. Angle 2θ (°) Interplanar distance Relative intensity (%) 1 6,507 13,5715 8.8 17 23,173 3,8352 2.8 2 8,653 10,2107 100 18 24,575 3,6195 28.2 3 11,414 7,7457 15.2 19 25,243 3,5252 11 4 13,132 6,7365 26.9 20 26,188 3,4001 6.7 5 14,217 6,2247 66.8 21 26,428 3,3697 6.9 6 16,211 5,4632 4.5 22 26,882 3,3139 1.7 7 17,354 5,1058 16.5 23 28,358 3,1446 5.4 8 18,065 4,9065 4.8 24 28,612 3,1173 2,2 9 18,341 4,8332 18.1 25 30,409 2,937 5.5 10 18,974 4,6733 1.8 26 31.1 2,8733 1.9 11 20.39 4,3518 22.8 27 31,688 2,8213 2.9 12 20,941 4,2386 23.4 28 32,322 2,7674 1.6 13 21,594 4,1119 9.6 29 32,874 2,7222 1.6 14 22,169 4,0065 5.5 30 33,876 2,6439 2 15 22.68 3,9174 9.9 31 34,711 2,5822 2,2 16 22,954 3,8713 4 32 35.02 2,5602 1.6

According to some embodiments of the present invention, crystalline form E has a differential scanning calorimetry curve comprising one endothermic peak onset at 121.57±3°C, 197.26±3°C, and 217.23±3°C, respectively; and one exothermic peak value at 168.31±3°C and 212.95±3°C, respectively.

According to some embodiments of the present invention, crystalline form E has a differential scanning calorimetry curve as shown in Fig. 14.

According to some embodiments of the present invention, crystalline form E has a thermogravimetric analysis curve in which the mass loss at 143.31±3°C is 6.775%; and the mass loss at 213.62±3°C is 0.3184%.

According to some embodiments of the present invention, crystalline form E has a thermogravimetric analysis curve, which is shown in Fig. 15.

In addition, a crystalline form F of the compound of formula (I) is proposed, wherein the crystalline form F has an X-ray powder diffraction pattern containing characteristic diffraction peaks at the following 2θ angles: 5.06±0.2°, 15.91±0.2° and 16.68±0.2°.

According to some embodiments of the present invention, crystalline form F has an X-ray powder diffraction pattern comprising 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°.

According to some embodiments of the present invention, crystalline form F has an X-ray powder diffraction pattern comprising 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°.

According to some embodiments of the present invention, crystalline form F has an X-ray powder diffraction pattern that is shown in Fig. 16.

According to some embodiments of the present invention, crystalline form F has an X-ray powder diffraction pattern, the analysis results of which are presented in Table 6:

Table 6. Results of the analysis of the X-ray powder diffraction pattern of the crystalline form F

No. Angle 2θ (°) Interplanar distance Relative intensity (%) No. Angle 2θ (°) Interplanar distance Relative intensity (%) 1 5,062 17,4414 100 13 21.44 4,1411 5.3 2 8,341 10,5913 3.3 14 22,006 4,0358 3.2 3 10,077 8,7708 1.8 15 23,328 3.81 1,2 4 10.98 8,0515 4.7 16 23,898 3,7204 2.7 5 15,128 5,8518 6.1 17 24,181 3,6776 3 6 15,911 5,5653 59 18 25,325 3,5139 3.1 7 16,685 5,3089 30 19 25,656 3,4694 2.5 8 17,628 5,027 12 20 26.96 3,3045 1,2 9 17,945 4,939 1.8 21 27,271 3,2674 1.9 10 18,869 4,6992 2.6 22 29,026 3,0738 1.8 11 19,719 4,4983 0.9 23 32,834 2,7254 0.8 12 20,332 4,3641 4 -

According to some embodiments of the present invention, crystalline form F has a differential scanning calorimetry curve containing a single endothermic peak onset at 48.69±3°C and 225.26±3°C, respectively.

According to some embodiments of the present invention, crystalline form F has a differential scanning calorimetry curve as shown in Fig. 17.

According to some embodiments of the present invention, crystalline form F has a thermogravimetric analysis curve in which the mass loss at 100±3°C is 3.404%.

According to some embodiments of the present invention, crystalline form F has a thermogravimetric analysis curve, which is shown in Fig. 18.

In addition, a method for obtaining a crystalline form F of the compound of formula (I) is proposed, comprising,

(a) adding a compound of formula (I) to an alcohol solvent while stirring and heating in an oil bath to 55-65°C;

(b) stirring at 47°C-53°C for 72 h;

(c) stop heating and continue stirring with spontaneous decrease in temperature over 1 h to 27°C;

(d) keeping at rest for 18 h, filtering and washing the filter cake with methanol;

(e) drying under vacuum at 60°C for 48 h.

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

In addition, the use of crystalline form A, crystalline form B, crystalline form C, crystalline form D, crystalline form E or crystalline form F in the manufacture of a medicament for the treatment of a Wee1-related disease is proposed.

Technical effect

Crystalline form A, crystalline form B, crystalline form C, crystalline form D, crystalline form E and crystalline form F of the compound according to the present invention exhibit good stability, are less affected by light, heat and moisture, have high solubility and are promising drugs.

Definitions

Unless otherwise stated, the following terms and expressions have the following definitions. Specific terms or expressions should not be considered as vague or unclear without a specific definition, but should be understood according to their ordinary meanings. Trade names used in this document refer to the corresponding product or active ingredient.

The intermediate compounds of the present invention can be prepared using a variety of synthetic methods that are well known to those skilled in the art, including specific embodiments presented below, embodiments by combination with other chemical synthetic methods, and equivalent alternatives well known to those skilled in the art. Preferred embodiments are exemplified below, but are not limited thereto.

The chemical reaction according to a particular embodiment is carried out in a suitable solvent, and this solvent must be suitable for the chemical transformations according to the present invention, as well as for the required reagents and materials. In order to obtain a compound according to the present invention, those skilled in the art can modify or select synthetic steps or reaction schemes based on available embodiments.

The present invention will be described in detail, and the examples given should not be considered as limiting the invention.

The solvents used according to the present invention are commercially available and can be used without further purification.

The solvents used in the present invention may be commercially available solvents. The following abbreviations are used herein: DCM is dichloromethane; DMF is N,N-dimethylformamide; DMSO is dimethyl sulfoxide; EtOH is ethanol; MeOH is methanol; TFA is trifluoroacetic acid; TsOH is p-toluenesulfonic acid; mp is melting point; EtSO ₃ H is ethanesulfonic acid; MeSO ₃ H is methanesulfonic acid; ATP is adenosine triphosphate; HEPES is 4-hydroxyethylpiperazineethanesulfonic acid; EGTA is ethylenebis(oxyethylenenitrilo)tetraacetic acid; MgCl ₂ is magnesium dichloride; MnCl ₂ is manganese dichloride; DTT is dithiothreitol; DCC is dicyclohexylcarbodiimide; DMAP is 4-dimethylaminopyridine; DIEA is N,N-diisopropylethylamine; wt% is weight percentage; THF is tetrahydrofuran.

Instruments and methods of analysis

1.1. X-ray powder diffractometry (XPD)

Device: X-ray powder diffractometer BRUKER D8 Advance

Test method: Approximately 10-20 mg of sample is used for analysis by X-ray powder diffraction.

The detailed parameters of X-ray powder diffractometry are shown below:

X-ray tube: Cu-Kα, (λ=1.54056 )

Tube voltage: 40 kV, tube current: 40 mA

Divergence gap: 0.60 mm

Detector slit: 10.50 mm

Anti-scatter gap: 7.10 mm

Scanning range 4-40°

Step size: 0.02°

Step duration: 0.12 s

Rotation speed of the sample table: 15 rpm

1.2. Differential scanning calorimetry (DSC)

Device: Differential scanning calorimeter TA Q2000

Test method: The sample (approximately 1 mg) is placed in an aluminum test boat and heated in a nitrogen flow (50 ml/min) from 30°C to 300°C at a heating rate of 10°C/min.

1.3. Thermogravimetric analysis (TGA)

Device: Thermogravimetric analyzer TA Q5000

Test method: The sample (2-5 mg) is placed in a platinum test boat and heated in a nitrogen flow (25 ml/min) from 30°C (room temperature) to 300°C at a heating rate of 10°C/min or until 20% mass loss.

Brief description of the figures

Fig. 1 shows the X-ray powder diffraction pattern (Cu-Kα radiation) of crystalline form A of the compound of formula (I);

Fig. 2 shows a differential scanning calorimetry curve of crystalline form A of the compound of formula (I);

Fig. 3 shows a thermogravimetric analysis curve of crystalline form A of the compound of formula (I);

Fig. 4 shows an X-ray powder diffraction pattern (Cu-Kα radiation) of crystalline form B of the compound of formula (I);

Fig. 5 shows a differential scanning calorimetry curve of crystalline form B of the compound of formula (I);

Fig. 6 shows a thermogravimetric analysis curve of the crystalline form B of the compound of formula (I);

Fig. 7 shows an X-ray powder diffraction pattern (Cu-Kα radiation) of the crystalline form C of the compound of formula (I);

Fig. 8 shows a differential scanning calorimetry curve of the crystalline form C of the compound of formula (I);

Fig. 9 shows a thermogravimetric analysis curve of the crystalline form C of the compound of formula (I);

Fig. 10 shows an X-ray powder diffraction pattern (Cu-Kα radiation) of the crystalline form D of the compound of formula (I);

Fig. 11 shows a differential scanning calorimetry curve of the crystalline form D of the compound of formula (I);

Fig. 12 shows a thermogravimetric analysis curve of the crystalline form D of the compound of formula (I);

Fig. 13 shows an X-ray powder diffraction pattern (Cu-Kα radiation) of the crystalline form E of the compound of formula (I);

Fig. 14 shows a differential scanning calorimetry curve of the crystalline form E of the compound of formula (I);

Fig. 15 shows a thermogravimetric analysis curve of the crystalline form E of the compound of formula (I);

Fig. 16 shows an X-ray powder diffraction pattern (Cu-Kα radiation) of the crystalline form F of the compound of formula (I);

Fig. 17 shows a differential scanning calorimetry curve of the crystalline form F of the compound of formula (I);

Fig. 18 shows a thermogravimetric analysis curve of the crystalline form F of the compound of formula (I).

Detailed disclosure of the present invention

The present invention will now be described in detail by way of examples, which does not imply any undesirable limitation. The present invention is described in detail herein, and specific embodiments thereof are also described. It should be obvious to those skilled in the art that various changes and improvements can be made to these specific embodiments without departing from the spirit and scope of the present invention.

Intermediate connection 1

The preparation was carried out using the synthesis method described in document WO2007126122.

Example 1. Compound of formula (I)

Formula (I)

Synthesis scheme:

Step 1. Synthesis of compound 1-A

At 0-15°C under nitrogen atmosphere, 3-butenylmagnesium bromide (1 M, 55.12 ml) was added dropwise to a solution of 2-acetyl-6-bromopyridine (7.35 g, 36.74 mmol) in THF (150 ml), and then the reaction solution was stirred at 10-20°C for 3 h. To quench the reaction, 100 ml of saturated ammonium chloride solution was added and liquid phase separation was performed to obtain an organic layer, which was washed with 50 ml of saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated to dryness on a rotary evaporator to obtain a brown oil. This brown oil was purified by silica gel column chromatography (PE/EA=7/1) to give compound 1-A. NMR ¹ H (400 MHz, DMSO-d ₆ ): δ (ppm) 7.73 (t, J=8.0 Hz, 1H), 7.64 (d, J=7.2 Hz, 1H), 7.46 (d, J=7.2 Hz, 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 compounds 1-A (3.47 g, 13.55 mmol) and 1-C (3.01 g, 13.55 mmol) in dioxane (150 mL) were added N,N''-dimethylethylenediamine (1.31 g, 14.90 mmol, 1.60 mL), copper iodide (2.58 g, 13.55 mmol) and potassium carbonate (2.62 g, 18.97 mmol), and the mixture was purged with nitrogen three times. The mixture was stirred at 95 °C under nitrogen atmosphere for 1.5 h and 200 mL of ammonia solution (28%) was added. The reaction mixture was extracted with ethyl acetate (2×300 ml), and the organic layers were combined, washed with 200 ml of saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure to dryness. The mixture was purified by silica gel column chromatography (PE/EA=3/1) to give compound 1-B. ^1H NMR (400 MHz, DMSO-d ₆ ): δ (ppm) 9.02 (s, 1H), 8.04 (t, J=8.0 Hz, 1H), 7.76 (d, J=7.2 Hz, 1H), 7.64 (d, J=7.6 Hz, 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

To a solution of compound 1-B (2.06 g, 5.18 mmol) in toluene (700 mL) was added Grubbs' second generation catalyst (1.32 g, 1.55 mmol), and the mixture was stirred for 6 h at 80 °C under nitrogen atmosphere. Further Grubbs' second generation catalyst (0.65 g, 0.775 mmol) was added, and the mixture was stirred at 80 °C under nitrogen atmosphere for 3 h. The mixture was cooled to room temperature and filtered. The filtrate was concentrated to dryness to give a brown precipitate, which was purified by silica gel column chromatography (PE/EA=1/1) to give compound 1-D. ¹ H NMR (400 MHz, DMSO-d ₆ ): δ (ppm) 9.07 (s, 1H), 8.06 (t, J=8.0 Hz, 1H), 7.79 (d, J=8.2 Hz, 1H), 7.70 (d, J=7.6 Hz, 1H), 5.39-5.25 (m, 3H), 4.66 (d, J=5.2 Hz, 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 solution of compound 1-D (360 mg, 974.45 μmol) in toluene (35 mL) was added meta-chlorobenzoic acid (265.09 mg, 1.31 mmol, purity 85%), and the reaction mixture was stirred at 25 °C for 2 h. To the reaction solution were added 4-(4-methylpiperazine)aniline (242.30 mg, 1.27 mmol) and N,N-diisopropylethylamine (503.76 mg, 3.90 mmol). The reaction solution was stirred at 25 °C for 12 h. To the reaction solution was added 25 mL of water while stirring, and the aqueous phase was extracted with ethyl acetate (3×30 mL). The organic phases were combined, washed with saturated sodium bicarbonate solution (30 ml) and saturated sodium chloride solution (30 ml), respectively, dried over anhydrous sodium sulfate, filtered and dried in vacuo to give the crude product, which was separated by preparative liquid chromatography (in neutral medium) to give compound 1-E. NMR ¹ H (400 MHz, CDCl ₃ ): δ (ppm) 1.70 (s, 3H) 1.78 (bd, J=13.54 Hz, 2H), 2.04 (bd, J=6.54 Hz, 1H), 2.08 -2.23 (m, 2 H), 2.39 (s, 3H), 2.62-2.64 (m, 4H), 3.21-3.24 (m, 4H), 4.24 (shs, 1H), 4.51 (shd, J=13.54 Hz, 1H), 5.61-5.88 (m, 2H), 6.95 (d, J=9.04 Hz, 2H) 7.49 (d, J=9.04 Hz, 3H), 7.81-7.90 (m, 2H) 8.87 (s, 1H); Mass spectrum (m/z): 513.1 [M+H] ⁺ .

Step 5. Synthesis of compound of formula (I)

Compound 1-E (100 mg, 195.08 μmol) was subjected to chiral separation by supercritical fluid chromatography (chromatography column: length 250 × internal diameter 50 mm, particle size 10 μm, mobile phase: A: supercritical CO ₂ , B: EtOH (0.1% NH ₃ ∙H ₂ O), A:B=55:45 at 200 mL/min), retention time: 21 minutes) to give the compound of formula (I). 1H NMR (400 MHz, CDCl ₃ ): δ (ppm) 1.60 (s, 3H), 1.68 (brs, 2H), 1.94 (bd, J=7.04 Hz, 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 (shs, 1H), 4.42 (shd, J=9.54 Hz, 1H), 5.65 (shs, 2H), 6.85 (d, J=9.04 Hz, 2H), 7.17 (s, 1H) 7.40 (d, J=9.04 Hz, 2H), 7.69-7.81 (m, 2H), 8.77 (s, 1H). Mass spectrum (m/z): 513.1 [M+H] ⁺ .

Example 2. Preparation of crystalline form A of the compound of formula (I)

Approximately 50 mg of the compound of formula (I) were placed in several glass vessels and 0.5 ml of acetone was added thereto, respectively, to obtain a suspension. The suspension samples were analyzed at a constant temperature (40°C) using a shaking device. The suspension samples were subjected to shaking at 40°C for 2 days and centrifuged. The upper liquid phase was removed and the residue samples were dried in a vacuum oven at 40°C overnight to obtain crystalline form A of the compound of formula (I).

Example 3. Preparation of crystalline form B of the compound of formula (I)

Approximately 50 mg of the compound of formula (I) were placed in several glass vessels and 0.3 ml of ethanol was added thereto, respectively, to obtain a suspension. The suspension samples were analyzed at a constant temperature (40°C) using a shaking device. The suspension samples were subjected to shaking at 40°C for 2 days and centrifuged. The upper liquid phase was removed and the residue samples were dried in a vacuum oven at 40°C overnight to obtain crystalline form B of the compound of formula (I).

Example 4. Preparation of crystalline form C of the compound of formula (I)

Approximately 50 mg of the compound of formula (I) were placed in several glass vessels and 0.2 ml of methanol was added thereto, respectively, to obtain a suspension. The suspension samples were analyzed at a constant temperature (40°C) using a shaking device. The suspension samples were subjected to shaking at 40°C for 2 days and centrifuged. The upper liquid phase was removed and the residue samples were dried in a vacuum oven at 40°C overnight to obtain crystalline form C of the compound of formula (I).

Example 5. Preparation of crystalline form D of the compound of formula (I)

Approximately 50 mg of the compound of formula (I) was placed in several glass vessels and 0.4 ml of a mixture of methanol and water in a ratio of 1/1 was added, respectively, to obtain a suspension. The suspension samples were analyzed at a constant temperature (40°C) using a shaking device. The suspension samples were subjected to shaking at 40°C for 2 days and centrifuged. The upper liquid phase was removed and the residue samples were dried in a vacuum oven at 40°C overnight to obtain crystalline form D of the compound of formula (I).

Example 6. Preparation of crystalline form E of the compound of formula (I)

Approximately 50 mg of the compound of formula (I) were placed in several glass vessels and 0.5 ml of acetonitrile was added thereto, respectively, to obtain a suspension. The suspension samples were analyzed at a constant temperature (40°C) using a shaking device. The suspension samples were subjected to shaking at 40°C for 2 days and centrifuged. The upper liquid phase was removed and the residue samples were dried in a vacuum oven at 40°C overnight to obtain crystalline form E of the compound of formula (I).

Example 7. Preparation of crystalline form F of the compound of formula (I)

At 25-26°C, the compound of formula (I) was added to a one-liter three-necked flask containing 560 ml of MeOH while stirring. The reaction system was heated in an oil bath to 55-65°C. After 20 minutes, the internal temperature was set to 47°C-53°C and the reaction system was stirred for 72 h at the set temperature. Heating was stopped and stirring was continued, and the temperature spontaneously decreased to 27°C within 1 h. The system was kept at rest for 18 h and filtered, and the filter cake was washed with 30 ml of MeOH. The filter cake was dried at 60°C in vacuo for 48 h to obtain crystalline form F of the compound of formula (I).

Experimental Example 1. Study of the stability of solid crystalline form F of the compound of formula (I) under conditions of high temperature, high humidity and illumination

According to the exposure factors and accelerated test conditions, accurately weighed portions (approximately 5 mg) of the crystalline form F of the compound of formula (I) were placed in two dry and clean glass vials and distributed in the form of a thin layer as formal test samples, which were exposed to normal test conditions (temperature 60°C, relative humidity 92.5%) and accelerated test conditions (temperature 40°C, relative humidity 75%; temperature 60°C, relative humidity 75%), and the samples were completely open. The test system was covered with aluminum foil with small holes. In addition, a small number of samples were collected and placed in 40 ml glass sample vials under the same conditions to determine the state of the crystal. Samples were collected and analyzed after 5 days, 10 days and one month. The analysis method is shown in Table 7, and the analysis results are shown in Table 8. The samples were placed under a light source (visible light 1200000 lux, ultraviolet light 200 W) in a fully open state at room temperature.

Table 7. Analysis of the relevant substances by high-performance liquid chromatography

Chromatographic column Waters XBridge Shield RP18 (4.6mm x 150mm, 3.5µm), Part Number: 186003045 Wavelength 230 nm Column temperature 40°C Flow rate 0.8 ml/min Injection volume 10 µl Mobile phase A: 5 mmol/L ammonium acetate solution (pH 4.5) (volume ratio) B: 100% ACN Mobile phase program Time (minutes) A% B% 0.01 95 5 10.00 95 5 50,00 30 70 51,00 10 90 55,00 10 90 56,00 95 5 62,00 95 5 62.01 Stop Data collection time 62 minutes Solvent a mixture of acetonitrile and water in a volume ratio of 50:50 Needle cleaning solvent a mixture of acetonitrile and water in a volume ratio of 50:50

Table 8. Results of the stability analysis of the solid sample content and the corresponding substance for the crystalline form F of the compound of formula (I) (5 days, 10 days, one month)

Conditions and duration of exposure Crystalline form Total impurity content, % Content, % 0 days Crystalline form F 0.63 99.38 60°C, 5 days Crystalline form F 0.74 99.39 60°C, 10 days Crystalline form F 0.84 99.98 humidity 92.5%, 5 days Crystalline form F 0.61 98.72 humidity 92.5%, 10 days Crystalline form F 0.64 98.38 Protection from light Crystalline form F 0.61 97.77 Lighting Crystalline form F 0.67 98.58 40°C, humidity 75%, 10 days Crystalline form F 0.63 98.01 40°C, humidity 75%, 1 month Crystalline form F 0.62 97.94 60°C, humidity 75%, 10 days Crystalline form F 0.69 100.43 60°C, humidity 75%, 1 month Crystalline form F 0.75 99.46

Conclusion: The crystalline form F of the compound of formula (I) does not change in the case of the crystalline form of the starting material compound during one month of exposure to stable factors and accelerated studies, showing good physical stability. In the analysis of related substances, the content of impurities increased slightly under high temperature (60°C) and accelerated conditions (temperature 60°C and relative humidity 75%), and the total amount of impurities remained almost unchanged under other conditions, which demonstrates little sensitivity to temperature.

Experimental conclusion: The crystalline form according to the present invention has good stability and suitability for the manufacture of a medicine.

Experimental Example 2. Activity of the compound of formula (I) in inhibiting enzymes under laboratory conditions

The experimental study was carried out at Eurofins and the experimental results were presented by this company.

A solution of 20 mM Tris-HCl, 0.2 mM EDTA, 500 μM polypeptide substrate (LSNLYHQGKFLQTFCGSPLYRRR), 10 mM magnesium acetate and 10 μM [γ- ³³ P]-ATP (intensity was 500 cpm/pmol) was added to the studied system at pH 8.5. After addition of Mg ²⁺ and ATP mixture, the reaction was initiated, which proceeded under incubation conditions at room temperature for 40 minutes. To stop the reaction, 3% phosphate buffer solution was added.

Ten microliters of the reaction solution were collected, filtered on a continuous P30 filter, and washed three times with 75 μM phosphate buffer solution and once with methanol, each wash lasting 5 minutes. After drying, the activity values were measured by scintillation counting. The results of the studies are presented in Table 9.

Table 9. Results of studies of the enzymatic activity of the compounds according to the present invention under laboratory conditions (IC ₅₀ )

Connection number Wee1 (IC ₅₀ nM) AZD1775 47 Compound of formula (I) 29

Experimental Example 3. Study of the penetrating ability of the compound according to the present invention under laboratory conditions

MDR1-MDCK II cells, approved by the Piet Borst laboratory of the Netherlands Cancer Institute, were used for the study; these cells are Madin-Darby canine kidney cells transfected with the human multidrug resistance gene (MDR1), which can robustly express the efflux transporter P-glycoprotein (P-gp) and are thus suitable for screening P-gp substrate or inhibitor and for predicting the permeability of compounds with high efflux barriers such as the duodenum, blood-brain barrier, liver cell nucleus and nephron. MDR1-MDCK II cells from channel 5-35 were used for the permeability study.

MDR1-MDCK II cells were cultured with α-MEM (minimal essential medium α) medium under the conditions of 37±1°C, 5% _CO2 , and saturated relative humidity. After that, the cells were seeded in a BD Transwell 96-well plate at a seeding density of 2.3× ¹⁰⁵ cells/ ^cm2 , and then the cells were cultured in a carbon dioxide incubator for 4-7 days for the transport experiment. The method for preparing the corresponding α-MEM medium was as follows.

The liquid nutrient base was prepared using powder (α-MEM powder from Gibco, catalog number 11900) dissolved in pure water, and then L-glutamine and NaHCO ₃ were added to the solution. Then, 10% FBS (fetal bovine serum), 1% PS (double antibody), and 1% NEAA (nonessential amino acids) were added when used to prepare the complete medium. The lot of α-MEM medium is shown in Table 10.

Table 10. Description of the α-MEM batch (1 l)

Description of the α-MEM batch (1 l) Connection (1 l) Molecular weight Concentration (mM) Quantity (mg/l) Powder of the environment / / 1 pack L-glutamine 146 2 292 _NaHCO3 84 17.85 1500

AZD1775 (or a compound of the present invention) and digoxin were administered at a concentration of 2 μM in two directions (directions A-B and B-A) with duplicate wells. During the study, fenoterol and propranolol were present at the same concentration of 2 μM and were administered in one direction (direction A-B) with duplicate wells.

The solutions used were pre-incubated at 37±1°C in a water bath for 30 minutes. The dosing solution and the receiving solution were added to the wells of the plate with the corresponding cells (75 and 250 μL for each upper and main well, respectively), respectively, to initiate the bidirectional transport experiment. After adding the samples, the cell plates were placed in an incubator at 37±1°C with 5% _CO2 and saturated relative humidity for incubation for 150 minutes. The information of the collected samples is shown in Table 11.

Table 11. Information on collected samples

Sample type Sample volume in each well (µl) Volume of stopping solution (µl) Volume of transport buffer solution (µl) Dosing end A-B 50 250 100 Receiving end A-B 150 250 0 Dosing end B-A 50 250 100 Receiving end B-A 50 250 100 T ₀ 50 250 100

Note: T ₀ means the initial dosing solution sample.

After all samples had been vortexed and centrifuged at 3220 g for 10 minutes, an appropriate volume of the supernatant was transferred to a sample assay plate. After sealing the plate, the sample should be stored at 2-8°C unless immediately analyzed using the liquid chromatography-tandem mass spectrometry analytical method.

Following completion of the transport experiment, the integrity of MDR1-MDCK II cells was examined using the Lucifer Yellow rejection assay. Following incubation for 30 min with Lucifer Yellow solution, a Lucifer Yellow-containing sample was collected and a 2e plate reader set to 425/528 nm (excitation/emission) was used to measure the relative fluorescence intensity of Lucifer Yellow in the sample.

A semi-quantitative analysis of the analyte AZD1775 (or a compound of the present invention) in which a standard substance (fenoterol, propranolol and digoxin) was present was performed, and the ratio of the peak area of the analyte to the peak area of the internal standard was used to measure the concentration of the analyte.

The experimental results are presented in Table 12.

Table 12. Penetration speed (10 ^-6 cm/s)

AZD1775 Compound of formula (I) A-B 2.83 4.55 B-A 29.3 17.38 Efflux ratio 10.37 3.82

Experimental conclusion: Compared with AZD1775, the penetrating property of the compound of formula (I) is significantly improved, which is favorable for the administration of the drug into the body.

Experimental Example 4. Pharmacokinetic Evaluation of Compound

This experiment was used to investigate the pharmacokinetics of AZD1775 (or a compound of the present invention) in the plasma of female BALB/c nude mice following a single intravenous administration and a single oral administration.

Twelve mice (provided by Lingchang) were randomly divided into two groups with six females in each group, and samples were collected by a cross-over blood sampling method. All the animals in the intravenous group received 1 mg/kg of AZD1775 (or the compound of the present invention) by intravenous injection, and the solvent composition was a clear solution of 5% HP-betaCD (Kunshan Ruisk Chemical Materials Co., Ltd.) containing 0.2 mg/ml of AZD1775 (or the compound of the present invention). The animals in the oral group received 10 mg/kg of AZD1775 (or the compound of the present invention) by gavage feeding, and the solvent composition was a homogeneous suspension of 0.5% methylcellulose containing 1 mg/ml of AZD1775 (or the compound of the present invention).

In the intravenous group, plasma samples were collected at nine time points: 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours and 24 hours after administration. In the oral group, plasma samples were collected at eight time points: 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours and 24 hours after administration. The samples were analyzed by liquid chromatography-tandem mass spectrometry to obtain the plasma concentration data of AZD1775 (or the compound of the present invention), and to calculate the pharmacokinetic parameters such as peak concentration, peak time, elimination rate, half-life, area under the drug concentration-time curve, bioavailability, etc.

The experimental results are presented in Table 13.

Table 13. Results of pharmacokinetic studies

Route of administration/dose Intravenous administration,
1 mg/kg Administration via tube feeding, 10 mg/kg Test product (the compound obtained in each example) Elimination rate (ml/min/kg) Elimination half-life T _1/2 (h) Area under the curve of drug concentration versus time AUC _0-last (nM∙h) Bioavailability F (%) AZD1775 85.7 0.252 1200 31 Compound of formula (I) 33.5 1.69 6037 62.4

Experimental conclusions: Compared with AZD1775, the compound of formula (I) can significantly improve multiple pharmacokinetic parameters in mice, with significant advantages demonstrated in terms of in vivo elimination rate, half-life, in vivo concentration integral, and bioavailability.

Experimental example 5. Study in the body

(1) In pharmacodynamic studies in vivo, the compound was tested in a BALB/c nude mouse model under the influence of a xenografted subcutaneous tumor from human LoVo colon cancer cells.

Experimental animals: The selected experimental animals (provided by Shanghai SIPPR-BK Experimental Animal Co., Ltd.) were 6-8 week-old BALB/c nude mice weighing 18-22 g.

LoVo human colon cancer cells were cultured in monolayer in vitro and the culture conditions were Ham's F-12 medium containing 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM glutamine, 37°C, 5% _CO2 . Trypsin-EDTA was used for routine digestion and passage twice a week. When the cell saturation was 80%-90%, the cells were harvested, counted and inoculated. LoVo cells (0.1 ml, 10× ¹⁰⁶ cells) were subcutaneously inoculated into the right dorsum of each nude mouse and the treatment group was started when the mean tumor volume reached 213 ^mm3 . The dosage for administration by tube feeding was 40 mg/kg twice daily. The experimental endpoint of the study was inhibition, attenuation, or cessation of tumor growth. Tumor diameters were measured twice weekly using a vernier caliper. The formula used to calculate tumor volume was V=0.5a× ^b2 , where a and b represent the major diameter and minor diameter of the tumor, respectively.

The antitumor efficacy of the compound was evaluated using TGI (%) or the relative tumor growth rate T/C (%). TGI (%) reflects the degree of tumor growth inhibition. TGI (%) was calculated as follows: TGI (%) = [(1 - (mean tumor volume at the end of drug administration for a particular treatment group - mean tumor volume at the beginning of drug administration for a treatment group)) / (mean tumor volume at the end of solvent administration for the control group - mean tumor volume at the beginning of solvent administration for the control group)] × 100%.

The final results of treatment over 16 days are presented in Table 14.

Table 14. Results of a pharmacodynamic study in a mouse model under the influence of a xenografted tumor from human colon cancer LoVo cell line

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

Conclusion: Compared with AZD1775, the compound of the present invention can significantly improve the tumor inhibitory effect in the mouse body, and the chirality of the compound produces an unexpected effect on the efficacy in the body.

(2) In pharmacodynamic studies in vivo, the compound was tested in a BALB/c nude mouse model under the influence of a xenografted subcutaneous tumor from human pancreatic cancer cell line BxPC-3.

Experimental animals: The selected experimental animals were BALB/c nude mice aged 6-8 weeks and weighing 18-22 g.

Tenth generation BxPC-3 cells were cultured in monolayer in vitro, and the culture conditions were RPMI 1640 medium (Gibco, catalog number 22400-089) containing 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM glutamine, 37°C, 5% CO ₂ , and 4 passages. The passaging method was performed twice a week using trypsin-EDTA for routine digestion and passaging. When the cell saturation reached 80%-90%, the cells were digested using trypsin-EDTA, counted, and resuspended in PBS at a density of 5 × 10 ⁷ cells/ml. Each animal was inoculated with 0.1 ml (5×10 ⁶ cells) of BxPC-3 in the right side of the back. When the average tumor volume reached 153 mm ³ , the animals were randomly divided into groups according to tumor volume and treatment was started. The dosage for tube feeding was 25 mg/kg once daily.

The experimental endpoint of the study was inhibition, attenuation, or cessation of tumor growth. Tumor diameters were measured twice a week using a vernier caliper. TGI (%) or the relative tumor growth rate T/C (%) was used to evaluate the antitumor efficacy of the compound. TGI (%) reflects the degree of tumor growth inhibition. TGI (%) was calculated as follows: TGI (%) = [(1 - (mean tumor volume at the end of drug administration for a given treatment group - mean tumor volume at the start of drug administration for a treatment group)) / (mean tumor volume at the end of vehicle administration for the control group - mean tumor volume at the start of vehicle administration for the control group)] x 100%.

The final experimental results of treatment for 27 days are presented in Table 15.

Table 15. Results of a pharmacodynamic study in a mouse model under the influence of a xenografted tumor from human pancreatic cancer BxPC-3 cells

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

Conclusion: As can be seen from Table 15, compared with AZD1775, the compound of formula (I) can significantly improve the inhibitory effect on tumors in the mouse body.

(3) The efficacy of the compound was studied in vivo using a model of a transplanted tumor from mouse colon cancer cell line CT26.

Experimental animals: The experimental animals selected were 7-week-old female BALB/c nude mice weighing 16-20 g.

Cells: Mouse colon cancer CT26 cells (Cell Bank of Type Culture Collection Committee of Chinese Academy of Sciences) were cultured in monolayer in vitro, and the culture conditions were RPMI 1640 medium containing 10% fetal bovine serum, 37°C, 5% _CO2 , incubator. Trypsin-EDTA was used for routine digestion and passaging. When the cells were in exponential growth phase and the saturation was 80%-90%, the cells were collected, counted and inoculated. 0.1 ml of DPBS solution containing 3× ¹⁰⁵ CT26 cells was subcutaneously inoculated into the right back of each mouse. When the average tumor volume reached 50-70 ^mm3 , random administration was performed according to the tumor volume. The dosage for tube feeding was 30 mg/kg twice a day.

The experimental endpoint of the study was inhibition, attenuation, or cessation of tumor growth. Tumor diameters were measured twice a week using a vernier caliper. TGI (%) or the relative tumor growth rate T/C (%) was used to evaluate the antitumor efficacy of the compound. TGI (%) reflects the degree of tumor growth inhibition. TGI (%) was calculated as follows: TGI (%) = [(1 - (mean tumor volume at the end of drug administration for a given treatment group - mean tumor volume at the start of drug administration for a treatment group)) / (mean tumor volume at the end of vehicle administration for the control group - mean tumor volume at the start of vehicle administration for the control group)] x 100%.

The final results of treatment over 18 days are presented in Table 16.

Table 16. Results of in vivo efficacy studies on the mouse colon cancer allograft cell line CT26 tumor model

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

Conclusion: As can be seen from Table 16, compared with AZD1775, the compound of formula (I) can significantly improve the inhibitory effect on tumors in the mouse body.

Claims (7)

1. Crystalline form A of the compound of formula (I), wherein crystalline form A has an X-ray powder diffraction pattern containing characteristic diffraction peaks at the following 2θ angles: 5.71±0.2°, 12.68±0.2°, 15.32±0.2°, 21.44±0.2°, 23.61±0.2° and 25.68±0.2° 2. Crystalline form A according to claim 1, wherein crystalline form A has an X-ray powder diffraction pattern containing 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. Crystalline form A according to claim 1, wherein crystalline form A has an X-ray powder diffraction pattern which is shown in Fig. 1. 4. Crystalline form A according to any one of claims 1 to 3, wherein crystalline form A has a differential scanning calorimetry curve containing one initial endothermic peak point at 34.95±3°C, 174.75±3°C and 219.12±3°C, respectively. 5. Crystalline form A according to any one of claims 1 to 3, wherein crystalline form A has a thermogravimetric analysis curve in which the mass loss at 70.33±3°C is 0.7367%, and the mass loss at 209.42±3°C is 3.123%. 6. Use of crystalline form A according to any one of claims 1-5 for the treatment of a Wee1-associated disease.