EA047813B1 - SALT FORM OF PYRROLOTRIAZINE COMPOUND, ITS CRYSTALLINE FORM AND METHOD OF ITS PRODUCTION - Google Patents

Abstract

The present invention relates to a salt form of a pyrrolotriazine compound, its crystalline form, a method for producing it and its use, and in particular to its use for producing MNK1/2 inhibitor drugs and/or for producing drugs for the treatment of colorectal cancer.

Description

Priority and Related Applications

The present disclosure claims priority based on a prior application, Patent Application No. 202110501179.9 and entitled Salt Form of Pyrrolotriazine Compound, Crystal Form Thereof, and Method for Preparing It, filed with the National Intellectual Property Administration of China on May 8, 2021, incorporated herein by reference in its entirety.

Field of technology

The present disclosure relates to a salt form of a pyrrolotriazine compound, a crystalline form thereof, a method for preparing it and a use thereof, and in particular to a compound of formula (II) and a crystalline form thereof.

State of the art

Mitogen-activated protein kinase-interacting kinase (MAP kinase-interacting kinase, or MNK for short) is a serine/threonine protein kinase. There are four subtypes of human MNK, namely MNK1a and MNK1b expressed by the MNK1 gene, and MNK2a and MNK2b expressed by the MNK2 gene, respectively. All four subtypes contain a nuclear localization signal (NLS) sequence and a eukaryotic translation initiation factor 4G (eIF4G) binding sequence at the N-terminus and are thus able to enter the cell nucleus to play a role and recognize and bind to downstream eIF4E. The MNK1a and MNK2a subtypes contain a MAPK binding site at the C-terminus and can be activated by phosphorylation of the upstream extracellular signal-regulated kinase (ERK) and mitogen-activated protein kinase p38. A nuclear export signal (NES) sequence at the C-terminus of MNK1a ensures its widespread presence in the cytoplasm, whereas the other 3 subtypes are primarily present in the nucleus.

Eukaryotic initiation factor 4E (eIF4E) is a cap-binding protein and can specifically recognize the cap structure at the 5' end of messenger RNA (mRNA) and is an important factor in the initiation of protein translation. Phosphorylated at S209, eIF4E can promote the translation of downstream proteins, mainly including c-MYC, cyclin D1, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and anti-apoptotic proteins such as mcl-1 and Bcl-2. eIF4E expression is up-regulated in lung cancer, colorectal cancer, gastric cancer, pancreatic ductal carcinoma, and other malignancies. MNK is the only known kinase capable of phosphorylating eIF4E. In addition, MNK is located at the intersection of multiple signaling pathways involved in tumor development and immune function, such as the RAS signaling pathway and the T-cell receptor (TCR), and can selectively control the transcription of regulators of antitumor immune responses. MNK activity and eIF4E activation are critical for tumor development and progression, but are not essential for normal cells. Therefore, a selective MNK inhibitor is expected to be an antitumor drug with low toxicity. EFT508 (WO 2015/200481; WO 2016/172010; WO 2017/075394; WO 2017/075412; WO 2017/087808; WO 2017/117052; WO 2018/152117; WO 2018/218038) is an orally administered selective MNK inhibitor developed by EFFECTOR TFIERAPEUTICS INC. The study demonstrated that eFT508 could selectively inhibit the expression of programmed cell death protein 1 (PD-1), lymphocyte activation gene 3 (LAG3), and interleukin 10 (TL-10) and improve the function of cytotoxic T cells without affecting the proliferation of normal T cells. Preclinical studies have shown that the combination of eFT508 and anti-PD-1 monoclonal antibody can enhance the efficacy and response rate. The drug has completed phase I clinical trials and demonstrated good safety; monotherapy for hematological tumors and common prostate cancer is currently in phase II clinical trials, combination therapy with monoclonal antibody avelumab for microsatellite-stable colorectal cancer (MSS CRC) is in phase II clinical trials, and combination therapy with PD-1/PD-L1 therapy (for patients previously treated with PD-1/PD-L1 therapy alone and who have had disease progression or no complete or partial relief) for solid tumors is in phase II clinical trials.

Brief description of the invention

The present disclosure provides a compound of formula (II),

- 1 047813

The present disclosure further provides a crystalline form A of the compound of formula (II), wherein the powder X-ray diffraction pattern of the crystalline form A has characteristic diffraction peaks at the following 2θ angles: 7.78±0.20°, 11.38±0.20° and

20.58±0.20°;

In some embodiments of the present disclosure, the powder X-ray diffraction pattern of crystalline form A described above has characteristic diffraction peaks at the following 2θ angles: 7.78±0.20°, 9.44±0.20°, 11.38±0.20°, 19.84±0.20°, 20.58±0.20°,

21.56±0.20°, 22.86±0.20° and 24.82±0.20°.

In some embodiments of the present disclosure, the powder X-ray diffraction pattern of crystalline Form A described above has characteristic diffraction peaks at the following 2θ angles: 6.58±0.20°, 7.78±0.20°, 9.44±0.20°, 11.38±0.20°, 14.38±0.20°, 18.66±0.20°, 19.84±0.20°, 20.58±0.20°, 21.56±0.20°, 22.86±0.20°, 23.54±0.20°, and 24.82±0.20°.

In some embodiments of the present disclosure, the powder X-ray diffraction pattern of crystalline Form A described above has characteristic diffraction peaks at the following 2θ angles: 4.78°±0.20°, 6.58°±0.20°, 7.78°±0.20°, 9.44°±0.20°, 11.38°±0.20°, 13.48°±0.20°, 14.38°±0.20°, 14.80°±0.20°, 16.42°±0.20°, 17.00°±0.20°, 17.32°±0.20°, 18.34°±0.20°,

18.66°±0.20°, 19.08°±0.20°, 19.60°±0.20°, 19.84°±0.20°, 20.28°±0.20°, 20.58°±0.20°, 21.56°±0.20°,

21.84°±0.20°, 22.52°±0.20°, 22.86°±0.20°, 23.26°±0.20°, 23.54°±0.20°, 24.46°±0.20°, 24.82°±0.20°,

25.50°±0.20°, 26.04°±0.20°, 26.58°±0.20°, 27.42°±0.20°, 27.82°±0.20°, 28.07°±0.20°, 28.42°±0.20°,

29.08°±0.20°, 29.66°±0.20°, 30.08°±0.20°, 31.20°±0.20°, 31.42°±0.20°, 38.22°±0.20° and 39.04°±0.20°.

In some embodiments of the present disclosure, the powder X-ray diffraction pattern of crystalline Form A described above has characteristic diffraction peaks at the following 2θ angles: 4.78°±0.10°, 6.58°±0.10°, 7.78°±0.10°, 9.44°±0.10°, 11.38°±0.10°, 13.48°±0.10°, 14.38°±0.10°, 14.80°±0.10°, 16.42°±0.10°, 17.00°±0.10°, 17.32°±0.10°, 18.34°±0.10°,

18.66°±0.10°, 19.08°±0.10°, 19.60°±0.10°, 19.84°±0.10°, 20.28°±0.10°, 20.58°±0.10°, 21.56°±0.10°,

21.84°±0.10°, 22.52°±0.10°, 22.86°±0.10°, 23.26°±0.10°, 23.54°±0.10°, 24.46°±0.10°, 24.82°±0.10°,

25.50°±0.10°, 26.04°±0.10°, 26.58°±0.10°, 27.42°±0.10°, 27.82°±0.10°, 28.07°±0.10°, 28.42°±0.10°,

29.08°±0.10°, 29.66°±0.10°, 30.08°±0.10°, 31.20°±0.10°, 31.42°±0.10°, 38.22°±0.10° and 39.04°±0.10°.

In some embodiments of the present disclosure, the X-ray powder diffraction pattern of crystalline Form A described above has characteristic diffraction peaks at the following 2θ angles: 4.78°, 6.58°, 7.78°, 9.44°, 11.38°, 13.48°, 14.38°, 14.80°, 16.42°, 17.00°, 17.32°, 18.34°, 18.66°, 19.08°, 19.60°, 19.84°, 20.28°, 20.58°, 21.56°, 21.84°, 22.52°, 22.86°, 23.26°, 23.54°, 24.46°, 24.82°, 25.50°, 26.04°, 26.58°, 27.42°, 27.82°, 28.07°, 28.42°, 29.08°, 29.66°, 30.08°, 31.20°, 31.42°, 38.22° and 39.04°.

In some embodiments of the present disclosure, the powder X-ray diffraction pattern of crystalline Form A described above has characteristic diffraction peaks at the following 2θ angles: 4.78°±0.20° and/or 6.58°±0.20° and/or 7.78°±0.20° and/or 9.44°±0.20° and/or 11.38°±0.20° and/or 13.48°±0.20° and/or 14.38°±0.20° and/or 14.80°±0.20° and/or 16.42°±0.20° and/or 17.00°±0.20° and/or 17.32°±0.20° and/or 18.34°±0.20°, and/or 18.66°±0.20°, and/or 19.08°±0.20°, and/or

19.60°±0.20°, and/or 19.84°±0.20°, and/or 20.28°±0.20°, and/or 20.58°±0.20°, and /or 21.56°±0.20°, and/or

21.84°±0.20°, and/or 22.52°±0.20°, and/or 22.86°±0.20°, and/or 23.26°±0.20°, and /or 23.54°±0.20°, and/or

24.46°±0.20°, and/or 24.82°±0.20°, and/or 25.50°±0.20°, and/or 26.04°±0.20°, and /or 26.58°±0.20°, and/or

27.42°±0.20°, and/or 27.82°±0.20°, and/or 28.07°±0.20°, and/or 28.42°±0.20°, and /or 29.08°±0.20°, and/or

29.66°±0.20°, and/or 30.08°±0.20°, and/or 31.20°±0.20°, and/or 31.42°±0.20°, and /or 38.22°±0.20°, and/or

39.04°±0.20°.

In some embodiments of the present disclosure, the powder X-ray diffraction pattern of crystalline Form A described above has characteristic diffraction peaks at the following 2θ angles: 4.78°±0.10° and/or 6.58°±0.10° and/or 7.78°±0.10° and/or 9.44°±0.10° and/or 11.38°±0.10° and/or 13.48°±0.10° and/or 14.38°±0.10° and/or 14.80°±0.10° and/or 16.42°±0.10° and/or 17.00°±0.10° and/or 17.32°±0.10° and/or 18.34°±0.10°, and/or 18.66°±0.10°, and/or 19.08°±0.10°, and/or

19.60°±0.10°, and/or 19.84°±0.10°, and/or 20.28°±0.10°, and/or 20.58°±0.10°, and /or 21.56°±0.10°, and/or

21.84°±0.10°, and/or 22.52°±0.10°, and/or 22.86°±0.10°, and/or 23.26°±0.10°, and /or 23.54°±0.10°, and/or

24.46°±0.10°, and/or 24.82°±0.10°, and/or 25.50°±0.10°, and/or 26.04°±0.10°, and /or 26.58°±0.10°, and/or

- 2 047813

27.42°±0.10°, and/or 27.82°±0.10°, and/or 28.07°±0.10°, and/or 28.42°±0.10°, and /or 29.08°±0.10°, and/or 29.66°±0.10°, and/or 30.08°±0.10°, and/or 31.20°±0.10° , and/or 31.42°±0.10°, and/or 38.22°±0.10°, and/or 39.04°±0.10°.

In some embodiments of the present disclosure, the X-ray powder diffraction (XRPD) pattern of crystalline form A described above is substantially as shown in Fig. 1.

In some embodiments of the present disclosure, the XRPD of crystalline form A described above is determined using Cu-Ka radiation.

In some embodiments of the present disclosure, XRPD analysis data for crystalline form A described above are shown in Table 1.

Table 1

XPRD analysis data for crystalline form A of compound of formula (II)

No. Angle 20 (°) Intensity Interplanetary Relative No. Angle 20 (°) Intensity Interplanetary Relative peak (imp/s) distance not (A) intensity (%) ь peak (imp/s) distance not (A) intensity (%) 1 4.78 124 18.46 9.8 21 22,52 240 3.94 19,0 2 6.58 104 13.42 8.2 22 22.86 640 3.89 50.6 3 7.78 357 11.35 28.2 23 23,26 301 3.82 23.8 4 9.44 245 9.36 19.3 24 23.54 426 3.78 33.6 5 11.38 1265 7.77 100,0 25 24.46 68 3.64 5.4 6 13.48 93 6.57 7.3 26 24.82 453 3.58 35.8 7 14.38 310 6.15 24.5 27 25,50 93 3.49 7.4 8 14.80 251 5.98 19.8 28 26.04 179 3.42 14.1 9 16.42 137 5.39 10.9 29 26.58 214 3.35 16.9 10 17,00 144 5.21 And,4 30 27,42 97 3.25 7,7 11 17.32 91 5,12 7.2 31 27.82 190 3.20 15,0 12 18.34 291 4.83 23,0 32 28.07 85 3.18 6,7 13 18.66 349 4.75 27.6 33 28,42 81 3.14 6.4 14 19.08 136 4.65 10.8 34 29.08 91 3.07 7.2 15 19,60 269 4.53 21.2 35 29.66 81 3.01 6.4 16 19.84 514 4.47 40.6 36 30.08 90 2.97 7.1 17 20,28 167 4.38 13.2 37 31,20 71 2.86 5,6 18 20.58 726 4.31 57.4 38 31,42 63 2.84 5.0 19 21,56 667 4.12 52.7 39 38.22 36 2.35 2.8 20 21.84 341 4.07 26.9 40 39.04 57 2.31 4,5

In some embodiments of the present disclosure, the differential scanning calorimetry curve of crystalline Form A described above has an endothermic peak at 287.17±3°C.

In some embodiments of the present disclosure, the differential scanning calorimetry (DSC) profile of crystalline Form A described above is shown in Fig. 2.

In some embodiments of the present disclosure, the thermogravimetric analysis (TGA) curve of crystalline form A described above exhibits a mass loss

- 3 047813

0.075% at 200.0±3°C.

In some embodiments of the present disclosure, the TGA profile of crystalline form A described above is shown in Fig. 3.

The present disclosure further provides a method for preparing crystalline form A of the compound of formula (II), comprising the following steps:

(a) adding a compound of formula (II) to a solvent to form a suspension;

(b) stirring the suspension at 40-55°C for 2-25 h; and (c) filtering the suspension and then drying the filter cake under vacuum at 30-45°C for 10-24 h;

where the solvent is selected from methanol, acetonitrile and tert-butyl methyl ether.

The present disclosure provides a crystalline form B of the compound of formula (II), wherein the X-ray powder diffraction (XRPD) diffraction pattern of crystalline form B has characteristic diffraction peaks at the following 2θ angles: 7.55±0.20°, 15.13±0.20° and 19.82±0.20°;

In some embodiments of the present disclosure, the powder X-ray diffraction pattern of crystalline form B described above has characteristic diffraction peaks at the following 2θ angles: 6.89±0.20°, 7.55±0.20°, 9.50±0.20°, 11.35±0.20°, 12.72±0.20°, 15.13±0.20°, 19.82±0.20°, and 26.63±0.20°.

In some embodiments of the present disclosure, the powder X-ray diffraction pattern of crystalline Form B described above has characteristic diffraction peaks at the following 2θ angles: 6.89±0.20°, 7.55±0.20°, 9.50±0.20°, 11.35±0.20°, 12.24±0.20°, 12.72±0.20°, 15.13±0.20°, 18.94±0.20°, 19.82±0.20°, 23.25±0.20°, 26.63±0.20°, and 27.27±0.20°.

In some embodiments of the present disclosure, the powder X-ray diffraction pattern of crystalline Form B described above has characteristic diffraction peaks at the following 2θ angles: 4.98°±0.20°, 6.89°±0.20°, 7.55°±0.20°, 8.46°±0.20°, 9.50°±0.20°, 10.12 ^° ±0.20°, 11.35°±0.20°, 12.24°±0.20°, 12.72°±0.20°, 14.05°±0.20°, 15.13°±0.20°, 15.65°±0.20°,

16.20°±0.20°, 17.79°±0.20°, 18.94°±0.20°, 19.82°±0.20°, 20.76°±0.20°, 21.61°±0.20°, 23.25°±0.20°,

23.87°±0.20°, 26.09°±0.20°, 26.63°±0.20°, 27.27°±0.20°, 28.45°±0.20°, 29.12°±0.20°, 30.95°±0.20°,

32.32°±0.20°, 34.62°±0.20° and 38.41°±0.20°.

In some embodiments of the present disclosure, the powder X-ray diffraction pattern of crystalline Form B described above has characteristic diffraction peaks at the following 2θ angles: 4.98°±0.10°, 6.89°±0.10°, 7.55°±0.10°, 8.46°±0.10°, 9.50°±0.10°, 10.12°±0.10°, 11.35°±0.10°, 12.24°±0.10°, 12.72°±0.10°, 14.05°±0.10°, 15.13°±0.10°, 15.65°±0.10°,

16.20°±0.10°, 17.79°±0.10°, 18.94°±0.10°, 19.82°±0.10°, 20.76°±0.10°, 21.61°±0.10°, 23.25°±0.10°,

23.87°±0.10°, 26.09°±0.10°, 26.63°±0.10°, 27.27°±0.10°, 28.45°±0.10°, 29.12°±0.10°, 30.95°±0.10°,

32.32°±0.10°, 34.62°±0.10° and 38.41°±0.10°.

In some embodiments of the present disclosure, the powder X-ray diffraction pattern of crystalline Form B described above has characteristic diffraction peaks at the following 2θ angles: 4.98°, 6.89°, 7.55°, 8.46°, 9.50°, 10.12°, 11.35°, 12.24°, 12.72°, 14.05°, 15.13°, 15.65°, 16.20°, 17.79°, 18.94°, 19.82°, 20.76°, 21.61°, 23.25°, 23.87°, 26.09°, 26.63°, 27.27°, 28.45°, 29.12°, 30.95°, 32.32°, 34.62° and 38.41°.

In some embodiments of the present disclosure, the powder X-ray diffraction pattern of crystalline Form B described above has characteristic diffraction peaks at the following 2θ angles: 4.98°±0.20° and/or 6.89°±0.20° and/or 7.55°±0.20° and/or 8.46°±0.20° and/or 9.50°±0.20° and/or 10.12°±0.20° and/or 11.35°±0.20° and/or 12.24°±0.20° and/or 12.72°±0.20° and/or 14.05°±0.20° and/or 15.13°±0.20° and/or 15.65°±0.20°, and/or 16.20°±0.20°, and/or 17.79°±0.20°, and/or

18.94°±0.20°, and/or 19.82°±0.20°, and/or 20.76°±0.20°, and/or 21.61°±0.20°, and /or 23.25°±0.20°, and/or

23.87°±0.20°, and/or 26.09°±0.20°, and/or 26.63°±0.20°, and/or 27.27°±0.20°, and /or 28.45°±0.20°, and/or

29.12°±0.20°, and/or 30.95°±0.20°, and/or 32.32°±0.20°, and/or 34.62°±0.20°, and /or 38.41°±0.20°.

In some embodiments of the present disclosure, the powder X-ray diffraction pattern of crystalline Form B described above has characteristic diffraction peaks at the following 2θ angles: 4.98°±0.10° and/or 6.89°±0.10° and/or 7.55°±0.10° and/or 8.46°±0.10° and/or 9.50°±0.10° and/or 10.12°±0.10° and/or 11.35°±0.10° and/or 12.24°±0.10° and/or 12.72°±0.10° and/or 14.05°±0.10° and/or 15.13°±0.10° and/or 15.65°±0.10°, and/or 16.20°±0.10°, and/or 17.79°±0.10°, and/or 18.94°±0.10°, and/or 19.82°±0.10°, and/or 20.76°±0.10°, and/or 21.61°±0.10°, and/or 23, 25°±0.10°, and/or

- 4 047813

23.87°±0.10°, and/or 26.09°±0.10°, and/or 26.63°±0.10°, and/or 27.27°±0.10°, and /or 28.45°±0.10°, and/or 29.12°±0.10°, and/or 30.95°±0.10°, and/or 32.32°±0.10° , and/or 34.62°±0.10°, and/or 38.41°±0.10°.

In some embodiments of the present disclosure, the XPRD pattern of crystalline Form B described above is substantially as shown in Fig. 4.

In some embodiments of the present disclosure, the XPRD of crystalline form B described above is determined using Cu-Ka radiation.

In some embodiments of the present disclosure, XPRD diffraction pattern analysis data for crystalline form B described above are shown in Table 2.

Table 2

XPRD analysis data for crystalline form B of compound of formula (II)

No. Angle 20 (°) Peak intensity (imp/s) Interplane distance (A) Relative intensity (%) No. Angle 20 (°) Peak intensity (imp/s) Interplanar distance (A) Relative intensity (%) 1 4.98 72,32 17.74 2.97 16 19.82 1532,5 9 4.48 62.93 2 6.89 896.22 12.82 36.80 17 20.76 150.45 4.28 6.18 3 7.55 1530.4 9 11.71 62.85 18 21.61 244.84 4,P 10.05 4 8.46 157.42 10.46 6.46 19 23,25 347,03 3.83 14.25 5 9.50 859,72 9.31 35.30 20 23.87 274.20 3.73 11.26 6 10,12 135.06 8.74 5.55 21 26.09 263,02 3.42 10.80 7 11.35 889,45 7.80 36.52 22 26.63 913,50 3.35 37,51 8 12.24 510,42 7.23 20.96 23 27,27 351,00 3.27 14.41 9 12.72 666,73 6.96 27,38 24 28.45 138.33 3.14 5.68 10 14.05 259.16 6.31 10.64 25 29.12 70.84 3.07 2.91 11 15,13 2435.2 5 5.86 100,00 26 30.95 54.09 2.89 2.22 12 15.65 242.34 5.66 9.95 27 32,32 32,02 2.77 1.31 13 16,20 272.35 5.47 11,18 28 34.62 16.98 2.59 0,70 14 17.79 95.51 4.99 3.92 29 38,41 68.55 2.34 2.81 15 18.94 579,19 4.69 23.78

In some embodiments of the present disclosure, the differential scanning calorimetry curve of crystalline Form B described above has an endothermic peak at 300.0±3°C.

In some embodiments of the present disclosure, the DSC profile of crystalline form B described above is shown in Fig. 5.

The present disclosure further provides a method for preparing crystalline form B of the compound of formula (II), comprising the following steps:

(a) adding a compound of formula (II) to a solvent to form a suspension;

(b) stirring the suspension at 40-55°C for 2-25 h; and (c) filtering the suspension and then drying the filter cake under vacuum at 30-45°C for 10-24 h; wherein the solvent is selected from ethanol and n-heptane.

The present disclosure further provides the use of a compound of the formula

- 5 047813 (II) described above, crystalline form A described above and crystalline form B described above, or crystalline forms obtained using the method described above, for the preparation of a medicinal product that is an MNK1/2 inhibitor.

The present disclosure further provides the use of a compound of formula (II) described above, crystalline form A described above, and crystalline form B described above, or crystalline forms obtained by the method described above, for the preparation of a medicament for the treatment of colorectal cancer.

Technical results

The compound of formula (I) according to the present disclosure has high selectivity for MNK1/2, and also has significant inhibitory activity against kinases; in addition, the compound has good membrane permeability and has excellent pharmacokinetic and pharmacodynamic properties. Each of the crystalline forms of the compound of formula (II) is stable, little affected by light, heat and humidity, and has good in vivo efficacy and a broad pharmaceutical prospect.

Definitions and description

Unless otherwise indicated, the following terms and phrases used in this specification are intended to have the following meanings. Unless otherwise specifically indicated, a particular phrase or term is not to be regarded as vague or unclear, but is to be interpreted in accordance with its generally accepted meaning. When a trademark is referred to, it is intended to refer to the corresponding commercial product or its active ingredient.

The intermediate compounds of the present disclosure can be prepared by various synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments obtained by combinations thereof with other chemical synthetic methods, and equivalent replacements thereof known to those skilled in the art. Preferred embodiments include the examples disclosed herein.

Chemical reactions according to particular embodiments of the present disclosure are carried out in a suitable solvent, which must be suitable for the chemical changes according to the present disclosure and the required reagents and materials. In order to prepare compounds according to the present disclosure, those skilled in the art sometimes need to modify or select a synthetic procedure or reaction scheme based on existing embodiments.

The structures of the compounds according to the present disclosure can be confirmed by conventional methods well known to those skilled in the art, and if the present disclosure refers to an absolute configuration of a compound, then said absolute configuration can be confirmed by conventional methods in the art. For example, in the single crystal X-ray diffraction (SXRD) method, diffraction intensity data of a grown single crystal are collected using a Bruker D8 venture diffractometer in which the light source is CuKa radiation, and the scanning mode is φ/ω scanning; after collecting the corresponding data, a direct method (Shelxs97) is further used to analyze the crystal structure, and thus the absolute configuration can be confirmed.

The present disclosure is described in detail below by way of examples, which are not intended to limit the present disclosure in any way.

All solvents used in the present disclosure are commercially available and can be used without further purification.

The following abbreviations are used in the present disclosure: DCM is dichloromethane; DMF is N.N-dimethylformamide; DMSO is dimethyl sulfoxide; EtOH is ethanol; MeOH is methanol; 2-MeTHF is 2-methyltetrahydrofuran; ACN is acetonitrile; toluene is methylbenzene; EtOAc is ethyl acetate; THF is tetrahydrofuran; H2O is water; TosOH is p-toluenesulfonic acid.

Compounds are named according to generally accepted nomenclature rules in the art or using ChemDraw® software, and for commercially available compounds, names are given according to suppliers' catalogues.

Brief description of the drawings

Fig. 1 shows the XRPD diffraction pattern of crystalline form A of the compound of formula (II).

Fig. 2 shows the DSC profile of crystalline form A of the compound of formula (II).

Fig. 3 shows the TGA profile of crystalline form A of the compound of formula (II).

Fig. 4 shows the XRPD diffraction pattern of crystalline form B of the compound

- 6 047813 formula (II).

Fig. 5 shows the DSC profile of crystalline form B of the compound of formula (II);

Fig. 6 shows the dynamic vapor sorption profile (DVS) of crystalline form A of the compound of formula (II).

Fig. 7 shows an ellipsoidal plot of a single crystal of the compound of formula (II).

Device parameters

The method for X-ray powder diffractometer (XRPD) for crystalline form A according to the present disclosure has the test parameters shown in Table 3.

Table 3

XRPD testing parameters for crystalline form A

XPRD Test Parameters Parameter Set value Model DX-2700BH X-ray radiation Si, k-α: 1.54184A; X-ray tube settings 40 kV, 30 mA Divergence gap 1 mm Primary scattering gap 28 mm Secondary gap 28 mm XPRD Test Parameters Parameter Set value Detector slit 0.3 mm Anti-scattering gap 1 mm Scan axis Os-Od Scanning range 2Θ (°) 3-40 Scanning time (s) 0,5 Scanning step size 2Θ (°) 0,02

The differential scanning calorimeter (DSC) method for crystalline form A according to the present disclosure has the test parameters shown in Table 4.

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Table 4

DSC testing parameters for crystalline form A

DSC Testing Parameters Device model Mettler DSC 1 Way Linear heating Crucible type Gold plated high pressure crucible, closed Temperature range 40-350°С Scanning speed (°C/min) 10 Protective gas Nitrogen

The method for a thermogravimetric analyzer (TGA) for crystalline form A according to the present disclosure has the test parameters shown in Table 5.

Table 5

TGA Test Parameters for Crystal Form A

TGA Test Parameters Device model TGA 550 Way Linear heating Sample tray Aluminum tray, open Temperature range 40-500 °C Scanning speed (°C/min) 10 Protective gas Nitrogen

The dynamic vapor sorption (DVS) method for crystalline form A according to the present disclosure has the test parameters shown in Table 6.

- 8 047813

Table 6

DVS Testing Parameters for Crystalline Form A

Manufacturer and model of the device SMS/DVS intrinsic Testing condition Weigh approximately 10 mg of sample for testing Temperature 25 °C Balancing Amass./Atemp.: 0.01%/min Drying Drying at 25°C and relative humidity (RH) 0% for 2 hours Testing gradient OR (%) 5% OV Range of RH testing gradient (%) 0%-95%-0% RH

The categories of hygroscopicity assessment are shown in Table 7 below.

Table 7

Table of categories for assessing hygroscopicity

Categories of hygroscopicity AW% Moisture absorption Absorbing enough water to form a solution Very hygroscopic Δ W%N I5% Hygroscopic Ι5%>Δ W%N2% Slightly hygroscopic 2%>Δ\ν%Ν0,2% Non-hygroscopic or barely hygroscopic AW%<0.2%

Note: AW% represents the hygroscopic mass increase of the test compound at 25±1°C and 80±2% RH.

The X-ray powder diffractometer (XRPD) method for crystalline form B according to the present disclosure has the testing parameters shown in Table 8.

- 9 047813

Table 8

XPRD testing parameters for crystalline form B

XPRD Test Parameters Parameter Set value Model X'Pert ³ X-ray radiation Si, ka, Καί (A): 1.54060; Ka2 (A): 1.54443; X-ray tube settings 45 kV, 40 mA Divergence gap Fixed 1/8° Anti-scattering gap P7.5 Scan mode Continuous Scanning range 3-40 20 (°) Scanning time of each step (s) 46,665 Scanning step size 2Θ (°) 0,0263

The differential scanning calorimeter (DSC) method for crystalline form B according to the present disclosure has the test parameters shown in Table 9.

Table 9

DSC testing parameters for crystalline form B

DSC Testing Parameters Device model TA Discovery DSC 2500 Way Linear heating Sample tray Aluminum tray, closed Temperature range 25-330°С Scanning speed (°C/min) 10 Protective gas Nitrogen

- 10 047813

Example 1. Preparation of a compound of formula (I). Synthetic route:

Stage 1.

Compound la (100 g, 462 mmol) was dissolved in ethanol (500 ml), and concentrated sulfuric acid (49.94 g, 509 mmol, purity: 98%) was added dropwise at 0 °C. The resulting reaction liquid was stirred at 95 °C for 16 h. After completion of the reaction, the reaction liquid was concentrated under reduced pressure to remove most of the ethanol. Water (300 ml) was added to the resulting concentrate, and the resulting mixed solution was extracted with ethyl acetate (250 ml x 3). The combined organic phase was washed with saturated sodium bicarbonate solution (300 ml), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure to give compound 1b. ¹ H Nuclear Magnetic Resonance (NMR) (400 MHz, CDCl3) δ 8.60 (s, 1H), 7.78 ( ^s , 1H), 4.48-4.42 (m, 2H), 2.58 (s, 3H), 1.43 (t, J=7.2 Hz, 3H).

Stage 2.

Compound 1b (10.0 g, 41.0 mmol) was dissolved in dichloromethane (200 mL), and trifluoroacetic anhydride (17.2 g, 81.9 mmol) and urea peroxyhydrate (8.09 g, 86.0 mmol) were added under stirring at 0 °C. The resulting reaction liquid was heated to 25 °C and stirred for 16 h. Water (200 mL) was added to the reaction liquid, and the mixed solution was extracted with dichloromethane (100 mL × 3). The organic phases were combined, washed successively with saturated sodium bicarbonate (200 mL × 2) and saturated brine (500 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 1c. Ή NMR (400 MHz, CDCl ₃ ) δ 8.23 (s, 1H),

- 11 047813

7.29 (s, 1H), 4.52-4.45 (m, 2H), 2.29 (s, 3H), 1.41 (t, J=7.2 Hz, 3H).

Stage 3.

Compound 1c (22.0 g, 84.6 mmol) was dissolved in NN-dimethylformamide (130 mL), and trifluoroacetic anhydride (35.5 g, 169 mmol) was added thereto under stirring at 0°C. The reaction liquid was stirred at 50°C for 1 h. Water (200 mL) was added to the reaction liquid, and the resulting mixed solution was extracted with ethyl acetate (100 mL×4). The organic phases were combined, washed successively with saturated sodium bicarbonate solution (200 mL×3) and saturated brine (150 mL×3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was added to a mixed liquid of petroleum ether and ethyl acetate (8/1, 90 ml), and the mixture was stirred at room temperature for 2 h and then filtered to give compound 1d. 1H NMR (400 MHz, _CDCl3 ) δ 9.89 (br. s, 1H), 7.75 (s, 1H), 4.46-4.41 (m, 2H), 2.44 (s, 3H), 1.43 (t, J=7.2 Hz, 3H).

Stage 4.

Compound 1d (2.00 g, 7.69 mmol) was dissolved in ethanol (20 mL), and aqueous ammonia (16.2 g, 115 mmol, purity: 25%) was added. The resulting reaction liquid was stirred at 40 °C for 16 h. The reaction liquid was concentrated under reduced pressure, and the crude product was added to a mixed liquid of methanol and dichloromethane (1/5, 48 mL). The resulting mixture was stirred overnight at room temperature, then filtered and washed with dichloromethane (5 mL×2). The filter cake was concentrated under reduced pressure to give compound 1e. Electron Spray Ionization Mass Spectrometry (MS-ESI), [M+H]+, calculated: 231 and 233, found: 231 and 233.

Stage 5.

Compound 1e (500 mg, 1.97 mmol) and cyclopentanone (664 mg, 7.89 mmol) were dissolved in anhydrous dioxane (6 mL), and concentrated sulfuric acid (98.7 mg, 0.986 mmol, purity: 98%) was added dropwise to the reaction liquid. The reaction liquid was stirred at 95°C for 3 h. The reaction liquid was concentrated under reduced pressure to remove a part of dioxane (about 3 mL), and then filtered. n-Hexane (10 mL) was added to the collected filter cake, and the mixture was stirred at room temperature for 2 h and filtered. The filter cake was dried under vacuum for 2 h to give compound 1f. MS-IRE, [M+H]+, calculated: 297 and 299, found: 297 and 299. 1H NMR (400 MHz, DMSOO δ 10.16 (s, 1H), 8.02 (s, 1H), 2.71-2.78 (m, 2H), 2.37 (s, 3H), 3 (m, 2H), 1.79-1.84 (m, 2H), 1.63-1.67 (m, 2H).

Stage 6.

Compound 1g (50 g, 0.442 mol), 1,8-diazabicyclo[5.4.0]undec-7-ene (67.3 g, 0.442 mol) was dissolved in tetrahydrofuran (500 mL). The reaction liquid was heated to 55 °C, and acetaldehyde (9.74 g, 0.221 mol) was added to the reaction liquid at this temperature. The reaction liquid was stirred at 55 °C for 18 h. Then, the reaction liquid was cooled to 22 °C and quenched with acetic acid (25 mL). The reaction liquid was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate (1000 mL) and diluted hydrochloric acid (1000 mL, 1 M). The organic phase was saved after liquid separation and the aqueous phase was extracted with ethyl acetate (300 ml x 3).

The organic phases were combined, washed successively with saturated sodium bicarbonate solution (100 ml) and brine (200 ml), dried over anhydrous sodium sulfate, filtered and concentrated, and the crude product was separated by column chromatography (4/1, petroleum ether/ethyl acetate, Rf = 0.56) to give compound 1h. MS-IRE, [M+H]+, calculated: 226, found: 226. 1H NMR (400 MHz, _CDCl3 ) δ 9.29 (br. s, 1H), 7.48 (d, J=3.2 Hz, 1H), 4.35 (q, J=7.2 Hz, 2H), 4.29 (q, J=7.2 Hz, 2H), 2.61 (s, 3H), 1.38 (t, J=7.2 Hz, 3H), 1.35 (m, J=7.2 Hz, 3H).

Stage 7.

Compound 1h (11.0 g, 48.8 mmol) was dissolved in N-methylpyrrolidone (60 mL), and potassium tert-butoxide (6.03 g, 53.7 mmol) was added to the reaction liquid. After the reaction liquid was stirred at 25°C for 0.5 h, a solution of compound 1i (9.78 g, 53.7 mmol) in N-methylpyrrolidone (30 mL) was added. The reaction liquid was further stirred for 20 h. The reaction liquid was washed with water (200 mL) and extracted with ethyl acetate (200 mL x 3). The organic phases were combined, washed with saturated brine (20 mL x 3), dried over anhydrous sodium sulfate, filtered and concentrated, and the crude product was separated by column chromatography (4/1, petroleum ether/ethyl acetate, Rf = 0.55) to give compound 1j. MS-IRE, [M+H]+, calculated: 241, found: 241. 1H NMR (400 MHz, _CDCl3 ) δ 7.49 (s, 1H), 4.35 (q, J=7.2 Hz, 2H), 4.27 (q, J=7.2 Hz, 2H), 2.57 (s, 3H), 1.40 (t, J=7.2 Hz, 3H), 1.34 (t, J=7.2 Hz, 3H).

Stage 8.

Compound lj (10.2 g, 42.5 mmol) was dissolved in formamide (120 mL), and phosphoric acid (832 mg, 8.49 mmol) was added to the reaction liquid. The reaction liquid was stirred at 125 °C for 16 h. Then, the reaction liquid was cooled to 22 °C, and a large amount of white solid precipitated from it. The mixture was filtered, and the collected filter cake was added to

- 12 047813 mixed solution of petroleum ether/ethyl acetate (1/1, 100 mL). The resulting mixture was stirred at 30 °C for 0.5 h and then filtered to give compound 1k. MS-IRE, [M+H]+, calculated: 222, found: 222. ¹ H NMR (400 MHz, DMSO-de) δ 7.90 (s, 1H), 7.84 (s, 1H), 4.23 (q, J=7.2 Hz, 2H), 2.61 (s, 3H), 1.28 (t, J=7.2 Hz, 3H).

Stage 9.

Compound 1k (4.00 g, 18.0 mmol) was dissolved in anhydrous tetrahydrofuran (50 mL). Methyl magnesium bromide (30.1 mL, 90.3 mmol) was added dropwise to the reaction liquid at 25 °C. After completion of the dropwise addition, the reaction liquid was heated to 25 °C and stirred for 15 h. The reaction liquid was quenched with saturated ammonium chloride solution (100 mL) and extracted with ethyl acetate (50 mL x 5). The organic phases were combined, washed with saturated brine (10 mL x 1), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and the crude product was separated by thin layer chromatography (2/1, petroleum ether/ethyl acetate, Rf = 0.39) to give compound 11.

MS-IRE, [M+H]+, calculated: 208, found: 208. ¹ H NMR (400 MHz, _CDCl3 ) δ 7.30 (br s, 1H), 7.24 (br s, 1H), 2.59 (s, 3H), 1.54 (s, 6H).

Stage 10.

Compound 11 (1.00 g, 4.83 mmol) and hydrogen peroxide (4.64 ml, 48.26 mmol, content: 30%) were dissolved in anhydrous tetrahydrofuran (30 ml). To the reaction liquid was added dropwise a cold solution of methanesulfonic acid (3.44 ml, 48.26 mmol) in water (10 ml) at 0 °C. The reaction liquid was stirred at 0 °C for 1 h. The reaction liquid was quenched with 10% aqueous sodium sulfite solution (15 ml) until starch iodine reaction paper showed a negative result. The solution was extracted with ethyl acetate (50 ml x 3). The organic phases were combined, washed with saturated brine (10 ml x 1), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and the crude product was separated by column chromatography (2/1, petroleum ether/ethyl acetate, Rf = 0.38) to give compound 1m. MS-IRE, [M+H]+, calculated: 166, found: 166.

Stage 11.

Compound 1m (400 mg, 2.42 mmol) was dissolved in anhydrous tetrahydrofuran (10 ml), and triethylamine (0.674 ml, 4.84 mmol) and pivaloyl chloride (350 mg, 4.84 mmol) were added to the reaction liquid. The reaction liquid was stirred at 0 °C for 1 h, washed with water (10 ml), and extracted with ethyl acetate (10 ml × 5). The organic phases were combined, washed with saturated brine (10 ml × 1), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and the crude product was separated by thin layer chromatography (2/1, petroleum ether/ethyl acetate, Rf = 0.63) to give compound 1n. MS-IRE, [M+H]+, calculated: 250, found: 250. 1H NMR (400 MHz, CD ₃ OD) δ 7.61 (s, 1H), 7.47 (s, 1H), 2.34 (s, 3H), 1.38 (s, 9H).

Stage 12.

Compound 1n (450 mg, 1.81 mmol) was dissolved in phosphorus oxychloride (8.85 mL). The reaction liquid was stirred at 100 °C for 1 h. Then, the reaction liquid was cooled to room temperature and poured into saturated ammonium bicarbonate solution (300 mL). The mixture was extracted with dichloromethane (50 mL x 3) and the organic phases were combined, washed with saturated brine (20 mL x 1), dried over anhydrous sodium sulfate, filtered and concentrated to give compound 1o. MS-IRE, [M+H]+, calculated: 268, found: 268.

Stage 13.

Compound 1o (2.00 g, 7.47 mmol), 2,4-dimethoxybenzylamine (1.87 g, 11.21 mmol) and triethylamine (2.27 g, 22.4 mmol) were dissolved in anhydrous tetrahydrofuran (30 mL). The reaction liquid was stirred at 70 °C for 1 h. Then, the reaction liquid was concentrated under reduced pressure to give crude compound 1p. MS-IRE, [M+H]+, calcd: 399, found: 399.

Stage 14.

Compound 1p (3.50 g, 8.78 mmol) was dissolved in methanol (3 mL) and tetrahydrofuran (20 mL), and a solution of sodium hydroxide (703 mg, 17.6 mmol) in water (20 mL) was added to the reaction liquid. The reaction liquid was stirred at 25 °C for 0.5 h. The reaction liquid was concentrated to remove the organic solvent, and the pH of the aqueous phase was adjusted to 7 with dilute aqueous hydrochloric acid (1 M), and the mixture was extracted with dichloromethane (50 mL x 3). The organic phases were combined, washed with saturated brine (10 ml x 1), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and the crude product was separated by column chromatography (2/1, petroleum ether/ethyl acetate, Rf = 0.32) to give compound 1q. MS-IRE, [M+H]+, calculated: 315, found: 315. 1H NMR (400 MHz, CD ₃ OD) δ 7.67 (s, 1H), 7.21 (d, J=8.4 Hz, 1H), 7.07 (s, 1H), 6.56 (d, J=2.4 Hz, 1H), 6.47 (dd, J=2.4, 8.4 Hz, 1H), 4.66 (s, 2H), 3.90 (s, 3H), 3.79 (s, 3H), 2.36 (s, 3H).

Stage 15.

Compound 1t (250 mg, 795 μmol) was dissolved in H.N-dimethylformamide (4 mL) and then added

- 13 047813 compound 1q (187 mg, 875 μmol) and sodium hydroxide (63.6 mg, 1.59 mmol). The reaction liquid was stirred at 50 °C for 0.5 h. After completion of the reaction, water (50 mL) was added to the reaction liquid for dilution, and the mixed solution was extracted with ethyl acetate (30 mL x 3). The combined organic phase was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The residue was separated by column chromatography (1:1, petroleum ether/ethyl acetate, Rf = 0.1) to give compound 1r. MS-IRE, [M+H]+, calcd: 448, found: 448.

Stage 16.

Compound 1r (300 mg, 670 μmol) was dissolved in trifluoroacetic acid (3.0 mL), and the reaction liquid was stirred at 100°C for 1 h. After completion of the reaction, the reaction liquid was concentrated, and the residue was purified by high performance liquid chromatography (hydrochloric acid system) to give the hydrochloride salt of compound 1s. MS-IRE, [M+H] ⁺ , calculated: 298, found: 298.

Stage 17.

Compound 1s hydrochloride (90 mg) and compound 1f (72 mg, 241 μmol) were dissolved in anhydrous dioxane (2 mL), and then cesium carbonate (250 mg, 766 μmol) and (2-dicyclohexylphosphino-3,6-dimethoxy-2,4,6-triisopropyl-1,1-biphenyl)(2-amino-1,1-biphenyl-2-yl)palladium(II) methanesulfonate (20 mg, 21.9 μmol) were added. The reaction liquid was stirred at 105 °C for 12 h under nitrogen atmosphere. The reaction liquid was concentrated under reduced pressure, and the residue was separated and purified by column chromatography (10:1, dichloromethane/methanol, Rf = 0.3) to give the crude compound. A mixed solution of methanol and ethanol (4/1, 10 ml) was added to the crude product, and the mixture was stirred at 20°C for 16 h and filtered. The filter cake was washed with methanol (2 ml x 2) and water (2 ml x 2), and then dried to obtain the compound of formula (I). IRE-MS, [M+H] ⁺ , calculated: 514, found: 514. ¹ H NMR (400 MHz, DMSO-06) δ 10.00 (s, 1H), 8.84 (s, 1H), 8.64 (s, 1H), 8.08 (s, 1H), 7.70 (s, 1H), 4.08 (t, J=5.6 Hz, 2H), 3.00 (t, J=13.5 Hz, 2H), 2.91-2.78 (m, 6H), 2.47 (s, 3H), 2.46 (s, 3H), 2.31-2.18 (m, 2H), 2.04-1.92 (m, 2H), 1.91-1.78 (m, 2H), 1.76-1.62 (m, 2H).

Example 2. Obtaining a compound of formula (II) and growing its single crystal.

The path of synthesis.

Stage 1.

The compound of formula (I) (2 g, 3.89 μmol) and hexafluoroisopropanol (40 ml) were stirred and mixed well, and p-toluenesulfonic acid monohydrate (814.89 mg, 4.28 mmol) was added to the solution. The reaction liquid was stirred at 40°C for 3 h and added dropwise to isopropanol (160 ml). The mixed solution was filtered, and the filter cake was dried under vacuum to obtain the compound of formula (II). 1H NMR (400 MHz, DMSO-06) δ 10.02 (s, 1H), 8.86 (br s, 1H), 8.64 (s, 1H), 8.11 (s, 1H), 7.78 (s, 1H), 7.48 (d, J=8.0 Hz, 2H), 7.10 (d, J= 8.0 Hz, 2H), 4.31 (br d, J=4.4 Hz, 2H), 4.08 - 3.62 (m, 6H), 2.89 - 2.78 (m, 2H), 2.72 - 2.57 (m, 2H), 2.51 (br s, 3H), 2.46 (s, 3H), 2.28 (s, 3H), 1.95-1.97 (m, 2H), 1.89 -1.79 (m, 2H), 1.73 - 1.64 (m, 2H). MS-IRE, [M+H] ⁺ , calculated: 514, detected: 514.

An appropriate amount of the compound of formula (II) was dissolved in 1 ml of dichloromethane/methanol (1:1) at room temperature, and the sample solution was placed in a semi-closed 4 ml sample vial to allow the solvents to evaporate slowly at room temperature. A colorless massive crystal was obtained on the following day, and it was tested by single crystal X-ray diffraction method. The results of the single crystal structure analysis are shown in Fig. 7.

Example 3. Obtaining crystalline form A of the compound of formula (II).

Acetonitrile (7.0 L), compound of formula (I) (700.22 g) and para-toluenesulfonic acid monohydrate (272.25 g) were added sequentially into the reactor while maintaining the internal temperature at 20-30°C. The internal temperature of the reactor was increased to 45-55°C, and the reaction liquid was stirred for 4.5 h and filtered. The filter cake was washed with acetonitrile (0.7 L). The resulting filter cake was transferred into the reactor, acetonitrile (7.0 L) was added, then the reaction liquid was stirred at 45-55°C for 20 h and filtered. The filter cake was washed successively with acetonitrile (0.7 L) and tert-butyl methyl ether (1.4 L) and dried under vacuum and reduced pressure at a temperature of not higher than 45°C and a pressure of not more than -0.1 MPa for 17 h to obtain crystalline form A of the compound of formula (II). The results of XRPD, DSC and TGA detection are shown in Fig. 1, Fig. 2 and Fig. 3.

- 14 047813

Example 4. Screening test of the crystalline form of the compound of formula (II) in various solvents.

Separately, approximately 50 mg of the compound of formula (II) were weighed and dissolved in various solvents, and the mixtures were stirred at 50°C for 3 h. The reaction liquids were cooled to room temperature, filtered for collection and dried under vacuum, and the resulting solids were tested by XRPD. The test results are shown in Table 10 below:

Table 10

Results of stability testing of compound of formula (II) in various solvents

No. Solvent Appearance Crystal form 1 Methanol Suspension Crystal form A 2 Acetonitrile Suspension Crystal form A 3 tertButyl methyl ether Suspension Crystal form A 4 Ethanol Suspension Crystal form B 5 //-Heptane Suspension Crystal form B

Conclusion.

The compound of formula (II) forms a crystalline form A in methanol, acetonitrile and tert-butyl methyl ether, and forms a crystalline form B in ethanol and n-heptane, wherein the results of XRPD and DSC detection of the crystalline form B are successively shown in Fig. 4 and Fig. 5.

Example 5. Study of the hygroscopicity of crystalline form A of the compound of formula (II).

Materials:

device for dynamic vapor sorption SMS DVS intrinsic.

Procedures:

Crystalline form A of compound of formula (II) (10-15 mg) was placed in the DVS sample tray for testing.

Results:

The DVS profile of crystalline form A of the compound of formula (II) is shown in Fig. 6, ΔW=1.179%.

Conclusion.

Crystalline form A of the compound of formula (II) had a hygroscopic mass increase of 1.179% at 25°C and 80% RH, had relatively weak hygroscopicity, and did not change before and after the hygroscopicity test, such that crystalline form A was a stable crystalline form.

Experimental Example 6: Testing the Solid State Stability of Crystalline Form A of the Compound According to the Present Disclosure.

The crystalline form A of the compound according to the present disclosure was subjected to the accelerated ascent stability test and the prolonged ascent stability study in accordance with the rules and requirements in the Guidelines for the Stability Test of APIs and Preparations (General Principles 9001 of the Four Parts of the Chinese Pharmacopoeia, 2020 Edition), Technical Guidelines for Stability Research of Chemical Drugs (APIs and Preparations) (Revised) issued by the Center for Drug Evaluation of the National Medical Products Administration, and ICH Q1B.

1. Testing stability with long and accelerated rise.

Each sample was individually placed in a two-layer LDPE bag, with each layer of the LDPE bag being closed with a buckle. The bag was then placed in an aluminum foil bag for thermal insulation and then placed in a container to ensure stability under the conditions of observation. The results of the solid state stability testing of crystalline Form A of the compound are shown in Table 11.

Table 11

Results of solid state stability testing of crystalline form A of the compound according to the present disclosure

Testing conditions Moment in time Crystal form 25 °C/60% OB (RH) Day 0 Crystal form A 12 months Crystal form A 40°С/75% OB (RH) Day 0 Crystal form A 6 months Crystal form A

Conclusion: Crystalline form A of the compound according to the present disclosure has good stability under the conditions of long-term and accelerated rise stability testing, and crystalline form A is a stable crystalline form.

Analysis of biological activity.

Assay Example 1: In vitro evaluation of the inhibitory activity of the compound according to the present disclosure against MNK2 protein kinase.

The aim of the experiment was to test the inhibitory activity of the compound against MNK2 protein kinase.

Experimental materials: Assay buffer: 8 mM 3-(N-morpholino)propanesulfonic acid, 0.2 mM disodium ethylenediaminetetraacetic acid, 0.01% polyoxyethylene lauryl ether, 5% glycerol, 0.1% β-mercaptoethanol, and 1 mg bovine serum albumin.

Experimental procedure: Mnk2 inhibitory activity assays were performed using the KinaseProfiler™ system from Eurofins Pharma Discovery Services UK Limited. Serially diluted DMSO solutions containing the test compound (3-fold serial dilution starting from 10 μM), MNK2 (pure) and 0.33 mg/ml myelin basic protein were added to freshly prepared buffer (pH 7.0) and then mixed until homogeneous. The reaction was initiated by adding a mixture of ^33P -adenosine triphosphate ( ^33P -ATP) (radioactivity intensity: 10 μCi/μl) and 10 ^mM magnesium acetate. After the resulting mixture was reacted at room temperature for 40 min, the reaction was stopped by adding phosphoric acid to dilute to a concentration of 0.5%. 10 μl of the reaction liquid was filtered using a P30 filtermat, and then the filtermat was washed four times with 0.425% phosphoric acid for 4 min each time, followed by one wash with methanol. After drying, the radioactivity intensity was determined using the filter binding method.

The inhibitory activity of the compound on protein kinase was expressed as the percentage of residual protein kinase activity compared to the substrate in the control sample (DMSO only). The Prism4 software package (GraphPad) was used to calculate the mean inhibitory concentration (IC ₅₀ ) value and curve. Specific information is shown in Table 12 below.

Table 12

Inhibitory activity (IC ₅₀ ) of the compound according to the present disclosure against protein kinase MNK2

Connection No. IC50 (nM) against MNK2 Compound of formula (I) 17

Conclusion from the experiment: The compound of formula (I) according to the present disclosure exhibits excellent inhibitory activity against protein kinase MNK2.

Assay Example 2: In vitro evaluation of the inhibitory activity of a compound according to this

- 16 047813 further disclosure of the invention with respect to the phosphorylation of eIF4E.

The aim of the experiment was to test the inhibition (IC50) of the compound on eIF4E phosphorylation for the HCT116 cell strain.

Experimental materials: HCT116 cells (American Type Culture Collection (ATCC)), RPMI1640 medium (Life Technology), fetal bovine serum (Hyclone), dual antibodies (penicillin, streptomycin) (Millipore), phosphate buffer (Corning), 384-well cell plate (PerkinElmer), and AlphaLISA® SureFire® Ultra™ p-eIF4E (Ser209) Assay Kit (PerkinElmer).

Experimental procedure: HCT116 cells were disrupted to obtain a cell suspension, which was then seeded into a 96-well plate. The cell plate was then placed in an incubator for overnight incubation. The compound was diluted to the appropriate concentration and added to the cell plate, and the resulting mixture was incubated for 3 h. The cells were then lysed using lysis buffer, and the lysate was transferred to a 384-well plate. Fresh mixed receptor was prepared according to kit directions and added to a 384-well plate and the mixture was incubated at room temperature for 1 h. Fresh mixed donor was then prepared according to kit directions and added to a 384-well plate and the resulting mixture was incubated at room temperature for 1 h. The signal was read using EnVision with standard AlphaLISA software, the curve was fitted using GraphPad Prism and the IC50 value was calculated. Specific information is shown in Table 13 below.

Table 13 Inhibitory activity (IC50) of the compound according to the present disclosure on eIF4E phosphorylation

Connection No. IC50 (nM) against the cell strain HCT116 p-eIF4E Compound of formula (I) 8.6

Conclusion from the experiment: The compound of formula (I) according to the present disclosure exhibits excellent inhibitory activity against eIF4E phosphorylation.

Assay Example 3: Experiment for evaluating the in vivo efficacy of the compound according to the present disclosure on CT-26 transplant tumor in mice.

The aim of the experiment was to determine the in vivo efficacy of the compound against CT-26 tumor transplanted into mice.

Experimental materials: CT-26 cells, RMPI-1640 medium containing 10% fetal bovine serum, and mice (female, Shanghai Sippe-Bk Lab Animal Co., Ltd.).

Experimental procedure: CT-26 cells were cultured in RMPI-1640 medium containing 10% fetal bovine serum in an incubator at 37°C/5% CO2. Tumor cells were subcultured, and after the appropriate concentration was reached and the tumor cells were in the logarithmic growth phase, they were collected, counted, and then resuspended in Dulbecco's phosphate-buffered saline (DPBS) (phosphate buffer), and the cell suspension concentration was adjusted to ^3x106 /mL for inoculation.

Preparation of transplanted colon cancer tumor in mice: The cells were collected and adjusted to a concentration of ^3×106 cells/mL (resuspension in DPBS to prepare a cell suspension), and 0.1 mL of tumor cells were subcutaneously injected into the right upper back of mice under sterile conditions, and each mouse was inoculated with 3× ¹⁰⁵ cells. After the tumor grew to a certain size, the length (a) and width (b) of said tumor were measured using a digital caliper, and the tumor volume (TV) was calculated, and the calculation formula was as follows: TV = axb2/2.

Inoculation of CT-26 tumor cells: On the day of inoculation, the animals were divided into groups (8 animals each) according to body weight and individually administered the drug, and the day of inoculation was regarded as DO. When the tumor size reached approximately 60 ^mm3 , the animals in the antibody-administered groups were divided into groups according to tumor size and body weight. The body weight and tumor size of the animals were measured three times a week during the experiment, while the clinical symptoms of the animals were observed and recorded daily, and the body weight of the animal measured last was referenced for each administration.

The inhibitory effect of the compound on colon cancer tumor transplanted into mice was determined after 21 days of administration at a dose of 30 mg/kg once a day (QD), 90 mg/kg QD and 200 mg/kg QD. The specific information is shown in Table 14 below.

The index for assessing antitumor activity is the relative tumor growth rate.

-

Claims (20)

proliferation T/C (%); if T/C (%)>40%, this indicates the ineffectiveness of the drug, and if T/C (%)<40% and P<0.05 after statistical processing, then the drug is considered effective. The formula for calculating T/C (%) is as follows: T/S (%)=(Trtv/Crtv)x100%. T _rtv is the relative tumor volume in the treated group and C _RTV is the relative tumor volume in the negative control group; TGI (%) ^a = (1 - mean tumor volume at the end of administration in the treated group/mean tumor volume at the end of treatment in the vehicle control group) x 100%. Table 14 Antitumor efficacy of the compound according to the present disclosure in vivo in the CT-26 transplant tumor model Compound Administered dose TGI% T/C% Compound of formula (I) 30 mg/kg, OD 63.57 36.43 Compound of formula (I) 90 mg/kg, OD 68.89 31.11 Compound of formula (I) 200 mg/kg, OD 68.51 33.31 Conclusion from the experiment: The compound of formula (I) according to the present disclosure has a significant effect on inhibiting the tumor of colon cancer transplant in mice. CLAUSE OF THE INVENTION 1. Compound of formula (II) 2. Crystalline form A of the compound of formula (II), wherein the powder X-ray diffraction pattern of crystalline form A contains characteristic diffraction peaks at the following 2θ angles: 7.78±0.20°, 11.38±0.20° and 20.58±0.20°; 3. Crystalline form A according to claim 2, wherein the powder X-ray diffraction pattern of crystalline form A contains characteristic diffraction peaks at the following 2θ angles: 7.78±0.20°, 9.44±0.20°, 11.38±0.20°, 19.84±0.20°, 20.58±0.20°, 21.56±0.20°, 22.86±0.20° and 24.82±0.20°. 4. Crystalline form A according to claim 3, wherein the powder X-ray diffraction pattern of crystalline form A contains characteristic diffraction peaks at the following 2θ angles: 6.58±0.20°, 7.78±0.20°, 9.44±0.20°, 11.38±0.20°, 14.38±0.20°, 18.66±0.20°, 19.84±0.20°, 20.58±0.20°, 21.56±0.20°, 22.86±0.20°, 23.54±0.20° and 24.82±0.20°. 5. Crystalline form A according to claim 4, wherein the powder X-ray diffraction pattern of crystalline form A comprises characteristic diffraction peaks at the following 2θ angles: 4.78°, 6.58°, 7.78°, 9.44°, 11.38°, 13.48°, 14.38°, 14.80°, 16.42°, 17.00°, 17.32°, 18.34°, 18.66°, 19.08°, 19.60°, 19.84°, 20.28°, 20.58°, 21.56°, 21.84°, 22.52°, 22.86°, 23.26°, 23.54°, 24.46°, 24.82°, 25.50°, 26.04°, 26.58°, 27.42°, 27.82°, 28.07°, 28.42°, 29.08°, 29.66°, 30.08°, 31.20°, 31.42°, 38.22° and 39.04°. 6. Crystalline form A according to claim 5, wherein the powder X-ray diffraction pattern of crystalline form A is substantially as shown in Fig. 1. 7. Crystalline form A according to any one of claims 2 to 6, wherein the differential scanning calorimetry curve of crystalline form A has an endothermic peak at 287.17±3°C. 8. Crystalline form A according to claim 7, wherein the differential scanning calorimetry profile of crystalline form A is shown in Fig. 2. 9. Crystalline form A according to any one of claims 2 to 6, wherein the thermogravimetric analysis curve of crystalline form A shows a mass loss of 0.075% at 200.0±3°C. 10. Crystalline form A according to claim 9, wherein the thermogravimetric analysis profile of the crystalline - 18 047813 of the A form is shown in Fig. 3. 11. A method for obtaining crystalline form A of the compound of formula (II), comprising the following stages: (a) adding a compound of formula (II) to a solvent to form a suspension; (b) stirring the suspension at 40-55°C for 2-25 h; and (c) filtering the suspension and then drying the filter cake in vacuum at 30-45°C for 10-24 h; where the solvent is selected from methanol, acetonitrile and tert-butyl methyl ether. 12. Crystalline form B of the compound of formula (II), wherein the powder X-ray diffraction pattern of crystalline form B contains characteristic diffraction peaks at the following 2θ angles: 7.55±0.20°, 15.13±0.20° and 19.82±0.20°; Me ₀ 13. Crystalline form B according to claim 12, wherein the powder X-ray diffraction pattern of crystalline form B contains characteristic diffraction peaks at the following 2θ angles: 6.89±0.20°, 7.55±0.20°, 9.50±0.20°, 11.35±0.20°, 12.72±0.20°, 15.13±0.20°, 19.82±0.20° and 26.63±0.20°. 14. Crystalline form B according to claim 13, wherein the powder X-ray diffraction pattern of crystalline form B comprises characteristic diffraction peaks at the following 2θ angles: 6.89±0.20°, 7.55±0.20°, 9.50±0.20°, 11.35±0.20°, 12.24±0.20°, 12.72±0.20°, 15.13±0.20°, 18.94±0.20°, 19.82±0.20°, 23.25±0.20°, 26.63±0.20° and 27.27±0.20°. 15. Crystalline form B according to claim 14, wherein the powder X-ray diffraction pattern of crystalline form B comprises characteristic diffraction peaks at the following 2θ angles: 4.98°, 6.89°, 7.55°, 8.46°, 9.50°, 10.12°, 11.35°, 12.24°, 12.72°, 14.05°, 15.13°, 15.65°, 16.20°, 17.79°, 18.94°, 19.82°, 20.76°, 21.61°, 23.25°, 23.87°, 26.09°, 26.63°, 27.27°, 28.45°, 29.12°, 30.95°, 32.32°, 34.62° and 38.41°. 16. Crystalline form B according to claim 15, wherein the powder X-ray diffraction pattern of crystalline form B is substantially as shown in Fig. 4. 17. Crystalline form B according to any one of claims 12 to 16, wherein the differential scanning calorimetry curve of crystalline form B has an endothermic peak at 300.0±3°C. 18. Crystalline form B according to claim 17, wherein the differential scanning calorimetry profile of crystalline form B is shown in Fig. 5. 19. Use of a compound of formula (II) according to claim 1, crystalline form A according to any one of claims 2 to 10, crystalline form B according to any one of claims 12 to 18 and crystalline forms obtained using the method according to claim 11, for the preparation of a medicinal product that is an MNK1/2 inhibitor. 20. Use of a compound of formula (II) according to claim 1, crystalline form A according to any one of claims 2 to 10, crystalline form B according to any one of claims 12 to 18 and crystalline forms obtained using the method according to claim 11, for the preparation of a medicinal product for the treatment of colorectal cancer. -