Salt form of pyrrolotriazine compound, its crystal form and its manufacturing method

Abstract

The present invention relates to a salt form of a pyrrolotriazine compound, its crystalline form, and its preparation method and application, in particular in the preparation of an MNK1 / 2 inhibitor drug and / or in the preparation of a drug for treating colorectal cancer. [Formula 1] JPEG2024516885000024.jpg30169

Description

Detailed Description of the Invention

[0001] Priority and Related Applications The present invention claims priority from a prior application, bearing patent application number 202110501179.9 and entitled "Salt forms of pyrrolotriazine compounds, their crystalline forms and preparation methods thereof," filed with the State Intellectual Property Office of the People's Republic of China on May 8, 2021, the entire text of which is incorporated herein by reference. [Technical field] The present invention relates to a salt form of a pyrrolotriazine compound, its crystalline form, and its preparation method and application, particularly to a compound of formula (II) and its crystalline form: [Background technology] Mitogen-activated protein kinase interacting kinase (abbreviated as MNK) belongs to serine / threonine protein kinases. Human MNK has four isoforms, MNK1a, MNK1b, MNK2a, and MNK2b, expressed by the MNK1 and MNK2 genes, respectively. The four isoforms contain a nuclear localization signal (NLS) and a sequence that binds to eIF4G at the N-terminus, so they can enter the cell nucleus and play a role in recognizing and binding to downstream eIF4E. The C-terminus of isoforms MNK1a and MNK2a has a site that binds to MAPK and can be activated by phosphorylation of upstream ERK and p38. MNK1a can be widely present in the cytoplasm due to the nuclear export signal (NES) at its C-terminus, while the other three isoforms are often present in the cell nucleus.

[0002] Eukaryotic translation initiation factor 4E (abbreviated as eIF4E) is a cap-binding protein that can specifically recognize the cap structure at the 5' end of mRNA and is an important initiation factor for the protein translation process. eIF4E phosphorylated at S209 can promote the translation process of downstream proteins, mainly including c-MYC, cyclin D1, VEGF, FGF, and anti-apoptotic proteins such as mcl-1 and Bcl-2. The expression of eIF4E is upregulated in multiple types of malignant tumors, including lung cancer, colorectal cancer, gastric cancer, and pancreatic ductal cancer. MNK is the only known kinase that can phosphorylate eIF4E. It is noted that MNK is located at multiple crossover sites related to tumor and immune signaling pathways, such as RAS and T cell receptor (TCR), and can selectively control the transcription of modulators of antitumor immune responses. MNK activity and activation of eIF4E are crucial for tumor initiation and progression, but are not essential for normal cells. Therefore, selective MNK inhibitors are expected to become antitumor drugs with low toxicity. EFT508 (WO2015 / 200481, WO2016 / 172010, WO2017 / 075394, WO2017 / 075412, WO2017 / 087808, WO2017 / 117052, WO2018 / 152117, WO2018 / 218038) is a selective oral MNK inhibitor developed by EFFECTOR THERAPEUTICS, INC. Research has shown that eFT508 can selectively inhibit the expression of PD-1, LAG3, and IL-10, improve the function of cytotoxic T cells, and has no effect on the proliferation of normal T cells. Preclinical studies have shown that the combination of eFT508 with a PD-1 monoclonal antibody can enhance efficacy and increase response rates.Currently, Phase I clinical trials have been completed with favorable safety results. In addition, its use as a single agent in hematological malignancies and castration-resistant prostate cancer is both undergoing Phase II clinical studies, its combination with Avelumab monoclonal antibody in the treatment of microsatellite-stable colorectal cancer (MSS CRC) is undergoing Phase II clinical studies, and its combination with PD-1 / PD-L1 therapy in the treatment of solid tumors (patients who have experienced disease progression or have not achieved complete or partial remission on PD-1 / PD-L1 therapy alone) is undergoing Phase II clinical studies. [Summary of the Invention] The present invention provides a compound of formula (II):

[0003] [ka]

[0004] The present invention further provides a crystalline form A of the compound of formula (II), characterized in that the X-ray powder diffraction pattern has characteristic diffraction peaks at 2θ angles of 7.78±0.20°, 11.38±0.20° and 20.58±0.20°.

[0005] [ka]

[0006] In some embodiments of the invention, the X-ray powder diffraction pattern of crystalline form A has characteristic diffraction peaks at 2θ angles of 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°.

[0007] In some embodiments of the invention, the X-ray powder diffraction pattern of crystalline Form A has characteristic diffraction peaks at 2θ angles of 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°.

[0008] In some embodiments of the present invention, the X-ray powder diffraction pattern of crystalline form A is 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°, 17.42°±0.20°, 18.42°±0.20°, 19.42°±0.20°, 20.42°±0.20°, 21.42°±0.20°, 22.42°±0.20°, 23.42°±0.20°, 24.42°±0.20°, 25.42°±0.20°, 26.42°±0.20°, 27.42°±0.20°, 28.42°±0.20°, 29.42°±0.20°, 30.42°±0.20°, 31.42°±0.20°, 32.42°±0.20°, 33.42°±0.20°, 34.42°±0.20°, 35.42°±0.20°, 36.42°±0.20°, 37.42°±0.20°, 38.42°±0.20°, 39.42°±0.20°, 40.42°±0.20°, 41.42°±0.20°, 42.42°±0.20°, 43. .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° It has characteristic diffraction peaks at 2θ angles of 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°.

[0009] In some embodiments of the present invention, the X-ray powder diffraction pattern of crystalline form A is 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°, 17.42°±0.10°, 18.42°±0.10°, 19.42°±0.10°, 20.42°±0.10°, 21.42°±0.10°, 22.42°±0.10°, 23.42°±0.10°, 24.42°±0.10°, 25.42°±0.10°, 26.42°±0.10°, 27.42°±0.10°, 28.42°±0.10°, 29.42°±0.10°, 30.42°±0.10°, 31.42°±0.10°, 32.42°±0.10°, 33.42°±0.10°, 34.42°±0.10°, 35.42°±0.10°, 36.42°±0.10°, 37.42°±0.10°, 38.42°±0.10°, 39.42°±0.10°, 40.42°±0.10°, 41.42°±0.10°, 42.42°±0.10°, 43. .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° It has characteristic diffraction peaks at 2θ angles of 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°.

[0010] In some embodiments of the present invention, the X-ray powder diffraction pattern of crystalline form A is 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°, 22.08°, 23.08°, 24.08°, 25.08°, 26.08°, 27.08°, 28.08°, 29.08°, 30.08°, 31.08°, 32.08°, 33.08°, 34.08°, 35.08°, 36.08°, 37.08°, 38.08°, 39.08°, 40.08°, 41.08°, 42.08°, 43.08°, 44.08°, 45.08°, 46.08°, 47.08°, 48.08°, 49.08°, 50.08°, 51.08°, 52.08°, 53.08°, 54.08°, 55.08°, 56.08°, 57.08°, 58.08°, 59.08°, 60.08°, 61.08°, 62.08°, 63.08°, 64.08 It has characteristic diffraction peaks at 2θ angles of 1.84°, 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°.

[0011] In some embodiments of the invention, the X-ray powder diffraction pattern of crystalline form A has an average angle of 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 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 27.5 ... 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°.

[0012] In some embodiments of the invention, the X-ray powder diffraction pattern of crystalline form A has an average angle of 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 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 27.42°±0.10°, and / or 27.52°±0.10°, and / or 27.52°±0.10°, and / or 27.52°±0.10°, and / or 27.52°±0.10°, and / or 27.52°±0.10°, and / or 27.54 ... 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°.

[0013] In some embodiments of the invention, the XRPD pattern of crystalline Form A is essentially as shown in FIG.

[0014] In some embodiments of the invention, the XRPD of crystalline form A is measured using Cu-Kα radiation.

[0015] In some embodiments of the present invention, the XRPD pattern analysis data for crystalline form A is as shown in Table 1.

[0016] [Table 1]

[0017] In some embodiments of the present invention, the differential scanning calorimetry curve for crystalline form A has an endothermic peak at 287.17±3° C.

[0018] In some embodiments of the present invention, the DSC pattern of crystalline form A is as shown in FIG.

[0019] In some embodiments of the present invention, the thermogravimetric analysis curve (TGA) of said crystalline form A reaches a weight loss of 0.075% at 200.0±3° C.

[0020] In some embodiments of the present invention, the TGA pattern of crystalline form A is as shown in FIG.

[0021] The present invention relates to (a) providing a suspension of a compound of formula (II) in a solvent; (b) stirring the suspension at 40-55° C. for 2-25 hours; and (c) a step of vacuum drying at 30 to 45° C. for 10 to 24 hours after filtration, There is further provided a process for preparing the crystalline form of Compound A of formula (II), wherein said solvent is selected from methanol, acetonitrile and tert-butyl methyl ether.

[0022] The present invention provides a crystalline form B of compound of formula (II), characterized in that the X-ray powder diffraction (XRPD) pattern has characteristic diffraction peaks at 2θ angles of 7.55±0.20°, 15.13±0.20° and 19.82±0.20°.

[0023] [ka]

[0024] In some embodiments of the invention, the X-ray powder diffraction pattern of crystalline form B has characteristic diffraction peaks at 2θ angles of 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°.

[0025] In some embodiments of the invention, the X-ray powder diffraction pattern of crystalline form B has characteristic diffraction peaks at 2θ angles of 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°.

[0026] In some embodiments of the invention, the X-ray powder diffraction pattern of crystalline form B is 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°, 18.77°±0.20°, 19.77°±0.20°, 20.77°±0.20°, 21.77°±0.20°, 22.77°±0.20°, 23.77°±0.20°, 24.77°±0.20°, 25.77°±0.20°, 26.77°±0.20°, 27.77°±0.20°, 28.77°±0.20°, 29.77°±0.20°, 30.77°±0.20°, 31.77°±0.20°, 32.77°±0.20°, 33.77°±0.20°, 34.77°±0.20°, 35.77°±0.20°, 36.77°±0.20°, 37.77°±0.20°, 38.77°±0.20°, 39.77 It has characteristic diffraction peaks at 2θ angles of 8.94°±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°.

[0027] In some embodiments of the invention, the X-ray powder diffraction pattern of crystalline form B is 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°, 18.77°±0.10°, 19.78°±0.10°, 20.0°±0.10°, 21.0°±0.10°, 22.0°±0.10°, 23.0°±0.10°, 24.0°±0.10°, 25.0°±0.10°, 26.0°±0.10°, 27.0°±0.10°, 28.0°±0.10°, 29.0°±0.10°, 30.0°±0.10°, 31.0°±0.10°, 32.0°±0.10°, 33.0°±0.10°, 34.0°±0.10°, 35.0°±0.10°, 36.0°±0.10°, 37.0°±0.10°, 38.0°±0.10°, 39.0°±0.10°, 40.0°±0.10° It has characteristic diffraction peaks at 2θ angles of 8.94°±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°.

[0028] In some embodiments of the invention, the X-ray powder diffraction pattern of crystalline form B has characteristic diffraction peaks at 2θ angles of 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°.

[0029] In some embodiments of the invention, the X-ray powder diffraction pattern of crystalline form B is at 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. 8.94°±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 having characteristic diffraction peaks at 2θ angles of 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°.

[0030] In some embodiments of the invention, the X-ray powder diffraction pattern of crystalline form B is at 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. 8.94°±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 23.87°±0.10°, and / or 26.09°±0.10°, and / or 26.63°±0.10°, and / or having characteristic diffraction peaks at 2θ angles of 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°.

[0031] In some embodiments of the invention, the XRPD pattern of crystalline form B is essentially as shown in FIG.

[0032] In some embodiments of the invention, the XRPD of the B crystalline form is measured using Cu-Kα radiation.

[0033] In some embodiments of the present invention, the XRPD pattern analysis data for crystalline form B is as shown in Table 2.

[0034] [Table 2]

[0035] In some embodiments of the present invention, the differential scanning calorimetry curve for the crystalline form B has an endothermic peak at 300.0±3° C.

[0036] In some embodiments of the present invention, the DSC pattern of crystalline form B is as shown in FIG.

[0037] The present invention relates to (a) providing a suspension of a compound of formula (II) in a solvent; (b) stirring the suspension at 40-55° C. for 2-25 hours; and (c) a step of vacuum drying at 30 to 45° C. for 10 to 24 hours after filtration, There is further provided a process for preparing crystalline form B of compound of formula (II), wherein said solvent is selected from ethanol and n-heptane.

[0038] The present invention further provides the use of the compound of formula (II), the crystalline form A and the crystalline form B, or the crystalline form prepared by the method, in the preparation of a MNK1 / 2 inhibitor medicament.

[0039] The present invention further provides the use of said compound of formula (II), said crystalline form A and said crystalline form B, or the crystalline form prepared by said process, in the manufacture of a medicament for treating colorectal cancer. [Effects of the invention] The compound of formula (I) of the present invention has very high selectivity against MNK1 / 2 and has significant inhibitory activity against the kinase, and also has relatively good membrane permeability and excellent pharmacokinetic and pharmacodynamic properties.In addition, the compound of formula (II) has stable crystal forms, is less affected by light, heat and humidity, and has good efficacy in the body, and has a broad pharmaceutical prospect.

[0040] Definitions and Explanations Unless otherwise specified, the following terms and phrases used in this application are intended to have the following meanings. A particular phrase or term, unless specifically defined, should not be considered unclear or indefinite but should be understood in its ordinary sense. When a trade name is mentioned in this application, it is intended to refer to the corresponding product or its active ingredient.

[0041] The intermediate compounds according to the present invention can be prepared by a number of synthetic methods known to those skilled in the art, including the specific embodiments given below, embodiments formed by combining with other chemical synthetic methods, and equivalent alternative methods known to those skilled in the art, and preferred embodiments include, but are not limited to, the examples of the present invention.

[0042] The chemical reactions of the specific embodiments of the present invention are completed in a suitable solvent, which must be suitable for the chemical reactions of the present invention and the necessary reagents and materials thereof. To obtain the compounds of the present invention, those skilled in the art may need to modify or select synthetic steps or reaction processes based on the existing embodiments.

[0043] The structure of the compound of the present invention can be confirmed by conventional methods well known to those skilled in the art, and when the present invention relates to the absolute configuration of a compound, the absolute configuration can be confirmed by conventional technical means in this field.For example, in single crystal X-ray diffraction (SXRD), the diffraction intensity data of the cultivated single crystal is collected by Bruker D8 venture diffractometer, the light source is CuKα radiation, the scanning method is φ / ω scanning, and after collecting correlation data, the crystal structure is further analyzed by direct method (Shelxs97) to confirm the absolute configuration.

[0044] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0045] All of the solvents used in the present invention are commercially available and can be used without further purification.

[0046] Regarding the abbreviations described in this invention, DCM stands for dichloromethane, DMF stands for N,N-dimethylformamide, DMSO stands for dimethylsulfoxide, EtOH stands for ethanol, MeOH stands for methanol, 2-MeTHF stands for 2-methyltetrahydrofuran, Dioxane stands for dioxane, ACN stands for acetonitrile, Toluene stands for toluene, Acetone stands for acetone, EtOAc stands for ethyl acetate, THF stands for tetrahydrofuran, HO stands for water, and TosOH stands for p-toluenesulfonic acid.

[0047] Compounds are named according to conventional naming principles in the art or by ChemDraw® software; commercially available compounds adopt the supplier's catalogue name. [Brief description of the drawings] [Figure 1] XRPD pattern of crystalline form A of compound of formula (II). [Figure 2] DSC pattern of crystalline form A of the compound of formula (II). [Figure 3] TGA pattern of crystalline form A of compound of formula (II). [Figure 4] XRPD pattern of crystalline form B of compound of formula (II). [Figure 5] DSC pattern of crystalline form B of compound of formula (II). [Figure 6] DVS pattern of crystal form A of the compound of formula (II). [Figure 7] Single crystal ellipsoid diagram of the compound of formula (II).

[0048] Equipment parameters The measurement parameters of the X-ray powder diffractometer (XRPD) method for the crystalline form A of the present invention are shown in Table 3.

[0049] [Table 3]

[0050] Table 4 shows the measurement parameters of the differential scanning calorimeter (DSC) method for the crystalline form A of the present invention.

[0051] [Table 4]

[0052] The measurement parameters of the thermogravimetric analysis (Thermal Gravimetric Analyzer, TGA) method for the crystalline form A of the present invention are shown in Table 5.

[0053] [Table 5]

[0054] The measurement parameters of the dynamic vapor sorption analysis (DVS) method for the crystalline form A of the present invention are shown in Table 6.

[0055] [Table 6]

[0056] The moisture absorption evaluation classification is as shown in Table 7 below.

[0057] [Table 7]

[0058] The measurement parameters of the X-ray powder diffractometer (XRPD) method for the crystalline form B of the present invention are shown in Table 8.

[0059] [Table 8]

[0060] The measurement parameters of the differential scanning calorimeter (DSC) method for the crystalline form B of the present invention are shown in Table 9.

[0061] [Table 9]

[0062] Example 1: Preparation of compounds of formula (I)

[0063] [ka]

[0064] Step 1 Compound 1a (100 g, 462 mmol) was dissolved in ethanol (500 mL), concentrated sulfuric acid (49.94 g, 509 mmol, purity: 98%) was added dropwise at 0° C., and the reaction solution was stirred at 95° C. for 16 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove most of the ethanol, water (300 mL) was added to the concentrated solution, and the solution was extracted with ethyl acetate (250 mL×3). The combined organic phase was washed with saturated sodium bicarbonate (300 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 1b. 1 H 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).

[0065] Step 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 hydrogen peroxide urea (8.09 g, 86.0 mmol) were added while stirring at 0°C. The reaction solution was heated to 25°C and stirred for 16 hours. Water (200 mL) was added to the reaction solution, and the mixture was extracted with dichloromethane (100 mL x 3). The organic phases were combined, washed with saturated sodium bicarbonate (200 mL x 2) and saturated saline (500 mL) in that order, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 1c. 1 H NMR (400 MHz, CDCl3) δ 8.23 ​​(s, 1H), 7.29 (s, 1H), 4.52-4.45 (m, 2H), 2.29 (s, 3H), 1.41 (t, J=7.2 Hz, 3H).

[0066] Step 3 Compound 1c (22.0 g, 84.6 mmol) was dissolved in N,N-dimethylformamide (130 mL), trifluoroacetic anhydride (35.5 g, 169 mmol) was added while stirring at 0°C, and the reaction solution was stirred at 50°C for 1 hour. Water (200 mL) was added to the reaction solution, and it was extracted with ethyl acetate (100 mL x 4). The organic phases were combined, washed with saturated sodium bicarbonate (200 mL x 3) and saturated saline (150 mL x 3) in that order, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was stirred at room temperature for 2 hours in a mixture of petroleum ether / ethyl acetate (8 / 1, 90 mL), and filtered to obtain compound 1d. 1 H 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).

[0067] Step 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, and the reaction solution was stirred at 40° C. for 16 hours. The reaction solution was concentrated under reduced pressure, and the crude product was stirred at room temperature overnight in a mixed solution of methanol / dichloromethane (1 / 5, 48 mL). It was then filtered and washed with dichloromethane (5 mL×2), and the filter cake was concentrated under reduced pressure to obtain compound 1e. MS-ESI calculated value [M+H] + were 231 and 233, and the actual values ​​were 231 and 233.

[0068] Step 5 Compound 1e (500 mg, 1.97 mmol) and cyclopentanone (664 mg, 7.89 mmol) were dissolved in anhydrous dioxane (6 mL), concentrated sulfuric acid (98.7 mg, 0.986 mmol, purity: 98%) was added dropwise to the reaction solution, and the reaction solution was stirred at 95° C. for 3 hours. The reaction solution was concentrated under reduced pressure to remove a portion of dioxane (about 3 mL) and filtered. n-Hexane (10 mL) was added to the collected filter cake, stirred at room temperature for 2 hours, filtered, and the filter cake was dried under vacuum for 2 hours to obtain compound 1f. MS-ESI calculated value [M+H] + The values ​​were 297 and 299, and the actual values ​​were 297 and 299. 1 H NMR (400 MHz, DMSO-d6) δ 10.16 (s, 1H), 8.02 (s, 1H), 2.71-2.78 (m, 2H), 2.37 (s, 3H), 1.91-1.93 (m, 2H), 1.79-1.84 (m, 2H), 1.63-1.67 (m, 2H).

[0069] Step 6 Compound 1g (50 g, 0.442 mol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (67.3 g, 0.442 mol) were dissolved in tetrahydrofuran (500 mL). The reaction solution was heated to 55°C, and acetaldehyde (9.74 g, 0.221 mol) was added to the reaction solution at this temperature. The reaction solution was stirred at 55°C for 18 hours. The reaction solution was cooled to 22°C and quenched with acetic acid (25 mL). The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate (1000 mL) and dilute hydrochloric acid (1000 mL, 1 M). After separation, the organic phase was kept, and the aqueous phase was extracted with ethyl acetate (300 mL x 3). The organic phases were combined, washed with saturated sodium bicarbonate solution (100 mL) and brine (200 mL) in sequence, dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was separated by column chromatography (4 / 1, petroleum ether / ethyl acetate, Rf=0.56) to give compound 1h. MS-ESI calculated value [M+H] + was 226, and the actual measured value was 226. 1 H NMR (400 MHz, CDCl3) δ9.29 (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).

[0070] Step 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 solution. After stirring the reaction solution at 25° C. for 0.5 hours, a solution of compound 1i (9.78 g, 53.7 mmol) in N-methylpyrrolidone (30 mL) was added. The reaction solution was then stirred for 20 hours. The reaction solution was washed with water (200 mL), extracted with ethyl acetate (200 mL×3), the organic phase was combined, the organic phase was washed with saturated saline (20 mL×3), dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was separated by column chromatography (4 / 1, petroleum ether / ethyl acetate, Rf=0.55) to obtain compound 1j. MS-ESI calculated value [M+H] + was 241, and the actual measured value was 241. 1 H NMR (400 MHz, CDCl3) δ7.49 (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).

[0071] Step 8 Compound 1j (10.2 g, 42.5 mmol) was dissolved in carboxamide (120 mL), and phosphoric acid (832 mg, 8.49 mmol) was added to the reaction solution. The reaction solution was stirred at 125° C. for 16 hours. The reaction solution was cooled to 22° C., at which time a large amount of white solid precipitated. The mixture was filtered, and the collected filter cake was added to a mixture of petroleum ether / ethyl acetate (1 / 1, 100 mL), and the mixture was stirred at 30° C. for 0.5 hours and filtered to obtain compound 1k. MS-ESI calculated value [M+H] + was 222, and the actual measured value was 222. 1 H NMR (400 MHz, DMSO-d6) δ7.90 (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).

[0072] Step 9 Compound 1k (4.00 g, 18.0 mmol) was dissolved in anhydrous tetrahydrofuran (50 mL). Methylmagnesium bromide (30.1 mL, 90.3 mmol) was added dropwise to the reaction solution at 25°C, and the reaction was then warmed to 25°C and stirred for 15 hours. The reaction solution was quenched with saturated ammonium chloride solution (100 mL), extracted with ethyl acetate (50 mL x 5), the organic phases were combined, the organic phase was washed with saturated brine (10 mL x 1), dried over anhydrous sodium sulfate, filtered, 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 1l.

[0073] MS-ESI calculated value [M+H] + 208, and the actual measured value was 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).

[0074] Step 10 Compound 1l (1.00 g, 4.83 mmol) and hydrogen peroxide (4.64 mL, 48.26 mmol, content: 30%) were dissolved in anhydrous tetrahydrofuran (30 mL). A solution of cold methanesulfonic acid (3.44 mL, 48.26 mmol) in water (10 mL) was added dropwise to the reaction solution at 0°C. The reaction solution was stirred at 0°C for 1 hour. The reaction solution was quenched with 10% aqueous sodium sulfite solution (15 mL) until potassium iodide starch paper showed negative. The solution was extracted with ethyl acetate (50 mL x 3), the organic phases were combined, the organic phase was washed with saturated saline (10 mL x 1), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the crude product was separated by column chromatography (2 / 1, petroleum ether / ethyl acetate, Rf = 0.38) to obtain compound 1m. MS-ESI calculated value [M+H] + was 166, and the actual measured value was 166.

[0075] Step 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 solution. The reaction solution was stirred at 0°C for 1 hour, the reaction solution was washed with water (10 mL), extracted with ethyl acetate (10 mL x 5), the organic phase was combined, the organic phase was washed with saturated saline (10 mL x 1), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the crude product was separated by thin layer chromatography (2 / 1, petroleum ether / ethyl acetate, Rf = 0.63) to obtain compound 1n. MS-ESI calculated value [M + H] + was 250 and the actual measured value was 250. 1 H NMR (400 MHz, CD3OD) δ 7.61 (s, 1H), 7.47 (s, 1H), 2.34 (s, 3H), 1.38 (s, 9H).

[0076] Step 12 Compound 1n (450 mg, 1.81 mmol) was dissolved in phosphorus oxychloride (8.85 mL). The reaction solution was stirred at 100° C. for 1 hour. The reaction solution was cooled to room temperature and poured into saturated ammonium bicarbonate solution (300 mL). The mixture was extracted with dichloromethane (50 mL×3), the organic phases were combined, the organic phase was washed with saturated brine (20 mL×1), dried over anhydrous sodium sulfate, filtered, and concentrated to give compound 1o. MS-ESI calculated value [M+H] + was 268, and the actual measured value was 268.

[0077] Step 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 solution was stirred at 70° C. for 1 hour. The reaction solution was concentrated under reduced pressure to give compound 1p as a crude product. MS-ESI calculated values ​​[M+H] + was 399, and the actual measured value was 399.

[0078] Step 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 solution. The reaction solution was stirred at 25°C for 0.5 hours. The reaction solution was concentrated to remove the organic solvent, and the aqueous phase was adjusted to pH = 7 with dilute aqueous hydrochloric acid (1 M), and the mixture was extracted with dichloromethane (50 mL x 3), and the organic phases were combined, washed with saturated aqueous sodium chloride (10 mL x 1), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the crude product was separated by column chromatography (2 / 1, petroleum ether / ethyl acetate, Rf = 0.32) to obtain compound 1q. MS-ESI calculated value [M + H] + was 315, and the actual measured value was 315. 1 H NMR (400 MHz, CD3OD) 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).

[0079] Step 15 Compound 1t (250 mg, 795 μmol) was dissolved in N,N-dimethylformamide (4 mL), and then compound 1q (187 mg, 875 μmol) and sodium hydroxide (63.6 mg, 1.59 mmol) were added and stirred at 50° C. for 0.5 hours. After the reaction was completed, the reaction solution was diluted with water (50 mL) and extracted with ethyl acetate (30 mL×3). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and separated by column chromatography (1:1, petroleum ether / ethyl acetate, Rf=0.1) to obtain compound 1r. MS-ESI calculated value [M+H] + was 448, and the actual measured value was 448.

[0080] Step 16 Compound 1r (300 mg, 670 μmol) was dissolved in trifluoroacetic acid (3.0 mL), and the reaction solution was stirred at 100° C. for 1 hour. After the reaction was completed, the reaction solution was concentrated, and the residue was purified by high performance liquid chromatography (hydrochloric acid system) to obtain the hydrochloride salt of compound 1s. MS-ESI calculated value [M+H] + was 298, and the actual measured value was 298.

[0081] Step 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, and the reaction solution was stirred at 105° C. for 12 hours under nitrogen gas protection. The reaction solution was concentrated under reduced pressure, separated by column chromatography (10:1, dichloromethane / methanol, Rf=0.3) and purified to obtain the compound as a crude product. The crude product was added with a mixture of methanol and ethanol (4 / 1, 10 mL), stirred at 20°C for 16 hours, filtered, and the filter cake was washed with methanol (2 mL x 2), washed with water (2 mL x 2), and dried to obtain compound of formula (I). MS-ESI calculated value [M+H] + was 514, and the actual measured value was 514. 1 H NMR (400 MHz, DMSO-d6) 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).

[0082] Example 2: Preparation of the compound of formula (II) and cultivation of its single crystal

[0083] [ka]

[0084] The compound of formula (I) (2 g, 3.89 μmol) was stirred with hexafluoroisopropanol (40 mL) to mix uniformly, p-toluenesulfonic acid monohydrate (814.89 mg, 4.28 mmol) was added to the solution, the reaction solution was stirred at 40° C. for 3 hours, the reaction solution was added dropwise to isopropanol (160 mL), filtered, and the filter cake was dried in vacuum to obtain the compound of formula (II). 1 H NMR (400 MHz, DMSO-d6) 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-ESI calculated value [M+H] + was 514, and the actual measured value was 514.

[0085] 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 4 mL semi-sealed sample bottle and allowed to slowly evaporate at room temperature. The next day, colorless bulk crystals were obtained and subjected to X-ray single crystal diffraction measurement. The results of the single crystal structure analysis are shown in Figure 7.

[0086] Example 3: Preparation of Crystalline Form A of Compound of Formula (II) The internal temperature was controlled at 20-30°C, and acetonitrile (7.0 L), the compound of formula (I) (700.22 g), and p-toluenesulfonic acid monohydrate (272.25 g) were added to the reaction vessel in that order, and the internal temperature of the reaction vessel was raised to 45-55°C and stirred for 4.5 hours. The mixture was filtered, and the filter cake was rinsed with acetonitrile (0.7 L). The obtained filter cake was transferred to the reaction vessel, and acetonitrile (7.0 L) was added, followed by stirring at 45-55°C for 20 hours. The mixture was filtered, and the filter cake was rinsed with acetonitrile (0.7 L) and tert-butyl methyl ether (1.4 L) in that order, and then vacuum-dried at a temperature of 45°C or less and a pressure of ≦-0.1 MPa for 17 hours to obtain crystalline form A of the compound of formula (II). The XRPD, DSC, and TGA detection results are shown in Figures 1, 2, and 3.

[0087] Example 4: Screening test of crystalline forms of compound of formula (II) in different solvents About 50 mg of compound of formula (II) was weighed into different solvents and stirred at 50°C for 3 hours, the reaction solution was cooled to room temperature, filtered, collected, and dried in vacuum to obtain solids, and XRPD measurement was performed, and the experimental results are shown in Table 10 below.

[0088] [Table 10]

[0089] Conclusion: The compound of formula (II) forms crystalline form A in methanol, acetonitrile and tert-butyl methyl ether, and forms crystalline form B in ethanol and n-heptane. The XRPD and DSC detection results of crystalline form B are shown in Figure 4 and Figure 5, respectively.

[0090] Example 5: Hygroscopicity study of crystalline form A of compound of formula (II) Test materials: SMS DVS intrinsic dynamic gas adsorption apparatus Testing Method: 10 to 15 mg of crystalline form A of the compound of formula (II) was placed in a DVS sample pan and subjected to measurement.

[0091] Test Results: The DVS pattern of crystalline form A of the compound of formula (II) is shown in FIG. 6, and ΔW=1.179%.

[0092] Test conclusion: The hygroscopic weight gain of the compound of formula (II) crystal form A at 25°C and 80% RH was 1.179%, and the hygroscopicity was relatively weak. Furthermore, the crystal form did not change before and after hygroscopicity, and therefore the crystal form was stable.

[0093] Experimental Example 6: Solid-state stability test study of crystalline form A of the compound according to the present invention For the crystalline form A of the compound of the present invention, accelerated release stability tests and long-term release stability studies were conducted with reference to the Chinese Pharmacopoeia 2020 Edition, Part 4, 9001 "Guideline for Stability Testing of Drug Substances and Preparations" and the "Technical Guideline for Stability Study of Chemical Drugs (Drug Substances and Preparations) (Revised)" issued by the Drug Evaluation Center of the China Food and Drug Administration, as well as the requirements stipulated by ICH Q1B.

[0094] 1.Long-term accelerated release stability test: Each sample was placed in a two-layer LDPE bag, each layer of the LDPE bag was sealed with a belt, and then placed in an aluminum foil bag and heat-sealed, and then placed in a stability box under the corresponding conditions for consideration. The results of the stability test of the crystalline form A of the compound are shown in Table 11.

[0095] [Table 11]

[0096] Conclusion: Crystalline form A of the compound of the present invention has good stability under long-term accelerated release stability test conditions, and crystalline form A belongs to stable crystal forms.

[0097] Biological activity measurement Measurement Example 1: In vitro evaluation of the inhibitory activity of the compound according to the present invention against MNK2 protein kinase Experimental objective: To detect the MNK2 protein kinase inhibitory activity of compounds Materials: Assay buffer solution: 8 mM 3-(N-morpholino)propanesulfonic acid, 0.2 mM disodium ethylenediaminetetraacetate, 0.01% polyoxyethylene lauryl ether, 5% glycerin, 0.1% β-mercaptoethanol, 1 mg bovine serum albumin. Experimental Procedure: The Mnk2 protein kinase inhibitory activity was measured using the KinaseProfilerTM service from Eurofins Pharma Discovery Services UK Limited. Serial DMSO dilutions (3-fold serial dilutions starting from 10 μM) containing the compounds to be tested, MNK2(h) protein kinase and 0.33 mg / mL myelin basic protein were added to freshly prepared buffer solution (pH 7.0) and mixed uniformly. 33 The reaction was started by adding a mixture of P-ATP (radiation intensity 10 μCi / μL) and 10 mM magnesium acetate, and was allowed to react at room temperature for 40 minutes. The reaction was stopped by adding phosphoric acid to dilute to a concentration of 0.5%. 10 μL of the reaction solution was filtered through a P30 filtermat, washed four times with 0.425% phosphoric acid for 4 minutes each time, washed once with methanol, and dried, and then the radiation intensity was detected by the filter-binding method.

[0098] The protein kinase inhibitory activity of the compounds is expressed as the percentage of protein kinase activity remaining relative to the blank primer (pure DMSO). IC 50 The values ​​and curves were calculated using Prism4 software packet (GraphPad), and the specific information is shown in Table 12 below.

[0099] [Table 12]

[0100] Experimental Conclusion: The compounds of formula (I) of the present invention exhibited excellent inhibitory activity against MNK2 protein kinase.

[0101] Measurement Example 2: In vitro evaluation of the inhibitory activity of the compounds according to the present invention against eIF4E phosphorylation Experimental objective: Inhibitory IC of compounds on eIF4E phosphorylation in HCT116 cell line 50 To detect a value.

[0102] Experimental materials: HCT116 cells (ATCC), RPM11640 medium (Life technologies), fetal bovine serum (Hyclone), dual antibiotics (penicillin, streptomycin) (Millipore), phosphate buffer solution (Corning), 384-well cell plates (PerkinElmer), AlphaLISA® SureFire® Ultra TM p-eIF4E(Ser209) Assay Kit(PerkinElmer) Experimental procedure: HCT116 cells were digested to prepare cell suspension and plated in a 96-well plate. The cell plate was then placed in an incubator and cultured overnight. Compounds were diluted to the appropriate concentration and added to the cell culture plate, followed by 3 hours of culture. Then, cells were lysed using lysis buffer, and the lysate was transferred to a 384-well plate. Mixed receptors were freshly prepared according to the instructions in the reagent kit and added to a 384-well plate and incubated at room temperature for 1 hour. Mixed feed was freshly prepared according to the instructions in the reagent kit and added to a 384-well plate and incubated at room temperature for 1 hour. The signals were read using the standard AlphaLISA program in EnVision, and curve fitting was performed using Graphpad prism, and IC 50 The specific information is shown in Table 13 below.

[0103] [Table 13]

[0104] Experimental Conclusion: The compound of formula (I) of the present invention showed excellent inhibitory activity against eIF4E phosphorylation.

[0105] Measurement Example 3: In vivo efficacy measurement of the compound according to the present invention in CT-26 mouse transplanted tumors Aim of the experiment: To determine the in vivo efficacy of the compound in CT-26 mouse tumor xenografts. Experimental materials: CT-26 cells, RMPI-1640 medium containing 10% fetal bovine serum, mice (female, Shanghai Bikai Laboratory Animal Co., Ltd.) Experimental procedure: CT-26 cells were cultured in RMPI-1640 medium containing 10% fetal bovine serum and incubated in a 5% CO2, 37°C incubator. When the tumor cells were grown to an appropriate concentration by subculture and were in logarithmic growth phase, the tumor cells were harvested, counted and resuspended in DPBS (phosphate buffered saline) to a concentration of 3 × 10 6 / mL and used for inoculation.

[0106] Establishment of mouse colon cancer transplant tumors: Cells were harvested and the concentration was adjusted to 3 × 10 6 The cells / milliliter (resuspended in DPBS) were adjusted and, under sterile conditions, 0.1 mL of tumor cells were injected subcutaneously into the right dorsal region of the mice, with the number of cells inoculated into each mouse being 3 × 10 5 After the tumor had grown to a certain size, the length (a) and width (b) of the tumor were measured with a digital caliper, and the tumor volume was calculated. The tumor volume (TV) was calculated as TV=a×b. 2 / 2.

[0107] Inoculation of CT-26 tumor cells: On the day of inoculation, animals were divided into groups according to their body weight, with 8 animals per group, and the day of inoculation was designated as D0. 3 When the tumors had grown to a certain extent, the antibody groups were divided according to the tumor size and body weight. During the experiment, the body weight and tumor size of the animals were measured three times a week, and the clinical symptoms of the animals were observed and recorded every day. The weight of the animals weighed the closest time was used as the basis for each administration. The inhibitory effect of the compounds on mouse colon cancer transplanted tumors was measured after 21 days of administration at doses of 30 mg / Kg QD (once a day), 90 mg / Kg QD (once a day), and 200 mg / Kg QD (once a day), and the specific information is shown in Table 14 below.

[0108] The evaluation index of antitumor activity was the relative tumor proliferation rate (T / C) (%), where T / C (%)>40% indicated ineffectiveness, and T / C (%)≦40% and P<0.05 indicated efficacy in statistical analysis. The formula for T / C (%) was T / C (%)=(T RTV / C RTV ) × 100%. T RTV is the relative tumor volume of the treatment group, and C RTV is the relative tumor volume in the negative control group, and TGI (%) a = (1-mean tumor volume at the end of treatment for the treatment group / tumor volume at the end of treatment for the vehicle control group) x 100%.

[0109] [Table 14]

[0110] Experimental conclusion: The compound of formula (I) of the present invention has significant inhibitory effect on mouse colon cancer xenograft tumors. [Brief description of the drawings]

[0111] [Figure 1] 1 is an XRPD pattern of crystalline form A of compound of formula (II). [Diagram 2] 1 is a DSC pattern of crystalline form A of compound of formula (II). [Diagram 3] 1 is a TGA pattern of crystalline form A of compound of formula (II). [Figure 4] FIG. 2 is an XRPD pattern of crystalline form B of compound of formula (II). [Diagram 5] 1 is a DSC pattern of crystalline form B of compound of formula (II). [Figure 6] 1 is a DVS pattern of crystalline form A of compound of formula (II). [Figure 7] FIG. 2 is a single crystal ellipsoid diagram of the compound of formula (II).

Claims

1. A compound of formula (II). 【Chemistry 1】

2. The X-ray powder diffraction pattern has characteristic diffraction peaks at 2θ angles of 7.78±0.20°, 11.38±0.20° and 20.58±0.20°. or an X-ray powder diffraction pattern having characteristic diffraction peaks at 2θ angles of 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°; or an X-ray powder diffraction pattern having characteristic diffraction peaks at 2θ angles of 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°; Or, the X-ray powder diffraction pattern is 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. having characteristic diffraction peaks at 2θ angles of 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°; Or, the XRPD pattern is as shown below: [Figure 1] 【change】 Crystal A of the compound of formula (II). 【Chemistry 2】

3. The differential scanning calorimetry curve has an endothermic peak at 287.17±3°C. Crystal A according to claim 2.

4. The DSC pattern is as shown below: Crystal A according to claim 3. [Figure 2]

5. Thermogravimetric analysis curve reaches weight loss of 0.075% at 200.0±3℃. Crystal A according to claim 2.

6. The TGA pattern is as shown below: Crystal A according to claim 5. FIG. 3

7. (a) providing a suspension of a compound of formula (II) in a solvent; (b) stirring the suspension at 40-55° C. for 2-25 hours; and (c) vacuum drying at 30-45° C. for 10-24 hours after filtration, The solvent is selected from methanol, acetonitrile and tert-butyl methyl ether; A method for producing crystalline form A of the compound of formula (II).

8. The X-ray powder diffraction pattern has characteristic diffraction peaks at 2θ angles of 7.55±0.20°, 15.13±0.20°, and 19.82±0.20°. or an X-ray powder diffraction pattern having characteristic diffraction peaks at 2θ angles of 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°; or an X-ray powder diffraction pattern having characteristic diffraction peaks at 2θ angles of 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°; or an X-ray powder diffraction pattern having characteristic diffraction peaks at 2θ angles of 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°; Or, the XRPD pattern is as shown below: FIG. 4 【change】 Crystal B of the compound of formula (II). 【Chemistry 3】

9. The differential scanning calorimeter curve has an endothermic peak at 300.0±3°C. The B crystal according to claim 8.

10. The DSC pattern is as shown below: The B crystal according to claim 9. FIG. 5

11. Use of the compound of formula (II) according to claim 1, crystalline A according to any one of claims 2 to 6, and crystalline B according to any one of claims 8 to 10 in the manufacture of an MNK1 / 2 inhibitor drug.

12. Use of the compound of formula (II) according to claim 1, crystalline A according to any one of claims 2 to 6, and crystalline B according to any one of claims 8 to 10 in the manufacture of a medicament for treating colorectal cancer.

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