Pyrolotriazine salts, crystalline compounds, and methods for preparing these compounds.

Abstract

The invention relates to the salt form of a pyrolotriazine compound, the crystalline form of the compound, methods of their preparation, and specifically to their application in the formulation of MNK1 / 2 inhibitors and / or in the formulation of drugs for the treatment of colorectal cancer.

Description

Salt form of pyrrolotriazine compound, its crystal form and preparation method thereof

[0001] Priority and related applications

[0002] This application claims priority to a prior application, patent application number 202110501179.9, filed with the State Intellectual Property Office of China on May 8, 2021, entitled “Salt forms of pyrrolotriazine compounds, crystal forms thereof, and methods for their preparation.” The entire text of that prior application is incorporated herein by reference. Technical Field

[0003] The present invention relates to salt forms of pyrrolotriazine compounds, their crystal forms, preparation methods and applications thereof, and particularly to compounds of formula (II) and their crystal forms. Background Art

[0004] Mitogen-activated protein kinase interacting kinases (MNKs) are serine / threonine protein kinases. Human MNKs have four subtypes: MNK1a, MNK1b, MNK2a, and MNK2b, expressed by the MNK1 and MNK2 genes, respectively. All four subtypes contain a nuclear localization signal (NLS) and an eIF4G binding sequence at their N-termini, enabling them to enter the nucleus and recognize and bind to downstream eIF4E. MNK1a and MNK2a subtypes possess MAPK binding sites at their C-termini, allowing them to be activated by upstream ERK and p38 phosphorylation. The nuclear export signal (NES) at the C-terminus of MNK1a allows it to be widely present in the cytoplasm, while the other three subtypes are primarily located in the nucleus.

[0005] Eukaryotic initiation factor 4E (eIF4E) is a cap-binding protein that specifically recognizes the cap structure at the 5' end of mRNA and is a crucial initiation factor for protein translation. Phosphorylated eIF4E at S209 promotes the translation of downstream proteins, including c-MYC, cyclin D1, VEGF, FGF, and anti-apoptotic proteins such as Mcl-1 and Bcl-2. eIF4E expression is upregulated in various malignancies, including lung cancer, colorectal cancer, gastric cancer, and pancreatic ductal carcinoma. MNK is the only known kinase that can phosphorylate eIF4E. Furthermore, MNK is located at the intersection of multiple tumor and immune signaling pathways, such as RAS and the T cell receptor (TCR), and can selectively control the transcription of regulators of the anti-tumor immune response. MNK activity and eIF4E activation are crucial for tumor development and progression but are not essential for normal cells. Therefore, selective MNK inhibitors hold promise as low-toxic anti-tumor drugs. 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. Studies have shown that eFT508 can selectively inhibit the expression of PD-1, LAG3, and IL-10, enhancing the function of cytotoxic T cells while leaving normal T cell proliferation unaffected. Preclinical studies have shown that combining eFT508 with a PD-1 monoclonal antibody can enhance efficacy and improve response rates. Phase I clinical trials have been completed with good safety. Currently, its monotherapy use in hematological tumors and prostate cancer is in Phase II clinical trials, its combination with avelumab monoclonal antibody for the treatment of microsatellite stable colorectal cancer (MSS CRC) is in Phase II clinical trials, and its combination with PD-1 / PD-L1 therapy (for patients who have experienced disease progression or have not achieved complete remission or partial remission with single PD-1 / PD-L1 therapy) for the treatment of solid tumors is in Phase II clinical trials.

[0006] Summary of the Invention

[0007] The present invention provides a compound of formula (II),

[0008]

[0009] The present invention also provides a crystalline form A of the compound of formula (II), characterized in that its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 7.78±0.20°, 11.38±0.20° and 20.58±0.20°;

[0010]

[0011] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A 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°.

[0012] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A 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°.

[0013] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A 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°.

[0014] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A 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°.

[0015] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A 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°.

[0016] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A 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 27.4 2°±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°.

[0017] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A 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 27.4 2°±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°.

[0018] In some embodiments of the present invention, the XRPD pattern of the above-mentioned crystal form A is basically as shown in Figure 1.

[0019] In some embodiments of the present invention, the XRPD of the above-mentioned Form A is measured using Cu-Kα radiation.

[0020] In some embodiments of the present invention, the XRPD pattern analysis data of the above-mentioned Form A is shown in Table 1.

[0021] Table 1 XRPD analysis data of the crystal form A of compound of formula (II)

[0022]

[0023] In some embodiments of the present invention, the differential scanning calorimetry curve of the above-mentioned crystal form A has an endothermic peak at 287.17±3°C.

[0024] In some embodiments of the present invention, the DSC spectrum of the above-mentioned crystal form A is shown in Figure 2.

[0025] In some embodiments of the present invention, the thermogravimetric analysis (TGA) curve of the above-mentioned crystal form A shows a weight loss of 0.075% at 200.0±3°C.

[0026] In some embodiments of the present invention, the TGA spectrum of the above-mentioned crystal form A is shown in Figure 3.

[0027] The present invention also provides a method for preparing the crystalline form A of compound of formula (II), comprising the following steps:

[0028] (a) adding the compound of formula (II) into a solvent to form a suspension;

[0029] (b) stirring the suspension at 40-55° C. for 2-25 hours;

[0030] (c) filtering and vacuum drying at 30-45° C. for 10-24 hours;

[0031] Wherein, the solvent is selected from methanol, acetonitrile and tert-butyl methyl ether.

[0032] The present invention provides a crystal form B of a compound of formula (II), characterized in that its X-ray powder diffraction (XRPD) pattern has characteristic diffraction peaks at the following 2θ angles: 7.55±0.20°, 15.13±0.20° and 19.82±0.20°;

[0033]

[0034] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned B crystal form has characteristic diffraction peaks at the following 2θ angles: 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°.

[0035] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned B crystal form has characteristic diffraction peaks at the following 2θ angles: 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°.

[0036] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form B 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°, 1 7.79°±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°.

[0037] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form B 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. 7.79°±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°.

[0038] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned B crystal form has characteristic diffraction peaks at the following 2θ angles: 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°.

[0039] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form B 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 17.7 9°±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°.

[0040] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form B 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 17.7 9°±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 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°.

[0041] In some embodiments of the present invention, the XRPD pattern of the above-mentioned Form B is substantially as shown in FIG4 .

[0042] In some embodiments of the present invention, the XRPD of the above-mentioned Form B is measured using Cu-Kα radiation.

[0043] In some embodiments of the present invention, the XRPD pattern analysis data of the above-mentioned Form B is shown in Table 2.

[0044] Table 2 XRPD analysis data of the crystal form B of compound of formula (II)

[0045]

[0046] In some embodiments of the present invention, the differential scanning calorimetry curve of the above-mentioned Form B has an endothermic peak at 300.0±3°C.

[0047] In some embodiments of the present invention, the DSC spectrum of the above-mentioned Form B is shown in Figure 5.

[0048] The present invention also provides a method for preparing the crystalline form B of compound of formula (II), comprising the following steps:

[0049] (a) adding the compound of formula (II) into a solvent to form a suspension;

[0050] (b) stirring the suspension at 40-55° C. for 2-25 hours;

[0051] (c) filtering and vacuum drying at 30-45° C. for 10-24 hours;

[0052] Wherein, the solvent is selected from ethanol and n-heptane.

[0053] The present invention also provides use of the compound of formula (II), the crystal form A and the crystal form B, or the crystal forms prepared according to the above method in the preparation of MNK1 / 2 inhibitor drugs.

[0054] The present invention also provides the use of the compound of formula (II), the crystal form A and the crystal form B, or the crystal forms prepared according to the above method in the preparation of drugs for treating colorectal cancer.

[0055] Technical Effects

[0056] The compound of formula (I) of the present invention has high selectivity for MNK1 / 2 and significant inhibitory activity against this kinase. It also has good membrane permeability and excellent pharmacokinetic and pharmacodynamic properties. Furthermore, the compound of formula (II) is stable in all its crystalline forms, is minimally affected by light, heat, and humidity, and exhibits good in vivo efficacy, suggesting broad prospects for drug development.

[0057] Definition and Description

[0058] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular phrase or term should not be construed as ambiguous or unclear unless specifically defined, but rather should be understood in accordance with its ordinary meaning. When a trade name appears herein, it is intended to refer to the corresponding commercial product or its active ingredient.

[0059] The intermediate compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent replacement methods well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention.

[0060] The chemical reactions of the present invention are carried out in suitable solvents that are compatible with the chemical transformations of the present invention and the reagents and materials required. To obtain the compounds of the present invention, it may sometimes be necessary for those skilled in the art to modify or select synthetic steps or reaction schemes based on existing embodiments.

[0061] The structures of the compounds of the present invention can be confirmed by conventional methods well known to those skilled in the art. If the present invention relates to the absolute configuration of the compounds, the absolute configuration can be confirmed by conventional techniques in the art. For example, single crystal X-ray diffraction (SXRD) is used to collect diffraction intensity data on the cultured single crystal using a Bruker D8 venture diffractometer, using CuKα radiation as the light source and scanning mode: After scanning and collecting relevant data, the crystal structure is further analyzed using the direct method (Shelxs97) to confirm the absolute configuration.

[0062] The present invention will be described in detail below through examples, which are not intended to limit the present invention in any way.

[0063] All solvents used in the present invention were commercially available and used without further purification.

[0064] The present invention uses the following abbreviations: DCM represents dichloromethane; DMF represents N,N-dimethylformamide; DMSO represents dimethyl sulfoxide; EtOH represents ethanol; MeOH represents methanol; 2-MeTHF represents 2-methyltetrahydrofuran; Dioxane represents dioxane; ACN represents acetonitrile; Toluene represents toluene; Acetone represents acetone; EtOAc represents ethyl acetate; THF represents tetrahydrofuran; H2O represents water; TosOH represents p-toluenesulfonic acid.

[0065] Compounds are named according to the conventional nomenclature in the art or using Software naming, commercially available compounds use supplier catalog names. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1: XRPD pattern of Form A of the compound of formula (II);

[0067] Figure 2: DSC spectrum of Form A of the compound of formula (II);

[0068] Figure 3: TGA spectrum of Form A of the compound of formula (II);

[0069] Figure 4: XRPD pattern of Form B of the compound of formula (II);

[0070] Figure 5: DSC spectrum of Form B of the compound of formula (II);

[0071] Figure 6: DVS spectrum of Form A of the compound of formula (II);

[0072] Figure 7: Single crystal ellipsoid diagram of the compound of formula (II).

[0073] Instrument parameters

[0074] The X-ray powder diffractometer (XRPD) method of the present invention's crystal form A has test parameters shown in Table 3.

[0075] Table 3 XRPD test parameters of Form A

[0076]

[0077] The differential scanning calorimeter (DSC) method of the present invention's crystal form A has test parameters shown in Table 4.

[0078] Table 4 DSC test parameters of Form A

[0079]

[0080]

[0081] Thermogravimetric analysis (TGA) method of Form A of the present invention, and its test parameters are shown in Table 5.

[0082] Table 5 TGA test parameters of Form A

[0083]

[0084] The dynamic vapor sorption analysis (DVS) method of the present invention's Form A, and its test parameters are shown in Table 6.

[0085] Table 6 DVS test parameters of Form A

[0086] Instrument Manufacturer Model SMS / DVS Intrinsic Test Conditions Weigh approximately 10 mg of sample and test Temperature: 25°C Equilibrium dm / dt: 0.01% / min Drying: 25°C, 0% RH Drying for 2 h RH (%) Test Step: 5% RH RH (%) Test Step Range: 0% to 95% to 0% RH

[0087] The classification of moisture absorption evaluation is shown in Table 7 below:

[0088] Table 7 Moisture absorption evaluation classification table

[0089] Hygroscopicity classification ΔW% deliquesces and absorbs sufficient water to form a liquid. Very hygroscopic ΔW% ≥ 15% Hygroscopic 15% > ΔW% ≥ 2% Slightly hygroscopic 2% > ΔW% ≥ 0.2% No or almost no hygroscopic ΔW% < 0.2%

[0090] Note: ΔW% indicates the weight gain of the test sample at 25±1℃ and 80±2%RH.

[0091] The X-ray powder diffractometer (XRPD) method of the present invention's Form B has test parameters shown in Table 8.

[0092] Table 8 XRPD test parameters of Form B

[0093]

[0094] The test parameters of the differential scanning calorimeter (DSC) method for Form B of the present invention are shown in Table 9.

[0095] Table 9 DSC test parameters of Form B

[0096]

[0097] Example 1: Preparation of compound of formula (I)

[0098] Synthesis route:

[0099]

[0100] first step

[0101] 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. The reaction solution was stirred at 95°C for 16 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure to remove most of the ethanol. Water (300 mL) was added to the concentrate, and the solution was extracted with ethyl acetate (250 mL x 3). The combined organic phases were 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 (400MHz, CDCl3) δ8.60 (s, 1H), 7.78 (s, 1H), 4.48-4.42 (m, 2H), 2.58 (s, 3H), 1.43 (t, J = 7.2Hz, 3H).

[0102] Step 2

[0103] Compound 1b (10.0 g, 41.0 mmol) was dissolved in dichloromethane (200 mL). Trifluoroacetic anhydride (17.2 g, 81.9 mmol) and urea hydrogen peroxide (8.09 g, 86.0 mmol) were added with 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 × 3). The organic phases were combined and washed sequentially with saturated sodium bicarbonate (200 mL × 2) and saturated brine (500 mL). The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 1c. 1 H NMR (400MHz, CDCl3) δ8.23 (s, 1H), 7.29 (s, 1H), 4.52-4.45 (m, 2H), 2.29 (s, 3H), 1.41 (t, J = 7.2Hz, 3H).

[0104] Step 3

[0105] 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 with stirring at 0°C. The reaction mixture was stirred at 50°C for 1 hour. Water (200 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (100 mL x 4). The organic phases were combined and washed sequentially with saturated sodium bicarbonate (200 mL x 3) and saturated brine (150 mL x 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was stirred in a mixture of petroleum ether / ethyl acetate (8 / 1, 90 mL) at room temperature for 2 hours and filtered to obtain compound 1d. 1 H NMR (400MHz, CDCl3) δ9.89 (br s, 1H), 7.75 (s, 1H), 4.46-4.41 (m, 2H), 2.44 (s, 3H), 1.43 (t, J = 7.2Hz, 3H).

[0106] Step 4

[0107] Compound 1d (2.00 g, 7.69 mmol) was dissolved in ethanol (20 mL), and aqueous ammonia (16.2 g, 115 mmol, 25% purity) was added. The reaction mixture was stirred at 40°C for 16 hours. The reaction mixture was concentrated under reduced pressure, and the crude product was stirred in a methanol / dichloromethane mixture (1 / 5, 48 mL) at room temperature overnight. The mixture was then filtered, washed with dichloromethane (5 mL x 2), and the filter cake was concentrated under reduced pressure to yield compound 1e. MS-ESI calculated value [M+H] + 231 and 233, measured values ​​231 and 233.

[0108] Step 5

[0109] 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 some dioxane (approximately 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 vacuum dried for 2 hours to obtain compound 1f. MS-ESI calculated value [M+H] + 297 and 299, measured values ​​297 and 299. 1 H NMR (400MHz, DMSO-d6) δ10.16(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).

[0110] Step 6

[0111] Dissolve compound 1g (50g, 0.442mol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (67.3g, 0.442mol) in tetrahydrofuran (500mL). Heat the reaction mixture to 55°C. At this temperature, add acetaldehyde (9.74g, 0.221mol). Stir the reaction mixture at 55°C for 18 hours. Cool the reaction mixture to 22°C and quench with acetic acid (25mL). 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 retained and the aqueous phase was extracted with ethyl acetate (300 mL × 3). The organic phases were combined and washed sequentially with saturated sodium bicarbonate solution (100 mL) and brine (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated by column chromatography (4 / 1, petroleum ether / ethyl acetate, Rf = 0.56) to obtain compound 1h. MS-ESI calculated value [M+H] + 226, measured value 226. 1 H NMR(400MHz, CDCl3)δ9.29(br s,1H),7.48(d,J=3.2Hz,1H),4.35(q,J=7.2Hz,2H),4.29(q,J=7.2Hz,2H),2.61(s,3H),1.38(t,J=7.2Hz,3H),1.35(m,J=7.2Hz,3H).

[0112] Step 7

[0113] 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 mixture. After stirring the reaction mixture at 25°C for 0.5 hour, a solution of compound 1i (9.78 g, 53.7 mmol) in N-methylpyrrolidone (30 mL) was added. The reaction mixture was stirred for 20 hours. The reaction mixture was washed with water (200 mL) and extracted with ethyl acetate (200 mL × 3). The organic phases were combined, washed with saturated brine (20 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated. 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] + 241, measured value 241. 1 H NMR (400MHz, CDCl3) δ7.49 (s, 1H), 4.35 (q, J = 7.2Hz, 2H), 4.27 (q, J = 7.2Hz, 2H), 2.57 (s, 3H), 1.40 (t, J = 7.2Hz, 3H), 1.34 (t, J = 7.2Hz, 3H).

[0114] Step 8

[0115] Compound 1j (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 mixture. The reaction mixture was stirred at 125°C for 16 hours. The reaction mixture was cooled to 22°C, at which point a large amount of white solid precipitated. The mixture was filtered, and the collected filter cake was added to a petroleum ether / ethyl acetate mixture (1 / 1, 100 mL). The mixture was stirred at 30°C for 0.5 hours and filtered to obtain compound 1k. MS-ESI calculated value [M+H] + 222, measured value 222. 1 H NMR (400MHz, DMSO-d6) δ7.90 (s, 1H), 7.84 (s, 1H), 4.23 (q, J = 7.2Hz, 2H), 2.61 (s, 3H), 1.28 (t, J = 7.2Hz, 3H).

[0116] Step 9

[0117] 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 mixture at 25°C. After completion of the addition, the reaction mixture was warmed to 25°C and stirred for 15 hours. The reaction mixture 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. The crude product was separated by thin-layer chromatography (2 / 1, petroleum ether / ethyl acetate, Rf = 0.39) to obtain compound 1l. MS-ESI calculated value [M+H] + 208, measured value 208. 1 H NMR (400MHz, CDCl3) δ7.30(br s,1H),7.24(br s,1H),2.59(s,3H),1.54(s,6H).

[0118] Step 10

[0119] Compound 1l (1.00 g, 4.83 mmol) and hydrogen peroxide (4.64 ml, 48.26 mmol, 30% content) 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 mixture at 0°C. The reaction mixture was stirred at 0°C for 1 hour. The reaction mixture was quenched with 10% aqueous sodium sulfite solution (15 mL) until the starch potassium iodide test paper showed a negative result. The solution was extracted with ethyl acetate (50 mL × 3). The organic phases were combined and washed with saturated brine (10 mL × 1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. 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] + 166, measured value 166.

[0120] Step 11

[0121] Compound 1m (400 mg, 2.42 mmol) was dissolved in anhydrous tetrahydrofuran (10 mL). 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, 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. 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] +250, actual measured value 250. 1 H NMR (400MHz, CD3OD) δ7.61(s,1H),7.47(s,1H),2.34(s,3H),1.38(s,9H).

[0122] Step 12

[0123] Compound 1n (450 mg, 1.81 mmol) was dissolved in phosphorus oxychloride (8.85 mL). The reaction mixture was stirred at 100°C for 1 hour. The reaction mixture 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, washed with saturated brine (20 mL × 1), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 1o. MS-ESI calculated value [M+H] + 268, measured value 268.

[0124] Step 13

[0125] 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 mixture was stirred at 70°C for 1 hour. The reaction mixture was concentrated under reduced pressure to obtain crude compound 1p. MS-ESI calculated value [M+H] + 399, actual measured value 399.

[0126] Step 14

[0127] Compound 1p (3.50 g, 8.78 mmol) was dissolved in methanol (3 mL) and tetrahydrofuran (20 mL). A solution of sodium hydroxide (703 mg, 17.6 mmol) in water (20 mL) was added to the reaction mixture. The reaction mixture was stirred at 25°C for 0.5 hours. The reaction mixture was concentrated to remove the organic solvent, and the aqueous phase was adjusted to pH 7 with dilute hydrochloric acid (1 M). The mixture was extracted with dichloromethane (50 mL × 3). The organic phases were combined, washed with saturated brine (10 mL × 1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. 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] + 315, measured value 315. 1H NMR (400MHz, CD3OD) δ7.67(s,1H),7.21(d,J=8.4Hz,1H),7.07(s,1H),6.56(d,J=2.4Hz ,1H),6.47(dd,J=2.4,8.4Hz,1H),4.66(s,2H),3.90(s,3H),3.79(s,3H),2.36(s,3H).

[0128] Step 15

[0129] Compound 1t (250 mg, 795 μmol) was dissolved in N,N-dimethylformamide (4 mL), followed by the addition of compound 1q (187 mg, 875 μmol) and sodium hydroxide (63.6 mg, 1.59 mmol), and stirred at 50°C for 0.5 h. After completion of the reaction, the reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (30 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Compound 1r was obtained by column chromatography (1:1, petroleum ether / ethyl acetate, Rf = 0.1). MS-ESI calculated value [M+H] + 448, measured value 448.

[0130] Step 16

[0131] Compound 1r (300 mg, 670 μmol) was dissolved in trifluoroacetic acid (3.0 mL), and the reaction mixture was stirred at 100°C for 1 hour. After the reaction was complete, the reaction mixture was concentrated, and the residue was purified by HPLC (hydrochloric acid system) to obtain the hydrochloride salt of compound 1s. MS-ESI calculated value [M+H] + 298, measured value 298.

[0132] Step 17

[0133] The hydrochloride salt of compound 1s (90 mg) and compound 1f (72 mg, 241 μmol) were dissolved in anhydrous dioxane (2 mL). Cesium carbonate (250 mg, 766 μmol) and methanesulfonic acid (2-dicyclohexylphosphino)-3,6-dimethoxy-2,4,6-triisopropyl-1,1-biphenyl)(2-amino-1,1-biphenyl-2-yl)palladium(II) (20 mg, 21.9 μmol) were then added. The reaction mixture was stirred at 105°C for 12 hours under nitrogen. The reaction mixture was concentrated under reduced pressure and purified by column chromatography (10:1, dichloromethane / methanol, Rf = 0.3) to obtain the crude product. A mixture of methanol and ethanol (4 / 1, 10 mL) was added to the crude product, stirred at 20°C for 16 hours, filtered, and the filter cake was washed with methanol (2 mL x 2) and water (2 mL x 2) before drying to obtain the compound of formula (I). MS-ESI calculated value [M+H]+ 514, measured value 514. 1 H NMR (400MHz, DMSO-d6) δ10.00(s,1H),8.84(s,1H),8.64(s,1H),8.08(s,1H),7.70(s,1H),4.08(t,J=5.6Hz,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).

[0134] Example 2: Preparation of the compound of formula (II) and its single crystal cultivation

[0135] Synthesis route:

[0136]

[0137] first step

[0138] The compound of formula (I) (2 g, 3.89 μmol) was stirred and mixed with hexafluoroisopropanol (40 mL). To the solution was added p-toluenesulfonic acid monohydrate (814.89 mg, 4.28 mmol). 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 under vacuum to obtain the compound of formula (II). 1 H NMR (400MHz, DMSO-d6) δ10.02(s,1H),8.86(br s,1H),8.64(s,1H),8.11(s,1H),7.78(s,1H),7.48(d,J=8.0Hz,2H),7.10(d,J=8.0Hz,2H),4.31(br d,J=4.4Hz,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] + 514, measured value 514.

[0139] Dissolve an appropriate amount of compound (II) in 1 mL of dichloromethane / methanol (1:1) at room temperature. Place the sample solution in a 4 mL semi-sealed vial and slowly evaporate at room temperature. The next day, colorless blocky crystals were obtained. Single crystal X-ray diffraction analysis revealed the single crystal structure, as shown in Figure 7.

[0140] Example 3: Preparation of Crystalline Form A of Compound (II)

[0141] To the reactor, acetonitrile (7.0 L), the compound of formula (I) (700.22 g), and p-toluenesulfonic acid monohydrate (272.25 g) were added sequentially, with the internal temperature controlled at 20-30°C. The reactor temperature 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 resulting filter cake was transferred to a reactor, acetonitrile (7.0 L) was added, and the mixture was stirred at 45-55°C for 20 hours. The mixture was filtered, and the filter cake was rinsed sequentially with acetonitrile (0.7 L) and tert-butyl methyl ether (1.4 L). The mixture was then dried under reduced pressure at a temperature not exceeding 45°C and a pressure ≤-0.1 MPa for 17 hours to obtain the crystalline form A of compound of formula (II). The XRPD, DSC, and TGA results are shown in Figures 1, 2, and 3.

[0142] Example 4: Crystal screening test of the compound of formula (II) in different solvents

[0143] About 50 mg of the compound of formula (II) and 2-(2-methyl-2-oxo-1-pyrrolidone) were weighed and added to different solvents, stirred at 50°C for 3 hours, and the reaction solution was cooled to room temperature. The solid obtained after filtration and vacuum drying was analyzed by XRPD. The experimental results are shown in Table 10 below:

[0144] Table 10 Stability test results of compound of formula (II) in different solvents

[0145] No. Solvent-displayed crystal form 1 Methanol suspension crystal form A 2 Acetonitrile suspension crystal form A 3 Tert-butyl methyl ether suspension crystal form A 4 Ethanol suspension crystal form B 5 n-heptane suspension crystal form B

[0146] Conclusion: The compound of formula (II) forms crystal form A in methanol, acetonitrile and tert-butyl methyl ether, and forms crystal form B in ethanol and n-heptane.

[0147] The XRPD and DSC test results of Form B are shown in Figures 4 and 5 respectively.

[0148] Example 5: Hygroscopicity study of the crystalline form of compound A of formula (II)

[0149] Experimental Materials:

[0150] SMS DVS intrinsic dynamic gas adsorption instrument

[0151] Experimental methods:

[0152] 10-15 mg of the crystalline form A of compound of formula (II) was placed in a DVS sample tray for testing.

[0153] Experimental results:

[0154] The DVS spectrum of the crystal form A of compound of formula (II) is shown in FIG6 , ΔW=1.179%.

[0155] Experimental conclusion:

[0156] The weight gain of the crystal form A of the compound of formula (II) at 25° C. and 80% RH is 1.179%, indicating weak hygroscopicity. Furthermore, the crystal form does not change before and after moisture absorption, so it is a stable crystal form.

[0157] Experimental Example 6: Study on the Solid Stability of Form A of the Compound of the Invention

[0158] With reference to the 9001 “Guidelines for Stability Testing of Active Pharmaceutical Ingredients and Preparations” of Part IV of the 2020 edition of the Chinese Pharmacopoeia, the “Technical Guidelines for Stability Studies of Chemical Drugs (APIs and Preparations) (Revised)” issued by the Center for Drug Evaluation of the State Food and Drug Administration, and the requirements of ICH Q1B, accelerated sampling stability tests and long-term sampling stability studies were conducted on Form A of the compound of the present invention.

[0159] 1. Long-term accelerated lofting stability test:

[0160] Each sample was placed in a double-layer LDPE bag, each LDPE bag was sealed with a ziplock, then placed in an aluminum foil bag and heat-sealed. The bag was then placed in a stability chamber under the appropriate conditions for testing. The results of the solid stability test of Form A of the compound are shown in Table 11.

[0161] Table 11 Solid stability test results of Form A of the compound of the present invention

[0162]

[0163] Conclusion: Crystal Form A of the compound of the present invention has good stability under the conditions of long-term accelerated storage stability test, and Crystal Form A is a stable crystal form.

[0164] Biological activity test

[0165] Test Example 1: In vitro evaluation of the inhibitory activity of the compounds of the present invention on MNK2 protein kinase

[0166] Experimental purpose: To detect the MNK2 protein kinase inhibitory activity of the compound

[0167] Experimental materials: Assay buffer solution: 8 mM 3-(N-morpholino)propanesulfonic acid, 0.2 mM disodium ethylenediaminetetraacetic acid, 0.01% lauryl polyoxyethylene ether, 5% glycerol, 0.1% β-mercaptoethanol, 1 mg bovine serum albumin

[0168] Experimental Procedure: The MNK2 protein kinase inhibitory activity assay was performed using the KinaseProfiler™ service of Eurofins Pharma Discovery Services UK Limited. A series of DMSO dilutions containing the test compound (starting at 10 μM, serially diluted 3-fold), MNK2(h) protein kinase, and 0.33 mg / mL myelin basic protein were added to a freshly prepared buffer solution (pH = 7.0) and stirred evenly. 33 The reaction was initiated with a mixture of P-ATP (radioactivity 10 μCi / μL) and 10 mM magnesium acetate for 40 minutes at room temperature. Phosphoric acid diluted to a 0.5% concentration was added to terminate the reaction. A 10 μL sample of the reaction solution was filtered through a P30 filtermat, washed four times with 0.425% phosphoric acid for 4 minutes each, washed once with methanol, and dried before being analyzed for radioactivity using the filter-binding method.

[0169] The protein kinase inhibitory activity of the compound was expressed as the percentage of the protein kinase activity remaining in the blank substrate (pure DMSO). IC was calculated using the Prism4 software package (GraphPad). 50 The specific information is shown in Table 12 below.

[0170] Table 12 IC values ​​of the compounds of the present invention for the inhibitory activity against MNK2 protein kinase 50

[0171] Compound number IC for MNK2 50 (nM) Compound 17 of formula (I)

[0172] Experimental conclusion: The compound of formula (I) of the present invention exhibits excellent inhibitory activity against MNK2 protein kinase.

[0173] Test Example 2: In vitro evaluation of the inhibitory activity of the compounds of the present invention on eIF4E phosphorylation

[0174] Experimental purpose: To detect the IC inhibitory effect of compounds on eIF4E phosphorylation in HCT116 cell lines 50 value.

[0175] Experimental materials: HCT116 cells (ATCC), RPMI 1640 medium (Life technology), fetal bovine serum (Hyclone), double antibody (penicillin, streptomycin) (Millipore), phosphate buffered saline (Corning), 384-well cell plates (PerkinElmer), p-eIF4E (Ser209) Assay Kit (PerkinElmer).

[0176] Experimental procedure: HCT116 cells were digested to prepare a cell suspension, which was then plated in a 96-well plate. The cell plate was then placed in an incubator and cultured overnight. The compound was diluted to the corresponding concentration and added to the cell culture plate, and the culture was continued for 3 hours. The cells were then lysed with lysis buffer, and the lysate was transferred to a 384-well plate. The mixed receptor was freshly prepared according to the kit instructions, added to the 384-well plate, and incubated at room temperature for 1 hour. The mixed donor was freshly prepared according to the kit instructions, added to the 384-well plate, and incubated at room temperature for 1 hour. The signal was read using the standard AlphaLISA program on EnVision, and the curve was fitted and the IC was calculated using Graphpad prism. 50 , the specific information is shown in Table 13 below.

[0177] Table 13 IC values ​​of the compounds of the present invention for the inhibition of eIF4E phosphorylation 50

[0178] Compound number IC for p-eIF4E in HCT116 cell line 50 (nM) Compound of formula (I) 8.6

[0179] Experimental conclusion: The compound of formula (I) of the present invention exhibits excellent inhibitory activity on eIF4E phosphorylation.

[0180] Test Example 3: In vivo efficacy study of the compound of the present invention in CT-26 mouse transplanted tumors

[0181] Experimental purpose: To test the efficacy of the compound in CT-26 mouse xenografts

[0182] Experimental materials: CT-26 cells, RMPI-1640 medium containing 10% fetal bovine serum, mice (female, Shanghai Bikai Laboratory Animal Co., Ltd.)

[0183] Experimental procedures: CT-26 cells were cultured in RMPI-1640 medium containing 10% fetal bovine serum in a 37°C incubator with 5% CO2. Tumor cells were collected after passage and grown to an appropriate density. When the tumor cells were in the logarithmic growth phase, the cells were counted and resuspended in DPBS (phosphate buffered saline). The cell suspension concentration was adjusted to 3×10 6 / mL for inoculation.

[0184] Establishment of mouse colon cancer xenografts: Collect cells and adjust the concentration to 3×10 6 cells / ml (resuspended in DPBS to form a cell suspension). Under sterile conditions, 0.1 mL of tumor cells were subcutaneously injected into the right back of the mouse. The number of cells inoculated per mouse was 3 × 10 5 When the tumor grows to a certain size, use a digital vernier caliper to measure the length (a) and width (b) of the tumor and calculate the tumor volume. The calculation formula for tumor volume (TV) is: TV = a × b 2 / 2.

[0185] CT-26 tumor cell inoculation: On the day of inoculation, the animals were divided into groups and dosed according to their weight, with 8 animals in each group. The day of inoculation was considered as D0. 3 The antibody groups were grouped according to tumor size and body weight. Animal body weight and tumor size were measured three times a week during the experiment. Clinical symptoms were observed and recorded daily. The most recently measured weight was used as a reference for each dosing. The inhibitory effect of the compound on colon cancer xenografts in mice was determined at doses of 30 mg / kg QD (once daily), 90 mg / kg QD (once daily), and 200 mg / kg QD (once daily) for 21 days. Specific information is shown in Table 14 below.

[0186] The evaluation index of antitumor activity is the relative tumor proliferation rate T / C (%). T / C (%)>40% is ineffective, T / C (%)≤40% and P<0.05 after statistical analysis is effective. The calculation formula of T / C (%) is: T / C (%)=(T RTV / C RTV )×100%. T RTV is the relative tumor volume of the treatment group, C RTV is the relative tumor volume of the negative control group; TGI (%) a =(1-average tumor volume of the treated group at the end of drug administration / average tumor volume of the solvent control group at the end of treatment)×100%.

[0187] Table 14 Antitumor efficacy of the compounds of the present invention in CT-26 transplanted tumor model

[0188] Compound dosage TGI% T / C% Compound of formula (I) 30 mg / Kg, QD 63.5736.43 Compound of formula (I) 90 mg / Kg, QD 68.8931.11 Compound of formula (I) 200 mg / Kg, QD 68.5133.31

[0189] Experimental conclusion: The compound of formula (I) of the present invention has a significant inhibitory effect on colon cancer transplanted tumors in mice.

Claims

1. A compound of formula (II), 2. The A polymorph of the compound of formula (II), having characteristic diffraction peaks at the following 2θ angles in its X-ray powder diffraction pattern: 7.78 ± 0.20°, 11.38 ± 0.20° and 20.58 ± 0.20°; 3. The A polymorph according to claim 2, having characteristic diffraction peaks at the following 2θ angles in its X-ray powder diffraction pattern: 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. The A polymorph according to claim 3, having characteristic diffraction peaks at the following 2θ angles in its X-ray powder diffraction pattern: 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. The A polymorph according to claim 4, having characteristic diffraction peaks at the following 2θ angles in its X-ray powder diffraction pattern: 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. The A polymorph according to claim 5, the XRPD pattern of which is substantially as shown in Figure 1.

7. The A polymorph according to any one of claims 2-6, having an endothermic peak at 287.17 ± 3 °C in its differential scanning calorimetry curve.

8. The A polymorph according to claim 7, the DSC pattern of which is as shown in Figure 2.

9. The A crystal form according to any one of claims 2-6 has a weight loss of 0.075% at 200.0 ± 3 °C in the thermogravimetric analysis curve.

10. The A crystal form according to claim 9 has a TGA spectrum as shown in Figure 3.

11. A method for preparing the A crystal form of the compound of formula (II), the steps comprising: (a) adding the compound of formula (II) to a solvent to form a suspension; (b) stirring the suspension at 40-55 °C for 2-25 hours; (c) filtering and then drying in vacuo at 30-45 °C for 10-24 hours; wherein, The solvent is selected from methanol, acetonitrile and tert-butyl methyl ether.

12. The B crystal form of the compound of formula (II) has characteristic diffraction peaks at the following 2θ angles in the X-ray powder diffraction pattern: 7.55 ± 0.20°, 15.13 ± 0.20°, and 19.82 ± 0.20°; 13. The B crystal form according to claim 12 has characteristic diffraction peaks at the following 2θ angles in the X-ray powder diffraction pattern: 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. The B crystal form according to claim 13 has characteristic diffraction peaks at the following 2θ angles in the X-ray powder diffraction pattern: 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. The B crystal form according to claim 14 has characteristic diffraction peaks at the following 2θ angles in the X-ray powder diffraction pattern: 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. The B crystal form according to claim 15 has an XRPD pattern substantially as shown in Figure 4.

17. The B crystal form according to any one of claims 12 - 16 has an endothermic peak at 300.0 ± 3 °C in its differential scanning calorimetry curve.

18. The DSC pattern of the B crystal form according to claim 17 is as shown in Figure 5.

19. Use of the compound of formula (II) according to claim 1, the A crystal form according to any one of claims 2 - 10, the B crystal form according to any one of claims 12 - 18, and the crystal form prepared by the method of claim 11 in the preparation of a drug for inhibiting MNK1 / 2.

20. Use of the compound of formula (II) according to claim 1, the A crystal form according to any one of claims 2 - 10, the B crystal form according to any one of claims 12 - 18, and the crystal form prepared by the method of claim 11 in the preparation of a drug for treating colorectal cancer.