Salt form, crystalline form, and preparation method and use of PLK1 kinase inhibitors

Abstract

This invention provides salt forms, crystalline forms, and preparation methods and uses of PLK1 kinase inhibitors. Specifically, it provides salt forms, crystalline forms, and preparation methods of compounds of formula I, as well as their use in the preparation of drugs for diseases caused by and / or related to abnormal PLK1 kinase activity. TIFF2026522276000035.tif39170

Description

[Technical Field]

[0001] This disclosure belongs to the field of pharmacochemistry and specifically relates to salt forms, crystalline forms, and methods for preparing and using PLK1 kinase inhibitors. [Background technology]

[0002] PLK1 is a serine / threonine kinase widely present in eukaryotic cells. PLK1 is involved in many processes of cell division, including biological processes such as the entry of cell division, rupture of the nuclear membrane, replication of centrioles, complete chromosome alignment, activation of the mitotic checkpoint, and cytokinesis.

[0003] During the G2 phase, PLK1 plays a crucial role in the activation of the cyclin B-CDK1 complex through at least two mechanisms. First, it activates CDC25C phosphatase, which then dephosphorylates CDK1 inhibitory phosphorylation, thereby activating CDK1. Second, PLK1 induces phosphorylation-dependent degradation of WEE1, thereby preventing further phosphorylation of CDK1 and transitioning the cell to the M phase. PLK1 is also involved in regulating the G2 and M phase DNA damage checkpoints.

[0004] Studies have demonstrated that PLK1 is overexpressed in various tumor tissues, and that excessively active PLK1 expression can allow cells to cross the G2 inhibitory checkpoint induced by DNA damage. Furthermore, PLK1 is closely associated with tumor development and progression, and PLK1 expression levels are linked to poor prognosis in clinical patients. Therefore, PLK1 inhibitors are expected to be effective antitumor drug targets.

[0005] Volasertib and Onvansertib are ATP-competitive PLK1 small molecule inhibitors that are currently progressing relatively rapidly in clinical trials and exhibit clear antitumor effects in various preclinical models and clinical studies, whether used as monotherapy or in combination. Volasertib is an injectable formulation and has been observed to have a relatively elevated incidence of high-grade adverse events in clinical trials. Onvansertib is an oral formulation and has been shown to have relatively low bioavailability in preclinical studies and a relatively small safety margin in clinical trials. Therefore, there is still a need in this field for compounds that are highly selective, highly active, and safer to meet clinical needs.

[0006] PCT / CN2022 / 137824 provides a PLK1 kinase inhibitor whose structural formula is shown below. As can be seen from the test results, the compound has a clear inhibitory effect on PLK1 kinase inhibition and can significantly inhibit tumor volume growth. All contents of the said application are incorporated herein by reference. TIFF2026522276000002.tif38170

[0007] The pharmacokinetic form of a compound (e.g., crystalline form, salt) tends to affect the chemical stability of the drug. Crystallization and storage conditions can alter the crystalline structure of a compound, sometimes leading to the formation of other crystalline forms. Generally, amorphous drug products lack a regular crystalline structure, tend to have relatively poor product stability, relatively fine precipitated crystals, are relatively difficult to filter, prone to caking, and have poor fluidity. Given the importance of the salt and crystalline forms and their stability of solid drugs in clinical therapy, in-depth research into the salt and crystalline forms of compounds of formula I is of great significance in developing drugs that are suitable for industrial production and exhibit good biological activity. [Overview of the Initiative]

[0008] In one aspect, the present disclosure provides an A-type crystal of a compound of formula I having characteristic diffraction peaks at 2θ angles of 4.807 ± 0.2°, 8.532 ± 0.2°, 11.645 ± 0.2°, 15.160 ± 0.2°, 18.347 ± 0.2°, and 25.514 ± 0.2° in an X-ray powder diffraction pattern using Cu-Kα radiation. TIFF2026522276000003.tif39170

[0009] In some embodiments, the A-type crystal of the compound of formula I has characteristic diffraction peaks at 2θ angles of 4.807 ± 0.2°, 8.532 ± 0.2°, 11.645 ± 0.2°, 15.160 ± 0.2°, 16.768 ± 0.2°, 18.347 ± 0.2°, 19.000 ± 0.2°, 19.247 ± 0.2°, and 25.514 ± 0.2° in an X-ray powder diffraction pattern using Cu-Kα radiation. In some embodiments, the X-ray powder diffraction pattern of the A-type crystal of the compound of formula I using Cu-Kα radiation is shown in Figure 1-1.

[0010] In one aspect, the present disclosure provides a pharmaceutically acceptable salt of the compound of formula I, wherein the pharmaceutically acceptable salt is selected from hydrochloride, sulfate, maleate, L-aspartic acid, L-tartrate, phosphate, fumarate, citrate, malate, glycolate, L-malate, hippurate, succinate, adipate, p-toluenesulfonate, methanesulfonate, benzenesulfonate, oxalate, malonate, gentisate, or hydrobromide.

[0011] In some embodiments, the pharmaceutically acceptable salt is maleate, fumarate, or succinate.

[0012] In some embodiments, the molar ratio of the acid molecule to the compound of formula I in the pharmaceutically acceptable salt is about 1:2 to 2:1, preferably about 1:1 or 1:2.

[0013] In some embodiments, the molar ratio of maleic acid to the compound of formula I in the pharmaceutically acceptable salt is about 1:1.

[0014] In some embodiments, the molar ratio of fumaric acid to compound I in the above-mentioned medicinal salt is about 1:1 or 1:2.

[0015] In some embodiments, the molar ratio of succinic acid to compound I in the above-mentioned medicinal salt is approximately 1:1.

[0016] In one embodiment, the disclosure provides a method for preparing a pharmaceutically acceptable salt of a compound of formula I, comprising the step of mixing the compound of formula I with an acid and a solvent. The solvent is one or more selected from ethanol, ethyl acetate, 2-methyltetrahydrofuran, tetrahydrofuran, acetone, water, dichloromethane, dimethyl sulfoxide, N-methylpyrrolidone, acetonitrile, toluene, N,N-dimethylformamide, and n-heptane.

[0017] In some embodiments, in a method for preparing a medicinal salt of the above-mentioned compound I, the molar ratio of the amount of compound I to the acid added is selected from 5:1 to 1:5, preferably 1:0.5 to 1:1.5, and more preferably 1:1.

[0018] In one embodiment, the disclosure provides a type A crystal of a maleate of a compound of formula I, having characteristic diffraction peaks at 2θ angles of 8.455±0.2°, 12.802±0.2°, 13.894±0.2°, 19.029±0.2°, 20.917±0.2°, and 21.383±0.2° in an X-ray powder diffraction pattern using Cu-Kα radiation.

[0019] In some embodiments, the A-type crystals of the maleate of the above formula I compound have characteristic diffraction peaks at 2θ angles of 8.455±0.2°, 12.313±0.2°, 12.802±0.2°, 13.894±0.2°, 17.051±0.2°, 17.713±0.2°, 19.029±0.2°, 20.917±0.2°, 21.383±0.2°, and 24.787±0.2° in the X-ray powder diffraction pattern using Cu-Kα radiation.

[0020] In some embodiments, the A-type crystals of the maleate of the above formula I compound have characteristic diffraction peaks at 2θ angles of 6.883±0.2°, 8.455±0.2°, 12.313±0.2°, 12.802±0.2°, 13.894±0.2°, 14.640±0.2°, 17.051±0.2°, 17.369±0.2°, 17.713±0.2°, 19.029±0.2°, 20.917±0.2°, 21.383±0.2°, 23.818±0.2°, 24.787±0.2°, and 25.823±0.2° in the X-ray powder diffraction pattern using Cu-Kα radiation.

[0021] In some embodiments, the X-ray powder diffraction pattern of the maleate of the above formula I compound, type A crystal, using Cu-Kα radiation, is shown in Figure 2-1.

[0022] In some embodiments, the molar ratio of maleic acid to compound I in the type A crystal of the maleate of the above-mentioned compound I is about 0.9 to 1.1:1, preferably 1.0:1.

[0023] In a selective embodiment, the disclosure provides a method for preparing type A crystals of a maleate of a compound of formula I, comprising the steps of mixing a compound of formula I, maleic acid, and a solvent and crystallizing them. In a selective embodiment, the solvent is selected from ethanol, ethyl acetate, 2-methyltetrahydrofuran, and acetone / water. The crystallization method includes the addition of a poor solvent, gas-solid diffusion, gas-liquid diffusion, stirring at room temperature, circulating stirring at temperature, slow volatilization, or polishing.

[0024] In one embodiment, the present disclosure provides a type A crystal of a fumarate of a compound of formula I, having characteristic diffraction peaks at 2θ angles of 7.931±0.2°, 15.752±0.2°, 16.012±0.2°, 20.426±0.2°, 21.636±0.2°, 22.902±0.2°, and 24.234±0.2° in an X-ray powder diffraction pattern using Cu-Kα radiation.

[0025] In some embodiments, the A-type crystals of the fumarate of the above formula I compound have characteristic diffraction peaks at 2θ angles of 7.931±0.2°, 12.635±0.2°, 13.761±0.2°, 15.752±0.2°, 16.012±0.2°, 16.972±0.2°, 19.481±0.2°, 20.426±0.2°, 21.636±0.2°, 22.285±0.2°, 22.641±0.2°, 22.902±0.2°, 23.597±0.2°, 24.234±0.2°, 27.948±0.2°, and 29.329±0.2° in the X-ray powder diffraction pattern using Cu-Kα radiation.

[0026] In some embodiments, the X-ray powder diffraction pattern of the type A crystal of the fumarate of the above formula I compound, using Cu-Kα radiation, is shown in Figure 3-1.

[0027] In some embodiments, the molar ratio of fumaric acid to compound I in the type A crystal of the fumarate of the compound of formula I is about 0.9 to 1.1:1, preferably 1.0:1.

[0028] In a selective embodiment, the disclosure provides a method for preparing type A crystals of a fumarate of a compound of formula I, comprising the steps of mixing a compound of formula I, fumaric acid, and solvent (I) and crystallizing them. In a selective embodiment, solvent (I) is selected from ethanol, ethyl acetate, 2-methyltetrahydrofuran, acetone / water, N-methylpyrrolidone, N,N-dimethylformamide, and tetrahydrofuran / water. The crystallization method includes the addition of a poor solvent, gas-solid diffusion, gas-liquid diffusion, stirring at room temperature, temperature-circulating stirring, slow volatilization, or polishing. The poor solvent is selected from methanol, isopropanol, methyl isobutyl ketone, isopropyl acetate, toluene, 2-butanol, butanone, ethyl acetate, or chloroform.

[0029] In one embodiment, the disclosure provides a type A crystal of succinate of compound I of formula, having characteristic diffraction peaks at 2θ angles of 7.771±0.2°, 13.539±0.2°, 15.519±0.2°, 20.105±0.2°, 21.347±0.2°, and 22.552±0.2° in an X-ray powder diffraction pattern using Cu-Kα radiation.

[0030] In some embodiments, the A-type crystal succinate of the above formula I compound has characteristic diffraction peaks at 2θ angles of 6.655±0.2°, 7.771±0.2°, 13.539±0.2°, 15.519±0.2°, 15.781±0.2°, 17.709±0.2°, 20.105±0.2°, 21.347±0.2°, 22.552±0.2°, and 23.887±0.2° in the X-ray powder diffraction pattern using Cu-Kα radiation.

[0031] In some embodiments, the X-ray powder diffraction pattern of the succinate type A crystal of the above formula I compound, using Cu-Kα radiation, is shown in Figure 4-1.

[0032] In some embodiments, the molar ratio of succinic acid to compound I in the type A crystal of succinate of the above-mentioned compound I is about 0.9 to 1.1:1, preferably 1.0:1.

[0033] In a selective embodiment, the disclosure provides a method for preparing type A crystals of succinate of a compound of formula I, comprising the steps of mixing a compound of formula I, succinic acid, and a solvent and crystallizing them. In a selective embodiment, the solvent is selected from ethanol, ethyl acetate, 2-methyltetrahydrofuran, and acetone / water. The crystallization method includes the addition of a poor solvent, gas-solid diffusion, gas-liquid diffusion, stirring at room temperature, temperature-circulating stirring, slow volatilization, or polishing.

[0034] In one embodiment, the disclosure provides a B-type crystal of a hemifumarate of a compound of formula I, having characteristic diffraction peaks at 2θ angles of 15.084±0.2°, 15.501±0.2°, 17.311±0.2°, 24.838±0.2°, 26.132±0.2°, and 26.736±0.2° in an X-ray powder diffraction pattern using Cu-Kα radiation.

[0035] In some embodiments, the B-type crystals of the hemifumarate of the above formula I compound have characteristic diffraction peaks at 2θ angles of 8.570±0.2°, 12.545±0.2°, 15.084±0.2°, 15.501±0.2°, 17.311±0.2°, 24.838±0.2°, 26.132±0.2°, and 26.736±0.2° in the X-ray powder diffraction pattern using Cu-Kα radiation.

[0036] In some embodiments, the X-ray powder diffraction pattern of the hemifumarate type B crystal of the above formula I compound using Cu-Kα radiation is shown in Figure 5-1.

[0037] In some embodiments, the molar ratio of fumaric acid to compound I in the type B crystal of the hemifumarate of compound I is about 0.5 to 0.7:1, preferably 0.6 to 0.7:1.

[0038] In some embodiments, the B-type crystal of the hemi-fumarate of the above-mentioned compound I is an NMP solvate, preferably with a molar ratio of NMP (i.e., N-methylpyrrolidone) to compound I of about 0.9 to 2.0:1, or a molar ratio of NMP to compound I of about 1.1:1 or 2:1.

[0039] In a selective embodiment, the disclosure provides a method for preparing type B crystals of a hemifumarate of a compound of formula I, comprising mixing a compound of formula I, fumaric acid, and solvent (I), and adding a poor solvent under stirring conditions. In a selective embodiment, solvent (I) is N-methylpyrrolidone, and the poor solvent is methyl tert-butyl ether. The crystallization method includes adding the poor solvent, gas-solid diffusion, gas-liquid diffusion, stirring at room temperature, temperature-circulating stirring, slow volatilization, or polishing.

[0040] In one embodiment, the disclosure provides a C-type crystal of a hemifumarate of a compound of formula I, having characteristic diffraction peaks at 2θ angles of 7.553±0.2°, 12.716±0.2°, 13.547±0.2°, 15.313±0.2°, 19.089±0.2°, and 22.755±0.2° in an X-ray powder diffraction pattern using Cu-Kα radiation.

[0041] In some embodiments, the C-type crystals of the hemifumarate of the above formula I compound have characteristic diffraction peaks at 2θ angles of 6.637±0.2°, 7.553±0.2°, 12.716±0.2°, 13.547±0.2°, 15.313±0.2°, 19.089±0.2°, 20.121±0.2°, 20.609±0.2°, and 22.755±0.2° in the X-ray powder diffraction pattern using Cu-Kα radiation.

[0042] In some embodiments, the C-type crystals of the hemifumarate of the above formula I compound have X-ray powder diffraction patterns using Cu-Kα radiation, as shown in Figure 6-1.

[0043] In some embodiments, the molar ratio of fumaric acid to compound I in the C-type crystal of the hemifumarate of the above-mentioned compound I is about 0.5 to 0.7:1, preferably 0.5:1.

[0044] In some embodiments, the C-type crystal of the hemifumarate of the above formula I compound is a hydrate.

[0045] In some embodiments, the disclosure provides a method for preparing C-type crystals of a hemifumarate of a compound of formula I, comprising the steps of mixing a compound of formula I, fumaric acid, and solvent (I) and crystallizing them. In selective embodiments, solvent (I) is selected from N-methylpyrrolidone, N,N-dimethylformamide, acetone / water, methanol / water, tetrahydrofuran / water, isopropanol / water, and acetonitrile / water. The crystallization method includes the addition of a poor solvent, gas-solid diffusion, gas-liquid diffusion, stirring at room temperature, temperature-circulating stirring, slow volatilization, or polishing.

[0046] In some embodiments, the Disclosure provides a method for preparing C-type crystals of a hemifumarate of a compound of formula I, comprising mixing a compound of formula I, fumaric acid, and solvent (I), and adding a poor solvent, wherein solvent (I) is selected from N-methylpyrrolidone and N,N-dimethylformamide, and the poor solvent is selected from water or acetone.

[0047] In some embodiments, the disclosure provides a method for preparing C-type crystals of hemifumarate of compound I, comprising adding A-type crystals of the fumarate of compound I to a solvent, then adding a PVP / PVC high polymer, and gradually volatilizing it under room temperature conditions. The solvent is selected from acetone / water, methanol / water, and tetrahydrofuran / water.

[0048] In one embodiment, the disclosure provides a D-type crystal of a hemifumarate of a compound of formula I, having characteristic diffraction peaks at 2θ angles of 6.881±0.2°, 8.103±0.2°, 12.572±0.2°, and 13.831±0.2° in an X-ray powder diffraction pattern using Cu-Kα radiation.

[0049] In some embodiments, the D-type crystals of the hemifumarate of the above formula I compound have characteristic diffraction peaks at 2θ angles of 6.881±0.2°, 8.103±0.2°, 12.572±0.2°, 13.831±0.2°, 19.569±0.2°, 20.533±0.2°, and 24.021±0.2° in the X-ray powder diffraction pattern using Cu-Kα radiation.

[0050] In some embodiments, the D-type crystals of the hemifumarate of the above formula I compound have X-ray powder diffraction patterns using Cu-Kα radiation, as shown in Figure 7-1.

[0051] In some embodiments, the molar ratio of fumaric acid to compound I in the D-type crystal of the hemifumarate of the above-mentioned compound I is about 0.5 to 0.7:1, preferably 0.5:1.

[0052] In some embodiments, the D-type crystal of the hemifumarate of the above formula I compound is a pipe hydrate or an anhydrous form.

[0053] In some embodiments, the disclosure provides a method for preparing D-type crystals of hemi-fumarates of compounds of formula I, which includes heating C-type crystals of the hemi-fumarate of a compound of formula I to 140°C.

[0054] In some embodiments, the present disclosure provides a method for preparing D-type crystals of the hemifumarate of the compound of formula I, comprising mixing the compound of formula I, fumaric acid, and tetrahydrofuran / water, adding acetone as a poor solvent, and then cooling to 5°C.

[0055] In one embodiment, the disclosure provides an E-type crystal of a hemi-fumarate of a compound of formula I, having characteristic diffraction peaks at 2θ angles of 6.188±0.2°, 12.492±0.2°, 15.473±0.2°, 17.729±0.2°, 25.017±0.2°, and 26.603±0.2° in an X-ray powder diffraction pattern using Cu-Kα radiation.

[0056] In some embodiments, the E-type crystals of the hemifumarate of the above formula I compound have characteristic diffraction peaks at 2θ angles of 6.188±0.2°, 12.492±0.2°, 15.473±0.2°, 17.729±0.2°, 25.017±0.2°, 25.275±0.2°, 26.603±0.2°, and 31.795±0.2° in the X-ray powder diffraction pattern using Cu-Kα radiation.

[0057] In some embodiments, the X-ray powder diffraction pattern of the hemifumarate of the above formula I compound, using Cu-Kα radiation, is shown in Figure 8-1.

[0058] In some embodiments, the molar ratio of fumaric acid to compound I in the E-type crystal of the hemifumarate of the above-mentioned compound I is about 0.5 to 0.7:1, preferably 0.7:1.

[0059] In some embodiments, the E-type crystal of the hemi-fumarate of the above formula I compound is a DMAc / CHCl3 cosolvate, where the molar ratio of DMAc (i.e., N,N-dimethylacetamide) to the formula I compound is about 0.4:1, and the molar ratio of CHCl3 to the formula I compound is about 0.3:1.

[0060] This disclosure provides a method for preparing type E crystals of hemifumarate of a compound of formula I, comprising dissolving type A crystals of the fumarate of a compound of formula I in N,N-dimethylacetamide, clarifying the mixture, adding chloroform, a poor solvent, sealing the container, and leaving it at room temperature until a solid precipitate forms.

[0061] In one embodiment, the disclosure provides an F-type crystal of a hemifumarate of a compound of formula I, having characteristic diffraction peaks at 2θ angles of 8.866±0.2°, 16.651±0.2°, 25.199±0.2°, and 26.157±0.2° in an X-ray powder diffraction pattern using Cu-Kα radiation.

[0062] In some embodiments, the F-type crystals of the hemifumarate of the above formula I compound have characteristic diffraction peaks at 2θ angles of 8.185±0.2°, 8.866±0.2°, 13.449±0.2°, 16.651±0.2°, 16.841±0.2°, 25.199±0.2°, and 26.157±0.2° in the X-ray powder diffraction pattern using Cu-Kα radiation.

[0063] In some embodiments, the X-ray powder diffraction pattern of the F-type crystal of the hemifumarate of the above formula I compound, using Cu-Kα radiation, is shown in Figure 9-1.

[0064] In some embodiments, the molar ratio of fumaric acid to compound I in the F-type crystal of the hemifumarate of the above-mentioned compound I is about 0.5 to 0.7:1, preferably 0.5:1 or 0.6:1.

[0065] In some embodiments, the F-type crystal of the hemi-fumarate of the above formula I compound is a DMAc (i.e., N,N-dimethylacetamide) solvate, where the molar ratio of DMAc to the formula I compound is approximately 0.5:1.

[0066] In some embodiments, the present disclosure provides a method for preparing F-type crystals of hemifumarate of a compound of formula I, comprising dissolving A-type crystals of the fumarate of a compound of formula I in N,N-dimethylacetamide, clarifying the mixture, adding a poor solvent, sealing the container, and allowing it to stand at room temperature until a solid precipitate forms, the poor solvent being selected from methanol, butanone, and methyl tert-butyl ether.

[0067] In one embodiment, the present disclosure provides a G-type crystal of a hemifumarate of a compound of formula I, having characteristic diffraction peaks at 2θ angles of 8.925±0.2°, 12.025±0.2°, 12.948±0.2°, 13.525±0.2°, 15.763±0.2°, 16.793±0.2°, 21.133±0.2°, 23.547±0.2°, 25.164±0.2°, and 26.101±0.2° in an X-ray powder diffraction pattern using Cu-Kα radiation.

[0068] In some embodiments, the G-type crystals of the hemifumarate of the above formula I compound have characteristic diffraction peaks at 2θ angles of 6.707±0.2°, 7.294±0.2°, 8.925±0.2°, 12.025±0.2°, 12.948±0.2°, 13.525±0.2°, 15.510±0.2°, 15.763±0.2°, 16.417±0.2°, 16.793±0.2°, 18.923±0.2°, 21.133±0.2°, 23.547±0.2°, 25.164±0.2°, and 26.101±0.2° in the X-ray powder diffraction pattern using Cu-Kα radiation.

[0069] In some embodiments, the G-type crystals of the hemifumarate of the above formula I compound have X-ray powder diffraction patterns using Cu-Kα radiation, as shown in Figure 10-1.

[0070] In some embodiments, the molar ratio of fumaric acid to compound I in the G-type crystal of the hemifumarate of the above-mentioned compound I is about 0.5 to 0.7:1, preferably 0.6:1.

[0071] In some embodiments, the G-type crystal of the hemifumarate of the above formula I compound is an NMP solvate, in which the molar ratio of NMP to the formula I compound is about 0.5:1.

[0072] In some embodiments, the method for preparing the G-type crystals of the hemi-fumarate of the compound of formula I includes heating the B-type crystals of the hemi-fumarate of the compound of formula I to about 140°C.

[0073] In a selective embodiment, in a method for preparing any one of the above crystal types, the crystallization method is one or more selected from room temperature crystallization, crystallization by gas-liquid diffusion, crystallization by gas-solid diffusion, crystallization induced by high polymer, cooling crystallization, crystallization by solvent evaporation, crystallization by addition of poor solvent, and crystallization induced by addition of seed crystal.

[0074] This disclosure further provides a drug composition comprising a type A crystal of a compound of formula I, any one of the above-mentioned pharmaceutically acceptable salts or crystalline forms thereof, and a pharmaceutically acceptable carrier. The drug composition can be prepared in various pharmaceutically acceptable dosage forms, such as tablets, capsules, oral solutions, granules, injections, or various sustained-release formulations. The drug composition can be administered orally or parenterally (e.g., intravenously, subcutaneously, or topically). The dosage can be appropriately adjusted according to the patient's age, sex, and disease type, and the usual daily dose is about 1 to 200 mg.

[0075] In one embodiment, the present disclosure further provides the use of a type A crystal of the compound of formula I, any one of the above pharmaceutically acceptable salts, crystalline form, or drug composition in the preparation of drugs for diseases caused by and / or related to abnormal PLK1 kinase activity.

[0076] In one embodiment, the disclosure further provides the use of a type A crystal of a compound of formula I, any one of the above pharmaceutically acceptable salts, crystalline form, or drug composition in the preparation of a drug, the drug being used to prevent and / or treat diseases caused by and / or related to abnormalities in protein kinase activity. These diseases include cancer, proliferative disorders, viral infections, autoimmune diseases, and neurodegenerative diseases.

[0077] The above cancers include cancers of the bladder, mammary glands, colon, kidneys, liver, lungs (including small cell lung cancer), esophagus, gallbladder, ovaries, pancreas, stomach, cervix, thyroid, prostate, and skin (including squamous cell carcinoma); leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, and non-Hodgkin's lymphoma. Lymphoid hematopoietic neoplasms, including lymphoma, hairy cell lymphoma and Burkitt lymphoma; myeloid hematopoietic neoplasms, including acute and chronic myeloid leukemia, myelodysplastic syndrome and promyelocytic leukemia; stromal tumors, including fibrosarcoma and rhabdomyosarcoma; central and peripheral nervous system tumors, including astrocytoma, neuroblastoma, glioma and schwannoma; and other tumors, including melanoma, seminomas, teratomas, osteosarcomas, xeroderma pigmentosum, keratoacanthoma, follicular carcinoma of the thyroid gland and Kaposi's sarcoma.

[0078] The cell proliferation disorders mentioned above include, for example, benign prostatic hyperplasia, familial adenomatous neoplasia, polyposis, neurofibroma, psoriasis, vascular smooth cell proliferation associated with atherosclerosis, pulmonary fibrosis, arthritis, glomerulonephritis, and postoperative stenosis and restenosis.

[0079] Definition and Description Unless otherwise specified, the following terms and phrases used herein are intended to have the meanings set forth below. No particular term or phrase should be considered uncertain or unclear unless specifically defined, and should be understood in a general sense. Where trade names appear herein, they are intended to refer to the corresponding product or its active ingredient.

[0080] The term "pharmaceutically acceptable" as used in this disclosure applies to compounds, compositions, and / or dosage forms that are within the bounds of reliable medical judgment, suitable for contact use with human and animal tissues, but free from excessive toxicity, irritation, allergic reactions, or other problems or complications, and commensurate with a reasonable benefit / risk ratio.

[0081] The compounds of this disclosure may have an asymmetric carbon atom (optical center) or a double bond. Racemates, diastereomers, geometric isomers, and individual isomers are all included within the scope of this disclosure.

[0082] The compounds of this disclosure may be in the form of specific geometric or stereoisomers. This disclosure includes all cis-trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures thereof, as well as other mixtures, such as mixtures rich in enantiomers or diastereomers, and all such compounds are intended to be within the scope of this disclosure. Substituents such as alkyl groups may have other chiral carbon atoms. All such isomers and mixtures thereof are within the scope of this disclosure.

[0083] The term "pharmaceutically acceptable carrier" refers to a representative carrier of any formulation or carrier medium that can deliver an effective amount of the active substance of this disclosure, does not interfere with the biological activity of the active substance, and is toxic and adverse to the host or patient, and includes, but is not limited to, binders, fillers, lubricants, disintegrants, wetting agents, dispersants, solubilizers, suspending agents, etc.

[0084] This disclosure is intended to include all isotopes of the atoms present in the compounds relating to this disclosure. An isotope is an atom that has the same number of atoms but a different mass number. As a general example, and not limited thereto, isotopes of hydrogen include deuterium and tritium. Isotopes of carbon include, 13 C and 14 Contains C. The isotope-labeled compounds of this disclosure can generally be prepared by conventional techniques known to those skilled in the art, or by methods similar to those described herein, using a suitable isotope-labeled reagent instead of an unlabeled reagent for further use. [Brief explanation of the drawing]

[0085] [Figure 1-1]These are the XRPD pattern, TGA / DSC pattern, and 1H NMR spectrum of the A-type crystal of compound I, respectively. [Figure 1-2] These are the XRPD pattern, TGA / DSC pattern, and 1H NMR spectrum of the A-type crystal of compound I, respectively. [Figure 1-3] These are the XRPD pattern, TGA / DSC pattern, and 1H NMR spectrum of the A-type crystal of compound I, respectively. [Figure 2-1] These are the XRPD pattern, 1H NMR spectrum, and TGA / DSC pattern of the maleate of the compound of formula I in type A crystal, respectively. [Figure 2-2] These are the XRPD pattern, 1H NMR spectrum, and TGA / DSC pattern of the maleate of the compound of formula I in type A crystal, respectively. [Figure 2-3] These are the XRPD pattern, 1H NMR spectrum, and TGA / DSC pattern of the maleate of the compound of formula I in type A crystal, respectively. [Figure 3-1] These are the XRPD pattern, 1H NMR spectrum, and TGA / DSC pattern of the A-type crystal of the fumarate of compound I, respectively. [Figure 3-2] These are the XRPD pattern, 1H NMR spectrum, and TGA / DSC pattern of the A-type crystal of the fumarate of compound I, respectively. [Figure 3-3] These are the XRPD pattern, 1H NMR spectrum, and TGA / DSC pattern of the A-type crystal of the fumarate of compound I, respectively. [Figure 4-1] These are the XRPD pattern, 1H NMR spectrum, and TGA / DSC pattern of the succinate A-type crystal of the compound of formula I, respectively. [Figure 4-2] These are the XRPD pattern, 1H NMR spectrum, and TGA / DSC pattern of the succinate A-type crystal of the compound of formula I, respectively. [Figure 4-3] These are the XRPD pattern, 1H NMR spectrum, and TGA / DSC pattern of the succinate A-type crystal of the compound of formula I, respectively. [Figure 5-1]These are the XRPD pattern, 1H NMR spectrum, and TGA / DSC pattern of the B-type crystal of the hemi-fumarate of compound I, respectively. [Figure 5-2] These are the XRPD pattern, 1H NMR spectrum, and TGA / DSC pattern of the B-type crystal of the hemi-fumarate of compound I, respectively. [Figure 5-3] These are the XRPD pattern, 1H NMR spectrum, and TGA / DSC pattern of the B-type crystal of the hemi-fumarate of compound I, respectively. [Figure 6-1] These are the XRPD pattern, 1H NMR spectrum, and TGA / DSC pattern of the C-type crystal of the hemi-fumarate of compound I, respectively. [Figure 6-2] These are the XRPD pattern, 1H NMR spectrum, and TGA / DSC pattern of the C-type crystal of the hemi-fumarate of compound I, respectively. [Figure 6-3] These are the XRPD pattern, 1H NMR spectrum, and TGA / DSC pattern of the C-type crystal of the hemi-fumarate of compound I, respectively. [Figure 7-1] These are the XRPD pattern, 1H NMR spectrum, and TGA / DSC pattern of the D-type crystal of the hemi-fumarate of compound I, respectively. [Figure 7-2] These are the XRPD pattern, 1H NMR spectrum, and TGA / DSC pattern of the D-type crystal of the hemi-fumarate of compound I, respectively. [Figure 7-3] These are the XRPD pattern, 1H NMR spectrum, and TGA / DSC pattern of the D-type crystal of the hemi-fumarate of compound I, respectively. [Figure 8-1] These are the XRPD pattern, 1H NMR spectrum, and TGA / DSC pattern of the E-type crystal of the hemi-fumarate of compound I, respectively. [Figure 8-2] These are the XRPD pattern, 1H NMR spectrum, and TGA / DSC pattern of the E-type crystal of the hemi-fumarate of compound I, respectively. [Figure 8-3]These are the XRPD pattern, 1H NMR spectrum, and TGA / DSC pattern of the E-type crystal of the hemi-fumarate of compound I, respectively. [Figure 8-4] These are the XRPD patterns of the E-type crystal of the hemi-fumarate of compound I before and after heating. [Figure 9-1] These are the XRPD pattern, 1H NMR spectrum, and TGA / DSC pattern of the F-type crystal of the hemi-fumarate of compound I, respectively. [Figure 9-2] These are the XRPD pattern, 1H NMR spectrum, and TGA / DSC pattern of the F-type crystal of the hemi-fumarate of compound I, respectively. [Figure 9-3] These are the XRPD pattern, 1H NMR spectrum, and TGA / DSC pattern of the F-type crystal of the hemi-fumarate of compound I, respectively. [Figure 10-1] These are the XRPD pattern, 1H NMR spectrum, and TGA / DSC pattern of the G-type crystal of the hemi-fumarate of compound I, respectively. [Figure 10-2] These are the XRPD pattern, 1H NMR spectrum, and TGA / DSC pattern of the G-type crystal of the hemi-fumarate of compound I, respectively. [Figure 10-3] These are the XRPD pattern, 1H NMR spectrum, and TGA / DSC pattern of the G-type crystal of the hemi-fumarate of compound I, respectively. [Figure 11] In vivo pharmacodynamic studies of compounds of formula I in mice - tumor growth curves in animals. [Figure 12] In vivo pharmacodynamic studies of compounds of formula I in mice - body weight of the animals during the test period. [Modes for carrying out the invention]

[0086] The present disclosure will be further explained below with specific examples and test cases, but the scope of this disclosure is not limited in any way.

[0087] Test conditions for the equipment used in the experiment: Powder X-ray diffraction (XRPD) (1) Except for the D-type crystal of the hemi-fumarate of compound I, X-ray powder diffraction data of the samples were collected under environmental conditions using a Bruker D2 powder X-ray diffractometer for all other crystal types. ~2 mg of sample was taken, laid flat on a high-purity silicon sample stage, pressed flat with a glass slide separated by weighing paper, and the sample was injected for detection. A Cu target (Kα) was used for the X-ray tube, the Kα2 / Kα1 intensity ratio was 0.50 (1.54439 Å / 1.5406 Å), the X-ray generator output was 300 W, the voltage was 30 kV, and the current was 10 mA. The divergence slit was 0.6 mm, and the solar slit was 4.0°. The step rate was 0.15 s / step, the step width was 0.02° (2θ), and the total number of steps was 1837.

[0088] (2) Rigaku Smart SE: A powder X-ray diffractometer of the Rigaku model, with a voltage of 40 kV and a current of 40 mA. The X-ray tube used a Cu target (Kα), and the intensity ratio of Kα2 / Kα1 was 0.50 (1.544414 Å / 1.540593 Å). The entrance slit was 0.5° and the receiving slit was 20.0 mm. A sample of 1-2 mg was collected under environmental conditions, and the sample was prepared by pressing it flat on a sample stage without background signal. X-ray powder diffraction data of the sample was collected under environmental conditions. The test range was 3-40° (2θ), the step rate was 8° / min, and the step width was 0.01° / step.

[0089] Thermogravimetric analyzer (TGA) Thermogravimetric data of the sample was collected using the TA Discovery series thermogravimeter TGA550. A few milligrams of sample were taken and placed in a Tzero aluminum pan (automatically weighed by the instrument during testing), and heated from room temperature to the target temperature under N2 protection, with an N2 flow rate of 60 mL / min and a heating rate of 10°C / min.

[0090] Differential scanning calorimeter (DSC) Thermal data of the sample was collected using a TA Discovery series differential scanning calorimeter DSC2500. Several mg of the sample was weighed into a Tzero aluminum pan and sealed with a Tzero airtight lid. Under N2 protection, it was heated to the target temperature (before decomposition temperature) with an N2 flow rate of 50 mL / min and a heating rate of 10°C / min.

[0091] Proton nuclear magnetic resonance ( 1 (H NMR) The samples were dissolved in DMSO-d6 to prepare solutions with concentrations of approximately 2–10 mg / mL, and proton nuclear magnetic resonance data of the samples were collected using a Bruker AVANCE NEO 400 MHz radio.

[0092] High-performance liquid chromatography (HPLC) Sample purity data was measured using an Agilent 1260 high-performance liquid chromatograph (with DAD detector) and a Zorbax Eclipse XDB-C18 column (4.6 × 250 mm, 5 μm).

[0093] Example 1: Synthesis and Characterization of Compound I 1.1 Synthesis of Intermediate 1-2 TIFF2026522276000004.tif24170

[0094] Intermediate 1-1 (20.0 g, 78.1 mmol), bis(dibenzylideneacetone)palladium (0.715 g, 0.78 mmol), and 2-dicyclohexylphosphino-2'-(N,N-dimethylamino)-biphenyl (0.615 g, 1.56 mmol) were dissolved in tetrahydrofuran (32 mL), and N-methylpiperazine (12.5 g, 125 mmol) and lithium bis(trimethylsilyl)amide (1.00 M, 187 mL) were added at 0°C. The reaction was carried out at 70°C for 3 hours. Water (200 mL) was added to quench the mixture, and dichloromethane (100 mL x 3) was added for extraction. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the organic phase was concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (methanol / dichloromethane = 1%~10%) to obtain intermediates 1-2 (15.5 g, yield 72.2%, brown solid). MS (ESI) m / z = 276.0 [M+H] + .

[0095] 1.2 Synthesis of Intermediates 1-3 TIFF2026522276000005.tif28170

[0096] Intermediate 1-2 (0.372 g, 1.35 mmol) was dissolved in 6 N HCl (2 mL), and cyanamide (0.455 g, 10.8 mmol) was added. The reaction was carried out at 80°C for 12 hours. The reaction mixture was concentrated under reduced pressure, and the residue was subjected to column chromatography (C18, acetonitrile / water = 0%~10%). Intermediate 1-3 (0.19 g, yield 45%, brown solid) was obtained. MS (ESI) m / z = 318.1 [M+H] + .

[0097] 1.3 Synthesis of Intermediates 1-5 TIFF2026522276000006.tif28170

[0098] Intermediate 1-4 (100 g, 892 mmol) was dissolved in toluene (1000 mL) and ethanol (123 g, 2680 mmol), PTSA (15.4 g, 89.2 mmol) was added, and the reaction was carried out at 120°C for 12 hours. Water (1000 mL x 2) was added and the mixture was extracted. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (ethyl acetate / petroleum ether = 0%~50%) to obtain intermediate 1-5 (57.9 g, yield 46.4%, oily substance).

[0099] 1.4 Synthesis of Intermediates 1-6 TIFF2026522276000007.tif30170

[0100] Intermediate 1-5 (30.0 g, 214 mmol) was dissolved in tetrahydrofuran (450 mL), and LiHMDS (1 M, 256 mL) was added dropwise at -50.0°C, stirring for 30 min. Diethyl oxalate (37.8 g, 258 mmol) was added, and the mixture was reacted at room temperature for 16 h. Saturated ammonium chloride (500 mL) was added to quench the mixture, and ethyl acetate (50 mL x 3) was added for extraction. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the organic phase was concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (ethyl acetate / petroleum ether = 0%~20%) to obtain intermediate 1-6 (25.0 g, yield 48.6%, oily substance). MS (ESI) m / z = 241.1 [M+H] + .

[0101] 1.5 Synthesis of Intermediates 1-7 TIFF2026522276000008.tif34170

[0102] Intermediate 1-6 (25.0 g, 104 mmol) was dissolved in acetic acid (120 mL), hydroxyethylhydrazine (8.16 g, 107 mmol) was added, and the mixture was reacted at room temperature for 15 hours. Water (500 mL) was added to quench the reaction, saturated Na2CO3 (800 mL) was added, and dichloromethane (800 mL x 3) was added for extraction. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the organic phase was concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (ethyl acetate / petroleum ether = 0%~5%) to obtain intermediate 1-7 (19.9 g, yield 76%, yellow solid). MS (ESI) m / z = 253.0 [M+H] + .

[0103] 1.6 Synthesis of Intermediates 1-8 TIFF2026522276000009.tif35170

[0104] Intermediate 1-7 (10.0 g, 39.6 mmol) was dissolved in DMF-DMA (100 mL) and reacted at 110°C for 12 hours. The reaction mixture was concentrated under reduced pressure, the residue was dissolved in methyl tert-butyl ether, and stirred for 3 hours. The mixture was filtered, the filtered cake was collected and dried to obtain intermediate 1-8 (6.0 g, crude product). This was used directly in the next step.

[0105] 1.7 Synthesis of Intermediates 1-9 TIFF2026522276000010.tif42170

[0106] Intermediates 1-8 (1.95 g, 6.34 mmol) and 1-3 (2.03 g, 6.41 mmol) were dissolved in DMF (20 mL) and reacted at 110°C for 12 hours. The reaction mixture was concentrated under reduced pressure, and the residue was subjected to silica gel column chromatography (ethyl acetate / petroleum ether = 0%~5%) to obtain intermediate 1-9 (1.90 g, yield 53.3%, yellow solid). MS (ESI) m / z = 562.2 [M+H] + .

[0107] 1.8 Synthesis of intermediates 1-10 TIFF2026522276000011.tif43170

[0108] Intermediate 1-9 (100 mg, 178 μmol) and DDQ (100 mg, 440 μmol) were dissolved in dioxane (20 mL) and reacted at 100 °C for 12 h. Saturated NaHCO3 (20 mL) was added for quenching, and ethyl acetate (20 mL × 3) was added for extraction. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the organic phase was concentrated under reduced pressure. The residue was purified by preparative liquid chromatography (C18 150×40 mm×10 μm, mobile phase: [water (TFA)-acetonitrile], B%: 25% - 55%, 10 min). Intermediate 1-10 (10 mg, yield 10%, brown solid) was obtained. MS (ESI) m / z = 560.1 [M+H] + 。

[0109] 1.9, Synthesis of Intermediate 1-11 TIFF2026522276000012.tif42170

[0110] Intermediate 1-10 (74 g, 132.25 mmol) was taken, acetonitrile (740 mL) was added, then 1 mol / L aqueous hydrochloric acid solution (264 mL, prepared by dissolving 22 mL of hydrochloric acid in 242 mL of water) was added, the temperature was raised to 60°C and the reaction was allowed to proceed for 1 hour, the temperature was lowered to room temperature and stirred for 1.5 hours, the mixture was filtered by suction, washed with an appropriate amount of 95% ethanol-water, and the filtered cake was dried under reduced pressure in an oven at 55°C to obtain the hydrochloride salt of 1-10 (37 g, yield 50%, off-white solid). Take the hydrochloride salt of intermediate 1-10 (102 g, 171.14 mmol), add methanol (1.15 L), then add 1 mol / L aqueous lithium hydroxide solution (856 mL, prepared by dissolving 35.9 g of lithium hydroxide monohydrate in 856 mL of water), raise the temperature to 60°C and react for 3 hours, then cool to room temperature, add 1 mol / L aqueous hydrochloric acid solution (740 mL, prepared by dissolving 61.7 mL of concentrated hydrochloric acid in 678.7 mL of water), stir for 0.5 hours, concentrate under reduced pressure to remove most of the methanol solvent, filter by suction, wash with 100 mL of water, and dry the filtered cake under reduced pressure in an oven at 40°C to obtain intermediate 1-11 (90 g, yield 98.9%, white solid).

[0111] 1.10 Synthesis and Characterization of Compound I TIFF2026522276000013.tif51170

[0112] Intermediate 1-11 (80 g, 154.28 mmol) was taken, ammonium chloride (24.76 g, 462.84 mmol) and N,N-diisopropylethylamine (59.8 g, 462.84 mmol) were added, and HATU (64.5 g, 169.7 mmol) was added under an ice bath. The mixture was stirred at room temperature for 0.5 hours, and an aqueous sodium hydroxide solution (3.28 L, prepared by dissolving 30.9 g of sodium hydroxide in 3.28 L of water) was added. After the addition was complete, the mixture was stirred at room temperature for 1.5 hours, filtered by suction, and the filtered cake was washed with 80 mL of water and 80 mL of methyl tert-butyl ether in sequence. The filtered cake was dried under reduced pressure in an oven at 50°C to obtain the crude product of compound I (76.6 g, yield 93.6%, white solid). The crude product of compound I (12 g, 22.6 mmol) was taken, 95% ethanol (120 mL) was added, the temperature was raised to 60°C and stirred for 1 hour, then cooled to room temperature and stirred for 3 hours, filtered by suction, washed with an appropriate amount of 95% ethanol-water, and the filtered cake was dried under reduced pressure in an oven at 55°C to obtain compound I (9.87 g, yield 82.3%, white solid). MS (ESI) m / z = 531.2 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.56 (s, 1H), 9.34 (s, 1H), 7.89 (d, J = 8.6 Hz, 1H), 7.67 (d, J = 8.8 Hz, 1H), 7.33 - 7.20 (m, 2H), 6.88 - 6.91 (m, 1H), 4.91 (t, J = 4.9 Hz, 2H), 4.65 (d, J = 5.3 Hz, 1H), 4.41 (q, J = 7.1 Hz, 2H), 3.70 - 3.54 (m, 3H), 3.28 - 3.10 (m, 3H), 3.10 - 3.09 (m, 1H), 3.16 - 3.09 (m, 1H), 2.34 - 2.19 (m, 3H), 1.39 (t, J = 7.1 Hz, 3H), 1.36 - 1.36 (m, 1H), 1.31 (t, J = 7.1 Hz, 1H), 1.26 - 1.12 (m, 1H).

[0113] As can be seen from the XRPD pattern of compound I (Figure 1-1), it is crystalline, with a relatively high degree of crystallinity, and was named type A crystal. Characteristic peaks are shown in Table 1, and the purity was 98.33 area%. As can be seen from the TGA / DSC results (Figure 1-2), the sample had one sharp endothermic peak at 226.5°C (starting temperature), was heated from room temperature to 200°C, and had a weight loss of 0.2%. 1 The 1H NMR spectrum (Figure 1-3, DMSO-d6) was consistent with the structure of compound I, and there was no clear solvent residue signal. Combined with the small weight loss of TGA and the single melting peak in the DSC, it was determined that the type A crystal was anhydrous.

[0114] TIFF2026522276000014.tif93170

[0115] Example 2: Type A crystal of maleate of compound I. 91.82 mg of maleic acid and 399.55 mg of compound I (with a molar ratio of maleic acid to compound I of 1.05) were weighed into a 20 mL glass bottle, 5 mL of ethyl acetate was added, and the mixture was suspended and stirred at room temperature for approximately 2 days. After suction filtration and washing with ethyl acetate, the filtration cake was vacuum-dried at 40°C for 3 hours to obtain 464.11 mg of type A crystals of maleate of compound I, with a chemical purity of 98.38 area%. As can be seen from the XRPD pattern (Figure 2-1), the degree of crystallinity was relatively high, and the position of characteristic peaks is shown in Table 2. As can be seen from the TGA / DSC results (Figure 2-2), the sample was heated from room temperature to 150°C, with a weight loss of 1.0% (weight loss corresponding to hemiwater was 1.4%), and there was one endothermic signal at 214.4°C (peak temperature). 1 As can be seen from the 1H NMR results (Figure 2-3), the molar ratio of maleic acid to compound I in the sample was 1.0:1, and the molar ratio of the residual solvent ethyl acetate to compound I was 0.04 (approximately 0.5 wt%). From the above characterization results, it was found that the A-type crystal of the maleate salt is the anhydrous crystalline form of the monomaleate salt.

[0116] TIFF2026522276000015.tif136170

[0117] Example 3: Type A crystal of fumarate of compound I 92.94 mg of fumaric acid and 404.63 mg of compound I (with a molar ratio of fumaric acid to compound I of 1.05) were weighed into a 20 mL glass bottle, 5 mL of 2-methyltetrahydrofuran was added, and the mixture was suspended and stirred at room temperature for approximately 2 days. After suction filtration and washing with 2-methyltetrahydrofuran, the filtration cake was vacuum-dried at 40°C for 3 hours to obtain 448.02 mg of type A crystals of fumarate of compound I, with a chemical purity of 98.36 area%. As can be seen from the XRPD pattern (Figure 3-1), the degree of crystallinity was high, and characteristic peaks are shown in Table 3. As can be seen from the TGA / DSC results (Figure 3-2), the sample was heated from room temperature to 180°C, with a weight loss of 0.3% (weight loss corresponding to hemiwater was 1.4%), and there was one endothermic signal at 266.6°C (peak temperature). 1 As can be seen from the 1H NMR results (Figure 3-3), the molar ratio of fumaric acid to compound I in the sample was 1.0:1, and the molar ratio of the residual solvent 2-methyltetrahydrofuran to compound I was 0.15 (approximately 2.0 wt%). From the above characterization results, it was found that the A-type crystal of the fumarate is the anhydrous crystalline form of the monofumarate.

[0118] TIFF2026522276000016.tif112170

[0119] Example 4: Type A crystal of succinate of compound I. 406.28 mg of compound I and 94.92 mg of succinic acid (molar ratio of succinic acid to compound I: 1.05) were weighed into a 20 mL glass bottle, 5 mL of 2-MeTHF was added, and the mixture was suspended and stirred at room temperature for approximately 2 days. After suction filtration and washing with 2-methyltetrahydrofuran, the filtered cake was vacuum-dried at 40°C for 3 hours to obtain 441.58 mg of type A crystals of succinate of compound I, with a chemical purity of 98.39 area%. As can be seen from the XRPD pattern (Figure 4-1), the degree of crystallinity was relatively low, and characteristic peaks are shown in Table 4. As can be seen from the TGA / DSC results (Figure 4-2), the sample was heated from room temperature to 150°C, with a weight loss of 0.4% (weight loss corresponding to hemiwater: 1.4%) and one endothermic signal at 223.3°C (peak temperature). 1 As can be seen from the 1H NMR results (Figure 4-3), the molar ratio of succinic acid to compound I in the sample was 1.0:1, and the molar ratio of the residual solvent 2-methyltetrahydrofuran to compound I was 0.08 (approximately 1.1 wt%). From the above characterization results, it was found that the succinate type A crystal is the anhydrous crystalline form of monosuccinate.

[0120] TIFF2026522276000017.tif99170

[0121] Example 5: Hemifumarate B-type crystal of compound I An appropriate amount of monofumarate type A crystals was weighed, dissolved in N-methylpyrrolidone (NMP), clarified, and filtered through a filtration membrane (nylon membrane, pore size 0.22 μm) to obtain the stock solution. The stock solution was dispensed into 20 mL glass bottles (each bottle containing 20 mg of the sample), and under magnetic stirring conditions, methyl tert-butyl ether was gradually added to the glass bottles until a solid appeared or the total volume of the solvent reached 9 mL. The solution was filtered by suction, and the filter cake was dried at room temperature to obtain hemifumarate type B crystals of the compound of formula I.

[0122] As can be seen from the XRPD pattern (Figure 5-1), the degree of crystallinity was relatively high, and the crystal form did not change after drying at room temperature, with characteristic peaks shown in Table 5. As can be seen from the TGA / DSC results (Figure 5-2), the sample was heated from room temperature to 140°C with a weight loss of 23.3%, and then heated to 240°C with a weight loss of 8.0%, and had four endothermic signals at 92.5°C, 105.1°C, 170.5°C, and 263.5°C (peak temperature). 1 As can be seen from the 1H NMR spectrum (Figure 5-3), the molar ratio of fumaric acid to compound I in the sample was 0.6:1, the molar ratio of N-methylpyrrolidone to compound I was 2.0:1 (approximately 24.7 wt%), and the molar ratio of methyl tert-butyl ether to compound I was 0.03 (approximately 0.3 wt%). After heating the B-type crystal of hemi-fumarate, XRPD was tested. The sample was converted to a G-type crystal of hemi-fumarate (see Example 10 for details of characterization) after heating to 140°C, and then converted to an A-type crystal of mono-fumarate after heating to 200°C. From the characterization results, it was found that the B-type crystal of hemi-fumarate was the NMP solvate of hemi-fumarate.

[0123] Repeated preparation of hemi-fumarate type B crystals: An appropriate amount of mono-fumarate type A crystals was weighed, dissolved in NMP, clarified, and filtered through a filtration membrane (nylon membrane, pore size 0.22 μm) to obtain the stock solution. The stock solution was dispensed into 4 mL glass bottles (each bottle containing 20 mg of the sample), and these were placed in a 20 mL glass bottle containing 4 mL of a poor solvent, either acetone or isopropyl acetate. The 20 mL glass bottle was tightly closed and left at room temperature until a solid precipitate formed. Then, it was filtered by suction, and the filtration cake was vacuum-dried at room temperature to obtain hemi-fumarate type B crystals. In the hemi-fumarate type B crystals obtained with acetone as the poor solvent, the molar ratio of fumaric acid to compound I was 0.7:1 in all cases, and the molar ratio of NMP to compound I was 0.9:1. In the B-type crystals of hemifumarate obtained with isopropyl acetate as the poor solvent, the molar ratio of fumaric acid to compound I was 0.7:1 in all cases, and the molar ratio of NMP to compound I was 1.1:1.

[0124] TIFF2026522276000018.tif74170

[0125] Example 6: C-type crystal of hemifumarate of compound I An appropriate amount of monofumarate type A crystals was weighed, dissolved in NMP, clarified, and filtered through a filtration membrane (nylon membrane, pore size 0.22 μm) to obtain the stock solution. The stock solution was dispensed into 20 mL glass bottles (each bottle containing approximately 20 mg of the sample), and under magnetic stirring conditions, water was gradually added to the glass bottles until a solid appeared or the total volume of the solvent reached 9 mL. The solution was filtered by suction, and the filter cake was dried at room temperature to obtain hemifumarate type C crystals of the compound of formula I.

[0126] As can be seen from the XRPD pattern (Figure 6-1), the degree of crystallinity was relatively high, and the crystal form did not change after drying at room temperature, with characteristic peaks shown in Table 6. As can be seen from the TGA / DSC results (Figure 6-2), the sample was heated from room temperature to 80°C with a weight loss of 5.8% (2.9% of which corresponds to 1 mole of water), and then heated to 140°C with a weight loss of 1.3%, and had four endothermic signals at 68.7°C, 115.6°C, 226.0°C, and 262.3°C (peak temperature), and one heat dissipation signal at 177.1°C (peak temperature). 1 As can be seen from the 1H NMR spectrum (Figure 6-3), the molar ratio of fumaric acid to compound I in the sample was 0.5:1, and no residual NMP was observed. From the characterization results, it was found that the C-type crystal of hemifumarate is the hydrate crystal form of hemifumarate.

[0127] Repeated preparation of C-type crystals of hemi-fumarate: An appropriate amount of A-type crystals of mono-fumarate was weighed, dissolved in tetrahydrofuran / water (1:1), clarified, and filtered through a filtration membrane (nylon membrane, pore size 0.22 μm) to obtain the stock solution. The stock solution was dispensed into 20 mL glass bottles (each bottle containing 20 mg of sample), and under magnetic stirring conditions, H2O, a poor solvent, was gradually added to the glass bottles until a solid appeared or the total volume of solvent reached 9 mL. The solution was filtered by suction, and the filtered cake was vacuum-dried at room temperature to obtain C-type crystals of hemi-fumarate. Using Rigaku Smart SE, XRPD tests were performed to convert the C-type crystals of hemi-fumarate to D-type crystals after heating at 90°C and 140°C, and to convert them to A-type crystals of fumarate and A-type crystals of compound I after heating at 200°C.

[0128] TIFF2026522276000019.tif112170

[0129] Example 7: D-type crystal of hemi-fumarate of compound I A sample of C-type hemi-fumarate crystals was heated to 140°C to obtain D-type hemi-fumarate crystals. XRPD testing was performed using Rigaku Smart SE, and the XRPD results are shown in Figure 7-1, with characteristic peaks shown in Table 7. 1 As can be seen from the 1H NMR spectrum (Figure 7-2), the molar ratio of fumaric acid to compound I in the sample was 0.5:1, and there was no residue of other solvents. As can be seen from the TGA / DSC results (Figure 7-3), the sample was heated from room temperature to 150°C, with a weight loss of 0.8% (1.5% weight loss corresponding to half a mole of water), and there were three endothermic signals at 62.5°C, 227.6°C, and 263.6°C (peak temperatures), and one heat dissipation signal at 170.8°C (peak temperature). After heating the D-type crystal of hemi-fumarate to 100°C, its crystal form did not change. From the above characterization results, it was found that the D-type crystal of hemi-fumarate is either the anhydrous crystalline form or the pipe hydrate of hemi-fumarate.

[0130] TIFF2026522276000020.tif37170

[0131] Example 8: E-type crystal of hemi-fumarate of compound I An appropriate amount of monofumarate type A crystals was weighed, dissolved in N,N-dimethylacetamide (DMAc), clarified, and filtered through a filtration membrane (nylon membrane, pore size 0.22 μm) to obtain the stock solution. The stock solution was dispensed into 4 mL glass bottles (each bottle containing 20 mg of the sample), and these were then placed into a 20 mL glass bottle containing 4 mL of CHCl3. The 20 mL glass bottle was tightly closed and left at room temperature until a solid precipitate formed.

[0132] The obtained solid was characterized using XRPD, and the results are shown in Figure 8-1. The sample's crystal structure remained unchanged after drying at room temperature, and characteristic peaks are shown in Table 8. As can be seen from the TGA / DSC results (Figure 8-2), the sample was heated from room temperature to 100°C with a weight loss of 3.5%, and then further heated to 200°C with a weight loss of 11.5%, and exhibited six endothermic signals at 62.6°C, 154.9°C, 185.7°C, 226.6°C, 235.5°C, and 264.5°C (peak temperature).

[0133] 1 As can be seen from the 1H NMR spectrum (Figure 8-3), the molar ratio of fumaric acid to compound I in the sample was 0.7:1, the molar ratio of DMAc to compound I was 0.4:1 (approximately 5.1 wt%), and the molar ratio of CHCl3 to compound I was 0.3:1 (approximately 5.2 wt%). After heating the E-type crystal of hemi-fumarate to 120°C, multiple peaks appeared, and after heating to 200°C, it converted to an A-type crystal of mono-fumarate + multiple peaks (all of which are indicated by asterisks), and after heating to 240°C, it converted to an A-type crystal of mono-fumarate (Figure 8-4). In conjunction with the above characterization results, it was found that the E-type crystal of hemi-fumarate is the DMAc / CHCl3 cosolvate of hemi-fumarate.

[0134] TIFF2026522276000021.tif112170

[0135] Example 9: F-type crystal of formula I compound hemifumarate An appropriate amount of monofumarate type A crystals was weighed, dissolved in DMAc, clarified, and filtered through a filtration membrane (nylon membrane, pore size 0.22 μm) to obtain the stock solution. The stock solution was dispensed into 4 mL glass bottles (each bottle containing 20 mg of the sample), and these were then placed into a 20 mL glass bottle containing 4 mL of the poor solvent, methyl tert-butyl ether. The 20 mL glass bottle was tightly closed and left at room temperature until a solid precipitate formed.

[0136] The obtained solid was characterized using XRPD, and the results are shown in Figure 9-1. The sample's crystal structure remained unchanged after drying at room temperature, and characteristic peaks are shown in Table 9. As can be seen from the TGA / DSC results (Figure 9-2), the sample was heated from room temperature to 100°C with a weight loss of 4.2%, and subsequently heated to 200°C with a weight loss of 7.7%, and exhibited four endothermic signals at 58.8°C, 199.0°C, 227.3°C, and 263.2°C (peak temperatures). 1 As can be seen from the 1H NMR spectrum (Figure 9-3), the molar ratio of fumaric acid to compound I in the sample was 0.5:1, and the molar ratio of DMAc to compound I was 0.5:1 (approximately 6.9 wt%). After heating the F-type crystal of hemi-fumarate to 120°C, the crystal form did not change. After heating to 210°C, it was converted to A-type crystal of mono-fumarate and A-type crystal of compound I, and after heating to 240°C, it was converted to A-type crystal of mono-fumarate. 1 ¹H NMR testing revealed that the molar ratio of DMAc to compound I in the sample was still 0.5. Crystallization occurred after heating the F-type crystal of hemi-fumarate to 210°C, and DMAc was still observed after heating to 120°C, indicating that the obtained F-type crystal of hemi-fumarate was the DMAc solvate of hemi-fumarate.

[0137] Repeated preparation of DMAc solvate of F-type crystals of hemi-fumarate: An appropriate amount of A-type crystals of mono-fumarate was weighed, dissolved in DMAc, clarified, and filtered through a filtration membrane (nylon membrane, pore size 0.22 μm) to obtain the stock solution. The stock solution was dispensed into 4 mL glass bottles (each bottle containing 20 mg of the sample), and these were placed in a 20 mL glass bottle containing 4 mL of ethanol, a poor solvent. The 20 mL glass bottle was tightly closed and left at room temperature. The precipitated solid was the DMAc solvate of F-type crystals of hemi-fumarate. Detection revealed that the molar ratio of fumaric acid to compound I was 0.6:1 in all cases.

[0138] TIFF2026522276000022.tif99170

[0139] Example 10: G-type crystal of hemifumarate of compound I The B-type crystals of hemi-fumarate prepared in Example 5 were heated to 140°C to obtain G-type crystals of hemi-fumarate. The XRPD of the sample is shown in Figure 10-1, and characteristic peaks are shown in Table 10. As can be seen from the TGA / DSC results (Figure 10-2), the sample was heated from room temperature to 220°C, with a weight loss of 6.4%, and exhibited three endothermic signals at 196.5°C, 225.9°C, and 263.3°C (peak temperatures). 1 As can be seen from the 1H NMR spectrum (Figure 10-3), the molar ratio of fumaric acid to compound I in the sample was 0.6:1, and the molar ratio of NMP to compound I was 0.5:1 (approximately 6.2 wt%). Combined with the TGA weight loss, the solvent in the NMR spectrum, and the conversion of the B-type crystal of hemi-fumarate to a G-type crystal by heating it to 140°C, and then to a A-type crystal of mono-fumarate by heating it to 200°C (see Example 5), it was determined that the G-type crystal of hemi-fumarate was the NMP solvate of hemi-fumarate.

[0140] TIFF2026522276000023.tif124170

[0141] Test Example 1. PLK1 kinase inhibitory activity 1. Test material: PLK1 kinase (Carnabio #05-157) Detection solution: PerkinElmer #TRF0218-M Detection equipment PerkinElmer EnVision 2. Test method: a. Using an Echo instrument, the test compound was serially diluted in a 1:3 ratio in a 384-well plate, with a maximum concentration of 1 μM, resulting in a total of 10 concentration points. Parallel well tests were performed for each concentration.

[0142] b. 5 μL / well of the PLK1 kinase and peptide substrate mixture was added to the measurement plates (including the control sample). GSK461364 was placed in each test plate as a positive control, and the DMSO solvent group was used as a negative control.

[0143] c. Centrifuge the test plate at 1000 rpm for approximately 15 seconds, and then incubate at 23°C for 15 minutes. d. Add 5 μL / well of ATP solution to start the reaction. The test plate is centrifuged at a rotation speed of e.1000 rpm for approximately 15 seconds, and then the test plate is sealed with a film and incubated at 23°C for a certain period of time. f. Add the detection solution to all wells of the measurement plate. The sample was centrifuged at 1000 rpm for approximately 15 seconds, then the film was sealed onto the measurement plate and incubated at 23°C for 60 minutes.

[0144] The test plates were read using h.EnVision. The test results were analyzed using XLFIT5 software.

[0145] 3. Test results: Inhibitory activity of the compounds related to this disclosure against PLK1 kinase IC 50 and I max (%) is shown in Table 11, I max This represented the maximum effect of the compound at the test concentration (S / B 8.52, Z factor 0.94).

[0146] TIFF2026522276000024.tif22170

[0147] Conclusion: Compound I has a clear inhibitory effect on PLK1 kinase.

[0148] Test Example 2. HCT116 cell proliferation inhibitory activity 1. Test materials: Cell line: HCT116 cells Cell culture medium: RPMI1640 + 10% FBS (GBICO) Cell culture plate: 96-well plate (Shanghai Jing'an Biotechnology Co., Ltd.) Detection reagent kit: ATPlite 1-step Luminescence (PerkinElmer) Detection equipment: BioTek multifunction microplate reader 2. Test method: HCT116 cells in the logarithmic growth phase were seeded at a density of 720 cells / well in a white-walled, clear-bottomed 96-well plate and cultured overnight in a 37°C, 5% CO2 incubator. The following day, the test compound was added to the cells at a concentration of 10 μM, diluted to nine concentrations using a 3-fold concentration gradient, with two parallel wells for each concentration. Blank medium was used as the negative control, and the same concentration of Onvansertib was used as the positive control. After adding the drug, the cells were cultured for 72 hours in a 37°C, 5% CO2 incubator. Then, an equal volume of ATPlite 1-step Luminescence reagent was added to the cells, incubated at room temperature in the dark for 3 minutes, shaken at 500 r for 2 minutes in a microshaker, and the luminescence intensity was detected using a microplate reader to calculate the cell inhibition rate.

[0149] Cell inhibition rate (%) = [100 - (Lum 被験試料 -Lum 培養液 ) / (Lum 陰性対照 -Lum 培養液 ) × 100%.

[0150] Data was processed using GraphPad Prism 7.0 to obtain cell inhibition rate curves and IC.50 I calculated it.

[0151] 3. Test results: Compound I is the half-inhibitory inhibitory concentration IC of HCT116 cell proliferation. 50 The concentration is 0.051 μM, which effectively inhibits HCT116 cell proliferation.

[0152] Test Example 3. Evaluation of the permeability and transporter substrate of the test substance. 1. Test materials: Caco-2 cells were seeded in Transwell-96 well plates at a seeding density of 1 × 10⁻⁶ 5 cells / cm 2 The cells were cultured in a carbon dioxide incubator for 21-28 days before being used in transport experiments, during which time the culture medium was changed once every 4-5 days.

[0153] 2. Test method: Hank's equilibrium salt buffer (pH 7.40±0.05) containing 10 mM HEPES was used as the transport buffer. The culture medium was removed from the culture plate, and the cells were washed twice with preheated transport buffer (approximately 75 μL and 40 mL of transport buffer were used each time for each apical well and bottom receiving plate). The inoculation and reception solutions were added to the corresponding cell plate wells (75 and 250 μL of sample were added to each apical and basal well, respectively), and bidirectional transport experiments (apical to basal (AB) and basal to apical (BA)) were initiated.

[0154] The test concentration of the test substance was 5.00 μM, and two parallel wells were prepared for each administration concentration. The test concentration of Digoxin was 10.0 μM, administered bidirectionally, while the test concentrations of Nadolol and Metoprolol were both 2.00 μM, administered unidirectionally (A-B direction), with two parallel wells prepared for each of the three control compounds. After adding the samples, the cell plates were incubated at 37±1°C, 5% CO2, and saturated humidity for 120 minutes.

[0155] The initial administration solution was the T0 sample. After adding the sample, it was taken and mixed with transport buffer and stop solution in a fixed ratio. After incubation for 120 minutes, the final sample was collected from both the administration and reception ends and similarly mixed with transport buffer and stop solution in a fixed ratio.

[0156] After shaking all samples with a vortex mixer, they were centrifuged at 3220 × g at 20°C for 20 minutes. An appropriate volume of supernatant was transferred to a sample analysis plate, and after blocking the plate, if the sample was not to be analyzed immediately, it was stored at 2-8°C and analyzed by LC-MS / MS.

[0157] The integrity of the Caco-2 cell layer was tested using the Lucifer Yellow Rejection Assay.

[0158] (1) Data analysis: The apparent permeability coefficient (P) is calculated using the following formula. app The following were calculated: flow rate (cm / s), efflux ratio (ER), and solution recovery rate (%). TIFF2026522276000025.tif32170

[0159] V R is the volume of the receiving end solution (0.075 mL for side A, 0.25 mL for side B), and Area is the relative surface area of ​​the cell monolayer (0.0804 cm²). 2 ) where Time is the incubation time (7200 s), C0 is the starting concentration (nM) of the test sample at the administration end or the ratio of the peak area of ​​the control compound, and V D This is the volume at the administration end (side A: 0.075 mL, side B: 0.25 mL), C D and C R These are the final concentrations (nM) of the test sample at the administration end and the receiving end, or the ratio of the peak areas of the control compound, respectively. The fluorescent yellow transmittance (%Lucifer Yellow) is calculated using the following formula: TIFF2026522276000026.tif10170

[0160] RFUApical and RFUBasolateral represent the relative fluorescence intensities at the apical and basal ends of fluorescent yellow, respectively. VApical and VBasolateral represent the loading volumes at the apical and basal ends (0.075 and 0.25 mL, respectively).

[0161] (2) Classification criteria: TIFF2026522276000027.tif38170

[0162] 3. Test results: The permeability results of compound I in Caco-2 cells are shown in Table 12.

[0163] TIFF2026522276000028.tif27170

[0164] Test Example 4. Evaluation of Liver Microsomal Metabolic Stability 1. Reagents: TIFF2026522276000029.tif51170

[0165] 2.Equipment TIFF2026522276000030.tif61170

[0166] 3. Test Method a. Preparation of phosphate buffer solution: A phosphate buffer solution with a pH of 7.4 and a concentration of 100 mM was prepared by dissolving phosphate buffer powder in 100 mL of pure water and stored in a refrigerator at 4°C for use.

[0167] b. All samples and the control testosterone were dissolved in DMSO to a 10 mM stock solution. The 10 mM stock solution was diluted with acetonitrile to 100 μM and prepared for use as a working fluid.

[0168] c. Preparation of liver microsome agonist solution: Stock solutions of liver microsomes (concentration 20 mg / mL) from mice, rats, and humans were taken and diluted to 0.56 mg / mL with 100 mM phosphate buffer solution to prepare liver microsome agonist solution.

[0169] d. Solution preparation: An appropriate amount of MgCl2 was weighed and prepared as a 60 mM MgCl2 solution with 100 mM phosphate buffer. An appropriate amount of NADPH was weighed and prepared as a 20 mM NADPH solution with 100 mM phosphate buffer. An equal volume of 60 mM MgCl2 solution was added to prepare an NADPH working solution containing 10 mM NADPH and 30 mM MgCl2. An acetonitrile solution containing 20 ng / mL tolbutamide was prepared as a stop solution.

[0170] e. Incubation of liver microsomes: 2 μL of each compound and the positive drug testosterone agonist were added to 178 μL of liver microsome agonist (0.56 mg / mL), gently and homogeneously mixed, and pre-incubated for 10 minutes in a 37°C shaking constant temperature water bath. After adding another 20 μL of NADPH agonist, the mixture was placed in a 37°C shaking constant temperature water bath, counting was started, and incubation was carried out for 5, 10, 20, 30, and 60 minutes.

[0171] f. Termination of the reaction: After incubation for the corresponding time, 400 μL of stop solution was added to terminate the reaction, and then the sample was immediately shaken for 1 minute. Centrifugation was performed at 10°C and 13500 rpm for 10 minutes. After centrifugation, the supernatant was transferred, and each compound was diluted with water as needed, and LC-MS analysis was performed. For the sample at minute 0, the stop solution was first added and mixed homogeneously with the liver microsomes, and then NADPH agonist was added. For the negative control sample, 20 μL of 30 mM MgCl2 solution was added instead of NADPH agonist.

[0172] 4. Data Analysis: The following formula T 1 / 2 and CL int(mic) Calculate C t =C0×e -k e t C t If =1 / 2C0, T 1 / 2 = ln2 / ke =0.693 / k e CL int(mic) =0.693 / T 1 / 2 Protein concentration of liver microsomes during incubation (mg / mL) CL int(liver) =CL int(mic) × Protein content of liver microsomes (mg) / Liver weight (g) × Ratio of liver weight to body weight The ratio of liver microsomal protein (mg) to liver weight (g) was 45 in both animals and humans.

[0173] The ratio of liver weight to body weight was 88, 40, and 20 g / kg in mice, rats, and humans, respectively.

[0174] 5. Test Results: The metabolic stability results of the compounds relating to this disclosure in mouse, rat, and human liver microsomes are shown in Table 13.

[0175] TIFF2026522276000031.tif39170

[0176] Study Example 5. In vivo pharmacodynamic studies of compounds of formula I in mice. 1. Test materials: Balb / C Nude mice, SPF grade, male, body weight 18-20 g, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0177] HCT116 cells were purchased from ATCC.

[0178] Matrigel Matrix, product number 354248, purchased from Becton, Dickinson and Company.

[0179] Kolliphor® HS 15, product number 42966, purchased from Sigma-Aldrich.

[0180] 2. Test method: a. Construction of a tumor-bearing mouse model: HCT116 cells in the logarithmic growth phase are taken, cultured, and the cells are collected and counted to 1 × 10⁻⁶. 6 Balb / C Nude mice were subcutaneously inoculated with one dose per mouse. Tumor volume and body weight of the mice were measured periodically. Seven days after inoculation, the tumor volume was 150 mm². 3 Mice of a certain size were selected, randomly divided into groups, and treated with the corresponding drugs.

[0181] b. Animal grouping and administration: All mice were randomly divided into groups: 6 mice in the negative control group (sham group, 20% Solutol HS15), 6 mice in the onvansertib monotherapy group (ONV-20 mg / kg group), and 6 mice in the formula I compound monotherapy group (T1-20 mg / kg group). The drugs were administered orally once daily for 21 consecutive days, and the tumor volume and body weight of the mice were measured periodically.

[0182] 3. Data Processing: All data was organized in Excel, and one-way ANOVA analysis and plotting were performed using GraphPad Prism 8.0 software. A p < 0.05 value indicates statistical significance. TIFF2026522276000032.tif21170

[0183] 4. Test results: As shown in Figure 11, when mice were treated with the drug for 11 days, the tumor inhibition rate in the T1-20 mg / kg group reached 82.0% (p<0.0001) compared to the sham group, while the tumor inhibition rate in the ONV-20 mg / kg group was 54.9% (p<0.05). After 16 consecutive days of administration, the tumor inhibition rate in the T1-20 mg / kg group reached 86.5% (p<0.001) compared to the sham group, while the tumor inhibition rate in the ONV-20 mg / kg group was 39.6% (p<0.05), and there was a significant difference in the data between the two groups, T1-20 mg / kg and ONV-20 mg / kg (p<0.01). After 21 consecutive days of administration, the tumor inhibition rate in the T1-20 mg / kg group reached 88.3% (p<0.0001) compared to the sham group, while the tumor inhibition rate in the ONV-20 mg / kg group was 37.0% (p<0.05). Furthermore, there was a significant difference in the data between the two groups, T1-20 mg / kg and ONV-20 mg / kg (p<0.01).

[0184] As shown in Figure 12, there was no significant difference in the body weight measured each time between the animals in each group during the study period.

[0185] 5. Test Conclusion: The compound of formula I relating to this disclosure has a significant inhibitory effect on the growth of colorectal tumor volume, and its efficacy at the same dose is superior to that of the control drug onvansertib.

[0186] Test Example 6. Hygroscopicity Study The hygroscopicity of type A crystals of compound I, maleate, fumarate, and succinate was evaluated by DVS at 25°C. All four samples were anhydrous crystalline form, and before testing, all samples were equilibrated under 0% RH conditions to remove any water or solvent adsorbed on the surface.

[0187] The water adsorption of the A-type crystals of compound I, maleate, fumarate, and succinate at 25°C / 80%RH was 0.78%, 1.43%, 0.86%, and 1.04%, respectively, indicating that the samples were slightly hygroscopic. After DVS testing, none of the crystal forms changed.

[0188] Test Example 7. Study on Solid Stability The solid stability of type A crystals of compound I, maleate, fumarate, and succinate was evaluated under three conditions: 25°C / 60%RH, 40°C / 75%RH, and 60°C. Approximately 5 mg of the sample was weighed into an HPLC vial, opened, and placed under stability conditions. After 1 week and 2 weeks, samples were taken, and the chemical purity of the crystal type was tested by HPLC, and the crystal type was detected by XRPD. The results of the solid stability are shown in Table 14. After 2 weeks under stability conditions, the crystal types of the type A crystals of compound I, maleate, fumarate, and succinate remained unchanged, and the purity of the samples did not significantly decrease.

[0189] TIFF2026522276000033.tif109170

Claims

1. A type A crystal of compound I, having characteristic diffraction peaks at 2θ angles of 4.807±0.2°, 8.532±0.2°, 11.645±0.2°, 15.160±0.2°, 18.347±0.2°, and 25.514±0.2° in an X-ray powder diffraction pattern using Cu-Kα radiation, preferably having characteristic diffraction peaks at 2θ angles of 4.807±0.2°, 8.532±0.2°, 11.645±0.2°, 15.160±0.2°, 16.768±0.2°, 18.347±0.2°, 19.000±0.2°, 19.247±0.2°, and 25.514±0.2°, more preferably having characteristic diffraction peaks at 2θ angles of 4.807±0.2°, 8.532±0.2°, 11.645±0.2°, 15.160±0.2°, 16.768±0.2°, 18.347±0.2°, 19.000±0.2°, 19.247±0.2°, and 25.514±0.2°, and more preferably having characteristic diffraction peaks at 2θ angles of 4.807±0.2°, 8.532±0.2°, and the X-ray powder diffraction pattern using Cu-Kα radiation is shown in Figure 1-1. A-type crystal of compound I.

2. A medicinal salt of a compound of formula I, wherein the medicinal salt is selected from hydrochloride, sulfate, maleate, L-aspartic acid, L-tartrate, phosphate, fumarate, citrate, malate, glycolate, L-malate, hippurate, succinate, adipine, p-toluenesulfonate, methanesulfonate, benzenesulfonate, oxalate, malonate, gentisinate, or hydrobromide, and is preferably maleate, fumarate, or succinate. A medicinal salt of a compound of formula I.

3. A type A crystal of the maleate of compound I, having characteristic diffraction peaks at 2θ angles of 8.455±0.2°, 12.802±0.2°, 13.894±0.2°, 19.029±0.2°, 20.917±0.2°, and 21.383±0.2° in an X-ray powder diffraction pattern using Cu-Kα radiation, preferably 8.455±0.2°, 12.313±0.2°, 12.802±0.2°, 13.894±0.2°, 17.051±0.2°, 17.713±0.2°, 19.029±0.2°, 20.917±0.2°, 21.383±0.2°, and 24.787±0.2°. The X-ray powder diffraction pattern using Cu-Kα radiation is most preferably characterized by diffraction peaks at the following 2θ angles: 6.883±0.2°, 8.455±0.2°, 12.313±0.2°, 12.802±0.2°, 13.894±0.2°, 14.640±0.2°, 17.051±0.2°, 17.369±0.2°, 17.713±0.2°, 19.029±0.2°, 20.917±0.2°, 21.383±0.2°, 23.818±0.2°, 24.787±0.2°, and 25.823±0.2°. A-type crystal of the maleate of compound I.

4. A type A crystal of the fumarate of compound I, having characteristic diffraction peaks at 2θ angles of 7.931±0.2°, 15.752±0.2°, 16.012±0.2°, 20.426±0.2°, 21.636±0.2°, 22.902±0.2°, and 24.234±0.2° in an X-ray powder diffraction pattern using Cu-Kα radiation, preferably 7.931±0.2°, 12.635±0.2°, 13.761±0.2°, and 15.752±0.2°. The X-ray powder diffraction pattern using Cu-Kα radiation is shown in Figure 3-1, and it is more preferably characterized by diffraction peaks at 2θ angles of 16.012±0.2°, 16.972±0.2°, 19.481±0.2°, 20.426±0.2°, 21.636±0.2°, 22.285±0.2°, 22.641±0.2°, 22.902±0.2°, 23.597±0.2°, 24.234±0.2°, 27.948±0.2°, and 29.329±0.2°. A-type crystal of the fumarate of compound I.

5. A type A crystal of succinate of compound I of formula I, having characteristic diffraction peaks at 2θ angles of 7.771±0.2°, 13.539±0.2°, 15.519±0.2°, 20.105±0.2°, 21.347±0.2°, and 22.552±0.2° in an X-ray powder diffraction pattern using Cu-Kα radiation, preferably 6.655±0.2° and 7.771± The X-ray powder diffraction pattern using Cu-Kα radiation is shown in Figure 4-1, and it exhibits characteristic diffraction peaks at 2θ angles of 0.2°, 13.539±0.2°, 15.519±0.2°, 15.781±0.2°, 17.709±0.2°, 20.105±0.2°, 21.347±0.2°, 22.552±0.2°, and 23.887±0.2°. A-type crystal of succinate of compound I.

6. A type B crystal of the hemifumarate of compound I, wherein the X-ray powder diffraction pattern using Cu-Kα radiation has characteristic diffraction peaks at 2θ angles of 15.084±0.2°, 15.501±0.2°, 17.311±0.2°, 24.838±0.2°, 26.132±0.2°, and 26.736±0.2°, preferably having characteristic diffraction peaks at 2θ angles of 8.570±0.2°, 12.545±0.2°, 15.084±0.2°, 15.501±0.2°, 17.311±0.2°, 24.838±0.2°, 26.132±0.2°, and 26.736±0.2°, and more preferably the X-ray powder diffraction pattern using Cu-Kα radiation is shown in Figure 5-1. B-type crystal of the hemi-fumarate of compound I.

7. A C-type crystal of the hemifumarate of compound I, having characteristic diffraction peaks at 2θ angles of 7.553±0.2°, 12.716±0.2°, 13.547±0.2°, 15.313±0.2°, 19.089±0.2°, and 22.755±0.2° in an X-ray powder diffraction pattern using Cu-Kα radiation, preferably 6.637±0.2°. The X-ray powder diffraction pattern using Cu-Kα radiation is more preferably shown in Figure 6-1, exhibiting characteristic diffraction peaks at 2θ angles of 7.553±0.2°, 12.716±0.2°, 13.547±0.2°, 15.313±0.2°, 19.089±0.2°, 20.121±0.2°, 20.609±0.2°, and 22.755±0.2°. C-type crystals of the hemi-fumarate of compound I.

8. A D-type crystal of the hemifumarate of compound I, having characteristic diffraction peaks at 2θ angles of 6.881±0.2°, 8.103±0.2°, 12.572±0.2°, and 13.831±0.2° in an X-ray powder diffraction pattern using Cu-Kα radiation, preferably having characteristic diffraction peaks at 2θ angles of 6.881±0.2°, 8.103±0.2°, 12.572±0.2°, 13.831±0.2°, 19.569±0.2°, 20.533±0.2°, and 24.021±0.2°, more preferably having characteristic diffraction peaks at 2θ angles of 6.881±0.2°, 8.103±0.2°, 12.572±0.2°, 13.831±0.2°, 19.569±0.2°, 20.533±0.2°, and 24.021±0.2°, and more preferably having the X-ray powder diffraction pattern using Cu-Kα radiation shown in Figure 7-1. D-type crystals of the hemi-fumarate of compound I.

9. An E-type crystal of the hemifumarate of compound I of formula I, having characteristic diffraction peaks at 2θ angles of 6.188±0.2°, 12.492±0.2°, 15.473±0.2°, 17.729±0.2°, 25.017±0.2°, and 26.603±0.2° in an X-ray powder diffraction pattern using Cu-Kα radiation, preferably having characteristic diffraction peaks at 2θ angles of 6.188±0.2°, 12.492±0.2°, 15.473±0.2°, 17.729±0.2°, 25.017±0.2°, 25.275±0.2°, 26.603±0.2°, and 31.795±0.2°, more preferably having characteristic diffraction peaks at 2θ angles of 6.188±0.2°, 12.492±0.2°, 15.473±0.2°, 17.729±0.2°, 25.017±0.2°, 25.275±0.2°, 26.603±0.2°, and 31.795±0.2°, and more preferably having the X-ray powder diffraction pattern using Cu-Kα radiation shown in Figure 8-1. E-type crystal of the hemi-fumarate of compound I.

10. A type F crystal of the hemifumarate of compound I, having characteristic diffraction peaks at 2θ angles of 8.866±0.2°, 16.651±0.2°, 25.199±0.2°, and 26.157±0.2° in an X-ray powder diffraction pattern using Cu-Kα radiation, preferably having characteristic diffraction peaks at 2θ angles of 8.185±0.2°, 8.866±0.2°, 13.449±0.2°, 16.651±0.2°, 16.841±0.2°, 25.199±0.2°, and 26.157±0.2°, more preferably having characteristic diffraction peaks at 2θ angles of 8.185±0.2°, 8.866±0.2°, 13.449±0.2°, 16.651±0.2°, 16.841±0.2°, 25.199±0.2°, and 26.157±0.2°, and more preferably having the X-ray powder diffraction pattern using Cu-Kα radiation shown in Figure 9-1. F-type crystal of the hemi-fumarate compound of formula I.

11. A G-type crystal of the hemifumarate of compound I, having characteristic diffraction peaks at 2θ angles of 8.925±0.2°, 12.025±0.2°, 12.948±0.2°, 13.525±0.2°, 15.763±0.2°, 16.793±0.2°, 21.133±0.2°, 23.547±0.2°, 25.164±0.2°, and 26.101±0.2° in an X-ray powder diffraction pattern using Cu-Kα radiation, preferably 6.707±0.2° and 7.294±0.2°. The X-ray powder diffraction pattern exhibits characteristic diffraction peaks at 2θ angles of 8.925±0.2°, 12.025±0.2°, 12.948±0.2°, 13.525±0.2°, 15.510±0.2°, 15.763±0.2°, 16.417±0.2°, 16.793±0.2°, 18.923±0.2°, 21.133±0.2°, 23.547±0.2°, 25.164±0.2°, and 26.101±0.2°, and more preferably, the X-ray powder diffraction pattern using Cu-Kα radiation is shown in Figure 10-1. G-type crystals of the hemi-fumarate of compound I.

12. A crystalline form according to any one of claims 1, 3 to 11 or a pharmaceutically acceptable salt according to claim 2, and a pharmaceutically acceptable carrier, Drug composition.

13. The use of a crystalline form according to any one of claims 1, 3 to 11, a medicinal salt according to claim 2, or a drug composition according to claim 12 in the preparation of a drug for preventing and / or treating diseases caused by and / or related to abnormal protein kinase activity.

14. Use of the crystalline form according to any one of claims 1, 3 to 11, the medicinal salt according to claim 2, or the drug composition according to claim 12 in the preparation of a drug for preventing or treating diseases caused by and / or related to abnormal PLK1 kinase activity.

15. The aforementioned diseases include cancer, cell proliferative disorders, viral infections, autoimmune diseases, and neurodegenerative diseases. The use described in claim 12 or 14.

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