Polymorph as protein kinase mek inhibitor, and preparation method therefor and use thereof

Abstract

The present invention relates to a polymorph of a benzothiazole compound as a protein kinase Mek inhibitor, and a preparation method therefor and the medical use thereof.

Description

Polymorph as protein kinase Mek inhibitor, preparation method and use thereof Technical Field

[0001] The present invention relates to polymorphs of a protein kinase Mek inhibitor benzothiazole compound, a preparation method thereof, and medical use thereof. Background Art

[0002] Overactivation of the Ras / Raf / Mek / Erk signaling pathway plays a crucial role in cancer cell proliferation and differentiation. Continuous or excessive activation of the Ras / Raf / Mek / Erk signaling pathway has been found in a variety of cancers, including pancreatic, colon, lung, bladder, kidney, skin, and breast cancers. Inhibiting the Ras / Raf / Mek / Erk signaling pathway could help treat these hyperproliferative diseases. Mek, a downstream target of Ras and Raf, plays a key role in this pathway. Its phosphorylation substrate is the MAP kinase Erk. Inhibition of Mek shuts down the Ras / Raf / Mek / Erk signaling pathway, inhibiting cancer cell proliferation. Therefore, Mek inhibitors can inhibit cancer cell growth, particularly in cancers driven by overactivation of Ras or Raf. Mek is also implicated in inflammatory diseases and conditions, including both acute and chronic inflammation.

[0003] Chinese Patent Application No. 201210190520.4 discloses a number of benzothiazole compounds that exhibit protein kinase Mek inhibitory activity, including the compound 4-fluoro-5-(2-fluoro-4-iodophenylamino)-1H-benzo[d]thiazole-6-carboxylic acid (2-hydroxy-ethoxy)-amide (hereinafter referred to as Compound 1). However, according to the preparation method of Chinese Patent Application No. 201210190520.4, the solid form of Compound 1 is amorphous. However, the purity of amorphous is generally difficult to control, the physical and chemical stability is generally poor, and the hygroscopicity is poor. Moreover, Compound 1 is unstable under light and easily degrades to form impurities (A).

[0004] This affects the efficacy and storage stability of compound 1 itself.

[0005] Therefore, it is necessary to develop a new crystal form of the compound, which is conducive to improving the purity control in process production and improving the stability and storage resistance of the compound.

[0006] Summary of the Invention

[0007] The present invention provides polymorphs of Compound 1, which exhibit advantages such as high purity, good solid-state stability, good powder properties, and mechanical stability. More specifically, the present invention provides seven crystalline forms of Compound 1, namely, Form I, Form II, Form IIIA, Form IIIB, Form IV, Form V, and Form VI. Forms I and IIIA exhibit improved solid-state stability, mechanical stability, hygroscopicity, and light stability compared to other crystalline forms or the amorphous form, thereby facilitating pharmaceutical processing.

[0008] Specifically, Forms I and IIIA of Compound 1 exhibit one or more of the following advantages over the amorphous form:

[0009] 1. The suspension competition results show that Form I is thermodynamically more stable than Form IIIA in the range from room temperature to 50°C;

[0010] 2. DVS results showed that Form I and Form IIIA were almost non-hygroscopic, and no crystal transformation occurred after DVS testing; the amorphous form transformed into Form I after DVS testing;

[0011] 3. Solid-state stability results showed that Form I did not undergo any crystal transformation or purity reduction after being stored in a closed container at 60°C for one day, or in an open container at 25°C / 60% RH or 40°C / 75% RH for one week, indicating that Form I has good physical and chemical stability under the evaluation conditions. Form IIIA maintained its purity after one day at 60°C, but diffraction peaks of Form I were observed. No crystal transformation or purity reduction occurred after being stored at 25°C / 60% RH or 40°C / 75% RH for one week. The purity of the amorphous form did not change significantly under all three evaluation conditions, but the crystal form transformed into Form I.

[0012] 4. The results of light stability show that Form I can withstand light (white light 5890Lux + ultraviolet 8.7W / m 2 ) after 24 hours under the conditions, the purity was significantly higher than that of Form IIIA, Form V and amorphous, showing better light stability;

[0013] 5. Dynamic solubility testing of Form I and the amorphous form in 1 M HCl and pH 1.0, 2.0, 4.5, and 7.4 buffers at room temperature showed that the solubility of the amorphous form was higher than that of Form I in all media within 10 minutes, with the solubility of the amorphous form first increasing and then decreasing. The highest solubility measured in 1 M HCl was higher than that in other pH buffers. Compared with the amorphous form, Form I dissolved slowly in all media (at different pH values), with a slightly higher solubility in 1 M HCl than in the other pH buffers. This ensures more stable dissolution of Form I in vivo and facilitates obtaining stable in vivo pharmacokinetics with stable blood drug concentrations, which helps avoid drug safety risks caused by excessive fluctuations in blood drug concentrations.

[0014] 6. Powder property test results show that Form I and amorphous samples have similar flowability. In addition, mechanical stability results show that after tableting (350 MPa) and manual grinding (approximately 3 minutes), Form I does not undergo a crystal transformation and the crystallinity does not decrease significantly, while the amorphous material transforms into Form I, and Form IIIA does not undergo a crystal transformation but has a decrease in crystallinity; and / or

[0015] 7. In pharmacokinetic experiments, Form I showed higher exposure and blood concentration than Form IIIA, indicating the potential for reducing the dosage.

[0016] According to the characterization data and evaluation results, Form I exhibited better light stability and no crystal transformation occurred under all evaluation conditions, while the amorphous form transformed into Form I after DVS, solid-state stability, solubility, and mechanical stability tests.

[0017] In a first aspect, the present invention provides a polymorph of formula (I)

[0018] wherein n is 0 or 1, and X is acetonitrile, water, 1,4-dioxane, ethanol, methanol, dimethylformamide, acetone, or a mixture thereof.

[0019] In some embodiments, n is 0. In some embodiments, n is 1; and X is acetonitrile, water, 1,4-dioxane, ethanol, or dimethylformamide.

[0020] In some embodiments, n is 0, and the polymorph is Form I, characterized in that the X-ray powder diffraction pattern of Form I includes the following characteristic diffraction peaks at 2θ positions: 16.71°±0.2°, 21.82°±0.2°, and 23.75°±0.2°, using Cu-Kα radiation. In some embodiments, the X-ray powder diffraction pattern of Form I also includes the following characteristic diffraction peaks at 2θ positions: 7.48°±0.2°, 22.36°±0.2°. In some embodiments, the X-ray powder diffraction pattern of Form I also includes the following characteristic diffraction peaks at 2θ positions: 5.3°±0.2°, 24.57°±0.2°, and 27.08°±0.2°. In some embodiments, the X-ray powder diffraction pattern of the crystalline form I further comprises characteristic diffraction peaks at the following 2θ positions: 11.81°±0.2°, 15.83°±0.2°, 17.92°±0.2°, 18.95°±0.2°, and 19.17°±0.2°. In some embodiments, the X-ray powder diffraction pattern of the crystalline form I comprises characteristic diffraction peaks at the following 2θ positions: 5.30°±0.2°, 7.48°±0.2°, 11.81°±0.2°, 14.85°±0.2°, 15.83°±0.2°, 16.71°±0.2°, 17.92°±0.2°, 18.95°±0.2°, 19.17°±0.2°, 19.43°±0.2°, 21.14°±0.2° , 21.82°±0.2°, 22.36°±0.2°, 23.75°±0.2°, 24.57°±0.2°, 27.08°±0.2°, 27.83°±0.2°, 28.88°±0.2°, 31.20°±0.2°, 31.92°±0.2°, 32.40°±0.2°, 33.91°±0.2°, 35.83°±0.2°, 37.51°±0.2°, and 39.04°±0.2°. In some embodiments, the X-ray powder diffraction pattern of Form I is substantially as shown in Fig. 3. In some embodiments, the Form I has a TGA pattern and / or DSC pattern as shown in Fig. 4. In some embodiments, the Form I is an anhydrate.

[0021] In some embodiments, n is 1, X is acetonitrile, and the polymorph is Form II, characterized in that the X-ray powder diffraction pattern of Form II includes the following characteristic diffraction peaks at 2θ positions: 24.99°±0.2°, 26.05°±0.2°, and 22.6°±0.2°, using Cu-Kα radiation. In some embodiments, the X-ray powder diffraction pattern of Form II also includes the following characteristic diffraction peaks at 2θ positions: 6.35°±0.2°, 20.34°±0.2°, 22.41°±0.2°, and 28.71°±0.2°. In some embodiments, the X-ray powder diffraction pattern of the crystalline Form II further comprises characteristic diffraction peaks at the following 2θ positions: 9.18°±0.2°, 16.03°±0.2°, 18.25°±0.2°, 27.07°±0.2°, 29.08°±0.2°, and 33.93°±0.2°. In some embodiments, the X-ray powder diffraction pattern of the crystalline Form II further comprises characteristic diffraction peaks at the following 2θ positions: 14.44°±0.2°, 24.64°±0.2°, 26.41°±0.2°, 32.27°±0.2°, 32.68°±0.2°, 37.07°±0.2°, and 39.51°±0.2°. In some embodiments, the X-ray powder diffraction pattern of the crystalline form II includes the following characteristic diffraction peaks at 2θ positions: 6.35°±0.2°, 9.18°±0.2°, 9.91°±0.2°, 14.44°±0.2°, 16.03°±0.2°, 18.25°±0.2°, 19.77°±0.2°, 20.34°±0.2°, 21.81°±0.2°, 22.41°±0.2°, 22.60°±0.2°, 23.84°±0.2°, 24.64°±0.2°, 24.99°±0.2°, 25.43 .2°, 34.19°±0.2°, 35.42°±0.2°, 37.07°±0.2°, 37.56°±0.2°, 38.69°±0.2°, and 39.51°±0.2°. In some embodiments, the X-ray powder diffraction pattern of Form II is substantially as shown in Fig. 6. In some embodiments, the crystalline Form II has a TGA graph and / or DSC graph as shown in Fig. 7 .

[0022] In some embodiments, n is 0, and the polymorph is Form IIIA, characterized in that the X-ray powder diffraction pattern of Form IIIA includes the following characteristic diffraction peaks at 2θ positions: 6.59°±0.2°, 22.69°±0.2°, 20.32°±0.2°, 23.62°±0.2°, 23.91°±0.2°, and 24.15°±0.2°, using Cu-Kα radiation. In some embodiments, the X-ray powder diffraction pattern of Form IIIA also includes the following characteristic diffraction peaks at 2θ positions: 10.8°±0.2°, 17.14°±0.2°, 13.75°±0.2°, 21.59°±0.2°, and 26.01°±0.2°. In some embodiments, the X-ray powder diffraction pattern of the crystalline form IIIA further includes characteristic diffraction peaks at the following 2θ positions: 18.71°±0.2°, 21.97°±0.2°, 25.54°±0.2°, 27.13°±0.2°, 27.59°±0.2° and 30.51°±0.2°. In some embodiments, the X-ray powder diffraction pattern of the crystalline form IIIA comprises characteristic diffraction peaks at the following 2θ positions: 6.59°±0.2°, 9.9°±0.2°, 10.8°±0.2°, 13.09°±0.2°, 13.75°±0.2°, 17.14°±0.2°, 17.87°±0.2°, 18.71°±0.2°, 19.19°±0.2°, 20.32°±0.2°, 21.59°±0.2°. °, 21.97°±0.2°, 22.69°±0.2°, 23.62°±0.2°, 23.91°±0.2°, 24.15°±0.2°, 25.54°±0.2°, 26.01°±0.2°, 27.13°±0.2°, 27.59°±0.2°, 28.83°±0.2°, 29.24°±0.2°, 30.51°±0.2°, 31.13°±0.2°, 31.79°±0.2°, 33.6°±0.2°, 34.14°±0.2°, 36.08°±0.2°, 36.67°±0.2° and 37.26°±0.2°. In some embodiments, the X-ray powder diffraction pattern of the crystalline Form IIIA is substantially as shown in Fig. 9. In some embodiments, the crystalline Form IIIA has a TGA chart and / or DSC chart as shown in Fig. 10. In some embodiments, the crystalline Form IIIA is an anhydrate.

[0023] In some embodiments, n is 0, and the polymorph is Form IIIB, characterized in that the Form IIIB has an X-ray powder diffraction pattern comprising the following characteristic diffraction peaks at 2θ positions: 6.53°±0.2°, 13.69°±0.2°, 18.6°±0.2°, 20.19°±0.2°, 21.52°±0.2°, and 22.64°±0.2°, using Cu-Kα radiation. In some embodiments, the Form IIIB has an X-ray powder diffraction pattern further comprising the following characteristic diffraction peaks at 2θ positions: 10.75°±0.2°, 17.07°±0.2°, 21.93°±0.2°, 26.13°±0.2°, 23.57°±0.2°, and 30.46°±0.2°. In some embodiments, the X-ray powder diffraction pattern of the crystalline form IIIB further includes characteristic diffraction peaks at the following 2θ positions: 13.05°±0.2°, 16.63°±0.2°, 20.82°±0.2°, 24.01°±0.2°, 27.55°±0.2°, and 31.79°±0.2°. In some embodiments, the X-ray powder diffraction pattern of the crystalline form IIIB includes the following characteristic diffraction peaks at 2θ positions: 6.53°±0.2°, 10.75°±0.2°, 12.62°±0.2°, 13.05°±0.2°, 13.69°±0.2°, 16.63°±0.2°, 17.07°±0.2°, 18.60°±0.2°, 19.59°±0.2°, 20.19°±0.2°, 20.82°±0.2°, 21. In some embodiments, the Form IIIB has an X-ray powder diffraction pattern substantially as shown in Fig. 13. In some embodiments, the Form IIIB has a TGA pattern and / or a DSC pattern as shown in Fig. 14.

[0024] In some embodiments, n is 1, X is 1,4-dioxane, and the polymorph is Form IV, characterized in that the X-ray powder diffraction pattern of Form IV includes the following characteristic diffraction peaks at 2θ positions: 8.56°±0.2°, 13.29°±0.2°, 17.69°±0.2°, 19.75°±0.2°, and 22.45°±0.2°, using Cu-Kα radiation. In some embodiments, the X-ray powder diffraction pattern of Form IV also includes the following characteristic diffraction peaks at 2θ positions: 5.26°±0.2°, 18.29°±0.2°, 31.83°±0.2°, 25.68°±0.2°, 22.86°±0.2°, 32.81°±0.2°, and 23.44°±0.2°. In some embodiments, the X-ray powder diffraction pattern of the crystalline form IV further includes characteristic diffraction peaks at the following 2θ positions: 126.57°±0.2°, 27.52°±0.2°, 35.69°±0.2°, 21.09°±0.2°, 20.35°±0.2° and 31.43°±0.2°. In some embodiments, the X-ray powder diffraction pattern of the crystalline form IV includes the following characteristic diffraction peaks at 2θ positions: 5.26°±0.2°, 8.56°±0.2°, 9.85°±0.2°, 13.29°±0.2°, 17.69°±0.2°, 18.29°±0.2°, 19.75°±0.2°, 20.35°±0.2°, 21.09°±0.2°, 22.45°±0.2°, 22.86 In some embodiments, the crystalline Form IV has an X-ray powder diffraction pattern substantially as shown in Fig. 17. In some embodiments, the crystalline Form IV has a TGA pattern and / or a DSC pattern as shown in Fig. 18.

[0025] In some embodiments, n is 1, X is ethanol, and the polymorph is Form V, characterized in that the X-ray powder diffraction pattern of Form V includes the following characteristic diffraction peaks at 2θ positions: 6.21°±0.2°, 8.47°±0.2°, 15.62°±0.2°, 21.73°±0.2°, 25.53°±0.2°, 25.94°±0.2°, and 28.05°±0.2°, using Cu-Kα radiation. In some embodiments, the X-ray powder diffraction pattern of Form V also includes the following characteristic diffraction peaks at 2θ positions: 9.61°±0.2°, 17.55°±0.2°, 19.25°±0.2°, 22.22°±0.2°, 23.12°±0.2°, 32.92°±0.2°, and 34.22°±0.2°. In some embodiments, the X-ray powder diffraction pattern of the crystalline Form V further includes characteristic diffraction peaks at the following 2θ positions: 9.06°±0.2°, 20.07°±0.2°, 28.49°±0.2°, 30.21°±0.2°, 31.25°±0.2°, 35.47°±0.2° and 38.94°±0.2°. In some embodiments, the X-ray powder diffraction pattern of the crystalline form V includes the following characteristic diffraction peaks at 2θ positions: 6.21°±0.2°, 8.47°±0.2°, 25.94°±0.2°, 15.62°±0.2°, 25.53°±0.2°, 28.05°±0.2°, 21.73°±0.2°, 17.55°±0.2°, 32.92°±0.2°, 23.12°±0.2°, 22.22°±0.2°, 19.25°±0.2°, 34.22°±0.2°, 9.61 In some embodiments, the X-ray powder diffraction pattern of Form V is substantially as shown in Fig. 20. In some embodiments, the crystalline Form V has a TGA graph and / or DSC graph as shown in Fig. 21.

[0026] In some embodiments, n is 1, X is dimethylformamide, and the polymorph is Form VI. In some embodiments, the X-ray powder diffraction pattern of Form VI is substantially as shown in Fig. 23. In some embodiments, the Form VI has a TGA pattern and / or DSC pattern as shown in Fig. 24.

[0027] In some embodiments, Form I, Form II, Form IIIA, Form IIIB, Form IV, Form V and Form VI each have a purity of about 85% or higher, for example, about 90% or higher, for example, about 95% or higher, for example, about 97% or higher, for example, about 99% or higher and including about 99.9% or higher, based on the weight of Compound 1, as determined by HPLC (high performance liquid chromatography). The remaining materials may include a crystalline form of Compound 1 and / or reaction impurities and / or processing impurities produced by its preparation, such as photodegradation impurity compound (A). Mixtures of Form I of Compound 1 with other solid forms (e.g., other crystalline forms and amorphous forms) are also within the scope of this disclosure.

[0028] In some embodiments, the polymorph of Compound 1 is substantially free of impurity (A). In some embodiments, the polymorph of Compound 1 contains less than 0.15% impurity (A) by weight relative to the polymorph, such as 0.10%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% impurity (A) by weight.

[0029] In some embodiments, Form I of Compound 1 is substantially free of impurity (A). In some embodiments, Form I of Compound 1 contains less than 0.15% by weight impurity (A), such as 0.10%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% by weight impurity (A), relative to Form I.

[0030] In some embodiments, Form I, Form II, Form IIIA, Form IIIB, Form IV, Form V, and Form VI each have a crystallinity of about 85% or greater, such as about 90% or greater, such as about 95% or greater, such as about 97% or greater, such as about 99% or greater, and including about 99.9% or greater.

[0031] In a second aspect, the present invention provides a pharmaceutical composition comprising any one of the polymorphs of Forms I to VI of the present invention, and a pharmaceutically acceptable carrier and / or excipient. In some embodiments, the pharmaceutical composition comprises Forms I or IIIA of the present invention, and a pharmaceutically acceptable carrier and / or excipient. In some embodiments, the pharmaceutical composition comprises Form I of the present invention, and a pharmaceutically acceptable carrier and / or excipient.

[0032] In a third aspect, the present invention provides a method for treating tumors, chronic inflammatory diseases, inflammatory bowel diseases, skin diseases, diabetes, eye diseases, diseases related to angiogenesis or angiogenesis in mammals, diseases related to chronic pain, and other diseases modulated by the Mek cascade in mammals, the method comprising administering to the mammal any one of the polymorphs of Forms I to VI of the present invention; the present invention provides any one of the polymorphs of Forms I to VI of the present invention for use in treating tumors, chronic inflammatory diseases, inflammatory bowel diseases, skin diseases, diabetes, eye diseases, diseases related to angiogenesis or angiogenesis in mammals, diseases related to chronic pain, and other diseases modulated by the Mek cascade in mammals; and use of any one of the polymorphs of Forms I to VI of the present invention in the preparation of a drug for treating tumors, chronic inflammatory diseases, inflammatory bowel diseases, skin diseases, diabetes, eye diseases, diseases related to angiogenesis or angiogenesis in mammals, diseases related to chronic pain, and other diseases modulated by the Mek cascade in mammals. In some embodiments, the mammal is a human.

[0033] In a fourth aspect, the present invention provides a method for treating RAS or RAF mutant cancer in a mammal, the method comprising administering 4-fluoro-5-(2-fluoro-4-iodophenylamino)-1H-benzo[d]thiazole-6-carboxylic acid (2-hydroxy-ethoxy)-amide (Compound 1) or a pharmaceutically acceptable salt thereof to the mammal. In some embodiments, the compound 1 is any one of the polymorphs of Form I to Form VI. In some embodiments, the RAS or RAF mutant cancer is, for example, a KRAS mutant cancer, a NRAS mutant cancer, a HRAS mutant cancer, or a BRAF mutant cancer. In some embodiments, the RAS mutant cancer is pancreatic cancer, colorectal cancer, lung cancer, melanoma, acute myeloid leukemia, bladder cancer, or head and neck cancer. In a preferred embodiment, the cancer is a NRAS mutant cancer. In some embodiments, the NRAS mutant cancer is a melanoma in which RNAS is mutated.

[0034] In one embodiment, KRAS comprises a mutation at one or more positions selected from codons 12, 13, 59, and 61. In one embodiment, the mutant form of KRAS has a mutation at one or more amino acid positions selected from G12, G13, S17, P34, A59, and Q61. In one embodiment, the mutant form of KRAS has one or more amino acid substitutions selected from the group consisting of G12C, G12S, G12R, G12F, G12L, G12N, G12A, G12D, G12V, G13C, G13S, G13D, G13V, G13P, S17G, P34S, A59E, A59G, A59T, Q61K, Q61L, Q61R, and Q61H. In one embodiment, the KRAS mutant form has a mutation at one or more amino acid positions selected from G12, G13, A59, Q61, K117, and A146. In one embodiment, the KRAS mutant form has one or more amino acid substitutions selected from the group consisting of G12C, G12R, G12S, G12A, G12D, G12V, G13C, G13R, G13S, G13A, G13D, G13V, A59E, A59G, A59T, Q61K, Q61L, Q61R, Q61H, K117N, K117R, K117E, A146P, A146T, and A146V. In one embodiment, the BRAF mutation is a BRAF V600E mutation.

[0035] In one embodiment, NRAS comprises a mutation at one or more positions selected from codons 12, 13, 59, 61, and 146. In some embodiments, the NRAS mutant form has a mutation at one or more amino acid positions selected from G12, G13, A59, Q61, K117, and A146. In some embodiments, the NRAS mutant form has one or more amino acid substitutions selected from the group consisting of G12C, G12R, G12S, G12A, G12D, G12V, G13C, G13R, G13S, G13A, G13D, G13V, A59D, A59T, Q61K, Q61L, Q61R, Q61H, K117N, K117R, K117E, A146P, A146T, and A146V.

[0036] In some embodiments, the cancer is an early, intermediate, or late stage cancer. The cancer can be locally advanced or metastatic. In some embodiments, the mammal has previously received immunotherapy. In some embodiments, the mammal has previously received immunotherapy and has an advanced melanoma with a NRAS mutation. In some embodiments, the melanoma is selected from the group consisting of: advanced melanoma, unresectable melanoma, metastatic melanoma, melanoma with a BRAF mutation, melanoma with a NRAS mutation, cutaneous melanoma, or intraocular melanoma.

[0037] In some embodiments, Compound 1 is in the form of a tablet, powder, granule, patch, inhaler, or capsule. In some embodiments, Compound 1 is in the form of a capsule. In some embodiments, Compound 1 is administered at a dose of 5-50 mg once or twice daily. In some embodiments, Compound 1 is administered at a dose of 12 mg twice daily.

[0038] In a fifth aspect, the present invention provides a method for preparing the crystalline form I of the present invention, the method comprising any one of the following:

[0039] a) adding an amorphous sample of Compound 1 to a solvent, then heating the solution at a temperature above about 70°C, cooling the resulting clear solution to room temperature, maintaining the solution at room temperature with continuous stirring, and allowing a solid to precipitate, which is then filtered and dried; or

[0040] b) dissolving an amorphous sample of Compound 1 in a good solvent, filtering to obtain a clear solution, and adding an antisolvent while stirring the clear solution until solids precipitate; or

[0041] c) Dissolve the amorphous sample of Compound 1 in a solvent, stir at about 50° C., and then filter the filtrate. Cool the filtrate to about 5° C. and collect the precipitated solid.

[0042] In some embodiments, the heating temperature in method 1) is from about 75°C to about 100°C; from about 80°C to about 90°C; or from about 75°C or about 85°C. In some embodiments, the room temperature in method 1) is from about 20°C to about 25°C. In some embodiments, stirring is continued for about 0.5-12 hours, about 1-12 hours, about 1-8 hours, about 1-5 hours, or longer, or stirring is continued for about 24-about 96 hours. In some embodiments, the supernatant in method 1) is cooled to room temperature within about 2 to about 5 hours or about 2.5 to about 3 hours. In some embodiments, after stirring at room temperature in method 1), the temperature may be further lowered to about 0-about 10°C and stirred. In some embodiments, stirring is continued at about 0-about 10°C for about 1-about 12 hours, about 1-about 8 hours, about 1-about 5 hours, or at about 0-about 10°C for longer, depending on the situation. In some embodiments, the solvent in method 1) is water, methanol, ethanol, isopropanol, acetone, methyl isobutyl ketone, 2-butanone, ethyl acetate, isopropyl acetate, methyl tert-butyl ether, tetrahydrofuran, anisole, 2-methyltetrahydrofuran, cyclopentyl methyl ether, 1,4-dioxane, acetonitrile, dichloromethane, toluene, m-xylene, n-heptane, n-hexane, n-pentane, dimethyl sulfoxide, dimethylacetamide, N-methylpyrrolidone, or a mixture thereof. In some embodiments, the solvent in method 1) is ethanol.

[0043] In some embodiments, the good solvent in method 2) is a solvent in which compound 1 is soluble, and the anti-solvent in method 2) is a solvent in which compound 1 is not soluble. In some embodiments, the good solvent in method 2) is MEK, 1,4-dioxane, or DMSO. In some embodiments, the anti-solvent in method 2) is MTBE, EtOAc, CHCl3, n-heptane, Anisole, EtOAc, H2O, IPAc, CPME, DCM, or toluene.

[0044] In some embodiments, the cooling rate in method 3) is about 0.1°C / min. In some embodiments, the solvent in method 3) is MIBK, Methyl acetate, 2-MeTHF, or acetone / EtOH (1:1).

[0045] The present invention provides a method for preparing Form V of the present invention, comprising: adding a sample of amorphous Compound 1 to a solvent, then heating at a temperature below about 70°C, cooling the resulting clear solution to about 0-10°C, standing at about 5°C until solids precipitate, and drying. In some embodiments, the method is heated at about 70°C, about 60°C, or about 50°C. In some embodiments, the cooling rate is about 0.1-0.5°C / minute. In some embodiments, the cooling rate is about 0.1°C / minute. In some embodiments, the resulting clear solution is cooled to about 5°C.

[0046] The present invention provides a method for preparing Form IIIA of the present invention, comprising: heating Form II to a first temperature and holding the temperature for about 3 to about 10 minutes, followed by cooling to obtain Form IIIA. In some embodiments, the first temperature is about 100 to about 140°C, for example, about 110 to about 130°C, for example, about 120°C. In some embodiments, the Form II is held at the first temperature for about 5 minutes. In some embodiments, after holding the temperature, the Form II is cooled to room temperature to about 50°C, for example, to room temperature.

[0047] Differential scanning calorimetry (DSC) is well known in the art. The melting peak height of a DSC curve depends on many factors related to sample preparation and instrument geometry, while the peak position is relatively insensitive to experimental details. Therefore, in some embodiments, the crystalline compound of the present invention has a DSC pattern with characteristic peak positions, has substantially the same properties as the DSC pattern provided in the accompanying drawings of the present invention, and has a measurement error tolerance of within ±5°C, generally required to be within ±3°C.

[0048] The numerical values ​​described and claimed in this invention are approximate. Variations in the numerical values ​​may be due to equipment calibration, equipment errors, crystal purity, crystal size, sample size, and other factors.

[0049] The crystal forms of the present invention are not limited to characteristic spectra that are exactly the same as those described in the drawings disclosed in the present invention, such as XRPD, DSC, TGA, DVS, and isothermal adsorption curves. Any crystal forms having characteristic spectra that are substantially the same or essentially the same as those described in the drawings fall within the scope of the present invention.

[0050] the term

[0051] Unless otherwise specifically defined in this document, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0052] As used herein, including in the claims, singular forms such as "a," "an," and "the" include their plural counterparts unless the context clearly indicates otherwise. Thus, for example, reference to "a crystal form" includes one or more such different crystal forms and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art, which may be modified or substituted for the methods described herein.

[0053] Throughout the following specification and claims, unless the context requires otherwise, the term "comprises" and variations such as "comprising" and "including" will be understood to imply the inclusion of a stated integer or step or set of integers or steps, but not the exclusion of any other integer or step or set of integers or steps. When used herein, the term "comprising" may be replaced with the term "containing" or, at times, with the term "having" when used.

[0054] The term "about" means plus or minus 10% or 5% or 2% of the value of the specified term.

[0055] A "therapeutically effective amount" refers to the amount of a compound that elicits the physiological or medical response of a tissue, system, or subject that is being treated, including an amount of a compound that, when administered to a subject, is sufficient to prevent the occurrence of, or to alleviate to some degree, one or more symptoms of the disorder or condition being treated. A "therapeutically effective amount" may vary depending on the compound, the disease, disorder, and / or symptoms of the disease or condition, the severity of the disease or condition, and / or symptoms of the disease or condition, the age of the subject being treated, and / or the weight of the subject being treated. The appropriate amount in any given situation will be apparent to one skilled in the art or can be determined by routine experimentation. In the context of combination therapy, a "therapeutically effective amount" refers to the total amount of the combined subject effective to treat the disease, disorder, or condition.

[0056] "Excipient" refers to a substance that is not a therapeutic agent itself but is used as a diluent, adjuvant, binder and / or vehicle and is added to a pharmaceutical composition to improve its handling or storage properties or to allow or facilitate the formation of a compound or pharmaceutical composition into a unit dosage form for administration.

[0057] "Crystal form" or "crystal" or "polymorph" refers to any solid material exhibiting a three-dimensional ordering, in contrast to amorphous solid material, which produces a characteristic XRPD pattern with well-defined peaks.

[0058] "Amorphous" refers to a non-crystalline molecular and / or ionic solid form. Amorphous solids do not exhibit X-ray diffraction patterns with sharp maxima.

[0059] "Hydrate" refers to a crystalline form of a molecule that further comprises water incorporated into the crystalline structure. The water molecules in a hydrate may exist in a regular arrangement and / or a disordered arrangement. Hydrates may comprise stoichiometric or non-stoichiometric amounts of water molecules.

[0060] "Anhydrate" refers to a crystalline form that contains substantially no water molecules in any form, for example, a crystalline form that contains substantially no water molecules in the crystal lattice or unit cell.

[0061] The term "solvate" refers to a crystalline form of a molecule further comprising one or more solvent molecules incorporated into the crystalline structure. The solvent molecules in the solvate can exist in a regular arrangement and / or a disordered arrangement. The solvate can comprise a stoichiometric or non-stoichiometric amount of solvent molecules. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methoxides, and isopropoxides, acetic acid. Methods of solvation are generally known in the art. It is noteworthy that in a solvate, the substance combined with the main molecule (e.g., active pharmaceutical ingredient) is liquid at room temperature, while in a eutectic, the substance is solid at room temperature.

[0062] The crystalline forms disclosed herein are substantially pure crystals. The term "substantially pure" as used herein refers to at least 85% by weight, preferably at least 95% by weight, more preferably at least 99% by weight of the crystalline forms disclosed herein, and also includes about 100% by weight of a crystalline form. The remaining material includes one or more other forms of the compound and / or reaction impurities and / or processing impurities resulting from its preparation. For example, a crystalline form of Compound 1 can be considered substantially pure because it has a purity greater than 90% by weight, as measured by means known and generally accepted in the art at the time, wherein the remaining less than 10% by weight of the material comprises an amorphous form of Compound 1 and / or one or more other forms and / or reaction impurities and / or processing impurities.

[0063] "X-ray powder diffraction pattern (XRPD pattern)" refers to an experimentally observed diffraction pattern or a parameter, data, or value derived therefrom. XRPD patterns are typically characterized by peak positions (abscissa) and / or peak intensities (ordinate). For the crystalline forms disclosed herein, only the principal peaks (i.e., the most characteristic, prominent, unique, and / or reproducible peaks) are summarized; other peaks can be obtained from the diffraction patterns by conventional methods. The principal peaks described above are reproducible within a margin of error (±2 of the last decimal place given, or ±0.2 of the given value).

[0064] "2θ" refers to the peak position expressed in degrees (°) based on the setup of an X-ray diffraction experiment and is typically the unit of the abscissa in a diffraction pattern. If the incident beam forms an angle θ with a certain lattice plane and the reflection is diffracted, the experimental setup requires that the reflected beam be recorded at an angle of 2θ. It should be understood that references to specific 2θ values ​​for specific crystalline forms in this application are intended to represent the 2θ value (expressed in degrees) measured using the X-ray diffraction experimental conditions described in this application.

[0065] The term "substantially the same" or "substantially as shown in Figure XX" with respect to X-ray diffraction peaks is intended to take into account variations in representative peak positions and intensities. For example, one skilled in the art will appreciate that peak positions (2θ) will exhibit some variation, typically as much as 0.1-0.2°, and that the instrument used to measure diffraction will also introduce some variation. Furthermore, one skilled in the art will appreciate that relative peak intensities will vary due to instrument-to-instrument differences, as well as the degree of crystallinity, preferred orientation, the surface of the sample being prepared, and other factors known to one skilled in the art, and should be considered merely qualitative measurements.

[0066] Pharmaceutical compositions comprising compounds disclosed herein can be administered orally, by inhalation, rectally, parenterally, or topically to a subject in need thereof. For oral administration, the pharmaceutical composition can be a regular solid formulation such as a tablet, powder, granule, capsule, or the like, or a liquid formulation such as an aqueous or oily suspension or other liquid formulation such as a syrup, solution, or suspension; for parenteral administration, the pharmaceutical composition can be a solution, aqueous solution, oily suspension concentrate, lyophilized powder, or the like. Preferably, the pharmaceutical composition is selected from the group consisting of tablets, coated tablets, capsules, suppositories, nasal sprays, or injections, more preferably tablets or capsules. The pharmaceutical composition can be administered as a single unit with a precise dosage. In addition, the pharmaceutical composition may further comprise additional active ingredients.

[0067] All preparations of the pharmaceutical composition disclosed in the present application can be prepared by conventional methods in the pharmaceutical field. For example, the active ingredient can be mixed with one or more excipients and then the desired preparation is prepared. "Pharmaceutically acceptable excipient" refers to a conventional pharmaceutical carrier suitable for the desired pharmaceutical preparation, for example: a diluent, a vehicle such as water, various organic solvents, etc., a filler such as starch, sucrose, etc., a binder such as cellulose derivatives, alginate, gelatin and polyvinylpyrrolidone (PVP); a wetting agent such as glycerol; a disintegrant such as agar, calcium carbonate and sodium bicarbonate; an absorption enhancer such as a quaternary ammonium compound; a surfactant such as hexadecanol; an absorption carrier such as kaolin and bentonite; a lubricant such as talc, calcium stearate, magnesium stearate, polyethylene glycol, etc. In addition, the pharmaceutical composition further comprises other pharmaceutically acceptable excipients such as a dispersant (decentralized agent), a stabilizer, a thickener, a complexing agent, a buffer, a penetration enhancer, a polymer, an aromatic compound, a sweetener and a dye.

[0068] "Pharmaceutical composition" refers to a composition comprising a crystalline form of a compound of the present invention and at least one additional pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" refers to a medium generally accepted in the art for delivering biologically active agents to animals, particularly mammals, including, i.e., adjuvants, excipients or vehicles, such as diluents, preservatives, fillers, flow regulators, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavorings, fragrances, antibacterial agents, antifungal agents, lubricants and dispensing agents, depending on the mode of administration and the nature of the dosage form.

[0069] Pharmaceutically acceptable carriers are formulated according to many factors within the scope of knowledge of those of ordinary skill in the art. These include, but are not limited to: the type and nature of the active agent being formulated; the subject to whom the composition containing the agent is to be administered; the intended route of administration of the composition; and the therapeutic indication being targeted. Pharmaceutically acceptable carriers include both aqueous and non-aqueous liquid media, as well as a variety of solid and semi-solid dosage forms. Such carriers may also include many different ingredients and additives in addition to the active agent, and such additional ingredients are included in the formulation for a variety of reasons well known to those of ordinary skill in the art (e.g., stabilization of the active agent, adhesive, etc.). Descriptions of suitable pharmaceutically acceptable carriers and the factors involved in their selection can be found in a variety of readily available sources, such as Allen, Jr., LV et al., Remington: The Science and Practice of Pharmacy (Volume 2), 22nd edition, Pharmaceutical Press (2012).

[0070] Of course, the dosage regimen of the solid form of the present application will vary according to known factors such as the pharmacodynamic characteristics of the specific agent and its mode and route of administration; the species, age, sex, health, medical condition and weight of the recipient; the nature and extent of the symptoms; the type of concurrent treatment; the frequency of treatment; the route of administration, the patient's renal and liver function and the desired effect. Typically, the daily oral dose range of each active ingredient will be between about 0.001 and about 5000 mg / day, preferably between about 0.01 and about 1000 mg / day, and most preferably between 0.1 and about 250 mg / day. Intravenously, during a constant rate infusion, the most preferred dosage range will be from about 0.01 to about 10 mg / kg / minute. The compounds of the present invention can be administered in a single daily dose, or the total daily dose can be administered in divided doses twice, three times or four times a day.

[0071] Dosage forms (pharmaceutical compositions) for administration may contain from about 1 mg to about 2000 mg of active ingredient per dosage unit. In these pharmaceutical compositions, the active ingredient will generally be present in an amount of about 0.1%-95% by weight based on the total weight of the composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Fig. 1 is the XRPD pattern of amorphous compound 1;

[0073] Fig. 2 shows the dynamic water sorption (DVS) of amorphous compound 1;

[0074] Fig.3 XRPD pattern of Form I of Example 2a;

[0075] Fig.4 TGA / DSC diagram of Form I of Example 2a;

[0076] Fig.5 Example 2a Form I 1 H NMR spectrum;

[0077] Fig. 6 is the XRPD pattern of Form II of Examples 3a and 3b;

[0078] Fig.7 is the TGA / DSC diagram of Form II of Example 3a;

[0079] Fig.8 is the crystal form II of Example 3a 1 H NMR spectrum;

[0080] Fig. 9 is the XRPD pattern of Form IIIA of Example 4a;

[0081] Fig. 10 is the TGA / DSC diagram of Form IIIA of Example 4a;

[0082] Fig.11 is the crystal form IIIA of Example 4a 1 H NMR spectrum;

[0083] Fig. 12 is an XRPD overlay of Form IIIA of Example 4a before and after heating;

[0084] Fig. 13 is the XRPD pattern of Form IIIB of Example 4b;

[0085] Fig. 14 is the TGA / DSC diagram of Form IIIB of Example 4b;

[0086] Fig.15 is the crystal form IIIB of Example 4b 1 H NMR spectrum;

[0087] Fig. 16 is a temperature-dependent XRPD pattern of Form IIIB of Example 4b;

[0088] Fig. 17 is the XRPD pattern of Form IV of Examples 5a and 5b;

[0089] Fig. 18 is the TGA / DSC graph of Form IV of Example 5a;

[0090] Fig.19 is the crystal form IV of Example 5a 1 H NMR spectrum;

[0091] Fig. 20 is the XRPD pattern of Form V of Example 6a;

[0092] Fig. 21 is the TGA / DSC graph of Form V of Example 6a;

[0093] Fig.22 is the crystal form V of Example 6a 1 H NMR spectrum;

[0094] Fig. 23 is the XRPD pattern of Form VI of Example 7a;

[0095] Fig. 24 is the DSC / TGA graph of Form VI of Example 7a;

[0096] Fig. 25 is the XRPD pattern of Form IV of Example 5a before and after heating;

[0097] Fig. 26 is an XRPD overlay of Form II of Example 3a before and after heating;

[0098] Fig. 27 is the XRPD pattern of Form II of Example 3b before and after heating;

[0099] Fig. 28 is the XRPD pattern of Form V of Example 6a before and after heating;

[0100] Fig. 29 is an XRPD overlay of the competition between Form I and IIIA suspended in EtOAc;

[0101] Fig. 30 is an XRPD overlay of the competition between Form I and IIIA in MIBK;

[0102] Fig. 31 is the XRPD overlay (I / II) of the competition between Form I and IIIA in acetone / H2O suspension;

[0103] Fig. 32 is the XRPD overlay (II / II) of the competition between Form I and IIIA in acetone / H2O suspension;

[0104] Fig. 33 is the DVS diagram of Form I;

[0105] Fig. 34 is the XRPD superposition of Form I before and after DVS testing;

[0106] Fig. 35 is the XRPD superposition of the amorphous sample before and after DVS testing;

[0107] Fig. 36 is the DVS diagram of Form IIIA in the moisture induction experiment;

[0108] Fig. 37 is the XRPD superposition of Form IIIA before and after DVS testing;

[0109] Fig. 38 is an XRPD overlay of Form I of Example 2a before and after solid-state stability evaluation;

[0110] Fig. 39 is an XRPD overlay before and after the amorphous solid state stability evaluation;

[0111] Fig. 40 is an XRPD overlay of Form IIIA before and after solid-state stability evaluation;

[0112] Fig.41 is the solubility diagram of amorphous at different pH values ​​(feed concentration 0.25 mg / mL);

[0113] Fig.42 is the solubility diagram of Form I at different pH values ​​(feed concentration 0.25 mg / mL);

[0114] Fig.43 is the solubility diagram of amorphous and crystalline form I at different pH values ​​(feed concentration 0.05 mg / mL);

[0115] Fig. 44 is an XRPD overlay of Form I before and after grinding;

[0116] Fig. 45 is an XRPD overlay of Form I before and after tableting;

[0117] Fig. 46 is an XRPD overlay of Form IIIA before and after grinding;

[0118] Fig. 47 is an XRPD overlay of Form IIIA before and after tableting;

[0119] Fig. 48 is the XRPD superposition of amorphous before and after grinding;

[0120] Fig. 49 is the XRPD superposition of amorphous before and after tableting;

[0121] Fig. 50 is an XRPD overlay for evaluating the photostability of Form I;

[0122] Fig. 51 is an XRPD overlay diagram for evaluating the photostability of Form IIIA;

[0123] Fig.52 is an XRPD overlay for evaluating the photostability of amorphous samples;

[0124] Fig. 53 is an XRPD overlay for evaluating the photostability of Form V;

[0125] Fig. 54 is an HPLC chart for evaluating the photostability of Form I;

[0126] Fig. 55 is an HPLC chart for evaluating the photostability of Form IIIA;

[0127] Fig.56 is a HPLC chart for evaluating the photostability of amorphous samples; and

[0128] Fig.57 is the HPLC chart for evaluating the photostability of Form V. Example

[0129] The following examples are only used to illustrate the present invention, and are not intended to limit the present invention in any way.

[0130] Solvent name correspondence table

[0131] The following instruments and methods are used in the present invention:

[0132] Instruments and methods

[0133] X-ray powder diffraction (XRPD): XRPD results are obtained from X'Pert 3 The data were collected on an Empyrean X-ray powder diffraction analyzer, and the scanning parameters are shown in Table 1.

[0134] Table 1: XRPD test parameters

[0135] Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC): TGA and DSC were collected on a TA Discovery 5500 thermogravimetric analyzer and a TA Discovery 2500 differential scanning calorimeter, respectively. Table 2 lists the TGA and DSC test parameters.

[0136] Table 2: TGA and DSC test parameters

[0137] 1H Solution NMR: 1H Solution NMR spectra were collected on a Bruker 400M NMR spectrometer using DMSO-d6 as the solvent.

[0138] Dynamic moisture sorption (DVS): Dynamic moisture sorption (DVS) curves were collected on an SMS (Surface Measurement Systems) DVS Intrinsic instrument. Relative humidity at 25°C was calibrated using the deliquescent points of LiCl, Mg(NO₃)₂, and KCl. DVS test parameters are listed in Table 3.

[0139] Table 3: DVS test parameters

[0140] High performance liquid chromatography (UPLC): Purity and solubility were tested by Agilent 1290 ultra performance liquid chromatography. The analysis conditions are shown in Tables 4 and 5.

[0141] Table 4: UPLC test conditions for purity testing

[0142] Table 5: ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY CONDITIONS FOR SOLUTION TEST

[0143] For instruments and methods not explicitly mentioned in the “Instruments and Methods” section, instruments and methods known in the art can be used.

[0144] Example 1a (Compound 1 is amorphous, repeating Example 1 of Chinese Patent Application No. 201210190520.4): According to the method of Example 1 of Chinese Patent Application No. 201210190520.4, the crude product obtained in step 13 of Example 1 was separated and purified by silica gel column chromatography using dichloromethane / methanol as eluent, and then rotary evaporation to obtain a white solid, which was found to be amorphous by XRPD analysis. Its XRPD pattern is shown in Fig. 1, and the dynamic water sorption (DVS) is shown in Fig. 2.

[0145] Example 2a: 4 mL of anhydrous ethanol was added to approximately 21 mg of the amorphous sample of Example 1a, and the mixture was heated and stirred in an 85°C oil bath for 5 minutes to dissolve the solution. Subsequently, the resulting clear solution was cooled to room temperature (25°C) with stirring over 2.5 hours, and stirring was continued at room temperature for 24 hours. The resulting solid sample was filtered and subjected to XRPD analysis.

[0146] The XRPD results are shown in Fig. 3. The TGA / DSC results (Fig. 4) show that the sample loses 0.77% of its weight when heated to 150°C, and there is a sharp endothermic peak at 221.3°C (onset temperature). 1 H NMR data were collected using DMSO-d6 as the solvent. No significant residual solvent was detected, as shown in Fig. 5. Based on the characterization results of Form I, which exhibited a small and gradual weight loss (less than the theoretical water content of 1.80% for a hemihydrate) and a single melting endotherm, it is speculated that Form I is an anhydrate or anhydrous crystal. XRPD diffraction peak data for Form I are shown in Table 6.

[0147] Table 6: XRPD diffraction peak data of Form I

[0148] Example 2b: Approximately 20 mg of each amorphous sample was weighed into a 20-mL vial. 0.4-1.0 mL of a good solvent (see Table 7) was added to dissolve the sample. The sample was filtered (through a 0.45 μm pore size PTFE filter) to obtain a clear solution. The antisolvent listed in Table 7 was added while stirring until a solid precipitated. The precipitated solid was separated by centrifugation and analyzed by XRPD. The results are shown in Table 7. The addition of the antisolvent produced a solid, which was confirmed to be Form I by XRPD.

[0149] Table 7: Summary of Antisolvent Addition Test

[0150] [1] : After adding the anti-solvent, the mixture was clarified and transferred to 5°C and stirred to precipitate the solid.

[0151] [2] : The solution became clear after adding the anti-solvent, and remained clear after stirring at 5°C. The solution was then stirred at -20°C to precipitate solids.

[0152] [3] : The solution becomes clear after adding the anti-solvent, and remains clear after stirring at 5°C and -20°C, and evaporates at room temperature.

[0153] [4] : After adding the anti-solvent, it turns into oil. After circulating suspension and stirring at 5-50℃, it still turns into oil. It evaporates at room temperature.

[0154] Example 2c: Approximately 20 mg of each amorphous sample from Example 1a was weighed into a 5-mL or HPLC vial. 0.7-4.0 mL of the solvent listed in Table 8 was added. The mixture was stirred at 50°C for approximately 2 hours, then filtered (through a 0.45 μm pore size PTFE membrane). The filtrate was placed in a biochemical incubator and cooled to 5°C at a rate of 0.1°C / min. The precipitated solid was collected and analyzed by XRPD. The test results are shown in Table 8. The slow cooling experiment yielded a solid, which was confirmed to be Form I by XRPD.

[0155] Table 8 Summary of 5℃ slow cooling test

[0156] [1] : Slowly cool to 5℃ and then clarify, transfer to -20℃ and let stand to get solid.

[0157] [2] : After slowly cooling to 5℃, it becomes clear. After being transferred to -20℃ and allowed to stand, no solid is precipitated. It is then transferred to room temperature for evaporation.

[0158] Example 3 Crystalline Form II

[0159] Example 3a: 71.6 mg of Form I from Example 2a was weighed into a 3 mL vial, 1.5 mL of ACN was added, and the mixture was magnetically stirred at room temperature for approximately 1 day. The solid was isolated by centrifugation and dried at ambient conditions (~21°C / 45% RH) for approximately 4 hours to obtain the final product, which was then analyzed by XRPD and TGA / DSC.

[0160] The XRPD results are shown in Fig. 6. The TGA results are shown in Fig. 7. TGA shows that the sample has a step-wise weight loss of 5.90% when heated to 120°C. The DSC results show two endothermic peaks at 94.1°C and 220.9°C (onset temperature), and one exothermic peak at 191.6°C (peak temperature). 1 H NMR results, shown in Fig. 8, indicate a molar ratio of ACN to API of 0.8 (5.9 wt %, consistent with the TGA weight loss). Based on characterization and heating results, Form II is hypothesized to be an ACN solvate, which undergoes desolvation and crystallization upon heating. XRPD diffraction peak data for Form II are shown in Table 9.

[0161] Table 9: XRPD diffraction peak data of Form II

[0162] Example 3b: 20.4 mg of Example 2a Form I sample was weighed into a 3 mL vial, and 2 mL of ACN / acetone (1:1, v / v) was added. After sonication, the solution was filtered through a 0.45 μm PTFE membrane to obtain a clear solution. The vial was sealed with parafilm, punctured with four small holes, and allowed to slowly evaporate in a fume hood for 5 days. Solid precipitation was observed. After being left at ambient conditions overnight, the solution was vacuum-dried at room temperature for approximately 1 day. The ACN / acetone (1:1, v / v) solution of Example 2a Form I was slowly evaporated, and the solid precipitated. The solution was then vacuum-dried at room temperature to obtain the final product, which was then subjected to XRPD analysis. The XRPD results are shown in Fig. 6.

[0163] Example 3c: Approximately 19.7 mg of the amorphous sample obtained in Example 1a was weighed into an HPLC vial. 0.5 mL of ACN / H₂O (19:1) solvent was added to the resulting suspension. The suspension was magnetically stirred (~1000 rpm) at 5°C for approximately 6 days. The solid was then isolated by centrifugation and analyzed by XRPD. The XRPD results indicated Form II.

[0164] Example 3d: Approximately 19.9 mg of the amorphous sample obtained in Example 1a was weighed into an HPLC vial. 0.5 mL of ACN / acetone (1:1) solvent was added to the resulting suspension. The suspension was magnetically stirred (~1000 rpm) at 5°C for approximately 6 days. The solid was then isolated by centrifugation and analyzed by XRPD. The XRPD results showed Form II.

[0165] Example 4 (Forms IIIA and IIIB)

[0166] Example 4a: A sample of Form II from Example 3a was heated to 120°C using a DSC, held at this temperature for 5 minutes, and then cooled to room temperature to obtain Form IIIA. XRPD results are shown in Fig. 9. TGA / DSC results (Fig. 10) show that the sample lost 0.26% weight upon heating to 200°C, which is lower than the theoretical water content of a hemihydrate (1.80%). DSC results revealed an exothermic signal at 200.3°C (peak temperature) and a sharp endothermic signal at 219.6°C (onset temperature). 1 H NMR results (Fig. 11) showed no significant residual solvent. Form IIIA was heated to 205°C, cooled to room temperature, and exposed to ambient conditions before and after conversion to Form I. XRPD results before and after heating are shown in Fig. 12. Based on the characterization results of Form IIIA, Form IIIA is presumed to be an anhydrate, and the exothermic signal at 198.8°C represents the thermal transition to Form I. XRPD diffraction peak data for Form IIIA are shown in Table 10.

[0167] Table 10 XRPD diffraction peak data of Form IIIA

[0168] Example 4b: 100.0 mg of Form I sample from Example 2a was weighed into a 20 mL vial, dissolved in 10 mL of THF, and filtered (through a 0.45 μm PTFE filter) to obtain a clear solution. The filtrate was rotary evaporated at 50°C and collected to obtain the final product. XRPD results are shown in Fig. 13. TGA / DSC results (Fig. 14) showed a 3.49% weight loss upon heating to 200°C. DSC results revealed a weak endothermic signal and an exothermic signal at 121.3°C and 144.6°C (peak temperatures), respectively, and a stronger endothermic signal at 218.3°C (onset temperature). 1 H NMR results (Fig. 15) show a molar ratio of residual solvent THF to API of 0.09 (1.3 wt%), presumably due to surface adsorption. Thermal signals at 121.3°C and 144.6°C for Form IIIB represent the transition to Form I. XRPD diffraction peak data for Form IIIB are shown in Table 11.

[0169] Table 11: XRPD diffraction peak data of Form IIIB

[0170] Example 5 (Form IV)

[0171] Example 5a: About 20 mg of the amorphous sample obtained in Example 1a was weighed into an HPLC vial, and 0.5 mL of 1,4-dioxane solvent was added. The resulting suspension was placed at room temperature with magnetic stirring (~1000 rpm) for about 6 days, and then the solid was separated by centrifugation and dried in vacuo at room temperature overnight to obtain the final product, which was then subjected to XRPD and TGA / DSC analysis. The XRPD and TGA / DSC results are shown in Figs. 17 and 17.

[0172] As shown in Fig. 18, TGA results showed that the sample had a step-wise weight loss of 16.80% when heated to 120°C; DSC results showed that there were two endothermic peaks at 95.6°C and 220.5°C (onset temperature). 1 The H NMR results are shown in Fig. 19. The molar ratio of 1,4-Dioxane to API in the sample was 1.2 (17.8 wt %, consistent with the TGA weight loss). Based on the above characterization results and the variable temperature XRPD results, Form IV is presumed to be a 1,4-Dioxane solvate. The XRPD diffraction peak data for Form IV are shown in Table 12.

[0173] Table 12: XRPD diffraction peak data of Form IV

[0174] Example 5b: A sample of Form I of Example 2a (500 mg) was stirred in 1,4-Dioxane (12.5 mL) at room temperature for 2 days, and the resulting solid was dried under vacuum at room temperature for 1 day. The XRPD results are shown in Fig. 17.

[0175] Example 6 (Form V)

[0176] Example 6a: 20.8 mg of Form I sample from Example 2a was weighed into a 5 mL vial and 4 mL of EtOH was added. The mixture was stirred at 70°C to dissolve, then cooled to 5°C over 650 minutes (at a rate of 0.1°C / min) and allowed to stand at 5°C. After removal of the solution, the solid was left to dry in an open container under ambient conditions (temperature: ~21°C, humidity: ~36% RH). XRPD results are shown in Fig. 20. TGA / DSC results (Fig. 21) show a 7.64% step-wise weight loss upon heating the sample to 120°C, with two endothermic peaks at 103.1°C and 223.3°C (onset temperature). 1 The H NMR results are shown in Fig. 22. The molar ratio of EtOH to API in the sample was 0.7 (6.2 wt%, consistent with the TGA weight loss). Based on the above characterization results and the temperature-dependent XRPD results, Form V is hypothesized to be an EtOH solvate, which, upon heating, desolvates and transforms into anhydrous Form I. The XRPD diffraction peak data for Form V are shown in Table 13.

[0177] Table 13: XRPD diffraction peak data of Form V

[0178] Example 7 (Crystal Form VI)

[0179] The amorphous sample (1 g) of Example 1a was dissolved in DMF (3 mL). After complete dissolution, IPA (6 mL) was added and stirred at room temperature overnight to precipitate a solid. The solid was filtered and the filter cake was washed with isopropanol and dried in vacuo to obtain a DMF solvate crystalline form, Form VI.

[0180] The XRPD results of Form VI are shown in Fig. 23. The DSC results show two endothermic peaks at 147.39°C and 208.56°C. The TGA results in Fig. 24 show that the weight loss is 0.67% when heated to 180°C.

[0181] Example 8 (Crystal Conversion)

[0182] Example 8a: Form IV of Example 5a was heated to 120°C, cooled to room temperature and exposed to ambient conditions to transform into Form I. The XRPD results are shown in Fig. 25.

[0183] Example 8b: The Form II of Example 3a was heated to 120°C and 210°C, respectively, cooled to room temperature and exposed to ambient conditions. The XRPD results are shown in Fig. 26. When heated to 120°C, it transformed into Form IIIB, and when heated to 210°C, it transformed into Form I.

[0184] Example 8c: Form II from Example 3b was heated to 120°C, cooled to room temperature, and exposed to ambient conditions before converting to Form I. XRPD results are shown in Fig. 27. It is speculated that the sample may also initially convert to Form IIIB upon heating, but then convert more rapidly to Form I under ambient conditions, as indicated by XRPD analysis.

[0185] Example 8d: Form IIIB of Example 4a was identified by variable temperature XRPD, as shown in Fig. 16. After N2 purging at 30°C for 20 minutes, the crystal form remained unchanged. Upon heating to 120°C under N2 protection, diffraction peaks of Form I were observed. Upon further heating to 170°C, Form I was predominant, with only minor diffraction peaks of Form IIIB. Cooling to 30°C under N2 protection resulted in no further transformation.

[0186] Example 8e: Form V of Example 6a was heated to 120°C, cooled to room temperature and exposed to ambient conditions to transform into Form I. The XRPD results are shown in Fig. 28.

[0187] Example 8f (Suspension competition experiment between Form I and IIIA)

[0188] This example relates to suspension competition experiments of Form I and Form IIIA in EtOAc and MIBK at room temperature and 50°C, as well as suspension competition experiments in acetone / H2O of different water activities at room temperature.

[0189] Weigh approximately 15 mg of Example 2a Form I sample into an HPLC vial, add 1 mL of the corresponding solvent, and stir at the appropriate temperature for 4 hours or overnight. Filter (0.45 μm PTFE filter) to obtain a saturated solution. Weigh equal masses of Example 2 Form I and Example 4a Form IIIA samples (approximately 5 mg each) into a new HPLC vial and add the saturated solution obtained in Step 1. Stir at the appropriate temperature, and perform XRPD analysis on the wet solid sample (covered with Kapton film to prevent potential crystallization due to solvent evaporation during the analysis).

[0190] The results of the suspension competition are summarized in Table 14. The XRPD results are shown in Figs. 29, 30, 31, and 32.

[0191] Table 14 Results of suspension competition experiments between Form I and IIIA

[0192] where a w : Theoretical water activity.

[0193] According to the XRPD comparison results, the physical mixture of Form I and IIIA transformed into Form I after suspension competition under all conditions, indicating that anhydrous Form I is thermodynamically more stable than Form IIIA under anhydrous conditions ranging from room temperature to 50°C and under water activity of 0 to 1 at room temperature.

[0194] Example 9 (Hygroscopicity of Crystal Form I)

[0195] The hygroscopicity of Form I of Example 2a, the amorphous form of Example 1a, and Form IIIA of Example 4a was evaluated by a dynamic moisture sorption (DVS) test at 25°C.

[0196] The DVS results for Form I are shown in Fig. 33. At 25°C / 80% RH, the water absorption increased by 0.047%, indicating near-hygroscopicity. XRPD comparison results (Fig. 34) show that Form I did not undergo any polymorphic transformation after DVS testing.

[0197] The DVS results for the amorphous form are shown in Fig. 2. As the humidity increases from 50% to 95% RH, the water absorption and weight gain decrease. This suggests that the amorphous sample undergoes crystalline transformation during this period of increasing humidity, with the adsorbed or entrapped water or organic solvent removed by the N2 purge. Comparative XRPD results (Fig. 35) show that the amorphous sample transforms to Form I after DVS testing.

[0198] The DVS results for Form IIIA are shown in Fig. 36. At 25°C / 80% RH, it absorbed 0.060% water, demonstrating near-hygroscopicity. XRPD comparison results (Fig. 37) show that Form IIIB did not undergo any polymorphic transformation after DVS testing.

[0199] Example 10 (Solid-state stability of Form I)

[0200] To evaluate the solid-state stability of Form I, Form IIIA, and the amorphous form, appropriate amounts of Form I from Example 2a, the amorphous form from Example 1a, and Form IIIA from Example 4a were weighed and subjected to stability tests at 60°C / closed for one day, 25°C / 60% RH / open for one week, and 40°C / 75% RH / open for one week. Stability samples tested under different conditions were evaluated for physical stability by XRPD and for purity by HPLC. The evaluation results are summarized in Table 15. The XRPD results are shown in Figs. 38, 39, and 40 (a faint diffraction peak of Form I is observed at the star mark in Fig. 40). The stability results showed that Form I did not undergo any crystal transformation or purity reduction under the three evaluation conditions, indicating that Form I had good physical and chemical stability under the evaluation conditions; the purity of the amorphous form did not change significantly under the three evaluation conditions, but all transformed into Form I; a faint diffraction peak of Form I was observed in Form IIIA after it was placed in a closed container at 60°C for one day, and the crystal form remained unchanged after being placed in an open container at 25°C / 60% RH and 40°C / 75% RH for one week.

[0201] Table 15 Summary of solid state stability evaluation results of Form I of Example 2a *: Amorphous samples were transformed into Form I after stability evaluation; #: Faint diffraction peaks of Form I were observed.

[0202] Example 11 (Solubility)

[0203] The dynamic solubility of Form I of Example 2a and the amorphous form of Example 1a was tested at room temperature under different pH conditions (1 M HCl and pH 2.0 / 4.5 / 6.8 / 7.4 buffers). The specific steps are as follows:

[0204] (1) Approximately 2.5 mg of Form I or amorphous sample was weighed into a 20 mL glass vial and 10 mL of buffer of different pH values ​​was added. Alternatively, approximately 2.0 mg of Form I or amorphous sample was weighed into a 20 mL glass vial and 8 mL of 1 M hydrochloric acid was added.

[0205] (2) Oscillate at room temperature (~500 rpm) for 3 min, draw 0.8-1 mL of sample with a syringe, filter (0.45 μm PTFE filter membrane), and then perform HPLC test. (3) For the solution obtained with the amorphous form as raw material, take out 100 μL and dilute 10 times with the corresponding buffer for later use. If solid precipitation occurs in the solution before dilution, test the diluted sample; if no solid precipitation occurs, test the sample before dilution; for the solution obtained with Form I as raw material, due to the estimated low solubility, it was not diluted and the clear solution after filtration was directly tested by HPLC.

[0206] The solubility of Form I and the amorphous form under different pH conditions is shown in Table 16. The results show that the solubility of Form I remains almost unchanged at different pH levels, while that of the amorphous form exhibits significant variations with pH changes (Fig. 41). Form I exhibits more stable dissolution in vivo (Fig. 42), making it easier to obtain pharmacokinetic results with stable blood concentrations, which helps avoid drug safety risks caused by excessive fluctuations in blood concentrations. Similar results were observed at a feed concentration of 0.05 mg / mL (Fig. 43).

[0207] Table 16 3 min solubility results of Form I and amorphous in different media (feeding concentration 0.25 mg / mL)

[0208] LOQ = 0.28 μg / mL.

[0209] Example 12 (Powder Properties)

[0210] The powder properties of the crystalline form I of Example 2a and the amorphous sample of Example 1a were evaluated, including angle of repose, bulk density, and tap density, to understand the powder flow properties of the crystalline form I and the amorphous form.

[0211] Bulk density and tap density: Add a specified mass of the sample to be evaluated to a 5-mL graduated cylinder and record the resulting volume. The bulk density is calculated by dividing the sample mass by the resulting volume. Tap the cylinder 200 times and record the final volume. The tap density is calculated by dividing the sample mass by the final volume. Each parameter should be tested in triplicate.

[0212] Angle of repose: Hold the funnel perpendicular to the bottom and slowly add material into the funnel. A well-proportioned cone of material will form on the bottom. Measure the height and bottom diameter of the cone. Repeat the measurement three times.

[0213] The results of bulk density / tap density are summarized in Table 17. The results show that the average bulk density and tap density of Form I are 0.34 g / cm 3 and 0.46g / cm 3 The calculated Carr index is 26%; the average bulk density and tap density of the amorphous form are 0.31 g / cm 3 and 0.46g / cm 3 , the calculated Carr index is 33%.

[0214] The results of the angle of repose are summarized in Table 18. The results show that the angles of repose for Form I and the amorphous sample are 27.7° and 26.7°, respectively. Comprehensive evaluation results show that Form I and the amorphous sample have similar Carr's index and angle of repose, indicating that Form I and the amorphous sample have similar flowability.

[0215] Table 17 Bulk density / tapped density test results

[0216] Carr's index = (tap density - bulk density) / tap density.

[0217] Table 18 Repose angle test results

[0218] The calculation formula of the angle of repose α is: α = tan -1 (h / D), h is the vertebral height, and D is the vertebral diameter.

[0219] Example 13 (Mechanical Stability)

[0220] Form I of Example 2a, Form IIIA of Example 4a, and the amorphous form of Example 1a were manually ground and compressed using a tabletting machine (350 MPa pressure). XRPD analysis was performed on the ground and compressed samples to evaluate their mechanical stability. The XRPD results are shown in Figs. 44, 45, 46, 47, 48, and 49. The evaluation results showed that after grinding and compression, Form I did not undergo a phase transition and its crystallinity did not decrease significantly. Form III maintained its crystallinity after grinding, but its crystallinity decreased slightly. After compression, its crystallinity remained unchanged, but a significant decrease in crystallinity was observed. The amorphous form transformed into Form I after grinding and compression.

[0221] Based on the results of crystal form characterization and identification, Form I was selected for evaluation of hygroscopicity, solid-state stability, solubility, powder properties, and mechanical stability. The same evaluations were also conducted on the amorphous form to compare its properties with Form I. DVS results showed that Form I had virtually no hygroscopicity and did not undergo a crystal phase transition after DVS testing. The amorphous form transformed into Form I after DVS testing. Solid-state stability results showed that Form I did not undergo a crystal phase transition or decrease in purity after being stored at 60°C for one day in a closed container, 25°C / 60% RH, or 40°C / 75% RH for one week in an open container, indicating that Form I exhibited good physical and chemical stability under the evaluated conditions. The amorphous form showed no significant change in purity under all three evaluation conditions, but transformed into Form I. Powder property testing of Form I demonstrated similar flow properties between Form I and the amorphous sample. The mechanical stability results showed that after tableting (350 MPa) and manual grinding (about 3 minutes), Form I did not undergo crystal transformation and the crystallinity did not decrease significantly, and the amorphous form transformed into Form I.

[0222] According to the characterization data and evaluation results, Form I did not undergo crystal form transformation under all evaluation conditions, while the amorphous form transformed into Form I after DVS, solid-state stability, solubility, and mechanical stability tests, indicating that Form I has better physical stability than the amorphous form.

[0223] Example 14 (Light Stability)

[0224] The crystal form I of Example 2a, the crystal form IIIA of Example 4a, the crystal form V of Example 6a and the amorphous form of Example 1a were respectively irradiated with light (white light 5800-5890 Lux + ultraviolet 7.9-8.7 W / m 2), samples were removed at 6 and 24 hours for XRPD and HPLC purity testing, assessing stability. The XRPD results are shown in Figs. 50, 51, 52, and 53: Form I, Form IIIA, Form V, and the amorphous form did not undergo crystalline transformation after 24 hours under light exposure. Under dark reference conditions, Form I, Form IIIA, and Form V did not undergo crystalline transformation after 24 hours, while faint diffraction peaks of Form I were observed for the amorphous form after 6 and 24 hours. The HPLC results are shown in Fig. 54, Fig. 55, Fig. 56, Fig. 57 and Table 19: After 24 h of illumination, the purity of Form I decreased slightly from 100.00 area % to 99.14 area % (impurity A content 0.79%) under illumination conditions, and the purity of Form IIIA, Form V and amorphous form decreased from 100.00 area % to 98.46 area % (impurity A content 1.34%), 92.05 area % (impurity A content 7.95%) and 90.42 area % (impurity A content 8.83%) under illumination conditions, respectively; the purity of the four samples did not change significantly under light-proof reference conditions (in Fig. 54, Fig. 55 and Fig. 56, the samples illuminated for 6 hours and the tests of photodegradation impurities were performed separately in two HPLC test sequences, so the retention times of the impurities may be slightly shifted). According to the results of light stability, Form I exhibits better light stability than Form IIIA, Form V and amorphous form.

[0225] Table 19 Light stability results of Form I / IIIA / V and amorphous

[0226] Example 15 (Pharmacokinetics Experiment in SD Rats)

[0227] 2.39 mg of Form I of Example 2a, 2.38 mg of Form IIIA of Example 4a, and 2.46 mg of the amorphous form of Example 1 were added to 7.967 mL, 7.906 mL, and 8.193 mL of 0.5% CMC-Na aqueous solution, respectively, to prepare oral formulations with a final concentration of 0.3 mg / mL. Three male SD rats were gavaged at a dose of 3 mg / kg body weight. Blood (0.15 mL) was collected from the jugular sinus at time points 0, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 10 h, and 24 h. The collected whole blood was placed in an EDTA-K2 anticoagulant tube and thoroughly mixed. The blood was centrifuged (1500-1600 g) for 10 min to separate the plasma for bioanalysis. The concentration of the test substance in the plasma samples was determined by LC-MS / MS analysis (instrument model: Triple Quad 5500; chromatographic column: Agilent ZORBAX XDB-C18; flow rate: 0.50 mL / min; injection volume: 2 μL; mobile phase A: water [0.1% formic acid + 5 mM ammonium acetate], mobile phase B: acetonitrile [0.1% formic acid]). The corresponding pharmacokinetic parameters (Table 20) were calculated using the non-compartmental model in Pharsight Phoenix 8.3. As can be seen from the data, the AUC of Form I and Form IIIA 0-t 9820ng·h / mL and 8890ng·h / mL, respectively, C max The drug concentrations in the blood are 1160 ng / mL and 1030 ng / mL, respectively, indicating sufficient drug plasma exposure in the body and good pharmacokinetic properties. In addition, Form I has higher exposure and blood concentration than Form IIIA, indicating the potential for reducing the dosage.

[0228] Table 20 Pharmacokinetic parameters of Form I, Form IIIA and amorphous form

[0229] Example 16: Inhibition of proliferation of NRAS-mutated melanoma cell lines

[0230] All cells were provided by Beijing Cancer Hospital. The human melanoma cell line SK-MEL-2 (NRAS Q61R) was cultured in MEM + 10% FBS + 1% penicillin-streptomycin at 37°C in a 5% carbon dioxide incubator. The human melanoma cell line HMVII (NRAS Q61K) was cultured in F12K + 10% FBS + 1% penicillin-streptomycin at 37°C in a 5% carbon dioxide incubator.

[0231] When the confluence of the cultured cells reached more than 80%, the adherent cells were trypsinized and the cell pellet was collected by centrifugation, counted, and 90 μL of the cell suspension was inoculated into a 96-well plate at an appropriate density. After 24 hours, a series of gradient dilutions of the compound (crystalline form I) (concentration range 0.15nM-10μM, 4-fold gradient dilution) were added to each well according to a volume of 10 μL. Three replicates were set for each concentration, for a total of 9 concentration points. The wells to which the same volume of 5% DMSO was added were used as controls, with a final DMSO concentration of 0.5%. After 3 days of drug treatment, cell viability was detected by MTT. 10 μL of MTT was added to each well and placed in an incubator for further 4 hours. The supernatant was discarded and 150 μL of DMSO was added to dissolve the crystalline formazan. The absorbance at 490 nM was detected using a microplate reader. The above experiment was repeated 3 times, and GraphPad Prism 8 software was used to prepare a dose-effect curve and calculate the IC50. The results are presented as the mean ± SD values ​​of the IC50 of the three experiments (Tables 21 and 22).

[0232] Table 21. IC of Compound 1 on SK-MEL-2 tumor cells 50 value

[0233] Table 22. IC of Compound 1 on HMVII tumor cells 50 value

[0234] Example 17: Inhibition of RAS or RAF mutations

[0235] The in vitro antiproliferative activity of compound 1 against RAS mutation or RAF mutation tumor cell lines and RAS / RAF wild-type tumor cell lines, as well as normal human cell lines, was detected using the tetrazolium salt (MTS) method.

[0236] Digest adherent cells in the logarithmic growth phase with trypsin or collect suspended cells by centrifugation, count, and inoculate 150 μL of cells in a 96-well plate. After 24 hours, add 50 μL / well of the compound (crystalline form I) diluted with culture medium at 4 times the final concentration (concentration range 0.15nM-1000nM, 3-fold gradient dilution). The wells to which the same volume of 2% DMSO was added were used as controls, and the final DMSO concentration was 0.5%. After the cells were cultured for another 72 hours, MTS was used to detect cell viability. The specific method is as follows: for adherent cells, discard the culture medium, add 20 μL of MTS and 100 μL of cell culture medium to each well, and directly add 20 μL of MTS to the suspended cells. Place in the incubator and continue to culture for 1-4 hours, then detect OD490, with OD650 value as a reference. GraphPad Prism software was used to prepare the dose-effect curve and calculate the IC 50 The results are shown in Table 23.

[0237] Table 23. In vitro anti-proliferative IC of compound 1 against RAS and RAF mutant tumor cells and human embryonic lung cells MRC-5 50 value

[0238] Example 18:

[0239] A multicenter, single-arm Phase II study evaluating the efficacy and safety of Compound 1 Form I in patients with advanced melanoma harboring NRAS mutations is underway. Patients received 12 mg of Compound 1 orally twice daily until unacceptable toxicity, disease progression (assessed by the investigator according to RECIST 1.1), withdrawal of consent, death, or when the investigator determined the risks outweighed the benefits. As of February 19, 2023, a total of 100 subjects were enrolled, of whom 95.0% (95 / 100) were included in the full analysis set (FAS).

[0240] Main efficacy indicators:

[0241] In the FAS population: the ORR (objective response rate) assessed by IRRC (Independent Imaging Review Committee) was 35.8% (34 / 95 cases) (95% CI: 26.2%, 46.3%).

[0242] In the FAS population, the IRRC-assessed results showed that the median PFS (progression-free survival) was 4.2 months (95% CI: 3.5, 5.6), the DCR was 72.6% (69 / 95 cases) (95% CI: 62.5%, 81.3%), and the median DoR was 6.1 months (95% CI: 3.9, 8.9).

[0243] Compound 1 has a good anti-tumor therapeutic effect on patients with advanced melanoma with NRAS mutations. The ORR evaluated by IRRC is 35.8%, and the efficacy data is significantly better than the clinical research data of similar drugs.

Claims

1. Polymorph of formula (I) where n is 0 or 1, and X is acetonitrile, water, 1,4 - Dioxane, ethanol, methanol, dimethylformamide, acetone or a mixture thereof.

2. The polymorph of claim 1, wherein n is 0.

3. The polymorph of claim 1, wherein n is 1; and X is acetonitrile, water, 1,4 - Dioxane or ethanol.

4. The polymorph of claim 1, wherein n is 0, and the polymorph is Form I, characterized in that The X - ray powder diffraction pattern of the crystalline form I comprises characteristic diffraction peaks at the following 2θ positions: 16.71° ± 0.2°, 21.82° ± 0.2° and 23.75° ± 0.2°, using Cu - Kα radiation.

5. The polymorph of claim 4, wherein the X - ray powder diffraction pattern of the crystalline form I further comprises characteristic diffraction peaks at the following 2θ positions: 7.48° ± 0.2° and 22.36° ± 0.2°.

6. The polymorph of claim 5, wherein the X - ray powder diffraction pattern of the crystalline form I further comprises characteristic diffraction peaks at the following 2θ positions: 5.3° ± 0.2°, 24.57° ± 0.2° and 27.08° ± 0.2°.

7. The polymorph of claim 6, wherein the X - ray powder diffraction pattern of the crystalline form I further comprises characteristic diffraction peaks at the following 2θ positions: 11.81° ± 0.2°, 15.83° ± 0.2°, 17.92° ± 0.2°, 18.95° ± 0.2° and 19.17° ± 0.2°.

8. The polymorph of claim 4, wherein the X - ray powder diffraction pattern of the crystalline form I comprises characteristic diffraction peaks at the following 2θ positions: 5.30° ± 0.2°, 7.48° ± 0.2°, 11.81° ± 0.2°, 14.85° ± 0.2°, 15.83° ± 0.2°, 16.71° ± 0.2°, 17.92° ± 0.2°, 18.95° ± 0.2°, 19.17° ± 0.2°, 19.43° ± 0.2°, 21.14° ± 0.2°, 21.82° ± 0.2°, 22.36° ± 0.2°, 23.75° ± 0.2°, 24.57° ± 0.2°, 27.08° ± 0.2°, 27.83° ± 0.2°, 28.88° ± 0.2°, 31.20° ± 0.2°, 31.92° ± 0.2°, 32.40° ± 0.2°, 33.91° ± 0.2°, 35.83° ± 0.2°, 37.51° ± 0.2° and 39.04° ± 0.2°.

9. The polymorph of claim 4, wherein the X - ray powder diffraction pattern of the crystalline form I is substantially as shown in Fig.

3.

10. The polymorph of claim 4, wherein the crystalline form I has a TGA graph and / or a DSC graph as shown in Fig.

4.

11. The polymorph of claim 1, wherein n is 1, X is acetonitrile, and the polymorph is Form II, characterized in that The X-ray powder diffraction pattern of the polymorphic form II includes characteristic diffraction peaks at the following 2θ positions: 6.35° ±0.2°, 20.34°±0.2°, 22.41°±0.2°, 22.6°±0.2°, 24.99°±0.2°, 26.05°±0.2°, and 28.71°±0.2°, using Cu-Kα radiation.

12. The polymorph of claim 11, wherein the X-ray powder diffraction pattern of the polymorphic form II further includes characteristic diffraction peaks at the following 2θ positions: 9.18°±0.2°, 16.03°±0.2°, 18.25°±0.2°, 27.07°±0.2°, 29.08°±0.2°, and 33.93°±0.2°.

13. The polymorph of claim 12, wherein the X-ray powder diffraction pattern of the polymorphic form II further includes characteristic diffraction peaks at the following 2θ positions: 14.44°±0.2°, 24.64°±0.2°, 26.41°±0.2°, 32.27°±0.2°, 32.68°±0.2°, 37.07°±0.2°, and 39.51°±0.2°.

14. The polymorph of claim 11, wherein the X-ray powder diffraction pattern of the polymorphic form II includes characteristic diffraction peaks at the following 2θ positions: 6.35°±0.2°, 9.18°±0.2°, 9.91°±0.2°, 14.44°±0.2°, 16.03°±0.2°, 18.25°±0.2°, 19.77°±0.2°, 20.34°±0.2°, 21.81°±0.2°, 22.41°±0.2°, 22.60°±0.2°, 23.84°±0.2°, 24.64°±0.2°, 24.99°±0.2°, 25.43°±0.2°, 26.05°±0.2°, 26.41°±0.2°, 27.07°±0.2°, 28.71°±0.2°, 29.08°±0.2°, 29.81°±0.2°, 31.16°±0.2°, 31.57°±0.2°, 32.27°±0.2°, 32.68°±0.2°, 33.93°±0.2°, 34.19°±0.2°, 35.42°±0.2°, 37.07°±0.2°, 37.56°±0.2°, 38.69°±0.2°, and 39.51°±0.2°.

15. The polymorph of claim 11, wherein the X-ray powder diffraction pattern of the polymorphic form II is substantially as shown in Fig.

6.

16. The polymorph of claim 11, wherein the polymorphic form II has a TGA graph and / or a DSC graph as shown in Fig.

7.

17. The polymorph of claim 1, wherein n is 0, and the polymorph is Form IIIA, characterized in that The X-ray powder diffraction pattern of Polymorph IIIA includes characteristic diffraction peaks at the following 2θ positions: 6.59° ± 0.2°, 22.69° ± 0.2°, 20.32° ± 0.2°, 23.62° ± 0.2°, 23.91° ± 0.2° and 24.15° ± 0.2°, using Cu-Kα radiation.

18. The polymorph of claim 17, wherein the X-ray powder diffraction pattern of Polymorph IIIA further includes characteristic diffraction peaks at the following 2θ positions: 10.8° ± 0.2°, 17.14° ± 0.2°, 13.75° ± 0.2°, 21.59° ± 0.2° and 26.01° ± 0.2°.

19. The polymorph of claim 18, wherein the X-ray powder diffraction pattern of Polymorph IIIA further includes characteristic diffraction peaks at the following 2θ positions: 18.71° ± 0.2°, 21.97° ± 0.2°, 25.54° ± 0.2°, 27.13° ± 0.2°, 27.59° ± 0.2° and 30.51° ± 0.2°.

20. The polymorph of claim 17, wherein the X-ray powder diffraction pattern of Polymorph IIIA includes characteristic diffraction peaks at the following 2θ positions: 6.59° ± 0.2°, 9.9° ± 0.2°, 10.8° ± 0.2°, 13.09° ± 0.2°, 13.75° ± 0.2°, 17.14° ± 0.2°, 17.87° ± 0.2°, 18.71° ± 0.2°, 19.19° ± 0.2°, 20.32° ± 0.2°, 21.59° ± 0.2°, 21.97° ± 0.2°, 22.69° ± 0.2°, 23.62° ± 0.2°, 23.91° ± 0.2°, 24.15° ± 0.2°, 25.54° ± 0.2°, 26.01° ± 0.2°, 27.13° ± 0.2°, 27.59° ± 0.2°, 28.83° ± 0.2°, 29.24° ± 0.2°, 30.51° ± 0.2°, 31.13° ± 0.2°, 31.79° ± 0.2°, 33.6° ± 0.2°, 34.14° ± 0.2°, 36.08° ± 0.2°, 36.67° ± 0.2° and 37.26° ± 0.2°.

21. The polymorph of claim 17, wherein the X-ray powder diffraction pattern of Polymorph IIIA is substantially as shown in Fig.

9.

22. The polymorph of claim 17, wherein Polymorph IIIA has a TGA graph and / or a DSC graph as shown in Fig.

10.

23. The polymorph of claim 1, wherein n is 0, and the polymorph is Form IIIB, characterized in that The X-ray powder diffraction pattern of Polymorph IIIB includes characteristic diffraction peaks at the following 2θ positions: 6.53° ± 0.2°, 13.69° ± 0.2°, 18.6° ± 0.2°, 20.19° ± 0.2°, 21.52° ± 0.2° and 22.64° ± 0.2°, using Cu-Kα radiation.

24. The polymorph of claim 23, wherein the X-ray powder diffraction pattern of the crystal form IIIB further comprises characteristic diffraction peaks at the following 2θ positions: 10.75° ± 0.2°, 17.07° ± 0.2°, 21.93° ± 0.2°, 26.13° ± 0.2°, 23.57° ± 0.2°, and 30.46° ± 0.2°.

25. The polymorph of claim 24, wherein the X-ray powder diffraction pattern of the crystal form IIIB further comprises characteristic diffraction peaks at the following 2θ positions: 13.05° ± 0.2°, 16.63° ± 0.2°, 20.82° ± 0.2°, 24.01° ± 0.2°, 27.55° ± 0.2°, 31.79° ± 0.2°.

26. The polymorph of claim 23, wherein the X-ray powder diffraction pattern of the crystal form IIIB comprises characteristic diffraction peaks at the following 2θ positions: 6.53° ± 0.2°, 10.75° ± 0.2°, 12.62° ± 0.2°, 13.05° ± 0.2°, 13.69° ± 0.2°, 16.63° ± 0.2°, 17.07° ± 0.2°, 18.60° ± 0.2°, 19.59° ± 0.2°, 20.19° ± 0.2°, 20.82° ± 0.2°, 21.52° ± 0.2°, 21.93° ± 0.2°, 22.64° ± 0.2°, 23.57° ± 0.2°, 24.01° ± 0.2°, 25.46° ± 0.2°, 26.13° ± 0.2°, 27.55° ± 0.2°, 30.46° ± 0.2°, 31.04° ± 0.2°, 31.79° ± 0.2°, 32.81° ± 0.2°, 33.54° ± 0.2°, 34.06° ± 0.2°, and 34.46° ± 0.2°.

27. The polymorph of claim 23, wherein the X-ray powder diffraction pattern of the crystal form IIIB is substantially as shown in Fig.

13.

28. The polymorph of claim 23, wherein the crystal form IIIB has a TGA graph and / or a DSC graph as shown in Fig.

14.

29. The polymorph of claim 1, wherein n is 1, X is 1,4-dioxane, and the polymorph is Form IV, characterized in that The X-ray powder diffraction pattern of the crystal form IV comprises characteristic diffraction peaks at the following 2θ positions: 8.56° ± 0.2°, 13.29° ± 0.2°, 17.69° ± 0.2°, 19.75° ± 0.2°, and 22.45° ± 0.2°, using Cu-Kα radiation.

30. The polymorph of claim 29, wherein the X-ray powder diffraction pattern of the crystal form IV further comprises characteristic diffraction peaks at the following 2θ positions: 5.26° ± 0.2°, 18.29° ± 0.2°, 31.83° ± 0.2°, 25.68° ± 0.2°, 22.86° ± 0.2°, 32.81° ± 0.2°, and 23.44° ± 0.2°.

31. The polymorph of claim 30, wherein the X-ray powder diffraction pattern of Form IV further comprises characteristic diffraction peaks at the following 2θ positions: 126.57° ± 0.2°, 27.52° ± 0.2°, 35.69° ± 0.2°, 21.09° ± 0.2°, 20.35° ± 0.2°, and 31.43° ± 0.2°.

32. The polymorph of claim 29, wherein the X-ray powder diffraction pattern of Form IV comprises characteristic diffraction peaks at the following 2θ positions: 5.26° ± 0.2°, 8.56° ± 0.2°, 9.85° ± 0.2°, 13.29° ± 0.2°, 17.69° ± 0.2°, 18.29° ± 0.2°, 19.75° ± 0.2°, 20.35° ± 0.2°, 21.09° ± 0.2°, 22.45° ± 0.2°, 22.86° ± 0.2°, 23.44° ± 0.2°, 24.44° ± 0.2°, 25.68° ± 0.2°, 26.57° ± 0.2°, 27.52° ± 0.2°, 28.40° ± 0.2°, 29.78° ± 0.2°, 31.43° ± 0.2°, 31.83° ± 0.2°, 32.81° ± 0.2°, 34.29° ± 0.2°, 35.69° ± 0.2°, and 37.72° ± 0.2°.

33. The polymorph of claim 29, wherein the X-ray powder diffraction pattern of Form IV is substantially as shown in Fig.

17.

34. The polymorph of claim 29, wherein Form IV has a TGA graph and / or a DSC graph as shown in Fig.

18. The polymorph of claim 1, wherein n is 1, X is ethanol, and the polymorph is Form V, characterized in that The X-ray powder diffraction pattern of Form V comprises characteristic diffraction peaks at the following 2θ positions: 6.21° ± 0.2°, 8.47° ± 0.2°, 15.62° ± 0.2°, 21.73° ± 0.2°, 25.53° ± 0.2°, 25.94° ± 0.2°, and 28.05° ± 0.2°, using Cu-Kα radiation.

36. The polymorph of claim 35, wherein the X-ray powder diffraction pattern of Form V further comprises characteristic diffraction peaks at the following 2θ positions: 9.61° ± 0.2°, 17.55° ± 0.2°, 19.25° ± 0.2°, 22.22° ± 0.2°, 23.12° ± 0.2°, 32.92° ± 0.2°, and 34.22° ± 0.2°.

37. The polymorph of claim 36, wherein the X-ray powder diffraction pattern of Form V further comprises characteristic diffraction peaks at the following 2θ positions: 9.06° ± 0.2°, 20.07° ± 0.2°, 28.49° ± 0.2°, 30.21° ± 0.2°, 31.25° ± 0.2°, 35.47° ± 0.2°, and 38.94° ± 0.2°.

38. The polymorph of claim 35, wherein the X-ray powder diffraction pattern of the crystalline form V comprises characteristic diffraction peaks at the following 2θ positions: 6.21° ± 0.2°, 8.47° ± 0.2°, 25.94° ± 0.2°, 15.62° ± 0.2°, 25.53° ± 0.2°, 28.05° ± 0.2°, 21.73° ± 0.2°, 17.55° ± 0.2°, 32.92° ± 0.2°, 23.12° ± 0.2°, 22.22° ± 0.2°, 19.25° ± 0.2°, 34.22° ± 0.2°, 9.61° ± 0.2°, 9.06° ± 0.2°, 38.94° ± 0.2°, 31.25° ± 0.2°, 35.47° ± 0.2°, 28.49° ± 0.2°, 30.21° ± 0.2°, 20.07° ± 0.2°, 39.78° ± 0.2°, 14.26° ± 0.2°, 24.32° ± 0.2°, 16.95° ± 0.2°, 32.33° ± 0.2°, 36.43° ± 0.2°, 24.94° ± 0.2°, 12.42° ± 0.2° and 37.86° ± 0.2°.

39. The polymorph of claim 35, wherein the X-ray powder diffraction pattern of the crystalline form V is substantially as shown in Fig.

20.

40. The polymorph of claim 35, wherein the crystalline form V has a TGA graph and / or a DSC graph as shown in Fig.

21.

41. The polymorph of any one of claims 1-40, wherein the polymorph substantially does not contain impurity (A).

42. The polymorph of claim 40, wherein the polymorph of compound 1 contains less than 0.15% by weight of impurity (A) relative to the polymorph.

43. The polymorph of claim 4, wherein the crystalline form I substantially does not contain impurity (A).

44. The polymorph of claim 43, wherein the crystalline form I contains less than 0.15% by weight of impurity (A) relative to the crystalline form I.

45. A pharmaceutical composition comprising the polymorph of any one of claims 1-44, and a pharmaceutically acceptable carrier and / or excipient.

46. The pharmaceutical composition of claim 45, wherein the polymorph is crystalline form I.

47. A method for treating tumors, chronic inflammatory diseases, inflammatory bowel diseases, skin diseases, diabetes, eye diseases, diseases related to angiogenesis or vascular regeneration in mammals, diseases related to chronic pain, and other diseases modulated by the Mek cascade in mammals, the method comprising administering to the mammal the polymorph of any one of claims 1-44.

48. The polymorph of any one of claims 1-44, which is used for treating tumors, chronic inflammatory diseases, inflammatory bowel diseases, skin diseases, diabetes, eye diseases, diseases related to angiogenesis or vascular regeneration in mammals, diseases related to chronic pain, and other diseases modulated by the Mek cascade in mammals. Use of the polymorph of any one of claims 1-44 for the preparation of a medicament for treating tumors, chronic inflammatory diseases, inflammatory bowel diseases, skin diseases, diabetes, eye diseases, diseases associated with angiogenesis or vascular regeneration in mammals, diseases associated with chronic pain, and other diseases modulated by the Mek cascade in mammals.

50. A method for preparing polymorphic form I of claim 4, said method comprising any one of the following: a) adding an amorphous sample of Compound 1 to a solvent, then heating at a temperature above about 70 °C, cooling the resulting clear solution to room temperature, maintaining the temperature at room temperature and continuing stirring, precipitating solids, filtering, and drying; or b) dissolving an amorphous sample of Compound 1 in a good solvent, filtering to obtain a clear solution, and adding an anti-solvent to the clear solution with stirring until solids precipitate; or c) dissolving an amorphous sample of Compound 1 in a solvent, stirring at about 50 °C, then filtering to obtain the filtrate, cooling the obtained filtrate to about 5 °C, and collecting the precipitated solids.

51. The method of claim 50, wherein in method 1), the heating temperature is about 75 °C to about 100 °C; about 80 °C to about 90 °C; or about 75 °C or about 85 °C.

52. The method of claim 50, wherein in method 1), the room temperature is about 20 - about 25 °C.

53. The method of claim 50, wherein in method 1), the time for continuing stirring is about 1 - about 12 hours, about 1 - about 8 hours, about 1 - about 5 hours or longer, or about 24 - about 96 hours or longer.

54. The method of claim 50, wherein in method 1), the clear solution is cooled to room temperature within about 2 to about 5 hours or within about 2.5 - about 3 hours.

55. The method of claim 50, wherein after maintaining the temperature at room temperature and continuing stirring in method 1), the temperature can optionally be further reduced to about 0 - about 10 °C, and the temperature is maintained with stirring.

56. The method of claim 50, wherein the temperature is maintained at about 0 - about 10 °C with stirring for about 1 - about 12 hours, about 1 - about 8 hours, about 1 - about 5 hours or longer.

57. The method of claim 50, wherein the solvent in method 1) is water, methanol, ethanol, isopropanol, acetone, methyl isobutyl ketone, 2-butanone, ethyl acetate, isopropyl acetate, methyl tert-butyl ether, tetrahydrofuran, anisole, 2-methyltetrahydrofuran, cyclopentyl methyl ether, 1,4-dioxane, acetonitrile, dichloromethane, toluene, m-xylene, n-heptane, n-hexane, n-pentane, dimethyl sulfoxide, dimethylacetamide, N-methylpyrrolidone, or a mixture thereof.

58. The method of claim 50, wherein the solvent in method 1) is ethanol.

59. The method of claim 50, wherein the good solvent in method 2) is a solvent in which Compound 1 can dissolve, and the anti-solvent in method 2) is a solvent in which Compound 1 cannot dissolve.

60. The method of claim 50, wherein the good solvent in method 2) is MEK, 1,4-dioxane, or DMSO; the anti-solvent in method 2) is MTBE, EtOAc, CHCl3, n-heptane, Anisole, EtOAc, H2O, IPAc, CPME, DCM, or toluene.

61. The method of claim 50, wherein the cooling rate in step 3) is 0.1 °C / minute.

62. The method of claim 50, wherein the solvent in step 3) is MIBK, Methyl acetate, 2-MeTHF, or acetone / EtOH (1:1).

63. A method for preparing polymorph V of claim 35, the method comprising: Add the amorphous sample of Compound 1 to a solvent, then heat at a temperature below about 70 °C. Cool the resulting clear solution to about 0 - about 10 °C, and let stand at this temperature until a solid precipitates, then dry.

64. The method of claim 63, wherein the method is heated at about 70 °C, about 60 °C, or about 50 °C.

65. The method of claim 63, wherein the cooling rate is about 0.1 - about 0.5 °C / min.

66. The method of claim 63, wherein the cooling rate is about 0.1 °C / min.

67. The method of claim 63, wherein the resulting clear solution is cooled to about 5 °C.

68. A method of treating a RAS or RAF mutant cancer in a mammal, the method comprising administering to the mammal 4-fluoro-5-(2-fluoro-4-iodophenylamino)-1H-benzo[d]thiazole-6-carboxylic acid (2-hydroxy-ethoxy)-amide (Compound 1) or a pharmaceutically acceptable salt thereof.

69. The method of claim 68, wherein the RAS or RAF mutant cancer is, for example, KRAS mutant cancer, NRAS mutant cancer, HRAS mutant cancer, or BRAF mutant cancer.

70. The method of claim 68 or 69, wherein the RAS mutant cancer is pancreatic cancer, colorectal cancer, lung cancer, melanoma, acute myeloid leukemia, bladder cancer, or head and neck cancer, etc.

71. The method of claims 68 to 70, wherein the cancer is NRAS mutant cancer.

72. The method of claim 71, wherein the NRAS mutant cancer is NRAS mutant melanoma.

73. The method of claim 72, wherein KRAS includes a mutation at one or more positions selected from codons 12, 13, 59, and 61.

74. The method of claims 68 to 70, wherein NRAS includes a mutation at one or more positions selected from codons 12, 13, 59, 61, and 146.

75. The method of claims 68 to 70, wherein the NRAS mutant form has a mutation at one or more amino acid positions selected from G12, G13, A59, Q61, K117, and A146.

76. The method of claim 75, wherein the NRAS mutant form has one or more amino acid substitutions selected from the following: G12C, G12R, G12S, G12A, G12D, G12V, G13C, G13R, G13S, G13A, G13D, G13V, A59D, A59T, Q61K, Q61L, Q61R, Q61H, K117N, K117R, K117E, A146P, A146T, and A146V.

77. The method of claims 68 to 70, wherein the cancer is in an early, intermediate or advanced stage. The cancer can be locally advanced or metastatic.

78. The method of claims 68 to 70, wherein the mammal has previously received immunotherapy.

79. The method of claim 78, wherein the mammal has previously received immunotherapy and has advanced melanoma with an NRAS mutation.

80. The method of claims 68 to 70, wherein the melanoma is selected from: advanced melanoma, unresectable melanoma, metastatic melanoma, melanoma with a BRAF mutation, melanoma with an NRAS mutation, cutaneous melanoma, or uveal melanoma.

81. The method of claims 68 to 80, wherein compound 1 is in the form of a capsule.

82. The method of claim 81, wherein compound 1 is administered at a dose of 5 - 50 mg per dose, once or twice daily.

83. The method of claim 82, wherein compound 1 is administered at a dose of 12 mg per dose, twice daily.

84. The method of claims 68 to 83, wherein compound 1 is any one of the polymorphs of polymorphic forms I to VI.

85. The method of claims 68 to 83, wherein compound 1 is polymorphic form I.

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