Polymorphs of a protein kinase mek inhibitor, processes for their production and uses thereof

Abstract

The present invention relates to polymorphs of a protein kinase Mek inhibitor benzothiazole compound, processes for their preparation, and their medical use.

Description

Technical Field

[0001] This invention relates to polymorphs of benzothiazole compounds, a protein kinase Mek inhibitor, methods for their preparation, and their pharmaceutical uses. Background Technology

[0002] Overactivation of the Ras / Raf / Mek / Erk signaling pathway plays a crucial role in the proliferation and differentiation of cancer cells. This pathway has been found to be continuously activated or overactivated in various cancers, such as pancreatic cancer, colon cancer, lung cancer, bladder cancer, kidney cancer, skin cancer, and breast cancer. Inhibiting the Ras / Raf / Mek / Erk signaling pathway may aid in the treatment of these hyperproliferative diseases. Mek, a downstream target of Ras and Raf, plays a key role in this pathway, and its phosphorylated substrate is the MAP kinase Erk. If Mek is inhibited, the Ras / Raf / Mek / Erk signaling pathway is shut down, thereby inhibiting cancer cell proliferation. Therefore, Mek inhibitors can suppress cancer cell growth, especially in cancers caused by overactivation of Ras or Raf. Meanwhile, Mek is also involved in inflammatory diseases and symptoms, including acute and chronic inflammation.

[0003] Chinese patent application No. 201210190520.4 discloses numerous benzothiazole compounds exhibiting protein kinase Mek inhibitory activity, including 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, compound 1 is in amorphous solid form. However, the purity of amorphous compounds is generally difficult to control, and their physical and chemical stability is usually poor, with poor hygroscopicity. Moreover, compound 1 is unstable under light and easily degrades to generate impurities (A).

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

[0005] Therefore, it is necessary to develop new crystal forms of this compound, which will help improve purity control in the production process and enhance the stability and storage resistance of the compound. Summary of the Invention

[0006] This invention provides polymorphs of compound 1, which possess advantages such as high purity, good solid-state stability, good powder properties, and mechanical stability. More specifically, this invention provides seven crystal forms of compound 1, namely crystal form I, crystal form II, crystal form IIIA, crystal form IIIB, crystal form IV, crystal form V, and VI. Among these, crystal forms I and IIIA exhibit better solid-state stability, mechanical stability, hygroscopicity, and light stability compared to other crystal forms or amorphous forms, thereby benefiting pharmaceutical processes.

[0007] Specifically, the crystal forms I and IIIA of compound 1 exhibit one or more of the following advantages compared to the amorphous form:

[0008] 1. The suspension competition results show that crystal form I is thermodynamically more stable than crystal form IIIA in the range of room temperature to 50°C;

[0009] 2. DVS results showed that crystal form I and crystal form IIIA were almost non-hygroscopic and did not undergo crystal form transformation after DVS testing; the amorphous form transformed into crystal form I after DVS testing.

[0010] 3. Solid-state stability results showed that crystal form I did not undergo crystal form transformation or purity decrease after being placed in a closed container at 60℃ for 1 day, or in an open container at 25℃ / 60%RH and 40℃ / 75%RH for 1 week, indicating that crystal form I has good physical and chemical stability under the evaluation conditions. Crystal form IIIA showed no change in purity after 1 day at 60℃, but diffraction peaks of crystal form I were observed. It did not undergo crystal form transformation or purity decrease after 1 week at 25℃ / 60%RH and 40℃ / 75%RH. The amorphous form did not show significant changes in purity under the three evaluation conditions, but its crystal form transformed into crystal form I.

[0011] 4. The light stability results show that crystal form I remains stable under illumination (white light 5890 Lux + ultraviolet 8.7 W / m). 2 Under these conditions, after 24 hours, the purity was significantly higher than that of crystal form IIIA, crystal form V, and amorphous, exhibiting better light stability.

[0012] 5. Dynamic solubility tests of crystalline form I and amorphous form in 1M HCl and pH 1.0 / 2.0 / 4.5 / 7.4 buffer solutions at room temperature showed that the solubility of amorphous form was higher than that of crystalline form I in all media within 10 minutes, and the solubility first increased and then decreased. The highest solubility measured in 1M HCl was higher than that in other pH buffer solutions. Compared with amorphous form, crystalline form I dissolved slowly in all media (different pH values), and its solubility in 1M HCl was slightly higher than that in other pH buffer solutions. This ensures that the dissolution of crystalline form I in vivo is more stable, making it easier to obtain stable in vivo pharmacokinetic concentrations and avoiding the drug safety risks caused by large fluctuations in blood drug concentrations.

[0013] 6. Powder properties tests showed that crystalline form I and the amorphous sample had similar flowability. Furthermore, mechanical stability tests indicated that after tableting (350 MPa) and manual grinding (approximately 3 minutes), crystalline form I did not undergo a crystalline transformation and its crystallinity did not decrease significantly; the amorphous sample transformed into crystalline form I. Crystalline form IIIA did not undergo a crystalline transformation but its crystallinity decreased; and / or

[0014] 7. In pharmacokinetic studies, crystal form I showed higher exposure and blood drug concentrations than crystal form IIIA, indicating a potential for lower dosage.

[0015] Based on the characterization data and evaluation results, crystal form I exhibited better light stability and did not undergo crystal form transformation under all evaluation conditions, while the amorphous form transformed into crystal form I after DVS, solid-state stability, solubility, and mechanical stability tests.

[0016] In a first aspect, the present invention provides a polymorph of formula (I).

[0017]

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

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

[0020] In some embodiments, n is 0, and the polymorph is crystal form I, characterized in that the X-ray powder diffraction pattern of crystal form I includes 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. In some embodiments, the X-ray powder diffraction pattern of crystal form I further includes characteristic diffraction peaks at the following 2θ positions: 7.48°±0.2° and 22.36°±0.2°. In some embodiments, the X-ray powder diffraction pattern of crystal form I further includes characteristic diffraction peaks at the following 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 crystal form I further includes 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 crystal form I includes 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°, and 21.14°±0.2°. The X-ray powder diffraction patterns of crystal form I are: 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 crystal form I is substantially as shown in Fig. 3. In some embodiments, crystal form I has a TGA pattern and / or DSC pattern as shown in Fig. 4. In some embodiments, crystal form I is an anhydrous.

[0021] In some embodiments, n is 1, X is acetonitrile, and the polymorph is crystal form II, characterized in that the X-ray powder diffraction pattern of crystal form II includes characteristic diffraction peaks at the following 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 crystal form II further includes characteristic diffraction peaks at the following 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 crystal 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°. In some embodiments, the X-ray powder diffraction pattern of crystal 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°. In some embodiments, the X-ray powder diffraction pattern of crystal 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°. In some embodiments, the X-ray powder diffraction pattern of crystal form II is substantially as shown in Fig. 6. In some embodiments, the crystal form II has a TGA diagram and / or DSC diagram as shown in Fig. 7.

[0022] In some embodiments, n is 0, and the polymorph is crystal form IIIA, characterized in that the X-ray powder diffraction pattern of crystal form 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. In some embodiments, the X-ray powder diffraction pattern of crystal form 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°. In some embodiments, the X-ray powder diffraction pattern of the crystal 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 crystal form 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°, 23.6°±0.2°. 2°±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 crystal form IIIA is substantially as shown in Fig. 9. In some embodiments, the crystal form IIIA has a TGA diagram and / or DSC diagram as shown in Fig. 10. In some embodiments, the crystal form IIIA is anhydrous.

[0023] In some embodiments, n is 0, and the polymorph is crystal form IIIB, characterized in that the X-ray powder diffraction pattern of crystal form 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. In some embodiments, the X-ray powder diffraction pattern of crystal form IIIB further includes 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°. In some embodiments, the X-ray powder diffraction pattern of the crystal 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 crystal form IIIB includes 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. The X-ray powder diffraction patterns of crystal form IIIB are 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°. In some embodiments, the X-ray powder diffraction pattern of crystal form IIIB is substantially as shown in Fig. 13. In some embodiments, crystal form IIIB has a TGA pattern and / or DSC pattern as shown in Fig. 14.

[0024] In some embodiments, n is 1, X is 1,4-dioxane, and the polymorph is crystal form IV. The polymorph IV is characterized by X-ray powder diffraction patterns comprising 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. In some embodiments, the X-ray powder diffraction patterns of crystal form IV further comprise 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°. In some embodiments, the X-ray powder diffraction pattern of crystal 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 crystal form IV includes 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°. The X-ray powder diffraction patterns of crystal form IV are 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°. In some embodiments, the X-ray powder diffraction pattern of crystal form IV is substantially as shown in Fig. 17. In some embodiments, crystal form IV has a TGA pattern and / or DSC pattern as shown in Fig. 18.

[0025] In some embodiments, n is 1, X is ethanol, and the polymorph is crystal form V. The polymorph V is characterized by having an X-ray powder diffraction pattern comprising 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. In some embodiments, the X-ray powder diffraction pattern of crystal 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°. In some embodiments, the X-ray powder diffraction pattern of crystal 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 crystal form V includes 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°. The X-ray powder diffraction patterns are 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°. In some embodiments, the X-ray powder diffraction pattern of the crystal form V is substantially as shown in Fig. 20. In some embodiments, the crystal form V has a TGA diagram and / or DSC diagram as shown in Fig. 21.

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

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

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

[0029] In some embodiments, crystal form I of compound 1 is substantially free of impurities (A). In some embodiments, crystal form I of compound 1 contains less than 0.15% by weight of impurities (A) relative to crystal form I, for example, 0.10%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% by weight of impurities (A).

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

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

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

[0033] Fourthly, the present invention provides a method for treating RAS or RAF mutant cancers in mammals, the method comprising administering to the mammal 4-fluoro-5-(2-fluoro-4-iodophenylamino)-1H-benzo[d]thiazol-6-carboxylic acid (2-hydroxy-ethoxy)-amide (compound 1) or a pharmaceutically acceptable salt thereof. In some embodiments, compound 1 is any polymorph of crystal form I to crystal form VI. In some embodiments, the RAS or RAF mutant cancer is, for example, KRAS mutant cancer, NRAS mutant cancer, HRAS mutant cancer, or 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, etc. In a preferred embodiment, the cancer is NRAS mutant cancer. In some embodiments, the NRAS mutant cancer is RNAS-mutated melanoma.

[0034] In one embodiment, KRAS includes mutations at one or more positions selected from codons 12, 13, 59, and 61. In one embodiment, the KRAS mutant form has mutations at one or more amino acid positions selected from G12, G13, S17, P34, A59, and Q61. In one embodiment, the KRAS mutant form has one or more amino acid substitutions selected from: 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 mutation has a mutation at one or more amino acid positions selected from G12, G13, A59, Q61, K117, and A146. In one embodiment, the KRAS mutation has one or more amino acid substitutions selected from: 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 the BRAF V600E mutation.

[0035] In one embodiment, the NRAS includes a mutation at one or more positions selected from codons 12, 13, 59, 61, and 146. In some embodiments, the NRAS mutation has a mutation at one or more amino acid positions selected from G12, G13, A59, Q61, K117, and A146. In some embodiments, the NRAS mutation has one or more amino acid substitutions selected from: 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 may 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 advanced melanoma with an NRAS mutation. In some embodiments, the melanoma is selected from: advanced melanoma, unresectable melanoma, metastatic melanoma, melanoma with a BRAF mutation, melanoma with an NRAS mutation, cutaneous melanoma, or intraocular melanoma.

[0037] In some embodiments, compound 1 is in the form of tablets, powders, granules, patches, inhalers, or capsules. In some embodiments, compound 1 is in the form of capsules. 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] Fifthly, the present invention provides a method for preparing crystal form I of the present invention, the method comprising any one of the following:

[0039] a) Add the amorphous sample of compound 1 to the solvent, then heat at a temperature above approximately 70°C. Cool the resulting supernatant to room temperature, maintain the temperature at room temperature while stirring, and allow the solid to precipitate. Filter and dry.

[0040] b) Dissolve the amorphous sample of compound 1 in a good solvent, filter to obtain a clear solution, and add the antisolvent while stirring the clear solution until a solid precipitates; or

[0041] c) Dissolve the amorphous sample of compound 1 in a solvent, stir at about 50°C, then filter and collect 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 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, the stirring time in method 1) is from about 0.5 to 12 hours, from about 1 to 12 hours, from about 1 to 8 hours, from about 1 to 5 hours, or longer, or the stirring time is from about 24 to about 96 hours. In some embodiments, the supernatant in method 1) is cooled to room temperature in about 2 to about 5 hours or from about 2.5 to about 3 hours. In some embodiments, after stirring at room temperature in method 1), the temperature may optionally be further reduced to about 0°C to about 10°C, and stirring is continued at this temperature. In some embodiments, stirring is continued at about 0°C to about 10°C for about 1 to about 12 hours, from about 1 to about 8 hours, from about 1 to about 5 hours, or at about 0°C to about 10°C for longer, as appropriate. 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 mixtures 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 antisolvent in method 2) is a solvent in which compound 1 is insoluble. In some embodiments, the good solvent in method 2) is MEK, 1,4-dioxane, or DMSO. In some embodiments, the antisolvent in method 2) is MTBE, EtOAc, CHCl3, n-heptane, Anisole, EtOAc, H2O, IPAc, CPME, DCM, or toluene.

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

[0045] This invention provides a method for preparing crystal form V of the present invention, the method comprising: adding an amorphous sample of compound 1 to a solvent, then heating at a temperature below about 70°C, cooling the resulting supernatant to about 0-about 10°C, allowing it to stand at about 5°C until a solid precipitates, and drying. In some embodiments, the method involves heating at about 70°C, about 60°C, or about 50°C. In some embodiments, the cooling rate is about 0.1-about 0.5°C / min. In some embodiments, the cooling rate is about 0.1°C / min. In some embodiments, the resulting supernatant is cooled to about 5°C.

[0046] This invention provides a method for preparing crystal form IIIA of the invention, the method comprising: heating crystal form II to a first temperature and holding it at that temperature for about 3 to about 10 minutes, and then cooling it to obtain crystal 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 crystal form II is held at the first temperature for about 5 minutes. In some embodiments, the crystal form II is held at that temperature and then cooled to room temperature to about 50°C, for example, cooled 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 compounds of the present invention have DSC plots with characteristic peak positions that have substantially the same properties as the DSC plots provided in the accompanying drawings of the present invention, with a measurement error tolerance of ±5°C, generally required to be within ±3°C.

[0048] The numerical values ​​described and protected in this invention are approximate. Variations within these values ​​may be attributed 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 those that are exactly the same as the characteristic spectra described in the accompanying drawings, such as XRPD, DSC, TGA, DVS, and isothermal adsorption curves. Any crystal form that has a characteristic spectra that is substantially the same or essentially the same as those spectra described in the accompanying drawings falls within the scope of the present invention.

[0050] the term

[0051] Unless otherwise specifically specified in this document, all other technical and scientific terms used in this application have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0052] This application includes singular terms such as “a,” “an,” and “the,” as used in the claims, including their corresponding plural references, unless the context clearly indicates otherwise. Thus, for example, reference to “crystalline form” includes one or more such different crystalline forms, and reference to “the method” includes reference to equivalent steps and methods known to those skilled in the art, which may be modified or substituted for the methods described in this application.

[0053] Throughout the following specification and claims, unless the context otherwise requires, the term "comprising" and variations such as "containing" and "including" will be understood to implicitly include the said integer or step or set of integers or steps, but not to exclude any other integer or step or set of integers or steps. When used in this application, the term "comprising" may be replaced by the term "containing," or sometimes by the term "having."

[0054] The term "about" refers to adding or subtracting 10%, 5%, or 2% of the value from the specified item.

[0055] "Therapeutic effective amount" refers to the amount of a compound that elicits a physiological or medical response in an tissue, system, or subject. This amount is sought, including the amount of a compound, when administered to the treated subject, sufficient to prevent the occurrence of one or more symptoms of the treated disease or condition or condition, or to alleviate them to some extent. "Therapeutic effective amount" can vary depending on the compound, the disease, condition, and / or the symptoms of the disease or condition, the severity of the disease or condition, and / or the age of the subject being treated, and / or the weight of the subject being treated. The appropriate amount in any given situation is obvious to those skilled in the art or can be determined by routine experimentation. In the case of combination therapy, "therapeutic effective amount" refers to the total amount of the combination of subjects that is effective in treating the disease, condition, or condition.

[0056] "Excipient" refers to an agent that is not itself a therapeutic agent but is used as a diluent, excipient, binder and / or medium to be added to a pharmaceutical composition to improve its disposal or storage properties or to allow or promote the formation of a unit dosage form of the compound or pharmaceutical composition for administration.

[0057] "Crystal form" or "polymorph" refers to any solid substance that exhibits a three-dimensional arrangement. In contrast to amorphous solid substances, it produces characteristic XRPD patterns with clearly defined peaks.

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

[0059] A "hydrate" refers to a crystalline form of a molecule that further contains water incorporated into its crystalline structure. Water molecules in a hydrate can exist in a regular and / or disordered arrangement. A hydrate can contain stoichiometric or non-stoichiometric amounts of water molecules.

[0060] "Anhydrous" refers to a crystal that contains virtually no water molecules of any kind, such as a crystal form in which there are essentially no water molecules in the crystal lattice or unit cell.

[0061] The term "solvate" refers to a crystalline form of a molecule that further comprises one or more solvent molecules incorporated into the crystalline structure. Solvent molecules in a solvate can be present in a regular and / or disordered arrangement. A solvate can contain stoichiometric or non-stoichiometric amounts of solvent molecules. Exemplary solvates include, but are not limited to, hydrates, ethoxides, methanols, isopropoxides, and acetic acid. Methods of solvation are generally known in the art. It is noteworthy that in a solvate, the substance bound to the major molecule (e.g., the active pharmaceutical ingredient) is liquid at room temperature, while in a eutectic, the substance is solid at room temperature.

[0062] The crystal form disclosed in this application is substantially pure crystal. As used in this application, "substantially pure" means at least 85% by weight, preferably at least 95% by weight, more preferably at least 99% by weight of the crystal form disclosed in this application, and also includes approximately 100% by weight of a certain crystal form. The remaining material includes one or more other forms of the compound and / or reactive and / or processing impurities arising from its preparation. For example, the 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 that time, wherein the remaining less than 10% by weight of material comprises amorphous and / or other forms of compound 1 and / or reactive and / or processing impurities.

[0063] "X-ray powder diffraction pattern (XRPD pattern)" refers to an experimentally observed diffraction pattern or the parameters, data, or values ​​derived from it. XRPD patterns are typically characterized by peak position (x-axis) and / or peak intensity (y-axis). For the crystal form disclosed in this application, only the main peaks (i.e., the most characteristic, significant, unique, and / or reproducible peaks) are summarized; other peaks can be obtained from the diffraction pattern using conventional methods. The aforementioned main peaks are reproducible within error limits (±2 decimal places given last, or ±0.2 of the given value).

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

[0065] The terms "substantially identical" or "substantially as shown in Figure XX" for X-ray diffraction peaks mean that representative peak positions and intensity variations are taken into account. For example, those skilled in the art will understand that peak positions (2θ) will show some variation, typically up to 0.1–0.2°, and that the instrument used to measure diffraction will also cause some variation. Furthermore, those skilled in the art will understand that relative peak intensities will vary due to differences between instruments, as well as the degree of crystallinity, preferred orientation, the surface of the prepared sample, and other factors known to those skilled in the art, and should be considered as qualitative measurements only.

[0066] Pharmaceutical compositions comprising the compounds disclosed in this application may be administered orally, by inhalation, rectally, parenterally, or topically to subjects in need. For oral administration, the pharmaceutical composition may be a regular solid dosage form such as tablets, powders, granules, capsules, etc., a liquid dosage form such as an aqueous or oil suspension, or other liquid dosage forms such as syrups, solutions, suspensions, etc. For parenteral administration, the pharmaceutical composition may be a solution, aqueous solution, oil suspension concentrate, lyophilized powder, etc. Preferably, the formulation of the pharmaceutical composition is selected from tablets, coated tablets, capsules, suppositories, nasal sprays, or injections, more preferably tablets or capsules. The pharmaceutical composition may be administered as a single unit with a precise dosage. Furthermore, the pharmaceutical composition may further contain additional active ingredients.

[0067] All formulations of the pharmaceutical compositions disclosed in this application can be prepared using conventional methods in the pharmaceutical industry. For example, the active ingredient can be mixed with one or more excipients to prepare the desired formulation. "Pharmaceutically acceptable excipients" refer to conventional pharmaceutical carriers suitable for the desired pharmaceutical formulation, such as: diluents, media such as water, various organic solvents, fillers such as starch, sucrose, binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone (PVP); wetting agents such as glycerin; disintegrants such as agar, calcium carbonate, and sodium bicarbonate; absorption enhancers such as quaternary ammonium compounds; surfactants such as cetyl alcohol; absorption carriers such as kaolin and soap clay; lubricants such as talc, calcium stearate, magnesium stearate, and polyethylene glycol. Furthermore, the pharmaceutical compositions further include other pharmaceutically acceptable excipients such as decentralized agents, stabilizers, thickeners, complexing agents, buffers, penetration enhancers, polymers, aromatic compounds, sweeteners, and dyes.

[0068] "Pharmaceutical composition" means a composition comprising a crystalline form of a compound of the present invention and at least one other pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" means a medium generally accepted in the art for delivering a bioactive agent to animals, particularly mammals, including, i.e., adjuvants, excipients, or mediators such as diluents, preservatives, fillers, flow conditioners, disintegrants, wetting agents, emulsifiers, suspensions, sweeteners, flavoring agents, aromatizers, antibacterial agents, antifungal agents, lubricants, and dispensing agents, depending on the manner of administration and the nature of the dosage form.

[0069] Pharmaceutically acceptable carriers are formulated based on a number of factors known to those skilled 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 pharmaceutical 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 a number of different components and additives in addition to the active agent, such additional components being included in the formulation for a variety of reasons well known to those skilled in the art (e.g., stabilization of the active agent, binder, 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 (Vol. 2), 22nd ed., Pharmaceutical Press (2012).

[0070] Of course, the dosage regimen of the solid form of this application will vary depending on known factors such as: the pharmacodynamic characteristics of the particular agent and its method and route of administration; the recipient's species, age, sex, health, medical condition, and weight; the nature and severity of symptoms; the type of concurrent treatment; the frequency of treatment; the route of administration; the patient's renal and hepatic function; and the desired effect. Typically, the daily oral dose range for 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. Intravenous infusion at a constant rate will preferably range from about 0.01 to about 10 mg / kg / minute. The compounds of the invention can be administered in a single daily dose, or the total daily dose can be administered in divided doses twice, three, or four times daily.

[0071] Dosage forms (pharmaceutical compositions) for administration may contain an active ingredient in amounts from about 1 mg to about 2000 mg per dose unit. In these pharmaceutical compositions, the active ingredient will typically be present in an amount of about 0.1% to 95% by weight based on the total weight of the composition. Attached Figure Description

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

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

[0074] Fig. 3 XRPD diagram of crystal form I in Example 2a;

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

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

[0077] Fig. 6 shows the XRPD diagrams of crystal form II in Examples 3a and 3b;

[0078] Fig. 7 shows the TGA / DSC diagram of crystal form II in Example 3a;

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

[0080] Fig. 9 is the XRPD diagram of crystal form IIIA in Example 4a;

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

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

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

[0084] Fig. 13 is the XRPD diagram of crystal form IIIB in Example 4b;

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

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

[0087] Fig. 16 is a temperature-dependent XRPD diagram of crystal form IIIB in Example 4b;

[0088] Fig. 17 shows the XRPD diagrams of crystal form IV for Examples 5a and 5b;

[0089] Fig. 18 is the TGA / DSC diagram of crystal form IV in Example 5a;

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

[0091] Fig. 20 is the XRPD diagram of crystal form V in Example 6a;

[0092] Fig. 21 is the TGA / DSC diagram of crystal form V in Example 6a;

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

[0094] Fig. 23 is the XRPD diagram of crystal form VI in Example 7a;

[0095] Fig. 24 is the DSC / TGA diagram of crystal form VI in Example 7a;

[0096] Fig. 25 shows the XRPD images of crystal form IV in Example 5a before and after heating;

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

[0098] Fig. 27 shows the XRPD images of crystal form II in Example 3b before and after heating;

[0099] Fig. 28 shows the XRPD images of crystal form V in Example 6a before and after heating;

[0100] Fig. 29 is an XRPD overlay of crystal forms I and IIIA competing for suspension in EtOAc;

[0101] Fig. 30 is an XRPD overlay of crystal forms I and IIIA competing for suspension in MIBK;

[0102] Fig. 31 is an XRPD overlay (I / II) of crystal forms I and IIIA competing for suspension in acetone / H2O;

[0103] Fig. 32 is an XRPD overlay (II / II) of crystal forms I and IIIA competing for suspension in acetone / H2O;

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

[0105] Fig. 34 shows the XRPD overlay images of crystal form I before and after DVS testing;

[0106] Fig. 35 shows the XRPD overlay images of the amorphous sample before and after the DVS test;

[0107] Fig. 36 shows the DVS diagram of crystal form IIIA in the humidification experiment;

[0108] Fig. 37 shows the XRPD overlay images of crystal form IIIA before and after DVS testing;

[0109] Fig. 38 shows the XRPD overlay before and after the solid-state stability assessment of crystal form I in Example 2a;

[0110] Fig. 39 shows the XRPD overlay before and after the stability assessment of the amorphous solid.

[0111] Fig. 40 shows the XRPD overlay before and after the solid-state stability assessment of crystal form IIIA;

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

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

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

[0115] Fig. 44 shows the XRPD overlay before and after grinding of crystal form I;

[0116] Fig. 45 shows the XRPD overlay images before and after compression of crystal form I;

[0117] Fig. 46 shows the XRPD overlay before and after grinding of crystal form IIIA;

[0118] Fig. 47 shows the XRPD overlay before and after compression of crystal form IIIA;

[0119] Fig. 48 shows the XRPD overlay before and after amorphous grinding;

[0120] Fig. 49 shows the XRPD overlay before and after amorphous tableting;

[0121] Fig. 50 is an XRPD overlay image evaluating the light stability of crystal form I;

[0122] Fig. 51 is an XRPD overlay image evaluating the photoluminescence stability of crystal form IIIA;

[0123] Fig. 52 is an XRPD overlay image evaluating the light stability of amorphous samples;

[0124] Fig. 53 is an XRPD overlay image evaluating the light-induced stability of crystal form V;

[0125] Fig. 54 is the HPLC chromatogram for evaluating the light stability of crystal form I;

[0126] Fig. 55 is the HPLC chromatogram for evaluating the light stability of crystal form IIIA;

[0127] Fig. 56 shows the HPLC chromatogram for evaluating the light stability of amorphous samples; and

[0128] Fig. 57 is an HPLC chromatogram for evaluating the photo-irradiation stability of crystal form V. Example

[0129] The following examples are for illustrative purposes only and are not intended to limit the invention in any way.

[0130] Solvent Name Correspondence Table

[0131]

[0132] The following instruments and methods are used in this invention:

[0133] Instruments and methods

[0134] X-ray powder diffraction (XRPD): XRPD results are obtained in X'Pert... 3 Data were collected using an Empyrean X-ray powder diffractometer, and the scanning parameters are shown in Table 1.

[0135] Table 1: XRPD Test Parameters

[0136]

[0137] Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC): TGA and DSC data 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.

[0138] Table 2: TGA and DSC Test Parameters

[0139]

[0140]

[0141] 1H Solution NMR: The 1H solution NMR spectrum was acquired on a Bruker 400M NMR spectrometer using DMSO-d6 as the solvent.

[0142] Dynamic moisture adsorption (DVS): Dynamic moisture adsorption (DVS) curves were acquired on the DVS Intrinsic of SMS (Surface Measurement Systems). The relative humidity at 25°C was corrected for the deliquescence points of LiCl, Mg(NO3)2, and KCl. The DVS test parameters are listed in Table 3.

[0143] Table 3: DVS Test Parameters

[0144]

[0145] High performance liquid chromatography (UPLC): Purity and solubility were tested using an Agilent 1290 ultra-high performance liquid chromatograph, and the analytical conditions are shown in Tables 4 and 5.

[0146] Table 4: Ultra-high performance liquid chromatography (UHPLC) test conditions for purity testing

[0147]

[0148]

[0149] Table 5: Ultra-high performance liquid chromatography test conditions for solubility testing

[0150]

[0151] For instruments and methods not explicitly specified in the "Instruments and Methods" section, instruments and methods known in the art may be used.

[0152] Example 1a (Compound 1 is amorphous, repeat of Example 1 of Chinese Patent Application No. 201210190520.4): Following 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 with dichloromethane / methanol as the eluent, and then rotary evaporated to obtain a white solid. XRPD analysis showed that it was amorphous, and its XRPD pattern is shown in Fig. 1, and its dynamic water adsorption (DVS) pattern is shown in Fig. 2.

[0153] Example 2a: 4 mL of anhydrous ethanol was added to approximately 21 mg of the amorphous sample from Example 1a, and the mixture was heated and stirred in an oil bath at 85°C for 5 minutes to dissolve it. Subsequently, the resulting supernatant was cooled to room temperature (25°C) with stirring over 2.5 h, and stirring was continued at room temperature for 24 h. The resulting solid sample was then filtered out and subjected to XRPD determination.

[0154] 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 (initial temperature).1 ¹H NMR data were acquired using DMSO-d6 as the solvent, and the results are shown in Fig. 5. No obvious solvent residue was detected. Based on the characterization results of crystal form I, which exhibits small and gradual weight loss (below the theoretical water content of 1.80% for hemihydrates) and a single melting endothermic signal, it is speculated that crystal form I is an hydrate or amorphous. The XRPD diffraction peak data of crystal form I are shown in Table 6.

[0155] Table 6: XRPD diffraction peak data of crystal form I

[0156]

[0157] Example 2b: Approximately 20 mg of each amorphous sample was weighed and added to a 20-mL vial. 0.4–1.0 mL of a good solvent (see Table 7) was added to dissolve the sample. The solution was filtered (using a 0.45 μm PTFE membrane) to obtain a clear solution. While stirring the clear solution, the antisolvent from Table 7 was added until a solid precipitated. The precipitated solid was separated by centrifugation and XRPD testing was performed. The results are shown in Table 7. The solid obtained by adding the antisolvent was identified as crystal form I by XRPD.

[0158] Table 7: Summary of Antisolvent Addition Experiments

[0159]

[0160]

[0161] [1] After adding the antisolvent, the mixture was clarified and then transferred to 5°C with stirring to precipitate a solid.

[0162] [2] After adding the antisolvent, the solution became clear. After stirring at 5°C, the solution remained clear. However, after stirring at -20°C, a solid precipitated.

[0163] [3] The solution clarified after the antisolvent was added, and remained clear after stirring at 5°C and -20°C. It then evaporated at room temperature.

[0164] [4] After the antisolvent is added, it becomes an oil. After circulating and stirring at a temperature of 5-50°C, it still becomes an oil. It then evaporates at room temperature.

[0165] Example 2c: Weigh approximately 20 mg of each amorphous sample from Example 1a into a 5-mL or HPLC vial, add 0.7–4.0 mL of the solvent listed in Table 8, stir at 50°C for approximately 2 hours, filter (using a 0.45 μm PTFE membrane), collect the filtrate, place the filtrate in a biochemical incubator, and cool to 5°C at a rate of 0.1°C / min. Collect the precipitated solid and perform XRPD testing. The results are shown in Table 8. The solid obtained from the slow cooling test was identified as crystal form I by XRPD.

[0166] Table 8 Summary of the 5℃ slow cooling test

[0167]

[0168] [1] After slowly cooling to 5°C, the solution becomes clear. Then, it is transferred to -20°C and allowed to stand before solidification occurs.

[0169] [2] After slowly cooling to 5°C, it became clear. After being transferred to -20°C and left to stand, no solid precipitated. It then evaporated at room temperature.

[0170] Example 3 Crystal Form II

[0171] Example 3a: 71.6 mg of the crystal form I sample from Example 2a was weighed into a 3 mL vial, and 1.5 mL of ACN was added. The sample was magnetically stirred at room temperature for about 1 day. The solid was separated by centrifugation and dried under ambient conditions (~21°C / 45% RH) for about 4 hours to obtain the final product. XRPD and TGA / DSC tests were then performed on the product.

[0172] The XRPD results are shown in Fig. 6. The TGA results are shown in Fig. 7. The TGA shows that the sample has a step-like weight loss of 5.90% when heated to 120℃. The DSC results show that there are two endothermic peaks at 94.1℃ and 220.9℃ (initial temperature) and one exothermic peak at 191.6℃ (peak temperature). 1 The 1H NMR results are shown in Fig. 8. The molar ratio of ACN to API in the sample is 0.8 (5.9 wt%, consistent with the TGA weight loss). Based on the characterization and heating experiments, it is speculated that crystal form II is an ACN solvate, which undergoes desolvation and crystal transformation upon heating. The XRPD diffraction peak data of crystal form II are shown in Table 9.

[0173] Table 9: XRPD diffraction peak data for crystal form II

[0174]

[0175] Example 3b: Weigh 20.4 mg of the crystal form I sample from Example 2a into a 3 mL vial, add 2 mL of ACN / acetone (1:1, v / v), sonicate, and filter (0.45 μm PTFE membrane) to obtain a clear solution. Seal with sealing film, poke 4 small holes, and place in a fume hood for slow evaporation for 5 days. Solid precipitation was observed. After being left under ambient conditions overnight, it was transferred to room temperature and vacuum dried for about 1 day. The ACN / acetone (1:1, v / v) solution of crystal form I from Example 2a was slowly evaporated, and after solid precipitation, it was transferred to room temperature and vacuum dried to obtain the final product, which was then subjected to XRPD testing. The XRPD results are shown in Fig. 6.

[0176] Example 3c: Approximately 19.7 mg of the amorphous sample obtained in Example 1a was weighed into an HPLC vial, and 0.5 mL of ACN / H2O (19:1) solvent was added. The resulting suspension was magnetically stirred at 5°C (~1000 rpm) for approximately 6 days. The solid was then separated by centrifugation and XRPD testing was performed. The XRPD result indicated crystal form II.

[0177] Example 3d: Approximately 19.9 mg of the amorphous sample obtained in Example 1a was weighed into an HPLC vial, and 0.5 mL of ACN / acetone (1:1) solvent was added. The resulting suspension was magnetically stirred at 5°C (~1000 rpm) for approximately 6 days. The solid was then separated by centrifugation and XRPD testing was performed. The XRPD result indicated crystal form II.

[0178] Example 4 (Crystal forms IIIA and IIIB)

[0179] Example 4a: The crystal form II sample from Example 3a was heated to 120°C using DSC and held at that temperature for 5 minutes before being cooled to room temperature to obtain crystal form IIIA. The XRPD results are shown in Fig. 9. The TGA / DSC results (Fig. 10) show that the sample lost 0.26% of its weight when heated to 200°C, which is lower than the theoretical water content of the hemihydrate (1.80%). The DSC results show an exothermic signal at 200.3°C (peak temperature) and a sharp endothermic signal at 219.6°C (initial temperature). 1 The 1H NMR results (Fig. 11) show no obvious solvent residue. Crystal form IIIA was transformed into crystal form I after being heated to 205℃, cooled to room temperature, and exposed to ambient conditions. The XRPD results before and after heating are shown in Fig. 12. Based on the characterization results of crystal form IIIA, it is inferred that crystal form IIIA is an anhydrous, and the exothermic signal at 198.8℃ is the thermal signal of the transformation to crystal form I. The XRPD diffraction peak data of this crystal form IIIA are shown in Table 10.

[0180] Table 10 XRPD diffraction peak data for crystal form IIIA

[0181]

[0182]

[0183] Example 4b: 100.0 mg of the crystal form I sample from Example 2a was weighed into a 20 mL vial, dissolved in 10 mL of THF, and filtered (using a 0.45 μm PTFE membrane) to obtain a clear solution. The filtrate was rotary evaporated at 50 °C, and the final product was collected. The XRPD results are shown in Fig. 13. The TGA / DSC results (Fig. 14) show that the sample lost 3.49% of its weight when heated to 200 °C; the DSC results show a weak endothermic signal at 121.3 °C and an exothermic signal at 144.6 °C (peak temperature), and a relatively strong endothermic signal at 218.3 °C (initial temperature). 1 The 1H NMR results (Fig. 15) show that the molar ratio of residual solvent THF to API is 0.09 (1.3 wt%), presumably due to surface adsorption. The thermal signals of crystal form IIIB at 121.3 °C and 144.6 °C are the thermal signals of the transition to crystal form I. The XRPD diffraction peak data of crystal form IIIB are shown in Table 11.

[0184] Table 11: XRPD diffraction peak data of crystal form IIIB

[0185]

[0186]

[0187] Example 5 (Crystal Form IV)

[0188] Example 5a: Approximately 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 magnetically stirred at room temperature (~1000 rpm) for approximately 6 days. The solid was then separated by centrifugation and vacuum dried overnight at room temperature to obtain the final product. XRPD and TGA / DSC tests were performed on the product. The XRPD and TGA / DSC results are shown in Fig. 17 and...

[0189] As shown in Fig. 18, TGA results show that the sample exhibits a step-like weight loss of 16.80% when heated to 120℃; DSC results indicate two endothermic peaks at 95.6℃ and 220.5℃ (initial temperature). 1The 1H NMR results are shown in Fig. 19. The molar ratio of 1,4-Dioxane to API in the sample is 1.2 (17.8 wt%, consistent with the TGA weight loss). Based on the above characterization results and temperature-dependent XRPD results, it is inferred that crystal form IV is a 1,4-Dioxane solvate. The XRPD diffraction peak data of this crystal form IV are shown in Table 12.

[0190] Table 12: XRPD diffraction peak data of crystal form IV

[0191]

[0192]

[0193] Example 5b: The crystal form I sample (500 mg) from Example 2a 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.

[0194] Example 6 (Crystal Form V)

[0195] Example 6a: Weigh 20.8 mg of the crystal form I sample from Example 2a into a 5 mL vial and add 4 mL of EtOH. Dissolve the sample by stirring at 70 °C, then cool to 5 °C over 650 minutes (cooling rate 0.1 °C / min) and allow it to stand at 5 °C. After removing the solution, dry the solid 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 step-like weight loss of 7.64% when the sample is heated to 120 °C, with two endothermic peaks at 103.1 °C and 223.3 °C (initial temperature). 1 The H NMR results are shown in Fig. 22. The molar ratio of EtOH to API in the sample is 0.7 (6.2 wt%), which is consistent with the weight loss observed by TGA. Based on the above characterization results and temperature-dependent XRPD results, it is speculated that crystal form V is an EtOH solvate, which transforms into the amorphous form I upon heating. The XRPD diffraction peak data of crystal form V are shown in Table 13.

[0196] Table 13: XRPD diffraction peak data for crystal form V

[0197]

[0198]

[0199] Example 7 (Crystal Form VI)

[0200] The amorphous sample (1g) of Example 1a was dissolved in DMF (3mL). After complete dissolution, IPA (6mL) was added, and the mixture was stirred overnight at room temperature. The solid precipitated, filtered, and the filter cake was washed with isopropanol and dried under vacuum to obtain the solvate crystal form of DMF, which is crystal form VI.

[0201] The XRPD results for crystal form VI are shown in Fig. 23. The DSC results show two endothermic peaks at 147.39℃ and 208.56℃. The TGA results in Fig. 24 show that the weight loss is 0.67% when heated to 180℃.

[0202] Example 8 (Crystal Conversion)

[0203] Example 8a: Crystal form IV of Example 5a was heated to 120°C, cooled to room temperature, and exposed to environmental conditions to transform into crystal form I. The XRPD results are shown in Fig. 25.

[0204] Example 8b: The crystal form II of Example 3a was heated to 120°C and 210°C respectively, cooled to room temperature and exposed to environmental conditions. The XRPD results are shown in Fig. 26. Heating to 120°C transformed it into crystal form IIIB, and heating to 210°C transformed it into crystal form I.

[0205] Example 8c: After heating crystal form II of Example 3b to 120°C, cooling to room temperature, and exposing it to ambient conditions, it transformed into crystal form I. The XRPD results are shown in Fig. 27. It is speculated that the sample may have initially transformed into crystal form IIIB after heating, but then rapidly transformed into crystal form I under ambient conditions, as shown in the XRPD test.

[0206] Example 8d: Crystal form IIIB of Example 4a was identified by variable-temperature XRPD, and the results are shown in Fig. 16. After purging with N2 at 30°C for 20 minutes, the crystal form remained unchanged. When heated to 120°C under N2 protection, diffraction peaks of crystal form I were observed. Upon further heating to 170°C, crystal form I was the dominant crystal form, with only a small number of diffraction peaks of crystal form IIIB. Cooling to 30°C under N2 protection did not result in further transformation.

[0207] Example 8e: The crystal form V of Example 6a was heated to 120°C, cooled to room temperature, and exposed to environmental conditions to transform into crystal form I. The XRPD results are shown in Fig. 28.

[0208] Example 8f (Competition Experiment of Crystal Forms I and IIIA Suspension)

[0209] This embodiment relates to a suspension competition experiment of crystal form I and crystal form IIIA in EtOAc and MIBK at room temperature and 50°C, and a suspension competition experiment of acetone / H2O with different water activities at room temperature.

[0210] Weigh approximately 15 mg of the Example 2a crystal form I sample into an HPLC bottle, add 1 mL of the corresponding solvent, and stir at the appropriate temperature for 4 hours or overnight. Filter (using a 0.45 μm PTFE membrane) to obtain a saturated solution. Weigh equal masses of Example 2a crystal form I and Example 4a crystal form IIIA samples (approximately 5 mg each) into a new HPLC bottle, and add the saturated solution obtained in step 1. Stir at the appropriate temperature, and perform XRPD testing on the solid wet sample (covered with a Kapton membrane to avoid potential crystal transformation due to solvent evaporation during the test).

[0211] The results of the suspension competition are summarized in Table 14. The XRPD results are shown in Fig. 29, Fig. 30, Fig. 31, and Fig. 32.

[0212] Table 14 Results of the suspension competition experiment for crystal types I and IIIA

[0213]

[0214] Where a w Theoretical water activity.

[0215] According to the XRPD comparison results, under all conditions, the physical mixture of crystal form I and IIIA transformed into crystal form I after suspension competition, indicating that under anhydrous conditions in the range of room temperature to 50°C and under water activity of 0 to 1 at room temperature, the ahydrous crystal form I is more thermodynamically stable than the crystal form IIIA.

[0216] Example 9 (Hygroscopicity of crystal form I)

[0217] The hygroscopicity of crystal form I in Example 2a, amorphous crystal in Example 1a, and crystal form IIIA in Example 4a was evaluated by dynamic moisture adsorption (DVS) test at 25°C.

[0218] The DVS results for crystal form I are shown in Fig. 33. At 25°C / 80% RH, the weight gain due to water absorption is 0.047%, indicating that it has almost no hygroscopicity. The XRPD comparison results (Fig. 34) show that crystal form I did not undergo a crystal form transformation after the DVS test.

[0219] The DVS results for the amorphous sample are shown in Fig. 2. As the humidity increased from 50% RH to 95% RH, the weight gain from water absorption decreased continuously. It is speculated that the amorphous sample underwent crystal transformation during the increase in humidity, and the water or organic solvent adsorbed or encapsulated in the original sample was removed under the action of N2 purging. The XRPD comparison results (Fig. 35) show that the amorphous sample transformed into crystal form I after the DVS test.

[0220] The DVS results for crystal form IIIA are shown in Fig. 36. At 25°C / 80% RH, the weight gain due to water absorption is 0.060%, indicating that it has almost no hygroscopicity. The XRPD comparison results (Fig. 37) show that crystal form IIIB did not undergo a crystal form transformation after the DVS test.

[0221] Example 10 (Solid-state stability of crystal form I)

[0222] To evaluate the solid-state stability of crystal forms I, IIIA, and amorphous samples, appropriate amounts of crystal form I from Example 2a, the amorphous sample from Example 1a, and crystal form IIIA from Example 4a were weighed and subjected to stability experiments under the following conditions: 60°C / closed / 1 day, 25°C / 60%RH / open / 1 week, and 40°C / 75%RH / open / 1 week. The physical stability of the samples under different conditions was assessed by XRPD testing of crystal form, and the chemical stability was assessed by HPLC testing of purity. The evaluation results are summarized in Table 15, and the XRPD results are shown in Fig. 38, Fig. 39, and Fig. 40 (a weak diffraction peak for crystal form I was observed at the starred location in Fig. 40). Stability results showed that crystal form I did not undergo crystal form transformation or purity reduction under the three evaluation conditions, indicating that crystal form I has good physical and chemical stability under the evaluation conditions; the amorphous form did not show significant purity changes under the three evaluation conditions, but all transformed into crystal form I; crystal form IIIA showed a weak diffraction peak of crystal form I after being placed in a closed container at 60℃ for 1 day, and the crystal form remained unchanged after being placed in an open container at 25℃ / 60%RH and 40℃ / 75%RH for 1 week.

[0223] Table 15 Summary of Solid-State Stability Evaluation Results for Crystal Form I in Example 2a

[0224]

[0225] *: Amorphous samples all transformed into crystal form I after stability assessment;

[0226] #: Weak diffraction peaks were observed for crystal form I.

[0227] Example 11 (Solubility)

[0228] The dynamic solubility of crystal form I from Example 2a and amorphous form from Example 1a in different pH conditions at room temperature (1M HCl and buffer solutions at pH 2.0 / 4.5 / 6.8 / 7.4) was tested. The specific steps are as follows:

[0229] (1) Weigh about 2.5 mg of crystalline form I or amorphous sample into a 20 mL glass bottle and add 10 mL of different pH buffer solution, or weigh about 2.0 mg of crystalline form I or amorphous sample into a 20 mL glass bottle and add 8 mL of hydrochloric acid (1 M).

[0230] (2) Shake 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 solutions obtained from amorphous raw materials, take 100 μL and dilute it 10 times with the corresponding buffer solution for later use. If there is solid precipitation in the solution before dilution, test the diluted sample; if there is no solid precipitation, test the sample before dilution. For solutions obtained from crystal form I raw materials, since its solubility is expected to be low, it is not diluted and the filtered clear solution is directly tested by HPLC.

[0231] Table 16 shows the solubility of crystalline form I and amorphous form under different pH conditions. The results indicate that the solubility of crystalline form I remained almost unchanged under different pH conditions, while the solubility of amorphous form showed a significant change with pH (e.g., Fig. 41). Crystalline form I exhibited more stable dissolution in vivo (e.g., Fig. 42), making it easier to obtain stable pharmacokinetic results in blood concentrations, which helps avoid the safety risks associated with large fluctuations in blood drug concentration. Similar results were observed when the dosage concentration was 0.05 mg / mL (e.g., Fig. 43).

[0232] Table 16. Solubility results of crystalline form I and amorphous form in different media over 3 minutes (feed concentration 0.25 mg / mL)

[0233]

[0234] LOQ = 0.28 μg / mL.

[0235] Example 12 (Powder Properties)

[0236] The powder properties of the crystalline I sample 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 crystalline I and amorphous samples.

[0237] Loose density and tapped density: A certain mass of the sample to be evaluated is added to a 5-mL graduated cylinder, and the volume is recorded. The loose density is calculated by dividing the mass of the sample by the volume at this point. The graduated cylinder is tapped 200 times, and the final volume is recorded. The tapped density is obtained by dividing the mass of the sample by the final volume. Each parameter is tested in triplicate.

[0238] Angle of repose: Fix the funnel perpendicular to the bottom surface and slowly add material into the funnel. A uniform cone of material will form at the bottom. Measure the height of the cone and the diameter of the bottom surface. Perform three parallel measurements.

[0239] The results of the loose density / tap density are summarized in Table 17. The results show that the average loose density and tap density of crystal form I are 0.34 g / cm³.3 and 0.46 g / cm 3 The Carr index was calculated to be 26%; the average loose density and tap density of the amorphous material were 0.31 g / cm³. 3 and 0.46 g / cm 3 The Cartesian exponent was calculated to be 33%.

[0240] The results of the angles of repose are summarized in Table 18. The results show that the angles of repose for crystalline I and amorphous samples are 27.7° and 26.7°, respectively. Based on the overall evaluation, crystalline I and amorphous samples have relatively similar Karl Fischer index and angles of repose, indicating that crystalline I and amorphous samples have similar flowability.

[0241] Table 17 Results of Loose Density / Tamped Density Tests

[0242]

[0243] Carr index = (tap density - loose density) / tap density.

[0244] Table 18 Results of Angle of Repose Test

[0245]

[0246] Formula for calculating the angle of repose α: α = tan -1 (h / D), where h is the height of the vertebral body and D is the diameter of the vertebral body.

[0247] Example 13 (Mechanical Stability)

[0248] The crystal form I of Example 2a, crystal form IIIA of Example 4a, and amorphous material of Example 1a were manually ground and tableted using a tablet press (350 MPa pressure). XRPD tests were performed on the ground and tableted samples to evaluate their mechanical stability. The XRPD results are shown in Fig. 44, Fig. 45, Fig. 46, Fig. 47, Fig. 48, and Fig. 49. The evaluation results show that after grinding and tableting, crystal form I did not undergo a crystal transformation and its crystallinity did not decrease significantly; crystal form III maintained its crystal form after grinding but its crystallinity decreased slightly, and after tableting, its crystal form remained unchanged but its crystallinity decreased significantly; the amorphous material transformed into crystal form I after grinding and tableting.

[0249] Based on the crystal form characterization and identification results, crystal form I was selected for hygroscopicity, solid-state stability, solubility, powder properties, and mechanical stability assessments. The same assessments were also performed on the amorphous form for property comparison with crystal form I. DVS results showed that crystal form I had almost no hygroscopicity and did not undergo crystal form transformation after DVS testing; the amorphous form transformed into crystal form I after DVS testing. Solid-state stability results showed that crystal form I did not undergo crystal form transformation or purity reduction after being placed in a closed container at 60℃ for 1 day, and in an open container at 25℃ / 60%RH and 40℃ / 75%RH for 1 week, indicating that crystal form I has good physical and chemical stability under the assessment conditions. The amorphous form showed no significant change in purity under all three assessment conditions, but transformed into crystal form I in all cases. Powder property tests of crystal form I showed that crystal form I and the amorphous sample have similar flowability. Mechanical stability results showed that after tableting (350 MPa) and manual grinding (about 3 minutes), crystal form I did not undergo crystal transformation and the crystallinity did not decrease significantly, and the amorphous form transformed into crystal form I.

[0250] Based on the characterization data and evaluation results, crystalline form I did not undergo crystalline transformation under all evaluation conditions, while the amorphous form transformed into crystalline form I after DVS, solid-state stability, solubility, and mechanical stability tests, indicating that crystalline form I has better physical stability than the amorphous form.

[0251] Example 14 (Light Stability)

[0252] The crystal forms I of Example 2a, IIIA of Example 4a, V of Example 6a, and amorphous material of Example 1a were subjected to illumination (white light 5800-5890 Lux + ultraviolet 7.9-8.7 W / m²) respectively. 2Samples were taken at 6 and 24 hours for XRPD and HPLC purity testing and stability assessment. The XRPD results are shown in Fig. 50, Fig. 51, Fig. 52, and Fig. 53: no crystal form transformation occurred in crystal form I, crystal form IIIA, crystal form V, and amorphous material after 24 hours of exposure to light; under the reference condition of being protected from light, no crystal transformation occurred in crystal form I, crystal form IIIA, and crystal form V after 24 hours of exposure to light, while a weak diffraction peak of crystal form I was observed in amorphous material after 6 and 24 hours of exposure. 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 crystal form I under illumination decreased slightly from 100.00 area % to 99.14 area % (impurity A content 0.79%). The purity of crystal form IIIA, crystal form V and amorphous under illumination 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%), respectively. The purity of the four samples under the light-protected reference conditions did not change significantly (the samples irradiated for 6 hours in Fig. 54, Fig. 55 and Fig. 56 and the tests of photodegradation impurities were performed in two separate HPLC test sequences, so the retention time of the impurities may have slightly shifted). Based on the results of light stability, crystal form I showed better light stability compared to crystal form IIIA, crystal form V and amorphous form.

[0253] Table 19 Results of the light stability of crystal forms I / IIIA / V and amorphous forms

[0254]

[0255] Example 15 (Pharmacokinetic Experiment in SD Rats)

[0256] 2.39 mg of crystalline form I from Example 2a, 2.38 mg of crystalline form IIIA from Example 4a, and 2.46 mg of amorphous form from Example 1 were used as test substances. 7.967 mL, 7.906 mL, and 8.193 mL of 0.5% CMC-Na aqueous solution were added, respectively, to prepare oral administration formulations with a final concentration of 0.3 mg / mL. Three male SD rats were administered the above three test substance solutions by gavage at a dose of 3 mg / kg body weight. 0.15 mL of blood was collected from the jugular sinus at time points of 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 mixture was centrifuged (1500–1600 g) for 10 min to separate the plasma for bioanalysis. The concentration of the test substance in plasma samples was determined by LC-MS / MS analysis (instrument model: Triple Quad 5500; 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 were calculated using a non-compartmental model in Pharsight Phoenix 8.3 (Table 20). The data show that the AUC of crystal form I and crystal form IIIA... 0-t 9820 ng·h / mL and 8890 ng·h / mL respectively, C max The concentrations were 1160 ng / mL and 1030 ng / mL, respectively, indicating adequate plasma exposure and good pharmacokinetic properties. Furthermore, crystal form I had higher exposure and plasma concentration than crystal form IIIA, suggesting the potential for lower dosage.

[0257] Table 20 Pharmacokinetic parameters for crystal form I, crystal form IIIA, and amorphous forms

[0258]

[0259] Example 16: Inhibition of proliferation of NRAS-mutant melanoma cell lines

[0260] 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 and streptomycin in a 37°C, 5% CO2 incubator. The human melanoma cell line HMVII (NRAS Q61K) was cultured in F12K + 10% FBS + 1% penicillin and streptomycin in a 37°C, 5% CO2 incubator.

[0261] When the confluence of cultured cells reached 80% or more, adherent cells were digested with trypsin, and the cell pellet was collected by centrifugation. Cells were counted, and 90 μL of cell suspension was seeded into 96-well plates at an appropriate density. After 24 hours, a series of serially diluted compounds (crystal form I) (concentration range 0.15 nM–10 μM, 4-fold serial dilution) were added to each well at a volume of 10 μL. Three replicates were set for each concentration, for a total of 9 concentration points. Wells containing the same volume of 5% DMSO served as controls, with a final DMSO concentration of 0.5%. Three days after drug treatment, cell viability was assessed using the MTT assay. 10 μL of MTT was added to each well, and the cells were incubated for another 4 hours. The supernatant was discarded, and 150 μL of DMSO was added to dissolve the formazan crystals. The absorbance at 490 nM was measured using a microplate reader. The above experiments were repeated three times. A dose-response curve was generated using GraphPadPrism 8 software, and the IC50 was calculated. Results are expressed as the mean IC50 ± SD value from the three experiments (Tables 21 and 22).

[0262] Table 21. IC50 of compound 1 on SK-MEL-2 tumor cells 50 value

[0263]

[0264]

[0265] Table 22. IC50 of compound 1 on HMVII tumor cells 50 value

[0266]

[0267] Example 17: Suppression of RAS or RAF mutations

[0268] The in vitro antiproliferative activity of compound 1 against RAS-mutated or RAF-mutated tumor cell lines, RAS / RAF wild-type tumor cell lines, and normal human cell lines was detected using the tetrazolium salt (MTS) method.

[0269] Adherent cells in logarithmic growth phase were digested with trypsin or suspension cells were collected by centrifugation, counted, and seeded into 150 μL of 96-well plates. After 24 hours, 50 μL / well of compound (crystal form I) diluted 4 times to its final concentration (concentration range 0.15 nM-1000 nM, serially diluted 3 times) was added to each well. Wells containing the same volume of 2% DMSO were used as controls (DMSO final concentration 0.5%). Cell viability was assessed by MTS after 72 hours of culture. The specific method was as follows: For adherent cells, the culture medium was discarded, and 20 μL of MTS and 100 μL of cell culture medium were added to each well; for suspension cells, 20 μL of MTS was added directly. After 1-4 hours of incubation, OD490 was measured, with OD650 as a reference. A dose-response curve was generated and IC50 was calculated using GraphPad Prism software. 50 The results are shown in Table 23.

[0270] Table 23. In vitro antiproliferative IC50 of compound 1 against RAS and RAF-mutant tumor cells and human embryonic lung cells MRC-5 50 value

[0271]

[0272] Example 18:

[0273] This multicenter, single-arm phase II study evaluated the efficacy and safety of compound 1, crystal form I, in patients with advanced melanoma exhibiting NRAS mutations. Patients received 12 mg orally twice daily until unacceptable toxicity, disease progression (assessed by the investigator according to RECIST 1.1), withdrawal of informed consent, death, or termination by the investigator at the discretion of the investigator when the risk outweighed the benefit. As of February 19, 2023, 100 subjects were enrolled, with 95.0% (95 / 100) of subjects included in the full analysis set (FAS).

[0274] Key therapeutic indicators:

[0275] In the FAS population: the ORR (objective response rate) assessed by the IRRC (Independent Imaging Review Committee) was 35.8% (34 / 95 cases) (95% CI: 26.2%, 46.3%).

[0276] In the FAS population, the IRRC assessment 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).

[0277] Compound 1 showed good anti-tumor therapeutic effects on patients with advanced melanoma with NRAS mutations, with an ORR of 35.8% as assessed by IRRC, which was significantly better than the clinical research data of similar drugs.

Claims

1. Polymorphs of Formula (I) (I) Where n is 0, the polymorph is crystal form I, characterized in that... The X-ray powder diffraction pattern of crystal form I includes characteristic diffraction peaks at the following 2θ positions: 5.3°±0.2°, 7.48°±0.2°, 11.81°±0.2°, 15.83°±0.2°, 16.71°±0.2°, 17.92°±0.2°, 18.95°±0.2°, 19.17°±0.2°, 21.82°±0.2°, 22.36°±0.2°, 23.75°±0.2°, 24.57°±0.2°, and 27.08°±0.2°, using Cu-Kα radiation.

2. The polymorph of claim 1, wherein the X-ray powder diffraction pattern of crystal form I includes 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°. 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°.

3. The polymorph of claim 1, wherein the X-ray powder diffraction pattern of said polymorph I is substantially as shown in Fig.

3.

4. The polymorph of claim 1, wherein the polymorph I has a TGA diagram and / or DSC diagram as shown in Fig.

4.

5. The polymorph of any one of claims 1-4, wherein the polymorph does not contain impurity A, and impurity A has the following structure: .

6. The polymorph of claim 1, wherein crystal form I contains less than 0.15% by weight of impurity A relative to crystal form I, said impurity A having the following structure: .

7. A pharmaceutical composition comprising a polymorph of any one of claims 1-6, and a pharmaceutically acceptable carrier and / or excipient.

8. Use of the polymorph of any one of claims 1-6 in the preparation of medicaments for treating tumors, chronic inflammatory diseases, inflammatory bowel diseases, skin diseases, diabetes, eye diseases, diseases related to angiogenesis or regeneration in mammals, diseases related to chronic pain, and other diseases modulated by the Mek cascade.

9. A method for preparing crystal form I of claim 1, the method comprising any one of the following: a) Add the amorphous sample of compound 1 to the solvent, then heat at a temperature above 70°C. Cool the resulting supernatant to room temperature, maintain the temperature at room temperature while stirring, and allow the solid to precipitate. Filter and dry. b) Dissolve the amorphous sample of compound 1 in a good solvent, filter to obtain a clear solution, and add the antisolvent while stirring the clear solution until a solid precipitates; or c) Dissolve the amorphous sample of compound 1 in a solvent, stir at 50 °C, then filter and collect the filtrate. Cool the filtrate to 5 °C and collect the precipitated solid. The compound 1 therein is 4-fluoro-5-(2-fluoro-4-iodophenylamino)-1H-benzo[d]thiazol-6-carboxylic acid (2-hydroxy-ethoxy)-amide.

10. The method of claim 9, wherein the heating temperature in method a) is 75°C to 100°C.

11. The method of claim 9, wherein the heating temperature in method a) is 80°C to 90°C.

12. The method of claim 9, wherein the heating temperature in method a) is 75°C or 85°C.

13. The method of claim 9, wherein the room temperature in method a) is 20-25°C.

14. The method of claim 9, wherein the stirring time in method a) is 1-12 hours.

15. The method of claim 9, wherein the stirring time in method a) is 1-8 hours.

16. The method of claim 9, wherein the stirring time in method a) is 1-5 hours or 24-96 hours.

17. The method of claim 9, wherein in method a), the supernatant is cooled to room temperature within 2 to 5 hours.

18. The method of claim 9, wherein in method a), the supernatant is cooled to room temperature within 2.5-3 hours.

19. The method of claim 9, wherein in method a), after stirring at room temperature, the temperature may optionally be further reduced to 0-10°C for stirring while maintaining the temperature.

20. The method of claim 19, wherein the mixture is stirred at 0-10°C for 1-12 hours.

21. The method of claim 19, wherein the mixture is stirred at 0-10°C for 1-8 hours.

22. The method of claim 19, wherein the mixture is stirred at 0-10°C for 1-5 hours.

23. The method of claim 9, wherein the solvent in method a) 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.

24. The method of claim 9, wherein the solvent in method a) is ethanol.

25. The method of claim 9, wherein the good solvent in method b) is a solvent in which compound 1 is soluble, and the antisolvent in method b) is a solvent in which compound 1 is insoluble.

26. The method of claim 9, wherein the good solvent in method b) is MEK, 1,4-dioxane or DMSO; and the antisolvent in method b) is MTBE, EtOAc, CHCl3, n-heptane, anisole, EtOAc, H2O, IPAc, CPME, DCM or toluene.

27. The method of claim 9, wherein the cooling rate in method c) is 0.1 °C / min.

28. The method of claim 9, wherein the solvent of method c) is MIBK, methyl acetate, 2-MeTHF or acetone / EtOH, wherein the ratio of acetone to EtOH is 1:

1.

29. Use of the polymorph of any one of claims 1-6 in the preparation of a medicament for treating RAS or RAF mutant cancers in mammals.

30. The use of claim 29, wherein the RAS or RAF mutant cancer is a KRAS mutant cancer, NRAS mutant cancer, HRAS mutant cancer, or BRAF mutant cancer.

31. The use of claim 29 or 30, wherein the RAS-mutant cancer is pancreatic cancer, colorectal cancer, lung cancer, melanoma, acute myeloid leukemia, bladder cancer, or head and neck cancer.

32. The use of claim 29 or 30, wherein the cancer is an NRAS-mutant cancer.

33. The use of claim 32, wherein the NRAS-mutant cancer is an NRAS-mutant melanoma.

34. The use of claim 30, wherein KRAS includes a mutation at one or more positions selected from codons 12, 13, 59 and 61.

35. The use of claim 33, wherein the NRAS includes mutations at one or more positions selected from codons 12, 13, 59, 61 and 146.

36. The use of claim 33, wherein the NRAS mutation is a mutation at one or more amino acid positions selected from G12, G13, A59, Q61, K117 and A146.

37. The use of claim 36, wherein the NRAS mutation 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.

38. The use of claim 29 or 30, wherein the cancer is an early, intermediate, or late-stage cancer.

39. The use of claim 29 or 30, wherein the cancer is locally advanced or metastatic.

40. The use of claim 29 or 30, wherein the mammal has previously received immunotherapy.

41. Use of claim 40, wherein the mammal has previously received immunotherapy and has advanced melanoma with NRAS mutations.

42. The use of claim 31, wherein the melanoma is advanced melanoma.

43. The use of claim 31, wherein the melanoma is an unresectable melanoma.

44. The use of claim 31, wherein the melanoma is a metastatic melanoma.

45. The use of claim 31, wherein the melanoma is a melanoma with a BRAF mutation.

46. ​​The use of claim 31, wherein the melanoma is a melanoma with an NRAS mutation.

47. The use of claim 31, wherein the melanoma is a cutaneous melanoma.

48. The use of claim 31, wherein the melanoma is an intraocular melanoma.

49. The use of claim 29 or 30, wherein the medicament is in the form of a capsule.

50. The use of claim 49, wherein crystal form I is administered at a dose of 5-50 mg once or twice daily.

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