A crystalline form of a cbl-b inhibitor

Abstract

The application provides a crystal form of a compound 4-chloro-6-(6-cyclopropyl-4-((1S,3R)-3-methyl-1-(4-methyl-4H-1,2,4-triazol-3-yl)cyclobutyl)pyridin-2-yl)-2-(((S)-3-methylpiperidin-1-yl)methyl)-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-7-one of formula (I), a preparation method and application thereof. The crystal form provided by the application has good chemical stability, physical stability and low hygroscopicity, is less affected by heat, humidity and light, and is convenient for storage and preparation development.

Description

Technical Field

[0001] This application discloses a crystal form of a CBL-B inhibitor, a method for preparing the inhibitor, and their application in the treatment of cancer. Background Technology

[0002] CBL-B is a cyclic E3 ligase and a member of the highly conserved CBL protein family, which in mammals consists of three CBL genes: CBL-A, CBL-B, and CBL-C. CBL proteins interact with target proteins through their protein-protein interaction domains, thereby regulating multiple signaling pathways, including tyrosine kinase (TK) signaling in various cell types. CBL-B ubiquitination of activated receptor TK regulates the assembly of endocytic proteins on the membrane and in sorting endosomes to promote lysosomal targeting, degradation, and signal termination. CBL-B is also important for reducing signal transduction from antigen and cytokine receptors via ubiquitination of receptor chains and associated cytoplasmic TKS, leading to inactivation and / or proteasome degradation. CBL-B is expressed in the immune cell lineage and acts as a key regulator of immune cell activation and maintenance of peripheral tolerance. Furthermore, ring domain mutations of the CBL protein and CBL-B linker sequence have been found in a variety of diseases and cancers, including luvenile myeloid leukemia (IMMF), precancerous leukemia (CMMF), spinal lipoprotein membrane leukemia, and acute myeloid leukemia (AMF). Therefore, CBL-B inhibition represents an opportunity for intrinsic and extratumor toxicity-based therapies.

[0003] Numerous studies have been conducted based on this mechanism of action, but no CBL-B inhibitors have been marketed. Therefore, there is an urgent need to develop effective CBL-B inhibitors for clinical application. Patent PCT / CN2024 / 098687 describes a small molecule inhibitor targeting CBL-B, with the structure shown in formula (Ⅰ), and its chemical name is 4-chloro-6-(6-cyclopropyl-4-((1S,3R)-3-methyl-1-(4-methyl-4H-1,2,4-triazol-3-yl)cyclobutyl)pyridin-2-yl)-2-(((S)-3-methylpiperidin-1-yl)methyl)-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-7-one. This small molecule inhibitor exhibits good cell proliferation inhibition activity and demonstrates good tumor suppression activity and tolerability in in vivo pharmacodynamic experiments, showing promise for development into a clinical drug.

[0004]

[0005] (I) Summary of the Invention This application discloses a polymorph of a CBL-B inhibitor, a method for preparing the polymorph, and its application in the treatment of cancer.

[0006] On the one hand, this application provides crystal form A of compound of formula (I), 4-chloro-6-(6-cyclopropyl-4-((1S,3R)-3-methyl-1-(4-methyl-4H-1,2,4-triazol-3-yl)cyclobutyl)pyridin-2-yl)-2-(((S)-3-methylpiperidin-1-yl)methyl)-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-7-one.

[0007] (I) The invention is characterized by using Cu-Kα radiation, and the X-ray powder diffraction pattern of the crystal form A has characteristic peaks at 2θ values ​​of 6.04, 9.76, 11.01, 12.18, 16.96, and 17.36, with a 2θ error range of ±0.2°.

[0008] In some embodiments of this application, the aforementioned crystal form A is characterized by using Cu-Kα radiation, wherein the X-ray powder diffraction pattern of crystal form A has characteristic peaks at 2θ values ​​of 6.04, 9.76, 11.01, 12.18, 16.56, 16.96, 17.36, 17.67, 20.67, 22.17, and 22.45, with a 2θ error range of ±0.2°.

[0009] In some embodiments of this application, the aforementioned crystal form A is characterized by using Cu-Kα radiation, wherein the X-ray powder diffraction pattern of crystal form A has characteristic peaks at 2θ values ​​of 5.44, 6.04, 9.76, 10.74, 11.01, 11.97, 12.18, 16.56, 16.96, 17.36, 17.67, 18.08, 18.59, 20.67, 21.20, 21.85, 22.17, 22.45, 23.70, 25.12, 25.56, and 26.04, with a 2θ error range of ±0.2°.

[0010] In some embodiments of this application, the aforementioned crystal form A is characterized by using Cu-Kα radiation, and the X-ray powder diffraction pattern of crystal form A is substantially as follows: Figure 1 As shown.

[0011] In some embodiments of this application, the aforementioned crystal form A is characterized in that the TGA-DSC spectrum of the crystal form is as follows: Figure 2 As shown.

[0012] In some embodiments of this application, the XRPD diffraction peak analysis data of crystal form A of the above formula (Ⅰ) are shown in Table 1.

[0013] Table 1. XRPD diffraction peak analysis data of crystal form A of compound (I)

[0014] This application provides crystal form B of compound (I) 4-chloro-6-(6-cyclopropyl-4-((1S,3R)-3-methyl-1-(4-methyl-4H-1,2,4-triazol-3-yl)cyclobutyl)pyridin-2-yl)-2-(((S)-3-methylpiperidin-1-yl)methyl)-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-7-one, characterized in that, using Cu-Kα radiation, the X-ray powder diffraction pattern of crystal form B has characteristic peaks at 2θ values ​​of 5.30, 11.01, 11.53, 12.58, 16.23, and 17.65, with a 2θ error range of ±0.2°.

[0015] In some embodiments of this application, the characteristic feature is that Cu-Kα radiation is used, and the X-ray powder diffraction pattern of the crystal form B has characteristic peaks at 2θ values ​​of 5.30, 7.23, 8.08, 11.01, 11.53, 12.58, 16.23, 16.59, 16.96, 17.65, 18.89, 21.78, 22.93, and 24.54, with a 2θ error range of ±0.2°.

[0016] In some embodiments of this application, the characteristic feature is that Cu-Kα radiation is used, and the X-ray powder diffraction pattern of crystal form B is substantially as follows: Figure 3 As shown.

[0017] In some embodiments of this application, the TGA-DSC spectrum of the crystal form is characterized as follows: Figure 4 As shown.

[0018] In some embodiments of this application, the XRPD diffraction peak analysis data of crystal form B of the above formula (Ⅰ) are shown in Table 2.

[0019] Table 2. XRPD diffraction peak analysis data of crystal form B of compound (I)

[0020] This application provides crystal form C of compound (I) 4-chloro-6-(6-cyclopropyl-4-((1S,3R)-3-methyl-1-(4-methyl-4H-1,2,4-triazol-3-yl)cyclobutyl)pyridin-2-yl)-2-(((S)-3-methylpiperidin-1-yl)methyl)-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-7-one, characterized in that the X-ray powder diffraction pattern of crystal form C has characteristic peaks at 2θ values ​​of 5.13, 9.48, 11.04, 16.51, 17.32, and 18.31, with a 2θ error range of ±0.2°.

[0021] In some embodiments of this application, the above-mentioned crystal form C has characteristic peaks in its X-ray powder diffraction pattern at 2θ values ​​of 5.13, 9.48, 11.04, 11.66, 15.16, 16.51, 17.32, 18.31, and 24.95, with a 2θ error range of ±0.2°.

[0022] In some embodiments of this application, the X-ray powder diffraction pattern of the aforementioned crystal form C is as follows: Figure 5 As shown.

[0023] In some embodiments of this application, the XRPD diffraction peak analysis data of the crystal form C of the above formula (Ⅰ) compound are shown in Table 3.

[0024] Table 3. XRPD diffraction peak analysis data of crystal form C of compound (I)

[0025] On the other hand, this application provides pharmaceutically acceptable salts formed by compounds of formula (I) and one or more acid molecules.

[0026] Formula (I) The acid is selected from organic acids or inorganic acids.

[0027] In some embodiments of this application, the pharmaceutically acceptable salt of the compound of formula (I) is characterized in that the molecular ratio of the compound of formula (I) to the organic acid or inorganic acid is 1:1 to 1:4; preferably, the molecular ratio of the compound of formula (I) to the organic acid or inorganic acid is 1:1 to 1:2.

[0028] In certain embodiments of this application, the pharmaceutically acceptable salt of the compound of formula (I) is characterized by being selected from hydrochloride, maleate, p-toluenesulfonate, methanesulfonate, hydrobromide, succinate, L-malate, L-tartrate, benzenesulfonate, phosphate, gentianate, oxalate, fumarate, and citrate.

[0029] In some embodiments of this application, the pharmaceutically acceptable salt of the compound of formula (I) is characterized in that the pharmaceutically acceptable salt is selected from maleate and hydrochloride.

[0030] In some embodiments of this application, the pharmaceutically acceptable salt of the compound of formula (I) is characterized in that the ratio of the compound of formula (I) to maleic acid is 1:2.

[0031] This application provides maleate crystal form I of compound (I) 4-chloro-6-(6-cyclopropyl-4-((1S,3R)-3-methyl-1-(4-methyl-4H-1,2,4-triazol-3-yl)cyclobutyl)pyridin-2-yl)-2-(((S)-3-methylpiperidin-1-yl)methyl)-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-7-one, characterized in that, using Cu-Kα radiation, the X-ray powder diffraction pattern of maleate crystal form I of compound (I) has characteristic peaks at 2θ values ​​of 8.67, 12.95, 15.00, 15.82, 17.91, and 22.59, with a 2θ error range of ±0.2°.

[0032] In some embodiments of this application, the X-ray powder diffraction pattern of the above-mentioned compound (I) maleate I has characteristic peaks at 2θ values ​​of 7.43, 8.67, 9.40, 12.19, 12.95, 14.37, 15.00, 15.82, 17.91, 19.07, 22.59, and 23.86, with a 2θ error range of ±0.2°.

[0033] In some embodiments of this application, the X-ray powder diffraction pattern of the aforementioned compound (Ⅰ) maleate I is basically as follows: Figure 6 As shown.

[0034] In some embodiments of this application, the above-mentioned (I) compound maleate I is characterized in that the TGA-DSC spectrum of the crystal form is as follows: Figure 7 As shown.

[0035] In some embodiments of this application, the XRPD diffraction peak analysis data of maleate I of the above formula (Ⅰ) are shown in Table 4.

[0036] Table 4. XRPD diffraction peak analysis data of maleate crystal form I of compound (Ⅰ).

[0037] This application provides maleate crystal form II of compound (I) 4-chloro-6-(6-cyclopropyl-4-((1S,3R)-3-methyl-1-(4-methyl-4H-1,2,4-triazol-3-yl)cyclobutyl)pyridin-2-yl)-2-(((S)-3-methylpiperidin-1-yl)methyl)-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-7-one, characterized in that the X-ray powder diffraction pattern of maleate crystal form II of compound (I) has characteristic peaks at 2θ values ​​of 6.94, 7.91, 8.82, 15.40, and 22.87, with a 2θ error range of ±0.2°.

[0038] In some embodiments of this application, the maleate crystal form II of the above-mentioned (I) compound has the following X-ray powder diffraction pattern: Figure 8 As shown.

[0039] In some embodiments of this application, the maleate crystal form II of the above-mentioned (I) compound is characterized in that the TGA-DSC spectrum of the crystal form is as follows: Figure 9 As shown.

[0040] In some embodiments of this application, the XRPD diffraction peak analysis data of maleate II of the above formula (I) are shown in Table 5.

[0041] Table 5. XRPD diffraction peak analysis data of maleate crystal form II of compound (I).

[0042] This application provides the hydrochloride crystal form I of compound (I) 4-chloro-6-(6-cyclopropyl-4-((1S,3R)-3-methyl-1-(4-methyl-4H-1,2,4-triazol-3-yl)cyclobutyl)pyridin-2-yl)-2-(((S)-3-methylpiperidin-1-yl)methyl)-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-7-one, characterized in that the X-ray powder diffraction pattern of the hydrochloride crystal form I of compound (I) has characteristic peaks at 2θ values ​​of 7.15, 8.96, 14.68, 19.28, 21.22, and 22.41, with a 2θ error range of ±0.2°.

[0043] In some embodiments of this application, the crystal form I of the above-mentioned (Ⅰ) compound hydrochloride has characteristic peaks in its X-ray powder diffraction pattern at 2θ values ​​of 7.15, 8.96, 10.72, 12.49, 14.68, 19.28, 20.56, 21.22, 22.41, 23.20, 25.11, and 27.83, with a 2θ error range of ±0.2°.

[0044] In some embodiments of this application, the crystal form I of the above-mentioned (Ⅰ) compound hydrochloride has characteristic peaks in its X-ray powder diffraction pattern at 2θ values ​​of 7.15, 8.96, 10.72, 11.44, 12.49, 13.51, 14.68, 15.57, 16.79, 18.00, 19.28, 20.56, 21.22, 22.41, 23.20, 25.11, 26.38, 27.83, and 29.28, with a 2θ error range of ±0.2°.

[0045] In some embodiments of this application, the crystal form I of the above-mentioned (Ⅰ) compound hydrochloride has an X-ray powder diffraction pattern that is basically as follows: Figure 10 As shown.

[0046] In some embodiments of this application, the XRPD diffraction peak analysis data of the hydrochloride crystal form I of the above formula (Ⅰ) are shown in Table 6.

[0047] Table 6. XRPD diffraction peak analysis data of crystal form I of compound (I) hydrochloride

[0048] This application also provides a pharmaceutical composition comprising any of the above-described crystal forms, a salt, and a pharmaceutically acceptable carrier.

[0049] This application also provides the use of the above-described crystal forms, salts, and pharmaceutical compositions in the preparation of medicaments for treating CBL-B-mediated diseases.

[0050] This application also provides the use of the above-mentioned crystal forms, salts, and pharmaceutical compositions in medicaments for treating CBL-B-mediated diseases.

[0051] In some of the schemes described in this application, the aforementioned CBL-B-mediated diseases include cancer.

[0052] Technical effect The crystal form in this application has good chemical stability, physical stability, and low hygroscopicity. It is less affected by temperature, humidity, and light, making it easy to store and develop formulations.

[0053] Definitions and Explanations Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered uncertain or unclear unless specifically defined, but should be understood in its ordinary sense.

[0054] The term "pharmaceutical-grade carrier" refers to a medium generally acceptable in the art for delivering a bioactive pharmaceutical agent to animals, particularly mammals. Depending on the route of administration and dosage form, this includes, for example, adjuvants, excipients, or excipients such as diluents, preservatives, fillers, flow modifiers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, aromatizers, antibacterial agents, antifungal agents, lubricants, and dispersants. Pharmaceutically acceptable carriers are formulated based on a multitude of factors, within the scope of those skilled in the art. These include, but are not limited to, the type and nature of the active pharmaceutical agent being formulated, the recipient to whom the composition containing the pharmaceutical agent is to be administered, the intended route of administration of the composition, and the target therapeutic indication. Pharmaceutically acceptable carriers include both aqueous and non-aqueous media, as well as various solid and semi-solid dosage forms. In addition to the active pharmaceutical agent, such carriers include many different components and additives, and the inclusion of such additional components in the formulation for various reasons (e.g., stabilizing active pharmaceutical agents, binders, etc.) is well known to those skilled in the art.

[0055] As is known in the art, X-ray powder diffraction patterns have one or more measurement errors due to minute variations in measurement conditions. The structures of the crystals, crystal forms, or crystalline forms disclosed or claimed in this application may exhibit similar but not identical analytical characteristics within a reasonable error range, depending on experimental conditions, purity, equipment, and other constant variables known to those skilled in the art. For example, the diffraction angle (2θ) in powder X-ray powder diffraction typically produces an error within ±0.20°. Therefore, this application includes not only crystals with completely consistent diffraction angles in powder X-ray powder diffraction, but also crystals with consistent diffraction angles within an error range of ±0.20°. The crystalline form of the compound of formula (I) in this application is not limited to crystals having the same X-ray powder diffraction pattern as shown in the accompanying drawings; any crystal having a substantially identical X-ray powder diffraction pattern as shown in the accompanying drawings is within the scope of this application.

[0056] The text refers to "X-ray powder diffraction patterns that are substantially the same as those shown in the accompanying figures." It should be understood that the term "substantially the same" used in this context also indicates that the 2θ angle values ​​of the X-ray powder diffraction patterns may vary slightly due to inherent experimental variations accompanying these measurements, and both are of the same crystalline form.

[0057] It should be understood that different types of equipment or different testing conditions may yield slightly different DSC spectra and endothermic transition temperature readings. DSC data can reflect changes in the state of matter; strong endothermic peaks can indicate that the substance has undergone dehydration or desolvation, or crystal transformation, or melting, etc.; when reflecting the molten state, the corresponding temperature is generally understood as the melting point of the substance. This value will be affected by compound purity, sample weight, heating rate, particle size, and calibration and maintenance of the testing equipment. Those skilled in the art will understand that the temperature at which a substance changes from a solid to a liquid state is usually a temperature range, not a fixed point value; therefore, the temperature corresponding to the endothermic peak or the melting point of the substance can be characterized by either the onset value, the peak value, or other reasonable values. The maximum endothermic transition temperature of the crystal form can be within the range of ±5.0℃ of the specific values ​​disclosed above, preferably within the range of ±2.0℃. For example, an endothermic peak around 253.5℃ in the DSC spectrum means an endothermic peak at 253.5℃ ±5.0℃, preferably 253.5℃ ±2.0℃.

[0058] This application also uses thermogravimetric analysis (TGA) to analyze the relationship between the degree of decomposition, sublimation, or evaporation of the crystal form (weight loss) and temperature. It should be understood that the values ​​obtained for the same crystal form may have some error due to factors such as sample purity, particle size, different types of equipment, and different testing methods. The temperature at which the crystal form decomposes, sublimates, or evaporates can be within ±3.0℃ of the specific values ​​disclosed above, for example, within ±2.0℃.

[0059] The "stability" of a crystal form includes "chemical stability" and / or "physical stability." "Chemical stability" refers to the degree to which the crystal form undergoes degradation reactions under certain temperature, humidity, and light conditions; it reflects the stability of the crystal form under storage conditions. "Physical stability" refers to the degree to which the crystal form undergoes a solid-state transformation under certain specific conditions, such as under conditions of high temperature, high humidity, grinding, tableting, solvent removal, or solvent adsorption, transforming into another crystal form. Therefore, "physical stability" can, to some extent, reflect the stability of the crystal form during its use in formulations and other processes.

[0060] The crystalline structure of this application can be prepared by various methods, including crystallization or recrystallization from a suitable solvent, sublimation, growth from a melt, solid-state transformation from another phase, crystallization from a supercritical fluid, and jet spraying. Techniques for crystallizing or recrystallizing the crystalline structure from a solvent mixture include solvent evaporation, lowering the temperature of the solvent mixture, crystallization of a supersaturated solvent mixture of the molecule and / or salt, lyophilizing the solvent mixture, and adding an antisolvent to the solvent mixture.

[0061] The term "drying" refers to the process of removing solvent from the resulting solid, including but not limited to air drying at room temperature, high-temperature drying, and vacuum drying.

[0062] The intermediate compounds of this application can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments of this application.

[0063] In the embodiments of this application, the title compound was named by converting the compound structure using Chemdraw.

[0064] Instruments and analytical methods: 1. X-ray powder diffraction (XRPD) Solid samples were analyzed using an X-ray powder diffractometer (Aeris). An appropriate amount of fine powder was taken and placed in the groove of the sample holder. A glass slide was used to press the powder into a flat and dense plane. The XRPD measurement parameters are shown in Table 7.

[0065] Table 7 XRPD Test Parameters

[0066] 2. Simultaneous thermal analysis (TGA-DSC) Alternatively, a Mettler Toledo simultaneous thermal analyzer can be used for thermogravimetric-differential scanning calorimetry (TGC) analysis of solids. Take an appropriate amount of the sample with a small spoon and place it in a crucible, spreading it evenly. Weigh the sample and heat it according to the parameters listed in Table 8. Analyze the data using STARE.

[0067] Table 8 Parameters of TGA-DSC Analysis Method Attached Figure Description Figure 1 The image shows the XRPD spectrum of crystal form A of compound (I).

[0068] Figure 2 The TGA-DSC spectrum of crystal form A of compound (Ⅰ) is shown.

[0069] Figure 3 The image shows the XRPD spectrum of crystal form B of compound (I).

[0070] Figure 4 The TGA-DSC spectrum of crystal form B of compound (Ⅰ) is shown.

[0071] Figure 5 The image shows the XRPD spectrum of crystal form C of compound (I).

[0072] Figure 6 The image shows the XRPD spectrum of maleate crystal form I of compound (Ⅰ).

[0073] Figure 7 The TGA-DSC spectrum of maleate crystal form I of compound (Ⅰ) is shown.

[0074] Figure 8 The image shows the XRPD spectrum of maleate crystal form II of compound (I).

[0075] Figure 9 The TGA-DSC spectrum of maleate crystal form II of compound (I) is shown.

[0076] Figure 10 The image shows the XRPD spectrum of the hydrochloride crystal form I of compound (Ⅰ). Detailed Implementation

[0077] The present application is described in detail below through examples, but this does not imply any adverse limitation on the present application. The compounds of this application can be prepared by various synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments of this application. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of this application without departing from the spirit and scope of this application. Detailed implementation method: Example 1 Preparation of compound (I)

[0079] Reaction route:

[0080] Operating steps: Step A: Under nitrogen protection at room temperature, 2-bromo-6-chloro-4-methylpyridine (5 g, 24.3 mmol), cyclopropylboronic acid (4.5 g, 52.0 mmol), [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloromethane complex (1 g, 1.2 mmol), and potassium phosphate (15 g, 71.9 mmol) were dissolved in toluene / water (60 mL / 6 mL). The reaction mixture was then stirred at 95 °C for 4 hours.

[0081] After LCMS monitoring showed the disappearance of the starting material, water (50 mL) was added to the reaction solution to quench the reaction. The mixture was extracted with ethyl acetate (30 mL × 2 times), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give 3.4 g of 2-chloro-6-cyclopropyl-4-methylpyridine.

[0082] MS (ESI) M / Z: 168.0 [M+H] + . Step B: Under nitrogen protection at -70 °C, 2-chloro-6-cyclopropyl-4-methylpyridine (3.4 g, 20.4 mmol) was dissolved in tetrahydrofuran (34 mL). Then, a 2 M solution of lithium diisopropylamino in tetrahydrofuran / n-heptane (25.5 mL, 51.0 mmol) was added dropwise, and the mixture was stirred for 1 hour. Next, dimethyl carbonate (2.7 g, 30.6 mmol) was added, the temperature was raised to 0 °C, and stirring was continued for 2 hours.

[0083] After LCMS monitoring showed the disappearance of the starting material, saturated ammonium chloride aqueous solution (30 mL) was added to the reaction solution to quench the reaction. The mixture was extracted with ethyl acetate (20 mL × 2 times), and the organic phases were combined and washed with saturated brine (30 mL × 2 times). The solution was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give 3.2 g of methyl 2-(2-chloro-6-cyclopropylpyridin-4-yl)acetate.

[0084] MS (ESI) M / Z: 226.0 [M+H] + . Step C: Under nitrogen protection at 0 °C, methyl 2-(2-chloro-6-cyclopropylpyridin-4-yl)acetate (3.2 g, 14.1 mmol) and 1,3-dibromo-2-methylpropane (3.1 g, 14.3 mmol) were dissolved in tetrahydrofuran (70 mL). Then, 60% wt sodium hydride (1.25 g, 31.0 mmol) was slowly added to the above solution. The reaction mixture was then stirred for 4 hours.

[0085] After LCMS monitoring showed the disappearance of the starting material, a saturated ammonium chloride aqueous solution (10 mL) was added to the above reaction solution to quench the reaction, followed by dilution with water (50 mL). The mixture was extracted with ethyl acetate (40 mL × 2 times), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography to obtain 3 g of methyl 1-(2-chloro-6-cyclopropylpyridin-4-yl)-3-methylcyclobutane-1-carboxylate.

[0086] MS (ESI) M / Z: MS 280.0 [M+H] + . Step D: At room temperature, methyl 1-(2-chloro-6-cyclopropylpyridin-4-yl)-3-methylcyclobutane-1-carboxylate (3 g, 10.7 mmol) was dissolved in methanol / tetrahydrofuran (40 mL / 10 mL). Then, an aqueous solution (20 mL) of lithium hydroxide (770 mg, 32.1 mmol) was added to the above solution. The reaction mixture was then stirred for 1 hour.

[0087] After LCMS monitoring showed the disappearance of the raw material, the reaction solution was concentrated under reduced pressure to remove the organic solvent, and then the pH was adjusted to 5 with 2 M dilute hydrochloric acid aqueous solution. The mixture was extracted with dichloromethane (40 mL × 2 times), and the organic phases were combined. Then, it was dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 2.2 g of crude 1-(2-chloro-6-cyclopropylpyridin-4-yl)-3-methylcyclobutane-1-carboxylic acid.

[0088] MS (ESI) M / Z: MS 266.0 [M+H] + . Step E: At room temperature, 1-(2-chloro-6-cyclopropylpyridin-4-yl)-3-methylcyclobutane-1-carboxylic acid (2.2 g, 8.27 mmol) was dissolved in N,N-dimethylformamide (36 mL). Then, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (4.08 g, 10.75 mmol) and N,N-diisopropylethylamine (3.2 g, 24.81 mmol) were added to the above solution, and the mixture was stirred for 10 minutes. Then, N-methylhydrazine thioamide (1.04 g, 9.92 mmol) was added, and the mixture was stirred for another hour.

[0089] After LCMS monitoring showed the disappearance of the starting material, water (50 mL) was added to the above reaction solution to quench the reaction. The mixture was extracted with ethyl acetate (50 mL × 2 times), the organic phases were combined, dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was slurried with petroleum ether / ethyl acetate (20 mL / 6 mL) to obtain 2.5 g of 2-(1-(2-chloro-6-cyclopropylpyridin-4-yl)-3-methylcyclobutane-1-carbonyl)-N-methylhydrazine-1-methylthioamide.

[0090] MS (ESI) M / Z: MS 353.0 [M+H] + . Step F: 2.5 g (7.08 mmol) of compound 2-(1-(2-chloro-6-cyclopropylpyridin-4-yl)-3-methylcyclobutane-1-carbonyl)-N-methylhydrazine-1-methylthioamide was dissolved in tetrahydrofuran (44 mL) at room temperature. Subsequently, an aqueous solution of sodium hydroxide (2.83 g, 70.8 mmol) (22 mL) was added to the above solution. The reaction mixture was then stirred at 80 °C for 4 hours.

[0091] After LCMS monitoring showed the disappearance of the starting material, 2M dilute hydrochloric acid aqueous solution was added dropwise to the reaction solution to adjust the pH to 5. The mixture was extracted with ethyl acetate (30 mL × 2 times), the organic phases were combined, dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 2.6 g of crude product 5-(1-(2-chloro-6-cyclopropylpyridin-4-yl)-3-methylcyclobutyl)-4-methyl-4H-1,2,4-triazol-3-thiol.

[0092] MS (ESI) M / Z: MS 335.0 [M+H] + . Step G: 5-(1-(2-chloro-6-cyclopropylpyridin-4-yl)-3-methylcyclobutyl)-4-methyl-4H-1,2,4-triazol-3-thiol (2.6 g, 7.78 mmol) was dissolved in dichloromethane / acetic acid (40 mL / 6 mL) at 0 °C. Then, 30% wt hydrogen peroxide (6 mL) was slowly added dropwise to the solution, and stirring was continued for 30 minutes.

[0093] After LCMS monitoring showed the disappearance of the starting material, the reaction solution was quenched in ice water (80 mL). The mixture was extracted with dichloromethane (30 mL × 2 times), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain 1 g of the mixture. 450 mg of the mixture was purified by preparative high-performance liquid chromatography to obtain 240 mg of compound P1 (short retention time) and 60 mg of compound P2 (long retention time).

[0094] Compound P1: 1 H NMR (400 MHz, CD3Cl) δ 8.05 (s, 1H), 7.07 (d, J = 1.6 Hz, 1H), 6.95(d, J= 1.2 Hz, 1H), 3.23 (s, 3H), 2.85 - 2.75 (m, 2H), 2.70 - 2.61 (m, 3H), 1.96 - 1.88 (m, 1H), 1.14 (d, J = 5.6 Hz, 3H), 1.07 - 1.01 (m, 2H), 1.00 - 0.95 (m, 2H). Compound P2: 1 H NMR (400 MHz, CD3Cl) δ 8.12 (s, 1H), 6.92 (d, J = 1.2 Hz, 1H), 6.81(d, J = 1.2 Hz, 1H), 3.27 (s, 3H), 3.19 - 3.06 (m, 2H), 2.62 - 2.51 (m, 1H), 2.32 - 2.19 (m, 2H), 1.95 - 1.86 (m, 1H), 1.13 (d, J = 6.4 Hz, 3H), 1.05 - 1.00 (m, 2H), 0.99 - 0.93 (m, 2H). Referring to the preparation method of a similar structure in patent WO2020264398, and combining a comprehensive analysis showing that the number of advantageous configurations significantly exceeds the number of disadvantageous configurations, compound P1 was determined to proceed to the next reaction step.

[0095] Step H: Under nitrogen protection at room temperature, compound (S)-4-chloro-2-((3-methylpiperidin-1-yl)methyl)-1-((2-(trimethylsilyl)ethoxy)methyl ester)-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-7-one (230 mg, 0.56 mmol), 2-chloro-6-cyclopropyl-4-((1S,3S)-3-methyl-1-(4-methyl-4H-1,2,4-triazol-3-yl)cyclobutyl)pyridine (compound P1, 170 mg, 0.56 mmol), N,N'-dimethylethylenediamine (59 mg, 0.67 mmol), cuprous iodide (106 mg, 0.56 mmol), and potassium carbonate (155 mg, 1.12 mmol) were dissolved in dimethyl sulfoxide (2.5 mL). The reaction system was then stirred at 150 °C for 6 hours.

[0096] After LCMS monitoring showed the disappearance of the starting material, water (10 mL) was added to the reaction solution to quench the reaction. The mixture was extracted with dichloromethane (10 mL × 2 times), and the organic phases were combined and washed with saturated brine (10 mL × 2 times). The solution was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain 250 mg of 4-chloro-6-(6-cyclopropyl-4-((1S,3R)-3-methyl-1-(4-methyl-4H-1,2,4-triazol-3-yl)cyclobutyl)pyridin-2-yl)-2-(((S)-3-methylpiperidin-1-yl)methyl)-1-((2-(trimethylsilyl)ethoxy)methyl ester)-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-7-one.

[0097] MS (ESI) M / Z: 676.3 [M+H] + . Step I: At room temperature, 250 mg (0.37 mmol) of compound 4-chloro-6-(6-cyclopropyl-4-((1S,3R)-3-methyl-1-(4-methyl-4H-1,2,4-triazol-3-yl)cyclobutyl)pyridin-2-yl)-2-(((S)-3-methylpiperidin-1-yl)methyl)-1-((2-(trimethylsilyl)ethoxy)methyl ester)-1,6-dihydro-7H-pyrrolo[2,3-c]pyridin-7-one (2.5 mL) was dissolved in dichloromethane. Trifluoroacetic acid (3.0 mL) was then added dropwise to the solution, and stirring was continued for 2 hours. After LCMS monitoring showed the starting material had disappeared, ammonia (5.5 mL) was added dropwise to the reaction solution until alkaline, and stirring was continued for 16 hours.

[0098] After LCMS monitoring showed the disappearance of the intermediate, water (10 mL) was added to the reaction solution to quench the reaction, and the mixture was extracted with dichloromethane (15 mL × 2 times). The organic phases were combined. The mixture was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography to obtain 90 mg of compound (Ⅰ).

[0099] MS (ESI) M / Z: 546.2 [M+H] + . 1 H NMR (400 MHz, DMSO- d 6) δ 12.43 (s, 1H), 8.34 (s, 1H), 7.60 (s, 1H), 7.47 (d, J = 1.2 Hz, 1H), 7.32 (d, J= 1.6 Hz, 1H), 6.25 (s, 1H), 3.59 (s, 2H),3.28 (s, 3H), 2.94 - 2.84 (m, 2H), 2.82 - 2.70 (m, 2H), 2.63 - 2.53 (m, 3H),2.24 - 2.15 (m, 1H), 1.94 - 1.83 (m, 1H), 1.66 - 1.53 (m, 4H), 1.50 - 1.39 (m, 1H), 1.08 (d, J = 5.6 Hz, 3H), 1.01 - 0.92 (m, 4H), 0.85 - 0.72 (m, 4H). Example 2 Biological test evaluation: Test Example 1: Jurkat T Activation Experiment Experimental objective: To test the effect of the compound on activating IL2 release in Jurkat T cells. Experimental Methods: 96-well Corning cell culture plates were coated overnight at 4°C with 100 μL of 5 μg / mL anti-human CD3 (BD) antibody dilution buffer. After washing once with PBS, the supernatant was removed, and 100 μL of 4 μg / mL anti-human CD28 (BD) antibody dilution buffer was added and mixed thoroughly. The compound was serially diluted 5-fold (final concentration selected from 1 μM to 0.32 nM, starting concentration selected from 1 μM, 5-fold dilution, 6 spots, the 6th spot being 0.32 nM), premixed with 2 x 10⁶ / mL Jurkat T cell suspension (ATCC) at a 1:1 ratio, and incubated at 37°C for 1 hour. 100 μL of the compound and cell mixture was transferred to the pre-treated wells, and cells were cultured at 37°C for 24 hours. The supernatant was collected, and IL-2 release was detected using an IL-TR FRET kit (bioauxilium). EC analysis was performed using Prism. 50 .

[0100] The structural formula of control compound 1, NX-1607, is as follows:

[0101] The structural formula of compound 2 is as follows:

[0102] Sources of control compounds: Control compound 1 was prepared with reference to patent WO2020264398; Control compound 2 was prepared with reference to patent WO2024020034.

[0103] Table 9. Activating activity of the compounds in this application on IL2 release from Jurkat T cells.

[0104] Based on the experimental data shown in Table 9, compared with the control group NX-1607, the compound of formula (I) of this application showed significantly stronger IL2 release activation activity in Jurkat T cells.

[0105] Test Example 2: Inhibition Test of CYP3A Experimental objective: To test the inhibitory effect of the compound on CYP3 enzyme. Experimental materials: a mixture of human liver microsomes, NADPH, ketoconazole, midazolam, and testosterone. Experimental methods: Weigh the required amount of the test compound or control compound powder and add the corresponding volume of DMSO to prepare a 30 mM stock solution. Then perform serial dilutions to obtain solutions with nominal DMSO concentrations of 20000, 6000, 2000, 600, 200, and 60 μM for each compound. The final concentrations of the compounds in the assay are 100, 30, 10, 3, 1, and 0.3 μM, and the final percentage of organic solvent introduced by the test compound or positive control inhibitor is 0.5%. The final concentrations of the positive control inhibitor are 0, 0.0015, 0.005, 0.015, 0.05, 0.15, and 0.5 μM. The final concentrations of the specific substrates midazolam and testosterone are 1 and 40 μM, respectively, and the final protein concentration of the mixed human liver microsomal solution is 0.2 mg / mL.

[0106] Incubation was performed in 96-well plates. 169 μL of microparticles and 1 μL of multiple concentrations of the test compound or positive control compound (DMSO) were added to each well. The plate was pre-incubated in a water bath at 37 °C for 5 minutes. Then, 10 μL of diluted substrate solution was added to the plate, and the mixture was vortexed for 15 seconds. Finally, 20 μL of 10 mM NADPH solution was added, bringing the final concentration to 1 mM to initiate the reaction.

[0107] At the predetermined time point, 300 μL of quenching solution (cold acetonitrile with 3% formic acid, 200 nM alprazolam, 200 nM labetalol hydrochloride, and 200 nM tolbutamide) was added to quench the reaction. The mixture was centrifuged at 3220 g for 40 minutes. 150 μL of the supernatant was transferred to a new plate. The supernatant could be diluted with 150 μL of pure water. After thorough mixing, the content of substrate metabolites was determined by LC / MS / MS.

[0108] Automated peak integration region checks were performed on all samples. Peak areas and internal standard peak areas were analyzed and exported to an Excel spreadsheet. Inhibition of CYP3A activity in human liver microsomes was measured as the percentage decrease in metabolite formation activity compared to a non-inhibited control (= 100% activity). IC50 was calculated as the logarithm of residual activity (%) and inhibitor concentration. 50 value.

[0109] The percentage of remaining activity is calculated as follows: Area ratio = Analyte peak area / Internal standard peak area Residual activity (%) = area ratio 待测药 / area ratio 空白对照 ×100% Calculate IC using Excel XLfit 5.5.1.3 50 value Table 10. Inhibitory activity of the compounds of this application against CYP3 enzymes

[0110] Cytochrome P450 3A (CYP3A) is one of the most important drug-metabolizing enzymes in the human body, participating in the metabolic clearance of approximately 50% of marketed drugs. Compared with the control compound 1, the compound of formula (I) in this application exhibits significantly weaker inhibitory activity against CYP3A, which can significantly reduce drug-drug interactions when used in combination. DDI )risk.

[0111] Test Example 3: In vitro evaluation of the compound's bidirectional permeability Experimental objective: To investigate the potential permeation and absorption of the test substance by detecting its permeability coefficient in a Caco-2 cell model.

[0112] Experimental materials: Caco-2 cells were purchased from the American Type Culture Collection (ATCC).

[0113] Test samples: Reference compound 2 and compound of formula (I) of this application. 1.1 Solution preparation: 1) Prepare 1 L HBSS (10 mM HEPES, pH 7.4). 2) Prepare a high-concentration DMSO stock solution of the test substance and dilute it to 1 mM with DMSO. Then dilute it accordingly with HBSS to obtain a test concentration of 5 μM; 3) Prepare high-concentration DMSO stock solutions of the control drugs digoxin and metoprolol, and dilute them to 2 mM stock solutions with DMSO. Then, dilute them accordingly with HBSS (10 mM HEPES, pH 7.4) to obtain a test concentration of 10 μM. 2.1 Drug penetration test: 1) Remove the Transwell culture plate from the incubator. Rinse the cell monolayer twice with HBSS (10 mM HEPES, pH 7.4) buffer and incubate at 37 °C for 30 minutes; 2) Determine the transport rate of the compound from the top to the base. Add 125 μL of the drug delivery solution to each well in the upper chamber (top), then transfer 50 μL of the sample to 200 μL of acetonitrile containing the internal standard as the 0-minute drug delivery sample from the top of the Transwell plate for detection. Add 235 μL of the receiver solution to each well in the lower chamber (base). 3) Determine the transport rate of the compound from the base to the top. Add 75 μL of receiving end solution to each well in the upper chamber (top) and 285 μL of dosing end solution to each well in the lower chamber (base). Then transfer 50 μL of sample to 200 μL of acetonitrile containing internal standard as the 0-minute dosing sample from the base of the Transwell plate for detection. 4) After merging the upper and lower transfer devices, incubate at 37 °C for 2 hours; 5) Transfer 50 μL of sample from the working solution preparation plate and add it to 200 μL of acetonitrile containing internal standard as the 0-minute dosing sample for detection; 6) After incubation, take 50 μL of sample from each well of the upper and lower chambers of the Transwell plate and add it to a new sample tube. Add 200 μL of acetonitrile containing the internal standard to the sample tube, vortex for 10 minutes, and then centrifuge at 3220 g for 30 minutes. Take 150 μL of the supernatant, dilute it with an equal volume of water, and then perform LC-MS / MS analysis. All samples were prepared in duplicate. 7) Evaluate the integrity of the cell monolayer after 2 hours of incubation using fluorescein leakage. Dilute the fluorescein stock solution to a final concentration of 100 μM using HBSS (10 mM HEPES, pH 7.4). Add 100 μL of fluorescein solution to each well of the upper Transwell plate and 300 μL of HBSS (10 mM HEPES, pH 7.4) to each well of the lower receiving plate. After incubation at 37°C for 30 minutes, aspirate 80 μL of solution from both the upper and lower layers of each well into a new 96-well plate. Perform fluorescence measurement using a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

[0114] The apparent permeability (Papp, unit: cm / s) of the compound in Caco-2 cells, based on the specific concentrations at the receiving and administering ends, is calculated using the following formula:

[0115] In the formula: VA is the volume of the receiving end solution (0.235 mL from the top to the base, and 0.075 mL from the base to the top), and Area is the area of ​​the Transwell-96-well plate membrane (0.143 cm²). 2 (time is the incubation time) (Unit: s).

[0116] The discharge rate is calculated using the following formula:

[0117] In the formula: Papp (BA) is the apparent permeability coefficient from the basal end to the apex; Papp (AB) is the apparent permeability coefficient from the top to the base.

[0118] 2. Experimental Results and Analysis The parameters for the permeability test are shown in Table 11 below.

[0119] Table 11. Permeability of the compounds in this application in the Caco-2 cell model

[0120] Based on the experimental data shown in Table 11, compared with control compound 2, compound (I) of this application has a lower efflux rate, indicating that the compound is better absorbed orally and has higher oral bioavailability.

[0121] Test Example 4: In vivo pharmacokinetic experiments of the compound of this application in beagle dogs Using male beagle dogs as test animals, the in vivo pharmacokinetic behavior of the compound of this application after injection at a dose of 1 mg / kg was studied.

[0122] 1. Test Protocol 1.1 Test sample: Control compound 1, control compound 2 and compound of formula (I).

[0123] 1.2 Experimental Animals Basic information about animals Species: Male Beagle (3 per group) Weight: Approximately 8~10 kg Source: Jiangsu Mars Biotechnology Co., Ltd. 1.3 Administration: Male beagle dogs were injected with control compound 1, control compound 2 and compound of formula (I) of this application at a dose of 1 mg / kg and a volume of 1 mL / kg.

[0124] Preparation of test solution: The test sample is dissolved in a solvent (10% DMSO + 10% Kolliphor HS15 + 80% Saline). DMSO, Kolliphor HS15 and Saline are added in sequence during preparation.

[0125] Control group: Control compound 1 and control compound 2, with the same structure as above.

[0126] Experimental group: Compound of formula (Ⅰ).

[0127] 1.4 Sample Collection: Data were collected at 0, 5 min, 15 min, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h before and after drug administration. Collect 0.5 mL of blood and place it in an EDTA-2K anticoagulant tube. Gently invert the tube to thoroughly mix the anticoagulant (EDTA-2K) with the blood, then immediately place it on wet ice. Centrifuge to separate the plasma within 15 minutes of blood collection. Centrifugation conditions: 4°C, 3000 g, 5 minutes. After centrifugation, separate the plasma and perform sample analysis.

[0128] 2. Experimental Results and Analysis The pharmacokinetic parameters are shown in Table 12 below. In vivo pharmacokinetic results in dogs showed that, compared with compound 1 (control group) and compound 2 (control group), compound (I) of this application had a slower in vivo clearance rate, a longer half-life, higher tissue distribution, and higher plasma exposure.

[0129] Table 12 Pharmacokinetic parameters of intravenous administration in beagle dogs

[0130] Example 3. Preparation of crystal form A Add 3 mL of methanol to 0.1137 g of compound (Ⅰ). Filter and slowly add 3 mL of purified water. Stir at room temperature for 1 day. After filtration, dry under vacuum at 50 °C for 2 h. Characterize by XRD; crystal form A.

[0131] Example 4. Preparation of crystal form B Add 5.5 mL of ethyl acetate to 25.7 mg of compound (I) and heat to 75 °C to dissolve. Filter into a new vial and stir at room temperature for 16 h to crystallize. Characterize by XRD; crystal form B.

[0132] Example 5. Preparation of Crystal Form C Add 0.5 mL of 1,4-dioxane to 32.4 mg of compound (I), and heat to 63 °C to dissolve. Filter into a new vial, slowly cool to room temperature over 2 hours to allow crystals to crystallize, and stir at room temperature for 2 days. Characterize by XRD; crystal form C.

[0133] Example 6. Maleate crystals I Preparation Add 45.2 mg of maleic acid to 211.5 mg of compound (Ⅰ), then add 2 mL of acetone and stir at room temperature. The solid dissolves and precipitates. After stirring for 24 h, filter and dry under vacuum at 50 °C for 3 h to obtain maleate crystals I. 1 H-NMR (400MHz, DMSO-d6): δ (ppm) 12.82 (s, 1H), 8.35 (s, 1H), 7.68 (s, 1H), 7.45-7.44 (d, J =1.2Hz, 1H), 7.342-7.339 (d, J =1.2Hz, 1H), 6.65 (s, 1H), 6.07 (s, 2H), 4.34 (s, 2H), 3.28(s, 3H), 2.93-2.83 (m, 2H), 2.80-2.70 (m, 1H), 2.64-2.56 (m, 3H), 2.25-2.21 (m, 1H), 1.89-1.56 (m, 4H), 1.10-0.89 (m, 11H).

[0134] 1 H-NMR results indicate that maleate crystal I contains approximately 2 equivalents of maleic acid.

[0135] Example 7. Preparation of maleate crystal II: Add 9.1 mg of maleic acid and 0.5 mL of ethyl acetate to 40.4 mg of compound (Ⅰ). Stir at room temperature for 4 days, filter, and dry under vacuum at 50 °C for 3 h to obtain maleate crystal II.

[0136] 1 H-NMR (400MHz, DMSO-d6): δ (ppm) 12.82 (s, 1H), 8.35 (s, 1H), 7.68 (s,1H), 7.44 (s, 1H), 7.34 (s, 1H), 6.65 (s, 1H), 6.07 (s, 2H), 4.36 (s, 2H), 3.28 (s, 3H), 2.97-2.67 (m, 4H), 2.64-2.56 (m, 3H), 2.28-2.17 (m, 1H), 1.89-1.56 (m, 4H), 1.14-0.82 (m, 11H). 1H-NMR results indicate that maleate crystal II contains approximately 2 equivalents of maleic acid.

[0137] Example 8. Preparation of hydrochloride crystal form I: Add 104.4 mg of a 25% isopropanol hydrochloride solution to 40.6 mg of compound (Ⅰ), add 0.5 mL of isopropanol, stir at room temperature for 3 days, filter, and vacuum dry at 50 °C for 7 h to obtain hydrochloride crystal form I.

[0138] 1 H-NMR (400MHz, DMSO-d6): δ (ppm) 12.80 (s, 1H), 11.81 (s, 1H), 9.32 (s,1H), 7.68 (s, 1H), 7..532-7.529 (d, J =1.6Hz, 1H), 7.47-7.46 (d, J =1.6Hz, 1H), 6.744-6.739 (d, J =2.0Hz, 1H), 4.39-4.38 (m, 2H), 3.44 (s, 3H), 3.37-3.34 (m,1H), 3.27-3.25 (m, 1H), 3.00-2.89 (m, 2H), 2.79-2.72 (m, 1H), 2.69-2.63 (m,3H), 2.49-2.42 (m, 1H), 2.27-2.20 (m, 1H), 2.00-1.98 (m, 1H), 1.88-1.77 (m,2H), 1.75-1.66 (m, 1H), 1.11-1.09 (m, 4H), 1.02-0.96 (m, 5H), 0.89-0.87 (m, 3H).

[0139] Example 9: Stability Experiment Following the "Guidelines for Stability Testing of Raw Materials and Preparations" in the Chinese Pharmacopoeia, the stability of compound (I) crystal form A and maleate crystal form I under different temperatures and humidity conditions was investigated. Purity was tested by HPLC on days 0, 5, and 10, and crystal form was tested by XRPD.

[0140] The experimental results are shown in Tables 13 and 14. Before and after the experiment, the physical properties of compound (Ⅰ) crystal form A and maleate crystal form Ⅰ remained stable under high temperature and high humidity conditions, their crystal forms did not change, and their chemical purity did not decrease significantly.

[0141] Table 13 Stability data of free alkali crystals A

[0142] Conclusion: The crystal form A of compound (I) has good stability under high temperature and high humidity conditions.

[0143] Table 14 Stability data for maleate crystal form I

[0144] Conclusion: Compound (Ⅰ) maleate crystal form I has good stability under high temperature and high humidity conditions.

Claims

1. The crystal form A of the compound of formula (Ⅰ), （Ⅰ） Its features are, Using Cu-Kα radiation, the X-ray powder diffraction pattern of crystal form A exhibits characteristic peaks at 2θ values ​​of 6.04, 9.76, 11.01, 12.18, 16.96, and 17.36, with a 2θ error range of ±0.2°; more preferably, the X-ray powder diffraction pattern of crystal form A exhibits characteristic peaks at 2θ values ​​of 6.04, 9.76, 11.01, 12.18, 16.56, 16.96, 17.36, 17.67, 20.67, 22.17, and 22.45 ... The powder diffraction pattern has characteristic peaks at 2θ values ​​of 5.44, 6.04, 9.76, 10.74, 11.01, 12.18, 16.56, 16.96, 17.36, 17.67, 18.08, 18.59, 20.67, 21.20, 21.85, 22.17, 22.45, 23.70, 25.12, 25.56, and 26.04, with a 2θ error range of ±0.2°. More preferably, the X-ray powder diffraction pattern of crystal form A is basically as shown in Figure 1. Even more preferably, the TGA-DSC pattern of the crystal form is shown in Figure 2.

2. Crystal form B of the compound of formula (I), characterized in that, Using Cu-Kα radiation, the X-ray powder diffraction pattern of crystal form B has characteristic peaks at 2θ values ​​of 5.30, 11.01, 11.53, 12.58, 16.23, and 17.65, with a 2θ error range of ±0.2°; more preferably, the X-ray powder diffraction pattern of crystal form B has characteristic peaks at 2θ values ​​of 5.30, 7.23, 8.08, 11.01, 11.53, 12.58, 16.23, 16.59, 16.96, 17.65, 18.89, 21.78, 22.93, and 24.54, with a 2θ error range of ±0.2°; more preferably, the X-ray powder diffraction pattern of crystal form B is shown in Figure 3; even more preferably, the TGA-DSC spectrum of crystal form B is shown in Figure 4.

3. The crystal form C of the compound of formula (I), characterized in that, Using Cu-Kα radiation, the X-ray powder diffraction pattern of crystalline form C exhibits characteristic peaks at 2θ values ​​of 5.13, 9.48, 11.04, 16.51, 17.32, and 18.31, with a 2θ error range of ±0.2°. More preferably, the X-ray powder diffraction pattern of crystalline form C exhibits characteristic peaks at 2θ values ​​of 5.13, 9.48, 11.04, 11.66, 15.16, 16.51, 17.32, 18.31, and 24.95, with a 2θ error range of ±0.2°. Even more preferably, its X-ray powder diffraction pattern is essentially as shown in Figure 5.

4. A pharmaceutically acceptable salt formed by a compound of formula (I) and one or more acid molecules, Formula (I) The acid is selected from organic acids or inorganic acids; more preferably, the molecular ratio of the compound of formula (I) to the organic acid or inorganic acid is 1:1 to 1:4; even more preferably, the molecular ratio of the compound of formula (I) to the organic acid or inorganic acid is 1:1 to 1:

2.

5. The pharmaceutically acceptable salt as described in claim 4, selected from hydrochloride, maleate, p-toluenesulfonate, methanesulfonate, hydrobromide, succinate, L-malate, L-tartrate, benzenesulfonate, phosphate, gentianate, oxalate, fumarate, citrate; further preferably selected from maleate and hydrochloride; even more preferably, the ratio of the compound of formula (I) to maleic acid is 1:

2.

6. The maleate crystal form I of the compound of formula (Ⅰ), characterized in that, Using Cu-Kα radiation, the X-ray powder diffraction pattern of maleate crystal form I of compound (Ⅰ) has characteristic peaks at 2θ values ​​of 8.67, 12.95, 15.00, 15.82, 17.91, and 22.59, with a 2θ error range of ±0.2°; more preferably, its X-ray powder diffraction pattern has characteristic peaks at 2θ values ​​of 7.43, 8.67, 9.40, 12.19, 12.95, 14.37, 15.00, 15.82, 17.91, 19.07, 22.59, and 23.86, with a 2θ error range of ±0.2°; more preferably, its X-ray powder diffraction pattern is shown in Figure 6; even more preferably, the TGA-DSC spectrum of the crystal form is shown in Figure 7.

7. The maleate crystal form II of the compound of formula (I), characterized in that, Using Cu-Kα radiation, the X-ray powder diffraction pattern of maleate crystal form II of compound (Ⅰ) has characteristic peaks at 2θ values ​​of 6.94, 7.91, 8.82, 15.40, and 22.87, with a 2θ error range of ±0.2°; more preferably, its X-ray powder diffraction pattern is shown in Figure 8; even more preferably, the TGA-DSC pattern of the crystal form is shown in Figure 9.

8. The hydrochloride crystal form I of the compound of formula (Ⅰ), characterized in that, Using Cu-Kα radiation, the X-ray powder diffraction pattern of the hydrochloride crystal form I exhibits characteristic peaks at 2θ values ​​of 7.15, 8.96, 14.68, 19.28, 21.22, and 22.41, with a 2θ error range of ±0.2°. More preferably, its X-ray powder diffraction pattern exhibits characteristic peaks at 2θ values ​​of 7.15, 8.96, 10.72, 12.49, 14.68, 19.28, 20.56, 21.22, 22.41, 23.20, 25.11, and 27.83, with a 2θ error range of ±0.2°. Further preferably, its X-ray powder diffraction pattern has characteristic peaks at 2θ values ​​of 7.15, 8.96, 10.72, 11.44, 12.49, 13.51, 14.68, 15.57, 16.79, 18.00, 19.28, 20.56, 21.22, 22.41, 23.20, 25.11, 26.38, 27.83, and 29.28, with a 2θ error range of ±0.2°; even more preferably, it is characterized by using Cu-Kα radiation, and its X-ray powder diffraction pattern is shown in Figure 10.

9. A pharmaceutical composition comprising the crystal form, salt, and pharmaceutically acceptable carrier as described in any one of claims 1-8.

10. Use of the crystal form, salt, or pharmaceutical composition of any one of claims 1-8 in the preparation of a medicament for treating CBL-B-mediated diseases.

Images