Crystalline form of aromatic heterocyclic compound, its composition, production method and application thereof

Abstract

The present invention discloses crystalline forms of an aromatic heterocyclic compound, their compositions, preparation methods, and applications. The crystalline forms include crystalline Form I and crystalline Form III, which show characteristic diffraction peaks at about 8.4°, 10.0°, 13.6°, 16.4°, and 19.7°, and at about 8.2°, 9.8°, 17.2°, and 26.8°, respectively, when measured by X-ray diffraction. The aromatic heterocyclic compound is stirred in a suitable solvent, filtered, and dried to obtain crystalline Form I or III. The present invention also discloses the use of crystalline Form I or III in the manufacture of drugs for the prevention or treatment of diseases caused by abnormalities in the JAK-STAT signaling pathway, which can be used to treat autoimmune diseases, cancer, and myeloproliferative disorders.

Description

[Technical Field]

[0001] The present invention belongs to the technical field of medicinal chemistry, and specifically relates to the crystalline form of aromatic heterocyclic compounds that inhibit the activity of Janus kinase (JAK), their compositions, preparation methods and applications. [Background technology]

[0002] Janus kinase (JAK) is a cytoplasmic tyrosine protein kinase that mediates the signaling of many inflammation-related cytokines from cytokine membrane receptors to STAT transcription factors. Abnormalities in JAK / STAT signaling are associated with numerous diseases, including immune-inflammatory disorders such as organ transplant rejection, multiple sclerosis, rheumatoid arthritis, type 1 diabetes, lupus, psoriasis, asthma, food allergies, atopic dermatitis and rhinitis, and skin rashes. JAK / STAT signaling is also reported to be closely associated with the onset and development of hematological malignancies and myeloproliferative disorders (including lung cancer, breast cancer, chronic spontaneous myelofibrosis, erythrocytosis, and idiopathic thrombocytosis).

[0003] JAK3 is mainly expressed in various hematopoietic tissue cells, including bone marrow cells, thymocytes, NK cells, activated B lymphocytes, and T lymphocytes, and its physiological effects are solely derived from the signal transduction process of the common gamma cytokine receptor family. Therefore, highly selective action on JAK3 kinase can avoid unnecessary side effects. This can improve the activity and selectivity of JAK3 kinase inhibitors, further enhancing the clinical efficacy of JAK3 kinase inhibitors, and providing significant clinical advantages over the Pan-JAK inhibitors and selective JAK1 and JAK2 inhibitors currently in clinical use.

[0004] [ka]

[0005] In the applicant's previous application (application number CN202211019771.6), the applicant studied the JAK3 kinase inhibitory activity and immunosuppressive activity of aromatic heterocyclic compound [Chemical Formula 1] (see paragraphs 425-510 of the specification and Figures 1-5). The aromatic heterocyclic compound has good JAK3 kinase inhibitory activity and significant immunosuppressive effect on SRBC-induced delayed allergic reaction in mice, can improve the symptoms of rheumatoid arthritis, alleviate colorectal damage in mice with inflammatory bowel disease, and significantly reduce the number of inflammatory cell infiltration in the lungs of mice induced by irradiation.

[0006] The phenomenon of drug polycrystalline form is one of the important factors that affect the quality and clinical therapeutic effect of a drug, and different crystalline forms will cause differences in stability, absorption and bioavailability, thereby affecting the clinical therapeutic effect of the drug. Therefore, the crystalline form of this aromatic heterocyclic compound and its preparation technology are of great importance to the application of this drug. Summary of the Invention

[0007] The present invention provides a crystalline form of an aromatic heterocyclic compound, its composition, preparation method, and application thereof, which can be used as a JAK kinase inhibitor for the treatment and prevention of clinical diseases related to abnormalities in the activity of these kinases, including autoimmune diseases, cancer, myeloproliferative disorders, and other diseases.

[0008] The present invention provides the following structural formula of an aromatic heterocyclic compound:

[0009] [ka]

[0010] The aromatic heterocyclic compound can have a variety of different crystal forms.

[0011] Crystalline Form I of the aromatic heterocyclic compound has characteristic diffraction peaks at 2θ angles of about 8.4°, 10.0°, 13.6°, 16.4°, and 19.7° using X-ray diffraction.

[0012] Crystalline Form II of the aromatic heterocyclic compound has characteristic diffraction peaks at 2θ angles of about 7.6°, 9.9°, 15.1°, 16.1°, and 19.7° using X-ray diffraction.

[0013] Crystalline Form III of the aromatic heterocyclic compound exhibits characteristic diffraction peaks at 2θ angles of approximately 8.2°, 9.8°, 17.2°, and 26.8° using X-ray diffraction.

[0014] Crystalline form IV of the aromatic heterocyclic compound has characteristic diffraction peaks at 2θ angles of about 7.9°, 9.4°, 15.7°, and 19.2° using X-ray diffraction.

[0015] Crystal form V of the aromatic heterocyclic compound has characteristic diffraction peaks at 2θ angles of about 12.8°, 13.0°, 17.2°, and 22.8° using X-ray diffraction.

[0016] Crystal form VI of the aromatic heterocyclic compound has characteristic diffraction peaks at 2θ angles of about 7.7°, 9.2°, and 19.7° using X-ray diffraction.

[0017] Crystalline Form VII of the aromatic heterocyclic compound has characteristic diffraction peaks at 2θ angles of about 7.9°, 8.3°, and 9.9° using X-ray diffraction.

[0018] Crystalline Form VIII of the aromatic heterocyclic compound has characteristic diffraction peaks at 2θ angles of about 8.0°, 9.6°, 18.8°, and 19.2° using X-ray diffraction.

[0019] Crystalline Form IX of an aromatic heterocyclic compound has characteristic diffraction peaks at 2θ angles of about 8.1° and 9.6° when measured by X-ray diffraction, with an error range of ±0.2° for the 2θ angle.

[0020] Furthermore, the crystalline form I has characteristic diffraction peaks at 2θ angles of about 15.2°, 18.1°, 24.1°, and 26.9° using X-ray diffraction; the crystalline form IV has characteristic diffraction peaks at 2θ angles of about 16.6° and 18.6° using X-ray diffraction; the crystalline form V has characteristic diffraction peaks at 2θ angles of about 18.6° and 20.2° using X-ray diffraction; and the crystalline form VII has characteristic diffraction peaks at 2θ angles of about 19.7°, 25.1°, and 33.5° using X-ray diffraction, where the error range of the 2θ angle is ±0.2°.

[0021] Furthermore, crystalline form I has an X-ray diffraction pattern characterized by the X-ray diffraction pattern shown in FIG. 1, crystalline form II has an X-ray diffraction pattern characterized by the X-ray diffraction pattern shown in FIG. 8, crystalline form III has an X-ray diffraction pattern characterized by the X-ray diffraction pattern shown in FIG. 17, crystalline form IV has an X-ray diffraction pattern characterized by the X-ray diffraction pattern shown in FIG. 25, crystalline form V has an X-ray diffraction pattern characterized by the X-ray diffraction pattern shown in FIG. 29, crystalline form VI has an X-ray diffraction pattern characterized by the X-ray diffraction pattern shown in FIG. 35, crystalline form VII has an X-ray diffraction pattern characterized by the X-ray diffraction pattern shown in FIG. 42, crystalline form VIII has an X-ray diffraction pattern characterized by the X-ray diffraction pattern shown in FIG. 51, and crystalline form IX has an X-ray diffraction pattern characterized by the X-ray diffraction pattern shown in FIG.

[0022] Furthermore, the melting point of crystalline form I is about 196°C. The melting point of crystalline form II is about 184.4°C. The melting point of crystalline form II is about 201°C. The melting point of crystalline form IV is about 202°C. The melting point of crystalline form V is about 223°C. The melting point of crystalline form VI is about 202°C. The melting point of crystalline form VII is about 194°C. The melting point of crystalline form VIII is about 204°C.

[0023] Another object of the present invention is to provide a combination of crystalline forms of aromatic heterocyclic compounds that can be used as JAK kinase inhibitors for the treatment and prevention of clinical diseases associated with abnormalities in the activity of these kinases, including autoimmune diseases, inflammatory diseases, cancer, and other diseases.

[0024] A crystalline composition of an aromatic heterocyclic compound, comprising one or a combination of two or more of crystalline forms I, II, III, IV, V, VI, VII, VIII, and IX, wherein crystalline form I and / or III account for 50% or more by weight of the crystalline composition. Preferably, the composition comprises one or two of crystalline forms I and III, wherein crystalline form I and / or III account for 50% or more by weight of the composition.

[0025] Another object of the present invention is to provide a combination of crystalline forms of aromatic heterocyclic compounds that can be used as JAK kinase inhibitors for the treatment and prevention of clinical diseases associated with abnormalities in the activity of these kinases, including autoimmune diseases, inflammatory diseases, cancer, and other diseases.

[0026] A crystalline composition of an aromatic heterocyclic compound, comprising one or a combination of two or more of crystalline forms I, II, III, IV, V, VI, VII, VIII, and IX, wherein crystalline form I and / or III account for 80% or more by weight of the crystalline composition. Preferably, the composition comprises one or two of crystalline forms I and III, wherein crystalline form I and / or III account for 80% or more by weight of the composition.

[0027] Another object of the present invention is to provide a combination of crystalline forms of aromatic heterocyclic compounds that can be used as JAK kinase inhibitors for the treatment and prevention of clinical diseases associated with abnormalities in the activity of these kinases, including autoimmune diseases, inflammatory diseases, cancer, and other diseases.

[0028] A crystalline composition of an aromatic heterocyclic compound, comprising one or a combination of two or more of crystalline forms I, II, III, IV, V, VI, VII, VIII, and IX, wherein crystalline form I and / or III account for 90% or more by weight of the crystalline composition. More preferably, the composition comprises one or two of crystalline forms I and III, wherein crystalline form I and / or III account for 90% or more by weight of the composition.

[0029] Another object of the present invention is to provide a pharmaceutical composition containing the above crystalline form for the treatment and prevention of clinical diseases associated with abnormal kinase activity, including autoimmune diseases, inflammatory diseases, cancer, and other diseases.

[0030] A pharmaceutical composition comprising said crystalline Form I and / or crystalline Form III and a pharmaceutically acceptable excipient.

[0031] Another object of the present invention is to provide a method for preparing crystalline Form I by adding a solvent to the aromatic heterocyclic compound to form a suspension, stirring at room temperature, filtering, and drying to obtain a white solid.

[0032] Furthermore, when producing crystalline I, the solvent is one or a combination of two or more of acetone, methanol, ethanol, water, ethyl acetate, toluene, methyl tert-butyl ether, and n-heptane, and the amount of the organic solvent used is 3 to 50 times the weight of the aromatic heterocyclic compound.

[0033] Preferably, in the method for preparing Crystalline Form I, a solvent is added to the aromatic heterocyclic compound to form a suspension, which is stirred at room temperature, filtered, and dried to obtain a white solid, Crystalline Form I, wherein the solvent is selected from water and ethanol. The volume of the solvent required for 1 mg of the aromatic heterocyclic compound is 0.01 to 0.2 mL.

[0034] Another object of the present invention is to provide a method for producing crystalline form I, which comprises adding a solvent to the aromatic heterocyclic compound to dissolve and clarify it, and then evaporating and drying it.

[0035] Furthermore, the solvent is one or a combination of two or more of acetone, chloroform, ethanol, tetrahydrofuran, 1,4-dioxane, and water.

[0036] Another object of the present invention is to provide a method for preparing crystalline Form I, which comprises adding a first organic solvent to the aromatic heterocyclic compound, dissolving the compound by heating, adding a second organic solvent, and then stirring to precipitate crystals, which are then filtered and dried.

[0037] Furthermore, the first organic solvent is one or a combination of two or more of methanol, ethanol, dichloromethane, trichloromethane, and dimethyl sulfoxide, more preferably methanol. The amount of the first organic solvent used is 10 to 50 times the weight of the aromatic heterocyclic compound. The second organic solvent is one or a combination of two or more of isopropyl ether, n-hexane, n-heptane, methyl tert-butyl ether, and water, more preferably isopropyl ether. The amount of the second organic solvent used is 10 to 50 times the weight of the aromatic heterocyclic compound. When methanol is selected as the first organic solvent, the volume of methanol required for 1 mg of the aromatic heterocyclic compound is 0.1 to 0.3 mL. When the second organic solvent is isopropyl ether, the volume of isopropyl ether required for 1 mg of the aromatic heterocyclic compound is 0.5 to 1.3 mL. The volume ratio of isopropyl ether to methanol is 4 to 7:1, more preferably 5 to 6:1.

[0038] Another object of the present invention is to provide a method for preparing crystalline Form III, which comprises adding a first organic solvent to the aromatic heterocyclic compound, dissolving the compound by heating, adding a second organic solvent, and then stirring to precipitate crystals, which are then filtered and dried.

[0039] Furthermore, when preparing Crystalline Form III, the first organic solvent is one or a combination of two or more of methanol, ethanol, tetrahydrofuran, 1,4-dioxane, and acetonitrile. The amount of the first organic solvent used is 10 to 50 times the weight of the aromatic heterocyclic compound. The second organic solvent is one or a combination of two or more of water and ethyl acetate, and the amount of the second organic solvent used is 10 to 50 times the weight of the aromatic heterocyclic compound.

[0040] More preferably, when preparing Crystalline Form III, the first organic solvent is selected from ethanol and methanol, and the second organic solvent is selected from water. The volume of the first organic solvent required for 1 mg of the aromatic heterocyclic compound is 0.1 to 0.3 mL, the volume of the second organic solvent required is 0.5 to 1.6 mL, and the volume ratio of the second organic solvent to the first organic solvent is 4 to 7:1, more preferably 5 to 6:1.

[0041] Another object of the present invention is to provide an application of the above crystals in the manufacture of drugs for the prevention or treatment of diseases caused by abnormalities in the JAK-STAT signaling pathway, which drugs can be used for the treatment of autoimmune diseases, cancers, and myeloproliferative disorders.

[0042] Furthermore, the autoimmune disease is one or more selected from alopecia areata, lupus, multiple sclerosis, amyotrophic lateral sclerosis, rheumatoid arthritis, rheumatoid arthritis, psoriasis, complications from organ transplantation, atopic dermatitis, autoimmune thyroid disease, ulcerative colitis, Crohn's disease, Sjögren's syndrome, vitiligo, autoimmune kidney damage, autoimmune liver damage, and chronic obstructive pulmonary disease; the cancer is one or more selected from colon cancer, gastric adenocarcinoma, bladder cancer, breast cancer, kidney cancer, liver cancer, lung cancer, thyroid cancer, head and neck cancer, prostate cancer, pancreatic cancer, cancer of the CNS (central nervous system), and malignant glioma; and the myeloproliferative disease is one or more selected from chronic myelomonocytic leukemia, atypical chronic myeloid leukemia, and juvenile myelomonocytic leukemia.

[0043] The aromatic heterocyclic compounds disclosed in the present invention have many crystalline forms. When researchers use hot-stage XRPD to study the crystalline form of the compound, they find that at 155 ℃, the sample transforms into another thermodynamically more stable anhydrous crystalline form, which we define as crystalline form I. At the same time, they also find crystalline form II, crystalline form III, crystalline form IV, crystalline form V, crystalline form VI, crystalline form VII, crystalline form VIII and crystalline form IX, a total of eight other crystalline forms, of which crystalline forms I and III are anhydrous crystalline forms. From this, it can be seen that the aromatic heterocyclic compounds disclosed in the present invention have the phenomenon of crystalline polymorphism, and most of the crystalline forms are solvates or hydrates, and under most preparation conditions, anhydrous crystalline forms cannot be obtained, so the preparation of anhydrous crystalline forms I and III is very difficult. Solvates and hydrates have the problem that they are prone to undergo crystal transformation due to desolvation during storage, and the crystalline form composition ratio of the compound varies with storage time, which may result in unstable compound quality. However, the crystalline forms I and III provided by the present invention are anhydrous crystalline forms that do not undergo crystal form transformation during storage, thereby overcoming the above-mentioned drawbacks of the compounds.

[0044] The researchers compared the nine crystalline forms using hot-stage XRPD, TG, DSC, DVS, and other methods. Hot-stage XRPD analysis showed that most crystalline forms transformed into crystalline form I at 150-170°C, and TG and DSC analyses showed that crystalline form I was the most stable during heating. DVS and isotherms demonstrated the structural stability of crystalline form I under desorption and adsorption conditions.

[0045] At the same time, researchers conducted competition tests of the above-mentioned crystalline forms in water and ethanol to examine the stability and transformation status of various crystalline forms in these solvents. The crystalline form competition experiments revealed that crystalline forms I and III have good crystalline form stability in water, while the other crystalline forms are unstable in water. Because water is the most commonly used solvent in drug formulations and solid powders are prone to absorbing water when exposed to air, crystalline forms I and III provided in this patent have good stability in the pharmaceutical application field and are suitable for development as medicinal crystalline forms. Crystalline form I has good crystalline stability in the presence of ethanol. Ethanol has wide applications in the manufacturing process of drug solvents and drug formulations, and the above experiments demonstrate that crystalline form I has significant stability advantages in the development of drug formulations in the presence of ethanol.

[0046] The researchers conducted a 15-day high temperature and humidity test on Form I, as well as a long-term, accelerated 3-month test, and all test results were in Form I, further demonstrating the stability of Form I during storage.

[0047] At the same time, the researchers conducted solubility tests of crystalline form I at different pH values, which showed that the solubility improved significantly with decreasing pH value, indicating that the sample could be rapidly dissolved in the human stomach. [Brief explanation of the drawings]

[0048] [Figure 1] FIG. 1 is an X-ray diffraction diagram of crystalline form I of the present invention. [Figure 2] FIG. 1 is a diagram showing X-ray diffraction peak data of crystalline form I of the present invention. [Figure 3] FIG. 1 is a TG diagram of crystalline form I of the present invention. [Figure 4] FIG. 1 is a DSC diagram of crystalline form I of the present invention. [Figure 5] FIG. 1 is a DVS diagram of crystalline form I of the present invention. [Figure 6] FIG. 1 is an isothermal curve diagram of crystalline form I of the present invention. [Figure 7] FIG. 1 is a PLM diagram of crystalline form I of the present invention. [Figure 8] FIG. 1 is an X-ray diffraction diagram of crystalline form II of the present invention. [Figure 9] FIG. 1 is a diagram showing X-ray diffraction peak data of crystalline form II of the present invention. [Figure 10] FIG. 1 is a TG diagram of crystalline form II of the present invention. [Figure 11] FIG. 1 is a DSC diagram of crystalline form II of the present invention. [Figure 12] FIG. 1 is a DVS diagram of crystalline form II of the present invention. [Figure 13] FIG. 1 is an isothermal curve diagram of crystalline form II of the present invention. [Figure 14] FIG. 1 is a PLM diagram of crystalline form II of the present invention. [Figure 15] FIG. 1 is a hot-stage XRPD diagram of crystalline form II in the present invention. [Figure 16] FIG. 1 is a hot-stage H-NMR diagram of crystalline form II of the present invention. [Figure 17] FIG. 1 is an X-ray diffraction diagram of crystalline form III of the present invention. [Figure 18] FIG. 1 is a diagram showing X-ray diffraction peak data of crystalline form III of the present invention. [Figure 19] FIG. 1 is a TG diagram of crystalline form III of the present invention. [Figure 20] FIG. 1 is a DSC diagram of crystalline form III of the present invention. [Figure 21] FIG. 1 is a DVS diagram of crystalline form III of the present invention. [Figure 22] FIG. 1 is an isothermal curve diagram of crystalline form III of the present invention. [Figure 23] FIG. 1 is a PLM diagram of crystalline form III of the present invention. [Figure 24] FIG. 1 is a hot-stage XRPD diagram of crystalline form III in the present invention. [Figure 25] FIG. 1 is an X-ray diffraction diagram of crystalline form IV of the present invention. [Figure 26] FIG. 1 is a diagram showing X-ray diffraction peak data of crystalline form IV of the present invention. [Figure 27] FIG. 1 is a TG diagram of crystalline form IV of the present invention. [Figure 28] FIG. 1 is a DSC diagram of crystalline form IV of the present invention. [Figure 29] FIG. 1 is an X-ray diffraction diagram of crystalline form V of the present invention. [Figure 30] FIG. 1 is a diagram showing X-ray diffraction peak data of crystalline form V of the present invention. [Figure 31] FIG. 1 is a TG diagram of Crystal V of the present invention. [Figure 32] FIG. 1 is a DSC diagram of Crystal V of the present invention. [Figure 33] FIG. 1 is a PLM diagram of crystal V in the present invention. [Figure 34] FIG. 1 is a hot-stage H-NMR diagram of crystalline form V in the present invention. [Figure 35] FIG. 1 is an X-ray diffraction diagram of crystalline form VI of the present invention. [Figure 36] FIG. 1 is a diagram showing X-ray diffraction peak data of crystalline form VI of the present invention. [Figure 37] FIG. 1 is a TG diagram of crystalline form VI of the present invention. [Figure 38] FIG. 1 is a DSC diagram of crystalline form VI in the present invention. [Figure 39] FIG. 1 is a PLM diagram of Crystal VI in the present invention. [Figure 40] FIG. 1 is a hot-stage XRPD diagram of Crystal VI in the present invention. [Figure 41] FIG. 1 is a hot-stage H-NMR diagram of crystalline form VI in the present invention. [Figure 42] FIG. 1 is an X-ray diffraction diagram of crystalline form VII of the present invention. [Figure 43] FIG. 1 is a diagram showing X-ray diffraction peak data of crystalline form VII of the present invention. [Figure 44] FIG. 1 is a TG diagram of Crystal VII of the present invention. [Figure 45] FIG. 1 is a DSC diagram of Crystal VII of the present invention. [Figure 46] FIG. 1 is a DVS diagram of Crystal VII of the present invention. [Figure 47] FIG. 1 is an isothermal curve diagram of crystalline form VII of the present invention. [Figure 48]FIG. 1 is a PLM diagram of Crystal VII of the present invention. [Figure 49] FIG. 1 is a hot-stage XRPD diagram of Crystal VII of the present invention. [Figure 50] FIG. 1 is a hot-stage H-NMR diagram of crystalline form VII of the present invention. [Figure 51] FIG. 1 is an X-ray diffraction diagram of crystalline form VIII of the present invention. [Figure 52] FIG. 1 is a diagram showing X-ray diffraction peak data of crystalline form VIII of the present invention. [Figure 53] FIG. 1 is a TG diagram of Crystal VIII of the present invention. [Figure 54] FIG. 1 is a DSC diagram of crystalline form VIII of the present invention. [Figure 55] FIG. 1 is a DVS diagram of crystalline form VIII of the present invention. [Figure 56] FIG. 1 is an isothermal curve diagram of crystalline form VIII of the present invention. [Figure 57] FIG. 1 is a PLM diagram of crystalline form VIII of the present invention. [Figure 58] FIG. 1 is a hot-stage XRPD diagram of crystalline form VIII of the present invention. [Figure 59] FIG. 1 is a hot-stage H-NMR diagram of crystalline form VIII of the present invention. [Figure 60] FIG. 1 is an X-ray diffraction diagram of crystalline form IX of the present invention. [Figure 61] FIG. 1 is a diagram showing X-ray diffraction peak data of crystalline form IX of the present invention. [Figure 62] FIG. 1 is a TG diagram of crystalline form IX of the present invention. [Figure 63] FIG. 1 is a DSC diagram of crystalline form IX of the present invention. [Figure 64] FIG. 1 is a PLM diagram of Crystal IX of the present invention. [Figure 65] FIG. 1 is a hot-stage XRPD diagram of Crystal IX of the present invention. [Figure 66] FIG. 1 is a hot-stage H-NMR diagram of crystalline form IX of the present invention. [Figure 67]FIG. 1 is an X-ray diffraction pattern of crystalline form I of the present invention at high temperature for 15 days. [Figure 68] FIG. 1 is a diagram showing X-ray diffraction peak data of crystalline form I of the present invention at high temperature for 15 days. [Figure 69] FIG. 1 is an X-ray diffraction pattern of crystalline form I of the present invention after 15 days at high humidity. [Figure 70] FIG. 1 is a diagram showing X-ray diffraction peak data of crystalline form I of the present invention after 15 days under high humidity conditions. [Figure 71] FIG. 1 is a long-term 3-month X-ray diffraction pattern of crystalline form I of the present invention. [Figure 72] FIG. 1 is a diagram showing long-term 3-month X-ray diffraction peak data of crystalline form I of the present invention. [Figure 73] FIG. 1 is a 3-month accelerated X-ray diffraction pattern according to the present invention. [Figure 74] FIG. 1 is a diagram showing X-ray diffraction peak data of a 3-month accelerated test according to the present invention. [Figure 75] 1 is a solubility curve of crystalline Form I samples at different pH values. [Figure 76] Immunosuppressive activity of compounds on the SRBC mouse model. [Figure 77] Immunosuppressive effect of compounds on a collagen-induced mouse arthritis model. [Figure 78] Immunosuppressive effects of compounds on dextran sulfate sodium (DSS)-induced inflammatory bowel disease model in mice. [Figure 79] Inhibitory effect of compounds on TNF-α levels in mice with acute radiation lung injury. [Figure 80] Compounds reduced the number of inflammatory cell infiltrates in the lungs of mice in acute radiation-induced lung injury. DETAILED DESCRIPTION OF THE INVENTION

[0049] In order to more clearly explain the present invention, the present invention will be further described below in connection with preferred embodiments. Those skilled in the art should understand that the specific description below is for illustrative purposes rather than restrictive, and should not limit the protection scope of the present invention.

[0050] All numerical designations herein (e.g., temperature, time, concentration, weight, etc., including their respective ranges) are typically approximated (+) or (-) by increments of 0.1 or 1.0. All numerical designations may be understood to be preceded by the term "about." [Example]

[0051] (Comparative Example): Aromatic Heterocyclic Compound

[0052] The aromatic heterocyclic compound was prepared by referring to the preparation process of Example 91 of application number CN202211019771.6, specifically as follows:

[0053] [ka]

[0054] Step 1: Intermediate SM1 (150 g, 0.50 mol), sodium hydrogen carbonate (25 g, 1.04 mol), and methyl iodide (73 g, 0.52 mol) were sequentially added to a 2000 mL three-neck flask containing 1000 mL of DMF, and the reaction mixture was stirred thoroughly for 2 hours at 0°C. After completion of the reaction, the reaction mixture was poured into water and extracted three times with dichloromethane. The organic layers were combined, washed twice with saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting crude product was purified by silica gel column chromatography to obtain 100 g of a yellow solid, intermediate IM1, in a 63.3% yield.

[0055] Step 2: Under nitrogen protection, intermediate IM1 (100 g, 0.32 mol), tetra(triphenylphosphorus)palladium (38 g, 33 mmol), intermediate SM3 (121 g, 0.5 mol), and potassium carbonate (137 g, 0.99 mol) were sequentially added to a 2000 mL three-neck flask containing 1000 mL of 1,4-dioxane. The reaction mixture was heated to 95 °C and stirred overnight. After cooling to room temperature, the reaction mixture was poured into 1000 mL of water and extracted three times with ethyl acetate. The organic layers were combined, washed twice with saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting crude product was purified by silica gel column chromatography to give 50 g of a pale yellow solid, intermediate IM2, in a 44.3% yield.

[0056] Step 3: Intermediate IM2 (50 g, 0.14 mol) was dissolved in dichloromethane, and an equal volume of trifluoroacetic acid was added. The mixture was allowed to react at room temperature overnight. After the reaction was completed, the pH was adjusted to 8-9, and the mixture was extracted three times with dichloromethane. The organic layers were combined, washed twice with saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting crude product was purified by silica gel column chromatography to obtain 20 g of a white solid, intermediate IM3, with a yield of 56.4%.

[0057] Step 4: Intermediate IM3 (20 g, 78.9 mmol) was dissolved in 4000 ml of dichloromethane, triethylamine (24.5 g, 243 mmol) was added, and acryloyl chloride (8.66 g, 95 mmol) was slowly added in an ice bath. The mixture was allowed to react at room temperature for 3 hours, after which saturated aqueous sodium bicarbonate solution was added, and the mixture was washed and extracted three times with dichloromethane. The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting crude product was purified by silica gel column chromatography using dichloromethane and methanol as eluents to obtain 15 g of the desired compound as a white solid, with a yield of 62%. 1H NMR(400MHz,DMSO-d6)δ11.65(s,1H),8.28(d,J=4.8Hz,1H),7.47(s,1H),7.10-7.03(m,1H),7.00(d,J=4.7Hz,1H),6.95-6.85(m,1H),6.1 3(d,J=15.1Hz,1H),5.73(d,J=12.3Hz,1H),4.81(d,J=14.2Hz,2H),3.91(d,J=5.2Hz,2H),3.83(s,3H),2.83(s,2H)ESI(M+H)+=308.14959.

[0058] The X-ray diffraction pattern of the resulting white solid showed characteristic diffraction peaks at 8.11° and 9.62° 2θ angles (see Figures 60 and 61). TGA showed a weight loss of 4.2% before 130°C and a weight loss of 3.7% between 130°C and 210°C. The sample decomposed at 361°C (see Figure 62). DSC showed a desolvation peak between 20°C and 140°C, an endothermic peak at 171°C, and a melting point of 199.8°C (see Figure 63). PLM revealed sheet-like crystals (see Figure 64). Hot-stage XRPD showed a transition to crystalline form I at 155°C and remained in crystalline form I at room temperature (see two XRD patterns marked at 155°C and RT in Figure 65). (In the figure, Form 1 is crystalline form I, prepared in Example 4). 1 According to the H-NMR diagram, the peaks are water and methanol peaks (see Figure 66), and it is a hydrate. The 1-(1-methyl-3-(1H-pyrroly[2,3-b]pyridin-4-yl)-1,4,6,7-tetrahydro-5H-pyrazoli[4,3-c]pyridin-5-yl)-2-en-1-one obtained by the above-mentioned production method is crystalline form IX.

[0059] As can be seen from this, crystalline form IX is not an anhydrous crystalline form and may be prone to crystalline form transition during storage, and its quality may become unstable if stored for a long period of time. [Example]

[0060] 20 mg of the aromatic heterocyclic compound produced in Example 1 was weighed, 4 ml of methanol and 2 ml of chloroform were added, the mixture was dissolved and clarified by ultrasonic waves, and the mixture was evaporated and dried at room temperature with the cap open to obtain a solid.

[0061] The X-ray diffraction pattern of the obtained solid showed it to be crystalline form IX. [Example]

[0062] 10 mg of the aromatic heterocyclic compound produced in Example 1 was weighed, 4 ml of methanol was added, the mixture was dissolved and clarified by ultrasonic waves, and the mixture was left open at room temperature to evaporate and dry, thereby obtaining a solid.

[0063] The X-ray diffraction pattern of the obtained solid showed it to be crystalline form IX. [Example]

[0064] 20 mg of the aromatic heterocyclic compound produced in Example 1 was weighed, and 2 ml of water was added to form a suspension. The suspension was stirred at room temperature for 4 days and centrifuged. The resulting solid was dried at 45°C to obtain a white solid.

[0065] The X-ray diffraction pattern of the resulting white solid showed characteristic diffraction peaks at 2θ angles of 8.39°, 9.96°, 13.58°, 15.21°, 16.38°, 18.11°, 19.73°, 24.09°, and 26.93° (see Figures 1 and 2). TGA revealed a weight loss of 0.93% before 150°C, and the sample decomposed at 354°C (see Figure 3). DSC indicated a melting point of approximately 196°C (see Figure 4). DVS and isothermal analysis revealed a moisture absorption of approximately 2.4% (see Figures 5 and 6). The desorption and adsorption curves overlapped, indicating no change in structure before and after. PLM revealed the sample to be fine particles (see Figure 7). The white solid obtained by this preparation method is crystalline I. [Example]

[0066] 20 mg of the aromatic heterocyclic compound produced in Example 1 and 4 ml of acetone were weighed, stirred, dissolved, and clarified, and then filtered. 14 ml of n-heptane was added dropwise to the filtrate, and the temperature was lowered to 4°C and the mixture was stirred overnight to precipitate crystals. The mixture was centrifuged, and the resulting solid was dried at 45°C to obtain a white solid.

[0067] The X-ray diffraction pattern of the obtained white solid showed characteristic diffraction peaks at 2θ angles of 6.68°, 26.86°, 7.59°, 9.91°, 15.06°, 16.15°, 19.08°, and 19.71° (see Figures 8 and 9). TGA showed a weight loss of 0.7% before 60°C and a weight loss of 8.02% between 60°C and 200°C. The sample decomposed at 355°C (see Figure 10). DSC showed desolvation peaks at 20°C-70°C and 70°C-160°C, with a melting point of around 184.4°C (see Figure 11). According to DVS and isotherm, the moisture absorption was about 2.4% (see Figures 12 and 13). According to PLM, the sample was a sheet crystal (see Figure 14). According to hot-stage XRPD, the sample transitioned to crystalline type I at 150°C and remained in crystalline type I even when cooled to room temperature (see Figure 15). 1 According to the H-NMR diagram, the peak is a water peak (see FIG. 16), which indicates that the white solid obtained by the above production method is crystalline II. [Example]

[0068] 20 mg of the aromatic heterocyclic compound produced in Example 1 and 3 ml of ethanol were weighed, stirred to dissolve and clarify, and filtered. 16 ml of water was added dropwise to the filtrate, and the temperature was lowered to 4°C and the mixture was stirred overnight to precipitate crystals. The mixture was centrifuged, and the resulting solid was dried at 45°C to obtain a white solid.

[0069] The X-ray diffraction pattern of the obtained white solid showed characteristic diffraction peaks at 2θ angles of 8.18°, 9.76°, 17.18°, and 26.81° (see Figures 17 and 18). TGA showed a weight loss of 2.38% at 125°C, and the sample decomposed at 359°C (see Figure 19). DSC showed a desolvation peak between 20°C and 140°C, an endothermic peak at 190°C, and a melting point of approximately 201°C (see Figure 20). DVS and isotherm analysis showed a moisture absorption of approximately 2.2% (see Figures 21 and 22). PLM showed that the sample was finely divided (see Figure 23). Hot-stage XRPD showed no morphological transition at 140°C (see Figure 24). The white solid obtained by the above preparation method is crystalline type III. [Example]

[0070] 20 mg of the aromatic heterocyclic compound produced in Example 1 and 3 ml of methanol were weighed, stirred, dissolved, and clarified, and then filtered. 16 ml of water was added dropwise to the filtrate, and the temperature was lowered to 4°C and the mixture was stirred overnight to precipitate crystals. The mixture was centrifuged, and the resulting solid was dried at 45°C to obtain a white solid.

[0071] The X-ray diffraction pattern of the obtained white solid showed that it was crystalline form III. [Example]

[0072] 50 mg of the aromatic heterocyclic compound produced in Example 1 and 10 ml of dichloromethane were stirred, dissolved, and clarified, and filtered. The filtrate was concentrated under reduced pressure and dried to obtain a white solid.

[0073] The X-ray diffraction pattern of the obtained white solid showed characteristic diffraction peaks at 2θ angles of 7.88°, 9.44°, 15.71°, and 19.24° (see Figures 25 and 26). TGA showed a weight loss of 11.97% at 160°C, the sample decomposed at 350°C, and contained 0.5 parts of dichloromethane solvate (see Figure 27). DSC showed a desolvation peak between 40°C and 170°C, and a melting point of approximately 202°C (see Figure 28). The X-ray diffraction pattern of the obtained white solid indicated that it was crystalline Form IV. [Example]

[0074] 10 mg of the aromatic heterocyclic compound prepared in Example 1 was weighed, 4 ml of 1,4-dioxane was added, and the mixture was dissolved and clarified by ultrasonic waves. The filtrate was then left at 40°C for evaporation and drying with an open cap, and then polished to obtain a white solid.

[0075] The X-ray diffraction pattern of the resulting white solid showed characteristic diffraction peaks at 2θ angles of 8.49°, 11.44°, 12.79°, 12.97°, 17.20°, 18.59°, 20.19°, 21.90°, 22.78°, 25.39°, and 28.52° (see Figures 29 and 30). TGA showed a weight loss of 9.65% before 125°C, with the sample decomposing at 354°C (see Figure 31). DSC showed a desolvation peak between 85°C and 140°C, with a melting point of approximately 223°C (see Figure 32). PLM revealed the sample to be fine particles (see Figure 33). 1 According to the H-NMR diagram, the peak is the 1,4-dioxane peak (see Figure 34), and the white solid obtained by the above production method is crystalline V. [Example]

[0076] 20 mg of the aromatic heterocyclic compound produced in Example 1 was weighed, dissolved and clarified by adding 3 ml of methanol, and a solid was precipitated by adding 16 ml of isopropyl ether. The solid was centrifuged and dried at 45°C to obtain a white solid.

[0077] The X-ray diffraction pattern of the obtained white solid showed that it was crystalline form I. [Example]

[0078] 10 mg of the aromatic heterocyclic compound prepared in Example 1 was weighed, and 4 ml of n-butanol and 5 ml of dichloromethane were added. The mixture was dissolved and clarified by ultrasonic waves, and then filtered. The filtrate was left at 40°C, opened, and evaporated to dryness. The mixture was then polished to obtain a white solid.

[0079] The X-ray diffraction pattern of the obtained white solid showed characteristic diffraction peaks at 2θ angles of 7.66°, 9.18°, and 19.66° (see Figures 35 and 36). TGA showed a weight loss of 10.9% before 150°C, and the sample decomposed at 357°C (see Figure 37). DSC showed a desolvation peak between 105°C and 155°C, with a melting point of approximately 202°C (see Figure 38). PLM revealed the sample to be blocky crystals (see Figure 39). Hot-stage XRPD showed a transition to crystalline phase I at 150°C, which remained crystalline phase I even at room temperature (see Figure 40). 1 According to the H-NMR diagram, the peak is the n-butanol peak (see Figure 41), and the n-butanol solvate is 0.5 parts. The white solid obtained by the above preparation method is crystalline VI. [Example]

[0080] 10 mg of the aromatic heterocyclic compound prepared in Example 1 was weighed and added to 4 ml of tetrahydrofuran, and the mixture was dissolved and clarified by ultrasonic waves and filtered. The filtrate was evaporated to dryness at a temperature of 50°C with the lid open and polished to obtain a white solid.

[0081] The X-ray diffraction pattern of the resulting white solid showed characteristic diffraction peaks at 2θ angles of 7.88°, 8.28°, 9.92°, 19.66°, 25.09°, and 33.45° (see Figures 42 and 43). TGA showed a weight loss of 6.0% before 200°C, with the sample decomposing at 358°C (see Figure 44). DSC showed a desolvation peak between 20°C and 145°C, with a melting point of approximately 194°C (see Figure 45). DVS and isotherm showed a moisture absorption of approximately 1.3% (see Figures 46 and 47). PLM showed the sample to be sheet-like crystals (see Figure 48). Hot-stage XRPD showed a transition to crystalline phase I at 150°C, which remained crystalline phase I even at room temperature (see Figure 49). 1 According to the H-NMR pattern, the peak is a water peak (see Figure 50), and the white solid obtained by the above production method is crystalline VII. [Example]

[0082] 10 mg of the aromatic heterocyclic compound prepared in Example 1 was weighed and added to 4 ml of acetonitrile, and the mixture was dissolved and clarified by ultrasound and filtered. The filtrate was left at 40°C, opened, and evaporated to dryness, and then polished to obtain a white solid.

[0083] The X-ray diffraction pattern of the obtained white solid showed characteristic diffraction peaks at 2θ angles of 8.03°, 9.59°, 18.78°, and 19.22° (see Figures 51 and 52). TGA showed a weight loss of 2.5% before 125°C and a weight loss of 4.0% between 125°C and 145°C. The sample decomposed at 362°C (see Figure 53). DSC showed desolvation peaks between 15°C and 130°C, between 130°C and 170°C, an endothermic peak at 181°C, and a melting point of approximately 204°C (see Figure 54). DVS and isotherm showed a moisture uptake of approximately 1.3% (see Figures 55 and 56). PLM showed the sample to be blocky crystals (see Figure 57). Hot-stage XRPD showed that the sample mostly transformed to Form I at 150°C (see Figure 58). 1 According to the H-NMR diagram, the peaks are those of water and solvent acetonitrile (see Figure 59), indicating that it is a hydrate. The white solid obtained by the above production method is crystalline VIII. [Example]

[0084] 30 mg of the aromatic heterocyclic compound produced in Example 1 was weighed, and 2 ml of ethanol was added to form a suspension. The suspension was stirred at room temperature for 4 days and centrifuged. The resulting solid was dried at 45°C to obtain a white solid.

[0085] The X-ray diffraction pattern of the obtained white solid showed that it was crystalline form I. [Example]

[0086] 10 g of the aromatic heterocyclic compound prepared in Example 1 was weighed, and 100 ml of ethanol was added to form a suspension, which was stirred at room temperature for 24 days, filtered, and dried to obtain 8.9 g of a white solid, with a yield of 89%.

[0087] The X-ray diffraction pattern of the obtained white solid showed that it was crystalline form I. [Example]

[0088] 2 mg each of the aromatic heterocyclic compounds of crystalline form I, crystalline form II, crystalline form III, crystalline form IV, crystalline form V, crystalline form VI, crystalline form VII, crystalline form VIII and crystalline form IX (total 18 mg) was taken, 3 ml of water was added to form a suspension, and the suspension was stirred at room temperature for 4 days and 7 days, and samples were taken on the 4th and 7th days, respectively, and X-ray diffraction tests were performed. The results showed that the suspensions were crystalline form I and crystalline form III, and no obvious changes were observed on the 4th and 7th days.

[0089] The above is a crystal form competition experiment, which shows that crystal forms I and III have good crystal form stability in water, while other crystal forms are unstable in water. Because water is the most commonly used solvent in drug formulations and solid powders are prone to absorbing water when exposed to air, crystal forms I and III provided by this patent have good stability in the field of pharmaceutical applications and are suitable for development as medicinal crystal forms. [Example]

[0090] 2 mg of each of the aromatic heterocyclic compounds of crystalline form I, crystalline form II, crystalline form III, crystalline form IV, crystalline form V, crystalline form VI, crystalline form VII, crystalline form VIII, and crystalline form IX was weighed out, and 3 ml of absolute ethanol was added to form a suspension. The suspension was stirred at room temperature for 4 days, and a sample was taken and subjected to X-ray diffraction measurement. The result showed that the suspension was crystalline form I, and the characteristic peaks of the other crystalline forms had disappeared.

[0091] The above crystal form competition experiment (ethanol stimulation) showed that crystalline form I has good crystal form stability in the presence of ethanol. Ethanol has wide applications in the manufacturing process of drug solvents and drug formulations, and the above experiment showed that crystalline form I has significant stability advantages in the development of drug formulations in the presence of ethanol. [Example]

[0092] Fifteen samples of crystalline form I prepared in Example 15 were weighed, weighing 50 mg each, and sealed in aluminum foil bags. The samples were subjected to stability studies under high temperature (60±2°C), high humidity (RH 90%±5%), long-term (25°C±2°C, RH 60%±5%), and accelerated (40°C±2°C, RH 75%±5%) conditions. The results showed that the crystalline form was stable for 15 days under the high temperature and humidity conditions (see Figures 67-70 for XRD diagram details). The crystalline form was also stable under the long-term and 3-month accelerated conditions (see Figures 71-74 for XRD diagram details). The influencing factors and stability test results fully demonstrated that crystalline form I can be stored stably.

[0093] Crystal form measurement results of influence factor test

[0094] [Table 1]

[0095] Crystal form measurement results of stability test

[0096] [Table 2] [Example]

[0097] 10mL of solutions with different pH values ​​were taken, and excess Form I sample was added. The solution was placed in a thermostatic rocker at 32℃ and shaken at 100 rpm for 24 hours. The saturated solution (with undissolved solids at the bottom) was then centrifuged and filtered. The content of linear solutions (prepared with the control) and the saturated solutions at different pH values ​​were measured. The solubility of the samples at different pH values ​​was calculated using the standard curve shown below. See Figure 75 for details.

[0098] [Table 3]

[0099] The test results showed that the solubility increased significantly with decreasing pH value, and in particular, the solubility of crystalline I reached 14531.36 μg / ml at pH 1.0, indicating that the sample could be rapidly dissolved in the human stomach, making crystalline I suitable for development as an oral solid dosage form. [Example]

[0100] Activity verification of aromatic heterocyclic compounds

[0101] The activities verified for the aromatic heterocyclic compound (produced in Example 91 of the prior application (application number: CN202211019771.6), abbreviated as YZ001052) include JAK3 kinase inhibitory activity and immune activity. See Examples 20A to 20F for details.

[0102] Example 20A: JAK3 Kinase Inhibitory Activity of Aromatic Heterocyclic Compounds (Experiments entrusted to SUNDIA Pharmaceutical Technology (Shanghai) Co., Ltd.)

[0103] Objective of the experiment:

[0104] Using the mobility shift assay, the in vitro inhibitory activity of test compounds (listed below) against JAK3 kinase activity was measured. Cerdulatinib (vendor: selleckchem, product number: S7634) was used as a positive control compound.

[0105] Experimental Method:

[0106] 1.Compound production

[0107] Compounds were dissolved in 100% DMSO to prepare 10 mM stock solutions and stored at -20°C in a refrigerator protected from light.

[0108] 2. Kinase reaction process

[0109] (1) 1x Kinase buffer was prepared.

[0110] (2) Preparation of compound concentration gradient: The test concentration of test compounds (including example compounds and PF-06651600) was 10,000 nM, 10-fold dilutions, 10 concentrations, and single-well measurements. A gradient dilution was performed in a 384-well plate to a 100-fold final concentration solution. Then, 250 nL was transferred to a 384-well reaction plate using an Echo 550 for use. 250 nL of 100% DMSO was added to the negative control wells and positive control wells, respectively.

[0111] (3) A kinase solution was prepared with 1× kinase buffer to a final concentration of 2.5 times.

[0112] (4) 10 μL of a kinase solution at 2.5 times the final concentration was added to each of the compound wells and the positive control wells, and 10 μL of 1× Kinase buffer was added to the negative control wells.

[0113] (5) The mixture was centrifuged at 1000 rpm for 30 seconds, shaken to mix evenly, and then incubated at room temperature for 10 minutes.

[0114] (6) A mixed solution of ATP and Kinase substrate 22 was prepared in 1× Kinase buffer at a final concentration of 25 / 15 times.

[0115] (7) 15 μL of a mixed solution of ATP and substrate at 25 / 15 times final concentration was added to each of the compound wells, positive control wells, and negative control wells of the 384 reaction plate to initiate the reaction.

[0116] (8) The 384-well plate was centrifuged at 1000 rpm for 30 seconds, shaken to mix evenly, and then incubated at room temperature for 30 minutes.

[0117] (9) The kinase reaction was stopped by adding 30 μL of stop detection solution, and the mixture was centrifuged at 1000 rpm for 30 seconds and shaken to mix evenly.

[0118] (10) The conversion rate is read using the Caliper EZ Reader.

[0119] 3. Data Analysis

[0120] (1) Calculation formula %Inhibition=(Conversion%_max-Conversion%_sample) / (Conversion%_max-Conversion%_min)×100

[0121] where Conversion%_sample is the conversion reading of the sample, Conversion%_min is the average value of the negative control wells and represents the conversion reading in the absence of enzyme activity wells, Conversion%_max is the average value of the positive control wells and represents the conversion reading in the absence of compound inhibition wells, and %Inhibition represents the percentage inhibition.

[0122] (2) Fitting dose-effect curves

[0123] The logarithmic value of the concentration was plotted on the X axis and the percentage inhibition rate on the Y axis. The IC50 value for each compound was calculated using the log(inhibitor) vs. response-variable slope fitting dose-effect curve in the analytical software GraphPad Prism 5. The calculation formula was Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50-X) * HillSlope)).

[0124] Test Results:

[0125] [Table 4]

[0126] The data in the above table demonstrate that YZ001052 has superior JAK3 in vitro kinase inhibitory activity to PF-06651600.

[0127] Example 20B Cellular Activity Experiments

[0128] Objective of the experiment:

[0129] Based on the mechanism of the JAK-STAT pathway, hPBMC cells were stimulated with the cytokine IL-15, and the downstream STAT5 phosphorylation level was used as a measurement index to evaluate the effects of compounds on the JAK-STAT pathway.

[0130] Experimental Method:

[0131] 1. Seeding plate for PBMC cell counting: 90 μL of PBMC was seeded into a 96-well plate (PBMC cell density: 80,000 / well).

[0132] 2. Test compound treatment: Immediately after seeding the cells on the plate, the test compound was administered at 5 μL / well (final concentrations: 1, 0.5, 0.1, 0.05, 0.01, 0.005 μM), and the cells were incubated in an incubation tank at 37°C for 45 minutes.

[0133] 3. Cytokine IL-15 stimulation: 5 μL IL-15 stimulation, incubated at 37°C for 30 minutes.

[0134] 4. Protein sample collection: Cells were collected in a centrifuge tube and centrifuged for 5 minutes. After centrifugation, the supernatant was discarded and the cells were digested with 1x Cell Extraction Buffer PTR.

[0135] 5. p-STAT5 measurement: Measurement was performed according to the requirements of the ELISA kit.

[0136] Experimental results: IC50 values ​​of compounds for p-STAT5 under IL-15 stimulation in PBMCs

[0137] [Table 5]

[0138] According to the data in the table above, our compounds have good effects in inhibiting the JAK3-STATs signal pathway at the cellular level, and some compounds have IC values ​​comparable to or lower than those of positive drugs. 50 showed.

[0139] Example 20C Immunosuppressive effect of compounds on delayed allergic reaction (DTH) in mice

[0140] Objective of the experiment:

[0141] The immunosuppressive activity of YZ001052, YZ001054, YZ001065, and YZ001085 was tested in an SRBC mouse model. PF-06651600 was used as a positive control compound. For the structures of YZ001054, YZ001065, and YZ001085, please refer to paragraph 49 of the specification of the prior application (application number CN202211019771.6).

[0142] Experimental Method:

[0143] Drug preparation: The compound was weighed and 0.5% CMC-Na solution was added to prepare a drug suspension (dosage 10 mg / mL).

[0144] Induction of delayed hypersensitivity (DTH) and drug administration: Thirty male Balb / c mice were weighed and randomly divided into six groups based on body weight, with five mice per group. The experimental period was 7 days in total. On day 0, sheep red blood cells (SRBCs) were injected subcutaneously for sensitization. 100 mg / kg (once daily) was administered intragastrically from day 0 to day 6. On day 6, SRBCs were injected into the right hind footpad for stimulation. Measurements and photographs were taken on day 7, and the experiment was terminated.

[0145] Pharmacological measurement parameters of the disease model: The measurement and observation parameters were the degree of redness and thickness of the nail pad of the SRBC-induced model mice. The right nail thickness was measured as a baseline before SRBC injection on day 6. The right nail thickness was measured again at the end of the experiment, and the difference in nail thickness before and after the two injections was calculated.

[0146] Statistical analysis: The experimental data were expressed as mean ± standard deviation (Mean ± SD). All data were analyzed using T-test, and P<0.05 was considered statistically significant. The results are shown in Figure 76.

[0147] Test Results:

[0148] As can be seen from Figure 76, after SRBC induction (model group), the thickness of the right nails of the mice increased significantly, and after drug treatment (including compounds YZ001052, YZ001054, YZ001065, and YZ001085), the thickness of the right nails of the mice all showed improvements to different degrees, indicating that compounds YZ001052, YZ001054, YZ001065, and YZ001085 have significant immunosuppressive effects on the delayed allergic reaction of SRBC-induced mice, and their effectiveness is not inferior to that of PF-06651600.

[0149] Example 20D: Immunosuppressive effects of compounds on collagen-induced murine arthritis model

[0150] 1. Preparation of collagen-induced murine arthritis model (mCIA)

[0151] Experimental Objective: To test the immunosuppressive activity of YZ001052 in an mCIA model. PF-06651600 was used as a positive control compound.

[0152] Experimental Method: Thirty-two female DBA / 1J mice were randomly divided into four groups: a blank control group, a model group, a model group treated with YZ001052, and a model group treated with PF-06651600. After successful establishment of the arthritis model, mice were administered 50 mg / kg of YZ001052 or PF-06651600 once daily. Complete and incomplete Freund's adjuvant were mixed with chicken type II collagen solution to form an emulsion. Except for the blank control group, mice were injected subcutaneously at the base of their tails and thighs with a 100 μL injection at the base of their tails and a 50 μL injection at the base of their thighs on Day 0 (primary immunization). On day 21, the mice received a subcutaneous injection of 200 μL of a mixture of incomplete Freund's adjuvant and chicken type II collagen solution as a booster injection (second immunization). Arthritis was considered to have occurred if at least one mouse had a clinical limb integral score of ≥2. Toenail thickness measurement: The thickness of the left and right hind paws of the mice, i.e., the swelling of the toenails, was measured using a vernier caliper and recorded. Measurements were taken every three days.

[0153] Experimental results: As shown in Figure 77, after the second immunization, inflammation in the toenail area of ​​the model group mice steadily worsened, swelling gradually spread to the entire toenail, and the arthritis score significantly increased on day 16. Meanwhile, after treatment with YZ001052 or PF-06651600, the degree of toenail swelling in the CIA mice improved to different degrees, and the arthritis score significantly decreased (compared to the model group).

[0154] Consistent with the arthritis scores, the mice in the model group showed obvious swelling in their toenails. However, after treatment with YZ001052 or PF-06651600, the swelling of the mice's toenails improved to different degrees. These results demonstrate that YZ001052 or PF-06651600 can improve the symptoms of rheumatoid arthritis.

[0155] Example 20E: Immunosuppressive effects of compounds on dextran sulfate sodium salt (DSS)-induced inflammatory bowel disease model in mice

[0156] Experimental Objective: To test the immunosuppressive activity of YZ001052 in a mouse model of DSS-induced inflammatory bowel disease. PF-06651600 was used as a positive control compound.

[0157] Experimental Method: Thirty-two female C57BL / 6 mice were randomly divided into four groups: a blank control group, a model group, a model group with YZ001052 treatment, and a model group with PF-06651600 treatment. At the start of the model preparation, drugs were administered simultaneously. YZ001052 and PF-06651600 were administered at a dose of 50 mg / kg once daily. 50 g of DSS was weighed and added to 1000 mL of sterile water to prepare a 5% DSS solution, which was then filtered through a 0.22 μm filter membrane. From day 0, the blank group received drinking water without DSS, while the remaining experimental groups received drinking water containing 5% DSS. All mice were sacrificed on day 8. After the experiment, colons and rectums were collected, photographed, and their lengths were measured for statistical analysis.

[0158] Experimental Results: As shown in Figure 78, compared with the normal control group, the colon and rectum length of the model mice was significantly shortened, suggesting that the colon and rectum of the inflammatory bowel disease mice was severely damaged. Compared with the model mice, the colon and rectum length of the YZ001052-treated mice was significantly increased, demonstrating that treatment with YZ001052 can alleviate colon and rectal damage in inflammatory bowel disease mice, with results superior to those of PF-06651600.

[0159] Example 20F Immunosuppressive Effects of Compounds on a Mouse Model of Radiation-Induced Lung Injury

[0160] 1. Preparation of a mouse model of acute radiation-induced lung injury

[0161] (1) Grouping of animals and drug administration

[0162] Eighteen female C57BL / 6 mice were randomly divided into three groups: a blank control group, a simple irradiation group, and an irradiation + drug YZ001052 (30 mg / kg qd) intervention group, each with six mice.

[0163] (2) Model creation method

[0164] Mice were anesthetized with 1% sodium pentobarbital via intraperitoneal injection and irradiated with 22.5 Gy of 220 KV X-rays to the whole lungs in a single dose at the Small Animal Precision Radiotherapy Research Platform (SARRP). After irradiation, mice were maintained under normal conditions.

[0165] 2. Index test

[0166] Three weeks after model preparation, mice were anesthetized, lung tissues were dissected and exposed, the left lung was ligated, an open tracheotomy was performed, the lungs were lavaged with cold PBS, and bronchoalveolar lavage fluid (BALF) was collected and cryocentrifuged at 4°C.

[0167] After collecting the supernatant, the content of the inflammatory factor TNF-α was measured by ELISA. As shown in Figure 79, after irradiation induction, the TNF-α level in the alveolar lavage fluid of the model group mice was significantly elevated. The drug YZ001052 has a good inhibitory effect on the level of TNF-α.

[0168] The alveolar lavage fluid was resuspended in 200 μL of PBS to precipitate the cells, and then used for white blood cell (WBC) counting. As shown in Figure 80, after irradiation induction, the number of white blood cells in the alveolar lavage fluid of the model group mice was significantly increased. The drug YZ001052 can significantly reduce the number of inflammatory cell infiltration in the lungs of mice caused by irradiation.

Claims

1. A crystalline form of an aromatic heterocyclic compound, wherein the structural formula of the aromatic heterocyclic compound is as follows: 【Chemical 1】 The crystalline form is as follows: Crystalline Form I exhibits characteristic diffraction peaks at 2θ angles of 8.4°, 10.0°, 13.6°, 16.4°, and 19.7° using an X-ray diffraction method. Crystalline Form III has characteristic diffraction peaks at 2θ angles of 8.2°, 9.8°, 17.2°, and 26.8° using X-ray diffraction. Here, the crystalline form of the aromatic heterocyclic compound is characterized in that the error range of the 2θ angle is ±0.2°.

2. The crystalline form I has characteristic diffraction peaks at 2θ angles of 15.2°, 18.1°, and 24.1° using an X-ray diffraction method. The crystalline form of claim 1, wherein the error range of the 2θ angle is ±0.2°.

3. A composition comprising one or two of crystalline I and crystalline III according to claim 1 or 2, and wherein the proportion of crystalline I and / or crystalline III in the total weight of the composition is 50% or more.

4. 4. The composition according to claim 3, wherein the proportion of Crystalline I and / or Crystalline III in the total weight of the composition is 80% or more.

5. 4. The composition according to claim 3, wherein the proportion of Crystalline I and / or Crystalline III in the total weight of the composition is 90% or more.

6. A pharmaceutical composition comprising one or both of Crystalline Form I and Crystalline Form III according to claim 1 or 2 and a pharmaceutically acceptable carrier or excipient.

7. 3. The method for producing Crystalline Form I according to claim 1 or 2, wherein the method comprises adding a solvent to the aromatic heterocyclic compound to form a suspension, stirring the suspension at room temperature, filtering, and drying to obtain Crystalline Form I, the solvent being one or a combination of two or more of acetone, methanol, ethanol, water, ethyl acetate, toluene, methyl tert-butyl ether, and n-heptane; or the method comprises adding a first organic solvent to the aromatic heterocyclic compound, dissolving the compound by heating, adding a second organic solvent, stirring to precipitate crystals, filtering, and drying to obtain Crystalline Form I, the first organic solvent being one or a combination of two or more of methanol, ethanol, dichloromethane, trichloromethane, and dimethyl sulfoxide, and the second organic solvent being one or a combination of two or more of isopropyl ether, n-hexane, n-heptane, methyl tert-butyl ether, and water.

8. The method for producing crystalline III according to claim 1, comprising adding a first organic solvent to the aromatic heterocyclic compound, dissolving the compound by heating, adding a second organic solvent, and subsequently stirring to precipitate crystals, followed by filtering and drying; the first organic solvent is one or a combination of two or more of methanol, ethanol, tetrahydrofuran, 1,4-dioxane, and acetonitrile; and the second organic solvent is one or a combination of two or more of water and ethyl acetate.

9. Use of the crystalline form of claim 1 or 2 in the manufacture of a drug for the prevention or treatment of a disease caused by an abnormality in the JAK-STAT signal pathway.

10. The use according to claim 9, wherein the abnormality of the JAK-STAT signal pathway refers to the overactivation or overexpression of JAK3 kinase.

11. 10. The use according to claim 9, wherein the disease is selected from one or more of the following: an autoimmune disease, a cancer, a myeloproliferative disorder.

12. 12. The application of claim 11, wherein the autoimmune disease is one or more selected from the group consisting of alopecia areata, lupus, multiple sclerosis, amyotrophic lateral sclerosis, rheumatoid arthritis, rheumatoid arthritis, psoriasis, organ transplant complications, atopic dermatitis, autoimmune thyroid disease, ulcerative colitis, Crohn's disease, Sjogren's syndrome, vitiligo, autoimmune kidney injury, autoimmune liver injury, and chronic obstructive pulmonary disease; the cancer is one or more selected from the group consisting of colon cancer, gastric adenocarcinoma, bladder cancer, breast cancer, kidney cancer, liver cancer, lung cancer, thyroid cancer, head and neck cancer, prostate cancer, pancreatic cancer, cancer of the CNS (central nervous system), and malignant glioma; and the myeloproliferative disorder is one or more selected from the group consisting of chronic myelomonocytic leukemia, atypical chronic myeloid leukemia, and juvenile myelomonocytic leukemia.

13. The application of claim 11, wherein the autoimmune disease is radiation lung injury, and the lung injury refers to acute radiation pneumonitis and radiation pulmonary fibrosis.

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