Crystalline forms of aromatic heterocyclic compounds, their compositions, methods of production, and applications 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 pharmaceutical chemistry, and specifically relates to a crystal form of an aromatic heterocyclic compound that inhibits the activity of Janus kinase (JAK), a composition thereof, a manufacturing method, and its application.

Background Art

[0002] Janus kinase (JAK) is a cytoplasmic tyrosine protein kinase that is responsible for the transmission of many inflammation-related cytokine signals from cytokine membrane receptors to STAT transcription factors. Abnormal JAK / STAT signal transduction is related to many diseases, and is involved in diseases related to immune inflammation, such as organ transplant rejection, multiple sclerosis, rheumatoid arthritis, type I diabetes, lupus, psoriasis, asthma, food allergy, atopic dermatitis and rhinitis, skin rash, etc. There are also reports closely related to the occurrence and development of solid and hematological malignancies and myeloproliferative disorders (including lung cancer, breast cancer, chronic idiopathic myelofibrosis, polycythemia vera, essential thrombocythemia, etc.).

[0003] JAK3 is mainly expressed in various hematopoietic tissue cells, including bone marrow cells, thymocytes, NK cells, activated B lymphocytes, T lymphocytes, etc. Since its physiological effect only comes from the signal transduction process of the common γ cytokine receptor family, acting highly selectively on JAK3 kinase can avoid unnecessary side effects. Therefore, the activity and selectivity of JAK3 kinase inhibitors can be enhanced, and the clinical use effect of JAK3 kinase inhibitors can be further enhanced, which has significant clinical advantages compared with the currently clinically applied Pan-JAK inhibitors and selective JAK1 and JAK2 inhibitors.

[0004]

Chemical Formula

[0005] In a prior application (application number CN202211019771.6), the applicant studied the JAK3 kinase inhibitory activity and immunosuppressive activity of an aromatic heterocyclic compound [Chemical Formula 1] (see paragraphs 425-510 and Figure 1-5 of the specification). This aromatic heterocyclic compound has good JAK3 kinase inhibitory activity and also exhibits a remarkable immunosuppressive effect against delayed allergic reactions in SRBC-induced mice, can improve symptoms of rheumatoid arthritis, can alleviate colorectal injury in mice with inflammatory bowel disease, and can significantly reduce the number of inflammatory cells infiltrating the lungs of mice induced by irradiation.

[0006] The phenomenon of polycrystalline form of drugs is a crucial factor influencing drug quality and clinical efficacy. Different crystalline forms lead to differences in stability, absorption, and bioavailability, thereby affecting the drug's clinical efficacy. Therefore, the crystalline form of this aromatic heterocyclic compound and its manufacturing technology are of great importance to the application of this drug. [Overview of the Initiative]

[0007] The object of the present invention is to provide a crystalline form of an aromatic heterocyclic compound, its composition, a method for producing it, and its applications. This crystalline form of aromatic heterocyclic compound can be used as a JAK kinase inhibitor for the treatment and prevention of clinical applications related to abnormal kinase activity, including autoimmune diseases, cancer, myeloproliferative disorders, and other diseases.

[0008] This invention provides the following structural formulas for aromatic heterocyclic compounds.

[0009] [ka]

[0010] The aforementioned aromatic heterocyclic compound can have various different crystalline forms.

[0011] This is a crystalline form I of an aromatic heterocyclic compound, and X-ray diffraction reveals characteristic diffraction peaks at 2θ angles of approximately 8.4°, 10.0°, 13.6°, 16.4°, and 19.7°.

[0012] This is the crystalline form II of an aromatic heterocyclic compound, and X-ray diffraction reveals characteristic diffraction peaks at 2θ angles of approximately 7.6°, 9.9°, 15.1°, 16.1°, and 19.7°.

[0013] This is a crystalline form III of an aromatic heterocyclic compound, and using X-ray diffraction, characteristic diffraction peaks are observed at 2θ angles of approximately 8.2°, 9.8°, 17.2°, and 26.8°.

[0014] This is a crystalline form IV of an aromatic heterocyclic compound, and X-ray diffraction reveals characteristic diffraction peaks at 2θ angles of approximately 7.9°, 9.4°, 15.7°, and 19.2°.

[0015] This is a crystalline form V of an aromatic heterocyclic compound, and using X-ray diffraction, characteristic diffraction peaks are observed at 2θ angles of approximately 12.8°, 13.0°, 17.2°, and 22.8°.

[0016] This is a crystalline form VI of an aromatic heterocyclic compound, which exhibits characteristic diffraction peaks at 2θ angles of approximately 7.7°, 9.2°, and 19.7°, as determined by X-ray diffraction.

[0017] This is a crystalline form VII of an aromatic heterocyclic compound, and X-ray diffraction reveals characteristic diffraction peaks at 2θ angles of approximately 7.9°, 8.3°, and 9.9°.

[0018] This aromatic heterocyclic compound is in crystalline form VIII, and X-ray diffraction reveals characteristic diffraction peaks at 2θ angles of approximately 8.0°, 9.6°, 18.8°, and 19.2°.

[0019] This is crystalline form IX of an aromatic heterocyclic compound, and using X-ray diffraction, characteristic diffraction peaks are observed at 2θ angles of approximately 8.1° and 9.6°, 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 approximately 15.2°, 18.1°, 24.1°, and 26.9° using X-ray diffraction method; the crystalline form IV has characteristic diffraction peaks at 2θ angles of approximately 16.6° and 18.6° using X-ray diffraction method; the crystalline form V has characteristic diffraction peaks at 2θ angles of approximately 18.6° and 20.2° using X-ray diffraction method; the crystalline form VII has characteristic diffraction peaks at 2θ angles of approximately 19.7°, 25.1°, and 33.5° using X-ray diffraction method, where the error range of the 2θ angle is ±0.2°.

[0021] Furthermore, the crystalline form I has the characteristics of the X-ray diffraction pattern shown in FIG. 1, the crystalline form II has the characteristics of the X-ray diffraction pattern shown in FIG. 8, the crystalline form III has the characteristics of the X-ray diffraction pattern shown in FIG. 17, the crystalline form IV has the characteristics of the X-ray diffraction pattern shown in FIG. 25, the crystalline form V has the characteristics of the X-ray diffraction pattern shown in FIG. 29, the crystalline form VI has the characteristics of the X-ray diffraction pattern shown in FIG. 35, the crystalline form VII has the characteristics of the X-ray diffraction pattern shown in FIG. 42, the crystalline form VIII has the characteristics of the X-ray diffraction pattern shown in FIG. 51, and the crystalline form IX has the characteristics of the X-ray diffraction pattern shown in FIG. 60.

[0022] Furthermore, the melting point of the crystalline form I is about 196°C. The melting point of the crystalline form II is about 184.4°C. The melting point of the crystalline form III is about 201°C. The melting point of the crystalline form IV is about 202°C. The melting point of the crystalline form V is about 223°C. The melting point of the crystalline form VI is about 202°C. The melting point of the crystalline form VII is about 194°C. The melting point of the 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 for the treatment and prevention of clinical applications related to abnormal kinase activities, including autoimmune diseases, inflammatory diseases, cancers, and other diseases, as JAK kinase inhibitors.

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

[0025] Another object of the present invention is to provide a combination of crystalline forms of an aromatic heterocyclic compound that can be used as a JAK kinase inhibitor for the treatment and prevention of clinical applications related to these kinase activity abnormalities, including autoimmune diseases, inflammatory diseases, cancer, and other diseases.

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

[0027] Another object of the present invention is to provide a combination of crystalline forms of an aromatic heterocyclic compound that can be used as a JAK kinase inhibitor for the treatment and prevention of clinical applications related to these kinase activity abnormalities, including autoimmune diseases, inflammatory diseases, cancer, and other diseases.

[0028] A crystalline aromatic heterocyclic compound composition comprising one or more combinations of crystalline forms I, II, III, IV, V, VI, VII, VIII, and IX, wherein crystalline form I and / or crystalline form III constitute 90% or more of the 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 crystalline form III constitute 90% or more of the weight of the composition.

[0029] Another object of the present invention is to provide a crystalline drug composition for the treatment and prevention of clinical applications related to abnormal kinase activity, including autoimmune diseases, inflammatory diseases, cancer, and other diseases.

[0030] A drug composition comprising the crystalline I and / or crystalline III, and pharmaceutically acceptable excipients.

[0031] Another object of the present invention is to provide a method for producing crystalline form I, which involves 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 more of the following: acetone, methanol, ethanol, water, ethyl acetate, toluene, methyl tert-butyl ether, and n-heptane, and the amount of organic solvent used is 3 to 50 times the weight of the aromatic heterocyclic compound.

[0033] Preferably, the method for producing crystalline form I involves adding a solvent to the aromatic heterocyclic compound to form a suspension, stirring at room temperature, filtering, and drying to obtain a white solid crystalline form I, wherein the solvent is selected from water and ethanol. The required solvent volume 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 by adding a solvent to the aromatic heterocyclic compound, dissolving and clarifying it, and then allowing it to volatilize and dry.

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

[0036] Another object of the present invention is to provide a method for producing crystalline form I, which involves adding a first organic solvent to the aromatic heterocyclic compound, heating and dissolving it, then adding a second organic solvent, subsequently stirring to precipitate crystals, and then filtering and drying the mixture.

[0037] Furthermore, the first organic solvent is one or more of methanol, ethanol, dichloromethane, trichloromethane, and dimethyl sulfoxide, and 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 more of isopropyl ether, n-hexane, n-heptane, methyl tert-butyl ether, and water, and 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, and 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, and more preferably 5 to 6:1.

[0038] Another object of the present invention is to provide a method for producing crystalline form III by adding a first organic solvent to the aromatic heterocyclic compound, heating and dissolving it, then adding a second organic solvent, subsequently stirring to precipitate crystals, and then filtering and drying.

[0039] Furthermore, when producing crystalline form III, the first organic solvent is one 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 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 producing 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 the above-mentioned crystalline properties in the manufacture of drugs for the prevention or treatment of diseases caused by abnormalities in the JAK-STAT signaling pathway. These drugs can be used for autoimmune diseases, cancer, 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 injury, autoimmune liver injury, 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, CNS (central nervous system) cancer, and malignant glioma; and the myeloproliferative disorder is one or more selected from chronic myelomonocytic leukemia, atypical chronic myeloid leukemia, and juvenile myelomonocytic leukemia.

[0043] The aromatic heterocyclic compounds described in this invention exist in various crystalline forms. When researchers studied the crystalline forms of the compounds using a hot stage XRPD, they discovered that at 155°C, the sample transforms into another thermodynamically more stable anhydrous crystalline form. We define this crystalline form as crystalline form I, and simultaneously discover a total of eight other crystalline forms: 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. Of these, crystalline forms I and III are anhydrous crystalline forms. As can be seen from this, the aromatic heterocyclic compounds described in this invention exhibit crystalline polymorphism, and many crystalline forms are solvates or hydrates. Since anhydrous crystalline forms cannot be obtained under many manufacturing conditions, the production of anhydrous crystalline forms I and III presents significant technical difficulties. Solvates or hydrates have the problem of being prone to crystallization transitions due to desolvation during storage, resulting in different crystalline form composition ratios for compounds at different storage times, which can lead to unstable compound quality. Crystallized forms I and III provided by the present invention are anhydrous crystalline forms, and do not undergo crystallization transitions during storage, thus overcoming the above-mentioned defects of the compounds.

[0044] The researchers compared the nine crystal forms using methods such as hot-stage XRPD, TG, DSC, and DVS. Hot-stage XRPD analysis revealed that most crystal forms converted to crystal form I at 150-170°C, and TG and DSC analysis showed that crystal form I was the most stable during the heating process. DVS and isothermal curves showed the structural stability of crystalline form I under desorption and adsorption conditions.

[0045] Simultaneously, the researchers conducted competitive tests of the aforementioned crystalline forms in water and ethanol, examining the stability and transformation status of each crystalline form in solvents. The competitive experiments revealed that crystalline forms I and III exhibited good crystalline stability in water, while the other crystalline forms were unstable in water. Since water is the most commonly used solvent in drug formulations, and solid powders readily absorb water when exposed to air, crystalline forms I and III provided by this patent possess good stability in the field of drug applications and are suitable for development as medicinal crystalline forms. Crystalline form I exhibits good crystalline stability in the presence of ethanol. Ethanol has extensive applications as a drug solvent and in the manufacturing process of drug formulations, and the experiments described above demonstrated that crystalline form I has a significant stability advantage in the development of drug formulations in the presence of ethanol.

[0046] The researchers conducted a 15-day high-temperature, high-humidity test on crystalline form I, followed by long-term and accelerated 3-month tests. All test results were for crystalline form I, and further demonstrated the storage stability of crystalline form I.

[0047] Simultaneously, researchers conducted solubility tests of crystalline form I at different pH values ​​and showed that solubility significantly increased as the pH value decreased, demonstrating that the sample could rapidly dissolve in the human stomach. [Brief explanation of the drawing]

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

[0049] To better illustrate the present invention, it will be further described below in relation to preferred embodiments. Those skilled in the art should understand that the following descriptions are illustrative and not restrictive, and should not limit the scope of protection of the present invention.

[0050] All numerical specifications in this invention (including their respective ranges, such as temperature, time, concentration, and weight) are typically appropriate as approximations, changing (+) or (-) in increments of 0.1 or 1.0. All numerical specifications can be understood as being preceded by the term "approximately". [Examples]

[0051] (Comparative example): Aromatic heterocyclic compound

[0052] The aromatic heterocyclic compound was manufactured by referring to the manufacturing 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 (25 g, 1.04 mol), and methyl iodide (73 g, 0.52 mol) were sequentially added to a 2000 mL three-necked flask containing 1000 mL of DMF, and the reaction system was allowed to react with sufficient stirring at 0°C for 2 hours. After the reaction was complete, the reaction mixture was poured into water, extracted three times with dichloromethane, the organic layer was combined, washed twice with saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the resulting crude product was purified by silica gel column chromatography to obtain intermediate IM1, which was 100 g of yellow solid, with a yield of 63.3%.

[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-necked flask containing 1000 ml of 1,4-dioxane. The reaction system was left at 95°C overnight with thorough stirring. After the reaction was complete, the mixture was cooled to room temperature, the reaction solution was poured into 1000 ml of water, extracted three times with ethyl acetate, the organic layers were combined, washed twice with saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the resulting crude product was purified by silica gel column chromatography to obtain intermediate IM2, which was 50 g of pale yellow solid, with a yield of 44.3%.

[0056] Step 3: Dissolve intermediate IM2 (50 g, 0.14 mol) in dichloromethane, add an equal volume of trifluoroacetic acid, and react overnight at room temperature. After the reaction is complete, adjust the pH to 8-9, extract three times with dichloromethane, combine the organic layers, wash twice with saturated sodium chloride, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and purify the resulting crude product by silica gel column chromatography to obtain intermediate IM3, which is 20 g of a white solid, with a yield of 56.4%.

[0057] Step 4: Dissolve intermediate IM3 (20 g, 78.9 mmol) in 4000 ml of dichloromethane, add triethylamine (24.5 g, 243 mmol), slowly add acryloyl chloride (8.66 g, 95 mmol) under ice bath, react at room temperature for 3 hours, then add saturated sodium bicarbonate aqueous solution, wash and extract three times with dichloromethane, combine the organic layers, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and purify the resulting crude product by silica gel column chromatography using dichloromethane and methanol as developing agents to obtain 15 g of the target 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] According to the X-ray diffraction pattern of the obtained white solid, characteristic diffraction peaks were observed at 2θ angles of 8.11° and 9.62° (see Figures 60 and 61). TGA showed a weight loss of 4.2% before 130°C and 3.7% before 130°C to 210°C. The sample decomposed at 361°C (see Figure 62). DSC showed a desolvation peak from 20°C to 140°C, with an endothermic peak at 171°C, and a melting point of 199.8°C (see Figure 63). PLM showed the sample was a sheet-like crystal (see Figure 64). Hot-stage XRD showed a transition to crystalline I at 155°C and remained crystalline I even at room temperature (see Figure 65 for two XRD patterns marked at 155°C and RT respectively) (in the figure, Form 1 is crystalline I and is produced in Example 4). 1 According to the 1H-NMR diagram, the peaking regions are water and methanol peaks (see Figure 66), indicating that it is a hydrate, and the 1-(1-methyl-3-(1H-pyrroly[2,3-b]pyridine-4-yl)-1,4,6,7-tetrahydro-5H-pyrazoly[4,3-c]pyridine-5-yl)-2-en-1-one obtained by the above production method is crystal form IX.

[0059] As can be seen from this, crystal form IX is not an anhydrous crystal form, and may be prone to crystal form transformation during storage, potentially leading to unstable quality with long-term storage. [Examples]

[0060] 20 mg of the aromatic heterocyclic compound prepared in Example 1 was weighed, 4 ml of methanol and 2 ml of chloroform were added, and the mixture was dissolved and clarified using sonication. The mixture was then opened and dried at room temperature to obtain a solid.

[0061] The X-ray diffraction pattern of the obtained solid indicated that it was crystal form IX. [Examples]

[0062] 10 mg of the aromatic heterocyclic compound prepared in Example 1 was weighed, 4 ml of methanol was added, and the mixture was dissolved and clarified using ultrasound. The mixture was then opened and dried at room temperature to obtain a solid.

[0063] The X-ray diffraction pattern of the obtained solid indicated that it was crystal form IX. [Examples]

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

[0065] According to the X-ray diffraction pattern of the obtained white solid, characteristic diffraction peaks were observed 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 indicated a weight loss of 0.93% before reaching 150°C, the sample decomposed at 354°C (see Figure 3), and it was anhydrous. DSC indicated a melting point of approximately 196°C (see Figure 4). DVS and isothermal curves showed a moisture absorption of approximately 2.4% (see Figures 5 and 6), the desorption and adsorption curves almost overlapped, indicating no change in structure before and after the desorption. PLM indicated the sample was fine particles (see Figure 7), and the white solid obtained by the above manufacturing method is crystalline I. [Examples]

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

[0067] According to the X-ray diffraction pattern of the obtained white solid, characteristic diffraction peaks were observed 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 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 approximately 184.4°C (see Figure 11). According to DVS and isothermal curves, the moisture absorption was approximately 2.4% (see Figures 12 and 13). According to PLM, the sample was sheet-like crystal (see Figure 14). According to hot-stage XRPD, it transitioned to crystalline state I at 150°C and remained crystalline state I even at room temperature (see Figure 15). 1 According to the 1H-NMR diagram, the peaking region is a water peak (see Figure 16), which is a hydrate, and the white solid obtained by the above manufacturing method is crystalline II. [Examples]

[0068] 20 mg of the aromatic heterocyclic compound prepared in Example 1 and 3 ml of ethanol were weighed, dissolved by stirring, and clarified. The mixture was filtered, 16 ml of water was added dropwise to the filtrate, and the mixture was cooled to 4°C and stirred overnight to precipitate crystals. The mixture was then 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). According to TGA, the weight loss was 2.38% at 125°C, and the sample decomposed at 359°C (see Figure 19). According to DSC, the desolvation peak was between 20°C and 140°C, with an endothermic peak at 190°C, and the melting point was approximately 201°C (see Figure 20). According to DVS and isothermal curves, the moisture absorption was approximately 2.2% (see Figures 21 and 22). According to PLM, the sample was fine particles (see Figure 23), and according to hot-stage XRPD, there was no crystal form transition at 140°C (see Figure 24). The white solid obtained by the above manufacturing method is crystalline III. [Examples]

[0070] 20 mg of the aromatic heterocyclic compound prepared in Example 1 and 3 ml of methanol were weighed, dissolved by stirring, and clarified. The mixture was filtered, 16 ml of water was added dropwise to the filtrate, and the mixture was cooled to 4°C and stirred overnight to precipitate crystals. The mixture was then 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 indicated that it was of crystal form III. [Examples]

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

[0073] According to the X-ray diffraction pattern of the obtained white solid, characteristic diffraction peaks were observed 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 the sample contained 0.5 parts of dichloromethane solvide (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 crystal form IV. [Examples]

[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 using ultrasound. The mixture was then filtered, and the filtrate was left open at 40°C to allow it to volatilize and dry. The mixture was then polished to obtain a white solid.

[0075] According to the X-ray diffraction pattern of the obtained white solid, characteristic diffraction peaks were observed 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). According to TGA, there was a weight loss of 9.65% before reaching 125°C, and the sample decomposed at 354°C (see Figure 31). According to DSC, the desolvation peak was at 85°C to 140°C, and the melting point was approximately 223°C (see Figure 32). According to PLM, the sample was fine particle (see Figure 33). 1 According to the 1H-NMR diagram, the peaking region is the 1,4-dioxane peak (see Figure 34), and the amount of 1,4-dioxane solvate is 0.5 parts. The white solid obtained by the above manufacturing method is crystalline V. [Examples]

[0076] 20 mg of the aromatic heterocyclic compound prepared in Example 1 was weighed, 3 ml of methanol was added to dissolve and clarify, 16 ml of isopropyl ether was added to precipitate the solid, centrifuged, and the resulting solid was dried at 45°C to obtain a white solid.

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

[0078] 10 mg of the aromatic heterocyclic compound prepared in Example 1 was weighed, 4 ml of n-butanol and 5 ml of dichloromethane were added, the mixture was dissolved and clarified using ultrasound, filtered, and the filtrate was left open at 40°C to allow it to volatilize and dry. The mixture was then polished to obtain a white solid.

[0079] According to the X-ray diffraction pattern of the obtained white solid, characteristic diffraction peaks were observed 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 reaching 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 indicated the sample was a block-like crystal (see Figure 39), and hot-stage XRPD showed a transition to crystalline state I at 150°C, remaining crystalline state I even at room temperature (see Figure 40). 1 According to the 1H-NMR spectrum, the peaking region is the n-butanol peak (see Figure 41), and the amount of n-butanol solvate is 0.5 parts. The white solid obtained by the above manufacturing method is crystalline VI. [Examples]

[0080] 10 mg of the aromatic heterocyclic compound prepared in Example 1 was weighed and added to 4 ml of tetrahydrofuran. The mixture was dissolved and clarified using ultrasound, filtered, and the filtrate was dried by volatilization at 50°C with the opening of the lid open. The filtrate was then polished to obtain a white solid.

[0081] According to the X-ray diffraction pattern of the obtained white solid, characteristic diffraction peaks were observed 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 reaching 200°C, and the sample decomposed 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 isothermal curves indicated a moisture absorption of approximately 1.3% (see Figures 46 and 47). PLM indicated the sample was sheet-like crystalline (see Figure 48). Hot-stage XRPD showed a transition to crystalline I at 150°C, and remained crystalline I even at room temperature (see Figure 49). 1 According to the 1H-NMR diagram, the peaking region is a water peak (see Figure 50), and it is a monohydrate. The white solid obtained by the above manufacturing method is crystalline VII. [Examples]

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

[0083] According to the X-ray diffraction pattern of the obtained white solid, characteristic diffraction peaks were observed 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 4.0% before 125°C to 145°C. The sample decomposed at 362°C (see Figure 53). DSC showed desolvation peaks at 15°C to 130°C and 130°C to 170°C, an endothermic peak at 181°C, and a melting point of approximately 204°C (see Figure 54). DVS and isothermal curves indicated a moisture absorption of approximately 1.3% (see Figures 55 and 56). PLM indicated the sample was a blocky crystalline form (see Figure 57). Hot-stage XRPD showed that most of the crystalline form transitioned to crystalline form I at 150°C (see Figure 58). 1 According to the 1H-NMR diagram, the peaking regions are water peaks and solvent acetonitrile peaks (see Figure 59), indicating a hydrate, and the white solid obtained by the above manufacturing method is crystalline VIII. [Examples]

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

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

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

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

[0088] Two mg each (18 mg total) of aromatic heterocyclic compounds of crystal forms I, II, III, IV, V, VI, VII, VIII, and IX were taken, and 3 ml of water was added to form suspensions. These suspensions were stirred at room temperature for 4 and 7 days, and samples were taken on the 4th and 7th days, respectively. X-ray diffraction analysis was performed, and the results showed that the compounds were crystal forms I and III, with no significant changes observed between the 4th and 7th days.

[0089] The above was a competitive experiment on crystal forms, demonstrating that crystal forms I and III exhibit good crystal stability in water, while the other crystal forms are unstable in water. Since water is the most commonly used solvent in drug formulations, and solid powders readily absorb water when exposed to air, crystal forms I and III provided by this patent have good stability in the field of drug applications and are suitable for development as medicinal crystal forms. [Examples]

[0090] Two mg each of aromatic heterocyclic compounds of crystal forms I, II, III, IV, V, VI, VII, VIII, and IX were weighed out, and 3 ml of anhydrous ethanol was added to form a suspension. The suspension was stirred at room temperature for four days, and samples were taken and X-ray diffraction measurements were performed. The results showed that the compound was of crystal form I, and the characteristic peaks of the other crystal forms had disappeared.

[0091] The above is a crystal form competition experiment (ethanol stimulation), which showed that crystal form I has good crystal form stability in the presence of ethanol. Ethanol has a wide range of applications as a drug solvent and in the manufacturing process of drug formulations, and the above experiment showed that crystal form I has a significant stability advantage in the development of drug formulations in the presence of ethanol. [Examples]

[0092] Samples of crystalline form I prepared in Example 15 were taken, and 15 samples totaling 50 mg each were weighed and sealed in aluminum foil bags. The stability of the crystalline form of each sample was studied under the following conditions: high temperature (60±2℃), high humidity (RH90%±5%), long-term test (25℃±2℃, RH60%±5%), and accelerated test (40℃±2℃, RH75%±5%). The test results showed that the crystalline form remained stable for 15 days under high temperature and high humidity conditions (see Figures 67-70 for details of the XRD diagram for 15 days under high temperature and high humidity conditions). The crystalline form remained stable under long-term and 3-month accelerated conditions (see Figures 71-74 for details of the XRD diagrams for long-term and 3-month accelerated conditions). The influencing factors and the results of the stability tests clearly demonstrated that crystalline form I can be stored stably.

[0093] Crystalline form measurement results of the influence factor test

[0094] [Table 1]

[0095] Crystal form measurement results from stability tests

[0096] [Table 2] [Examples]

[0097] Ten mL of solutions with different pH values ​​were taken, an excess of the sample in crystalline form I was added, and the saturated solutions (with undissolved solids at the bottom) were placed in a constant temperature rocker at 32°C and vibrated at a rotation speed of 100 rpm for 24 hours. After centrifugal filtration, the content of the saturated solutions at different pH values ​​was measured in linear solutions of different concentrations (prepared with the control sample), and the solubility of the samples at different pH values ​​was calculated using a standard curve. The results are as follows; please refer to Figure 75 for details.

[0098] [Table 3]

[0099] The test results showed a significant improvement in solubility as the pH value decreased. In particular, the solubility of crystalline I reached 14531.36 μg / ml under pH 1.0 conditions, indicating that the sample can rapidly dissolve in the human stomach, making crystalline I suitable for development as an oral solid dosage form. [Examples]

[0100] Activity verification of aromatic heterocyclic compounds

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

[0102] Example 20A: JAK3 kinase inhibitory activity of aromatic heterocyclic compounds (Experiments conducted by SUNDIA Pharmaceutical Technology (Shanghai) Co., Ltd.)

[0103] Experimental objective:

[0104] The in vitro inhibitory activity of the test compounds (listed below) against JAK3 kinase activity was measured using the Mobility Shift Assay. Cerdulatinib (vendor: selleckchem, product number: S7634) was used as the positive control compound.

[0105] Experimental method:

[0106] 1.Compound production

[0107] The compound was dissolved in 100% DMSO to prepare a 10 mM stock solution, which was stored in a refrigerator at -20°C, protected from light.

[0108] 2. Kinase reaction process

[0109] (1) Prepared 1× Kinase buffer.

[0110] (2) Preparation of compound concentration gradient: The test concentration of the test compounds (including the example compound and PF-06651600) was 10,000 nM, 10-fold dilution, 10 concentrations, and measured in single wells. A gradient dilution was performed in a 384-well plate to a final concentration solution 100-fold. Then, 250 nL was transferred to the 384 reaction plate using Echo550 for use. 250 nL of 100% DMSO was added to the negative control well and the positive control well, respectively.

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

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

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

[0114] (6) A mixed solution of ATP and Kinase substrate 22 was prepared using 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 the final concentration was added to each of the 384 reaction plate compound wells, positive control well, and negative control well, and the reaction was started.

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

[0117] (9) Add 30 μL of termination detection solution to stop the kinase reaction, centrifuge at 1000 rpm for 30 seconds, and shake to mix uniformly.

[0118] (10) Read the conversion rate using Caliper EZ Reader.

[0119] 3. Data Analysis

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

[0121] Here, Conversion%_sample is the sample conversion rate, Conversion%_min is the mean value of the negative control wells and represents the conversion rate without enzyme activity wells, Conversion%_max is the mean value of the positive control wells and represents the conversion rate without compound inhibition wells, and %Inhibition represents the percentage inhibition rate.

[0122] (2) Fitting dose-effect curve

[0123] The logarithm of the concentration was plotted on the X-axis, and the percentage inhibition rate on the Y-axis. The IC50 values ​​for the enzyme activity of each compound were obtained using the log(inhibitor)vs.response-Variable slope fitting dose-effect curve in the analysis software GraphPad Prism 5. The calculation formula is Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50 - X) * HillSlope)).

[0124] Experimental results:

[0125] [Table 4]

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

[0127] Example 20B: Cell activity experiment

[0128] Experimental objective:

[0129] Based on the mechanism of the JAK-STAT pathway, hPBMC cells were stimulated using the cytokine IL-15, and the effects of compounds on the JAK-STAT pathway were evaluated using downstream STAT5 phosphorylation levels as a measurement indicator.

[0130] Experimental method:

[0131] 1. Inoculation plate for PBMC cell counting: 90 μL of PBMCs were inoculated into a 96-well plate (PBMC cell density 80,000 / well).

[0132] 2. Treatment with test compounds: Immediately after planting cells in plates, drug administration was performed. 5 μL / well of the target drug (final concentrations of 1, 0.5, 0.1, 0.05, 0.01, and 0.005 μM) was incubated in a 37°C incubation tank for 45 minutes.

[0133] 3. Cytokine IL-15 stimulation: 5 μL of IL-15 was administered and 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 cleaved with 1× 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 p-STAT5 in PBMC of compounds under IL-15 stimulation.

[0137] [Table 5]

[0138] According to the data in the table above, our compounds exhibit good inhibitory effects on the JAK3-STATs signaling pathway at the cellular level, and some compounds have equivalent or lower IC50 than positive drugs. 50 This was shown.

[0139] Example 20C: Immunosuppressive effect of the compound on delayed-to-thoracic allergic reaction (DTH) in mice.

[0140] Experimental objective:

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

[0142] Experimental method:

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

[0144] Induction of delayed-tolerant reactions (DTH) and drug administration: Thirty male Balb / c mice were weighed and randomly divided into six groups based on body weight, with each group consisting of five mice. The experiment lasted a total of seven days. On day 0, sheep red blood cells (SRBCs) were subcutaneously injected to induce sensitization. 100 mg / kg (once daily) was administered intragastricly from day 0 to day 6. On day 6, SRBCs were injected into the right hind leg pad of the mice to induce excitation. Measurements were taken and photographs were taken on day 7 of the experiment, and the experiment was concluded.

[0145] Pharmacological measurement indicators for the disease model: The measurement and observation indicators were the degree of erythromatosis and thickness of the nail pads of SRBC-induced model mice. The thickness of the right nail was measured as baseline before SRBC injection excitation on day 6. The thickness of the right nail 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: Experimental data were statistically analyzed using the mean ± standard deviation (Mean ± SD). All data were statistically analyzed using a t-test, with P < 0.05 considered a statistically significant difference. See Figure 76 for results.

[0147] Experimental results:

[0148] As is clear from Figure 76, the thickness of the right claw of mice after SRBC induction (model group) increased significantly, and the thickness of the right claw of mice treated with drugs (containing compounds YZ001052, YZ001054, YZ001065, and YZ001085) all showed varying degrees of improvement. This indicates that compounds YZ001052, YZ001054, YZ001065, and YZ001085 have a significant immunosuppressive effect on delayed allergic reactions in SRBC-induced mice, and that their effect is not inferior to that of PF-06651600.

[0149] Example 20D: Immunosuppressive effect of the compound on a collagen-induced mouse arthritis model

[0150] 1. Creation of a collagen-induced mouse arthritis model (mCIA)

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

[0152] Experimental Method: 32 female DBA / 1J mice were randomly divided into four groups: a blank control group, a model group, a model + YZ001052 administration group, and a model + PF-06651600 administration group. After successfully creating an arthritis model, drug intervention with YZ001052 or PF-06651600 was administered at a dose of 50 mg / kg once daily. Complete and incomplete Freund's adjuvant were mixed with chicken type II collagen solution to form emulsifiers. Except for the blank control group, on Day 0, the mixture of complete Freund's adjuvant and chicken type II collagen solution was subcutaneously injected into the tail root and thigh root of the mice. 100 μL was injected subcutaneously into the tail root of each mouse, and 50 μL was injected subcutaneously into both thigh roots (primary immunization). On day 21, mice were subcutaneously injected with 200 μL of a mixture of incomplete Freund's adjuvant and chicken type II collagen solution as a reinforcement injection (second immunization). Arthritis was judged to have occurred if at least one mouse had a limb clinical integral of ≥2 points. Measurement of toenail thickness: The thickness of the toenails on both the left and right hind feet of the mice, i.e., the degree of swelling of the toenails, was measured and recorded using calipers, and the measurement frequency was once every three days.

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

[0154] Consistent with arthritis scores, the model mice showed clear swelling of the toenails. However, after treatment with YZ001052 or PF-06651600, varying degrees of improvement were observed in the swelling of the mice's toenails. These results indicate that YZ001052 or PF-06651600 can improve the symptoms of rheumatoid arthritis.

[0155] Example 20E: Immunosuppressive effect of the compound on a dextran sulfate sodium salt (DSS)-induced mouse inflammatory bowel disease model.

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

[0157] Experimental Method: 32 female C57BL / 6 mice were randomly divided into four groups: a blank control group, a model group, a model group with YZ001052, and a model group with PF-06651600. The drugs were administered simultaneously at the start of model preparation, with YZ001052 and PF-06651600 administered at a dose of 50 mg / kg once daily. 50 g of DSS was weighed, 1000 mL of sterile water was added, and a 5% DSS solution was prepared, which was filtered through a 0.22 μm filtration membrane. From day 0, the blank group was given drinking water without DSS, while the remaining experimental groups were given drinking water containing 5% DSS. All mice were sacrificed on day 8. After the experiment, the colorectum was collected, photographed, its length measured, and statistical analysis was performed.

[0158] Experimental results: As shown in Figure 78, the colorectal length of the model group mice was significantly shortened compared to the normal control group, suggesting that the colorectal region of inflammatory bowel disease mice suffers severe damage. Compared to the model group mice, the colorectal length of the YZ001052-treated mice was clearly increased, indicating that treatment with YZ001052 can mitigate colorectal damage in inflammatory bowel disease mice, and the results are superior to those of PF-06651600.

[0159] Example 20F Immunosuppressive effect of the compound on a mouse model of radiation-induced lung injury.

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

[0161] (1) Animal grouping 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, with six mice in each group.

[0163] (2) Model creation method

[0164] Mice were anesthetized by intraperitoneal injection with 1% pentobarbital sodium and irradiated with a single dose of 22.5 Gy of 220 kV X-rays to the entire lung using a Small Animal Precision Radiotherapy Research Platform (SARRP). After irradiation, the mice were kept in normal living conditions.

[0165] 2. Indicator testing

[0166] Three weeks after model preparation, mice were anesthetized, lung tissue was dissected and exposed, the left lung was ligated, an open tracheostomy was performed, the lungs were washed with cold PBS, and bronchoalveolar lavage fluid (BALF) was collected and frozen-centrifuged 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, TNF-α levels in the bronchoalveolar lavage fluid of the model mice were clearly elevated after irradiation induction. The drug YZ001052 has a good inhibitory effect on TNF-α levels.

[0168] The bronchoalveolar lavage fluid was resuspended in 200 μL of PBS to precipitate cells, which were then used for leukocyte (WBC) counting. As shown in Figure 80, the number of white blood cells in the bronchoalveolar lavage fluid of the model mice clearly increased after irradiation induction. The drug YZ001052 can significantly reduce the number of inflammatory cells infiltrating mouse pneumonia induced by irradiation.

Claims

1. The aromatic heterocyclic compound crystal is as follows: 【Chemistry 1】 The aforementioned crystal is either crystal I or crystal III. Crystal I exhibits characteristic diffraction peaks at 2θ angles of 8.4°, 10.0°, 13.6°, 16.4°, and 19.7°, as determined by X-ray diffraction. The aforementioned crystal III exhibits characteristic diffraction peaks at 2θ angles of 8.2°, 9.8°, 17.2°, and 26.8°, as determined by X-ray diffraction. Here, we have a crystal of an aromatic heterocyclic compound characterized by an error range of ±0.2° for the 2θ angle.

2. Crystal I, when analyzed by X-ray diffraction, exhibits characteristic diffraction peaks at 2θ angles of 15.2°, 18.1°, and 24.1°. The crystal according to claim 1, characterized in that the error range of the 2θ angle is ±0.2°.

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

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

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

6. A drug composition comprising one or two of the crystals I and III described in claim 1 or 2, and a pharmaceutically acceptable carrier or excipient.

7. A method for producing crystal I according to claim 1 or 2, wherein a solvent is added to the aromatic heterocyclic compound to form a suspension, which is stirred at room temperature, filtered, and dried to obtain crystal I, wherein the solvent is one or more combinations of acetone, methanol, ethanol, water, ethyl acetate, toluene, methyl tert-butyl ether, and n-heptane, or a first organic solvent is added to the aromatic heterocyclic compound, which is heated and dissolved, then a second organic solvent is added, which is stirred to precipitate crystals, which is filtered, and dried to obtain crystal I, wherein the first organic solvent is one or more combinations of methanol, ethanol, dichloromethane, trichloromethane, and dimethyl sulfoxide, and the second organic solvent is one or more combinations of isopropyl ether, n-hexane, n-heptane, methyl tert-butyl ether, and water.

8. A method for producing crystal III according to claim 1, characterized in that a first organic solvent is added to the aromatic heterocyclic compound, heated and dissolved, then a second organic solvent is added, followed by stirring to precipitate crystals, then filtered and dried, wherein the first organic solvent is one or more of methanol, ethanol, tetrahydrofuran, 1,4-dioxane, and acetonitrile, and the second organic solvent is one or more of water and ethyl acetate.

9. A method of using the crystal described in claim 1 or 2 in the manufacture of a drug for the prevention or treatment of a disease caused by abnormalities in the JAK-STAT signaling pathway.

10. The method according to claim 9, characterized in that the JAK-STAT signaling pathway abnormality refers to the overactivation or overexpression of JAK3 kinase.

11. The method according to 9, characterized in that the disease is selected from one or more of the following: autoimmune diseases, cancer, and myeloproliferative disorders.

12. The method according to 11, characterized in that 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 injury, autoimmune liver injury, 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 disorder is one or more selected from chronic myelomonocytic leukemia, atypical chronic myeloid leukemia, and juvenile myelomonocytic leukemia.

13. The method according to 11, characterized in that the autoimmune disease is radiation lung injury, and the lung injury refers to acute radiation pneumonitis and radiation lung fibrosis.

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