Crystalline form of the compound, as well as its preparation method and use.

Abstract

The present invention discloses a crystalline form of a compound, as well as its preparation and use. The compound has the structure shown in Formula I. The crystalline form has relatively good solid-state stability and flowability, relatively low hygroscopicity, and relatively good solubility, making it advantageous for the preparation and storage of pharmaceutical formulations. Furthermore, the crystalline form has excellent pharmacokinetic properties, relatively excellent safety during repeated administration, and can be administered via multiple administration routes (e.g., injection, oral administration). It is available in a wide range of formulations, and has particularly high oral bioavailability, making it highly valuable for practical applications. The crystalline form can be used as a JAK inhibitor to develop drugs for the prevention and / or treatment of inflammatory diseases, tumors, autoimmune diseases, and allergic diseases in humans and / or animals. [Formula 1] JPEG2025542332000054.jpg45170

Description

[Technical Field]

[0001] This invention relates to the field of research on chemical crystalline polymorphisms of drugs, and more specifically to the crystalline form of a compound represented by formula I, as well as methods for preparing and using the same. [Background technology]

[0002] The JAK-STAT signaling pathway is a cytokine-stimulated signaling pathway involved in many important biological processes, including cell proliferation, differentiation, apoptosis, and immunomodulation. Compared to other signaling pathways, the signaling process of this pathway is relatively simple. It mainly consists of three elements: a tyrosine kinase-associated receptor, the tyrosine kinase JAK, and the transcription factor STAT.

[0003] JAK inhibitors can selectively inhibit JAK kinase and block the JAK-STAT pathway. Currently, FDA-approved JAK inhibitors include tofacitinib, ruxolitinib, oclacitinib, and baricitinib. Oclacitinib has relatively good therapeutic effects on allergic skin diseases in pet dogs, but it has little effect on cytokines that are not involved in JAK1 activation. Its effect on allergic reactions is limited to suppressing the release of allergy mediators, and it cannot fundamentally block the binding of allergy mediators to related receptors. Therefore, it cannot fundamentally inhibit the onset and progression of allergic skin diseases, which limits the range of applications of oclacitinib. Another JAK inhibitor, baricitinib, is a selective JAK1 and JAK2 inhibitor with IC50 values ​​of 5.9 nM and 5.7 nM, respectively. Its selectivity is approximately 70 times and 10 times higher than that of inhibitors acting on JAK3 and Tyk2, but it does not inhibit c-Met or Chk2, and its indications are relatively limited.

[0004] Chinese patent CN111499641B discloses a JAK inhibitor having relatively good JAK inhibitory activity and expected to be used for the prevention and / or treatment of inflammatory diseases and cancer in humans and / or animals, as well as a method for preparing the same. While therapeutic activity is the most important property for a therapeutic agent, the solid form of the drug candidate (i.e., crystalline or amorphous form) is also important for the pharmacological characterization and development of its practical API. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] To overcome the shortcomings of the prior art, the present invention provides a crystalline form of the compound represented by formula I, as well as a method for preparing and using the same. [Means for solving the problem]

[0006] This invention provides crystalline form I of the compound represented by formula I. [ka]

[0007] The XRPD pattern of crystal form I (using Cu-Kα emission) has characteristic peaks at at least three (e.g., three, four, five, six, seven, or especially all) positions among the 2θ values ​​of approximately 12.4°±0.2°, 14.6°±0.2°, 16.7°±0.2°, 17.2°±0.2°, 20.3°±0.2°, 24.8°±0.2°, and 25.0°±0.2°.

[0008] Furthermore, the XRPD pattern of crystal form I (using Cu-Kα emission) also has characteristic peaks at at least three (e.g., three, four, five, six, seven, or especially all) positions among the 2θ values ​​of approximately 13.2°±0.2°, 15.2°±0.2°, 19.3°±0.2°, 21.2°±0.2°, 21.6°±0.2°, 24.0°±0.2°, and 27.0°±0.2°.

[0009] Further, said crystalline Form I has an XRPD pattern substantially as shown in Figure 1.

[0010] Further, the DSC pattern of said crystalline Form I has an endothermic peak at 195 to 210°C (for example, around 195°C, 200°C, 205°C, 210°C).

[0011] Further, said crystalline Form I has a DSC pattern substantially as shown in Figure 2.

[0012] Further, in the TGA pattern of said crystalline Form I, the weight loss when heated from 30°C to 180°C is 1.7% (for example, 1.0%, 1.5%, 1.7%, 2.0%, 2.5%, 3.0%).

[0013] Further, said crystalline Form I has a TGA pattern substantially as shown in Figure 2.

[0014] Further, said crystalline Form I is an anhydrous crystalline form.

[0015] Further, when observed under a polarizing microscope, said crystalline Form I is acicular and / or elongated plate-like crystals.

[0016] The present invention further provides a method for preparing crystalline Form I, wherein crystalline Form I is prepared by using an amorphous form of the compound represented by formula I as a starting material, through one or a combination of more of the anti-solvent addition method, gas-solid diffusion method, suspension stirring method, slow volatilization method, slow cooling method, gas-liquid permeation method, or anti-anti solvent addition method.

[0017] Further, the specific steps of said anti-solvent addition method comprise: dissolving the amorphous raw material of the compound represented by formula I in a good solvent, and stirring while adding the anti-solvent dropwise until a solid precipitates.

[0018] Further, the good solvent in said anti-solvent addition method is one selected from methanol, 1,4-dioxane, chloroform, or dimethyl sulfoxide.

[0019] Further, the poor solvent in the poor solvent addition method is one selected from methyl isobutyl ketone, isopropyl acetate, methyl tert-butyl ether, n-heptane, ethyl acetate, 2-butanone, m-xylene, cyclopentyl methyl ether, toluene, anisole, or water.

[0020] In some embodiments of the present invention, in the poor solvent addition method, the good solvent is methanol, and the poor solvent is selected from methyl isobutyl ketone, isopropyl acetate, and methyl tert-butyl ether.

[0021] In some other embodiments of the present invention, in the poor solvent addition method, the good solvent is 1,4-dioxane, and the poor solvent is selected from n-heptane and ethyl acetate.

[0022] In some other embodiments of the present invention, in the poor solvent addition method, the good solvent is chloroform, and the poor solvent is selected from n-heptane, m-xylene, and cyclopentyl methyl ether.

[0023] In some other embodiments of the present invention, in the poor solvent addition method, the good solvent is dimethyl sulfoxide, and the poor solvent is selected from toluene, anisole, and water.

[0024] Further, the specific steps of the gas-solid diffusion method comprise: mixing an amorphous raw material of the compound represented by formula I with a solvent, sealing the mixture, and allowing it to stand at room temperature until a solid precipitates.

[0025] Further, the suspension stirring method is selected from a room temperature suspension stirring method, a 50°C suspension stirring method, or a temperature cycle suspension stirring method.

[0026] Further, the specific steps of the room temperature suspension stirring method comprise: suspending an amorphous raw material of the compound represented by formula I in a solvent, and stirring the suspension at room temperature until a solid precipitates.

[0027] Furthermore, the specific steps of the 50°C suspension stirring method include suspending the amorphous raw material of the compound represented by formula I in a solvent, suspending and stirring it for 2 to 4 days in a temperature cycle of 50 to 5°C, and then subjecting it to another 50°C suspension stirring until a solid precipitates.

[0028] Furthermore, the specific steps of the temperature cycle suspension stirring method include suspending the amorphous raw material of the compound represented by formula I in a solvent, stirring at 40-60°C, cooling from 40-60°C to 0-10°C within 400-500 minutes, stirring at 0-10°C for 1-3 hours, then raising the temperature to 40-60°C within 20-40 minutes, stirring at 40-60°C for 1-3 hours, repeating the above steps (for example, twice), then cooling down to 0-10°C within 400-500 minutes, maintaining the temperature at 0-10°C, and stirring until a solid precipitates.

[0029] Furthermore, the specific steps of the slow volatilization method include dissolving an amorphous raw material of the compound represented by formula I in a solvent, shaking and filtering it, collecting the filtrate, and slowly volatilizing it at room temperature until a solid precipitates.

[0030] Furthermore, the specific steps of the slow cooling method include dissolving the amorphous raw material of the compound represented by formula I in a solvent, stirring at 40-60°C, filtering the solution once it becomes clear, slowly cooling the filtrate from 40-60°C to 0-10°C, and collecting the precipitated solid.

[0031] Furthermore, the specific steps of the gas-liquid permeation method include dissolving an amorphous raw material of the compound represented by formula I in a solvent in an open container, then placing the open container in a sealed container containing a poor solvent, and allowing it to stand at room temperature until a solid precipitates.

[0032] Furthermore, the specific steps of the poor-poor solvent addition method include dissolving an amorphous raw material of the compound represented by formula I in a good solvent, adding the resulting solution to a poor solvent, and precipitating a solid.

[0033] The present invention further provides hydrochloride crystalline form I of the compound represented by formula I.

[0034] Furthermore, the XRPD pattern of the hydrochloride crystalline form I (using Cu-Kα emission) has characteristic peaks at at least three (e.g., four, five, six, seven, eight, nine, or especially all) positions among the 2θ values ​​of approximately 6.2°±0.2°, 10.9°±0.2°, 12.3°±0.2°, 16.3°±0.2°, 17.2°±0.2°, 18.9°±0.2°, 19.4°±0.2°, 24.7°±0.2°, and 27.5°±0.2°.

[0035] Furthermore, the XRPD pattern of the hydrochloride crystalline form I (using Cu-Kα emission) also has characteristic peaks at at least three (e.g., four, five, six, seven, eight, nine, or especially all) positions among the 2θ values ​​of approximately 11.6°±0.2°, 15.0°±0.2°, 18.4°±0.2°, 21.3°±0.2°, 23.3°±0.2°, 24.3°±0.2°, 25.6°±0.2°, 26.8°±0.2°, and 30.0°±0.2°.

[0036] Furthermore, the hydrochloride crystalline form I has an XRPD pattern that is approximately as shown in Figure 4.

[0037] Furthermore, the DSC pattern of the hydrochloride crystalline form I has endothermic peaks at 210-220°C (for example, around 210°C, 213°C, 216°C, and 210°C).

[0038] Furthermore, the hydrochloride crystalline form I has a DSC pattern that is approximately as shown in Figure 5.

[0039] Furthermore, in the TGA pattern of the hydrochloride crystalline form I, the weight loss when heated from 25°C to 150°C is 0.5% (e.g., 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%).

[0040] Furthermore, the hydrochloride crystalline form I has a TGA pattern that is approximately as shown in Figure 5.

[0041] Furthermore, observation with a polarizing microscope revealed that the hydrochloride crystal form I is a rod-shaped crystal.

[0042] The present invention further provides a method for preparing the hydrochloride salt crystal form I of a compound represented by formula I (for example, as described in the present invention), which involves mixing the crystalline form I of the compound represented by formula I (for example, as described in the present invention) with hydrochloric acid, adding a solvent (stirring, centrifugation), and obtaining the hydrochloride salt crystal form I.

[0043] Furthermore, the molar ratio of crystalline form I to hydrochloric acid is 1:1.

[0044] Furthermore, the solvent is selected from ethanol, acetone / water (e.g., 19:1, v / v), ethyl acetate, or 2-methyltetrahydrofuran.

[0045] The present invention further provides a pharmaceutical composition comprising crystalline form I or hydrochloride crystalline form I of a compound represented by formula I, and one or more pharmaceutically acceptable auxiliary materials.

[0046] Furthermore, the auxiliary material is one or more selected from carriers, diluents, binders, lubricants, and wetting agents. Preferably, the pharmaceutical composition contains a therapeutically effective amount of crystalline form I or hydrochloride crystalline form I of the compound represented by formula I.

[0047] Preferably, the pharmaceutical composition can be administered to humans and / or animals.

[0048] Furthermore, the pharmaceutical composition is suitable for enteral or non-enteral administration, such as intravenous, intramuscular, intradermal, and subcutaneous administration. Therefore, preferably, the pharmaceutical composition also includes aqueous and non-aqueous sterile suspensions which may contain antioxidants, buffers, bactericides, and solutes that make the formulation isotonic with the recipient's blood, as well as suspension aids, solubilizers, thickeners, stabilizers, and preservatives.

[0049] Furthermore, the pharmaceutical composition can be prepared into pharmaceutical formulations in the form of syrups, elixirs, suspensions, powders, granules, tablets, capsules, lozenges, aqueous solutions, creams, ointments, lotions, gels, emulsions, and the like.

[0050] Furthermore, the pharmaceutical formulation is preferably in unit dosage form. In this form, the formulation may be further divided into unit doses containing an appropriate amount of the active ingredient. The unit dosage form may be a capsule, a tablet, or any other dosage form. Alternatively, the unit dosage form may be a packaged formulation such as tablets, capsules, and powders packaged in vials or ampoules.

[0051] The amount of the active ingredient in the aforementioned unit dose formulation may vary or be adjusted from 0.1 mg to 1000 mg depending on the specific use and potency of the active ingredient. If necessary, the composition may also include other suitable therapeutic agents.

[0052] The present invention further relates to the use of crystalline form I or hydrochloride crystalline form I of the compound represented by formula I in the preparation of drugs for treating JAK inhibitors, Janus-activated kinase signaling transducers and activators of transcriprion (JAK-STAT) pathway-related diseases. provide.

[0053] The present invention further provides the use of crystalline form I or hydrochloride crystalline form I of the compound represented by formula I in the treatment of JAK inhibition, Janus-activated kinase signaling factor, and activator-transcriptional pathway-related diseases.

[0054] Furthermore, the aforementioned diseases are selected from inflammatory diseases, tumors, autoimmune diseases, and allergic diseases.

[0055] The present invention further provides the use of crystalline form I or hydrochloride crystalline form I of the compound represented by formula I in the preparation of drugs for preventing and / or treating inflammatory diseases, tumors, autoimmune diseases and allergic diseases in humans and / or animals.

[0056] The present invention further provides the use of crystalline form I or hydrochloride crystalline form I of the compound represented by formula I in the prevention and / or treatment of inflammatory diseases, tumors, autoimmune diseases and allergic diseases in humans and / or animals.

[0057] Furthermore, the inflammatory disease is selected from rheumatoid arthritis, canine dermatitis, psoriasis, ulcerative colitis, or Crohn's disease.

[0058] Furthermore, the tumor is a malignant tumor (cancer), and the cancer is further selected from myelofibrosis, polycythemia vera, essential thrombocythemia, chronic myeloid leukemia, breast cancer, lung cancer, or pancreatic cancer.

[0059] Furthermore, the autoimmune disease is selected from systemic lupus erythematosus, type 1 diabetes, rheumatoid arthritis, multiple sclerosis, ankylosing spondylitis, psoriasis, celiac disease, ulcerative colitis, or Crohn's disease.

[0060] Furthermore, the allergic disease is selected from allergic dermatitis, allergic conjunctivitis, allergic asthma, or allergic rhinitis.

[0061] In some embodiments of the present invention, the allergic disease is allergic dermatitis in dogs and cats, which includes one or more symptoms such as itching, red rash, hair loss, desquamation, edema, and ulcers, and may sometimes be accompanied by symptoms such as otitis, sneezing, and lacrimation, particularly itching. In one embodiment of the present invention, the disease is allergic pruritus in dogs. [Effects of the Invention]

[0062] The present invention has the following technical effects.

[0063] Based on previous research findings, the inventors conducted experimental studies and obtained crystals of the free crystalline form and hydrochloride crystalline form of the compound represented by formula I. These crystals exhibit sharp diffraction peaks, relatively high crystallinity, relatively low solvent residue, a single DSC thermal signal, and minimal TGA weight loss. Furthermore, the ligand safety level is relatively high, resulting in relatively good solid stability and fluidity, relatively low hygroscopicity, and relatively good solubility. These properties are advantageous for the preparation and storage of pharmaceutical formulations, and are expected to have excellent application value and drugability. In addition, clinical trial results showed that the free crystalline form and hydrochloride crystalline form described in the present invention have excellent pharmacokinetic properties. Systemic exposure in experimental animals increased linearly in a dose-dependent manner, blood drug concentration rapidly reached a peak, stability in the body was high, there was no apparent drug accumulation after continuous administration, clinical toxicity was low, and safety was relatively excellent, demonstrating significant superiority over the positive control drug. Furthermore, the free crystalline form and hydrochloride crystalline form described in the present invention have high oral bioavailability and excellent therapeutic effects on various diseases, including excellent inhibitory and antipruritic effects on allergic pruritic symptoms in dogs. Moreover, the free crystalline form and hydrochloride crystalline form described in the present invention can be administered via various routes of administration (e.g., injection, oral administration) and can take various formulation forms, making their future application potential very promising. [Brief explanation of the drawing]

[0064] [Figure 1] The XRPD chart for free crystal form A is shown. [Figure 2] The TGA / DSC chart for free crystal form A is shown. [Figure 3] The 1H NMR spectrum of free crystal form A is shown. [Figure 4] The XRPD chart for hydrochloride crystalline form A is shown. [Figure 5] The TGA / DSC chart for hydrochloride crystalline form A is shown. [Figure 6] The 1H NMR spectrum of hydrochloride crystalline form A is shown. [Figure 7]The XRPD chart for sulfate crystal form A is shown. [Figure 8] The TGA / DSC chart for sulfate crystal form A is shown. [Figure 9] The 1H NMR spectrum of sulfate crystal form A is shown. [Figure 10] The XRPD chart for phosphate crystal form A is shown. [Figure 11] The TGA / DSC chart for phosphate crystal form A is shown. [Figure 12] The 1H NMR spectrum of phosphate crystal form A is shown. [Figure 13] The XRPD chart for the solubility of free crystalline form A in H2O is shown. [Figure 14] The XRPD chart for the solubility of free crystalline form A in SGF is shown. [Figure 15] The XRPD chart for the solubility of free crystalline form A in FaSSIF is shown. [Figure 16] The XRPD chart for the solubility of free crystalline form A in FeSSIF is shown. [Figure 17] The XRPD chart for the solubility of hydrochloride crystalline form A in pH buffer is shown. [Figure 18] The XRPD chart for the solubility of free crystalline form A in pH buffer is shown. [Figure 19] The DVS chart for hydrochloride crystalline form A is shown. [Figure 20] The DVS chart for free crystal form A is shown. [Figure 21] The XRPD overlays of hydrochloride crystalline form A before and after the DVS test are shown. [Figure 22] The XRPD overlays of free crystal form A before and after DVS testing are shown. [Figure 23] The XRPD overlays of hydrochloride crystalline form A before and after stability evaluation are shown. [Figure 24] The XRPD overlays of free crystal form A before and after stability evaluation are shown. [Figure 25]The UPLC diagram for the stability evaluation of hydrochloride crystal form A is shown (Note: Shifts in the main peak appearance time may occur due to changes in system pressure during testing at different times. In each test, the starting sample is tested against its API peak appearance time). [Figure 26] The UPLC diagram for the stability evaluation of free crystal form A is shown (Note: Shifts in the main peak appearance time may occur due to changes in system pressure during testing at different times. In each test, the starting sample is tested against its API peak appearance time). [Figure 27] The PLM chart for hydrochloride crystalline form A is shown. [Figure 28] The PLM chart for free crystal form A is shown. [Figure 29] The XRPD charts for hydrochloride crystalline form A before and after grinding and tableting are shown. [Figure 30] The XRPD charts of free crystalline form A before and after grinding and tableting are shown. [Figure 31] This shows the blood concentration-time curve (logarithmic coordinate) in beagle dogs administered 2 mg / kg of free crystalline form A by intravenous injection. [Figure 32] The blood concentration-time curves for beagle dogs administered intragastricly at various doses of free crystalline form A are shown. [Figure 33] This shows the blood concentration-time curve for beagle dogs that received multiple doses of 6 mg / kg free crystalline form A (intragastric administration at the 1st and 7th doses). [Figure 34] This shows the blood concentration-time curve in male beagle dogs after intragastric administration of 6 mg / kg hydrochloride crystalline form A. [Modes for carrying out the invention]

[0065] Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as those commonly understood by those skilled in the art.

[0066] In this invention, the term "crystalline form" is confirmed by characterization of the X-ray powder diffraction pattern. Those skilled in the art will understand that the physicochemical properties discussed herein may be characterized, and that experimental errors are due to instrument conditions, sample preparation, and sample purity, etc. In particular, it is well known to those skilled in the art that X-ray diffraction patterns usually change depending on instrument conditions. Of particular importance is that the relative intensity of the X-ray powder diffraction pattern may also change depending on changes in experimental conditions, so the order of peak intensities cannot be used as the sole determinative factor. In practice, the relative intensity of the diffraction peaks in the XRPD pattern is related to the preferred orientation of the crystal, and the peak intensities shown herein are for illustrative purposes only, not absolute comparison. Furthermore, experimental errors in peak angles are usually less than 5%, and these angular errors must also be taken into consideration, with errors of ±0.2° usually being acceptable. In addition, the overall shift in peak angles occurs due to the influence of experimental factors such as sample thickness, but a certain degree of shift is usually acceptable. Therefore, the X-ray powder diffraction patterns of the crystal forms of the present invention do not necessarily perfectly match the X-ray powder diffraction patterns of the examples referred to herein, and it will be understood by those skilled in the art that “the XRPD patterns are the same” as described herein does not mean they are exactly the same, the positions of the same peaks may differ by ±0.2°, and some variation in peak intensity is permissible. All crystal forms having the same or similar characteristic peaks as these patterns are included within the scope of the present invention. Those skilled in the art can compare the patterns described in the present invention with those of unknown crystal forms to determine whether the two sets of patterns reflect the same crystal form or different crystal forms. In some embodiments, crystal form A of the present invention is pure and monolithic and substantially not mixed with other crystal forms. In the present invention, “substantially none” for a new crystal form means that this crystal form contains less than 20% (by weight), particularly less than 10% (by weight), further less than 5% (by weight), and further less than 1% (by weight) of other crystal forms.

[0067] Furthermore, the numerical values ​​and ranges of numerical values ​​described in this invention should not be understood narrowly as the numerical values ​​or ranges of numerical values ​​themselves. Those skilled in the art should understand that specific numerical values ​​can vary based on different specific technical environments without departing from the spirit and principles of this invention. In this invention, the range of variation that those skilled in the art can predict is mainly expressed using the term "approximately". When the term "approximately" is used before a numerical value in this invention and refers to that value, it means any value within ±10%, preferably within ±5%, more preferably within ±2%, and even more preferably within ±1% of that value. For example, "approximately 10" should be interpreted as meaning 9 to 11, preferably 9.5 to 10.5, more preferably 9.8 to 10.2, and more preferably 9.9 to 10.1.

[0068] Furthermore, in X-ray diffraction patterns of powder samples, the diffraction spectrum obtained from crystalline compounds is often characterized by a specific crystal form. The relative intensity of spectral bands (especially at low angles) can change due to dominant orientation effects caused by differences in crystallization conditions, particle size, relative content of the mixture, and other test conditions. Therefore, the relative intensity of the diffraction peaks is not characteristic of the crystal in question, and when determining whether it is the same as a known crystal form, more attention should be paid to the position of the peaks than to the relative intensity of the peaks.

[0069] In this invention, the term "room temperature" means that the temperature of an object is close to or the same as the temperature of the surrounding space (for example, inside the fume hood in which the object is placed). Typically, room temperature is about 20°C to 30°C, or about 22°C to 27°C, or about 25°C.

[0070] Poor solvent crystallization (also called poor solvent addition, precipitation crystallization, salting out, or forced crystallization) is a method in which one or more poor solvents are added to a solution in which the desired product is dissolved in a good solvent, causing the product to be slightly dissolved in the solution, and then the solution is brought to a supersaturated state to precipitate crystals. Poor-to-poor solvent crystallization is a method in which one or more poor solvents are added to a solution in which the desired product is dissolved in a good solvent, causing the product to be slightly dissolved in the solution, and then the solution is brought to a supersaturated state to precipitate crystals.

[0071] Poor solvents are relative in a system because they have less ability to dissolve the desired product than good solvents, for example, by 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% or more. Good and poor solvents can be polar or nonpolar solvents, and may be one or more selected from, for example, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), water, alcoholic solvents, ether solvents, ketone solvents, esteric solvents, alkane solvents, aromatic hydrocarbon solvents, and nitrile solvents. Here, alcoholic solvents include, but are not limited to, methanol, ethanol, propanol, isopropanol, 1,3-propylene glycol, 1,2-propylene glycol, trichloro-t-butanol, or combinations thereof. Etheric solvents include, but are not limited to, tetrahydrofuran, methyl tert-butyl ether, 1,4-dioxane, or combinations thereof. Ketone solvents include, but are not limited to, acetone, methyl ethyl ketone, 4-methyl-2-pentanone, or combinations thereof. Ester solvents include, but are not limited to, ethyl acetate, isopropyl acetate, n-butyl acetate, tert-butyl acetate, or combinations thereof. Alkane solvents include, but are not limited to, dichloromethane, chloroform, n-hexane, cyclohexane, pentane, n-heptane, or combinations thereof. Aromatic hydrocarbon solvents include, but are not limited to, benzene, toluene, or combinations thereof. Nitrile solvents include, but are not limited to, acetonitrile and malononitrile.

[0072] Poor solvent crystallization and poor-to-poor solvent crystallization can be performed by batch, semi-batch, or continuous crystallization operations. When adding a poor solvent to a solution (poor solvent crystallization) or a product solution to a poor solvent (poor-to-poor solvent crystallization), the solvent can be added dropwise at a constant rate, or initially slowly and then gradually increased.

[0073] In the present invention, unless otherwise specified, the term "animal" as used herein refers to animals other than humans, particularly mammals such as monkeys, pigs, cows, sheep, horses, donkeys, dogs, cats, rabbits, mice, foxes, raccoons, minks, and camels. In some embodiments of the present invention, the animals are pets such as dogs, cats, and rodents (e.g., chinchillas, pet rabbits, hamsters, guinea pigs, mice, gerbils, chipmunks, squirrels, flying squirrels, curly-haired guinea pigs, degus, etc.).

[0074] The term "treatment" means preventing, curing, reversing, reducing, alleviating, minimizing, inhibiting, blocking, and / or stopping one or more clinical symptoms of a disease after its onset.

[0075] The term "prevention" means treating a disease before it develops, thereby avoiding, minimizing, or making it difficult for the disease to occur or progress.

[0076] The term "inflammation" refers to the body's defensive response to a stimulus, which can manifest as redness, swelling, fever, pain, and dysfunction, and may be infectious inflammation caused by an infection, or non-infectious inflammation caused by an infection, such as inflammation caused by an immune response (such as inflammation caused by various types of hypersensitivity reactions or certain autoimmune diseases). The term "inflammatory disease" refers to a disease that involves inflammation.

[0077] The term "tumor" refers to an abnormal mass of tissue whose growth exceeds and is not in harmony with the growth of normal tissue. Tumors can be either "benign" or "malignant" depending on their characteristics, such as the degree of cellular differentiation (morphological and functional), growth rate, local invasion, and metastasis. "Benign tumors" are typically well-differentiated, grow more slowly than malignant tumors, and remain confined to the site of origin. Furthermore, benign tumors lack the ability to invade, invade, or metastasize to distant sites. In some cases, certain "benign" tumors may later develop into malignant tumors due to further genetic modification in subpopulations of tumor cells; these tumors are called "precancerous tumors." "Malignant tumors" are typically poorly differentiated (undifferentiated), exhibit characteristic rapid growth, and involve progressive invasion, invasion, and destruction of surrounding tissues. Furthermore, malignant tumors usually have the ability to metastasize to distant sites.

[0078] The term "cancer" refers to malignant tumors (Stedman's Medical Dictionary, 25th ed.; Hensyl ed.; Williams & Wilkins: Philadelphia, 1990).

[0079] The term "autoimmune disease" refers to a condition in which the body's immune response to its own antigens causes damage to its own tissues. The American Autoimmune Related Diseases Association compiles a comprehensive list of autoimmune diseases.

[0080] The various publications, patents, and disclosures of published patent specifications cited herein are incorporated herein by reference as a whole.

[0081] Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are for illustrative purposes only and do not limit the scope of the present invention. In the examples, unless specific conditions are specified, the procedures were followed according to conventional conditions or conditions recommended by the manufacturer. Reagents or equipment whose manufacturers are not specified are all commercially available products.

[0082] The Chinese and English names of the solvents used in the examples are as follows:

[0083] MeOH: methanol; EtOH: ethanol; IPA: isopropanol; n-BuOH: n-butanol; Acetone: acetone; MIBK: methyl isobutyl ketone; MEK: 2-butanone; ԅ: ethyl acetate; IPAc: isopropyl acetate; n-Butyl acetate: n-butyl acetate; MTBE: methyl tert-butyl ether; CPME: cyclopentyl methyl ether; THF: tetrahydrofuran; 2-MeTHF: 2-methyltetrahydrofuran; 1,4-Dioxane: 1,4-dioxane; ACN: acetonitrile; DCM: dichloromethane; Toluene: toluene; n-Heptane: n-heptane; DMSO: dimethyl sulfoxide; Anisole: anisole; Cyclohexane: cyclohexane; n-Hexane: n-hexane The structure of compound A in the example is as follows: [ka]

[0084] The preparation is as follows:

[0085] Step 1: Synthesis of Compound 2 [ka] Under room temperature conditions, compound 1 (500g, 2.58mol), dichloromethane (1L), and purified water (1L) are sequentially added to a 5L three-necked flask, and mechanical stirring is started. Then, sodium bicarbonate (400g, 4.76mol) and di-tert-butyl dicarbonate (650g, 2.98mol) are sequentially added. After the addition is complete, the mixture is stirred at room temperature for 4 hours, then dichloromethane (1L) and purified water (1L) are added, and the mixture is separated. The aqueous phase was extracted with dichloromethane (800 ml x 2), and the organic phase was added and washed with saturated brine (1000 ml x 1). The organic phase was dried with anhydrous sodium sulfate, filtered, concentrated at 40°C under reduced pressure until no more distillate evaporated, petroleum ether (500 ml) was added, slurryed at room temperature for 0.5 hours, filtered under reduced pressure, the filtered cake was collected, and air-dried at 25°C for 16 hours to obtain 513 g of white solid (yield 77%).

[0086] Step 2: Synthesis of Compound 3 [ka] Under room temperature conditions, compound 2 (513 g, 2.58 mol) and anhydrous DMF (2 L) were added to a 5 L three-necked flask. Mechanical stirring was initiated, followed by three nitrogen purging cycles under reduced pressure. The temperature was controlled to 10°C using an ice bath, and sodium hydride (103.8 g, 2.59 mol) was added batch by batch. No exothermic reaction was observed. After the addition was complete, the mixture was stirred for 0.5 hours, heated with warm water, and returned to room temperature. Methyl iodide (340 g, 2.40 mol) was added, and no heat dissipation was observed. After the dropwise addition was completed over 100 minutes, the mixture was slowly heated to 36-38°C, resulting in a vigorous reaction and the release of a large amount of heat (uncontrollable). The mixture was cooled to room temperature and stirred overnight. The reaction mixture was slowly added to 2 L of saturated NH4Cl solution (temperature controlled to less than 20°C), then extracted with EA (1 L x 3), the organic phase was washed with saturated NaCl (500 mL x 3), and the organic phase was concentrated to obtain 550 g of oil. This was then subjected to wet column chromatography, eluted at PE / EA = 10 / 1-5 / 1, and 517 g of colorless oil (yield 95.9%) was obtained.

[0087] Step 3: Synthesis of Compound 4 [ka] Add anhydrous tetrahydrofuran (300 mL) to a 5 L three-necked flask, control the temperature to 10-15°C using an ice bath, begin stirring under N2 protection, and add lithium aluminum tetrahydride (46 g, 1.21 mol) decomposingly, controlling the temperature to below -5°C. compound 3 (516 g, 1.90 mol) anhydrous tetrahydrofuran solution (1.5 L) was added dropwise, and after the addition was complete, the reaction was allowed to proceed for 2.5 hours. The temperature was controlled to 0-10°C, and 46 mL of 15% sodium hydroxide solution and 135 mL of water were added dropwise. After the addition was complete, the mixture was filtered by suction under reduced pressure, the filter cake was rinsed with 500 mL of ethyl acetate, and after suction filtration, the filtrate was collected. 500 mL of ethyl acetate and 1000 mL of water were added to the filtrate, and the liquid-liquid separation was performed to collect the organic phase. The aqueous phase was further extracted with EA (500 mL x 3), the organic phases were combined and washed with saturated sodium chloride solution (500 mL x 3), dried, and concentrated to obtain 407 g of a colorless oily substance (yield 88.1%).

[0088] Step 4: Synthesis of Compound 5 [ka] Under room temperature conditions, compound 4 (407 g, 1.67 mol), anhydrous dichloromethane (1000 ml), and pyridine (304.3 g, 3.9 mol) were sequentially added to a 3 L three-necked flask. Stirring was started under nitrogen protection, and the temperature was controlled to 10-15°C using an ice bath. Methanesulfonyl chloride (250 g, 2.18 mol) was added dropwise. After the addition was complete, the mixture was stirred overnight at room temperature, and 500 mL of water was added to quench the reaction. The aqueous phase was extracted with dichloromethane (500 mL x 3), washed with saturated NaCl solution (1000 ml x 1), dried, and concentrated to obtain 510.2 g of a yellow oily substance (yield 94.5%).

[0089] Step 5: Synthesis of Compound 6 [ka] At room temperature, compound 5 (510 g, 1.59 mol), acetone (3000 ml), and NaI (310 g, 2.07 mol) were sequentially added to a 10 L three-necked flask, stirring was started, and the mixture was heated and refluxed overnight. After cooling to room temperature, water (1000 ml) was added and quetzing was performed. Subsequently, the mixture was extracted with ethyl acetate (1 L x 3), washed with 2 L saturated NaCl, dried, and concentrated to obtain 423 g of oily substance, which was then used directly in the next step. The yield was 75.8%.

[0090] Step 6: Synthesis of Compound 8 [ka] At room temperature, compound 7 (35 g, 0.289 mol) and anhydrous tetrahydrofuran (1100 ml) were added to a 5 L three-necked flask, and stirring was started. When the temperature was reduced to 0-5°C, sodium hydride (27.5 g, 0.687 mol) was added batch by batch, a significant heat dissipation occurred. After the addition was complete, a THF solution (1000 ml) of pivalic acid anhydride (51.2 g, 0.275 mol) was added dropwise, the temperature was controlled to below 20°C, and the reaction was allowed to proceed at room temperature for 2 hours. Then, 100 mL of methanol was added to the system, the temperature was controlled to 0-20°C, and 600 mL of NH4Cl aqueous solution and 600 mL of saturated brine were added dropwise to quench the reaction. Subsequently, the mixture was extracted with ethyl acetate (500 mL x 3), the organic phase was washed with 1 L of saturated NaCl, dried, concentrated to obtain an oily substance, and subjected to column chromatography PE / EA = 5 / 1, 3 / 1. The obtained solid was slurryed with 50 mL of petroleum ether to obtain 45.8 g of white solid (yield 81.3%).

[0091] Step 7: Synthesis of Compound 9 [ka] At room temperature, compound 8 (45 g, 0.22 mol), 1,4-dioxane (200 ml), and 15-crown-5 (58 g) were sequentially added to a 500 mL three-necked flask, and stirring was started. The temperature was then lowered to 10-20 °C, and sodium hydride (10.5 g, 0.26 mol) was added batch by batch. After the addition was complete, ethyl iodide (68.4 g, 1.44 mol) was added dropwise at room temperature. After the addition was complete, the mixture was stirred overnight at 40 °C. Then, aqueous ammonium chloride solution (100 mL) was added to the system and quetzing was performed. The aqueous phase was extracted with ethyl acetate (100 mL x 3), then washed with 200 mL of saturated NaCl, dried, concentrated to obtain an oily substance, and subjected to column chromatography PE / EA = 10 / 1, 7 / 1, 5 / 1, 3 / 1 to obtain 45.26 g of white solid (yield 88.3%).

[0092] Step 8: Synthesis of Compound 10 [ka] At room temperature, compound 9 (45 g, 0.19 mol) and dichloromethane (200 ml) were added to a 1 L three-necked flask, and stirring was started. Trifluoroacetic acid (33 g, 0.29 mol) was added at room temperature, and after the addition was complete, the mixture was stirred at room temperature for 3 hours. Then, saturated sodium chloride aqueous solution (100 mL) was added to the system and quenched, and the mixture was extracted with dichloromethane (50 mL x 3). Next, the mixture was washed with 100 mL of saturated sodium bicarbonate, dried, and concentrated to obtain a crude solid product. Column chromatography was performed to sequentially elute the product at PE / EA = 5 / 1, 3 / 1, and 1 / 1, yielding 27 g of a white solid (yield 80.2%).

[0093] Step 9: Synthesis of Compound 11 [ka] At room temperature, compound 10 (26 g, 0.146 mol), 1,4-dioxane (146 ml), and 15-crown-5 (48.5 g) were sequentially added to a 500 mL three-necked flask, and stirring was started. The temperature was then lowered to 10-20°C, and sodium hydride (8.8 g, 0.22 mol) was added batch by batch. After the addition was complete, a THF solution (50 ml) of compound 6 (103.8 g, 0.293 mol) was added dropwise at room temperature. After the addition was complete, the mixture was stirred at 50°C for 5 days. Then, 100 mL of aqueous ammonium chloride solution was added to the system and quenched. Extraction with EA (100 mL x 3) was performed, the organic phase was washed with saturated NaCl (200 ml), dried, and concentrated to obtain an oily substance. Column chromatography was performed to sequentially elute the PE / EA at 10 / 1, 7 / 1, 5 / 1, and 3 / 1, yielding 31.4 g of a white solid (yield 53.5%).

[0094] Step 10: Synthesis of Compound 12 [ka] Compound 11 (31 g, 0.077 mol) and anhydrous tetrahydrofuran (500 mL) were added to a 1 L three-necked flask. The temperature was controlled to 10°C in an ice bath, and stirring was started under N2 protection. Lithium aluminum tetrahydride (8.8 g, 0.24 mol) was added batch by batch. After the addition was complete, the mixture was reacted for 1 hour. The temperature was controlled to 0-10°C, and saturated sodium sulfate solution (24 ml) was added dropwise for quetching. Then, 200 ml of ethyl acetate was added to dilute the mixture. The mixture was filtered by suction under reduced pressure, and the filter cake was rinsed with 50 ml of ethyl acetate. After suction filtration was complete, the filtrate was collected, 100 ml of water was added, and the mixture was separated. The aqueous phase was extracted with ethyl acetate (50 ml x 2), and the organic phase was combined and washed with saturated sodium chloride solution (100 ml x 1). The mixture was dried and concentrated to obtain a colorless oil. The oil was eluted with ethyl acetate by column chromatography to obtain 18.5 g of oil (yield 80%).

[0095] Step 11: Synthesis of Compound 13 [ka] At room temperature, compound 12 (18.5 g, 0.058 mol) and dichloromethane (45 ml) were added to a 250 mL three-necked flask, and stirring was started. Trifluoroacetic acid (20 ml) was then added at room temperature, and after the addition was complete, the mixture was stirred overnight at room temperature and concentrated until the distillate no longer evaporated, yielding 13.3 g of crude product, which was then used directly in the next step.

[0096] Step 12: Synthesis of Compound 15 [ka] Compound 13 (13.3g, 0.058mol), anhydrous DMF (100ml), potassium carbonate (33.4g, 0.24mol), and compound 14 (18.7g, 0.058mol) were sequentially added to a 250mL three-necked flask, and stirring was started. The temperature was raised to 115°C and the reaction was allowed to proceed overnight. After cooling to room temperature, water (150ml) was added to quench the reaction, and the mixture was extracted with ethyl acetate (100mL x 6). Next, it was washed with saturated NaCl (500ml x 3), the organic phase was dried, concentrated, and an oily substance was obtained. A semi-solid was obtained by column chromatography PE / EA=1 / 1, DCM / MeOH=25 / 1, and 10ml of ethyl acetate and 50ml of n-hexane were added to form a slurry, yielding 22.4g of a nearly white solid (yield 76.5%).

[0097] Step 13: Synthesis of Compound A [ka] Compound 15 (22.4 g, 0.045 mol), anhydrous THF (250 ml), and TBAF solution (93 ml, 1 M / L) were added to a 500 mL three-necked flask. Stirring was started, and the reaction was carried out under reflux overnight. The reaction mixture was cooled and concentrated until the fraction no longer evaporated. Then, water (150 ml) and ethyl acetate (200 ml) were sequentially added to the system to separate the organic phase. Next, the aqueous phase was extracted with ethyl acetate (100 ml x 3), washed with saturated NaCl (500 ml x 1), dried with anhydrous sodium sulfate, concentrated to obtain a crude solid, which was separated by column chromatography and eluted with DCM / MeOH = 25 / 1. Subsequently, it was slurryed with ethyl acetate (20 ml) to obtain 11 g of white solid (yield 73.3%). Ms: 336.2 [M+H] + . 1 H NMR (400 MHz, DMSO) δ 11.60 (s, 1H), 8.08 (s, 1H), 7.11 (s, 1H), 6.53 (s, 1H), 4.67 (s, 1H), 3.57 (s, 1H), 3.16 (s, 3H), 2.97 (q, J = 7.6 Hz, 2H), 2.93(d, J = 6.0 Hz, 2H), 2.12-1.99 (m, 3H), 1.75-1.68 (m, 4H), 1.33-1.24 (m, 2H), 1.22 (t, J = 7.6 Hz, 3H). Example 1: Preparation and characterization of the free crystalline form of compound A.

[0098] 1.1 Apparatus and Methods 1.1.1 X-ray Powder Diffraction (XRPD) XRPD results were collected using PANalytical X'Pert3 and Empyrean X-ray powder diffraction analyzers. Samples were tested by placing them in the center of a background-free silicon wafer, and the test parameters are shown in Table 1.

[0099] [Table 1] 1.1.2 Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC) TGA and DSC charts were collected using a TA Q5000 thermogravimetric analyzer and a TA Discovery 2500 differential scanning calorimetry analyzer, respectively. Test parameters are shown in Table 2. [Table 2] 1.1.3 Hydrogen Spectroscopy Liquid Nuclear Magnetic Resonance ( 1 (H NMR)

[0100] Hydrogen spectra and liquid nuclear magnetic resonance spectra were collected using a Bruker 400M NMR spectrometer with DMSO-d6 as the solvent. 1.1.4 Dynamic Moisture Adsorption (DVS)

[0101] Dynamic moisture adsorption (DVS) curves were collected using a Surface Measurement Systems (SMS) DVS Intrinsic. Relative humidity at 25°C was corrected using the deliquescence points of LiCl, Mg(NO3)2, and KCl. Test parameters are shown in Table 3.

[0102] [Table 3] 1.1.5 Ultra-high performance liquid chromatography (UPLC)

[0103] Purity in the tests was measured using Waters H-Class ultra-high performance liquid chromatography, and the analytical conditions are shown in Table 4.

[0104] [Table 4] 1.2 Preparation and Characterization of Free Crystalline Form A of Compound A

[0105] Using MeOH as a good solvent and MIBK as a poor solvent, free crystalline form A was obtained by the poor solvent addition method (test number 02-A1 shown in Table 6). The XRPD pattern of free crystal form A is shown in Figure 1, and the XRPD diffraction peak data is shown in Table 5. Figure 2 shows the TGA / DSC pattern of free crystal form A. The TGA results showed a weight loss of 1.7% when heated from room temperature to 180°C. The DSC results showed a sharp endothermic peak at 197.4°C (start temperature), which is presumed to be a melting signal. Free crystal form A 1 The 1H NMR pattern is shown in Figure 3. Based on the above characterization results, the free crystalline form A of compound A shows relatively little TGA weight loss and a single DSC signal, suggesting that it is an anhydrous crystalline form.

[0106] [Table 5]

[0107] The specific experimental method and results for preparing the above crystals are as follows.

[0108] Approximately 20 mg of compound A sample was weighed into a 20 mL vial and dissolved in 0.3–1.0 mL of a good solvent (see Table 6). The poor solvents listed in Table 6 were added dropwise to the clear solution and mixed until a solid precipitated. If no solid precipitated after adding 5 mL of the poor solvent, the addition of the poor solvent was stopped. The precipitated solid was separated by centrifugation and subjected to an XRPD test. If no solid precipitated, the sample was transferred to 5°C and stirred, or allowed to volatilize at room temperature. The test results are shown in Table 6. In the poor solvent addition test, free crystalline form A and an oily sample were obtained.

[0109] [Table 6] Example 2: Preparation and characterization of the salt-type crystalline form of compound A.

[0110] 2.1 Apparatus and Methods 2.1.1 X-ray Powder Diffraction (XRPD) XRPD results were collected using X'Pert3 and Empyrean X-ray powder diffraction analyzers, and the test parameters are shown in Table 7.

[0111] [Table 7] 2.1.2 Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC)

[0112] TGA results were collected using a TA Discovery 5500 thermogravimetric analyzer, and DSC results were collected using a TA Discovery 2500 differential scanning calorimetry analyzer. The test parameters are shown in Table 8.

[0113] [Table 8] 2.1.3 Hydrogen Spectroscopy Liquid Nuclear Magnetic Resonance (1H NMR)

[0114] Hydrogen spectra and liquid nuclear magnetic resonance spectra were collected using a Bruker 400M NMR spectrometer with DMSO-d6 as the solvent. 2.1.4 Dynamic Moisture Adsorption (DVS)

[0115] Dynamic moisture adsorption (DVS) curves were collected using the DVS Intrinsic of Surface Measurement Systems (SMS). Relative humidity at 25°C was corrected using the deliquescence points of LiCl, Mg(NO3)2, and KCl. DVS test parameters are shown in Table 9.

[0116] [Table 9] 2.1.5 Ultra-high performance liquid chromatography (UPLC)

[0117] In the experiment, purity, solubility, and molar ratio were tested by Waters H-Class UPLC ultra-high performance liquid chromatography. The analytical conditions are shown in Table 10.

[0118] [Table 10] 2.1.6 PLM

[0119] PLM images were collected using a ZEISS Scope. A1 polarizing microscope. 2.1.7 Ion Chromatography (IC)

[0120] In the experiment, ion content was analyzed using a Thermo Fisher ICS-1100 ion chromatograph. The specific conditions are shown in Table 11.

[0121] [Table 11] 2.2 Preparation and Characterization of Salt Crystal Form of Compound A

[0122] In this experiment, we investigated the possibility of free crystalline form A of compound A forming corresponding salts with 23 different acids under four different solvents, using free crystalline form A of compound A as the starting material. The specific experiments are summarized in Table 12. The specific steps were as follows: First, approximately 20 mg of free crystalline form A of compound A and an equimolar amount of the corresponding acid ligand were weighed into an HPLC vial, 0.5 mL of solvent was added, and the mixture was magnetically stirred (approximately 1000 rpm) at room temperature for about 4 days to separate the solid, which was then detected by XRPD. If no solid precipitated, the mixture was transferred to 5°C / -20°C and stirred, and a poor solvent was added or the mixture was evaporated at room temperature.

[0123] The study revealed that, under 92 different experimental conditions, it is not easy to obtain the corresponding salt crystal form of compound A from its free crystalline form A in acidic systems. XRPD comparisons showed that only three salt crystal forms—hydrochloride crystal form A, sulfate crystal form A, and phosphate crystal form A—can exist stably in four different solvents.

[0124] [Table 12-1] [Table 12-2] 2.2.1 Hydrochloride crystal form A

[0125] The XRPD results are shown in Figure 4, and the XRPD diffraction peak data are shown in Table 13. The TGA / DSC results are shown in Figure 5. The sample showed a weight loss of 0.53% when heated to 150°C. The DSC results showed a sharp endothermic peak at 212.9°C (starting temperature). 1 The results of the 1H NMR spectrum are shown in Figure 6. The molar ratio of the residual solvent EtOH to API was 0.06 (0.7 wt%).

[0126] [Table 13-1] [Table 13-2] 2.2.2 Sulfate crystal form A

[0127] The XRPD results are shown in Figure 7, and the XRPD diffraction peak data are shown in Table 14. The TGA / DSC results are shown in Figure 8. The TGA results show that the sample undergoes a stepwise weight loss of 14.83% when heated to 100°C. The DSC results show two endothermic signals at 56.9°C and 96.1°C (peak temperature). 1 The results of the 1H NMR spectrum are shown in Figure 9. The residual solvent, acetone, was clearly not detected.

[0128] [Table 14-1] [Table 14-2] 2.2.3 Phosphate Crystal Form A

[0129] The XRPD results are shown in Figure 10, and the XRPD diffraction peak data are shown in Table 15. The TGA / DSC results are shown in Figure 11. The TGA results showed that the sample lost 2.82% of its weight when heated to 150°C. The DSC results showed three endothermic signals at 69.0°C, 149.0°C, and 163.2°C (peak temperature). 1 The results of the 1H NMR spectrum are shown in Figure 12. The residual solvent, acetone, was clearly not detected.

[0130] [Table 15]

[0131] A comparison of the TGA / DSC charts for free crystalline form A, hydrochloride crystalline form A, sulfate crystalline form A, and phosphate crystalline form A revealed that free crystalline form A and hydrochloride crystalline form A exhibited relatively superior thermal stability. Free base crystalline form A had a dry weight loss of 1.7% when heated to 180°C, while hydrochloride crystalline form A had a dry weight loss of 0.53% when heated to 150°C. Their melting points were 201.5°C and 216.4°C, respectively. A sample of sulfate crystalline form A showed a stepwise weight loss of 14.83% when heated to 100°C. DSC results showed two endothermic signals at 56.9°C and 96.1°C, indicating that sulfate crystalline form A decomposes or melts relatively easily at low temperatures and has relatively poor thermal stability. The phosphate crystal form A sample showed a weight loss of 2.82% when heated to 150°C. However, DSC results showed three endothermic signals at 69.0°C, 149.0°C, and 163.2°C, indicating slightly inferior thermal stability.

[0132] Based on the above, compared to sulfate crystal form A and phosphate crystal form A, free crystal form A and hydrochloride crystal form A have advantages such as sharp crystal diffraction peaks, relatively high crystallinity, relatively low solvent residue, a single DSC thermal signal, and small TGA weight loss, as well as a relatively high level of ligand safety. In this invention, tests conducted under more than 100 experimental conditions revealed that among the obtained crystal forms, only free crystal form A and hydrochloride crystal form A possessed relatively good solid-state properties and could be developed as potential drug crystal forms. Example 3 Evaluation of Dynamic Solubility

[0133] The dynamic solubility of hydrochloride crystalline form A and free crystalline form A in H2O and biological solvents (SGF, FaSSIF, and FeSSIF) was evaluated at 37°C. The specific steps were as follows: Approximately 40 mg of hydrochloride crystalline form A and free crystalline form A were weighed into 5 mL glass vials and added to 4 mL of the corresponding media (H2O, SGF, FaSSIF, and FeSSIF), respectively. The mixtures were mixed in a rotating incubator at 25 rpm at 37°C, with sampling points at 1, 2, 4, and 24 hours. At each sampling point, approximately 0.8 mL of the suspension was taken into a centrifuge tube and centrifuged (12000 rpm, 3 min, 37°C). The supernatant was filtered through a PTFE filter membrane (pore size 0.45 μm), the solubility and pH of the filtrate were tested, and the solid was measured by XRPD (see Example 2 for the equipment and methods used).

[0134] The results of the dynamic solubility evaluation are summarized in Table 16. The hydrochloride crystalline form A became clear in all of the following solutions: H2O, SGF, FaSSIF, and FeSSIF, with a solubility exceeding 9 mg / mL. Free crystalline form A showed a solubility of approximately 9-10 mg / mL in SGF and FeSSIF (the sample did not become clear), while its solubility in H2O and FaSSIF was relatively low (2.1-2.4 mg / mL).

[0135] Figures 13 to 16 show the XRPD results for the solid samples separated at each sampling point. These results show that free crystal form A did not undergo crystallization after 1, 2, 4, and 24 hours in H2O, FaSSIF, and FeSSIF. A diffraction peak of hydrochloride crystal form A was observed after 24 hours in SGF, and some of the free form was absorbed by the Cl medium. - It was presumed that it reacted with another substance to form a hydrochloride salt.

[0136] [Table 16] Example 4 Evaluation of 24-hour solubility

[0137] The solubility of hydrochloride crystalline form A and free crystalline form A in pH 2.0, pH 4.5, and pH 7.4 buffers was evaluated at room temperature over 24 hours. The specific steps were as follows: 10 mg each of hydrochloride crystalline form A and free crystalline form A were weighed into HPLC vials and added to 1 mL of the corresponding buffer. Magnetic stirring was performed at room temperature for 24 hours (approximately 500 rpm). The suspensions were centrifuged (10000 rpm, 2 min, room temperature). The supernatant was filtered through a PTFE filter membrane (pore size 0.22 μm), and the solubility and pH of the filtrate were tested. The solid was measured by XRPD (see Example 2 for the equipment and methods used).

[0138] Table 17 summarizes the 24-hour solubility results, and Figures 17 and 18 show the XRPD results for the separated solid samples. These results indicate that (1) hydrochloride crystalline form A showed relatively high solubility at pH 2.0 and pH 4.5 (>9.2 mg / mL, the sample was clear). At pH 7.4, it was converted to free crystalline form A. (2) The solubility of free crystalline form A in pH 4.5 buffer was relatively high (>9.1 mg / mL, the sample was clear). It did not dissolve at pH 2.0 and pH 7.4, with solubility of 6.2 mg / mL and 1.7 mg / mL, respectively. The XRPD results showed that the crystalline form remained unchanged even after 24 hours of stirring.

[0139] [Table 17] Example 5: Evaluation of hygroscopicity

[0140] The hygroscopic properties of hydrochloride crystalline form A and free crystalline form A were evaluated by dynamic moisture adsorption experiments at 25°C between 0%RH and 95%RH, and the results are summarized in Table 18 (see Example 2 for the equipment and methods used).

[0141] The DVS results are shown in Figures 19 and 20. The moisture-absorbing weight increases of hydrochloride crystalline form A and free crystalline form A at 25°C / 80%RH were 0.47% and 0.39%, respectively, indicating slight hygroscopicity. Figures 21 and 22 show the XRPD comparison results of samples before and after the DVS test. The XRPD results indicate that no crystal transition occurred in either of the two samples after the DVS test.

[0142] [Table 18] Example 6 Evaluation of Solid Stability

[0143] To evaluate the solid stability of hydrochloride crystalline form A and free crystalline form A, appropriate amounts of each sample were weighed and subjected to stability experiments for 1 day in a sealed state at 80°C, and for 4 weeks in an open state at 25°C / 60%RH and 40°C / 75%RH. Solid samples separated under different conditions were tested for crystalline form by XRPD to evaluate physical stability and for purity by ULC to evaluate chemical stability (see Example 2 for the equipment and methods used). The evaluation results are summarized in Table 19.

[0144] The XRPD comparison results are shown in Figures 23 and 24, and the ULC results are shown in Table 20. tableThe results are summarized in section 21, and the UPLC patterns are shown in Figures 25 and 26. The solid-state stability results show that under the three evaluation conditions, neither the hydrochloride crystal form A nor the free crystal form A undergoes crystallization transitions or purity degradation, indicating that they possess relatively good physicochemical stability under the evaluation conditions.

[0145] [Table 19]

[0146] [Table 20]

[0147] [Table 21] Example 7 Evaluation of powder properties

[0148] To understand the basic powder properties of each salt type and free crystal form A, the powder properties of hydrochloride crystal form A and free crystal form A, including fluidity and morphology, were evaluated.

[0149] Fluidity was evaluated by testing the bulk density / tap density and angle of repose of the sample (see Example 2 for the equipment and methods used). The methods for testing bulk density and tap density were as follows: (1) A constant mass of the sample to be evaluated was added to a 5 mL graduated cylinder and the volume was recorded. The bulk density was calculated by dividing the mass of the sample by the volume at that time. (2) The graduated cylinder was tapped 200 times and the final volume was recorded. The tap density was calculated by dividing the mass of the sample by the final volume. (3) The test was performed three times in parallel. Method for testing the angle of repose: (1) A funnel was fixed perpendicular to the base and the substance was slowly poured into the funnel. (2) The base formed a symmetrical cone shape. The height of the cone and the diameter of the base were measured. (3) The test was performed three times in parallel.

[0150] The results of bulk density / tap density are summarized in Table 22. From these results, the bulk density and tap density of the hydrochloride crystalline form A sample are 0.14 g / cm 3 and 0.24 g / cm 3 respectively, and the calculated Carr index is 41%; the bulk density and tap density of the free crystalline form A sample are 0.11 g / cm 3 and 0.23 g / cm 3 respectively, and the calculated Carr index is 51%.

[0151] The results of angle of repose are summarized in Table 23. From these results, it is shown that the angles of repose of the hydrochloride crystalline form A and free crystalline form A powder samples are 33.9° and 47.1° respectively. The results of comprehensive evaluation show that both the hydrochloride crystalline form A sample and the free crystalline form A sample have relatively small Carr index and angle of repose, and relatively good flowability.

[0152] The PLM results are shown in Figure 27 and Figure 28. Under a polarized light microscope, it is observed that hydrochloride crystalline form A is rod-shaped crystals, and free crystalline form A is acicular or long plate-shaped crystals.

[0153]

Table 22

[0154]

Table 23

[0155] Hydrochloride crystalline form A and free crystalline form A were used as starting samples, manually pulverized respectively, and tableted with a tableting machine (pressure 350 MPa). The pulverized and tableted samples were subjected to XRPD testing to evaluate mechanical stability (see Example 2 for the equipment and methods used). The specific results are summarized in Table 24.

[0156] Figures 29 and 30 show the XRPD results before and after crushing and tableting. From these results, it was inferred that (1) after crushing, neither the crystal form nor the degree of crystallinity of hydrochloride crystal form A changed, and although the crystal form of free crystal form A did not change, some diffraction peaks broadened, suggesting a slight decrease in the degree of crystallinity. (2) After tableting, neither the crystal form of hydrochloride crystal form A nor free crystal form A changed, but the degree of crystallinity decreased slightly in all cases (some diffraction peaks broadened, and some weak diffraction peaks disappeared).

[0157] [Table 24]

[0158] In summary, the evaluation results above indicate that both hydrochloride crystalline form A and free crystalline form A possess excellent overall physicochemical properties, including relatively good solid stability and fluidity, relatively low hygroscopicity, and relatively good solubility. Therefore, they can be developed as potential drug crystalline forms and have promising future application potential. Example 9: Pharmacokinetic study of free crystalline form A

[0159] 1. Objective of the experiment This experiment aims to study the pharmacokinetic properties of free crystalline form A (prepared in Example 1, sometimes simply referred to as the drug in this example) in male and female beagle dogs, and includes: 1) a single intravenous (iv) injection (iv) study of 2 mg / kg; 2) dose escalation studies with intragastric (ig) administration of 2, 6, and 20 mg / kg; and 3) a study of continuous intragastric oral administration of 6 mg / kg for 7 days.

[0160] 2. Laboratory animals The ordinary beagle dogs used in this experiment were purchased from Beijing Mars Biotechnology Co., Ltd., with license number SCXK (Jing) 2016-0001, animal qualification certificate numbers 1103182011000078 and 1103182011000079, and laboratory animal use license number SYXK (Su) 2021-0045. The animal room was well-ventilated and fully air-conditioned, with temperature maintained at 16-26°C and humidity maintained at 40%-70%. Artificial lighting was used, with 12-hour light period and 12-hour dark period. The body weight of male beagle dogs at the initial administration ranged from 8.0 kg to 11.6 kg, and the body weight of female beagle dogs ranged from 6.9 kg to 11.3 kg.

[0161] 3. Preparation of Formulation

[0162] (1) Intravenous injection administration The solvent for the intravenous injection group was DMA:30% Solutol-HS15:physiological saline=10:10:80, v / v / v. 150.34 mg of free crystalline form A was weighed into a glass bottle, 7.517 mL of DMA was added, and vortexing and sonication were performed until the particles were dissolved. Then 7.517 mL of 30% (w / v) Solutol-HS aqueous solution was added and mixed by vortexing, followed by addition of 60.136 mL of physiological saline and mixing by vortexing to obtain a colorless solution with a final concentration of 2 mg / mL. The solution was filtered through a filter membrane (PALL, nylon, 0.45 µm), and administered to animals in group A.

[0163] (2) Oral intragastric administration Solvent for the intragastric administration group: 0.5% CMC-Na aqueous solution. 150.43 mg of free crystalline form A was weighed into a glass bottle, 376.075 mL of 0.5% CMC-Na aqueous solution was added, and vortexing and sonication were performed until the particles were dissolved to obtain a colorless solution with a final concentration of 0.4 mg / mL, which was administered to animals in group B. 450.58 mg of free crystalline form A was weighed into a glass bottle, 375.483 mL of 0.5% CMC-Na aqueous solution was added, and the mixture was vortexed and sonicated until the particles dispersed, yielding a white suspension with a final concentration of 1.2 mg / mL. This suspension was administered to animals in group C on day 1. The preparation process for the administered drugs in group C from day 2 to day 7 was the same as on day 1. 1500.94 mg of free crystalline form A was weighed into a glass bottle, 375.235 mL of 0.5% CMC-Na aqueous solution was added, and the mixture was vortexed and sonicated until the particles dispersed, yielding a white suspension with a final concentration of 4 mg / mL. This suspension was then administered to animals in group D. All administered formulations were freshly prepared on the day of administration, and samples were retained for measurement of formulation concentration. In group D, only samples of the administered formulations on days 1, 4, and 7 were retained for measurement of formulation concentration.

[0164] 4. Experimental group assignment In this experiment, all animals were randomly divided into four groups (3 animals / sex / group). Group assignments and administration information are shown in Table 25. Animals in groups A, B, and D were fasted before administration, and animals in group C were fasted before the first and last doses. All animals were fasted for at least 12 hours before administration and fed again 4 hours after administration. Throughout the experiment, all animals had free access to water and food.

[0165] [Table 25]

[0166] 5. Sample collection and processing Animals in Group A were intravenously injected with 2 mg / kg of the drug, and blood was collected from the jugular vein before administration and at 0.033, 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours after administration. Animals in Groups B and D were administered a single intragastric dose of 2 and 20 mg / kg of the drug, respectively, and blood was collected from the jugular vein before administration and at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours after administration. Animals in Group C were administered 6 mg / kg of the drug intragastricly once daily for 7 consecutive days, and blood was collected from the jugular vein before the first dose, before the seventh dose, and at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours after administration. During the period from the second to the sixth administration, blood samples (from the jugular vein) were collected before administration and 0.5 hours after administration. The sample taken before the second administration was the same as the sample taken 24 hours after the first administration. Blood samples from the animals described above were collected via the jugular vein in 0.5 mL / dose volumes. All collected whole blood samples were placed in a centrifuge tube containing EDTA-K2, and the tube was inverted to thoroughly mix the anticoagulant with the blood. All samples were placed on moist ice before centrifugation and centrifuged at 1524 g for 10 minutes to separate the plasma. The plasma samples were transferred to a new centrifuge tube and stored at -90 to -60°C until analysis. Drug concentrations in the plasma of beagle dogs were detected by LC-MS / MS. The obtained blood drug concentration data were used to calculate relevant pharmacokinetic parameters using the WinNonlin 8.0 non-compartment model pharmacokinetic processing software.

[0167] 6. Experimental Results Clinical observations of the animals before administration, before and after each blood sampling time, and after administration revealed no significant abnormalities. The drug concentration-time curves for each group are shown in Figures 31 to 33, and the main pharmacokinetic parameters are shown in Tables 26 to 27.

[0168] [Table 26] When a dose of 2 mg / kg was administered intravenously to beagle dogs, the C0 and AUC of the drug in male and female beagle dogs were observed. 0-t The ratios were 0.758 and 0.748, respectively (within the range of 0.5 to 2 times), and no significant difference was observed in systemic exposure. Drug clearance (CL) in male and female beagle dogs was equivalent to 0.110 times the hepatic blood flow of beagle dogs (approximately 31 mL / min / kg, Davies and Morris (1993)), indicating that clearance in the body of beagle dogs is slow. Steady-state volume of distribution (V dss This figure is 1.57 times the total body fluid volume of a beagle (approximately 0.60 L / kg, Davies and Morris (1993)), indicating a tendency for it to be distributed in tissues.

[0169] [Table 27]

[0170] When Beagle dogs were administered 2, 6, and 20 mg / kg as a single intragastric dose, the average time to peak was 0.792–1.33 hours after administration. After dose adjustment, AUC 0-t The average oral utilization rates F% were 88.0%, 100.0%, and 101.0%, respectively.

[0171] When Beagle dogs were given a single intragastric dose of 2, 6, and 20 mg / kg of the drug, the C levels of the drug differed between male and female dogs at the three dose levels. max The ratios are 1.36, 0.810, and 0.894, respectively, and the AUC 0-t The ratios were 1.10, 0.716, and 0.877, respectively. No significant differences were observed between sexes in terms of total body exposure (the ratios were all within the range of 0.5 to 2 times).

[0172] When male and female beagle dogs were administered intragastric doses of 2, 6, and 20 mg / kg, the unit dose C max These values ​​are 675, 785, and 665 kg*ng / ml / mg, respectively, and the unit dose AUC0-t These values ​​were 4380, 4990, and 5030 ng*h*kg / mL / mg, respectively. In the dose range of 2-20 mg / kg, Beagle The systemic exposure to the drug in the body increased linearly in a dose-dependent manner (unit doses of 20 mg / kg and 2 mg / kg C). max Ratio and Unit Dose AUC 0-t The ratios were 0.985 and 1.15, respectively, both within the range of 0.5 to 2 times.

[0173] When beagle dogs were given 6 mg / kg of the drug intragastricly for 7 consecutive days, males and females showed different results. Beagle C at the 7th and 1st doses max and AUC 0-t The ratios were 0.964 and 0.887, respectively, and no significant drug accumulation was observed.

[0174] Based on the above, when beagle dogs were administered intragastricly at doses of 2, 6, and 20 mg / kg, no significant difference in systemic exposure was observed between males and females. In the dose range of 2 to 20 mg / kg, systemic exposure in the experimental animals increased linearly in a dose-dependent manner, and no significant drug accumulation was observed after 7 consecutive days of administration. Example 10: Pharmacokinetic study of hydrochloride crystalline form A

[0175] 1. Objective of the experiment This experiment aims to study the pharmacokinetic properties of hydrochloride crystalline form A (prepared in Example 2, sometimes simply referred to as the drug in this example) in the bodies of male and female beagle dogs.

[0176] 2. Laboratory animals The beagle dogs (male, n=3, 31 months old) used in this experiment were purchased from Mas Biotechnology Co., Ltd., and their animal quality certificate number is 110318201100056783.

[0177] 3. Preparation of the formulation Solvent for the intragastric administration group: 0.5% CMC-Na aqueous solution 260.20 mg of hydrochloride crystal form A (prepared in Example 2) was weighed into a glass bottle, 193.827 mL of 0.5% CMC-Na aqueous solution was added, vortexing and ultrasonic treatment were performed until the particles were dissolved to obtain a colorless solution with a final concentration of 1.2 mg / mL, which was administered to animals in Group A.

[0178] 4. Grouping of experimental groups In this experiment, 3 male Beagle dogs were randomly selected and administered with the drug, and the administration method is shown in Table 28. All animals were fasted for at least 12 hours before administration, and were fed again 4 hours after administration. During the experiment, all animals had free access to water.

[0179]

Table 28

[0180] Animals in Group A were intragastrically administered with 6 mg / kg of the drug once, and blood was collected from the jugular vein before administration and 0.083, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 hours after administration.

[0181] Blood samples from the above animals were collected through the jugular vein at a volume of 0.5 mL per collection. All collected whole blood samples were placed into centrifuge tubes containing EDTA-K2, and the centrifuge tubes were inverted to thoroughly mix the anticoagulant and blood. All samples were placed on wet ice before centrifugation, and centrifuged at 1500 g for 10 minutes to separate plasma. Plasma samples were transferred into sample tubes and stored at -40 to -20°C until analysis.

[0182] The drug concentration in the plasma of Beagle dogs was detected by LC-MS / MS method. The LC-MS / MS detection method is as follows.

[0183] Instrument: LC-MS / MS (Triple Quad 5500+: LC-MS-MS-023). MS conditions: ESI positive ion mode; multiple reaction monitoring (MRM), m / z 336.2 / 149.1 Chromatography column: Waters Xselect HSS T3, 3.5 μm, 2.1 × 50 mm Mobile phase A: H2O (0.1% formic acid + 5 mM NH4OAc) Mobile phase B:MeOH:ACN=1:1 (0.1% formic acid) Flow rate: 0.50mL / min Injection volume: 5μL Holding time: 1.24min The elution program is shown in Table 29.

[0184] [Table 29] 6. Experimental Results

[0185] Clinical observations of the animals before administration, before and after each blood sampling point, and after administration revealed no obvious abnormalities. The drug concentration-time curve is shown in Figure 34, and the main pharmacokinetic parameters are shown in Table 30.

[0186] [Table 30]

[0187] As shown in the table above, when a single intragastric administration of 6 mg / kg of the drug was given to beagle dogs, T max The time interval is 0.417 hours, which indicates that the hydrochloride crystalline form A of the present invention reaches its peak more quickly in the body. In the case of the commercially available drug Apoquel (Apoquel®, active ingredient: oclacitinib), the time interval after oral administration to experimental dogs was 0.417 hours. max The incubation period was less than one hour (P7, CVMP assessment report for APOQUEL (EMEA / V / C / 002688 / 0000), EMA / 481054 / 2013). Compared to the commercially available drug Apoquel, the hydrochloride crystalline form A of this application was found to be superior or at least equivalent in terms of the time it takes to reach the peak in vivo. Example 11: Acute toxicity test of free crystalline form A

[0188] 1. Purpose of the Experiment After a single oral administration of the free crystalline form A of the test substance (prepared in Example 1), the toxic reactions produced by the experimental animals were observed in a short period of time, to obtain a preliminary understanding of the toxicity characteristics and dose-response relationship of the test substance.

[0189] 2. Experimental Animals The Sprague-Dawley rats (SPF grade) used in this experiment were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., with production license number SCXK (Zhejiang) 2019-0001, animal certificate numbers 20220107Aazz0619000738 and 20220107Aazz0619000691. The body weight range of male rats was 206.6~234.5g, and the body weight range of female rats was 188.4~205.9g.

[0190] 3. Preparation of Formulation Solvent for intragastric administration: 0.5% CMC-Na aqueous solution: Weigh 17.5031 g of CMC-Na (800~1200 mPa·s) into a suitable container, add 3500 mL of deionized water, stir until uniform, to obtain a colorless clear liquid, which was stored at room temperature until use. 100 mg / mL free crystalline form A solution: Weigh 9000.4 mg of free crystalline form A, place it in a wide-mouth bottle marked with a 90 mL line, add an appropriate amount of the above 0.5% CMC-Na (800~1200 mPa·s) aqueous solution to the container, stir until uniform and perform ultrasonic treatment, then continue to add an appropriate amount of 0.5% CMC-Na (800~1200 mPa·s) aqueous solution to reach a volume of 90 mL, stir until uniform and perform ultrasonic treatment. Sampling was performed as needed to obtain a milky white suspension. 55.9 mg / mL free crystalline form A solution: Place 44.7 mL of 100 mg / mL free crystalline form A solution into a suitable container, add 35.3 mL of 0.5% CMC-Na (800~1200 mPa·s) aqueous solution to the above container, and vortex until uniform. Sampling was performed as needed to obtain a milky white suspension. 44.7 mL of a 31.2 mg / mL free crystalline form A solution and a 55.9 mg / mL free crystalline form A solution were placed in a suitable container. 35.3 mL of a 0.5% CMC-Na (800-1200 mPa.s) aqueous solution was added to the container, and the mixture was vortexed until homogeneous. Sampling was performed as needed to obtain a milky white suspension. 44.7 mL of a 17.4 mg / mL free crystalline form A solution and a 31.2 mg / mL free crystalline form A solution were placed in a suitable container. 35.3 mL of a 0.5% CMC-Na (800-1200 mPa.s) aqueous solution was added to the container, and the mixture was vortexed until homogeneous. Sampling was performed as needed to obtain a milky white suspension. 9.7 mg / mL free crystalline form A solution: 44.7 mL of 17.4 mg / mL free crystalline form A solution was placed in a suitable container, and 35.3 mL of 0.5% CMC-Na (800~1200 mPa.s) aqueous solution was added to the container and vortexed until homogeneous. Sampling was performed as needed to obtain a white suspension.

[0191] 4. Experimental group assignment In this experiment, all animals were randomly divided into five groups (5 animals / sex / group) and administered 97, 174, 312, 559, and 1000 mg / kg of the drug as a single intragastric dose, followed by observation for 7 days. Grouping and administration information is shown in Table 31. All animals were administered based on their most recent body weight. The drug formulations needed to be stirred for at least 10 minutes before administration and must be continued throughout the administration process until the administration of samples at this concentration was complete. Animals were fasted for 11-12 hours prior to administration and re-fed approximately 2 hours after administration, but water intake was not restricted. All animals received a comprehensive physical examination by a veterinarian prior to dosing. During the acclimation period, cage-side observations were performed 1 to 2 times a day, and detailed clinical observations were performed once a day. During the experimental period, cage-side observations were performed twice a day (once on the day of dissection), and detailed clinical observations were performed 1 to 2 times a day. All surviving animals were weighed once randomly on the day of grouping (Day 1), before dosing on Day 1, Day 3, Day 5, Day 7, and Day 8. Food intake was measured once for 24±1 hours on Day 1 (Day 1 to Day 2), Day 2 (Day 2 to Day 3), and Day 6 (Day 6 to Day 7). At the end of the experimental period (Day 8), all surviving animals were euthanized by carbon dioxide inhalation and active abdominal blood collection / venous blood collection, followed by gross anatomical examination. Animals that died during the experiment also underwent gross anatomical examination.

[0192] [Table 31] 5. Experimental Results

[0193] The average recovery rates of the drug in test product preparations of various concentrations ranged from 105.8% to 109.8%, and the %RSD of recovery rates in the upper, middle and lower layers of low-concentration and high-concentration samples were 0.89 or less, which met the acceptance criteria of an average recovery rate of 100±15% at the theoretical concentration and %RSD≦10.

[0194] Under this experimental condition, after a single oral administration of the drug to SD rats, the LD50 in male rats was 592 mg / kg, the 95% confidence interval was 429 to 818 mg / kg, and the maximal tolerance dose (MTD) was 312 mg / kg. For female rats, the LD50 was 418 mg / kg, the 95% confidence interval was 313 to 558 mg / kg, and the maximal tolerance dose was 174 mg / kg. The LD50 of the commercially available drug Apoquel in rats is 310 mg / kg (P9, CVMP assessment report for APOQUEL (EMEA / V / C / 002688 / 0000), EMA / 481054 / 2013), indicating that the free crystal form A of the present invention has lower toxicity. Example 12 Study on urinary and fecal excretion after a single intragastric administration of free crystalline form A to beagle dogs

[0195] 1. Purpose of the experiment This experiment aims to study the excretion process of free crystalline form A (prepared in Example 1) into feces and urine of beagle dogs after a single oral administration to beagle dogs.

[0196] 2. Experimental animals The conventional beagle dogs used in this experiment were purchased from Beijing Mars Biotechnology Co., Ltd., with production license number: SCXK (Jing) 2016-0001, quality certificate numbers: 1103182011000078 (male) and 1103182011000079 (female). The experimental animals were housed in the animal facility of Suzhou Shengsu New Drug Development Co., Ltd. (license number: SYXK (Su) 2021-0045). The animal facility is well-ventilated, fully equipped with air conditioners, maintained at a temperature of 16~26°C and a humidity of 40%~70%, and uses artificial lighting with a 12-hour light period and a 12-hour dark period. On the day of the experiment, at the time of administration, the body weight of male beagle dogs ranged from 8.6 to 10.5 kg, and the body weight of female beagle dogs ranged from 7.6 to 9.0 kg.

[0197] 3. Preparation of formulation Solvent for intragastric administration: 0.5% CMC-Na aqueous solution Approximately 450.72 mg of free crystalline form A was weighed into a glass bottle, 378.605 mL of 0.5% CMC-Na aqueous solution was added, and the mixture was stirred and sonicated until the particles were dispersed, to obtain a white suspension with a final concentration of 1.2 mg / mL. The administration formulation was freshly prepared on the day of administration, and a sample was stored for the determination of formulation concentration.

[0198] 4. Grouping of experiments On the day before the experiment, 6 beagle dogs (half male and half female) were fasted overnight in metabolic cages. Before administration, urine and feces from each animal were collected as pre-dose 0-point samples. On the day of administration, the beagle dogs received a single intragastric administration of the administration formulation at a dose of 6 mg / kg. Four hours after administration, the animals were fed again, and water was provided ad libitum throughout the experiment.

[0199] 5. Sample collection and processing Urine and fecal excretion experiment group: Urine and feces were collected before administration and at 0-4, 4-8, 8-12, 12-24, 24-48, 48-72, 72-96, and 96-120 hours after administration. After urine collection was complete, the volume of urine was measured and recorded. A portion of the collected urine was diluted with methanol to a volume ratio of urine to methanol of 4:1 (v / v). After vortexing to homogenize, it was divided into two parts. After collecting fecal samples at different time periods, food residue on the surface was removed and the weight of the feces was measured. Depending on the weight of the feces, a certain volume of homogenate (20% methanol in water) was added, and the feces were homogenized to a ratio of feces to homogenate of 1:10 (fecal weight:homogenate volume, w / v), and then divided into two parts. After completing the collection of the final time point for the urine and fecal excretion experimental groups, the metabolic cages were washed with 2000 mL of water:ethanol (1:1, v:v), and the washing solution from each cage was collected separately. After shaking the washing solution well, 1 mL was immediately transferred to a 1.5 mL EP tube and stored in a refrigerator at -90 to 60°C along with the urine and fecal homogenates until sample analysis. Using LC-MS / MS, drug concentrations in excrement samples from each group of animals were detected over different periods, and the excretion rate and cumulative excretion rate for each period were calculated.

[0200] 6. Experimental Results

[0201] [Table 32]

[0202] When a single intragastric dose of 6 mg / kg was administered to beagle dogs, the cumulative excretion rate of the drug in urine within 120 hours was 43.8 ± 9.97%, and the cumulative excretion rate in feces within 120 hours was 3.48 ± 0.663%. The total cumulative excretion rate of the drug in the feces and urine of beagle dogs within 120 hours was 47.3 ± 9.62%. After 120 hours, the amount of drug in the cage cleaning solution of beagle dogs was 4.87 ± 3.09% of the administered dose. When experimental dogs orally ingested the drug Apoquel, the cumulative excretion rate in the urine of dogs within 24 hours was only 3.6%. P8, CVMP assessment report for APOQUEL (EMEA / V / C / 002688 / 0000), EMA / 481054 / 2013).

[0203] Based on the above, after a single intragastric administration of 6 mg / kg, the total excretion rate of the drug of the present invention in the body of beagle dogs was 52.2% (feces, urine, and lavage solution), and free crystalline form A was more stable in the body of dogs.

[0204] The above embodiments are used solely to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments can be modified or some or all of their technical features can be replaced with equivalents. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A crystal of crystalline form I of the compound represented by formula I, The crystal is characterized in that the XRPD pattern of the crystal form I has characteristic peaks at at least three positions among the positions where the 2θ value is 12.4°±0.2°, 14.6°±0.2°, 16.7°±0.2°, 17.2°±0.2°, 20.3°±0.2°, 24.8°±0.2°, and 25.0°±0.2°. 【Chemistry 1】

2. The crystal according to claim 1, wherein the XRPD pattern of the crystal form I further has characteristic peaks at at least three positions among the positions where the 2θ value is 13.2°±0.2°, 15.2°±0.2°, 19.3°±0.2°, 21.2°±0.2°, 21.6°±0.2°, 24.0°±0.2°, and 27.0°±0.2°.

3. The aforementioned crystal form I is, 【Chemistry 2】 The crystal according to claim 1, characterized by having the XRPD pattern shown.

4. A method for preparing crystals according to any one of claims 1 to 3, The preparation method is characterized by comprising the step of preparing crystalline form I from an amorphous compound represented by formula I as a starting material, and being one or more combinations selected from poor solvent addition, gas-solid diffusion, suspension stirring, slow volatilization, slow cooling, gas-liquid permeation, or poor-poor solvent addition.

5. A crystal of the hydrochloride salt of the compound represented by formula I, The XRPD pattern of the hydrochloride crystal form I is characterized by having characteristic peaks at at least three positions among the 2θ values ​​of 6.2°±0.2°, 10.9°±0.2°, 12.3°±0.2°, 16.3°±0.2°, 17.2°±0.2°, 18.9°±0.2°, 19.4°±0.2°, 24.7°±0.2°, and 27.5°±0.2°. 【Transformation 3】

6. The crystal according to claim 5, wherein the XRPD pattern of the hydrochloride crystal form I further has characteristic peaks at at least three positions among the positions where the 2θ values ​​are 11.6°±0.2°, 15.0°±0.2°, 18.4°±0.2°, 21.3°±0.2°, 23.3°±0.2°, 24.3°±0.2°, 25.6°±0.2°, 26.8°±0.2°, and 30.0°±0.2°.

7. The hydrochloride crystalline form I is, 【Chemistry 4】 The crystal according to claim 5, characterized by having the XRPD pattern shown.

8. The hydrochloride crystalline form I is, 【Transformation 5】 The crystal according to claim 5, characterized by having the DSC pattern and TGA pattern shown.

9. A method for preparing crystals according to any one of claims 5 to 8, The process includes the step of mixing the crystal described in claim 1 with hydrochloric acid, adding a solvent, and obtaining crystals of hydrochloride salt crystal form I. A preparation method characterized by the above.

10. A pharmaceutical composition comprising a crystal according to any one of claims 1 to 3 or a crystal according to any one of claims 5 to 8, and one or more pharmaceutically acceptable auxiliary materials.

11. Use of the crystal according to any one of claims 1 to 3 or the crystal according to any one of claims 5 to 8 in the preparation of a drug for treating a JAK-STAT pathway-related disease.

12. Use of the crystal according to any one of claims 1 to 3 or the crystal according to any one of claims 5 to 8 in the preparation of a drug for preventing and / or treating inflammatory diseases, tumors, autoimmune diseases and allergic diseases in humans and / or animals.

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