Crystalline forms of compounds, and methods for their preparation 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] The present invention relates to the field of research into chemical crystalline polymorphism of drugs, and in particular to crystalline forms of the compound of formula I, as well as methods for their preparation and use. [Background technology]

[0002] The JAK-STAT signaling pathway is a cytokine-stimulated signaling pathway that is involved in many important biological processes, such as cell proliferation, differentiation, apoptosis, and immune regulation. Compared with other signaling pathways, the transduction process of this pathway is relatively simple. It is mainly composed of three components: tyrosine kinase-associated receptors, tyrosine kinase JAK, and transcription factors STAT.

[0003] JAK inhibitors selectively inhibit JAK kinases and block the JAK-STAT pathway. Currently, FDA-approved JAK inhibitor drugs include tofacitinib, ruxolitinib, oclacitinib, and baricitinib. While oclacitinib has a relatively good therapeutic effect on allergic skin diseases in pet dogs, it has little effect on cytokines not involved in JAK1 activation. Its effect on allergic responses is limited to suppressing the release of allergic mediators, but it cannot fundamentally block the binding of allergic mediators to their associated receptors. This prevents it from fundamentally inhibiting the development and progression of allergic skin diseases, limiting its application. Another JAK inhibitor, baricitinib, is a selective inhibitor of JAK1 and JAK2 with IC50 values ​​of 5.9 nM and 5.7 nM, respectively, which makes it approximately 70-fold and 10-fold more selective than its action on JAK3 and Tyk2, respectively. However, it has no inhibitory effect on c-Met or Chk2, and its indication is relatively single.

[0004] Chinese Patent CN111499641B discloses a JAK inhibitor and a method for preparing the same, which has relatively excellent JAK inhibitory activity and is expected to be used for the prevention and / or treatment of inflammatory diseases and cancer in humans and / or animals. While therapeutic activity is the most important property for a therapeutic agent, the solid form (i.e., crystalline or amorphous form) of a drug candidate is also important for the pharmacological properties and development of its practical API. Summary of the Invention [Problem to be solved by the invention]

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

[0006] The present invention provides crystalline Form I of the compound of formula I. [ka]

[0007] The XRPD pattern of Form I (using Cu-Kα radiation) has characteristic peaks at at least three (e.g., three, four, five, six, seven, particularly all) of the following 2θ values: about 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] Additionally, the XRPD pattern (using Cu-Kα radiation) of Form I further comprises characteristic peaks at at least three (e.g., three, four, five, six, seven, particularly all) of the following 2θ values: about 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] Furthermore, the crystalline form I has an XRPD pattern approximately as shown in FIG.

[0010] Furthermore, the DSC pattern of the crystalline form I has endothermic peaks at 195 to 210°C (for example, around 195°C, 200°C, 205°C, and 210°C).

[0011] Furthermore, the crystalline form I has a DSC pattern approximately as shown in FIG.

[0012] Furthermore, the TGA pattern of the crystalline form I shows a weight loss of 1.7% (e.g., 1.0%, 1.5%, 1.7%, 2.0%, 2.5%, 3.0%) when heated from 30°C to 180°C.

[0013] Furthermore, the crystalline form I has a TGA pattern approximately as shown in FIG.

[0014] Furthermore, the crystalline form I is an anhydrous crystalline form.

[0015] Furthermore, when observed under a polarizing microscope, the crystalline form I appears as needle-like and / or long plate-like crystals.

[0016] The present invention further provides a method for preparing crystalline Form I, starting from an amorphous form of the compound of Formula I, by one or more combinations of anti-solvent addition method, gas-solid diffusion method, suspension stirring method, slow evaporation method, slow cooling method, gas-liquid permeation method, or anti-anti-solvent addition method to prepare crystalline Form I.

[0017] Furthermore, the specific steps of the anti-solvent addition method include 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] Furthermore, the good solvent in the poor solvent addition method is one selected from methanol, 1,4-dioxane, chloroform, and dimethyl sulfoxide.

[0019] Furthermore, 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, and water.

[0020] In some embodiments of the present invention, in the anti-solvent addition method, the good solvent is methanol, and the anti-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 anti-solvent addition method, the good solvent is 1,4-dioxane, and the anti-solvent is selected from n-heptane and ethyl acetate.

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

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

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

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

[0026] Furthermore, the specific steps of the room temperature suspension stirring method include suspending the amorphous raw material of the compound of Formula I in a solvent and stirring at room temperature until a solid precipitates.

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

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

[0029] Furthermore, the specific steps of the slow evaporation method include dissolving the amorphous raw material of the compound of Formula I in a solvent, shaking and filtering, and then collecting the filtrate and slowly evaporating it at room temperature until a solid precipitates.

[0030] Furthermore, specific steps of the slow cooling method include dissolving an amorphous raw material of the compound represented by Formula I in a solvent, stirring the solution at 40-60°C, filtering the solution when 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 placing an amorphous raw material of the compound represented by Formula I in an open container, dissolving it in a solvent, and then placing the open container in a sealed container containing a poor solvent and leaving it to stand at room temperature until a solid precipitates.

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

[0033] The present invention further provides crystalline Form I of the hydrochloride salt of the compound of formula I.

[0034] Additionally, the XRPD pattern of the hydrochloride salt crystalline Form I (using Cu-Kα radiation) has characteristic peaks at at least three (e.g., four, five, six, seven, eight, nine, particularly all) of the following 2θ values: 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] Additionally, the XRPD pattern (using Cu-Kα radiation) of the hydrochloride salt crystalline Form I further has characteristic peaks at at least three (e.g., four, five, six, seven, eight, nine, particularly all) of the following 2θ values: about 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 salt crystalline form I has an XRPD pattern approximately as shown in FIG.

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

[0038] Furthermore, the hydrochloride salt crystalline form I has a DSC pattern approximately as shown in FIG.

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

[0040] Furthermore, the hydrochloride salt crystalline form I has a TGA pattern approximately as shown in FIG.

[0041] Furthermore, when observed under a polarizing microscope, the hydrochloride salt crystalline form I is a rod-shaped crystal.

[0042] The present invention further provides a process for preparing crystalline Form I of the hydrochloride salt of compound of formula I, which comprises mixing crystalline Form I of compound of formula I (e.g., as described in the present invention) with hydrochloric acid and adding a solvent (stirring, centrifugation) to obtain crystalline Form I of the hydrochloride salt.

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

[0044] Furthermore, the solvent is one selected from ethanol, acetone / water (for example, 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 the compound of Formula I and one or more pharmaceutically acceptable auxiliary materials.

[0046] Furthermore, the auxiliary material is one or more selected from the group consisting of a carrier, a diluent, a binder, a lubricant, and a wetting agent. Preferably, the pharmaceutical composition comprises a therapeutically effective amount of crystalline Form I or hydrochloride crystalline Form I of the compound of Formula I.

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

[0048] Furthermore, the pharmaceutical compositions are suitable for enteral or parenteral administration, such as intravenous, intramuscular, intradermal, and subcutaneous administration. Thus, the pharmaceutical compositions preferably also include aqueous and non-aqueous sterile suspensions, which may contain antioxidants, buffers, bactericides, and solutes that render the formulation isotonic with the recipient's blood, etc., as well as suspending aids, solubilizers, thickeners, stabilizers, and preservatives.

[0049] Furthermore, the pharmaceutical composition may be prepared into a pharmaceutical preparation in the form of a syrup, elixir, suspension, powder, granules, tablets, capsules, lozenges, aqueous solution, cream, ointment, lotion, gel, emulsion, or the like.

[0050] Furthermore, the pharmaceutical preparation is preferably in unit dosage form.In this form, the preparation can be further divided into unit doses containing appropriate amounts of active ingredients.The unit dosage form can be capsules, tablets or any dosage form.The unit dosage form can also be packaged preparations such as tablets, capsules and powders packaged in vials or ampoules.

[0051] The quantity of active ingredient in said unit dose preparation may be varied or adjusted from 0.1 mg to 1000 mg depending on the particular application and potency of the active ingredient. If desired, the composition may also contain other suitable therapeutic agents.

[0052] The present invention further relates to the use of crystalline Form I or the hydrochloride crystalline Form I of the compound of formula I in the preparation of a medicament for treating a JAK inhibitor, Janus-activated kinase signal transducers and activators of transcription (JAK-STAT) pathway-related disease. provide.

[0053] The present invention further provides the use of crystalline Form I or the hydrochloride crystalline Form I of the compound of Formula I in the treatment of JAK inhibition, Janus-activated kinase signal transducer and activator of transcription pathway-related disorders.

[0054] Furthermore, the disease is 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 of formula I in the preparation of a medicament for the prevention and / or treatment of 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 of formula I in the prevention and / or treatment of inflammatory diseases, tumors, autoimmune diseases and allergic diseases in humans and / or animals.

[0057] Further, 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 further, the cancer is selected from myelofibrosis, polycythemia vera, essential thrombocythemia, chronic myeloid leukemia cells, breast cancer, lung cancer, or pancreatic cancer.

[0059] Further, 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 itchy skin, red rash, hair loss, scaling, edema, ulcers, etc., and may sometimes be accompanied by symptoms such as otitis, sneezing, and tearing, particularly itchy skin. In one embodiment of the present invention, the disease is allergic itchy skin in dogs. [Effects of the Invention]

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

[0063] Based on previous research findings, the present inventors further conducted experimental studies to obtain the free crystalline form and hydrochloride crystalline form of the compound of Formula I. These crystals not only exhibit sharp diffraction peaks, relatively high crystallinity, relatively little residual solvent, a single DSC thermal signature, and low TGA weight loss, but also have a relatively high ligand safety level. Therefore, they all have relatively good solid-state stability and flowability, relatively low hygroscopicity, and relatively good solubility, which are advantageous for the preparation and storage of pharmaceutical formulations and are expected to have excellent application value and drugability. Furthermore, clinical trials have shown that the free crystalline form and hydrochloride crystalline form described in the present invention have excellent pharmacokinetic properties, with systemic exposure increasing linearly in a dose-dependent manner in experimental animals, rapid peak blood drug concentrations, high in-vivo stability, no significant drug accumulation after repeated administration, low clinical toxicity, and relatively excellent safety, significantly superior to positive control drugs. In addition, the free crystalline form and hydrochloride crystalline form described in the present invention have high oral bioavailability and excellent therapeutic effects against various diseases, such as excellent inhibitory and antipruritic effects against allergic pruritus symptoms in dogs. Furthermore, the free crystalline form and hydrochloride crystalline form described in the present invention can be administered by various routes (e.g., injection, oral administration) and can be formulated into various dosage forms, and their future applications are highly promising. [Brief explanation of the drawings]

[0064] [Figure 1] 1 shows an XRPD chart of free crystalline form A. [Figure 2] 1 shows a TGA / DSC chart of free crystalline form A. [Figure 3] 1 shows the 1H NMR spectrum of free crystalline form A. [Figure 4] 1 shows an XRPD chart of crystalline form A of the hydrochloride salt. [Figure 5] 1 shows a TGA / DSC chart of crystalline form A of the hydrochloride salt. [Figure 6] 1 shows the 1H NMR spectrum of the hydrochloride salt crystalline form A. [Figure 7]1 shows an XRPD chart of crystalline form A of the sulfate salt. [Figure 8] 1 shows a TGA / DSC chart of crystalline form A of the sulfate salt. [Figure 9] 1 shows the 1H NMR spectrum of sulfate salt crystalline form A. [Figure 10] 1 shows an XRPD chart of crystalline form A of the phosphate salt. [Figure 11] 1 shows a TGA / DSC chart of crystalline form A of the phosphate salt. [Figure 12] 1 shows the 1H NMR spectrum of phosphate salt crystalline form A. [Figure 13] 1 shows a sample XRPD chart for the solubility of free crystalline form A in HO. [Figure 14] 1 shows a sample XRPD chart for the solubility of free crystalline form A in SGF. [Figure 15] 1 shows a sample XRPD chart for the solubility of free crystalline form A in FaSSIF. [Figure 16] 1 shows an XRPD chart of a sample for the solubility of free crystalline form A in FeSSIF. [Figure 17] 1 shows an XRPD chart of a sample relating to the solubility of hydrochloride salt crystalline form A in pH buffer solutions. [Figure 18] 1 shows a sample XRPD chart for the solubility of free crystalline form A in pH buffer solutions. [Figure 19] 1 shows a DVS chart of the hydrochloride salt crystalline form A. [Figure 20] 1 shows a DVS chart of free crystalline form A. [Figure 21] 1 shows an XRPD overlay of the hydrochloride salt crystalline form A before and after DVS testing. [Figure 22] 1 shows an XRPD overlay of free crystalline form A before and after DVS testing. [Figure 23] 1 shows XRPD overlays of hydrochloride salt crystalline form A before and after stability evaluation. [Figure 24] 1 shows XRPD overlays of free crystalline form A before and after stability evaluation. [Figure 25]Figure 1 shows the UPLC diagram of stability evaluation of hydrochloride crystalline form A (Note: Shifts in the main peak appearance time may occur due to changes in system pressure during testing at different times. For each test, the starting sample is tested for its API peak appearance time.) [Figure 26] Figure 1 shows the UPLC diagram of stability evaluation of free crystalline Form A (Note: Shifts in the appearance time of the main peak may occur due to changes in system pressure during testing at different times. For each test, the starting sample is tested for its API peak appearance time.) [Figure 27] 1 shows a PLM chart of crystalline form A of the hydrochloride salt. [Figure 28] 1 shows a PLM chart of free crystalline form A. [Figure 29] 1 shows XRPD charts of hydrochloride salt crystalline form A before and after milling and tableting. [Figure 30] 1 shows XRPD charts of free crystalline form A before and after milling and tableting. [Figure 31] 1 shows the blood concentration-time curve (logarithmic coordinate) in beagle dogs administered 2 mg / kg of free crystalline form A intravenously. [Figure 32] 1 shows the blood concentration-time curves in beagle dogs after intragastric administration of various doses of free crystalline form A. [Figure 33] 1 shows the blood concentration-time curves in beagle dogs administered 6 mg / kg of free crystalline form A multiple times (intragastric administration at the 1st and 7th times). [Figure 34] 1 shows the blood concentration-time curves in male beagle dogs after intragastric administration of 6 mg / kg of crystalline hydrochloride form A. DETAILED DESCRIPTION OF THE INVENTION

[0065] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0066] In the present 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 error is due to instrument conditions, sample preparation, sample purity, and the like. In particular, those skilled in the art are well aware that X-ray diffraction patterns typically vary depending on instrument conditions. It is particularly important to note that the relative intensities of X-ray powder diffraction patterns may also change depending on changes in experimental conditions, so the order of peak intensities cannot be used as the sole determinant. In practice, the relative intensities of diffraction peaks in an XRPD pattern are related to the preferred orientation of the crystal, and the peak intensities shown herein are for illustrative purposes, not absolute comparisons. Furthermore, experimental errors in peak angles are typically less than 5%, and errors in these angles must also be taken into account; an error of ±0.2° is typically acceptable. Experimental factors such as sample thickness can cause an overall shift in peak angle, but a certain degree of shift is typically acceptable. Therefore, the X-ray powder diffraction pattern of the crystalline form of the present invention may not be completely identical to the X-ray powder diffraction patterns of the examples mentioned herein. It is understood by those skilled in the art that the "same XRPD pattern" described herein does not mean that the XRPD patterns are completely identical; the positions of the same peaks may differ by ±0.2°, and some degree of variation in peak intensity is acceptable. All crystalline 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 an unknown crystalline form and determine whether the two sets of patterns reflect the same crystalline form or different crystalline forms. In some embodiments, crystalline form A of the present invention is pure and single, and is substantially not mixed with other crystalline forms. In the present invention, "substantially free" of a new crystalline form means that this crystalline 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 crystalline forms.

[0067] It should be noted that the numerical values ​​and numerical ranges stated in the present invention should not be narrowly interpreted as the numerical values ​​or numerical ranges themselves. Those skilled in the art should understand that specific numerical values ​​can be varied based on different specific technical environments without departing from the spirit and principles of the present invention. In the present invention, the range of variation that a person skilled in the art can predict is primarily expressed by the term "about." When the term "about" is used before a numerical value in the present invention to refer to the numerical value, it means any value within a range of ±10%, preferably within a range of ±5%, more preferably within a range of ±2%, and even more preferably within a range of ±1% of the numerical value. For example, "about 10" should be interpreted as meaning 9 to 11, preferably 9.5 to 10.5, more preferably 9.8 to 10.2, and even more preferably 9.9 to 10.1.

[0068] In X-ray diffraction patterns of powder samples, the diffraction spectrum obtained from a crystalline compound is often characteristic of a specific crystalline form. However, the relative intensities of spectral bands (especially at low angles) can vary due to predominant orientation effects caused by differences in crystallization conditions, particle size, relative content of the mixture, and other test conditions. Therefore, the relative intensities of diffraction peaks are not characteristic of the target crystal, and when determining whether a known crystalline form is the same, more attention should be paid to the peak positions rather than the relative intensities of the peaks.

[0069] In the present invention, the term "room temperature" means that the temperature of an object is close to or the same as the temperature of the space (e.g., the temperature of the draft chamber in which the object is placed). Typically, room temperature is about 20°C to about 30°C, or about 22°C to 27°C, or about 25°C.

[0070] Anti-solvent crystallization (also known as anti-solvent addition, precipitation crystallization, salting out, or forced crystallization) is a method in which one or more anti-solvents are added to a solution of a desired product in a good solvent, causing the product to become slightly dissolved in the solution, resulting in the solution becoming supersaturated and crystals being precipitated. Anti-anti-solvent crystallization is a method in which one or more anti-solvents are added to a solution of a desired product in a good solvent, causing the product to become slightly dissolved in the solution, resulting in the solution becoming supersaturated and crystals being precipitated.

[0071] The term "poor solvent" in a system is relative; a poor solvent has a lower ability to dissolve the target product than a good solvent, e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% or more. The good solvent and poor solvent can be polar or nonpolar solvents, such as dimethylformamide (DMF), dimethyl sulfoxide (DMSO), water, alcoholic solvents, ether solvents, ketone solvents, ester solvents, alkane solvents, aromatic hydrocarbon solvents, and nitrile solvents. 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. Ether 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] Antisolvent and anti-antisolvent crystallization can be performed in batch, semi-batch, or continuous crystallization procedures. The antisolvent can be added to the solution (antisolvent crystallization) or the product solution to the antisolvent (anti-antisolvent crystallization) at a constant rate or slowly at first and then gradually increasing the rate.

[0073] In the present invention, unless otherwise specified, the term "animal" as used herein refers to non-human animals, particularly mammals such as monkeys, pigs, cows, sheep, horses, donkeys, dogs, cats, rabbits, rats, foxes, raccoon dogs, minks, camels, etc. In some embodiments of the present invention, the animal is a pet such as a dog, cat, or rodent (e.g., chinchilla, pet rabbit, hamster, guinea pig, mouse, gerbil, chipmunk, squirrel, flying glider, chipmunk, curly-haired guinea pig, degu, etc.).

[0074] The term "treatment" means preventing, curing, reversing, alleviating, ameliorating, minimizing, inhibiting, arresting, and / or halting one or more clinical symptoms of a disease after the onset of the disease.

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

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

[0077] The term "tumor" refers to an abnormal mass of tissue whose growth exceeds and is not in sync with normal tissue growth. Tumors can be either "benign" or "malignant" depending on characteristics such as the degree of cellular differentiation (morphological and functional), growth rate, local invasion, and metastasis. Benign tumors are typically well differentiated, grow slower 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 can later become malignant due to further genetic alterations in a subpopulation of tumor cells; these tumors are called "premalignant tumors." Malignant tumors are typically poorly differentiated (anaplastic) and exhibit characteristically rapid growth, accompanied by progressive infiltration, invasion, and destruction of surrounding tissue. Furthermore, malignant tumors typically have the ability to metastasize to distant sites.

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

[0079] The term "autoimmune disease" refers to diseases caused by the body's immune response to its own antigens, resulting in damage to its own tissues. The American Autoimmune Related Diseases Association has compiled a comprehensive list of autoimmune diseases.

[0080] The disclosures of various publications, patents and published patent specifications cited herein are hereby incorporated by reference in their entireties.

[0081] Hereinafter, embodiments of the present invention will be described in detail with reference to examples. However, it will be understood by those skilled in the art that the following examples are for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. Unless specific conditions are specified, the examples were carried out according to conventional conditions or conditions recommended by the manufacturer. All reagents or instruments for which the manufacturer is not specified are commercially available.

[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; EtOAc: 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 examples is as follows: [ka]

[0084] Its preparation is as follows.

[0085] Step 1: Synthesis of Compound 2 [ka] Compound 1 (500 g, 2.58 mol), dichloromethane (1 L), and purified water (1 L) were sequentially added to a 5 L three-neck flask at room temperature, and mechanical stirring was initiated. Sodium bicarbonate (400 g, 4.76 mol) and di-tert-butyl dicarbonate (650 g, 2.98 mol) were then sequentially added. After the addition was complete, the mixture was stirred at room temperature for 4 hours, and dichloromethane (1 L) and purified water (1 L) were added, followed by separation. The aqueous phase was extracted with dichloromethane (800 ml × 2), the combined organic phases were washed with saturated brine (1000 ml × 1), the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure at 40 ° C until no more distillates evaporated, petroleum ether (500 ml) was added, the mixture was slurried at room temperature for 0.5 hours, filtered under reduced pressure, the filter cake was collected and air-dried at 25 ° C for 16 hours to obtain 513 g of a white solid (yield 77%).

[0086] Second step: Synthesis of compound 3 [ka] Compound 2 (513 g, 2.58 mol) and anhydrous DMF (2 L) were added to a 5 L three-neck flask at room temperature and mechanical stirring was initiated. The mixture was purged with nitrogen under reduced pressure three times. The temperature was controlled at 10°C using an ice bath, and sodium hydride (103.8 g, 2.59 mol) was added batchwise without any significant exothermic reaction. After the addition was complete, the mixture was stirred for 0.5 hours, heated in hot water, and then returned to room temperature. Methyl iodide (340 g, 2.40 mol) was added without any significant exothermic reaction. After the dropwise addition was completed over 100 minutes, the mixture was slowly heated to 36-38°C. The reaction proceeded vigorously, releasing a large amount of heat (uncontrollably). The mixture was then cooled to room temperature and stirred overnight. The reaction mixture was slowly added to 2 L of saturated NH4Cl solution (temperature controlled below 20°C), extracted with EA (1 L x 3), the organic phase was washed with saturated NaCl (500 mL x 3), and concentrated to give 550 g of an oily substance, which was subjected to wet column chromatography and eluted with PE / EA = 10 / 1 to 5 / 1 to give 517 g of a colorless oily substance (yield 95.9%).

[0087] Third step: Synthesis of compound 4 [ka] Anhydrous tetrahydrofuran (300 mL) was added to a 5 L three-necked flask, and the temperature was controlled at 10-15°C using an ice bath. Stirring was started under N2 protection, and lithium aluminum tetrahydride (46 g, 1.21 mol) was decomposedly added. The temperature was controlled below -5°C, and a solution of 3 (516 g, 1.90 mol) in anhydrous tetrahydrofuran (1.5 L) was added dropwise. After the addition was complete, the reaction was continued for 2.5 h. The temperature was controlled at 0-10°C, and 15% sodium hydroxide solution (46 After completion of the addition, the mixture was suction filtered under reduced pressure, and the filter cake was rinsed with 500 mL of ethyl acetate. After completion of the suction filtration, the filtrate was collected, and 500 mL of ethyl acetate and 1,000 mL of water were added to the filtrate, followed by separation. The organic phase was collected, and the aqueous phase was further extracted with EA (500 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (500 mL × 3), dried, and concentrated to obtain 407 g of a colorless oil (yield 88.1%).

[0088] Step 4: Synthesis of Compound 5 [ka] 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-neck flask at room temperature, and stirring was initiated under nitrogen protection. The temperature was controlled at 10-15°C using an ice bath, and methanesulfonyl chloride (250 g, 2.18 mol) was added dropwise. After the addition was complete, the mixture was stirred at room temperature overnight, 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 give 510.2 g of a yellow oil (yield 94.5%).

[0089] Step 5: Synthesis of Compound 6 [ka] 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-neck flask at room temperature, stirred, heated to reflux overnight, cooled to room temperature, quenched by adding water (1000 ml), extracted with ethyl acetate (1 L x 3), washed with 2 L saturated NaCl, dried, and concentrated to give 423 g of oil, which was used directly in the next step. The yield was 75.8%.

[0090] Step 6: Synthesis of Compound 8 [ka] Compound 7 (35 g, 0.289 mol) and anhydrous tetrahydrofuran (1100 ml) were added to a 5 L three-neck flask at room temperature, and stirring was initiated. The temperature was lowered to 0-5°C, and sodium hydride (27.5 g, 0.687 mol) was added in batches, resulting in vigorous heat release. After the addition was completed, a THF solution (1000 mL) of pivalic anhydride (51.2 g, 0.275 mol) was added dropwise, and the temperature was controlled below 20°C. The reaction was allowed to proceed at room temperature for 2 hours. 100 mL of methanol was then added to the reaction mixture. The temperature was controlled at 0-20°C, and 600 mL of aqueous NH4Cl and 600 mL of saturated saline were added dropwise to quench the reaction. The mixture was then extracted with ethyl acetate (500 mL x 3). The organic phase was washed with 1 L of saturated NaCl, dried, and concentrated to obtain an oily substance. This was then subjected to column chromatography (PE / EA = 5 / 1, 3 / 1). The resulting solid was slurried with 50 mL of petroleum ether to obtain 45.8 g of a 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 initiated. The temperature was lowered to 10-20 ° C., and sodium hydride (10.5 g, 0.26 mol) was added batchwise. After the addition was completed, ethyl iodide (68.4 g, 1.44 mol) was added dropwise at room temperature. After the addition was completed, the mixture was stirred at 40 ° C. overnight, and then an aqueous ammonium chloride solution (100 mL) was added to the system for quenching. The aqueous phase was extracted with ethyl acetate (100 mL × 3), washed with 200 mL of saturated NaCl, dried, and concentrated to obtain an oily substance. Column chromatography (PE / EA) was performed at PE / EA = 10 / 1, 7 / 1, 5 / 1, and 3 / 1 to obtain 45.26 g of a white solid (88.3% yield).

[0092] Step 8: Synthesis of Compound 10 [ka] Compound 9 (45 g, 0.19 mol) and dichloromethane (200 mL) were added to a 1 L three-neck flask at room temperature, and stirring was started. Trifluoroacetic acid (33 g, 0.29 mol) was added at room temperature. After the addition was completed, the mixture was stirred at room temperature for 3 h. Then, saturated aqueous sodium chloride solution (100 mL) was added to the mixture to quench, and the mixture was extracted with dichloromethane (50 mL × 3). The mixture was then washed with 100 mL of saturated sodium bicarbonate, dried, and concentrated to obtain a solid crude product. The solid was subjected to column chromatography to obtain 27 g of a white solid (yield 80.2%).

[0093] Step 9: Synthesis of Compound 11 [ka] 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-neck flask at room temperature, and stirring was initiated. The temperature was lowered to 10-20°C, and sodium hydride (8.8 g, 0.22 mol) was added batchwise. After the addition was completed, a THF solution (50 ml) of compound 6 (103.8 g, 0.293 mol) was added dropwise at room temperature. After the addition was completed, the mixture was stirred at 50°C for 5 days, and then quenched with 100 mL of aqueous ammonium chloride solution. The mixture was extracted with EA (100 mL x 3). The organic phase was washed with saturated NaCl (200 ml), dried, and concentrated to obtain an oily product. The product was subjected to column chromatography with PE / EA = 10 / 1, 7 / 1, 5 / 1, and 3 / 1 sequentially to obtain 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-neck flask, and the temperature was controlled at 10 °C using an ice bath. Stirring was started under N2 protection, and lithium aluminum tetrahydride (8.8 g, 0.24 mol) was added batchwise. After the addition was completed, the mixture was reacted for 1 h. The temperature was controlled at 0-10 °C, and saturated sodium sulfate solution (24 mL) was added dropwise to quench the mixture. Then, 200 mL of ethyl acetate was added to dilute the mixture. The mixture was suction filtered under reduced pressure, and the filter cake was rinsed with 50 mL of ethyl acetate. After the suction filtration was completed, the filtrate was collected and 100 mL of water was added. The liquid was separated. The aqueous phase was extracted with ethyl acetate (50 mL × 2). The combined organic phases were washed with saturated sodium chloride solution (100 mL × 1), dried, and concentrated to obtain a colorless oil. This was eluted with ethyl acetate by column chromatography to obtain 18.5 g of an oil (yield 80%).

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

[0096] Step 12: Synthesis of Compound 15 [ka] Compound 13 (13.3 g, 0.058 mol), anhydrous DMF (100 ml), potassium carbonate (33.4 g, 0.24 mol), and compound 14 (18.7 g, 0.058 mol) were sequentially added to a 250 mL three-neck flask, and stirring was initiated. The mixture was heated to 115°C and reacted overnight. After cooling to room temperature, water (150 ml) was added to quench the reaction. The mixture was extracted with ethyl acetate (100 ml x 6), washed with saturated NaCl (500 ml x 3), and the organic phase was dried and concentrated to give an oil. This was then subjected to column chromatography using PE / EA = 1 / 1 and DCM / MeOH = 25 / 1 to give a semi-solid. This was slurried with 10 ml of ethyl acetate and 50 ml of n-hexane to give 22.4 g of an almost white solid (76.5% yield).

[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-neck flask, stirred, and refluxed overnight. The reaction mixture was cooled and concentrated until no further distillates were evaporated. Water (150 ml) and ethyl acetate (200 ml) were added sequentially to the system to separate the organic phase. The aqueous phase was then extracted with ethyl acetate (100 ml × 3), washed with saturated NaCl (500 ml × 1), dried over anhydrous sodium sulfate, and concentrated to obtain a solid crude product. The solid was separated by column chromatography and eluted with DCM / MeOH = 25 / 1. The mixture was then slurried with ethyl acetate (20 ml) to obtain 11 g of a white solid (73.3% yield). 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 Equipment and Methods 1.1.1 X-ray powder diffraction (XRPD) XRPD results were collected on a PANalytical X'Pert3 and Empyrean X-ray powder diffraction analyzer. Samples were tested by placing them in the center of a background-free silicon wafer, and the test parameters are listed in Table 1.

[0099] [Table 1] 1.1.2 Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC) TGA and DSC charts were collected on a TA Q5000 thermogravimetric analyzer and a TA Discovery 2500 differential scanning calorimeter, respectively. The test parameters are listed in Table 2. [Table 2] 1.1.3 Hydrogen Spectrum Liquid Nuclear Magnetic Resonance ( 1 H NMR)

[0100] Hydrogen Spectra Liquid nuclear magnetic resonance spectra were collected on a Bruker 400M NMR instrument using DMSO-d6 as the solvent. 1.1.4 Dynamic Water Sorption (DVS)

[0101] Dynamic moisture sorption (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 listed in Table 3.

[0102] [Table 3] 1.1.5 Ultra Performance Liquid Chromatography (UPLC)

[0103] The purity in the study was tested by 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, the free crystalline form A was obtained by the poor solvent addition method (Test No. 02-A1 shown in Table 6). The XRPD pattern of free crystalline form A is shown in FIG. 1, and the XRPD diffraction peak data is shown in Table 5. The TGA / DSC pattern of free crystalline form A is shown in Figure 2. The TGA results showed that the weight loss was 1.7% when heated from room temperature to 180°C. The DSC results showed a sharp endothermic peak at 197.4°C (onset temperature), which is presumed to be the melting signal. Free crystalline form A 1 The 1 H NMR pattern is shown in Figure 3. From the above characterization results, the free crystalline form A of Compound A showed relatively little TGA weight loss and a single DSC signal, which is inferred to be an anhydrous crystalline form.

[0106] [Table 5]

[0107] The specific experimental methods 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 to 1.0 mL of a good solvent (see Table 6). The anti-solvent in Table 6 was added to the clear solution and mixed dropwise until a solid precipitated. If no solid precipitated even after adding 5 mL of anti-solvent, the addition of the anti-solvent was discontinued. The precipitated solid was separated by centrifugation and subjected to XRPD testing. If no solid precipitated, the solution was transferred to 5°C and stirred, or allowed to evaporate at room temperature. The test results are shown in Table 6. In the anti-solvent addition test, free crystalline form A and an oily sample were obtained.

[0109] [Table 6] Example 2 Preparation and Characterization of Salt Crystal Forms of Compound A

[0110] 2.1 Equipment and Methods 2.1.1 X-ray powder diffraction (XRPD) XRPD results were collected on 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 on a TA Discovery 5500 thermogravimetric analyzer, and DSC results were collected on a TA Discovery 2500 differential scanning calorimeter. Table 8 shows the test parameters.

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

[0114] Hydrogen Spectra Liquid nuclear magnetic resonance spectra were collected on a Bruker 400M NMR instrument using DMSO-d6 as the solvent. 2.1.4 Dynamic Water Sorption (DVS)

[0115] Dynamic moisture sorption (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. DVS test parameters are shown in Table 9.

[0116] [Table 9] 2.1.5 Ultra Performance Liquid Chromatography (UPLC)

[0117] In the experiment, the 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 on a ZEISS Scope. A1 polarized light microscope. 2.1.7 Ion Chromatography (IC)

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

[0121] [Table 11] 2.2 Preparation and characterization of salt-type crystalline forms of Compound A

[0122] In this experiment, the free crystalline form A of Compound A was used as the starting material to investigate the possibility of forming corresponding salts with 23 different acids in four different solvents. The specific experiment is summarized in Table 12. The specific steps are as follows: First, approximately 20 mg of the 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 approximately 4 days to separate the solid, followed by XRPD detection. If a solid did not precipitate, the mixture was transferred to 5°C / -20°C and stirred, and either an anti-solvent was added or the mixture was evaporated at room temperature.

[0123] The study found that under 92 different experimental conditions, the free crystalline form A of Compound A was not easily converted into the corresponding salt crystalline form in an acidic system. XRPD comparison revealed that only three salt crystalline forms, hydrochloride crystalline form A, sulfate crystalline form A, and phosphate crystalline form A, could stably exist in four different solvents.

[0124] [Table 12-1] [Table 12-2] 2.2.1 Hydrochloride Crystalline Form A

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

[0126] [Table 13-1] [Table 13-2] 2.2.2 Sulfate Crystalline Form A

[0127] The XRPD results are shown in FIG. 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 exhibits a stepwise weight loss of 14.83% when heated to 100°C. The DSC results show that there are two endothermic signals at 56.9°C and 96.1°C (peak temperatures). 1 The H NMR results are shown in Figure 9. No residual solvent, acetone, was clearly detected.

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

[0129] The XRPD results are shown in FIG. 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% 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 temperatures). 1 The H NMR results are shown in Figure 12. No residual solvent, acetone, was clearly detected.

[0130] [Table 15]

[0131] Comparing the TGA / DSC charts of the free base crystalline form A, hydrochloride crystalline form A, sulfate crystalline form A, and phosphate crystalline form A, it was found that the free base crystalline form A and hydrochloride crystalline form A have relatively better thermal stability. The free base crystalline form A exhibited a dry weight loss of 1.7% when heated to 180°C, while the hydrochloride crystalline form A exhibited a dry weight loss of 0.53% when heated to 150°C. The melting points of both crystalline forms were 201.5°C and 216.4°C, respectively. A sample of sulfate crystalline form A exhibited a gradual weight loss of 14.83% when heated to 100°C. The DSC results showed two endothermic signals at 56.9°C and 96.1°C, indicating that sulfate crystalline form A is prone to decomposition or melting at relatively low temperatures and has relatively poor thermal stability. A sample of phosphate crystalline form A lost 2.82% weight when heated to 150°C, but DSC results showed three endothermic signals at 69.0°C, 149.0°C, and 163.2°C, indicating slightly poorer thermal stability.

[0132] From the above, compared with the sulfate crystalline form A and the phosphate crystalline form A, the free crystalline form A and the hydrochloride crystalline form A have the advantages of sharper crystal diffraction peaks, higher crystallinity, less residual solvent, single DSC thermal signal, and small TGA weight loss, as well as a higher ligand safety level. In the present invention, after testing under more than 100 experimental conditions, it was found that among the obtained crystalline forms, only the free crystalline form A and the hydrochloride crystalline form A have relatively good solid-state properties and can be developed as potential pharmaceutical crystalline forms. Example 3: Evaluation of dynamic solubility

[0133] The dynamic solubility of hydrochloride crystalline Form A and free crystalline Form A in HO and biological solvents (SGF, FaSSIF, and FeSSIF) was evaluated at 37°C. The specific steps are 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 medium (HO, SGF, FaSSIF, and FeSSIF), respectively. The mixture was mixed by rotation at 25 rpm in a rotary incubator at 37°C, and sampling points were 1, 2, 4, and 24 hours. At each sampling point, approximately 0.8 mL of the suspension was collected in a centrifuge tube and centrifuged (12,000 rpm, 3 min, 37°C). The supernatant was filtered through a PTFE filter membrane (pore size 0.45 μm), and the solubility and pH of the filtrate were tested, and the solid was measured by XRPD (see Example 2 for the equipment and method used).

[0134] The results of the dynamic solubility evaluation are summarized in Table 16. The hydrochloride crystalline form A was clear in HO, SGF, FaSSIF, and FeSSIF, with a solubility of over 9 mg / mL, while the free crystalline form A had a solubility of approximately 9-10 mg / mL in SGF and FeSSIF (the sample did not clear), and a relatively low solubility (2.1-2.4 mg / mL) in HO and FaSSIF.

[0135] The XRPD results for the solid samples separated at each sampling point are shown in Figures 13 to 16. These results show that the free crystalline form A did not undergo crystal transformation after 1, 2, 4, or 24 hours in HO, FaSSIF, and FeSSIF, while the diffraction peaks of the hydrochloride crystalline form A were observed after 24 hours in SGF. It was found that some of the free form A was due to Cl in the medium. - It was presumed that it reacted with the

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

[0137] The 24-hour solubilities of hydrochloride crystalline form A and free crystalline form A in pH 2.0, pH 4.5, and pH 7.4 buffer solutions were evaluated at room temperature. The specific steps were as follows: 10 mg of hydrochloride crystalline form A and free crystalline form A were weighed into HPLC vials and added to 1 mL of the corresponding buffer solution. The mixture was magnetically stirred at room temperature for 24 hours (approximately 500 rpm). The suspension was centrifuged (10,000 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, and the solid was measured by XRPD (see Example 2 for the equipment and method used).

[0138] The 24-hour solubility results are summarized in Table 17, and the XRPD results of the isolated solid samples are shown in Figures 17 and 18. These results show that (1) hydrochloride crystalline form A exhibited relatively high solubility at pH 2.0 and pH 4.5 (>9.2 mg / mL, the sample was clear). It converted to free crystalline form A at pH 7.4. (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 or pH 7.4, with solubilities of 6.2 mg / mL and 1.7 mg / mL, respectively. The XRPD results showed that the crystalline form remained unchanged after 24 hours of stirring.

[0139] [Table 17] Example 5 Evaluation of moisture absorption

[0140] The hygroscopicity of the hydrochloride crystalline form A and the free crystalline form A was evaluated by dynamic moisture sorption 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 method used).

[0141] The DVS results are shown in Figures 19 and 20. The hygroscopic weight gains of hydrochloride crystalline Form A and free crystalline Form A at 25°C / 80% RH were 0.47% and 0.39%, respectively, indicating that they are slightly hygroscopic. The XRPD comparison results of the samples before and after the DVS test are shown in Figures 21 and 22. The XRPD results showed that no crystalline transition occurred in either of the two samples after the DVS test.

[0142] [Table 18] Example 6 Evaluation of solid state stability

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

[0144] The XRPD comparison results are shown in Figures 23 and 24, the UPLC results are summarized in Tables 20 to 21, and the UPLC patterns are shown in Figures 25 and 26. The solid-state stability results showed that neither hydrochloride crystalline Form A nor free crystalline Form A underwent crystal transition or purity loss under the three evaluation conditions, 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 form and free crystalline form A, the powder properties of hydrochloride crystalline form A and free crystalline form A, including flowability and morphology, were evaluated.

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

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

[0151] The results of the angle of repose are summarized in Table 23. These results showed that the angles of repose of the hydrochloride crystalline form A and the free crystalline form A powder samples were 33.9° and 47.1°, respectively. Overall evaluation showed that both the hydrochloride crystalline form A and the free crystalline form A samples had relatively small Carr indices and angles of repose, indicating relatively good flowability.

[0152] The results of PLM are shown in Figures 27 and 28. Under a polarized microscope, hydrochloride crystalline form A was observed to be rod-shaped crystals, while free crystalline form A was observed to be needle-like or long plate-like crystals.

[0153] [Table 22]

[0154] [Table 23] Example 8 Evaluation of mechanical stability

[0155] The hydrochloride crystalline form A and the free crystalline form A were used as starting samples, which were manually crushed and compressed into tablets using a tablet press (pressure 350 MPa), respectively. The crushed and compressed samples were subjected to XRPD testing to evaluate their mechanical stability (see Example 2 for the equipment and method used). Specific results are summarized in Table 24.

[0156] The XRPD results before and after milling and tableting are shown in Figures 29 and 30. From these results, it was inferred that (1) after milling, the crystalline form and crystallinity of hydrochloride crystalline form A did not change, while the crystalline form of free crystalline form A did not change, but some diffraction peaks broadened, and the crystallinity may have slightly decreased. (2) After tableting, the crystalline forms of hydrochloride crystalline form A and free crystalline form A did not change, but the crystallinity of all of them slightly decreased (some diffraction peaks broadened and some weak diffraction peaks disappeared).

[0157] [Table 24]

[0158] Based on the above evaluation results, it was found that both hydrochloride crystalline form A and free crystalline form A have excellent overall physicochemical properties, such as relatively good solid stability and fluidity, relatively low hygroscopicity, and relatively good solubility, and can be developed as potential drug crystalline forms with promising future applications. Example 9 Pharmacokinetic study of free crystalline form A

[0159] 1. Purpose of the experiment This experiment was designed to study the pharmacokinetic properties of free crystalline Form A (prepared in Example 1 and sometimes referred to simply as the drug in this example) in male and female Beagle dogs and included: 1) a single intravenous (iv) study at 2 mg / kg; 2) a dose escalation study using intragastric (ig) administration at 2, 6, and 20 mg / kg; and 3) a 7-day intragastric oral administration study at a dose of 6 mg / kg.

[0160] 2. Experimental Animals The normal beagle dogs used in this experiment were purchased from Beijing Masu Biotechnology Co., Ltd. with license number SCXK(King)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 air-conditioned, with a temperature maintained between 16 and 26°C and humidity between 40 and 70%. Artificial lighting was used, with a 12-hour light and 12-hour dark cycle. At the time of initial dosing, male beagles weighed between 8.0 and 11.6 kg, while female beagles weighed between 6.9 and 11.3 kg.

[0161] 3. Preparation of Formulations

[0162] (1) Intravenous 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 the mixture was vortexed and sonicated until the particles were dissolved. Then, 7.517 mL of a 30% (w / v) aqueous solution of Solutol-HS was added and mixed by vortexing. Next, 60.136 mL of saline was added and mixed 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 the animals in Group A.

[0163] (2) Oral intragastric administration Vehicle 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, and 376.075 mL of 0.5% CMC-Na aqueous solution was added, followed by vortexing and sonication until the particles were dissolved, resulting in 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, and 375.483 mL of 0.5% CMC-Na aqueous solution was added, followed by vortexing and ultrasonication until the particles were dispersed, resulting in a white suspension with a final concentration of 1.2 mg / mL, which was administered to the animals in Group C on Day 1. The preparation process for the drugs administered to Group C on Days 2 to 7 was the same as that on Day 1. 1500.94 mg of free crystalline form A was weighed into a glass bottle, and 375.235 mL of 0.5% CMC-Na aqueous solution was added, followed by vortexing and ultrasonication until the particles were dispersed, resulting in a white suspension with a final concentration of 4 mg / mL, which was administered to animals in Group D. All dosage forms were prepared fresh on the day of administration, and samples were retained for determination of formulation concentration. In Group D, only samples of dosage forms on days 1, 4, and 7 were retained for determination of formulation concentration.

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

[0165] [Table 25]

[0166] 5. Sample Collection and Processing Animals in group A received an intravenous injection of 2 mg / kg of the drug, and blood samples were 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 received a single intragastric injection of 2 and 20 mg / kg of the drug, respectively. Blood samples were 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 received a single intragastric injection of 6 mg / kg of the drug for 7 consecutive days. Blood samples were collected from the jugular vein before the first and seventh doses and at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours after administration. Blood samples (jugular vein) were collected pre-dose and 0.5 hours post-dose during the second to sixth dose periods, and the pre-dose sample for the second dose was the sample 24 hours after the first dose. Blood samples from the animals were collected via the jugular vein at a volume of 0.5 mL per sample. All collected whole blood samples were placed in centrifuge tubes containing EDTA-K2 and inverted to thoroughly mix the anticoagulant with the blood. All samples were placed on wet ice before centrifugation and centrifuged at 1524 g for 10 minutes to separate the plasma. Plasma samples were transferred to new centrifuge tubes 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-compartmental model.

[0167] 6. Experimental Results The animals were clinically observed before administration, before and after each blood sampling time point, and after administration, and no obvious abnormalities were observed. 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 and 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 0-t The ratios were 0.758 and 0.748 (within the range of 0.5 to 2 times), respectively, and no significant difference was observed in the systemic exposure. The drug clearance (CL) in male and female beagle dogs was equivalent to 0.110 times the hepatic blood flow in beagles (approximately 31 mL / min / kg, Davies and Morris (1993)), indicating that clearance in the body of beagles is slow. The steady-state volume of distribution (V dss ) is 1.57 times the total body fluid volume of a beagle dog (approximately 0.60 L / kg, Davies and Morris (1993)), indicating a tendency for distribution to tissues.

[0169] [Table 27]

[0170] Following a single intragastric dose of 2, 6, or 20 mg / kg in beagle dogs, the mean time to peak concentration ranged from 0.792 to 1.33 hours after administration. After dose adjustment, the AUC 0-t The oral availability F% calculated from the mean values ​​was 88.0%, 100.0%, and 101.0%, respectively.

[0171] Beagle dogs were given a single intragastric dose of 2, 6, and 20 mg / kg of the drug, and the C max The ratios were 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 clear gender differences were observed in terms of systemic exposure (all ratios were within the range of 0.5-2).

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

[0173] When 6mg / kg of the drug was administered intragastrically to beagle dogs for 7 consecutive days, the C max and AUC 0-t The ratios were 0.964 and 0.887, respectively, and no obvious drug accumulation was observed.

[0174] In conclusion, no significant difference in systemic exposure was observed between males and females after intragastric administration of 2, 6, and 20 mg / kg to beagle dogs. Within the dose range of 2 to 20 mg / kg, systemic exposure in the bodies of experimental animals increased linearly in a dose-dependent manner, and no clear drug accumulation was observed after 7 consecutive days of administration. Example 10 Pharmacokinetic Study of Hydrochloride Crystalline Form A

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

[0176] 2. Experimental Animals The beagle dogs (male, n = 3, 31 months old) used in this experiment were purchased from Mas Biotechnology Co., Ltd., with the animal quality certificate number 110318201100056783.

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

[0178] 4. Experimental Grouping In this experiment, three male beagle dogs were randomly selected and administered the drug. The administration method is shown in Table 28. All animals were fasted for at least 12 hours before administration and re-fed 4 hours after administration. All animals had free access to water during the experiment.

[0179] [Table 28] 5. Sample Collection and Processing

[0180] The 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 animals were collected via the jugular vein at a volume of 0.5 mL per sample. All collected whole blood samples were placed in centrifuge tubes containing EDTA-K2, which were then inverted to thoroughly mix the anticoagulant with the blood. All samples were placed on wet ice before centrifugation and centrifuged at 1500 g for 10 minutes to separate the plasma. Plasma samples were transferred to 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. The LC-MS / MS detection method was 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: HO (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 dissolution program is shown in Table 29.

[0184] [Table 29] 6. Experimental Results

[0185] The animals were clinically observed before administration, before and after each blood sampling time point, and after administration, and no obvious abnormalities were observed. The drug concentration-time curves are 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 dose of 6 mg / kg of the drug was administered intragastrically to beagle dogs, T max The T was 0.417 hours, which indicates that the hydrochloride crystalline form A of the present application reaches its peak faster in the body. In the case of the commercially available drug Apoquel® (active ingredient: oclacitinib), the T after oral administration to laboratory dogs was max The time to peak in vivo was less than 1 hour (P7, CVMP assessment report for APOQUEL (EMEA / V / C / 002688 / 0000), EMA / 481054 / 2013). Compared with the commercially available drug APOQUEL, the hydrochloride crystalline form A of the present application was found to be superior or at least equivalent in terms of the time to 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 over a short period of time to obtain a preliminary understanding of the toxicological properties and dose-response relationship of the test substance.

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

[0190] 3. Preparation of Formulations Solvent for intragastric administration: 0.5% CMC-Na aqueous solution: 17.5031 g of CMC-Na (800-1200 mPa·s) was weighed into an appropriate container, 3500 mL of deionized water was added, and the mixture was stirred until homogeneous to obtain a colorless, clear liquid. This was then stored at room temperature until use. 100mg / mL free crystalline form A solution: 9000.4mg of free crystalline form A was weighed and placed in a 90mL wide-mouth bottle. An appropriate amount of 0.5% CMC-Na (800-1200mPa s) aqueous solution was added to the above container, and the solution was stirred and sonicated until homogenous. Further, appropriate amounts of 0.5% CMC-Na (800-1200mPa s) aqueous solution were added until the solution reached 90mL, and the solution was stirred and sonicated until homogenous. Sampling was performed as necessary to obtain a milky white suspension. 55.9 mg / mL free crystalline Form A solution: 44.7 mL of 100 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 above container and vortexed until homogenous. Sampling was performed as necessary to obtain a milky white suspension. 31.2 mg / mL free crystalline form A solution: 44.7 mL of 55.9 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 above container and vortexed until homogenous. Sampling was performed as necessary to obtain a milky white suspension. 17.4 mg / mL free crystalline form A solution: 44.7 mL of 31.2 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 above container and vortexed until homogenous. Sampling was performed as necessary 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 above container and vortexed until homogenous. Sampling was performed as necessary to obtain a white suspension.

[0191] 4. Experimental Grouping In this experiment, all animals were randomly divided into five groups (five animals per sex per group) and administered 97, 174, 312, 559, and 1000 mg / kg of drug, respectively, by single intragastric administration, followed by seven-day observation. Grouping and dosing information are shown in Table 31. All animals were dosed based on their most recent body weight. Pharmaceutical formulations must be stirred for at least 10 minutes before administration and should continue to be stirred throughout the administration process until the administration of the sample at this concentration is complete. Animals were fasted for 11–12 hours before administration and were re-fed approximately 2 hours after administration, but water intake was not restricted. All animals underwent a comprehensive physical examination by a veterinarian before administration. During the acclimation period, cageside observations were conducted once or twice daily, and detailed clinical observations were conducted once daily. During the experimental period, cageside observations were conducted twice daily (once on the day of necropsy), and detailed clinical observations were conducted once or twice daily. All surviving animals were weighed once on the day of randomization (day 1), day 1 (pre-administration), days 3, 5, 7, and 8. Food intake was measured 24 ± 1 h on days 1 (days 1–2), 2 (days 2–3), and 6 (days 6–7). At the end of the experimental period (day 8), all surviving animals were euthanized by carbon dioxide inhalation and active abdominal / venous blood sampling, followed by gross anatomical examination. Animals that died during the experiment also underwent gross anatomical examination.

[0192] [Table 31] 5. Experimental Results

[0193] The mean recoveries of the drug in the test product formulations at various concentrations ranged from 105.8% to 109.8%, and the recovery %RSDs for the upper, middle, and lower layers of the low- and high-concentration samples were ≤0.89, meeting the acceptance criteria of a mean recovery of 100±15% at the theoretical concentration and a %RSD ≤10.

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

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

[0196] 2. Experimental Animals The common beagle dogs used in this experiment were purchased from Beijing Masu Biotechnology Co., Ltd., with production license number SCXK(King)2016-0001 and quality certificate numbers 1103182011000078 (male) and 1103182011000079 (female). Experimental animals were housed in the animal room of Suzhou Shengsu New Drug Development Co., Ltd. (license number SYXK(Su)2021-0045). The animal room was well-ventilated and air-conditioned, with a temperature maintained between 16 and 26°C and humidity between 40 and 70%. Artificial lighting was used, with a 12-hour light and 12-hour dark cycle. At the time of administration on the experimental day, the weights of male beagles ranged from 8.6 to 10.5 kg, and those of female beagles ranged from 7.6 to 9.0 kg.

[0197] 3. Preparation of Formulations Vehicle for intragastric administration: 0.5% CMC-Na aqueous solution Approximately 450.72 mg of free crystalline Form A was weighed into a glass vial, and 378.605 mL of 0.5% CMC-Na aqueous solution was added. The mixture was stirred and sonicated until the particles were dispersed, resulting in a white suspension with a final concentration of 1.2 mg / mL. The dosage formulation was freshly prepared on the day of administration, and a sample was saved for measurement of the formulation concentration.

[0198] 4. Experimental Grouping The day before the experiment, six beagle dogs (half male, half female) were fasted overnight in metabolic cages. Before administration, urine and feces from each animal were collected as pre-administration zero samples. On the day of administration, the beagle dogs were intragastrically administered a single dose of 6 mg / kg of the administration formulation. Four hours after administration, the animals were fed again and had free access to water throughout the experiment.

[0199] 5. Sample Collection and Processing Urine and fecal excretion: Experimental groups: 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. Upon completion of urine collection, urine volume was measured and recorded. A portion of the collected urine was diluted with methanol to a urine-to-methanol volume ratio of 4:1 (v / v). The sample was vortexed to homogenize it and then divided into two aliquots. After collecting fecal samples at different time points, surface food residues were removed and the feces were weighed. Depending on the fecal weight, a fixed volume of homogenate (20% methanol in water) was added and homogenized at a feces-to-homogenate ratio of 1:10 (feces weight:homogenate volume, w / v). The samples were then divided into two aliquots. After completing the final time point collection of urine and fecal excretion for each experimental group, the metabolic cages were washed with 2000 mL of water:ethanol (1:1, v:v). The washings from each cage were collected separately. After shaking the washings thoroughly, 1 mL of each 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 analysis. The LC-MS / MS method was used to detect the drug concentrations in the excreta samples of animals from each group at different periods, and the excretion rate and cumulative excretion rate for each period were calculated.

[0200] 6. Experimental Results

[0201] [Table 32]

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

[0203] From 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 fluid), and the free crystalline form A was more stable in the body of dogs.

[0204] It should be noted that the above embodiments are only used to explain the technical solutions of the present invention, and are not intended to limit the same. 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 the technical features can be replaced with equivalents. These modifications or substitutions do not cause the essence of the corresponding technical solutions to depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Crystalline Form I of the compound of formula I, The XRPD pattern of crystalline form I is characterized by having characteristic peaks at at least three positions among the 2θ values ​​of about 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. 2. The crystalline form I of claim 1, wherein the XRPD pattern of the crystalline form I further comprises characteristic peaks at at least three of the following 2θ values: 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°.

3. 2. The crystalline form I of claim 1, wherein the crystalline form I has an XRPD pattern approximately as shown in FIG. 1, and preferably, the crystalline form I has a DSC pattern and a TGA pattern approximately as shown in FIG.

2.

4. A process for preparing crystalline form I according to any one of claims 1 to 3, comprising the steps of: The preparation method includes preparing crystalline Form I from an amorphous compound of Formula I as a starting material, and the preparation method is one or more combinations selected from an anti-solvent addition method, a gas-solid diffusion method, a suspension stirring method, a slow evaporation method, a slow temperature reduction method, a gas-liquid permeation method, and a poor-anti-solvent addition method; Preferably, the anti-solvent addition method comprises the steps of dissolving an amorphous raw material of the compound represented by formula I in a good solvent and adding an anti-solvent; Preferably, the good solvent is one selected from methanol, 1,4-dioxane, chloroform, and dimethyl sulfoxide; Preferably, the anti-solvent is one selected from methyl isobutyl ketone, isopropyl acetate, methyl tert-butyl ether, n-heptane, ethyl acetate, m-xylene, cyclopentyl methyl ether, toluene, anisole, or water; More preferably, the good solvent is methanol and the poor solvent is selected from methyl isobutyl ketone, isopropyl acetate, and methyl tert-butyl ether; alternatively, the good solvent is 1,4-dioxane and the poor solvent is selected from n-heptane and ethyl acetate; alternatively, the good solvent is chloroform and the poor solvent is selected from n-heptane, m-xylene, and cyclopentyl methyl ether; or alternatively, the good solvent is dimethyl sulfoxide and the poor solvent is selected from toluene, anisole, and water.

5. Crystalline Form I of the hydrochloride salt of the compound of formula I, The XRPD pattern of the hydrochloride crystalline form I is characterized by having characteristic peaks at at least three of the following 2θ values: 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°. 【Chemistry 2】

6. 6. The hydrochloride crystalline Form I of claim 5, wherein the XRPD pattern of the hydrochloride crystalline Form I further comprises characteristic peaks at at least three of the following 2θ values: about 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. 6. The hydrochloride salt crystalline form I of claim 5, wherein the hydrochloride salt crystalline form I has an XRPD pattern substantially as shown in FIG.

4.

8. 6. The hydrochloride salt crystalline form I of claim 5, wherein the hydrochloride salt crystalline form I has a DSC pattern and a TGA pattern substantially as shown in FIG.

9. A process for preparing the hydrochloride crystalline form I according to any one of claims 5 to 8, comprising the steps of: mixing crystalline Form I of claim 1 with hydrochloric acid, and adding a solvent to obtain hydrochloride crystalline Form I; Preferably, the solvent is one selected from ethanol, acetone / water, ethyl acetate, or 2-methyltetrahydrofuran.

10. A pharmaceutical composition comprising crystalline form I as defined in any one of claims 1 to 3 or hydrochloride crystalline form I as defined in any one of claims 5 to 8, and one or more pharmaceutically acceptable auxiliary materials.

11. Use of crystalline form I according to any one of claims 1 to 3 or hydrochloride crystalline form I according to any one of claims 5 to 8 in the preparation of a medicament for treating a JAK-STAT pathway related disease.

12. Use of crystalline form I according to any one of claims 1 to 3 or hydrochloride crystalline form I according to any one of claims 5 to 8 in the preparation of a medicament for the prevention and / or treatment of inflammatory diseases, tumors, autoimmune diseases and allergic diseases in humans and / or animals, comprising Preferably, the inflammatory disease is selected from rheumatoid arthritis, canine dermatitis, psoriasis, ulcerative colitis, or Crohn's disease; Preferably, the cancer is selected from myelofibrosis, polycythemia vera, essential thrombocythemia, chronic myeloid leukemia, breast cancer, lung cancer, or pancreatic cancer; Preferably, 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; Preferably, the allergic disease is selected from allergic dermatitis, allergic conjunctivitis, allergic asthma or allergic rhinitis; More preferably, the allergic disease is allergic dermatitis in dogs and cats, and includes one or more of the symptoms of skin itching, red rash, hair loss, scaling, edema, ulcers, particularly allergic skin itching in dogs.

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