WO2024160203A1 - 一种化合物的晶型及其制备方法和用途 - Google Patents

一种化合物的晶型及其制备方法和用途 Download PDF

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WO2024160203A1
WO2024160203A1 PCT/CN2024/074718 CN2024074718W WO2024160203A1 WO 2024160203 A1 WO2024160203 A1 WO 2024160203A1 CN 2024074718 W CN2024074718 W CN 2024074718W WO 2024160203 A1 WO2024160203 A1 WO 2024160203A1
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solvent
crystalline form
hydrochloride
crystal form
compound
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English (en)
French (fr)
Inventor
安娜
李丹妮
宋敏
林星雨
陆婷婷
张雪
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Felicamed Biotechnology Co Ltd
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Felicamed Biotechnology Co Ltd
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Priority to AU2024214810A priority Critical patent/AU2024214810A1/en
Priority to EP24749682.1A priority patent/EP4660196A1/en
Priority to JP2025536606A priority patent/JP2025542332A/ja
Publication of WO2024160203A1 publication Critical patent/WO2024160203A1/zh
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/13—Crystalline forms, e.g. polymorphs

Definitions

  • the present invention belongs to the field of pharmaceutical chemistry polymorph research, and specifically relates to a crystal form of a compound represented by formula I, a preparation method and use thereof.
  • the JAK-STAT signaling pathway is a signal transduction pathway stimulated by cytokines, which is involved in many important biological processes such as cell proliferation, differentiation, apoptosis and immune regulation. Compared with other signaling pathways, the transmission process of this signaling pathway is relatively simple, and it mainly consists of three components, namely tyrosine kinase-related receptors, tyrosine kinase JAK and transcription factor STAT.
  • JAK inhibitors can selectively inhibit JAK kinases and block the JAK-STAT pathway.
  • the FDA-approved JAK inhibitors include Tofacitinib (tofacitinib citrate), Ruxolitinib, Oclacitinib (oclacitinib), and Baricitinib (baricitinib).
  • Tofacitinib tofacitinib citrate
  • Ruxolitinib tofacitinib citrate
  • Oclacitinib oclacitinib
  • Baricitinib baricitinib
  • oclacitinib has a good effect on allergic skin diseases in pet dogs, it has little effect on cytokines that are not involved in activating JAK1, and its effect on allergic reactions is only to the stage of inhibiting the release of allergic mediators.
  • JAK inhibitor Baricitinib
  • IC50 5.9nM and 5.7nM, respectively, which is about 70 and 10 times more selective than JAK3 and Tyk2. It has no inhibitory effect on c-Met and Chk2, and its indications are relatively single.
  • Chinese patent CN111499641B discloses a JAK inhibitor and a preparation method thereof, which has good JAK inhibitory activity and is expected to be used for preventing and/or treating inflammatory diseases and cancers in humans and/or animals. While therapeutic activity is the property of primary concern for therapeutic agents, the solid state form (i.e., crystalline or amorphous form) of a drug candidate is also critical to its pharmacological properties and development as a viable API.
  • the present invention provides a crystalline form of a compound represented by formula I and a preparation method and use thereof.
  • the present invention provides a crystalline form I of a compound represented by formula I,
  • the XRPD spectrum of the crystalline form I (using Cu-K ⁇ radiation) has characteristic peaks at at least three (e.g., three, four, five, six, seven, and especially all) positions at 2 ⁇ values of approximately 12.4° ⁇ 0.2°, 14.6° ⁇ 0.2°, 16.7° ⁇ 0.2°, 17.2° ⁇ 0.2°, 20.3° ⁇ 0.2°, 24.8° ⁇ 0.2°, and 25.0° ⁇ 0.2°.
  • the XRPD spectrum of the crystalline form I also has characteristic peaks at at least three (e.g., three, four, five, six, seven, and especially all) positions at 2 ⁇ values of approximately 13.2° ⁇ 0.2°, 15.2° ⁇ 0.2°, 19.3° ⁇ 0.2°, 21.2° ⁇ 0.2°, 21.6° ⁇ 0.2°, 24.0° ⁇ 0.2°, and 27.0° ⁇ 0.2°.
  • Form I has an XRPD pattern substantially as shown in FIG1 .
  • the DSC spectrum of the crystal form I has an endothermic peak at 195-210°C (such as around 195°C, 200°C, 205°C, and 210°C).
  • the crystalline form I has a DSC spectrum substantially as shown in FIG2 .
  • the TGA spectrum 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.
  • the crystalline form I has a TGA spectrum substantially as shown in FIG2 .
  • the crystalline form I is an anhydrous crystalline form.
  • the crystal form I is needle-shaped and/or long plate-shaped crystals.
  • the present invention also provides a method for preparing the crystalline form I, using the amorphous compound of formula I as the starting material, and adopting one or more of the anti-solvent addition method, gas-solid diffusion method, suspension stirring method, slow volatilization method, slow cooling method, gas-liquid permeation method or anti-anti-solvent addition method.
  • the combination of the two kinds of compounds was used to prepare Form I.
  • the specific steps of the anti-solvent addition method include dissolving the amorphous raw material of the compound represented by Formula I with a good solvent, adding the anti-solvent, and stirring while adding dropwise until solid precipitates.
  • the good solvent in the anti-solvent addition method is selected from one of methanol, 1,4-dioxane, chloroform or dimethyl sulfoxide.
  • the anti-solvent in the anti-solvent addition method is selected from one of methyl isobutyl ketone, isopropyl acetate, methyl tert-butyl ether, n-heptane, ethyl acetate, 2-butanone, m-xylene, cyclopentyl methyl ether, toluene, anisole or water.
  • the good solvent is methanol
  • the anti-solvent is selected from methyl isobutyl ketone, isopropyl acetate, and methyl tert-butyl ether.
  • the good solvent is 1,4-dioxane
  • the anti-solvent is selected from n-heptane and ethyl acetate.
  • the good solvent is chloroform
  • the anti-solvent is selected from n-heptane, m-xylene, and cyclopentyl methyl ether.
  • the good solvent is dimethyl sulfoxide
  • the anti-solvent is selected from toluene, anisole, and water.
  • 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, and standing at room temperature until a solid precipitates.
  • suspension stirring method is selected from: room temperature suspension stirring method, 50°C suspension stirring method or temperature cycle suspension stirring method.
  • the specific steps of the room temperature suspension stirring method include suspending the amorphous raw material of the compound represented by formula I with a solvent, and stirring at room temperature until solid is precipitated.
  • the specific steps of the 50°C suspension stirring method include suspending the amorphous raw material of the compound represented by Formula I with a solvent, cyclically suspending and stirring at a temperature of 50-5°C for 2-4 days, and then transferring to 50°C for suspension and stirring until solids precipitate.
  • the specific steps of the temperature cycle suspension stirring method include suspending the amorphous raw material of the compound represented by Formula I with a solvent, stirring at 40-60°C, cooling from 40-60°C to 0-10°C within 400-500 minutes, and stirring at 0-10°C for 1-3 hours, heating to 40-60°C within 20-40 minutes, and stirring at 40-60°C for 1-3 hours, repeating the above steps (for example, 2 times), cooling to 0-10°C within 400-500 minutes, and maintaining stirring at 0-10°C until solid precipitates.
  • the specific steps of the slow volatilization method include dissolving the amorphous raw material of the compound represented by Formula I with a solvent, taking the filtrate after shaking and filtering, and slowly volatilizing it at room temperature until solid precipitates.
  • the specific steps of the slow cooling method include dissolving the amorphous raw material of the compound represented by Formula I with a solvent, stirring at 40-60°C, filtering the solution after it is clarified, slowly cooling the filtrate from 40-60°C to 0-10°C, and collecting the precipitated solid.
  • the specific steps of the gas-liquid permeation method include placing the amorphous raw material of the compound represented by Formula I in an open container and dissolving it with a solvent, then placing the open container in a sealed container containing an anti-solvent, and standing at room temperature until solid precipitates.
  • the specific steps of the anti-antisolvent addition method include dissolving the amorphous raw material of the compound represented by Formula I with a good solvent, and adding the resulting solution into the antisolvent until a solid is precipitated.
  • the present invention also provides a hydrochloride crystal form I of the compound represented by formula I.
  • the XRPD spectrum of the hydrochloride salt form I (using Cu-K ⁇ radiation) has characteristic peaks at at least three (e.g., four, five, six, seven, eight, nine, and especially all) positions at 2 ⁇ values of approximately 6.2° ⁇ 0.2°, 10.9° ⁇ 0.2°, 12.3° ⁇ 0.2°, 16.3° ⁇ 0.2°, 17.2° ⁇ 0.2°, 18.9° ⁇ 0.2°, 19.4° ⁇ 0.2°, 24.7° ⁇ 0.2°, and 27.5° ⁇ 0.2°.
  • the XRPD spectrum of the hydrochloride salt form I also has characteristic peaks at at least three (e.g., four, five, six, seven, eight, nine, and especially all) positions at 2 ⁇ values of approximately 11.6° ⁇ 0.2°, 15.0° ⁇ 0.2°, 18.4° ⁇ 0.2°, 21.3° ⁇ 0.2°, 23.3° ⁇ 0.2°, 24.3° ⁇ 0.2°, 25.6° ⁇ 0.2°, 26.8° ⁇ 0.2°, and 30.0° ⁇ 0.2°.
  • hydrochloride salt form I has an XRPD pattern substantially as shown in FIG. 4 .
  • the DSC spectrum of the hydrochloride salt form I has an endothermic peak at 210-220°C (such as 210°C, 213°C, 216°C, and around 210°C).
  • hydrochloride salt form I has a DSC spectrum substantially as shown in FIG5 .
  • the TGA spectrum of the hydrochloride salt 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.
  • hydrochloride salt form I has a TGA spectrum substantially as shown in FIG5 .
  • the hydrochloride salt form I is a rod-shaped crystal.
  • the present invention also provides a method for preparing the hydrochloride crystal form I of the compound shown in formula I, comprising mixing the crystal form I of the compound shown in formula I (as described in the present invention) and hydrochloric acid, adding a solvent (stirring, centrifuging), and obtaining the hydrochloride crystal form I.
  • the molar ratio of the crystal form I to hydrochloric acid is 1:1.
  • the solvent is selected from one of ethanol, acetone/water (eg, 19:1, v/v), ethyl acetate or 2-methyltetrahydrofuran.
  • the present invention also provides a pharmaceutical composition, which comprises the crystalline form I or the hydrochloride crystalline form I of the compound represented by formula I, and one or more pharmaceutically acceptable excipients.
  • the auxiliary material is selected from one or more of a carrier, a diluent, a binder, a lubricant, and a wetting agent.
  • the pharmaceutical composition comprises a therapeutically effective amount of the crystalline form I or the hydrochloride crystalline form I of the compound of formula I.
  • the pharmaceutical composition can be administered to humans and/or animals.
  • the pharmaceutical composition is suitable for enteral or parenteral administration, such as administration by intravenous, intramuscular, intradermal and subcutaneous routes. Therefore, preferably, the pharmaceutical composition also includes antioxidants, buffers, bacteriostats, and solutes that make the preparation isotonic with the blood of the recipient, as well as aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers and preservatives.
  • the pharmaceutical composition can be formulated into pharmaceutical preparations in the following forms: syrups, elixirs, suspensions, powders, granules, tablets, capsules, lozenges, aqueous solutions, creams, ointments, lotions, gels, emulsions, etc.
  • the pharmaceutical preparation is preferably in unit dosage form.
  • the preparation is subdivided into unit doses containing appropriate amounts of active ingredients.
  • the unit dosage form can be a capsule, tablet or any dosage form; in addition, the unit dosage form can also be a packaged preparation, such as tablets, capsules and powders packaged in vials or ampoules.
  • the amount of the active ingredient in the unit dose formulation can be varied or adjusted from 0.1 mg to 1000 mg, depending on the specific application and efficacy of the active ingredient. If desired, the composition may also contain other suitable therapeutic agents.
  • the present invention also provides a use of a crystalline form I or a hydrochloride form I of a compound shown in formula I in the preparation of JAK inhibitors and drugs for treating diseases related to the Janus kinase signaling and transcription activator (JAK-STAT) pathway.
  • a crystalline form I or a hydrochloride form I of a compound shown in formula I in the preparation of JAK inhibitors and drugs for treating diseases related to the Janus kinase signaling and transcription activator (JAK-STAT) pathway.
  • the present invention also provides a use of a crystalline form I or a hydrochloride crystalline form I of a compound represented by formula I in inhibiting JAK and treating diseases related to Janus kinase, signal transduction and transcription activator pathways.
  • the disease is selected from: inflammatory diseases, tumors, autoimmune diseases and allergic diseases.
  • the present invention also provides a use of the crystalline form I or the hydrochloride crystalline form I of the compound represented by formula I in the preparation of a drug for preventing and/or treating inflammatory diseases, tumors, autoimmune diseases and allergic diseases in humans and/or animals.
  • the present invention also provides a use of the crystalline form I or the hydrochloride crystalline form I of the compound represented by formula I in preventing and/or treating inflammatory diseases, tumors, autoimmune diseases and allergic diseases in humans and/or animals.
  • the inflammatory disease is selected from: rheumatoid arthritis, canine dermatitis, psoriasis, ulcerative colitis or Crohn's disease.
  • the tumor is a malignant tumor (cancer); further, the cancer is selected from: myelofibrosis, polycythemia vera, essential thrombocythemia, chronic myeloid leukemia, breast cancer, lung cancer or pancreatic cancer.
  • 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.
  • the allergic disease is selected from: allergic dermatitis, allergic conjunctivitis, allergic asthma or allergic rhinitis.
  • the allergic disease is canine or feline allergic dermatitis, which may include one or more of the following symptoms: itchy skin, rash, hair loss, desquamation, edema, ulcers, etc., sometimes accompanied by symptoms such as otitis, sneezing, and tearing, especially itchy skin.
  • the disease is canine allergic skin itching.
  • the present invention is based on the previous research results of the inventors. After experimental research, the free crystal form and the hydrochloride crystal form of the compound shown in Formula I are further obtained.
  • the crystals not only have sharp diffraction peaks, high crystallinity, less solvent residue, a single DSC thermal signal, small TGA weight loss, and a high ligand safety level, but also have good solid-state stability and fluidity, low hygroscopicity, and good solubility, which are conducive to the preparation and storage of pharmaceutical preparations, and have very good application value and drug development prospects.
  • the clinical trial results show that the free crystal form and the hydrochloride crystal form described in the present invention have very good pharmacokinetic properties, and their systemic exposure increases linearly in a dose-related manner in experimental animals, the blood drug concentration reaches a peak quickly, the in vivo stability is high, there is no obvious drug accumulation after continuous administration, the clinical toxicity is low, the safety is good, and it is obvious
  • the free crystal form and the hydrochloride crystal form of the present invention have high oral bioavailability and good therapeutic effects on various diseases, such as good inhibitory and antipruritic effects on canine allergic pruritus.
  • the free crystal form and the hydrochloride crystal form of the present invention can be administered through various routes of administration (such as injection, oral administration) and can be used in various preparation forms, which have very good application prospects.
  • FIG1 shows the XRPD pattern of free crystalline form A.
  • FIG2 shows the TGA/DSC graph of free crystalline form A.
  • FIG3 shows the 1 H NMR spectrum of free crystalline form A.
  • FIG4 shows the XRPD pattern of hydrochloride salt form A.
  • FIG. 5 shows the TGA/DSC graph of hydrochloride form A.
  • FIG6 shows the 1 H NMR spectrum of hydrochloride form A.
  • FIG. 7 shows the XRPD pattern of sulfate salt form A.
  • FIG8 shows the TGA/DSC graph of sulfate crystal form A.
  • FIG9 shows the 1 H NMR spectrum of sulfate salt form A.
  • FIG. 10 shows the XRPD pattern of phosphate crystal form A.
  • FIG. 11 shows the TGA/DSC graph of phosphate crystal form A.
  • FIG. 12 shows the 1 H NMR spectrum of phosphate crystal form A.
  • FIG. 13 shows the XRPD pattern of the solubility sample of free form A in H 2 O.
  • FIG. 14 shows the XRPD pattern of the solubility sample of free form A in SGF.
  • FIG. 15 shows the XRPD pattern of the solubility sample of free form A in FaSSIF.
  • FIG. 16 shows the XRPD pattern of the solubility sample of free form A in FeSSIF.
  • FIG. 17 shows the XRPD pattern of the solubility sample of hydrochloride Form A in pH buffer.
  • FIG. 18 shows the XRPD pattern of the solubility sample of free form A in pH buffer.
  • FIG. 19 shows the DVS diagram of hydrochloride form A.
  • FIG. 20 shows the DVS diagram of free-state crystalline form A.
  • FIG. 21 shows the XRPD overlay of hydrochloride salt Form A before and after DVS testing.
  • FIG. 22 shows the XRPD overlay of free form A before and after DVS testing.
  • FIG. 23 shows an XRPD overlay of hydrochloride Form A before and after stability evaluation.
  • FIG. 24 shows an XRPD overlay of free crystalline Form A before and after stability evaluation.
  • Figure 25 shows the UPLC chart of the stability evaluation of hydrochloride form A (Note: the deviation of the main peak elution time may be caused by the change of system pressure during testing at different times. The starting sample was tested at the API elution time each time).
  • FIG26 shows the UPLC chart of the stability evaluation of free form A (Note: the deviation of the main peak elution time may be caused by the change of system pressure during the test at different time. The starting sample was tested for each test to determine the API elution time).
  • FIG. 27 shows a PLM diagram of hydrochloride salt form A.
  • FIG. 28 shows the PLM image of free crystalline form A.
  • FIG. 29 shows the XRPD pattern of hydrochloride Form A before and after grinding and tableting.
  • FIG30 shows the XRPD pattern of free-state Form A before and after grinding and tableting.
  • FIG31 shows the blood drug concentration-time curve (logarithmic coordinate) of beagle dogs after intravenous administration of 2 mg/kg free-state crystalline form A.
  • FIG32 shows the blood drug concentration-time curve of beagle dogs given different doses of free form A by oral gavage.
  • FIG33 shows the blood drug concentration-time curves of beagle dogs after multiple oral administration of 6 mg/kg free crystalline form A for the first and seventh times.
  • FIG34 shows the blood drug concentration-time curve of male beagle dogs given 6 mg/kg hydrochloride crystal form A by oral gavage.
  • the term "crystalline form" is confirmed by X-ray powder diffraction pattern characterization.
  • X-ray powder diffraction pattern characterization Those skilled in the art will understand that the physicochemical properties discussed here can be characterized, and the experimental errors depend on the conditions of the instrument, the preparation of the sample, the purity of the sample, etc.
  • the present invention It is well known to those skilled in the art that X-ray diffraction patterns usually change with the conditions of the instrument. It is particularly important to point out that the relative intensity of the X-ray powder diffraction pattern may also change with changes in experimental conditions, so the order of peak intensity cannot be used as the only or decisive factor.
  • the relative intensity of the diffraction peaks in the XRPD spectrum is related to the preferred orientation of the crystal, and the peak intensity shown herein is illustrative rather than for absolute comparison.
  • the experimental error of the peak angle is usually 5% or less, and the error of these angles should also be taken into account, and an error of ⁇ 0.2° is usually allowed.
  • the overall shift of the peak angle will be caused, and a certain shift is usually allowed.
  • the X-ray powder diffraction pattern of a crystal form in the present invention does not have to be completely consistent with the X-ray powder diffraction pattern in the embodiment referred to herein, and the "same XRPD pattern" described herein does not mean absolutely the same, the same peak position can differ by ⁇ 0.2° and the peak intensity allows a certain variability.
  • Any crystal form having a pattern that is the same or similar to the characteristic peaks in these patterns belongs to the scope of the present invention.
  • a person skilled in the art can compare the spectra listed in the present invention with the spectra of an unknown crystal form to confirm whether the two sets of spectra reflect the same or different crystal forms.
  • the crystal form A of the present invention is pure and single, and is substantially free of any other crystal forms.
  • substantially free when used to refer to a new crystal form means that the crystal form contains less than 20% (by weight) of other crystal forms, especially less than 10% (by weight) of other crystal forms, more preferably less than 5% (by weight) of other crystal forms, and more preferably less than 1% (by weight) of other crystal forms.
  • the relative intensity of the diffraction peak is not characteristic of the crystal in question, and when judging whether it is the same as a known crystal form, more attention should be paid to the position of the peak rather than their relative intensity.
  • the term "room temperature” refers to that the temperature of an article is close to or the same as the temperature of a space (e.g., a place in a fume hood in which the article is located). Typically, the room temperature is about 20°C to about 30°C, or about 22°C to 27°C, or about 25°C.
  • the anti-solvent crystallization method (also known as anti-solvent addition, precipitation crystallization, salting out or forced crystallization) is generally a method of adding one or more anti-solvents to a solution in which the target product is dissolved in a positive solvent, and the product is slightly soluble in the solution, so that the solution reaches a supersaturated state and crystals are precipitated.
  • the anti-anti-solvent crystallization is generally a method of adding one or more anti-solvents to a solution in which the target product is dissolved in a positive solvent, and the product is slightly soluble in the solution, so that the solution reaches a supersaturated state and crystals are precipitated.
  • the anti-solvent has a poorer ability to dissolve the target product than the positive solvent, such as a poorer ability of more than 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80%, so the anti-solvent in the system is relative.
  • the positive solvent and the anti-solvent can be polar solvents or non-polar solvents, such as can be selected from: dimethylformamide (DMF), dimethyl sulfoxide (DMSO), water, alcohol solvents, ether solvents, ketone solvents, ester solvents, alkane solvents, aromatic solvents, nitrile solvents One or more.
  • alcohol solvents include but are not limited to methanol, ethanol, propanol, isopropanol or 1,3-propylene glycol, 1,2-propylene glycol or trichlorobutanol or a combination thereof; ether solvents include but are not limited to tetrahydrofuran, methyl tert-butyl ether or 1,4-dioxane or a combination thereof; ketone solvents include but are not limited to acetone, methyl ethyl ketone or 4-methyl-2-pentanone or a combination thereof; ester solvents include but are not limited to ethyl acetate, isopropyl acetate, n-butyl acetate or tert-butyl acetate or a combination thereof; alkane solvents include but are not limited to dichloromethane, chloroform, n-hexane, cyclohexane or pentane or n-heptane or
  • Antisolvent crystallization and anti-antisolvent crystallization can be performed by batch, semi-batch or continuous crystallization.
  • the antisolvent is added to the solution (antisolvent crystallization) or the product solution is added to the antisolvent (anti-antisolvent crystallization), and it can be added dropwise at a constant rate or it can be added dropwise slowly at the beginning and then gradually increased.
  • the "animal” described herein is a non-human animal, in particular a mammal, such as a monkey, pig, cow, sheep, horse, donkey, dog, cat, rabbit, mouse, fox, raccoon dog, mink, camel, etc.
  • the animal is a pet, such as a dog, cat, rodent (e.g., chinchilla, pet rabbit, hamster, guinea pig, mouse, gerbil, chipmunk, squirrel, flying squirrel, chipmunk, reverse plush guinea pig, degu, etc.).
  • treating refers to preventing, curing, reversing, attenuating, alleviating, minimizing, inhibiting, suppressing and/or halting one or more clinical symptoms of a disease after onset of the disease.
  • prevent refers to avoiding, minimizing or making the onset or development of a disease difficult by treating it before it occurs.
  • inflammation is the body's defense response to stimulation, manifested as redness, swelling, heat, pain, and dysfunction, etc. It can be infectious inflammation caused by infection, or non-infectious inflammation not caused by infection, such as inflammation caused by immune response (such as various types of hypersensitivity reactions, inflammation caused by some autoimmune diseases).
  • inflammatory disease refers to a disease with inflammation.
  • tumor refers to an abnormal mass of tissue, wherein the growth of the mass exceeds the growth of normal tissue and is not coordinated with the growth of normal tissue.
  • Tumors can be “benign” or “malignant”, depending on the following characteristics: degree of cell differentiation (including morphology and function), growth rate, local invasion and metastasis.
  • benign tumors are usually well-differentiated, characterized by slower growth than malignant tumors, and remain confined to the site of origin. In addition, benign tumors do not have the ability to infiltrate, invade or metastasize to distant sites.
  • tumors may later lead to malignant tumors, which may be caused by additional genetic changes in a subpopulation of the neoplastic cells of the tumor, and these tumors are called “precancerous tumors".
  • malignant tumors are usually poorly differentiated (anaplastic) and have a characteristic rapid growth, accompanied by progressive infiltration, invasion and destruction of surrounding tissues.
  • malignant tumors usually have the ability to metastasize to distant sites.
  • cancer refers to a malignant tumor (Stedman’s Medical Dictionary, 25th ed.; Hensyl ed.; Williams & Wilkins: Philadelphia, 1990).
  • autoimmune disease refers to diseases caused by the body's immune response to its own antigens, which leads to damage to its own tissues.
  • the American Autoimmune Related Diseases Association has a comprehensive list of autoimmune diseases.
  • 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:
  • the aqueous phase was extracted with dichloromethane (800 ml x 2), the organic phases were combined, washed with saturated brine (1000 ml x 1), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure at 40 degrees to no distillation. The residue was evaporated, petroleum ether (500 ml) was added, and the mixture was slurried at room temperature for 0.5 hour. The residue was filtered under reduced pressure, and the filter cake was collected and dried under forced air at 25° C. for 16 hours to obtain 513 g of a white solid with a yield of 77%.
  • the aqueous phase was extracted with dichloromethane (500 mL ⁇ 3), washed with saturated NaCl solution (1000 ml X 1), dried and concentrated to obtain 510.2 g of a yellow oil with a yield of 94.5%.
  • TGA Thermogravimetric analysis
  • DSC differential scanning calorimetry
  • TGA and DSC graphs were collected on TA Q5000 thermogravimetric analyzer and TA Discovery 2500 differential scanning calorimeter, respectively.
  • the test parameters are shown in Table 2.
  • Dynamic moisture sorption (DVS) curves were collected on the DVS Intrinsic of SMS (Surface Measurement Systems). The relative humidity at 25°C was corrected using the deliquescent points of LiCl, Mg(NO 3 ) 2 , and KCl. The test parameters are shown in Table 3.
  • the TGA/DSC spectrum of free crystalline form A is shown in Figure 2.
  • the TGA results show that it loses 1.7% of weight when heated from room temperature to 180°C; the DSC results show that it has a sharp endothermic peak at 197.4°C (starting temperature), which is presumed to be a melting signal.
  • Anti-solvent was added to obtain a clear solution, which remained clear after stirring at room temperature/5°C and was transferred to room temperature to evaporate and precipitate solids.
  • TGA Thermogravimetric analysis
  • DSC differential scanning calorimetry
  • TGA results were collected on a TA Discovery 5500 thermogravimetric analyzer, and the DSC results were collected on a TA Discovery 2500 differential scanning calorimeter. Table 8 lists the test parameters.
  • Dynamic moisture sorption (DVS) curves were collected on the DVS Intrinsic of SMS (Surface Measurement Systems). The relative humidity at 25°C was corrected using the deliquescent points of LiCl, Mg(NO 3 ) 2 , and KCl. The DVS test parameters are listed in Table 9.
  • PLM images were acquired on a ZEISS Scope.A1 polarizing microscope.
  • ThermoFisher ICS-1100 ion chromatograph was used to analyze the ion content in the experiment. The specific conditions are shown in Table 11.
  • This experiment uses compound A free form A as raw material to study the possibility of compound A free form A forming corresponding salt forms under 23 different acids and 4 different solvents.
  • the specific experiments are summarized in Table 12. The specific steps are as follows: First, weigh about 20 mg of compound A free form A and equimolar corresponding acid ligands in an HPLC vial, add 0.5 mL of solvent, stir magnetically ( ⁇ 1000 rpm) at room temperature for about 4 days, and separate the solid for XRPD detection; if no solid precipitates, transfer to 5°C/-20°C for stirring, add anti-solvent or evaporate at room temperature.
  • the TGA/DSC results are shown in FIG5 .
  • the sample loses 0.53% of its weight when heated to 150° C., and the DSC results show a sharp endothermic peak at 212.9° C. (starting temperature).
  • the TGA/DSC results are shown in Figure 8.
  • the TGA results show that the sample has a step-wise 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 temperature).
  • the TGA/DSC results are shown in Figure 11.
  • the TGA results show that the sample loses 2.82% of its weight when heated to 150°C; the DSC results show that there are three endothermic signals at 69.0°C, 149.0°C and 163.2°C (peak temperature).
  • the DSC results show that it has two endothermic signals at 56.9°C and 96.1°C, indicating that sulfate crystalline form A is prone to decomposition or melting at lower temperatures and has poor thermal stability.
  • the phosphate crystal form A sample lost 2.82% of its weight when heated to 150°C, but the DSC results showed that it had three endothermic signals at 69.0°C, 149.0°C and 163.2°C, indicating that its thermal stability was slightly poor.
  • free crystal form A and hydrochloride crystal form A not only have sharp crystal diffraction peaks, higher crystallinity, less solvent residue, a single DSC thermal signal, small TGA weight loss, etc., but also have a higher ligand safety level. It can be seen that after more than 100 kinds of experimental conditions, only free crystal form A and hydrochloride crystal form A among the obtained crystal forms have good solid-state properties and can be developed as potential drug crystal forms.
  • the dynamic solubility of hydrochloride crystal form A and free crystal form A in H 2 O and biological solvents was evaluated at 37°C.
  • the specific steps are as follows: weigh about 40 mg of hydrochloride crystal form A and free crystal form A into 5 mL glass vials, add 4 mL of the corresponding medium (H 2 O, SGF, FaSSIF and FeSSIF) respectively; rotate and mix at a rate of 25 rpm on a rotating incubator at 37°C, and the sampling points are 1, 2, 4, and 24 hours; take about 0.8 mL of the suspension at each sampling point into a centrifuge tube, centrifuge (12000 rpm, 3 min, 37°C); filter the supernatant with a PTFE filter membrane (pore size 0.45 ⁇ m), test the solubility and pH of the filtrate, and measure XRPD on the solid (see Example 2 for the instrument and method used).
  • the 24-hour solubility of hydrochloride form A and free form A in pH 2.0, pH 4.5 and pH 7.4 buffers was evaluated at room temperature.
  • the specific steps are as follows: weigh about 10 mg of hydrochloride form A and free form A into HPLC vials, add 1 mL of the corresponding buffer, respectively; stir magnetically at room temperature for 24 hours ( ⁇ 500 rpm).
  • the suspension was centrifuged (10000 rpm, 2 min, room temperature); the supernatant was filtered with a PTFE filter membrane (pore size 0.22 ⁇ m), the filtrate was tested for solubility and pH, and the solid was measured by XRPD (see Example 2 for the instrument and method used).
  • the 24-hour solubility results are summarized in Table 17, and the XRPD results of the separated solid samples are shown in Figures 17 and 18.
  • the results show that: (1) the hydrochloride crystal form A has a high solubility in pH 2.0 and pH 4.5 (>9.2 mg/mL, the sample is clear); it is converted into free crystal form A at pH 7.4; (2) the free crystal form A has a high solubility in pH 4.5 buffer (>9.1 mg/mL, the sample is clear); it is not clear in pH 2.0 and pH 7.4, with solubilities of 6.2 mg/mL and 1.7 mg/mL, respectively.
  • the XRPD results show that the crystal form remains unchanged after stirring for 24 hours.
  • hydrochloride form A and free form A were evaluated, including fluidity and morphology, to understand the basic powder properties of each salt form and free form A.
  • the fluidity is evaluated by testing the bulk density/tapped density and angle of repose of the sample (see Example 2 for the instrument and method used).
  • the test method for bulk density and tapped density is as follows: (1) Add a certain mass of the sample to be evaluated into a 5 mL measuring cylinder and record the volume at this time. The bulk density is calculated by dividing the mass of the sample by the volume at this time. (2) Tap the measuring cylinder 200 times and record the final volume. The tapped density is calculated by dividing the mass of the sample by the final volume. (3) Test three times in parallel.
  • the test method for the angle of repose is as follows: (1) Fix the funnel vertically to the bottom surface and slowly add the material to the funnel. (2) The bottom surface forms a symmetrical cone of the material. Measure the height of the cone and the bottom diameter. (3) Measure three times in parallel.
  • Carr's index (tapped density - bulk density) / tapped density.
  • the hydrochloride crystal form A and the free crystal form A were used as the starting samples, and they were ground manually and pressed by a tablet press (350 MPa pressure), and the ground and pressed samples were subjected to XRPD testing to evaluate their mechanical stability (the instruments and methods used are shown in Example 2). The specific results are summarized in Table 24.
  • hydrochloride form A and free form A have good solid-state stability and fluidity, low hygroscopicity, good solubility, etc. Both have excellent comprehensive physical and chemical properties and can be developed as potential drug crystal forms with good application prospects.
  • the purpose of this experiment is to study the pharmacokinetic properties of free crystalline Form A (prepared in Example 1, sometimes referred to as the drug in this example) in male and female beagle dogs, including 1) a single intravenous injection (i.v.) study at 2 mg/kg; 2) a dose escalation study of 2, 6 and 20 mg/kg oral gavage (i.g.); 3) a 7-day oral gavage study at a dose of 6 mg/kg.
  • the ordinary beagle dogs used in this experiment were purchased from Beijing Mas Biotechnology Co., Ltd., license number: SCXK (Beijing) 2016-0001, animal qualification certificate number 1103182011000078 and 1103182011000079, experimental animal use license number: SYXK (Su) 2021-0045, the animal room is well ventilated, equipped with air conditioning, the temperature is maintained at 16-26 ° C, the humidity is maintained at 40%-70%, artificial lighting is used, and the light and dark are 12 hours each.
  • the weight range of male beagles at the first dose was 8.0-11.6 kg
  • the weight range of female beagles was 6.9-11.3 kg.
  • Solvent for intragastric administration group 0.5% CMC-Na aqueous solution.
  • mice were randomly divided into 4 groups (3/sex/group), and the grouping and dosing information are 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 dosing. All animals were fasted for at least 12 hours before dosing, and food was resumed 4 hours after dosing. All animals were free to drink water and eat during the experiment.
  • Animals in group A were intravenously injected with 2 mg/kg of the drug, and blood was collected from the jugular vein before and 0.033, 0.083, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h after administration.
  • Animals in groups B and D were given 2 and 20 mg/kg of the drug by single oral gavage, respectively, and blood was collected from the jugular vein before and 0.083, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h after administration.
  • Animals in group C were given 6 mg/kg of the drug by oral gavage once a day for seven consecutive days, and blood was collected from the jugular vein before the first and seventh administrations and 0.083, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h after administration.
  • Blood samples were collected (jugular vein) before and 0.5 h after administration during the second to sixth administrations, and the sample before the second administration was the sample 24 h after the first administration.
  • the blood samples of the above animals were collected from the jugular vein, with a blood volume of 0.5 mL/time. All the whole blood samples collected were placed in a EDTA-K2 Place the sample in a centrifuge tube and invert the tube to mix the anticoagulant and blood thoroughly. Place the tube on wet ice before centrifugation. Centrifuge at 1524g for 10 min to separate the plasma. Transfer the plasma sample to a new centrifuge tube and store it at -90 to -60°C until analysis.
  • the drug concentration in beagle dog plasma was detected by LC-MS/MS method.
  • the obtained blood drug concentration data were used to calculate the relevant pharmacokinetic parameters using the pharmacokinetic processing software WinNonlin8.0 non-compartmental model.
  • the animals were observed clinically before administration, before and after each blood sampling time point, and after administration, and no obvious abnormalities were found.
  • the drug concentration-time curves of each group are shown in Figures 31 to 33, and the main pharmacokinetic parameters are shown in Tables 26 to 27.
  • the C 0 and AUC 0-t ratios of the drug in male and female beagles were 0.758 and 0.748, respectively (within the range of 0.5-2 times), and no significant gender difference in systemic exposure was observed.
  • the drug clearance (CL) in male and female beagles was equivalent to 0.110 times the liver blood flow of beagles (about 31 mL/min/kg, Davies and Morris (1993)), indicating that it was cleared slowly in beagles; the steady-state distribution volume (V dss ) was 1.57 times the total body fluid volume of beagles (about 0.60 L/kg, Davies and Morris (1993)), indicating that it tended to be distributed in tissues.
  • the average peak time was between 0.792 and 1.33 h after administration.
  • the oral availability F% calculated by the average AUC 0-t was 88.0%, 100.0% and 101.0%, respectively.
  • the C max ratios of the drug in males and females at the three dose levels were 1.36, 0.810 and 0.894, respectively, and the AUC 0-t ratios were 1.10, 0.716 and 0.877, respectively. There was no obvious gender difference in systemic exposure (the ratios were all within the range of 0.5-2 times).
  • the unit dose C max was 675, 785 and 665 kg*ng/ml/mg
  • the unit dose AUC 0-t was 4380, 4990 and 5030 ng*h*kg/mL/mg, respectively. This indicates that within the dose range of 2 to 20 mg/kg, the systemic exposure of the drug in rats showed a dose-related linear increase (the unit dose C max ratio and unit dose AUC 0-t ratio of 20 and 2 mg/kg were 0.985 and 1.15, respectively, both within the range of 0.5-2 times).
  • Solvent for intragastric administration group 0.5% CMC-Na aqueous solution.
  • hydrochloride crystal form A prepared in Example 2
  • the animals in group A were given a single oral administration of 6 mg/kg of the drug, and blood was collected from the jugular vein before administration and 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h after administration.
  • Blood samples from the above animals were collected through the jugular vein, with a blood volume of 0.5 mL/time. All collected whole blood samples were placed in centrifuge tubes containing EDTA-K2, and the centrifuge tubes were turned upside down to fully mix the anticoagulant and blood. They were placed on wet ice before centrifugation, centrifuged at 1500g for 10 minutes to separate plasma, and the plasma samples were transferred to sample tubes and stored at -40 to -20°C until analysis.
  • the drug concentration in beagle dog plasma was detected by LC-MS/MS method.
  • the LC-MS/MS detection method is as follows:
  • MS conditions ESI positive ion mode; multiple reaction monitoring (MRM); m/z 336.2/149.1.
  • Injection volume 5 ⁇ L.
  • the animals were observed clinically before administration, before and after each blood sampling time point, and after administration, and no obvious abnormalities were found.
  • the drug concentration-time curve is shown in Figure 34, and the main pharmacokinetic parameters are shown in Table 30.
  • T max was 0.417 h, indicating that the hydrochloride crystal form A of the present application reaches the peak in vivo faster.
  • the active ingredient is oclacitinib
  • T max is less than 1h (P7, CVMP assessment report for APOQUEL (EMEA/V/C/002688/0000), EMA/481054/2013). It can be seen that compared with the marketed drug APOQUEL, the hydrochloride crystal form A of the present application has better or at least equivalent advantages in terms of peak time in vivo.
  • the toxicity reaction produced by the experimental animals was observed within a short period of time to preliminarily understand the toxicity characteristics and dose-response relationship of the test substance.
  • the Sprague-Dawely rats (SPF grade) used in this experiment were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd. with a production license number of SCXK (Zhejiang) 2019-0001 and animal certificate numbers of 20220107Aazz0619000738 and 20220107Aazz0619000691, respectively.
  • the weight range of males was 206.6-234.5 g, and the weight range of females was 188.4-205.9 g.
  • Oral administration solvent 0.5% CMC-Na aqueous solution: weigh 17.5031 g of CMC-Na (800-1200 mPa.s) into a suitable container, add 3500 mL of deionized water, stir until uniform to obtain a colorless clear liquid, and store at room temperature until use.
  • 100mg/mL free crystal form A solution Weigh 9000.4mg free crystal form A and place it in a wide-mouth bottle with a line of 90mL, add an appropriate amount of 0.5% CMC-Na (800-1200mPa.s) aqueous solution to the above container, stir and ultrasonicate until uniform, continue to add an appropriate amount of 0.5% CMC-Na (800-1200mPa.s) aqueous solution to 90mL, stir and ultrasonicate until uniform. Take samples as required to obtain a milky white suspension.
  • CMC-Na 800-1200mPa.s
  • a solution Take 44.7 mL of 100 mg/mL free form A solution into a suitable container, add 35.3 mL of 0.5% CMC-Na (800-1200 mPa.s) aqueous solution into the container, vortex until uniform. Take samples as required to obtain a milky white suspension.
  • CMC-Na 800-1200 mPa.s
  • mice were randomly divided into 5 groups (5/sex/group), and were given 97, 174, 312, 559 and 1000 mg/kg of drugs, respectively, and then given once by oral gavage. The animals were observed for 7 days. The grouping and dosing information are shown in Table 31. All animals were dosed according to the latest weighed body weight. The drug preparation was stirred for at least 10 minutes before administration, and the dosing process was also stirred until the end of the dosing of the sample at this concentration. The animals were fasted for 11 to 12 hours before administration. About 2 hours after administration, the diet was resumed, and drinking water was not restricted.
  • the average recovery of the drug in the test preparations of various concentrations was in the range of 105.8%-109.8%, and the %RSD of the upper, middle and lower layers of the low and high concentration samples was ⁇ 0.89, which met the acceptance criteria of average recovery at theoretical concentration of 100 ⁇ 15% and %RSD ⁇ 10.
  • the LD50 of male rats was 592 mg/kg, the 95% confidence interval was 429-818 mg/kg, and the maximum tolerated dose (MTD) was 312 mg/kg; the LD50 of female rats was 418 mg/kg, the 95% confidence interval was 313-558 mg/kg, and the maximum tolerated dose was 174 mg/kg.
  • the LD50 of the marketed drug APOQUEL for rats is 310 mg/kg (P9, CVMP assessment report for APOQUEL (EMEA/V/C/002688/0000), EMA/481054/2013), indicating that the free crystalline form A of the present invention has lower toxicity.
  • Example 12 Study on urine and fecal excretion of free form A in beagle dogs after single oral administration
  • This experiment aims to study the excretion process of the drug in feces and urine of beagle dogs after a single oral administration of free crystalline form A (prepared in Example 1) to male and female beagle dogs.
  • the ordinary beagle dogs used in this experiment were purchased from Beijing Mas Biotechnology Co., Ltd., with production license number: SCXK (Beijing) 2016-0001, and quality certificate numbers 1103182011000078 (male) and 1103182011000079 (female).
  • the experimental animals were kept in the animal room of Suzhou Shengsu New Drug Development Co., Ltd., with license number: SYXK (Suzhou) 2021-0045.
  • the animal room was well ventilated and equipped with air conditioning. The temperature was maintained at 16-26°C, the humidity was maintained at 40%-70%, and artificial lighting was used, with 12 hours of light and dark.
  • the weight range of male beagles on the day of the experiment was 8.6-10.5 kg, and the weight range of female beagles was 7.6-9.0 kg.
  • Intragastric administration solvent 0.5% CMC-Na aqueous solution.
  • Urine and feces excretion experimental group Urine and feces were collected before administration and 0-4, 4-8, 8-12, 12-24, 24-48, 48-72, 72-96 and 96-120h after administration. After urine collection, the volume was measured and recorded, and part of the collected urine was added with methanol for dilution, so that the urine: methanol volume ratio was 4:1 (v/v), vortexed and divided into 2 portions. After the feces samples were collected at each time period, the surface food residues were picked up and weighed.
  • feces weight homogenate volume, w/v
  • homogenized and divided into 2 portions.
  • the metabolic cage was rinsed with 2000mL of water:ethanol (1:1, v:v) and the rinse fluid of each cage was collected separately. After the rinse fluid was thoroughly shaken, 1mL was immediately taken out and placed in a 1.5mL EP tube, and stored in a -90 ⁇ 60°C refrigerator together with the urine and feces homogenate until the sample analysis.
  • the LC-MS/MS method was used to detect the drug concentration in the excretion samples of each group of animals at different time periods, and the excretion rate and cumulative excretion rate in each time period were calculated.
  • the cumulative excretion rate of the drug in the urine of beagles within 120 hours was 43.8 ⁇ 9.97%, and the cumulative excretion rate in the feces within 120 hours was 3.48 ⁇ 0.663%.
  • the total cumulative excretion rate of the drug in the feces and urine of beagles within 120 hours was 47.3 ⁇ 9.62%.
  • the amount of drug in the cage flushing fluid of the beagles was 4.87 ⁇ 3.09% of the dose.
  • the total excretion rate of the drug of the present invention in beagle dogs was 52.2% (feces, urine and flushing fluid), and the free crystal form A was more stable in dogs.

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Abstract

本发明公开了一种化合物的晶型及其制备方法和用途，所述化合物具有式I所示结构，所述晶型具有较好的固态稳定性和流动性、较低的引湿性、较佳的溶解度，有利于药物制剂的制备和储存。而且，所述晶型具有非常好的药代动力学特性，连续给药安全性较佳，且其可通过多种给药途径(例如注射、口服)施用，制剂形式丰富，特别是口服生物利用度高，具有非常好的应用价值。所述晶型可作为JAK抑制剂，用于开发预防和/或治疗人和/或动物的炎症性疾病、肿瘤、自身免疫性疾病及过敏性疾病的药物。

Description

一种化合物的晶型及其制备方法和用途 技术领域
本发明属于药物化学多晶型研究领域，具体涉及一种式I所示化合物的晶型及其制备方法和用途。
背景技术
JAK-STAT信号通路是一条由细胞因子刺激的信号转导通路，参与细胞的增殖、分化、凋亡以及免疫调节等许多重要的生物学过程。与其它信号通路相比，这条信号通路的传递过程相对简单，它主要由三个成分组成，即酪氨酸激酶相关受体、酪氨酸激酶JAK和转录因子STAT。
JAK抑制剂可选择性抑制JAK激酶，阻断JAK-STAT通路。目前，已获FDA批准JAK抑制剂类药物有Tofacitinib(枸橼酸托法替尼)、Ruxolitinib、Oclacitinib(奥拉替尼)、Baricitinib(巴瑞克替尼)。尽管奥拉替尼对宠物犬过敏性皮肤病的疗效较好，但是，由于奥拉替尼对不参与激活JAK1的细胞因子影响甚微，对过敏反应的作用仅到抑制过敏介质的释放的阶段，并不能从根本上直接阻断过敏介质与相关受体的结合，进而不能从根本上阻断过敏性皮肤病的发生发展，从而限制了奥拉替尼的应用范围。另一种JAK抑制剂Baricitinib是一种选择性的JAK1和JAK2抑制剂，IC50分别为5.9nM和5.7nM，比作用于JAK3和Tyk2选择性高70和10倍左右，对c-Met和Chk2没有抑制作用，其适应症比较单一。
中国专利CN111499641B公开了一种JAK抑制剂及其制备方法，其具有较佳的JAK抑制活性，预期可用于预防和/或治疗人和/或动物的炎症性疾病及癌症。尽量治疗活性对于治疗剂是主要关心的性质，药物候选物的固态形式(即，结晶或无定形形式)对其作为可行API的药理性质和其开发也很关键。
发明内容
为克服现有技术的不足，本发明提供了一种式I所示化合物的晶型及其制备方法和用途。
本发明提供了一种式I所示化合物的晶型I，
所述的晶型I的XRPD图谱(使用Cu-Kα辐射)在2θ值约为12.4°±0.2°、14.6°±0.2°、16.7°±0.2°、17.2°±0.2°、20.3°±0.2°、24.8°±0.2°和25.0°±0.2°的位置中至少三个(例如三个、四个、五个、六个、七个，特别是全部)位置处具有特征峰。
进一步地，所述的晶型I的XRPD图谱(使用Cu-Kα辐射)还在2θ值约为13.2°±0.2°、15.2°±0.2°、19.3°±0.2°、21.2°±0.2°、21.6°±0.2°、24.0°±0.2°和27.0°±0.2°的位置中至少三个(例如三个、四个、五个、六个、七个，特别是全部)位置处具有特征峰。
进一步地，所述的晶型I具有基本上如图1所示的XRPD图谱。
进一步地，所述的晶型I的DSC图谱，在195～210℃(如195℃、200℃、205℃、210℃附近)具有吸热峰。
进一步地，所述的晶型I具有基本上如图2所示的DSC图谱。
进一步地，所述的晶型I的TGA图谱从30℃加热至180℃失重1.7％(如1.0％、1.5％、1.7％、2.0％、2.5％、3.0％)。
进一步地，所述的晶型I具有基本上如图2所示的TGA图谱。
进一步地，所述的晶型I为无水晶型。
进一步地，在偏光显微镜下观察，所述的晶型I为针状和/或长片状晶体。
本发明还提供了一种晶型I的制备方法，以无定形形式的式I所示化合物为起始原料，可以采用反溶剂添加法、气固扩散法、悬浮搅拌法、缓慢挥发法、缓慢降温法、气液渗透法或反-反溶剂添加法的一种或多 种的组合制备得到晶型I。
进一步地，所述的反溶剂添加法的具体步骤包括将式I所示化合物无定形原料用良溶剂溶解，加入反溶剂，边滴加边搅拌至有固体析出。
进一步地，所述的反溶剂添加法中的良溶剂选自甲醇、1，4-二氧六环、三氯甲烷或二甲亚砜中的一种。
进一步地，所述的反溶剂添加法中的反溶剂选自甲基异丁基酮、乙酸异丙酯、甲基叔丁基醚、正庚烷、乙酸乙酯、2-丁酮、间二甲苯、环戊基甲醚、甲苯、苯甲醚或水中的一种。
在本发明的一些实施例中，所述反溶剂添加法中，良溶剂为甲醇，反溶剂选自甲基异丁基酮、乙酸异丙酯、甲基叔丁基醚。
在本发明另一些实施例中，所述反溶剂添加法中，良溶剂为1，4-二氧六环，反溶剂选自正庚烷、乙酸乙酯。
在本发明另一些实施例中，所述反溶剂添加法中，良溶剂为三氯甲烷，反溶剂选自正庚烷、间二甲苯、环戊基甲醚。
在本发明另一些实施例中，所述反溶剂添加法中，良溶剂为二甲亚砜，反溶剂选自甲苯、苯甲醚、水。
进一步地，所述的气固扩散法的具体步骤包括将式I所示化合物无定形原料与溶剂混合，密封，室温下静置至固体析出。
进一步地，所述的悬浮搅拌法选自：室温悬浮搅拌法、50℃悬浮搅拌法或温度循环悬浮搅拌法。
进一步地，所述的室温悬浮搅拌法的具体步骤包括将式I所示化合物无定形原料与溶剂混悬，在室温搅拌至有固体析出。
进一步地，所述的50℃悬浮搅拌法的具体步骤包括将式I所示化合物无定形原料与溶剂混悬，在50～5℃温度循环悬浮搅拌2～4天后，重新转移至50℃悬浮搅拌至有固体析出。
进一步地，所述的温度循环悬浮搅拌法的具体步骤包括将式I所示化合物无定形原料与溶剂混悬，在40～60℃下搅拌，400～500分钟内由40～60℃降温至0～10℃，并在0～10℃下搅拌1～3小时后，20～40分钟内升温至40～60℃，并在40～60℃下搅拌1～3小时，重复以上步骤(例如2次)后，在400～500分钟内降温至0～10℃，并保持在0～10℃下搅拌至有固体析出。
进一步地，所述的缓慢挥发法的具体步骤包括将式I所示化合物无定形原料用溶剂溶解，经震荡过滤后取其滤液，在室温下缓慢挥发至有固体析出。
进一步地，所述的缓慢降温法的具体步骤包括将式I所示化合物无定形原料用溶剂溶解，在40～60℃下搅拌，溶液澄清后过滤，将滤液从40～60℃缓慢降温至0～10℃，收集析出的固体。
进一步地，所述的气液渗透法的具体步骤包括将式I所示化合物无定形原料放置在敞口容器中并用溶剂溶解，然后将敞口容器放置在装有反溶剂的密封容器中，室温下静置至有固体析出。
进一步地，所述的反-反溶剂添加法的具体步骤包括将式I所示化合物无定形原料用良溶剂溶解，将所得溶液加入反溶剂中至析出固体。
本发明还提供了一种式I所示化合物的盐酸盐晶型I。
进一步地，所述的盐酸盐晶型I的XRPD图谱(使用Cu-Kα辐射)在2θ值约为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°和27.5°±0.2°的位置中至少三个(例如四个、五个、六个、七个、八个、九个，特别是全部)位置处具有特征峰。
进一步地，所述的盐酸盐晶型I的XRPD图谱(使用Cu-Kα辐射)还在2θ值约为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°和30.0°±0.2°的位置中至少三个(例如四个、五个、六个、七个、八个、九个，特别是全部)位置处具有特征峰。
进一步地，所述的盐酸盐晶型I具有基本上如图4所示的XRPD图谱。
进一步地，所述的盐酸盐晶型I的DSC图谱，在210～220℃(如210℃、213℃、216℃、210℃附近)具有吸热峰。
进一步地，所述的盐酸盐晶型I具有基本上如图5所示的DSC图谱。
进一步地，所述的盐酸盐晶型I的TGA图谱从25℃加热至150℃失重0.5％(如0.5％、1.0％、1.5％、2.0％、2.5％、3.0％)。
进一步地，所述的盐酸盐晶型I具有基本上如图5所示的TGA图谱。
进一步地，在偏光显微镜下观察，所述盐酸盐晶型I为棒状晶体。
本发明还提供了一种式I所示化合物的盐酸盐晶型I的制备方法，将式I所示化合物的晶型I(如本发明所述)和盐酸混合，加入溶剂，(搅拌，离心)，得到盐酸盐晶型I。
进一步地，所述晶型I与盐酸的摩尔比为1∶1。
进一步地，所述的溶剂选自乙醇、丙酮/水(例如19∶1，v/v)、乙酸乙酯或2-甲基四氢呋喃中的一种。
本发明还提供了一种药物组合物，其包含式I所示化合物的晶型I或盐酸盐晶型I，以及一种或多种药学上可接受的辅料。
进一步地，所述的辅料选自：载体、稀释剂、粘合剂、润滑剂、润湿剂中的一种或多种。优选地，所述的药物组合物包含治疗有效量的式I所示化合物的晶型I或盐酸盐晶型I。
优选的，所述的药物组合物可施用于人和/或动物。
进一步地，所述的药物组合物适于胃肠给药或非胃肠给药，如通过静脉内、肌内、皮内和皮下途径给药，因此，优选的，所述的药物组合物还包括抗氧化剂，缓冲剂，抑菌剂，和使制剂与接受者的血液等渗的溶质，以及含水和非水的无菌悬浮剂，其可包含助悬剂，增溶剂，增稠剂，稳定剂和防腐剂。
进一步地，所述的药物组合物可以配制为以下形式的药物制剂：糖浆剂，酏剂，悬浮剂，粉剂，颗粒剂，片剂，胶囊，锭剂，水溶液，霜剂，膏剂，洗液剂，凝胶剂，乳剂等。
进一步地，所述的药物制剂优选为单位剂型。在这种形式中，该制剂被再分成包含适当量的活性组分的单位剂量。单位剂型可以是胶囊，片剂或者任意剂型；另外，单位剂型也可以是包装好的制剂，诸如包装在小瓶或者安瓿中的片剂、胶囊和粉剂等。
所述的单位剂量制剂中活性组分的量可从0.1毫克到1000毫克之间改变或调整，根据活性组分的具体应用和效力而定。如果需要，组合物还可包含其它适合的治疗剂。
本发明还提供了一种式I所示化合物的晶型I或盐酸盐晶型I在制备JAK抑制剂、治疗Janus激酶\信号传导及转录激活因子(Janus-activated kinase Singal transducers and activators of transcription，JAK-STAT)通路相关的疾病的药物中的应用。
本发明还提供了一种式I所示化合物的晶型I或盐酸盐晶型I在抑制JAK、治疗Janus激酶\信号传导及转录激活因子通路相关的疾病中的应用。
进一步地，所述疾病选自：炎症性疾病、肿瘤、自身免疫性疾病及过敏性疾病。
本发明还提供了一种式I所示化合物的晶型I或盐酸盐晶型I在制备预防和/或治疗人和/或动物的炎症性疾病、肿瘤、自身免疫性疾病及过敏性疾病的药物中的应用。
本发明还提供了一种式I所示化合物的晶型I或盐酸盐晶型I在预防和/或治疗人和/或动物的炎症性疾病、肿瘤、自身免疫性疾病及过敏性疾病中的应用。
进一步地，所述炎症性疾病选自：类风湿性关节炎、犬皮炎、银屑病、溃疡性结肠炎或克罗恩病。
进一步地，所述肿瘤为恶性肿瘤(癌症)；更进一步地，所述癌症选自：骨髓纤维化、真性红细胞增多症、特发性血小板增多症、慢性粒细胞白血病、乳腺癌、肺癌或胰腺癌。
进一步地，所述自身免疫性疾病选自：系统性红斑狼疮、1型糖尿病、类风湿性关节炎、多发性硬化、强直性脊柱炎、银屑病、乳糜泻、溃疡性结肠炎或克罗恩病。
进一步地，所述过敏性疾病选自：过敏性皮炎、过敏性结膜炎、过敏性哮喘或过敏性鼻炎。
在本发明的一些实施例中，所述过敏性疾病为犬、猫过敏性皮炎，其可包括如下症状中的一种或多种：皮肤瘙痒、红疹、脱毛、脱屑、水肿、溃疡等，有时伴有耳炎、打喷嚏、流泪等症状，特别是皮肤瘙痒。在本发明的一个实施例中，所述疾病为犬过敏性皮肤瘙痒。
本发明具有以下技术效果：
本发明基于发明人之前的研究成果，经过实验研究，进一步得到式I所示化合物的游离态晶型和盐酸盐晶型的晶体，其不仅衍射峰尖锐，结晶度较高，溶剂残留较少，有单一的DSC热信号，TGA失重小，且配体安全等级较高，可见其均具有较好的固态稳定性和流动性、较低的引湿性、较佳的溶解度，有利于药物制剂的制备和储存，具有非常好的应用价值和成药前景。此外，临床试验结果更是表明，本发明所述的游离态晶型和盐酸盐晶型具有非常好的药代动力学特性，其系统暴露量在实验动物体内呈剂量相关性线性增长，血药浓度达峰快，在体内稳定性高，经连续给药无明显药物蓄积，临床毒性低，安全性较佳，明显 优于阳性对照药物。且本发明所述的游离态晶型和盐酸盐晶型的口服生物利用度高，且对多种病症具有良好的治疗效果，例如犬过敏性瘙痒症状具有良好的抑制和止痒效果。此外，本发明所述的游离态晶型和盐酸盐晶型可通过多种给药途径(例如注射、口服)施用，可采用多种制剂形式，具有非常好的应用前景。
附图说明
图1所示为游离态晶型A的XRPD图。
图2所示为游离态晶型A的TGA/DSC图。
图3所示为游离态晶型A的1H NMR图。
图4所示为盐酸盐晶型A的XRPD图。
图5所示为盐酸盐晶型A的TGA/DSC图。
图6所示为盐酸盐晶型A的1H NMR图。
图7所示为硫酸盐晶型A的XRPD图。
图8所示为硫酸盐晶型A的TGA/DSC图。
图9所示为硫酸盐晶型A的1H NMR图。
图10所示为磷酸盐晶型A的XRPD图。
图11所示为磷酸盐晶型A的TGA/DSC图。
图12所示为磷酸盐晶型A的1H NMR图。
图13所示为游离态晶型A在H2O中溶解度样品的XRPD图。
图14所示为游离态晶型A在SGF中溶解度样品的XRPD图。
图15所示为游离态晶型A在FaSSIF中溶解度样品的XRPD图。
图16所示为游离态晶型A在FeSSIF中溶解度样品的XRPD图。
图17所示为盐酸盐晶型A在pH缓冲液中溶解度样品的XRPD图。
图18所示为游离态晶型A在pH缓冲液中溶解度样品的XRPD图。
图19所示为盐酸盐晶型A的DVS图。
图20所示为游离态晶型A的DVS图。
图21所示为盐酸盐晶型A在DVS测试前后的XRPD叠图。
图22所示为游离态晶型A在DVS测试前后的XRPD叠图。
图23所示为盐酸盐晶型A在稳定性评估前后的XRPD叠图。
图24所示为游离态晶型A在稳定性评估前后的XRPD叠图。
图25所示为盐酸盐晶型A稳定性评估的UPLC图(注：主峰出峰时间偏移可能是由于不同时间测试时系统压力变化导致的。每次测试时将起始样品进行测试以那个API出峰时间)。
图26所示为游离态晶型A稳定性评估的UPLC图(注：主峰出峰时间偏移可能是由于不同时间测试时系统压力变化导致的。每次测试时将起始样品进行测试以那个API出峰时间)。
图27所示为盐酸盐盐晶型A的PLM图。
图28所示为游离态晶型A的PLM图。
图29所示为盐酸盐晶型A研磨及压片前后的XRPD图。
图30所示为游离态晶型A研磨及压片前后的XRPD图。
图31所示为比格犬静脉注射给予2mg/kg游离态晶型A后的血药浓度-时间曲线(对数坐标)。
图32所示为比格犬灌胃给予不同剂量游离态晶型A的血药浓度-时间曲线。
图33所示为比格犬多次给药第1次与第7次灌胃给予6mg/kg游离态晶型A后的血药浓度-时间曲线。
图34所示为雄性比格犬灌胃给予6mg/kg盐酸盐晶型A的血药浓度-时间曲线。
具体实施方式
除非另有定义，本发明中所使用的所有科学和技术术语具有与本发明涉及技术领域的技术人员通常理解的相同的含义。
在本发明中，术语“晶型”是通过X射线粉末衍射图表征证实的。本领域技术人员能够理解，这里所讨论的理化性质可以被表征，其中的实验误差取决于仪器的条件、样品的准备和样品的纯度等。特别是，本 领域技术人员公知，X射线衍射图通常会随着仪器的条件而有所改变。特别需要指出的是，X射线粉末衍射图的相对强度也可能随着实验条件的变化而变化，所以峰强度的顺序不能作为唯一或决定性因素。事实上，XRPD图谱中衍射峰的相对强度与晶体的择优取向有关，本文所示的峰强度为说明性而非用于绝对比较。另外，峰角度的实验误差通常在5％或更少，这些角度的误差也应该被考虑进去，通常允许有±0.2°的误差。另外，由于样品厚度等实验因素的影响，会造成峰角度的整体偏移，通常允许一定的偏移。因而，本领域技术人员可以理解的是，本发明中一个晶型的X射线粉末衍射图不必和本文所指的实施例中的X射线粉末衍射图完全一致，本文所述“XRPD图相同”并非指绝对相同，相同峰位置可相差±0.2°且峰强度允许一定可变性。任何具有和这些图谱中的特征峰相同或相似的图的晶型均属于本发明的范围之内。本领域技术人员能够将本发明所列的图谱和一个未知晶型的图谱相比较，以证实这两组图谱反映的是相同还是不同的晶型。在一些实施方案中，本发明的晶型A是纯的、单一的，基本没有混合任何其他晶型。本发明中，“基本没有”当用来指新晶型时指这个晶型含有少于20％(重量)的其他晶型，尤其指少于10％(重量)的其他晶型，更指少于5％(重量)的其他晶型，更指少于1％(重量)的其他晶型。
需要说明的是，本发明中提及的数值及数值范围不应被狭隘地理解为数值或数值范围本身，本领域技术人员应当理解其可以根据具体技术环境的不同，在不背离本发明精神和原则的基础上围绕具体数值有所浮动，本发明中，这种本领域技术人员可预见的浮动范围多以术语“约”来表示。当在本发明数值前使用术语“约”并且指代所述数值时，其意指该值的±10％的范围内，优选±5％的范围内，更优选±2％的范围内，更优选±1％的范围内的任意值。例如，“约10”应解释为意指9-11，优选为9.5-10.5，更优选为9.8-10.2，更优选为9.9-10.1。
还需要说明的是，在粉末样品X射线衍射图谱中，由晶体化合物得到的衍射谱图特定的晶型往往是特征性的，其中谱带(尤其是在低角度)的相对强度可能会因为结晶条件、粒径、混合物的相对含量和其它测试条件的差异而产生的优势取向效果而变化。因此，衍射峰的相对强度对所针对的晶体并非是特征性的，判断是否与已知的晶型相同时，更应该注意的是峰的位置而不是它们的相对强度。
在本发明中，术语“室温”是指物品的温度与空间(例如所述物品位于其中的通风橱的场所)的温度接近或相同。通常，室温为约20℃至约30℃，或约22℃至27℃，或约25℃。
反溶剂结晶(也称为反溶剂添加、沉淀析晶、盐析或逼晶)法通常是通过在用正溶剂溶解了目标产物的溶液中加入一种或几种反溶剂，产品在所述溶液中处于微溶状态，从而使溶液达到过饱和状态后析出结晶的方法。反-反溶剂结晶通常是通过在用正溶剂溶解了目标产物的溶液后，将其加入一种或几种反溶剂，产品在所述溶液中处于微溶状态，从而使溶液达到过饱和状态后析出结晶的方法。
反溶剂溶解目标产物的能力比正溶剂差，比如差超过10％、20％、30％、40％、50％、60％、70％或者80％，所以，体系中的反溶剂是相对而言。正溶剂和反溶剂可以为极性溶剂或非极性溶剂，如可以选自：二甲基甲酰胺(DMF)、二甲基亚砜(二甲基亚砜)、水、醇类溶剂、醚溶剂、酮类溶剂、酯类溶剂、烷烃类溶剂、芳烃类溶剂、腈类溶剂的一种或多种。其中，醇类溶剂包括但不限于甲醇、乙醇、丙醇、异丙醇或1，3-丙二醇、1，2-丙二醇或三氯叔丁醇或其组合；醚溶剂包括但不限于诸如四氢呋喃、甲基叔丁基醚或1，4-二氧六环或其组合；酮类溶剂包括但不限于丙酮、甲乙酮或4-甲基-2-戊酮或其组合；酯类溶剂包括但不限于乙酸乙酯、乙酸异丙酯、乙酸正丁酯或乙酸叔丁酯或其组合；烷烃类溶剂包括但不限于二氯甲烷、氯仿、正己烷、环己烷或戊烷或正庚烷或其组合；芳烃类溶剂包括但不限于苯、甲苯或其组合；腈类溶剂包括但不限于乙腈、丙二腈。
反溶剂结晶、反-反溶剂结晶可以通过间歇、半间歇或连续结晶操作。反溶剂加入溶液(反溶剂结晶)中或产品溶液加入反溶剂(反-反溶剂结晶)中，可以以恒定速率滴加，也可以是起始时缓慢滴加，然后逐渐递加速率。
在本发明中，除非另外指出，否则本文中所述的“动物”为非人类动物，特别是哺乳动物，例如猴、猪、牛、羊、马、驴、狗、猫、兔、鼠、狐、貉、貂、骆驼等。在本发明的一些实施方式中，所述动物为宠物，如狗、猫、啮齿动物(例如龙猫、宠物兔、仓鼠、荷兰猪、小白鼠、沙鼠、金花鼠、松鼠、复齿鼯鼠、花栗鼠、逆毛绒豚、八齿鼠等)。
术语“治疗”是指在疾病发作之后预防、治愈、逆转、减弱、减轻、最小化、抑制、制止和/或停止疾病的一种或多种临床症状。
术语“预防”指在疾病发作之前，通过治疗以避免、最小化或令疾病难于发作或发展。
术语“炎症”是机体对于刺激的防御反应，表现为红、肿、热、痛和功能障碍等；其可以为感染引起的感染性炎症，也可以为不是由于感染引起的非感染性炎症，例如免疫反应引起的炎症(如各种类型的超敏反应、一些自身免疫性疾病引起的炎症)。术语“炎症性疾病”是指具有炎症的疾病。
术语“肿瘤”指组织的异常块，其中所述块的生长超过正常组织的生长并且不与正常组织的生长相协调。肿瘤可以是“良性的”或“恶性的”，这取决于以下特征：细胞分化程度(包括形态和功能)、生长速率、局部侵袭和转移。“良性肿瘤”通常是分化良好的，其特征在于生长比恶性肿瘤慢，并且保持局限于起源部位。此外，良性肿瘤不具有浸润、侵袭或转移到远处部位的能力。在一些情况下，某些“良性”肿瘤后来可能会导致恶性肿瘤，这可能是由于肿瘤的赘生性细胞的亚群中的额外遗传改变造成的，且这些肿瘤称为“癌前肿瘤”。“恶性肿瘤”通常是低分化的(间变性)，并且具有特征性的快速生长，伴随着周围组织的进行性浸润、侵袭和破坏。此外，恶性肿瘤通常具有转移到远处部位的能力。
术语“癌症”是指恶性肿瘤(Stedman’s Medical Dictionary，25th ed.；Hensyl ed.；Williams&Wilkins：Philadelphia，1990)。
术语“自身免疫性疾病”是指机体对自身抗原发生免疫反应而导致自身组织损害所引起的疾病。美国自身免疫相关疾病协会(AmericanAutoimmune Related Diseases Association)列出了一份比较全面的自身免疫性疾病清单。
本文所引用的各种出版物、专利和公开的专利说明书，其公开内容通过引用整体并入本文。
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限制本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例中所用到的溶剂名称的中英文如下：
MeOH：甲醇；EtOH：乙醇；IPA：异丙醇；n-BuOH：正丁醇；Acetone：丙酮；MIBK：甲基异丁基酮；MEK：2-丁酮；EtOAc：乙酸乙酯；IPAc：乙酸异丙酯；n-Butyl acetate：乙酸正丁酯；MTBE：甲基叔丁基醚；CPME：环戊基甲醚；THF：四氢呋喃；2-MeTHF：2-甲基四氢呋喃；1，4-Dioxane：1，4-二氧六环；ACN：乙腈；DCM：二氯甲烷；Toluene：甲苯；n-Heptane：正庚烷；DMSO：二甲亚砜；Anisole：苯甲醚；Cyclohexane：环己烷；n-Hexane：正己烷。
实施例中所述化合物A的结构如下：
其制备如下：
第1步：化合物2的合成
室温条件下，向5L的三口瓶中依次加入化合物1(500g，2.58mol)、二氯甲烷(1L)和纯净水(1L)，开启机械搅拌，然后依次加入碳酸氢钠(400g，4.76mol)、二碳酸二叔丁酯(650g，2.98mol)，加毕，室温下搅拌4小时后，加入二氯甲烷(1L)和纯净水(1L)，分液，水相用二氯甲烷萃取(800mlX 2)，合并有机相，用饱和食盐水洗涤(1000ml X 1)，有机相用无水硫酸钠干燥，过滤，40度减压浓缩至无馏 分蒸出，加入石油醚(500ml)，室温打浆0.5小时，减压过滤，收集滤饼，25℃鼓风干燥16小时得到白色固体513g，收率77％。
第2步：化合物3的合成
室温条件下，向5L的三口瓶中加入化合物2(513g，2.58mol)和无水DMF(2L)，开启机械搅拌，氮气减压置换3次，冰浴控温在10℃，开始分批加入氢化钠(103.8g，2.59mol)，放热不明显，加毕，搅拌0.5小时用温水加热恢复到室温，滴加碘甲烷(340g，2.40mol)，放热不明显，100分钟滴加完毕，缓慢加热到36-38℃后开始剧烈反应，放出大量热量(不可控)，降至室温搅拌过夜。将反应液缓慢倒入2L饱和NH4Cl溶液中(控温小于20℃)，然后用EA萃取(1L×3)，有机相用饱和NaCl洗涤(500mL×3)，浓缩有机相得到油状物550g，湿法柱层析，PE/EA＝10/1-5/1洗脱，得到无色油状物517g，收率95.9％。
第3步：化合物4的合成
向5L三口瓶中无水四氢呋喃(300mL)，冰浴控温10-15℃，N2保护下，开启搅拌，分批加入四氢铝锂(46g，1.21mol)控温小于-5℃，滴加3(516g，1.90mol)的无水四氢呋喃溶液(1.5L)，加毕反应2.5h，控温0-10℃滴加15％氢氧化钠溶液46mL，水135mL，滴加完毕，减压抽滤，滤饼用500mL乙酸乙酯淋洗，抽滤完毕，收集滤液，向滤液中分别加入乙酸乙酯500ml，水1000ml，分液，收集有机相，水相用再EA萃取(500ml×3)，合并有机相并用饱和氯化钠溶液洗涤(500mL×3)，干燥，浓缩得到407g无色油状物，收率88.1％。
第4步：化合物5的合成
在室温条件下，向3L三口瓶中依次加入化合物4(407g，1.67mol)、无水二氯甲烷(1000ml)、吡啶(304.3g，3.9mol)，氮气保护下开启搅拌，冰浴控温10-15℃条件下滴加甲基磺酰氯(250g，2.18mol)，滴加完毕，室温搅拌过夜，加入500mL水淬灭反应，水相用二氯甲烷萃取(500mL×3)，饱和NaCl溶液洗涤(1000ml X 1)，干燥，浓缩得到黄色油状物510.2g，收率94.5％。
第5步：化合物6的合成
在室温下，向10L三口瓶中依次加入化合物5(510g，1.59mol)、丙酮(3000ml)、NaI(310g，2.07mol)，开启搅拌，加热回流反应过夜，降温到室温，加入水(1000ml)淬灭，然后用乙酸乙酯萃取(1L×3)，再用2L饱和NaCl洗涤，干燥，浓缩得到油状物423g，直接用于下一步，收率75.8％。
第6步：化合物8的合成
在室温下，向5L三口瓶中加入化合物7(35g，0.289mol)和无水四氢呋喃(1100ml)，开启搅拌，降温至0-5℃，分批加入氢化钠(27.5g，0.687mol)，放热剧烈，加毕，滴加特戊酸酐(51.2g，0.275mol)的THF溶液(1000ml)，控温小于20℃，室温反应2h，然后向体系中加入甲醇100mL，控温0-20℃滴加NH4Cl水溶液600mL和饱和食盐水600mL淬灭反应，然后用乙酸乙酯萃取(500mL×3)，有机相用1L饱和NaCl洗涤，干燥，浓缩得到油状物，柱层析PE/EA＝5/1，3/1，得到固体用50mL石油醚打浆，得到白色固体45.8g，收率81.3％。
第7步：化合物9的合成
在室温下，向500mL三口瓶中依次加入化合物8(45g，0.22mol)、1，4-二氧六环(200ml)、15-crown-5(58g)，开启搅拌，降温至10-20℃，分批加入氢化钠(10.5g，0.26mol)加毕，室温滴加碘乙烷(68.4g，1.44mol)，加毕40℃搅拌过夜，然后向体系中加入氯化铵水溶液(100mL)淬灭，水相用乙酸乙酯萃取(100mL×3)，再用200mL饱和NaCl洗涤，干燥，浓缩得到油状物，柱层析PE/EA＝10/1，7/1，5/1，3/1得到白色固体45.26g，收率88.3％。
第8步：化合物10的合成
在室温下，向1L三口瓶中加入化合物9(45g，0.19mol)和二氯甲烷(200ml)，开启搅拌，室温下加入三氟乙酸(33g，0.29mol)，加毕，室温搅拌3h，然后向体系中加入饱和氯化钠水溶液(100mL)淬灭，用二氯甲烷萃取(50mL×3)，再用饱和碳酸氢钠100ml洗涤，干燥，浓缩得到固体粗品，柱层析依次用PE/EA＝5/1，3/1，1/1洗脱得到白色固体27g，收率80.2％。
第9步：化合物11的合成
室温下，向500mL三口瓶中依次加入化合物10(26g，0.146mol)、1，4-二氧六环(146ml)、15-crown-5(48.5g)，开启搅拌，降温至10-20℃，分批加入氢化钠(8.8g，0.22mol)，加毕，室温滴加化合物6(103.8g，0.293mol)的THF溶液(50ml)，加毕50℃搅拌5天，然后向体系中加入氯化铵水溶液100mL淬灭，EA萃取(100mL×3)，有机相用饱和NaCl(200ml)洗涤，干燥，浓缩得到油状物，柱层析依次用PE/EA＝10/1，7/1，5/1，3/1洗脱得到白色固体31.4g，收率53.5％。
第10步：化合物12的合成
向1L三口瓶中加入化合物11(31g，0.077mol)和无水四氢呋喃(500mL)，冰浴控温10℃，N2保护下，开启搅拌，分批加入四氢铝锂(8.8g，0.24mol)加毕反应1h，控温0-10℃滴加饱和硫酸钠溶液(24ml)淬灭，然后加乙酸乙酯200ml稀释并减压抽滤，滤饼用50mL乙酸乙酯淋洗，抽滤完毕，收集滤液并加水100ml分液，水相用乙酸乙酯萃取(50ml×2)，合并有机相并用饱和氯化钠溶液洗涤(100ml×1)，干燥，浓缩得到无色油状物，柱层析用乙酸乙酯洗脱得到油状物18.5g，收率80％。
第11步：化合物13的合成
在室温下，向250mL三口瓶中加入化合物12(18.5g，0.058mol)和二氯甲烷(45ml)，开启搅拌，室温下加入三氟乙酸(20ml)，加毕，室温搅拌过夜，浓缩至无馏分蒸出，得到粗品13.3g，直接投下一步。
第12步：化合物15的合成
向250mL三口瓶中依次加入化合物13(13.3g，0.058mol)、无水DMF(100ml)、碳酸钾(33.4g，0.24mol)和化合物14(18.7.0.058mol)开启搅拌，升温至115℃，反应过夜，降温到室温，然后加入水(150ml)淬灭反应，乙酸乙酯萃取(100mL×6)，再用饱和NaCl洗涤(500ml X 3)，有机相干燥，浓缩得到油状物，柱层析PE/EA＝1/1，DCM/MeOH＝25/1得到半固态固体，加入乙酸乙酯10ml和正己烷50ml打浆得到类白的固体22.4g，收率76.5％。
第13步：化合物A的合成
向500mL三口瓶中加入化合物15(22.4g，0.045mol)、无水THF(250ml)和TBAF溶液(93ml， 1M/L)开启搅拌，回流反应过夜，降温浓缩反应液无馏分蒸出，然后向体系中依次加入水(150ml)，乙酸乙酯(200ml)，分出有机相，水相再用乙酸乙酯(100mlX 3)萃取，再用饱和NaCl洗涤(500mlX 1)，无水硫酸钠干燥，浓缩得到固态粗品，柱层析分离，用DCM/MeOH＝25/1洗脱，然后用乙酸乙酯(20ml)打浆得到白色的固体11g，收率73.3％。
Ms：336.2[M+H]+.1H NMR(400MHz，DMSO)δ11.60(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＝7.6Hz，2H)，2.93(d，J＝6.0Hz，2H)，2.12-1.99(m，3H)，1.75-1.68(m，4H)，1.33-1.24(m，2H)，1.22(t，J＝7.6Hz，3H).
实施例1化合物A游离态晶型的制备和表征
1.1仪器和方法
1.1.1 X射线粉末衍射(XRPD)
XRPD结果是在帕纳科X’Pert3和EmpyreanX射线粉末衍射分析仪上采集，样品放置在无背景硅片中央进行测试，测试参数如表1所示。
表1 XRPD测试参数
1.1.2热重分析(TGA)和差式扫描量热(DSC)
TGA和DSC图分别在TA Q5000热重分析仪和TA Discovery 2500差示扫描量热仪上采集，测试参数如表2所示。
表2 TGA和DSC测试参数
1.1.3氢谱液态核磁(1H NMR)
氢谱液态核磁谱图在Bruker 400M核磁共振仪上采集，DMSO-d6作为溶剂。
1.1.4动态水分吸附(DVS)
动态水分吸附(DVS)曲线在SMS(Surface Measurement Systems)的DVS Intrinsic上采集。在25℃时的相对湿度用LiCl、Mg(NO3)2和KCl的潮解点校正。测试参数如表3所示。
表3 DVS测试参数
1.1.5超高效液相色谱(UPLC)
试验中纯度由Waters H-Class超高效液相色谱仪测试，分析条件如表4。
表4超高效液相色谱测试条件
1.2化合物A游离态晶型A的制备和表征
以MeOH为良溶剂，以MIBK为反溶剂，通过反溶剂添加法(表6中所示试验编号02-A1)，得到游离态晶型A。
游离态晶型A的XRPD图谱如图1所示，XRPD衍射峰数据如表5所示。
游离态晶型A的TGA/DSC图谱如图2所示。TGA结果表明，其从室温加热至180℃失重1.7％；DSC结果表明，其在197.4℃(起始温度)处有一个尖锐的吸热峰，推测为熔化信号。
游离态晶型A的1H NMR图谱如图3所示。
上述表征结果表明，化合物A游离态晶型A具有较小的TGA失重及单一的DSC信号，推测其为无水晶型。
表错误！文档中没有指定样式的文字。游离态晶型A的XRPD衍射峰数据
上述晶型制备的具体实验方法和结果如下：
分别称取约20mg化合物A样品加至20mL的小瓶内，用0.3～1.0mL的良溶剂(见表6)溶解后，向该澄清溶液中加入表6中的反溶剂，边滴加边搅拌至有固体析出，若加入5mL反溶剂后仍无固体析出则停止加入反溶剂。离心分离析出的固体并进行XRPD测试。若无固体析出则转移至5℃下搅拌或室温下挥发。 试验结果见表6，反溶剂添加试验中得到游离态晶型A和成油样品。
表6试验结果
*：反溶剂添加后得到澄清溶液或油状样品，在室温下搅拌过夜析出固体。
#：反溶剂添加得到澄清溶液，在室温下搅拌过夜后仍澄清，转移至5℃继续搅拌7天析出固体。
&：反溶剂添加得到澄清溶液，在室温/5℃下搅拌后仍澄清，转移至室温挥发析出固体。
实施例2化合物A盐型晶型的制备和表征
2.1仪器和方法
2.1.1 X射线粉末衍射(XRPD)
XRPD结果在X’Pert3和Empyrean X射线粉末衍射分析仪上采集，测试参数如表7所示。
表7 XRPD测试参数
2.1.2热重分析(TGA)和差式扫描量热(DSC)
TGA结果是在TA Discovery 5500热重分析仪上采集，DSC结果是在TA Discovery 2500差示扫描量热仪上采集，表8列出了测试参数。
表8 TGA和DSC测试参数

2.1.3氢谱液态核磁(1H NMR)
氢谱液态核磁谱图在Bruker 400M核磁共振仪上采集，DMSO-d6作为溶剂。
2.1.4动态水分吸附(DVS)
动态水分吸附(DVS)曲线在SMS(Surface Measurement Systems)的DVS Intrinsic上采集。在25℃时的相对湿度用LiCl、Mg(NO3)2和KCl的潮解点校正。DVS测试参数列于表9。
表9 DVS测试参数
2.1.5超高效液相色谱(UPLC)
实验中纯度、溶解度和摩尔比由Waters H-Class UPLC超高效液相色谱仪测试，分析条件如表10所示。
表10超高效液相色谱测试条件

2.1.6 PLM
PLM图片是在ZEISS Scope.A1偏光显微镜上采集。
2.1.7离子色谱(IC)
实验中使用ThermoFisher ICS-1100离子色谱仪分析离子含量，具体条件见表11。
表11离子色谱条件和参数
2.2化合物A盐型晶型的制备和表征
本实验以化合物A游离态晶型A为原料，研究了化合物A游离态晶型A与23种不同的酸和4种不同的溶剂下，形成相应盐型晶型的可能性。具体实验汇总于表12。具体步骤如下：首先称取约20mg的化合物A游离态晶型A和等摩尔的相应酸配体于HPLC小瓶中，加入0.5mL溶剂，室温下磁力搅拌(～1000rpm)约4天后分离固体用于XRPD检测；若无固体析出则转移至5℃/-20℃搅拌、添加反溶剂或室温挥发。
研究发现，92种不同的实验条件下，化合物A游离态晶型A在酸体系里不易得到相应的盐型晶型。经XRPD比对后，仅有3种盐型晶型能在4种不同溶剂下都稳定存在，分别为盐酸盐晶型A、硫酸盐晶型A和磷酸盐晶型A。
表12盐型筛选实验结果汇总

[1]：室温搅拌后成油或成胶，转移至50～5℃温度循环搅拌后仍成油或成胶，转移至室温真空干燥。
[2]：室温搅拌后澄清，转移至5℃及-20℃搅拌后仍澄清，添加反溶剂n-Heptane后成油，转移至50～5℃温度循环搅拌后析出固体。
[3]：室温搅拌后澄清，转移至5℃及-20℃搅拌后仍澄清，添加反溶剂n-Heptane后成油，转移至50～5℃温度循环搅拌后成胶，转移至室温真空干燥。
2.2.1盐酸盐晶型A
XRPD结果如图4所示，XRPD衍射峰数据如表13所示。
TGA/DSC结果如图5所示，样品加热至150℃失重0.53％，DSC结果显示在212.9℃(起始温度)处有1个尖锐的吸热峰。
1H NMR结果如图6所示，残留溶剂EtOH与API的摩尔比为0.06(0.7wt％)。
表13盐酸盐晶型A的XRPD衍射峰数据

2.2.2硫酸盐晶型A
XRPD结果如图7所示，XRPD衍射峰数据如表14所示。
TGA/DSC结果如图8所示。TGA结果显示，样品加热至100℃有14.83％的台阶式失重；DSC结果显示，在56.9℃和96.1℃(峰值温度)处有2个吸热信号。
1H NMR结果如图9所示，未检测到明显的残留溶剂Acetone。
表14硫酸盐晶型A的XRPD衍射峰数据

2.2.3磷酸盐晶型A
XRPD结果如图10所示，XRPD衍射峰数据如表15所示。
TGA/DSC结果如图11所示。TGA结果显示，样品加热至150℃失重2.82％；DSC结果显示，在69.0℃、149.0℃和163.2℃(峰值温度)处有3个吸热信号。
1H NMR结果如图12所示，未检测到明显的残留溶剂Acetone。
表15磷酸盐晶型A的XRPD衍射峰数据

对比游离态晶型A、盐酸盐晶型A、硫酸盐晶型A和磷酸盐晶型A的TGA/DSC图可以看出，游离态晶型A和盐酸盐晶型A的热稳定性较好。游离碱晶型A加热到180℃干燥失重为1.7％，盐酸盐晶型A加热到150℃干燥失重为0.53％，两者的熔点分别为201.5℃和216.4℃。硫酸盐晶型A样品加热至100℃有14.83％的台阶式失重，DSC结果显示，其在56.9℃和96.1℃处有2个吸热信号，表明硫酸盐晶型A在较低温度下容易发生分解或融化，热稳定性较差。磷酸盐晶型A样品加热至150℃失重为2.82％，但DSC结果显示，其在69.0℃、149.0℃和163.2℃处有3个吸热信号，表明其热稳定性略差。
综上所述，相对硫酸盐晶型A和磷酸盐晶型A，游离态晶型A和盐酸盐晶型A不仅晶体衍射峰尖锐，结晶度较高，溶剂残留较少，具有单一的DSC热信号，TGA失重小等，且配体安全等级较高，可见，本发明经过百余种试验条件的测试，所获得晶型中仅游离态晶型A和盐酸盐晶型A具有较好的固态性质，可以作为潜在的药物晶型进行开发。
实施例3动态溶解度评估
在37℃下对盐酸盐晶型A和游离态晶型A在H2O及生物溶媒(SGF、FaSSIF和FeSSIF)中的动态溶解度进行了评估。具体步骤如下：分别称取约40mg的盐酸盐晶型A、和游离态晶型A至5mL玻璃小瓶中，分别加入4mL的相应介质(H2O、SGF、FaSSIF及FeSSIF)；37℃下在旋转培养器上以25rpm的速率旋转混合，取样点为1、2、4、24小时；在每个取样点取约0.8mL的悬浊液到离心管中，离心分离(12000rpm，3min，37℃)；上清液用PTFE滤膜(孔径为0.45μm)过滤，滤液测试溶解度和pH，固体测XRPD(使用仪器和方法见实施例2)。
动态溶解度评估结果汇总于表16中，结果显示：盐酸盐晶型A在H2O、SGF、FaSSIF和FeSSIF中均溶清，溶解度＞9mg/mL；游离态晶型A在SGF和FeSSIF中的溶解度约9～10mg/mL(样品未溶清)，在H2O和FaSSIF中的溶解度相对较低(2.1～2.4mg/mL)。
各取样点分离出的固体样品的XRPD结果见图13至图16，结果显示：游离态晶型A在H2O、FaSSIF和FeSSIF中1、2、4、24小时后均未发生晶型转变；在SGF中24小时后观察到盐酸盐晶型A的衍射峰，推测部分游离态与介质中Cl-反应生成盐酸盐。
表16动态溶解度评估结果

S：溶解度(mg/mL)，以游离态计。FC：晶型转变。--：样品溶清，无固体用于XRPD测试。
实施例424小时溶解度评估
在室温下对盐酸盐晶型A和游离态晶型A在pH 2.0、pH 4.5和pH 7.4缓冲液中的24小时溶解度进行了评估。具体步骤如下：分别称取约10mg的盐酸盐晶型A和游离态晶型A至HPLC小瓶中，分别加入1mL的相应缓冲液；室温下磁力搅拌24小时(～500rpm)。将悬浊液离心分离(10000rpm，2min，室温)；上清液用PTFE滤膜(孔径为0.22μm)过滤，滤液测试溶解度和pH，固体测XRPD(使用仪器和方法见实施例2)。
24小时溶解度结果汇总于表17中，分离出的固体样品的XRPD结果见图17和图18。结果显示：(1)盐酸盐晶型A在pH 2.0和pH 4.5中溶解度较高(＞9.2mg/mL，样品溶清)；在pH 7.4中转变为游离态晶型A；(2)游离态晶型A在pH 4.5缓冲液中溶解度较高(＞9.1mg/mL，样品溶清)；在pH 2.0和pH 7.4中未溶清，溶解度分别为6.2mg/mL和1.7mg/mL，XRPD结果显示搅拌24小时后晶型不变。
表17盐酸盐晶型A与游离态晶型A在pH缓冲液中的溶解度结果
S：溶解度(mg/mL)，以游离态计。FC：晶型转变。
--：样品溶清，无固体用于XRPD测试。
实施例5引湿性评估
通过25℃下0％RH～95％RH之间的动态水分吸附实验对盐酸盐晶型A和游离态晶型A的引湿性进行了评估，结果汇总在表18中(使用仪器和方法见实施例2)。
DVS结果如图19和图20所示，盐酸盐晶型A和游离态晶型A在25℃/80％RH时吸湿增重分别为0.47％和0.39％，表明其略有引湿性。
DVS测试前后样品的XRPD对比结果如图21和图22所示，XRPD结果表明，两种样品在DVS测试后均未发生晶型转变。
表18盐酸盐晶型A与游离态晶型A的引湿性结果
实施例6固态稳定性评估
为了评估盐酸盐晶型A和游离态晶型A的固态稳定性，分别称取适量样品，设置在80℃条件下闭口放置1天以及25℃/60％RH和40℃/75％RH条件下敞口放置4周的稳定性实验。将不同条件下分离出的固体样品，分别通过XRPD测试晶型评估物理稳定性，UPLC测试纯度评估化学稳定性(使用仪器和方法见实施例2)。评估结果汇总于表19。
XRPD对比结果如图23和图24所示，UPLC结果汇总在表20至表21，UPLC图谱如图25和图26。固态稳定性结果显示：盐酸盐晶型A和游离态晶型A在三种评估条件下均未发生晶型转变或纯度降低，表明其在评估条件下具有较好的物理和化学稳定性。
表19盐酸盐晶型A和游离态晶型A的固态稳定性结果
*：结晶度有所降低。
表20盐酸盐晶型A稳定性评估的UPLC结果
表21游离态晶型A稳定性评估的UPLC结果
实施例7粉体学性质评估
对盐酸盐晶型A和游离态晶型A开展粉体学性质的评估，包括流动性和形貌，以了解各盐型和游离态晶型A的粉体学基本性质。
通过测试样品的松密度/振实密度和休止角的以评估流动性(使用仪器和方法见实施例2)。松密度与振实密度的测试方法：(1)将一定质量的待评估样品加入一个5mL量筒中，记录此时体积。松密度由样品的质量除以此时的体积计算得到。(2)将量筒轻叩200次，记录最终体积，振实密度由样品质量除以最终体积得到。(3)平行测试三次。休止角的测试方法：(1)将漏斗与底面垂直固定，将物料缓慢加入到漏斗中。(2)底面形成一个匀称的物料的椎体。测量椎体的高度和底面直径。(3)平行测定三次。
松密度/振实密度的结果汇总于表22，结果显示，盐酸盐晶型A样品的松密度和振实密度分别为0.14g/cm3和0.24g/cm3，计算得到卡尔指数为41％；游离态晶型A样品的松密度和振实密度分别为0.11g/cm3和0.23g/cm3，计算得到卡尔指数为51％。
休止角的结果汇总于表23，结果显示，盐酸盐晶型A和游离态晶型A粉末样品的休止角分别为33.9°和47.1°。综合评估的结果，盐酸盐晶型A和游离态晶型A样品均具有较小的卡尔指数和休止角，具有较好的流动性。
PLM结果如图27和图28所示，在偏光显微镜下观察到盐酸盐晶型A为棒状晶体；游离态晶型A为针状或长片状晶体。
表22松密度/振实密度测试结果
卡尔指数＝(振实密度-松密度)/振实密度。
表23休止角测试结果
休止角α计算公式：α＝tan-1(h/D)，h为椎体高度，D为椎体直径。
实施例8机械力稳定性评估
以盐酸盐晶型A和游离态晶型A为起始样品，分别对其进行手动研磨和压片机压片(350MPa压力)，并对研磨及压片后的样品进行XRPD测试以评估其机械力稳定性(使用仪器和方法见实施例2)。具体结果汇总在表24中。
研磨及压片前后的XRPD结果如图29和图30所示，结果显示：(1)在研磨后，盐酸盐晶型A晶型及结晶度均不变，游离态晶型A晶型不变但部分衍射峰展宽，推测结晶度可能略有降低；(2)在压片后，盐酸盐晶型A和游离态晶型A晶型均不变，结晶度均略有降低(部分衍射峰展宽，部分微弱的衍射峰消失)。
表24机械力稳定性评估结果
综合上述评估结果可知，盐酸盐晶型A和游离态晶型A均具有较好的固态稳定性和流动性、较低的引湿性、较佳的溶解度等，二者均具备优异的综合理化性质，可作为潜在的药物晶型进行开发，具有良好的应用前景。
实施例9游离态晶型A的药代动力学研究
1、实验目的
本实验旨在研究游离态晶型A(实施例1制备，在本实施例中有时简称为药物)在雌雄比格犬体内的药代动力学性质，包括1)2mg/kg单次静脉注射(i.v.)研究；2)2、6及20mg/kg灌胃(i.g.)给药剂量递增研究；3)6mg/kg剂量下连续7天灌胃给药研究。
2、实验动物
本实验所用的普通级比格犬，购自于北京玛斯生物技术有限公司，许可证号：SCXK(京)2016-0001，动物合格证号为1103182011000078和1103182011000079，实验动物使用许可证号：SYXK(苏)2021-0045，动物房通风良好，装备空调，温度保持在16-26℃，湿度保持在40％-70％，采用人工照明，明暗各12小时。第一次给药时雄性比格犬的体重范围为8.0-11.6kg，雌性比格犬的体重范围为6.9-11.3kg。
3、制剂配制
(1)静脉注射给药
静注给药组溶媒：DMA∶30％Solutol-HS 15∶Saline＝10∶10∶80，v/v/v。
称量150.34mg游离态晶型A至玻璃瓶中，加入7.517mL的DMA，涡旋并超声至颗粒溶解后，加入7.517mL的30％(w/v)Solutol-HS水溶液，涡旋混匀后，再加入60.136mL的Saline，涡旋混匀，得终浓度为2mg/mL的无色溶液，使用过滤膜(PALL，Nylon，0.45μm)过滤后，用于A组动物给药。
(2)口服灌胃给药
灌胃给药组溶媒：0.5％CMC-Na水溶液。
称量150.43mg游离态晶型A至玻璃瓶中，加入376.075mL的0.5％CMC-Na水溶液，涡旋并超声至颗粒溶解，即得终浓度为0.4mg/mL的无色溶液，用于B组动物给药。
称量450.58mg游离态晶型A至玻璃瓶中，加入375.483mL的0.5％CMC-Na水溶液，涡旋并超声至颗粒分散，即得终浓度为1.2mg/mL的白色混悬液，用于C组第1天动物给药。C组第2-7天给药制剂配制过程同第1天。
称量1500.94mg游离态晶型A至玻璃瓶中，加入375.235mL的0.5％CMC-Na水溶液，涡旋并超声至颗粒分散，即得终浓度为4mg/mL的白色混悬液，用于D组动物给药。
所有的给药制剂均于给药当天新鲜配制，并留样用于制剂浓度测定，其中D组仅对第1、4、7天的给药制剂进行留样用于制剂浓度测定。
4、实验分组
本实验中所有动物随机分组为4组(3只/性别/组)，分组以及给药信息如表25。A、B、D组动物给药前禁食，C组首、末两次给药前禁食，所有动物给药前禁食时间不少于12h，且于给药后4h恢复给食。所有动物在实验过程中均可自由饮水和进食。
表25动物分组及给药方式
5、样品采集及处理
A组动物静脉注射给予2mg/kg药物，分别在给药前及给药后0.033、0.083、0.25、0.5、1、2、4、8、12和24h于颈静脉采血。B、D组动物分别单次灌胃给予2和20mg/kg药物，分别在给药前及给药后0.083、0.25、0.5、1、2、4、8、12和24h于颈静脉采血。C组动物每天一次灌胃给予6mg/kg药物，连续七天，分别在第一次和第七次给药前及给药后0.083、0.25、0.5、1、2、4、8、12和24h于颈静脉采血，第二到第六次给药期间分别采集(颈静脉)给药前及给药后0.5h血样，其中第二次给药前样品即为第一次给药后24h样品。
上述动物血样均通过颈静脉采集，每次采血量为0.5mL/次，采集的所有全血样品均置于含EDTA-K2 的离心管中，上下颠倒离心管使抗凝剂与血液充分混合。在离心前都放置于湿冰上，1524g条件下离心10min分离血浆，转移血浆样品至新的离心管中，保存在-90～-60℃条件，直至分析。
通过LC-MS/MS方法检测比格犬血浆中药物浓度。所得血药浓度数据采用药动学处理软件WinNonlin8.0非房室模型计算相关药代动力学参数。
6、实验结果
动物在给药前、每个采血时间点前后以及给药后进行临床观察，未见明显异常。各组药物浓度-时间曲线如图31至图33所示，主要药代动力学参数如表26至表27所示。
表26比格犬静脉注射2mg/kg药物后的主要药代动力学参数
比格犬按2mg/kg剂量静脉注射给药后，药物在雄性与雌性比格犬体内的C0和AUC0-t比值分别为0.758和0.748(在0.5-2倍的范围内)，系统暴露量未见明显的性别差异。药物在雌雄比格犬体内清除率(CL)相当于比格犬肝脏血流量(约31mL/min/kg，Davies and Morris(1993))的0.110倍，提示其在比格犬体内清除较慢；稳态分布容积(Vdss)为比格犬体液总量(约0.60L/kg，Davies and Morris(1993))的1.57倍，提示其较倾向分布于组织中。
表27比格犬灌胃给予不同剂量药物的药代动力学参数
比格犬单次灌胃给予2、6和20mg/kg药物后，平均达峰时间在给药后0.792到1.33h之间。经剂量校正后，以AUC0-t平均值计算，口服利用度F％分别为88.0％，100.0％以及101.0％。
比格犬单次灌胃给予2、6和20mg/kg药物后，药物在三个剂量水平下雄性与雌性的Cmax比值分别为1.36、0.810和0.894，AUC0-t比值分别为1.10、0.716和0.877，系统暴露量未见明显的性别差异(比值均在0.5-2倍的范围内)。
雌雄比格犬按2、6和20mg/kg剂量灌胃给药后，单位剂量Cmax分别为675、785和665kg*ng/ml/mg，单位剂量AUC0-t分别为4380、4990和5030ng*h*kg/mL/mg。表明在2到20mg/kg剂量范围内，药物在大鼠体内的系统暴露量呈现剂量相关性线性增长(20和2mg/kg的单位剂量Cmax比率和单位剂量AUC0-t比率分别为0.985和1.15，均在0.5-2倍的范围内)。
比格犬连续7天灌胃给予6mg/kg药物后，雌雄大鼠体内第7次和第1次的Cmax和AUC0-t比值分别为0.964和0.887，未观察到明显药物蓄积。
综上所述，比格犬按2、6和20mg/kg剂量灌胃给药后，系统暴露量无明显雌雄差异。在2-20mg/kg剂量范围内，系统暴露量在实验动物体内呈剂量相关性线性增长，且连续7天给药无明显药物蓄积。
实施例10盐酸盐晶型A的药代动力学研究
1、实验目的
本实验旨在研究盐酸盐晶型A(实施例2制备，在本实施例中有时简称为药物)在雌雄比格犬体内的药代动力学性质。
2、实验动物
本实验所用比格犬，雄，n＝3，31个月，购自玛斯生物技术有限公司，动物质量合格证号：110318201100056783。
3、制剂配制
灌胃给药组溶媒：0.5％CMC-Na水溶液。
称量260.20mg盐酸盐晶型A(实施例2制备)至玻璃瓶中，加入193.827mL0.5％CMC-Na水溶液，涡旋并超声至颗粒溶解，即得终浓度为1.2mg/mL得无色溶液，用于A组动物给药。
4、实验分组
本实验随机选取3只雄性比格犬给药，给药方式如表28。所有动物给药前禁食，禁食时间不少于12h，于给药后4h恢复给食。所有动物在实验过程中均可自由饮水。
表28动物给药方式
5、样品采集及处理
A组动物单次灌胃给予6mg/kg药物，分别在给药前及给药后0.083、0.25、0.5、1、2、4、8、12、24h于颈静脉采血。
上述动物血样均通过颈静脉采集，每次采血量为0.5mL/次，采集的所有全血样品均置于含EDTA-K2的离心管中，上下颠倒离心管使抗凝剂与血液充分混合。在离心前都放置于湿冰上，1500g条件下离心10min分离血浆，转移血浆样品至样品管中，保存在-40～-20℃条件，直至分析。
通过LC-MS/MS方法检测比格犬血浆中药物浓度，LC-MS/MS检测方法如下所示：
仪器：LC-MS/MS(Triple Quad 5500+：LC-MS-MS-023)。
MS条件：ESI正离子模式；多反应监测(MRM)；m/z 336.2/149.1。
色谱柱：Waters Xselect HSS T3，3.5μm，2.1×50mm。
流动相A：H2O(0.1％甲酸+5mM NH4OAc)。
流动相B：MeOH∶ACN＝1∶1(0.1％甲酸)。
流速：0.50mL/min。
进样体积：5μL。
保留时间：1.24min。
洗脱程序如表29所示。
表29洗脱程序
6、实验结果
动物在给药前、每个采血时间点前后以及给药后进行临床观察，未见明显异常。药物浓度-时间曲线如图34所示，主要药代动力学参数如表30所示。
表30比格犬单次灌胃给予6mg/kg药物的药代动力学参数
如上表所示，比格犬单次灌胃给予6mg/kg药物后，Tmax为0.417h，可见本申请盐酸盐晶型A在体内达峰更快。而对于已上市药品爱波克(活性成分为奥拉替尼)，实验犬口服该药物后，Tmax为小于1h(P7，CVMP assessment report for APOQUEL(EMEA/V/C/002688/0000)，EMA/481054/2013)。可见，相对于已上市药品爱波克，本申请盐酸盐晶型A在体内达峰时间方面具备更好或至少相当的优势。
实施例11游离态晶型A的急性毒性试验
1、实验目的
经口服一次性给予受试物游离态晶型A(实施例1制备)后，在短时间内观察实验动物所产生的毒性反应，初步了解受试物的毒性特征和剂量-反应关系。
2、实验动物
本实验所用的Sprague-Dawely大鼠(SPF级)，购自于浙江维通利华实验动物技术有限公司，生产许可证号码为SCXK(浙)2019-0001，动物合格证编号分别为20220107Aazz0619000738，20220107Aazz0619000691，雄性体重范围为206.6～234.5g，雌性体重范围为188.4～205.9g。
3、制剂配制
灌胃给药溶媒：0.5％CMC-Na水溶液：称取17.5031g的CMC-Na(800-1200mPa.s)至合适容器，加入3500mL去离子水，搅拌至均一，得到无色澄清液体，室温保存，直至使用。
100mg/mL游离态晶型A溶液：称取9000.4mg游离态晶型A置于已划线至90mL的广口瓶，加入适量的0.5％CMC-Na(800-1200mPa.s)水溶液至上述容器中，搅拌超声至均一，继续加入适量的0.5％CMC-Na(800-1200mPa.s)水溶液至90mL，搅拌超声至均一。按要求取样，得到乳白色混悬液。
55.9mg/mL游离态晶型A溶液：取44.7mL的100mg/mL游离态晶型A溶液至合适容器中，加入35.3mL的0.5％CMC-Na(800-1200mPa.s)水溶液至上述容器，涡旋至均一。按要求取样，得到乳白色混悬液。
31.2mg/mL游离态晶型A溶液：取44.7mL的55.9mg/mL游离态晶型A溶液至合适容器中，加入35.3mL的0.5％CMC-Na(800-1200mPa.s)水溶液至上述容器，涡旋至均一。按要求取样，得到乳白色混悬液。
17.4mg/mL游离态晶型A溶液：取44.7mL的31.2mg/mL游离态晶型A溶液至合适容器中，加入35.3mL的0.5％CMC-Na(800-1200mPa.s)水溶液至上述容器，涡旋至均一。按要求取样，得到乳白色混悬液。
9.7mg/mL游离态晶型A溶液：取44.7mL的17.4mg/mL游离态晶型A溶液至合适容器中，加入35.3mL的0.5％CMC-Na(800-1200mPa.s)水溶液至上述容器，涡旋至均一。按要求取样，得到白色混悬液。
4、实验分组
本实用中所有动物随机分组为5组(5只/性别/组)，分别给予97、174、312、559和1000mg/kg药物后，经口灌胃给药1次，观察7天，分组及给药信息如表31。所有动物按照最新称量的体重进行给药。药制剂给药前至少搅拌10min，且给药过程也持续搅拌至本浓度样品给药结束。动物给药前禁食11～12h， 给药后约2h恢复饮食，不限制饮水。
所有动物在给药前由兽医进行一次全面的体检。适应期每天进行1～2次笼边观察和1次详细临床观察。实验期间每天进行2次笼边观察(解剖当天1次)，每天1～2次详细临床观察。所有存活动物分别在随机分组当天(Day-1)、Day 1(给药前)、Day 3、Day 5、Day 7及Day 8称量一次体重；分别在Day 1(Day 1～Day 2)、Day 2(Day 2～Day 3)、Day 6(Day 6～Day 7)测定1次24±1小时的耗食量。实验期末(Day 8)通过二氧化碳吸入加腹主动/静脉放血的方式对所有存活动物实施安乐死后进行大体解剖检查，试验过程中死亡动物进行大体解剖检查。
表31动物分组及给药方式
5、实验结果
各浓度的供试品制剂中药物的平均回收率在105.8％-109.8％范围内，低、高浓度样品上中下三层回收率％RSD≤0.89，符合平均回收率在理论浓度100±15％及％RSD≤10的接受标准。
在本试验条件下，SD大鼠单次经口灌胃给予药物后，雄性大鼠LD50为592mg/kg，95％可信区间为429～818mg/kg，最大耐受剂量(Maximal tolerance dose，MTD)为312mg/kg；雌性大鼠LD50为418mg/kg，95％可信区间为313～558mg/kg，最大耐受剂量为174mg/kg。而已上市药品爱波克对大鼠的LD50为310mg/kg(P9，CVMP assessment report for APOQUEL(EMEA/V/C/002688/0000)，EMA/481054/2013)，表明本发明的游离态晶型A的毒性更低。
实施例12比格犬单次灌胃给予游离态晶型A后尿液和粪便排泄研究
1、实验目的
本实验旨在研究游离态晶型A(实施例1制备)在雌雄比格犬单次灌胃给药后，药物在比格犬粪便和尿液中的排泄过程。
2、实验动物
本实验使用的普通级比格犬，购自于北京玛斯生物技术有限公司，生产许可证号：SCXK(京)2016-0001，质量合格证号为1103182011000078(雄)和1103182011000079(雌)。实验动物饲养在苏州圣苏新药开发有限公司动物房内，使用许可证号：SYXK(苏)2021-0045，动物房通风良好，装备空调，温度保持在16-26℃，湿度保持在40％-70％，采用人工照明，明暗各12小时。实验当天给药时雄性比格犬的体重范围为8.6-10.5kg，雌性比格犬的体重范围为7.6-9.0kg。
3、制剂配制
灌胃给药溶媒：0.5％CMC-Na水溶液。
称量450.72mg左右的游离态晶型A至玻璃瓶中，加入378.605mL的0.5％CMC-Na水溶液，搅拌并超声至颗粒分散，即得终浓度为1.2mg/mL的白色混悬液。给药制剂于给药当天新鲜配制，并留样用于制剂浓度的测定。
4、实验分组
6只比格犬(雌雄各半)于实验前一天置于代谢笼中禁食过夜。于给药前收集各动物尿液和粪便作为给药前0点样品。给药当天，比格犬单次灌胃给予6mg/kg的给药制剂。给药4小时后，动物恢复给食，实验过程中动物自由饮水。
5、样品采集及处理
尿液和粪便排泄实验组：于给药前以及给药后0～4、4～8、8～12、12～24、24～48、48～72、72～96和96～120h收集尿液和粪便。尿液收集完成后量取并记录体积，取部分收集的尿液加入甲醇进行稀释，使尿液∶甲醇体积比4∶1(v/v)，涡旋摇匀后，分装2份。各时间段粪便样品分别收集后，捡去表面食物残渣，称重。根据粪便重量加入一定体积匀浆液(20％甲醇水)，粪便与匀浆液比例为1∶10(粪便重量∶匀浆液体积，w/v)，匀浆，分装2份。尿液和粪便排泄实验组采集完最后一个点，用2000mL水∶乙醇(1∶1，v∶v)冲洗代谢笼并单独收集每个笼子的冲洗液，将冲洗液充分摇匀后立即取1mL装入1.5mL EP管中，与尿液和粪便匀浆液一起保存在-90～60℃冰箱中直至样品分析。
运用LC-MS/MS方法检测各组动物不同时间段的排泄样品中的药物浓度，计算各时间段的排泄率和累积排泄率。
6、实验结果
表32比格犬单次灌胃给药6mg/kg药物后的累积排泄率
比格犬按6mg/kg单次灌胃给药后，药物在比格犬尿液中120h内的累积排泄率为43.8±9.97％，在粪便中120h内的累积排泄率为3.48±0.663％。药物在比格犬120h内粪便加尿液的总累积排泄率为47.3±9.62％。120h后比格犬的笼具冲洗液中药物量为给药量的4.87±3.09％。而实验犬口服药物爱波克后，其在犬尿液中24h内的累积排泄率仅为3.6％(P8，CVMP assessment report for APOQUEL(EMEA/V/C/002688/0000)，EMA/481054/2013)。
综上，按6mg/kg单次灌胃给药后，本发明的药物在比格犬体内的总排泄率为52.2％(粪便、尿液加冲洗液)，游离态晶型A在犬体内更加稳定。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，但本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。

Claims (12)

  1. 一种式I所示化合物的晶型I，其特征在于，所述的晶型I的XRPD图谱在2θ值约为12.4°±0.2°、14.6°±0.2°、16.7°±0.2°、17.2°±0.2°、20.3°±0.2°、24.8°±0.2°和25.0°±0.2°的位置中至少三个位置处具有特征峰
  2. 根据权利要求1所述的晶型I，其特征在于，所述的晶型I的XRPD图谱还在2θ值约为13.2°±0.2°、15.2°±0.2°、19.3°±0.2°、21.2°±0.2°、21.6°±0.2°、24.0°±0.2°和27.0°±0.2°的位置中至少三个位置处具有特征峰。
  3. 根据权利要求1所述的晶型I，其特征在于，所述的晶型I具有基本上如图1所示的XRPD图谱；优选地，所述的晶型I具有基本上如图2所示的DSC图谱和TGA图谱。
  4. 一种权利要求1-3任一项所述的晶型I的制备方法，其特征在于，所述的制备方法包括以无定形形式的式I所示化合物为起始原料制备得到晶型I，其选自：反溶剂添加法、气固扩散法、悬浮搅拌法、缓慢挥发法、缓慢降温法、气液渗透法或反-反溶剂添加法中一种或多种的组合；
    优选地，所述的反溶剂添加法包括：将式I所示化合物无定形原料用良溶剂溶解，加入反溶剂；
    优选地，所述的良溶剂选自甲醇、1，4-二氧六环、三氯甲烷或二甲亚砜中的一种；
    优选地，所述的反溶剂选自甲基异丁基酮、乙酸异丙酯、甲基叔丁基醚、正庚烷、乙酸乙酯、间二甲苯、环戊基甲醚、甲苯、苯甲醚或水中的一种；
    更优选地，所述良溶剂为甲醇，反溶剂选自甲基异丁基酮、乙酸异丙酯、甲基叔丁基醚；或，所述良溶剂为1，4-二氧六环，反溶剂选自正庚烷、乙酸乙酯；或，所述良溶剂为三氯甲烷，反溶剂选自正庚烷、间二甲苯、环戊基甲醚；或，所述良溶剂为二甲亚砜，反溶剂选自甲苯、苯甲醚、水。
  5. 一种式I所示化合物的盐酸盐晶型I，其特征在于，所述的盐酸盐晶型I的XRPD图谱在2θ值约为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°和27.5°±0.2°的位置中至少三个位置处具有特征峰
  6. 根据权利要求5所述的盐酸盐晶型I，其特征在于，所述的盐酸盐晶型I的XRPD图谱还在2θ值约为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°和30.0°±0.2°的位置中至少三个位置处具有特征峰。
  7. 根据权利要求5所述的盐酸盐晶型I，其特征在于，所述的盐酸盐晶型I具有基本上如图4所示的XRPD图谱。
  8. 根据权利要求5所述的盐酸盐晶型I，其特征在于，所述的盐酸盐晶型I具有基本上如图5所示的DSC图谱和TGA图谱。
  9. 一种如权利要求5-8任一项所述的盐酸盐晶型I的制备方法，其特征在于，将权利要求1所述的晶型I和盐酸混合，加入溶剂，得到盐酸盐晶型I；
    优选地，所述的溶剂选自乙醇、丙酮/水、乙酸乙酯或2-甲基四氢呋喃中的一种。
  10. 一种药物组合物，其特征在于，其包含如权利要求1-3任一项所述的晶型I或权利要求5-8任一项所述的盐酸盐晶型I，以及一种或多种药学上可接受的辅料。
  11. 一种如权利要求1-3任一项所述的晶型I或权利要求5-8任一项所述的盐酸盐晶型I在制备治疗JAK-STAT通路相关的疾病的药物中的应用。
  12. 一种如权利要求1-3任一项所述的晶型I或权利要求5-8任一项所述的盐酸盐晶型I在制备预防和/或治疗人和/或动物的炎症性疾病、肿瘤、自身免疫性疾病及过敏性疾病的药物中的应用；
    优选地，所述炎症性疾病选自：类风湿性关节炎、犬皮炎、银屑病、溃疡性结肠炎或克罗恩病；
    优选地，所述癌症选自：骨髓纤维化、真性红细胞增多症、特发性血小板增多症、慢性粒细胞白血病、乳腺癌、肺癌或胰腺癌；
    优选地，所述自身免疫性疾病选自：系统性红斑狼疮、1型糖尿病、类风湿性关节炎、多发性硬化、强直性脊柱炎、银屑病、乳糜泻、溃疡性结肠炎或克罗恩病；
    优选地，所述过敏性疾病选自：过敏性皮炎、过敏性结膜炎、过敏性哮喘或过敏性鼻炎；
    更优选地，所述过敏性疾病为犬、猫过敏性皮炎，其包括如下症状中的一种或多种：皮肤瘙痒、红疹、脱毛、脱屑、水肿、溃疡，特别是犬过敏性皮肤瘙痒。
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