Novel crystal forms of compounds, their preparation methods, pharmaceutical compositions containing them, veterinary drug compositions containing them, and their uses.

Abstract

This invention relates to the field of chemical and pharmaceutical technology, and provides a crystal form of a compound of formula (I), a method for preparing the crystal form, a pharmaceutical composition containing the crystal form, and their use in the preparation of medicaments for treating diseases, symptoms or conditions, or methods for treating diseases, symptoms or conditions.

Description

Technical Field

[0001] This invention relates to the field of chemical and pharmaceutical technology, and in particular to a novel crystal form of a compound, a method for preparing the crystal form, a pharmaceutical composition containing the crystal form, and the use of the crystal form and the pharmaceutical composition. Prior Technology

[0002] Polymorphism, or polymorphism, is a unique property of certain molecules and molecular compositions. The same molecule can form different crystals due to different arrangements, and these crystals possess different crystal structures and physical properties, such as solubility, stability, hygroscopicity, and X-ray diffraction patterns. The polymorphism of drug molecules is increasingly attracting widespread attention. Many pharmaceutically active organic compounds can crystallize in more than one three-dimensional crystal structure. However, it is generally impossible to predict whether a specific organic drug compound will form different crystal forms, let alone predict the structure and properties of the crystal form itself. Exploring new crystal forms or polymorphs of pharmaceutically usable compounds can provide opportunities to improve the overall performance of pharmaceutical products.

[0003] The compound (R)-2-(1-(2-(1-hydroxyethyl)imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)piperidin-4-yl)acetonitrile has the structural formula shown in formula (I): (I)

[0004] Currently, research on the crystal form of this compound is still in its infancy. The novel crystal form of the compound of formula (Ⅰ) provided by this invention has the characteristics of good stability, high solubility, and high bioavailability, and has broad application prospects. Summary of the Invention

[0005] The purpose of this invention is to provide a new crystal form of compound (R)-2-(1-(2-(1-hydroxyethyl)imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)piperidin-4-yl)acetonitrile (hereinafter referred to as compound (I)).

[0006] This invention provides a crystal form A of compound of formula (I). (I) The X-ray powder diffraction pattern of crystal form A has characteristic peaks at 2θ angles of 8.6°±0.2°, 9.8°±0.2°, 10.7°±0.2°, 11.2°±0.2°, 12.3°±0.2°, 12.9°±0.2°, 16.0°±0.2°, and 19.0°±0.2°.

[0007] In some embodiments, the X-ray powder diffraction pattern of crystal form A has characteristic peaks at 2θ angles of 8.6°±0.2°, 9.8°±0.2°, 10.7°±0.2°, 11.2°±0.2°, 12.3°±0.2°, 12.9°±0.2°, 16.0°±0.2°, 17.0°±0.2°, 17.4°±0.2°, 17.8°±0.2°, 19.0°±0.2°, 19.9°±0.2°, and 21.2°±0.2°.

[0008] In some embodiments, key information regarding the X-ray powder diffraction pattern of crystal form A is shown in Table 1.

[0009] [Table 1] serial number Diffraction angle 2θ (°) Interplanar spacing (Å) relative strength 1 8.6 10.28 15.8% 2 9.8 8.98 100.0% 3 10.7 8.23 11.6% 4 11.2 7.86 28.1% 5 12.3 7.19 21.3% 6 12.9 6.85 7.5% 7 13.7 6.44 7.3% 8 13.9 6.35 10.0% 9 16.0 5.52 6.4% 10 17.0 5.21 8.8% 11 17.4 5.09 27.4% 12 17.8 4.98 51.4% 13 18.4 4.82 2.2% 14 18.6 4.77 2.5% 15 19.0 4.68 31.1% 16 19.9 4.46 7.3% 17 20.4 4.34 22.8% 18 20.6 4.30 26.0% 19 21.2 4.20 11.3% 20 22.8 3.90 6.0% 21 23.4 3.81 4.7% 22 24.7 3.60 2.5% 23 25.0 3.55 2.8% 24 26.3 3.39 2.7% 25 26.6 3.35 3.7% 26 27.0 3.31 4.9% 27 27.6 3.23 4.4% 28 27.8 3.20 5.1% 29 28.3 3.15 3.6% 30 28.6 3.12 3.1% 31 29.0 3.07 1.5% 32 30.2 2.96 1.8% 33 31.2 2.87 5.3% 34 31.5 2.84 1.7% 35 31.9 2.80 2.1% 36 32.7 2.74 2.2% 37 36.3 2.47 1.3% 38 37.4 2.40 1.2% 39 37.8 2.38 2.5% 40 38.1 2.36 5.3% 41 38.7 2.33 1.0%

[0010] In some embodiments, the crystal form A has an X-ray powder diffraction pattern substantially as shown in FIG2.

[0011] In some embodiments, the crystal form A has an overlay of differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) spectra, which are substantially as shown in Figure 3.

[0012] In some embodiments, the three-dimensional structure of crystal form A is shown in Figure 4.

[0013] In some embodiments, crystal form A is amorphous.

[0014] This invention further provides a method for preparing crystal form A, the method comprising the following steps: 1) Dissolve the compound of formula (I) in a crystallization solvent; 2) Concentration and filtration; The crystallization solvent is selected from ethanol (EtOH), n-heptane, isopropanol (IPA), 2-butanol (2-Butanol), butanone (MEK), ethyl acetate (EtOAc), 2-methyltetrahydrofuran (2-MeTHF), tetrahydrofuran (THF), methyl tert-butyl ether (MTBE), or any mixture of two or more solvents.

[0015] In some embodiments of the present invention, the crystallization solvent is selected from ethanol.

[0016] The present invention further provides a method for preparing crystal form A, comprising: dissolving the compound of formula (I) in ethanol until clear, and then concentrating it; stirring the obtained concentrate to precipitate a solid; filtering and drying to obtain the crystal form A.

[0017] In some embodiments, the concentration temperature is 35°C-55°C.

[0018] In some embodiments, the concentration of the concentrate is 0.5-2 g / mL.

[0019] In some embodiments, the stirring temperature is 5°C-15°C.

[0020] The present invention further provides a crystal form H of the compound of formula (I), wherein the X-ray powder diffraction pattern of the crystal form H has characteristic peaks at 2θ angles of 9.4°±0.2°, 11.0°±0.2°, 14.1°±0.2°, 16.0°±0.2°, 17.8°±0.2°, 19.3°±0.2°, and 21.9°±0.2°.

[0021] In some embodiments, the X-ray powder diffraction pattern of the crystal form H has characteristic peaks at 2θ angles of 9.4°±0.2°, 11.0°±0.2°, 11.7°±0.2°, 14.1°±0.2°, 15.4°±0.2°, 16.0°±0.2°, 17.8°±0.2°, 19.3°±0.2°, 20.9°±0.2°, and 21.9°±0.2°.

[0022] In some embodiments, the crystal form H has an X-ray powder diffraction pattern substantially as shown in FIG5.

[0023] In some embodiments, the crystal form H has a superimposed image of differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) as shown in FIG6.

[0024] In some embodiments, the crystal form H is amorphous.

[0025] The present invention further provides a crystal form S of the compound of formula (I), wherein the X-ray powder diffraction pattern of the crystal form S has characteristic peaks at 2θ angles of 9.1°±0.2°, 10.1°±0.2°, 13.4°±0.2°, 15.5°±0.2°, 16.0°±0.2°, 17.8°±0.2°, 19.7°±0.2°, and 21.8°±0.2°.

[0026] In some embodiments, the X-ray powder diffraction pattern of the crystal form S has characteristic peaks at 2θ angles of 9.1°±0.2°, 10.1°±0.2°, 13.4°±0.2°, 15.5°±0.2°, 16.0°±0.2°, 17.8°±0.2°, 19.7°±0.2°, 21.0°±0.2°, 21.8°±0.2°, 23.3°±0.2°, 24.0°±0.2°, and 24.8°±0.2°.

[0027] In some embodiments, the crystal form S has an X-ray powder diffraction pattern substantially as shown in FIG7.

[0028] In some embodiments, the crystal form S has a superimposed image of differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) as shown in FIG8.

[0029] In some embodiments, the crystal form S is amorphous.

[0030] The present invention further provides a crystal form O of the compound shown in formula (Ⅰ), wherein the X-ray powder diffraction pattern of the crystal form O has characteristic peaks at 2θ angles of 10.3°±0.2°, 11.9°±0.2°, 14.2°±0.2°, 15.3°±0.2°, 19.6°±0.2°, 20.3°±0.2°, 20.5°±0.2°, and 21.4°±0.2°.

[0031] In some embodiments, the X-ray powder diffraction pattern of the crystal form O has characteristic peaks at 2θ angles of 10.3°±0.2°, 11.9°±0.2°, 14.2°±0.2°, 15.3°±0.2°, 19.6°±0.2°, 20.3°±0.2°, 20.5°±0.2°, 21.4°±0.2°, 25.6°±0.2°, 28.6°±0.2°, 29.4°±0.2°, and 30.0°±0.2°.

[0032] In some embodiments, the crystal form O has an X-ray powder diffraction pattern substantially as shown in FIG9.

[0033] In some embodiments, the crystal form O has a superimposed image of differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) as shown in FIG10.

[0034] In some embodiments, the crystal form O is amorphous.

[0035] The present invention further provides a crystal form P of the compound of formula (I), wherein the X-ray powder diffraction pattern of the crystal form P has characteristic peaks at 2θ angles of 9.5°±0.2°, 16.5°±0.2°, 19.6°±0.2°, 20.3°±0.2°, 23.9°±0.2°, and 25.1°±0.2°.

[0036] In some embodiments, the X-ray powder diffraction pattern of the crystal form P has characteristic peaks at 2θ angles of 9.5°±0.2°, 16.5°±0.2°, 19.6°±0.2°, 20.3°±0.2°, 23.9°±0.2°, 25.1°±0.2°, 26.3°±0.2°, and 28.9°±0.2°.

[0037] In some embodiments, the crystal form P has an X-ray powder diffraction pattern substantially as shown in FIG11.

[0038] In some embodiments, the crystal form P has a superimposed image of differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) as shown in FIG12.

[0039] In some embodiments, the crystal form P is amorphous.

[0040] The present invention further provides a crystal form Q of the compound of formula (I), wherein the X-ray powder diffraction pattern of the crystal form Q has characteristic peaks at 2θ angles of 8.5°±0.2°, 14.5°±0.2°, 15.0°±0.2°, 16.8°±0.2°, 19.4°±0.2°, 23.6°±0.2°, 24.8°±0.2°, 26.3°±0.2°, and 27.4°±0.2°.

[0041] In some embodiments, the X-ray powder diffraction pattern of the crystal form Q has characteristic peaks at 2θ angles of 8.5°±0.2°, 14.5°±0.2°, 15.0°±0.2°, 15.8°±0.2°, 16.8°±0.2°, 18.8°±0.2°, 19.4°±0.2°, 20.1°±0.2°, 23.6°±0.2°, 24.8°±0.2°, 26.3°±0.2°, and 27.4°±0.2°.

[0042] In some embodiments, key information regarding the X-ray powder diffraction pattern of the crystal form Q is shown in Table 2.

[0043] [Table 2] serial number diffraction angle 2θ(°) Interplanar spacing (Å) relative strength (%) 1 8.5 10.37 100 2 14.5 6.13 39.46 3 15.0 5.91 48.64 4 15.8 5.60 12.22 5 16.8 5.26 17.81 6 17.2 5.16 5.43 7 17.5 5.07 4.04 8 18.8 4.71 9.87 9 19.4 4.57 44 10 20.1 4.42 12.97 11 21.3 4.18 2.44 12 22.6 3.94 5.51 13 23.2 3.84 2.04 14 23.6 3.78 18.48 15 24.8 3.59 80.59 16 26.3 3.39 37.28 17 27.4 3.26 16.8 18 28.8 3.10 7.1 19 30.4 2.94 10.21 20 33.5 2.67 4.11 twenty one 38.0 2.37 3.57

[0044] In some embodiments, the crystal form Q has an X-ray powder diffraction pattern substantially as shown in FIG13.

[0045] In some embodiments, the crystal form Q has a polarized microscope image that is substantially as shown in FIG14.

[0046] In some embodiments, the crystal form Q has a superimposed image of differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) as shown in FIG15.

[0047] In some embodiments, the crystal form Q has a nuclear magnetic resonance hydrogen spectrum (1H-NMR spectrum) substantially as shown in FIG16.

[0048] In some embodiments, the crystal form Q has a dynamic gas-phase adsorption pattern substantially as shown in FIG17.

[0049] In some embodiments, the crystal form Q is the hydrate crystal form of the compound of formula (I).

[0050] The present invention further provides a method for preparing crystal form Q, comprising: stirring crystal form A in an aqueous ethanol solution; centrifuging and drying to obtain crystal form Q.

[0051] In some embodiments, the water activity of the ethanol aqueous solution is 0.6-0.8.

[0052] The present invention further provides a crystal form M of the compound of formula (I), wherein the X-ray powder diffraction pattern of the crystal form M has characteristic peaks at 2θ angles of 8.7°±0.2°, 9.9°±0.2°, 17.4°±0.2°, 18.6°±0.2°, 18.9°±0.2°, 20.6°±0.2°, and 26.5°±0.2°.

[0053] In some embodiments, the X-ray powder diffraction pattern of the crystal form M has characteristic peaks at 2θ angles of 8.7°±0.2°, 9.9°±0.2°, 13.1°±0.2°, 17.4°±0.2°, 18.6°±0.2°, 18.9°±0.2°, 20.6°±0.2°, 26.5°±0.2°, 28.1°±0.2°, and 31.1°±0.2°.

[0054] In some embodiments, the crystal form M has an X-ray powder diffraction pattern substantially as shown in FIG18.

[0055] In some embodiments, the crystal form M has a superimposed image of differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) as shown in FIG19.

[0056] In some embodiments, the crystal form M has a hydrogen nuclear magnetic resonance spectrum (1H-NMR spectrum) substantially as shown in FIG20.

[0057] In some embodiments, the crystal form M is the hydrate crystal form of the compound of formula (I).

[0058] The present invention further provides a crystal form composition of the aforementioned crystal form. In some embodiments of the present invention, the crystal form A accounts for more than 50% by weight of the crystal form composition, preferably more than 80%, more preferably more than 90%, and most preferably more than 95%.

[0059] The present invention further provides a pharmaceutical composition comprising an effective therapeutic amount of crystal form A or crystal form Q or a mixture thereof and a pharmaceutically acceptable carrier, diluent or excipient.

[0060] The present invention further provides a veterinary drug composition comprising an effective therapeutic amount of crystal form A or crystal form Q or a mixture thereof and a pharmaceutically acceptable carrier, diluent or excipient.

[0061] The present invention further provides the use of crystal form A or crystal form Q or a mixture thereof in the preparation of pharmaceuticals.

[0062] In some embodiments, the drug is used to treat, prevent, or delay allergic reactions, allergic dermatitis, atopic dermatitis, eczema, pruritus, or psoriasis.

[0063] In some embodiments, the drug is used to treat, prevent, or delay allergic reactions, allergic dermatitis, atopic dermatitis, eczema, pruritus, or psoriasis in mammals.

[0064] In some embodiments, the mammal includes companion animals.

[0065] In some embodiments, the mammals include domestic animals.

[0066] In some embodiments, the drug is used to treat diseases mediated by JAK kinase (JAK1).

[0067] The present invention further provides a method for treating and / or preventing diseases, the method comprising administering a therapeutically effective amount of crystal form A or crystal form Q or a mixture thereof to a mammal in need.

[0068] In some embodiments, the effective therapeutic dose ranges from 0.01 mg / kg body weight / day to 100 mg / kg body weight / day.

[0069] In some embodiments, the effective therapeutic dose ranges from 0.1 mg / kg body weight / day to 30 mg / kg body weight / day.

[0070] In some embodiments, the disease is mediated by JAK kinase (JAK1).

[0071] In some embodiments, the mammal includes companion animals.

[0072] In some embodiments, the mammals include domestic animals.

[0073] In some embodiments, crystal form A or crystal form Q or a mixture thereof are applied orally, parenterally, or topically.

[0074] The novel crystalline form of the compound (R)-2-(1-(2-(1-hydroxyethyl)imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)piperidin-4-yl)acetonitrile provided by this invention exhibits good stability, high solubility, and high bioavailability, and has broad application prospects. Of course, implementing any product or method of this invention does not necessarily require achieving all of the advantages described above simultaneously.

[0075] Relevant definitions and explanations

[0076] In this invention, the term "substantially" used in phrases such as "having an X-ray powder diffraction pattern substantially as shown in Figure 1" or "having an X-ray powder diffraction pattern substantially as shown in Figure 1" indicates the precise location of the peaks in the pattern and should not be interpreted as absolute values. This is because, as those skilled in the art will recognize, the 2θ values ​​of an X-ray powder diffraction pattern can be subject to errors due to different measurement conditions (such as the equipment and instruments used) and different samples. The measurement error of the diffraction angle in an X-ray powder diffraction pattern is 5% or less, and generally, a difference of ±0.2° for a given value is considered appropriate. It should also be understood that the relative intensity of the peaks may fluctuate with experimental conditions and sample preparation, such as the preferred orientation of the particles in the sample. The use of automated or fixed divergence slits will also affect the calculation of relative intensity. The intensities shown in the XRPD curves included herein are merely exemplary and should not be used for absolute comparisons.

[0077] The water activity described in this invention refers to the ratio of the equilibrium vapor pressure of water in an ethanol aqueous solution to the saturated vapor pressure of pure water at the same temperature.

[0078] This invention does not limit the drying temperature, as long as the purpose of this invention can be achieved. For example, drying can be carried out in the temperature range of 10℃-30℃.

[0079] The therapeutically effective amount described in this invention refers to the amount of a drug, when used to treat an object, sufficient to affect the treatment of a disease, condition, or symptom, or at least one clinical symptom of a disease or condition. The therapeutically effective amount can vary depending on the drug, the symptoms of the disease or condition, the severity of the symptoms, etc. Wherever possible, a suitable dosage may be obvious to those skilled in the art or may be determined by conventional experiments.

[0080] The pharmaceutically acceptable carriers, diluents, or excipients described in this invention refer to non-toxic carriers, diluents, or excipients that do not adversely affect the pharmacological activity of the crystal form with which they are formulated and are safe for animal use. Pharmaceutically acceptable carriers, diluents, or excipients that can be used in the crystal forms of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, magnesium stearate, lecithin, serum proteins, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, mixtures of saturated plant fatty acid metaglycerides, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose substances (e.g., microcrystalline cellulose, hydroxypropyl methylcellulose, lactose monohydrate, sodium lauryl sulfate, and croscarmellose sodium), polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polypropylene block polymers, polyethylene glycol, and lanolin, etc.

[0081] Pharmaceutical compositions containing the crystal form of the present invention can be administered orally, nasally, rectally, parenterally, or topically. For oral administration, the pharmaceutical composition can be formulated into conventional solid dosage forms such as tablets, powders, granules, and capsules; liquid dosage forms such as aqueous or oil suspensions; or other liquid dosage forms such as syrups, solutions, and suspensions. For parenterally administration, the pharmaceutical composition can be formulated into solutions, aqueous solutions, oil suspensions, lyophilized powders for injection, etc. Simple Explanation of the Diagram

[0082] To more clearly illustrate the technical solutions of the embodiments of the present invention and the prior art, the drawings used in the embodiments and the prior art are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0083] Figure 1 shows the X-ray powder diffraction pattern of the amorphous compound.

[0084] Figure 2 shows the X-ray powder diffraction pattern of crystal form A.

[0085] Figure 3 is an overlay of the differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) spectra of crystal form A.

[0086] Figure 4 shows the three-dimensional structure of crystal form A.

[0087] Figure 5 shows the X-ray powder diffraction pattern of crystal form H.

[0088] Figure 6 is an overlay of the differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) spectra of crystal form H.

[0089] Figure 7 shows the X-ray powder diffraction pattern of crystal form S.

[0090] Figure 8 is an overlay of the differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) spectra of crystal form S.

[0091] Figure 9 shows the X-ray powder diffraction pattern of crystal form O.

[0092] Figure 10 is an overlay of the differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) spectra of crystal form O.

[0093] Figure 11 shows the X-ray powder diffraction pattern of crystal form P.

[0094] Figure 12 is an overlay of the differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) spectra of crystal form P.

[0095] Figure 13 shows the X-ray powder diffraction pattern of crystal form Q.

[0096] Figure 14 shows a polarizing microscope image of crystal form Q.

[0097] Figure 15 is an overlay of the differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) spectra of crystal form Q.

[0098] Figure 16 shows the 1H NMR spectrum of crystal form Q.

[0099] Figure 17 shows the dynamic gas-phase adsorption diagram of crystal form Q.

[0100] Figure 18 shows the X-ray powder diffraction pattern of crystal form M.

[0101] Figure 19 is an overlay of the differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) spectra of crystal form M.

[0102] Figure 20 shows the 1H NMR spectrum of crystal form M.

[0103] Figure 21 shows the changes in skin thickness in different groups during the treatment of atopic dermatitis animal models with crystal type A.

[0104] Figure 22 shows the changes in average auricular thickness in each group during the treatment of atopic dermatitis animal model with crystal type A.

[0105] Figure 23 shows the changes in the severity index scores of skin lesions in each group during the treatment of atopic dermatitis animal model with crystal type A.

[0106] Figure 24 shows the changes in skin thickness in different groups during the treatment of psoriasis animal models with crystal type A.

[0107] Figure 25 shows the changes in psoriasis area and severity index scores in each group during the treatment of psoriasis animal models with crystal form A. Implementation

[0108] [Abbreviations] XRPD: X-ray powder diffraction; TGA: Thermogravimetric analysis; DSC: Differential Scan Calorimetry; 1H-NMR: Proton nuclear magnetic resonance spectrum; PLM: Polarizing microscope; DVS: Dynamic Steam Adsorption Analyzer; SXRD: Single-crystal X-ray diffraction; HPLC: High Performance Liquid Chromatography; LC-MS / MS: Liquid Chromatography-Tandem Mass Spectrometry; ACN: Acetonitrile; DCM: Dichloromethane; n-Heptane: n-Heptane; MeOH: Methanol; EtOH: Ethanol; EtOAc: Ethyl acetate; IPA: Isopropyl alcohol; 2-MeTHF:2-Methyltetrahydrofuran; 2-Butanol; MEK: Butyl ketone; THF: Tetrahydrofuran; MTBE: Methyl tert-butyl ether; FaSSIF: Simulated intestinal fluid during fasting; SIF: Simulated intestinal fluid; FeSSIF: Simulated intestinal fluid during feeding; SGF: Simulated gastric juice; RH: Relative Humidity; AD: Atopic dermatitis; SI: Skin lesion severity index; PASI: Psoriasis Area and Severity Index; Mean ± SD: Mean ± Standard Deviation; Mean±SEM: Mean ± Standard Error; AAALAC: International Committee for Assessment and Accreditation of Laboratory Animals; SPF: Specific Pathogen-Free Animals; IVC: Independent Ventilation Cage Box.

[0109] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to drawings and embodiments. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention are within the scope of protection of this invention.

[0110] Unless otherwise stated, the information on the testing instruments and testing methods used in this invention is as follows.

[0111] (1) X-ray powder diffraction analysis

[0112] X-ray powder diffraction data of the samples were collected under ambient conditions using a Bruker D2 X-ray powder diffractometer with an X-ray emitter power of 300 W. The sample stage had no background signal. The step speed was 0.15 s / step, with a total of 1837 steps and a step size of 2θ = 0.02°. The voltage was 30 kV, and the current was 10 mA. The X-ray tube used a Cu target (Kα) with a Kα2 / Kα1 intensity ratio of 0.50 (1.54439 Å / 1.5406 Å).

[0113] (2) Thermogravimetric analysis

[0114] Thermogravimetric data of the samples were collected using a TA Discovery series thermogravimetric analyzer TGA550. Several milligrams of sample were placed in a Tzero aluminum pan and heated from room temperature to 300.0 ºC under N 2 protection at a flow rate of 25 mL / min and a heating rate of 10 ºC / min.

[0115] (3) Differential scanning calorimetry

[0116] Thermal data of the sample were collected using a TA Discovery series differential scanning calorimeter DSC2500. Several milligrams of sample were weighed into a Tzero aluminum pan and sealed with a Tzero cap. The sample was heated to 300.0 ºC under N2 protection at a flow rate of 50 mL / min and a heating rate of 10 ºC / min.

[0117] (4) Polarizing microscope

[0118] The shape of the crystal form was observed and PLM images were taken using an Olympus BX3M-KMA-S polarizing microscope at room temperature.

[0119] (5) Dynamic gas phase adsorption instrument

[0120] Hygroscopicity data of the samples were collected using an ADVENTURE series dynamic gas phase adsorption analyzer under N2 protection. The sample volume was approximately 30 mg.

[0121] The present invention will be described in detail below with reference to specific embodiments.

[0122] Example 1: Preparation of the amorphous form of compound (I)

[0123] Step 1: Triethyloxoboron tetrafluoride (251 mg, 1.32 mmol) and R-lactamine (118 mg, 1.32 mmol) were dissolved in 8 mL of tetrahydrofuran and reacted with the solution at room temperature for 2 hours. The solution was concentrated under reduced pressure to obtain a colorless oil, which was dissolved in 5 mL of anhydrous ethanol and added to 5 mL of anhydrous ethanol containing compound 2-(1-((5-amino-1-p-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-4-yl)amino)piperidin-4-yl)acetonitrile (189 mg, 0.44 mmol). The mixture was heated to 75 °C and stirred for 1 hour. After the reaction was complete, the solution was quenched with an aqueous sodium bicarbonate solution, 30 mL of water was added, and the mixture was extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed with 50 mL of saturated brine and dried over anhydrous sodium sulfate. The filtrate was filtered under reduced pressure and concentrated. The filtrate was then subjected to silica gel column chromatography to obtain compound (R)-2-(1-(2-(1-hydroxyethyl)-6-p-toluenesulfonamide[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)piperidin-4-yl)acetonitrile (110 mg, yield 52%). LCMS ESI(+) m / z: 479.1 (M+1).

[0124] Step 2: Compound (R)-2-(1-(2-(1-hydroxyethyl)-6-p-toluenesulfonamide[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)piridin-4-yl)acetonitrile (110 mg, 0.23 mmol) was dissolved in 9 mL of methanol, and 3 mL of 1 N sodium hydroxide aqueous solution was added. The mixture was stirred at room temperature for 16 hours. After the reaction was completed, the pH was adjusted to 7-8 with acetic acid, the mixture was concentrated under reduced pressure, and the product was prepared by silica gel column chromatography and high performance liquid chromatography to obtain compound (R)-2-(1-(2-(1-hydroxyethyl)-imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)piridin-4-yl)acetonitrile (30 mg, 40% yield). 1H NMR(400MHz,DMSO-d 6) δ11.92(s,1H),8.55(s,1H),7.50(d,J=3.4Hz,1H),6.73(d,J=3.4Hz,1H),5.31(s,1H),5.16(q,J=6.6Hz,1H),3.65-3.53(m,2H),3. 15(dd,J=30.2,10.0Hz,2H),2.64(d,J6.4Hz,2H),2.17-2.04(m,1H),1.98-1.89(m,2H),1.72-1.60(m,2H),1.56(d,J=6.6Hz,3H).LCMS ESI(+)m / z:325.0(M+1). The XRPD spectrum was determined and is shown in Figure 1. The final product obtained was an amorphous substance.

[0125] (The preparation method is described on page 11 of the specification of the applicant's compound patent WO2021051899A1.)

[0126] Example 2: Preparation of crystal form A of compound (I)

[0127] Weigh 15 mg of the amorphous compound of formula (I) into a 4 mL glass bottle, add ethanol until the sample is dissolved, and then place it into a 20 mL glass bottle containing 4 mL of n-heptane. Tightly seal the 20 mL glass bottle and place it at room temperature until a solid precipitates. Separate the solid and determine it by XRPD, TGA, and DSC. The obtained solid is crystal form A.

[0128] Example 3: Preparation of crystal form A of compound (I)

[0129] Dissolve 1.5 kg of the amorphous product of formula (I) in 15 L of ethanol and stir at 75℃-85℃ until the product is completely dissolved. Then concentrate the product to 1.5 L-3 L at 35℃-55℃. Stir the concentrated solution at 5℃-15℃ for 1-2 hours to precipitate a solid. Filter and dry to obtain 0.95 kg of solid. XRPD, TGA, and DSC analysis showed that the solid was crystal form A.

[0130] Example 4: Preparation of crystal form H of compound (I)

[0131] Weigh 15 mg of crystal form A sample, add 0.4 mL of ACN, and magnetically stir at room temperature to obtain a suspension. After about one week, separate the solid, and XRPD, TGA, and DSC analysis showed that the obtained solid was crystal form H.

[0132] Example 5: Preparation of crystal form S of compound (I)

[0133] Crystal form Q (Example 8) was heated to 165 ºC to obtain a solid. The solid was determined to be crystal form S by XRPD, TGA and DSC.

[0134] Example 6: Preparation of crystal form O of compound (I)

[0135] Weigh 15 mg of crystal form A sample, add 0.4 mL of DCM / n-heptane solution (2:1, v / v), and magnetically stir at room temperature to obtain a suspension. After about one week, separate the solid, heat the obtained solid to 165 ºC under N 2 protection, and then cool to room temperature. XRPD, TGA, and DSC analysis showed that the obtained solid was crystal form O.

[0136] Example 7: Preparation of crystal form P of compound (I)

[0137] Weigh 15 mg of crystal form A sample, add MeOH solvent, and stir at room temperature to obtain a clear stock solution. Dispense the stock solution into 20 mL glass bottles, and under magnetic stirring, gradually add 0.2 mL to 0.5 mL of water until a solid appears. Separate the solid, heat it to 120 °C, and obtain a solid. XRPD, TGA, and DSC analysis show that the obtained solid is crystal form P.

[0138] Example 8: Preparation of crystal form Q of compound (I)

[0139] Crystal form A was stirred in an ethanol aqueous solution with a water activity of 0.6-0.8 until a solid was produced. The solid was then separated by centrifugation, dried, and determined by XRPD, TGA, and DSC to be crystal form Q.

[0140] Example 9: Preparation of crystal form M of compound (I)

[0141] Weigh 15 mg of crystal form A sample, add EtOH solvent, and stir at room temperature to obtain a clear stock solution. Dispense the stock solution into 20 mL glass bottles, and under magnetic stirring, gradually add 0.2 mL to 0.5 mL of the corresponding EtOAc solution to the glass bottles. Allow it to evaporate slowly at room temperature. XRPD, TGA, and DSC analysis showed that the obtained solid was crystal form M.

[0142] Example 10: Single Crystal X-ray Diffraction (SXRD) Experiment

[0143] Crystal preparation: Weigh 15 mg of the compound of formula (I) crystal form A into a 15 mL test tube, add 1 mL of dichloromethane to dissolve it, and then add 2 mL of ethanol. Cover the mouth of the test tube with a sealing film, and poke 5-10 small holes in the sealing film with a needle, allowing it to evaporate. After about three weeks, the solution will have completely evaporated, and colorless blocky crystals will be obtained at the bottom of the test tube.

[0144] Single-crystal X-ray diffraction analysis was performed on the crystal. The results showed that the crystal was colorless and blocky. The crystal size used for diffraction analysis was 0.09×0.09×0.08 mm3, belonging to the monoclinic crystal system, space group P1211, with the following cell parameters: a=9.6796(4) Å, α=90°, b=14.0338(6) Å, β=108.829.218(2)°, c=14.5745(6) Å, γ=90°, cell volume V=1873.87(14) Å3, number of molecules in the cell Z=4, and one molecule in an independent region of the unit lattice.

[0145] Example 11: Thermodynamic Analysis Experiment

[0146] Crystal form H and crystal form S were heated to appropriate temperatures under N2 protection, cooled to room temperature, and then the XRPD of the solids was measured.

[0147] Results: 1) When crystal form H was heated to 162 ºC, the sample partially transformed into crystal form A; when heated to 210 ºC, the sample completely transformed into crystal form A. 2) When crystal form S was heated to 215 ºC, the sample transformed into crystal form A.

[0148] Analysis: 1) Based on the TGA / DSC spectrum of crystal form H, it can be seen that the sample loses 0.6% weight when heated to 210 ºC, and there are two exothermic peaks in the DSC before melting. According to the Burger-Ramberger rule, crystal form H and crystal form A have a single variable relationship.

[0149] 2) Based on the TGA / DSC spectrum of crystal form S, it can be seen that the sample loses 1.6% of its weight when heated to 210 ºC, and there is an exothermic peak in the DSC before melting. According to the Burger-Ramberger rule, crystal form S and crystal form A have a single variable relationship.

[0150] This experiment demonstrates that crystal form A is more thermodynamically stable than crystal forms H and S.

[0151] Example 12: Suspension Competition Experiment

[0152] Under two conditions, room temperature and 60 °C, a suspension competition experiment was conducted with crystal form A, crystal form O, or crystal form P using IPA and 2-MeTHF as solvents.

[0153] The specific procedure was as follows: Weigh four excess portions of crystal form A into four HPLC vials. Add 0.5 mL of IPA to two vials and 0.5 mL of 2-MeTHF to the other two vials. Place them at room temperature and 60 ºC and stir magnetically for about 1 hour, respectively. Filter the solutions using a preheated nylon membrane (22 μm pore size) to obtain saturated solutions. Add 1 mg-2 mg each of crystal form A, crystal form O, and crystal form P to the saturated filtrate. Stir magnetically at room temperature or 60 ºC for about 1 day. Centrifuge to separate the solids for XRPD testing. The results of the suspension competition experiment are shown in Table 3.

[0154] [Table 3] Results of the suspension competition experiment Initial crystal form solvent Temperature (ºC) The resulting crystal form Crystal form A / Crystal form O / Crystal form P IPA room temperature Crystal form A 2-MeTHF Crystal form A IPA 60 Crystal form A 2-MeTHF Crystal form A

[0155] The results showed that the solids obtained in both solvents at room temperature and 60 °C were of crystal form A, indicating that crystal form A is more stable at both room temperature and 60 °C compared to crystal forms O and P.

[0156] Example 13: Dynamic solubility

[0157] The dynamic solubility of crystal form A and crystal form Q in three biological solvents (simulated gastric juice, simulated fasting intestinal juice, and analogous feeding intestinal juice) was tested at 37 ºC to evaluate the solubility and stability of the two crystal forms.

[0158] The specific steps for preparing biological solvents are as follows.

[0159] (1) Preparation method of FaSSIF: a. Weigh 42 mg NaOH, 395 mg NaH 2PO 4·H 2O and 619 mg NaCl into a 100 mL beaker and dissolve them in 90 mL of deionized water; b. Adjust the pH to 6.5 with 1N NaOH or 1N HCl; make up to 100 mL with deionized water to obtain FaSSIF blank solvent; c. Weigh 224 mg of simulated intestinal fluid (SIF powder, hereinafter referred to as SIF powder) and add 50 mL of the above FaSSIF blank solvent, stir until completely dissolved; make up to 100 mL with FaSSIF blank solvent to obtain solvent FaSSIF; d. Let stand at room temperature for 2 hours before use.

[0160] (2) Preparation method of FeSSIF: a. Weigh 404 mg NaOH, 865 mg glacial acetic acid and 1189 mg NaCl into a 100 mL beaker and dissolve in 90 mL of deionized water; b. Adjust the pH to 5.0 with 1N NaOH or 1N HCl; make up to 100 mL with deionized water to obtain FeSSIF blank solvent; c. Weigh 1120 mg SIF powder and add to 50 mL of the above FeSSIF blank solvent, stir until dissolved; make up to 100 mL with FeSSIF blank solvent to obtain solvent FeSSIF; d. Let stand at room temperature before use to ensure that it is a clear solution.

[0161] (3) Preparation method of SGF: a. Weigh 200 mg NaCl into a 100 mL beaker and dissolve it in 90 mL of deionized water; b. Adjust the pH to 1.6 with 1N HCl; make up to 100 mL with deionized water to obtain SGF blank solvent; c. Weigh 6 mg SIF powder and add it to 50 mL of the above SGF blank solvent, stir until dissolved; make up to 100 mL with SGF blank solvent to obtain solvent SGF; d. Let stand at room temperature before use to ensure that it is a clear solution.

[0162] The specific method for dynamic solubility testing is as follows: Approximately 40 mg of crystal form A or crystal form Q is added to 4 mL of each of the three biological solvents, and the mixture is shaken at a constant temperature of 37 ºC (100 rpm). At 1, 4, and 24 hours, 1.3 mL of the suspension (or clear solution) is collected, and the suspension is centrifuged for 5 minutes. The resulting solid is used for crystal form detection (XRPD), and the supernatant / clarity is filtered and used for solubility and pH testing.

[0163] Solubility data of the samples were collected using a high-performance liquid chromatograph (equipped with an image diode array detector).

[0164] Test results: The results of the dynamic solubility test are shown in Table 4.

[0165] [Table 4] Results of dynamic solubility determination Dynamic solubility of SGF at 37 °C sample 1 hour 4 hours 24 hours solubility pH Crystal form changes solubility pH Crystal form changes solubility pH Crystal form changes Crystal form A 0.72 2.92 No change 0.79 2.99 -- 0.82 2.95 No change Crystal form Q 0.62 2.60 No change 0.69 2.98 -- 0.67 2.71 No change Dynamic solubility of FaSSIF at 37 °C sample 1 hour 4 hours 24 hours solubility pH Crystal form changes solubility pH Crystal form changes solubility pH Crystal form changes Crystal form A 0.05 6.41 No change 0.06 6.49 -- 0.05 6.54 No change Crystal form Q 0.03 6.45 No change 0.04 6.40 -- 0.03 6.49 No change Dynamic solubility of FeSSIF at 37 °C sample 1 hour 4 hours 24 hours solubility pH Crystal form changes solubility pH Crystal form changes solubility pH Crystal form changes Crystal form A 0.08 4.92 No change 0.08 4.95 -- 0.09 4.99 No change Crystal form Q 0.05 4.96 -- 0.04 4.98 -- 0.05 5.00 No change Note: Starting samples: Crystal form A, Crystal form Q --: The crystal form of the undissolved sample after 24 hours is the same as that of the starting sample, therefore the crystal form of the undissolved sample after 1 hour or 4 hours was not tested.

[0166] The results showed that: at 37 °C for 24 hours, 1) the solubility of crystal form A in all three solvents (SGF / FaSSIF / FeSSIF) was greater than that of crystal form Q; 2) crystal forms A and Q remained stable after being suspended and stirred in SGF / FaSSIF / FeSSIF for 24 hours.

[0167] Example 14: Solid-state stability

[0168] Test method: Weigh 17-20 mg of crystal form A and crystal form Q samples into HPLC vials, and place them open under three conditions: 1) 25 ºC / 60%RH, 2) 40 ºC / 75%RH, and 3) 60 ºC. Purity tests (by HPLC) and crystal form detection (by XRPD) were performed on the starting samples, samples stored for one week, and samples stored for two weeks. Specific results are shown in Table 5.

[0169] [Table 5] 2-week stability study data for crystal form A and crystal form Q starting sample condition 1 week 2 weeks Crystal form purity(%) purity(%) Crystal form changes purity(%) Crystal form changes Crystal form A 99.59 25ºC / 60%RH 99.64 No change 99.60 No change 40 ºC / 75%RH 99.62 99.60 60 ºC 99.62 99.66 Crystal form Q 99.87 25 ºC / 60%RH 99.80 No change 99.87 No change 40 ºC / 75%RH 99.93 99.87 60 ºC 99.91 99.92

[0170] The results showed that: 1) Purity: After one and two weeks of storage under the selected conditions, the purity of crystal form A and crystal form Q did not change significantly. 2) Crystal form change: The XRPD comparison results of crystal forms A and Q stored under three different conditions for 0, 1 and 2 weeks showed that the crystal forms of crystal forms A and Q did not change after one and two weeks of storage under the selected conditions.

[0171] Conclusion: Both crystal forms A and Q exhibit good solid-state stability.

[0172] Example 15: Crystal form stability test

[0173] Crystal form M was stored at 25 ºC and 55%RH for 35 days. XRPD of the solid was tested, and it was found that crystal form M transformed into crystal form A.

[0174] The results show that under ambient temperature and humidity, crystal form M is unstable and easily transforms into the stable crystal form A.

[0175] Example 16: Hygroscopicity Test

[0176] The hygroscopicity of the above crystal forms was determined in accordance with the guidelines for hygroscopicity testing of drugs in Chapter 9103 of the General Section IV of the 2020 edition of the Chinese Pharmacopoeia.

[0177] The criteria for judging hygroscopicity are: Deliquescence: Absorbs sufficient moisture to form a liquid; Extremely hygroscopic: The weight gain due to hygroscopic absorption is no less than 15%; It has hygroscopic properties: the weight gain due to moisture absorption is less than 15%, but not less than 2%; Slightly hygroscopic: weight gain due to moisture absorption is less than 2%, but not less than 0.2%; It has little or no hygroscopicity: the weight gain due to moisture absorption is less than 0.2%.

[0178] The test results show that crystal form A has a moisture absorption weight gain of 0.05%, indicating that it has no or almost no moisture absorption. Compared with other crystal forms described in this patent, it is more suitable for the preparation of solid dosage forms.

[0179] Example 17: Influencing Factor Experiment

[0180] The stability of the crystal forms of the present invention under accelerated testing conditions was determined. Crystal forms A, H, S, O, P, Q, and M were subjected to stability tests under 5000 Lx light, 60°C high temperature, and 90%RH±5%RH high humidity conditions, respectively. Samples were taken on days 0, 5, 10, and 30. The content, total impurities, and XRPD plots of the samples were recorded and compared with the initial data. The comparison results showed that crystal forms A, H, S, O, P, Q, and M exhibited good stability, especially crystal form A. The experimental data on the influencing factors of crystal form A are shown in Table 5a below:

[0181] [Table 5a] Experimental data on factors affecting crystal form A Investigation Project 0d High temperature test High humidity test Light test (60℃) (25℃, 90±5%RH) (Illuminance 5000Lx and UV lamp energy 0.9W / m2) 5d 10d 30d 5d 10d 30d 5d 10d 30d Total impurities 0.325% 0.34% 0.33% 0.35% 0.32% 0.31% 0.32% 0.34% 0.36% 0.43% content(%) 99.1% 100.2% 100.4% 100.3% 100.1% 100.1% 100.1% 99.5% 98.9% 99.4% XRPD test Crystal form A Crystal form A Crystal form A Crystal form A Crystal form A Crystal form A Crystal form A Crystal form A Crystal form A Crystal form A

[0182] Example 18: Pharmacokinetic experiment

[0183] Twelve SD rats were divided into two groups, with 6 rats in each group, half male and half female. They were given a single oral administration of the drug respectively. Blood was collected from the fundus venous plexus at the designated time points, plasma was separated and stored in a -80°C refrigerator.

[0184] For the above plasma samples, after precipitating proteins with acetonitrile, the supernatant was diluted with water, and 5 μL was taken for LC-MS / MS detection.

[0185] It was found that the bioavailability of the crystal form A sample in vivo was high, meeting the requirements of drug development.

[0186] Pharmacological and efficacy experiment

[0187] Example A: Pharmacodynamic effect on atopic dermatitis animal model.

[0188] 1. Experimental materials

[0189] (1) Experimental animals and feeding methods

[0190] 55 female BALB / C mice, 6 weeks old at the time of purchase and 8 weeks old at the time of experiment, weighing 18 g - 20 g. The animals were sourced from Shanghai Lingchang Biotechnology Co., Ltd. (SCXK(Shanghai)2018 - 00...), the production qualification certificate number of the experimental animals was 20180003012342; the license number for the use of experimental animals was: SYXK(Shanghai)2020 - 0001 (Via Biotechnology (Shanghai) Co., Ltd.).

[0191] All mice were housed in an SPF-grade IVC (Indoor Ventilation and Control) animal facility with a constant temperature and pressure system, maintaining a temperature of 20°C-26°C, humidity of 40%-70%, and a 12-hour light-dark cycle. Six mice were housed in each cage, which measured 325 mm × 210 mm × 180 mm. Bedding in the cages was changed twice weekly. Throughout the experiment, all mice had free access to food and water. Feed and water were autoclaved and changed twice weekly.

[0192] (2) Test sample a. Tofacitinib, free base, purity 98%, total amount 200 mg, storage temperature 4℃. b. Upadacitinib, free base, purity 98%, total amount 70mg, storage temperature 4℃. c. Crystal form A, free alkali, purity 99.7%, total amount 300 mg, storage temperature 4℃.

[0193] 2. Experimental Procedures and Methods

[0194] Modeling and drug administration method: Hair removal cream was applied to a 5cm x 2cm area from the head to the back of each animal. The application time of the hair removal cream was controlled within 2 minutes. The hair removal area was washed with warm water to remove the hair removal cream. The animal's hair was immediately dried with a hair dryer, and the animal was placed on a 37℃ constant temperature electric blanket for 1-2 hours. After the animal was able to move around normally, it was placed in its cage for normal feeding and recovery for 1 day (animals with red, swollen, or broken skin after hair removal were removed). On the second day, 42 animals with normal skin were randomly divided into 7 groups according to their weight, with 6 animals in each group. Except for the G1 normal control group, each animal in the other groups was induced by applying 200 μL of 0.5 wt% dinitrochlorobenzene solution (solvent: acetone and olive oil in a mass ratio of 3:1) evenly to its back and auricle for three consecutive days. The second stage of induction began on day 14, with 1 wt% dinitrochlorobenzene solution (solvent: acetone and olive oil in a mass ratio of 3:1) every three days until the end of the experiment. At the same time, treatment drugs were started. The drugs administered to the groups are shown in Table 6.

[0195] [Table 6] Route of administration, dosage and grouping in the pharmacodynamic experiment of the AD model Group number Animal number Dinitrochlorobenzene Drug administration group Dosage (mg / kg) route of administration Dosage frequency 1 6 - Blank control group - - - 2 6 + negative control group - oral Twice a day for 14 days 3 6 + Tofacitinib 30 oral Twice a day for 14 days 4 6 + Upatinib 10 oral Twice a day for 14 days 5 6 + Crystal form A 3 oral Twice a day for 14 days 6 6 + Crystal form A 10 oral Twice a day for 14 days 7 6 + Crystal form A 30 oral Twice a day for 14 days

[0196] Preparation method of the test drug: as shown in Table 7.

[0197] [Table 7] Preparation methods of the test drugs Sample Name Preparation method Dosage concentration (mg / mL) store Tofacitinib 0.5 g methylcellulose + 0.25 mL Tween 20 Add sterile deionized water to 100 mL, then add 300 mg tofacitinib and sonicate with ultrasound until completely dissolved. 3 mg / mL, twice daily 4℃ Crystal form A Take three 0.5 g portions of methylcellulose. Add sterile deionized water to 100 mL, then add 300 mg, 100 mg, and 30 mg respectively. Crystal form A was ultrasonically oscillated until fully dissolved. 3 mg / mL, 1 mg / mL, 0.3 mg / mL Twice a day 4℃ Upatinib 0.5 g methylcellulose +0.25 mL Tween 20, Add sterile deionized water to 100 mL, then add 100 mg of utpatinib and sonicate with ultrasound until completely dissolved. 1 mg / mL, twice daily 4℃

[0198] 3. Experimental Observation

[0199] Throughout the experiment, the use and observation of laboratory animals were conducted in accordance with the relevant regulations of AAALAC on the use and management of animals.

[0200] 4. Evaluation Indicators

[0201] Skin thickness (double layer): The double layer thickness of the back skin was measured using calipers 1 day before modeling and on days 14, 17, 20, 23, 26 and 28 after modeling.

[0202] Auricle thickness: The thickness of the right auricle was measured using calipers 1 day before modeling and on days 14, 17, 20, 23, 26 and 28 after modeling.

[0203] AD-SI Score: The disease condition score is based on the clinical SI scoring criteria, and is scored on three indicators: skin color / erythema, scaly erythema, scaling / peeling / lichenification, thickening, and degree of thickening / edema. The total score is 12 points, and the scoring criteria are as follows: Erythema / Skin Color: 0 points, none; 1 point, mild: pale red; 2 points, moderate: red; 3 points, significant: dark red; 4 points, very obvious: extremely deep red. Scaling / Peeling / Lichenification: 0 points, none; 1 point, mild: some epidermis has scaling / peeling / lichenification, mainly fine; 2 points, moderate: most skin surface is completely or partially covered with scaling / peeling / lichenification, in flakes; 3 points, significant: almost all skin is covered with scaling / peeling / lichenification, the scaling / peeling / lichenification is relatively thick and layered; 4 points, very obvious: all skin is covered with scaling / peeling / lichenification, very thick and layered. Thickening / edema: 0 points, none: level with normal skin; 1 point, mild: slightly raised above the normal skin surface; 2 points, moderate: moderately raised, with round or sloping edges; 3 points, significant: thickened plaque with obvious elevation; 4 points, very significant: highly thickened with very obvious elevation. Skin / auricular hyperplasia inhibition rate (%) = 1 - [(skin / auricular thickness in treatment group - skin / auricular thickness in blank control group) / skin / auricular thickness in negative control group - skin / auricular thickness in blank control group)] %.

[0204] 5. Experiment terminated

[0205] The experiment ended after 14 days of treatment. The animals were euthanized, and the skin on the back and ears of the mice were dissected and separated. Half of the skin was fixed and preserved in formalin, and the other half was stored at -80°C.

[0206] 6. Data Analysis

[0207] All data are expressed as Mean ± SD or Mean ± SEM. SPSS 23.0 was used to perform normality and homogeneity of variance tests. One-way ANOVA was then used based on the results. *P < 0.05 was considered statistically significant.

[0208] 7. Experimental Results

[0209] (1) Effect of the test drug on skin thickness

[0210] On day 10 of treatment, the changes in average skin thickness and the inhibitory effect of each group are shown in Tables 8 and 9 and Figure 21. Figure 21 shows the changes in skin thickness of each group during the treatment period (Note: *P<0.05, **P<0.01, ***P<0.001 are all compared with the G2 negative control group).

[0211] [Table 8] Data on mean skin thickness (double layer) (Mean±SEM) (n=6) Group Average skin thickness (double layer) (mm) Day 0 Day 1 Day 4 Day 7 Day 10 G1 blank control group 0.61±0.01 0.87±0.02 0.81±0.02 0.57±0.01 0.63±0.01 G2 negative control group 0.60±0.02 1.04±0.02 1.66±0.17 2.67±0.3 2.64±0.2 G3. Tofacitinib 30mg / kg 0.63±0.03 0.99±0.03 1.13±0.1* 1.78±0.11 2.08±0.13 G4. Upatinib 10mg / kg 0.60±0.02 0.89±0.04 1.07±0.05* 2.06±0.25 2.23±0.14 G5. Crystal form A 3mg / kg 0.63±0.01 0.98±0.01 1.08±0.01** 1.85±0.12 2.35±0.15 G6. Crystal form A 10mg / kg 0.60±0.02 0.94±0.02 1.14±0.04 1.49±0.09* 2.23±0.17 G7. Crystal form A 30mg / kg 0.62±0.02 0.79±0.05 1.05±0.03** 1.01±0.08*** 1.56±0.21* Note: *P<0.05, **P<0.01, ***P<0.001 are all compared with the G2 negative control group.

[0212] [Table 9] Inhibition of skin thickening in each treatment group on day 10 of treatment (n=6) Group Average skin thickness on day 10 (mm) Inhibition rate p-value G1 blank control group 0.63 / G2 negative control group 2.64 / G3. Tofacitinib 30mg / kg 2.08 27.86% 0.118 G4. Upatinib 10mg / kg 2.23 20.40% 0.390 G5. Crystal form A 3mg / kg 2.35 14.43% 0.580 G6. Crystal form A 10mg / kg 2.23 20.40% 0.312 G7. Crystal form A 30mg / kg 1.56 53.73% 0.007** Note: *P<0.05, **P<0.01, ***P<0.001 are all compared with the G2 negative control group.

[0213] (2) Effect of the test drug on auricle thickness

[0214] On day 10 of treatment, the changes in average auricle thickness and the inhibitory effect in each group are shown in Tables 10 and 11 and Figure 22. Figure 22 shows the changes in average auricle thickness in each group during the treatment period (Note: *P<0.05, **P<0.01, ***P<0.001 are all compared with the G2 negative control group).

[0215] [Table 10] Mean auricular thickness data (Mean±SEM) (n=6) Group Average skin thickness (double layer) (mm) Day 0 Day 1 Day 4 Day 7 Day 10 G1 blank control group 0.21±0.00 0.24±0.00 0.23±0.01 0.20±0.00 0.21±0.01 G2 negative control group 0.20±0.00 0.23±0.00 0.41±0.03 0.58±0.04 0.79±0.09 G3. Tofacitinib 30mg / kg 0.19±0.01 0.22±0.01 0.28±0.01*** 0.44±0.03 0.64±0.05 G4. Upatinib 10mg / kg 0.20±0.01 0.24±0.01 0.32±0.03** 0.51±0.05 0.65±0.03 G5. Crystal form A 3mg / kg 0.20±0.00 0.22±0.01 0.30±0.01*** 0.39±0.03* 0.57±0.04 G6. Crystal form A 10mg / kg 0.20±0.01 0.23±0.01 0.32±0.01** 0.43±0.03 0.54±0.07* G7. Crystal form A 30mg / kg 0.21±0.01 0.22±0.01 0.29±0.02*** 0.32±0.01*** 0.50±0.03** Note: *P<0.05, **P<0.01, ***P<0.001 are all compared with the G2 negative control group.

[0216] [Table 11] Suppression of auricular thickening in each treatment group on day 10 of treatment (n=6) Group Average skin thickness on day 10 (mm) Inhibition rate p-value G1 blank control group 0.21 / G2 negative control group 0.79 / G3. Tofacitinib 30mg / kg 0.64 25.86% 0.289 G4. Upatinib 10mg / kg 0.65 24.14% 0.410 G5. Crystal form A 3mg / kg 0.57 37.93% 0.056 G6. Crystal form A 10mg / kg 0.54 43.10% 0.035* G7. Crystal form A 30mg / kg 0.50 50.00% 0.006** Note: *P<0.05, **P<0.01, ***P<0.001 are all compared with the G2 negative control group.

[0217] (3) Effects of the test drug on AD-SI scores in AD model animals

[0218] By day 10 of treatment, the changes in AD-SI scores for each group (n=6) are shown in Table 12 and Figure 23. Figure 23 shows the changes in AD-SI scores for each group during the treatment period.

[0219] [Table 12] AD-SI score data during treatment Group AD-SI score (Mean ± SD) Day 0 Day 1 Day 4 Day 7 Day 10 G1 blank control group 0.00 0.00 0.00 0.00 0.00 G2 negative control group 0.00 0.00 4.17±1.17 11.50±0.55 10.67±0.52 G3. Tofacitinib 30mg / kg 0.00 0.00 0.17±0.41*** 7.17±0.98 6.33±1.37 G4. Upatinib 10mg / kg 0.00 0.00 1.00±0.00 6.50±1.05* 7.17±1.47 G5. Crystal form A 3mg / kg 0.00 0.00 1.00±0.00 6.50±1.05* 5.83±0.75* G6. Crystal form A 10mg / kg 0.00 0.00 0.50±0.55** 5.83±1.17** 4.67±0.52** G7. Crystal form A 30mg / kg 0.00 0.00 0.50±0.55** 3.00±2.19*** 3.50±1.39*** Note: *P<0.05, **P<0.01, ***P<0.001 are all compared with the G2 negative control group.

[0220] Result analysis: On the 10th day of treatment, the average double-layer skin thickness of the back in Group G7 treated with polymorph A (30 mg / kg, twice a day) was 1.56 mm, the average auricle thickness was 0.5 mm, and the average score was 3.5 points. Compared with the G2 negative control group (the average double-layer skin thickness of the back was 2.64 mm, the average auricle thickness was 0.79 mm, and the average AD-SI score was 10.67 points), it had an obvious effect of inhibiting the inflammatory thickening of the back skin and significantly improving the manifestations of AD. The inhibition rate of the back skin thickness was 53.73%, and the inhibition rate of the auricle thickness was 50%. Moreover, there were significant differences statistically (P < 0.01).

[0221] On the 10th day of treatment, in Group G6 treated with polymorph A (10 mg / kg, twice a day), the average auricle thickness was 0.54 mm, and the average AD-SI score was 4.67 points. Compared with the G2 negative control group (the average auricle thickness was 0.79 mm, and the average AD-SI score was 10.67 points), it had an obvious effect of inhibiting the inflammatory thickening of the auricle and improving the manifestations of AD. The inhibition rate of the auricle thickness was 43.1%, and there were significant differences statistically (P < 0.05); the average double-layer skin thickness of the back in the treatment group with polymorph A (10 mg / kg, twice a day) was 2.23 mm. Compared with the G2 negative control group, it had an effect of inhibiting the inflammatory thickening of the back skin, and the inhibition rate was 20.4%.

[0222] Example B: Pharmacodynamic effect of psoriasis animal model.

[0223] 1. Experimental materials

[0224] (1) Experimental animals and feeding methods

[0225] 75 C57 / BL 6J female mice, 7 - 8 weeks old, weighing 18 g - 20 g. The animals were from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. (SCXK(Zhe)2019 - 0001), and the experimental animal production qualification certificate number was 20210107Abzz0619000247; the experimental animal use permit number: SYXK(Shanghai)2020 - 0001 (Viva Biotech (Shanghai) Co., Ltd.).

[0226] All mice were housed in an SPF-grade IVC (Indoor Ventilation and Control) animal facility with a constant temperature and pressure system, maintaining a temperature of 20°C-26°C, humidity of 40%-70%, and a 12-hour light-dark cycle. Each cage contained 3-5 mice and measured 325 mm × 210 mm × 180 mm. Bedding was changed twice weekly. Throughout the experiment, all mice had free access to food and water. Feed and water were autoclaved and changed twice weekly.

[0227] (2) Test sample a. Tofacitinib, free base, purity 98%, total amount 100 mg, storage temperature 4℃. b. Crystal form A, purity 99.7%, total amount 200 mg, storage temperature 4℃.

[0228] 2. Experimental Procedures and Methods

[0229] Modeling and drug administration methods: Hair was removed from a 2cm × 3cm area on the back of each animal using depilatory cream. The application time was controlled within 2 minutes. The area was then washed with warm water to remove the depilatory cream. The animal's hair was immediately dried with a hair dryer, and the animal was placed on a 37℃ constant temperature electric blanket for 1-2 hours. After the animals were allowed to move freely, they were placed in their cages for normal feeding and recovery for 1 day (animals with red, swollen, or broken skin after hair removal were removed were discarded). On the second day, 54 animals with normal skin were randomly divided into 6 groups of 9 animals each, according to their weight. In groups G2-G6, 60mg (60μL) of 5wt% imiquimod cream (Aldara) was evenly applied to the back of each animal daily for seven consecutive days. The route of administration, dosage, and grouping for the psoriasis model efficacy experiment are shown in Table 13.

[0230] [Table 13] Route of administration, dosage and grouping in the psoriasis model drug efficacy experiment Group number Animal number Imiquimod cream Drug administration group Dosage (mg / kg) route of administration Dosage frequency 1 9 - Blank control group - - - 2 9 + negative control group - oral 2 times a day * 7 3 9 + Tofacitinib 30 oral 2 times a day * 7 4 9 + Crystal form A 0.5 oral 2 times a day * 7 5 9 + Crystal form A 1.5 oral 2 times a day * 7 6 9 + Crystal form A 5 oral 2 times a day * 7

[0231] Preparation method of the test drug: as shown in Table 14.

[0232] [Table 14] Preparation methods of the test drugs Sample Name Preparation method Dosage concentration (mg / mL) store Tofacitinib 0.5g methylcellulose + 0.25mL Tween 20 Add sterile deionized water to 100 mL, then add 600 mg tofacitinib and sonicate with ultrasound until completely dissolved. 6 mg / mL, twice daily 4℃ Crystal form A Take three 0.5 g portions of methylcellulose. Add sterile deionized water to 100 mL, then add 10 mg, 30 mg, and 100 mg of the solution respectively. Crystal form A was ultrasonically oscillated until fully dissolved. 0.1 mg / mL, 0.3 mg / mL, 1 mg / mL, twice daily 4℃

[0233] 3. Experimental Observation

[0234] Throughout the experiment, the use and observation of laboratory animals were conducted in accordance with the relevant regulations of AAALAC on the use and management of animals.

[0235] 4. Evaluation Indicators

[0236] Skin thickness (bilayer): The thickness of the back skin bilayer was measured using calipers before drug administration and modeling, and on days 1, 3, 5, and 7 after drug administration.

[0237] PASI Score: The condition score is based on the clinical PASI scoring criteria, assessing three indicators: erythema, scaling, and thickening. The total score is 12 points, and the scoring criteria are as follows: Erythema: 0 points, none; 1 point, mild: pale red; 2 points, moderate: red; 3 points, significant: dark red; 4 points, very obvious: extremely deep red. Scales: 0 points, none; 1 point, mild: scales on some parts of the epidermis, mainly fine scales; 2 points, moderate: scales completely or partially cover most of the skin surface, in flakes; 3 points, significant: scales cover almost all of the skin, the scales are relatively thick and layered; 4 points, very obvious: scales cover all of the skin, the scales are very thick and layered. Plaque thickening degree: 0 points, none: level with normal skin; 1 point, slight: slightly raised above the skin surface; 2 points, moderate: moderately raised, with round or sloping edges; 3 points, significant: thickened plaque with obvious elevation; 4 points, very significant: highly thickened with very obvious elevation. Skin proliferation inhibition rate (%) = 1 - [(skin thickness on day 7 in the treatment group - skin thickness on day 7 in the blank control group) / skin thickness on day 7 in the negative control group - skin thickness on day 7 in the blank control group)] %.

[0238] After the experiment, the skin on the back of the mice was dissected and separated, and then fixed and preserved in formalin.

[0239] 5. Experiment terminated

[0240] Seven days after treatment, the animals were euthanized, and the skin on the back of the mice was dissected and preserved in formalin.

[0241] 6. Data Analysis

[0242] All data are expressed as mean ± SEM. SPSS 23.0 was used to perform normality and homogeneity of variance tests. One-way ANOVA was then used based on the results. *P < 0.05 was considered statistically significant.

[0243] 7. Experimental Results

[0244] (1) Effect of the test drug on skin thickness

[0245] On day 7 of treatment, the changes in average skin thickness and the inhibitory effect in each group are shown in Tables 15 and 16 and Figure 24. Figure 24 shows the changes in skin thickness in each group during the treatment period.

[0246] [Table 15] Data on mean skin thickness (double layer) variation (Mean±SEM) (n=9) Group Average skin thickness (double layer) (mm) Day 0 Day 1 Day 3 Day 5 Day 7 G1 blank control group 0.51±0.01 0.54±0.01 0.54 ±0.01 0.67 ±0.02 0.81±0.03 G2 negative control group 0.51±0.01 0.62±0.02 1.73 ±0.11 2.04 ±0.13 1.59±0.12 G3. Tofacitinib 30mg / kg 0.51±0.01 0.66±0.02 1.28±0.09** 1.09±0.06*** 1.05±0.04*** G4. Crystal form A 0.5 mg / kg 0.50±0.01 0.60±0.01 1.57 ±0.11 1.27±0.1*** 1.32±0.09 G5. Crystal form A 1.5 mg / kg 0.50±0.01 0.61±0.02 1.76 ±0.15 1.53±0.08** 1.15±0.04** G6. Crystal form A 5mg / kg 0.50±0.01 0.64±0.01 1.35 ±0.10* 1.24±0.11*** 1.03±0.03*** Note: *P<0.05, **P<0.01, ***P<0.001 are all compared with the G2 negative control group.

[0247] [Table 16] Skin thickening inhibition in each treatment group on day 7 of treatment (n=9) Group Average skin thickness on day 7 (mm) Inhibition rate p-value G1 Blank Control Group 0.81 / G2 negative control group 1.59 / G3. Tofacitinib 30mg / kg 1.05 69.2% 0.000 G4. Crystal form A 0.5 mg / kg 1.32 34.6% 0.159 G5. Crystal form A 1.5 mg / kg 1.15 56.4% 0.017 G6. Crystal form A 5mg / kg 1.03 71.8% 0.000 Note: *P<0.05, **P<0.01, ***P<0.001 are all compared with the G2 negative control group.

[0248] (2) Effect of the test drug on the Disease Severity Index (PASI) score in psoriasis model animals

[0249] On day 7 of treatment, the changes in PASI scores for each group (n=9) are shown in Table 17 and Figure 25. Figure 25 shows the changes in PASI scores for each group during the treatment period.

[0250] [Table 17] PASI score data during treatment Group PASI score (Mean±SEM) Day 0 Day 1 Day 3 Day 5 Day 7 G1 blank control group 0.0 0.0 0.0 0.0 0.0 G2 negative control group 0.0 0.0 6.1±0.59 8.3±0.65 9.6±0.29 G3. Tofacitinib 30mg / kg 0.0 0.0 4.6±0.50 4.9±0.42*** 2.8±0.43*** G4. Crystal form A 0.5 mg / kg 0.0 0.0 6.6±0.69 5.9±0.79** 6.1±0.56*** G5. Crystal form A 1.5 mg / kg 0.0 0.0 6.1±0.56 7.0±0.53 4.9±0.39*** G6. Crystal form A 5mg / kg 0.0 0.0 4.9±0.48 4.7±0.58*** 3.6±0.44*** Note: *P<0.05, **P<0.01, ***P<0.001 are all compared with the G2 negative control group.

[0251] Results analysis: On day 7 of treatment, the G4 group treated with crystal form A (0.5 mg / kg, twice daily) had a mean PASI score of 6.1, which was significantly better than that of the G2 negative control group, and the difference was statistically significant (P<0.05).

[0252] On day 7, the mean double skin thickness on the back of the G5 group treated with crystalline A (1.5 mg / kg, twice daily) and the G6 group treated with crystalline A (5 mg / kg, twice daily) was 1.15 mm and 1.03 mm, respectively, and the mean PASI scores were 4.9 and 3.6, respectively. Compared with the G2 negative control group, they significantly inhibited inflammatory thickening of the back skin and significantly improved the symptoms of psoriasis. The inhibition rates of back skin thickening were 56.4% and 71.8%, respectively, which were statistically highly significant (P<0.001).

[0253] As can be seen from the above, the compound (R)-2-(1-(2-(1-hydroxyethyl)imidazo[4,5-d]pyrrolo[2,3-b]pyridin-1(6H)-yl)piperidin-4-yl)acetonitrile free base provided by the present invention has good stability and high solubility, and has the effect of inhibiting inflammatory thickening of the skin on the back of mice and inflammatory thickening of the auricle, which can effectively improve the incidence of atopic dermatitis and psoriasis; the preparation method is simple to operate and has good reproducibility, which can meet the requirements of large-scale industrial production.

[0254] The various embodiments in this specification are described in a related manner. The same or similar parts between the various embodiments can be referred to each other. Each embodiment focuses on describing the differences from other embodiments.

[0255] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0256] none

Claims

1. A crystal form A of a compound of formula (I), characterized in that the X-ray powder diffraction pattern of (I) has characteristic peaks at 2θ angles of 8.6°±0.2°, 9.8°±0.2°, 10.7°±0.2°, 11.2°±0.2°, 12.3°±0.2°, 12.9°±0.2°, 16.0°±0.2° and 19.0°±0.2°.

2. Crystal form A as described in claim 1, wherein, The X-ray powder diffraction pattern shows characteristic peaks at 2θ angles of 8.6°±0.2°, 9.8°±0.2°, 10.7°±0.2°, 11.2°±0.2°, 12.3°±0.2°, 12.9°±0.2°, 16.0°±0.2°, 17.0°±0.2°, 17.4°±0.2°, 17.8°±0.2°, 19.0°±0.2°, 19.9°±0.2°, and 21.2°±0.2°.

3. Crystal form A as described in claim 1 or 2, wherein, The X-ray powder diffraction patterns are basically shown in Table 1.

4. Crystal form A as described in claim 1 or 2, wherein, The X-ray powder diffraction pattern is basically as shown in Figure 2.

5. A crystal form Q of a compound of formula (I), characterized in that the X-ray powder diffraction pattern of (I) has characteristic peaks at 2θ angles of 8.5°±0.2°, 14.5°±0.2°, 15.0°±0.2°, 16.8°±0.2°, 19.4°±0.2°, 23.6°±0.2°, 24.8°±0.2°, 26.3°±0.2°, and 27.4°±0.2°.

6. Crystal form Q as described in claim 5, wherein, The X-ray powder diffraction pattern shows characteristic peaks at 2θ angles of 8.5°±0.2°, 14.5°±0.2°, 15.0°±0.2°, 15.8°±0.2°, 16.8°±0.2°, 18.8°±0.2°, 19.4°±0.2°, 20.1°±0.2°, 23.6°±0.2°, 24.8°±0.2°, 26.3°±0.2°, and 27.4°±0.2°.

7. Crystal form Q as described in claim 5 or 6, wherein, The X-ray powder diffraction pattern is basically as shown in Figure 13.

8. A pharmaceutical composition comprising an effective therapeutic amount of crystal form A as described in any one of claims 1 to 4 or crystal form Q as described in any one of claims 5 to 7, or a mixture thereof; and a pharmaceutically acceptable carrier, diluent, or excipient.

9. A veterinary drug composition comprising an effective therapeutic amount of crystal form A as described in any one of claims 1 to 4 or crystal form Q as described in any one of claims 5 to 7, or a mixture thereof; and a pharmaceutically acceptable carrier, diluent, or excipient.

10. A method for preparing crystal form A as described in any one of claims 1 to 4, comprising the following steps: The compound of formula (I) was dissolved in ethanol until clear, and then concentrated; the resulting concentrate was stirred to precipitate a solid; and then filtered and dried to obtain the crystal form A.

11. Use of a crystal form A as described in any one of claims 1 to 4, or a crystal form Q as described in any one of claims 5 to 7, or a pharmaceutical composition as described in claim 8, or a veterinary composition as described in claim 9, in the preparation of a medicament for treating diseases mediated by JAK kinase.

12. The use as described in claim 11, wherein, The drug is used to treat and prevent delayed allergic reactions, allergic dermatitis, atopic dermatitis, eczema, pruritus, or psoriasis.

13. The use as described in claim 12, wherein, The drug is used to treat and prevent delayed hypersensitivity reactions, allergic dermatitis, atopic dermatitis, eczema, pruritus, or psoriasis in mammals.

14. The use as described in claim 13, wherein, The mammals mentioned include companion animals.

15. The use as described in claim 13 or 14, wherein, The mammals mentioned include livestock.

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