WO2021164786A1 - Jak抑制剂的晶型及其应用 - Google Patents

Jak抑制剂的晶型及其应用 Download PDF

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WO2021164786A1
WO2021164786A1 PCT/CN2021/077228 CN2021077228W WO2021164786A1 WO 2021164786 A1 WO2021164786 A1 WO 2021164786A1 CN 2021077228 W CN2021077228 W CN 2021077228W WO 2021164786 A1 WO2021164786 A1 WO 2021164786A1
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crystal form
compound
angles
ray powder
formula
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PCT/CN2021/077228
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English (en)
French (fr)
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毛魏魏
钱文远
韦昌青
方亮
穆利伟
陈曙辉
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珠海联邦制药股份有限公司
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Priority to AU2021222686A priority Critical patent/AU2021222686B2/en
Priority to CN202180010731.2A priority patent/CN115038701B/zh
Priority to CA3166743A priority patent/CA3166743A1/en
Priority to US17/796,613 priority patent/US20230091250A1/en
Priority to KR1020227032610A priority patent/KR20220157398A/ko
Priority to EP21757247.8A priority patent/EP4108664A4/en
Priority to JP2022549366A priority patent/JP2023514291A/ja
Publication of WO2021164786A1 publication Critical patent/WO2021164786A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • 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]
    • 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 invention relates to a crystal form as a JAK inhibitor and its application in the preparation of drugs for treating JAK1 or/and TYK2 related diseases.
  • JAK kinase is a family of intracellular non-receptor tyrosine kinases, which has 4 members: JAK1, JAK2, JAK3 and TYK2 (Robert Roskoski Jr. (2016)). JAK1, JAK2 and TYK2 are expressed in all tissue cells of the human body. JAK3 is mainly expressed in various hematopoietic tissue cells, mainly in bone marrow cells, thymocytes, NK cells and activated B lymphocytes and T lymphocytes. From the perspective of gain-of-function expression or mutation analysis, JAK1 and JAK3 are more related to immune regulation, while JAK2 is directly related to the production of red blood cells and platelets.
  • JAK1 and JAK2 functions can cause mouse embryonic death. No diseases related to the loss of JAK1 and JAK2 functions have been found in humans, which indirectly indicates the importance of the physiological functions of JAK1 and JAK2. Loss of JAK3 function can cause severe comprehensive immune deficiency. There are few studies on the function of TYK2, and it has been reported that it can cause deficiencies related to intrinsic immunity. (James D. Clark, (2014)).
  • JAK-STAT pathway Downstream of JAKs is the family of signal transducers and activators of transcription (STAT).
  • the JAK-STAT pathway transmits extracellular signals from a variety of cytokines, growth factors and hormones to the nucleus, and is responsible for the expression of thousands of protein-coding genes.
  • cytokines bind to their receptors, JAK family members autophosphorylate and/or transphosphorylate each other, and then STATs are phosphorylated and then migrate to the nucleus to regulate transcription.
  • JAK STAT intracellular signal transduction is suitable for interferons, most interleukins, and a variety of cytokines and endocrine factors, such as EPO, TPO, GH, OSM, LIF, CNTF, GM CSF and PRL (Vainchenker W. et al. ( 2008)).
  • cytokines and endocrine factors such as EPO, TPO, GH, OSM, LIF, CNTF, GM CSF and PRL (Vainchenker W. et al. ( 2008)).
  • Different JAK family members selectively bind to different cytokine receptors, giving signal transduction specificity, thereby exerting different physiological effects. This selective mode of action allows JAK inhibitors to be relatively specifically applied to diseases treatment.
  • the IL-2 or IL-4 receptors together with the common ⁇ chain bind to JAK1 and JAK3, while the type I receptor with the same ⁇ chain binds to JAK2; the type I receptor using gp130 (glycoprotein 130) and the other Type I receptors activated by aggregate cytokines preferentially bind to JAK1/2 and TYK2; Type I receptors activated by hormone-like cytokines bind to and activate JAK2 kinase; Type II receptors of interferon bind to JAK1 and TYK2, while IL- Receptors of the 10 cytokine family bind to JAK1/2 and TYK2.
  • the various specific bindings of the above-mentioned cytokines and their receptors with JAK family members trigger different physiological effects, which provide the possibility for the treatment of different diseases.
  • JAK-STAT signaling pathway is involved in many important biological processes such as cell proliferation, differentiation, apoptosis and immune regulation.
  • Existing clinical data show that long-term inhibition of JAK2 will inhibit cytokines such as G-CSF, TPO and EPO, thereby affecting the proliferation and differentiation of hematopoietic stem cells; JAK3 inhibition will down-regulate the number of NK cells and increase the probability of infection. Therefore, being a JAK inhibitor may affect the number and function of white blood cells, red blood cells and lymphocytes to varying degrees.
  • selective JAK1 or/and TYK2 inhibitors with a narrow range of action on cytokines can theoretically maintain efficacy and improve safety (Daniella M. Schwartz, et al. (2017)).
  • US2009220688 discloses Filgotinib, which is a drug used by Galapagos Company for the treatment of rheumatoid arthritis in Phase III clinical trials.
  • the present invention provides crystal form A of the compound of formula (I), and its X-ray powder diffraction (XRPD) pattern has characteristic diffraction peaks at the following 2 ⁇ angles: 6.91 ⁇ 0.20°, 12.21 ⁇ 0.20° and 19.06 ⁇ 0.20°.
  • the X-ray powder diffraction pattern of the above crystal form A has characteristic diffraction peaks at the following 2 ⁇ angles: 6.91 ⁇ 0.20°, 12.21 ⁇ 0.20°, 13.69 ⁇ 0.20°, 19.06 ⁇ 0.20°, 19.86 ⁇ 0.20 °, 20.59 ⁇ 0.20°, 22.06 ⁇ 0.20° and 27.52 ⁇ 0.20°.
  • the X-ray powder diffraction pattern of the above crystal form A has characteristic diffraction peaks at the following 2 ⁇ angles: 6.91 ⁇ 0.20°, 10.34 ⁇ 0.20°, 12.21 ⁇ 0.20°, 13.69 ⁇ 0.20°, 18.11 ⁇ 0.20 °, 19.06 ⁇ 0.20°, 19.86 ⁇ 0.20°, 20.59 ⁇ 0.20°, 22.06 ⁇ 0.20° and 27.52 ⁇ 0.20°.
  • the X-ray powder diffraction pattern of the above crystal form A has characteristic diffraction peaks at the following 2 ⁇ angles: 6.91 ⁇ 0.20°, 10.34 ⁇ 0.20°, 12.21 ⁇ 0.20°, 13.69 ⁇ 0.20°, 17.44 ⁇ 0.20 °, 18.11 ⁇ 0.20°, 19.06 ⁇ 0.20°, 19.86 ⁇ 0.20°, 20.59 ⁇ 0.20°, 22.06 ⁇ 0.20°, 24.46 ⁇ 0.20° and 27.52 ⁇ 0.20°.
  • the XRPD pattern of the above-mentioned crystal form A is shown in FIG. 1.
  • the XRPD pattern analysis data of the above-mentioned crystal form A is shown in Table 1.
  • the differential scanning calorimetry curve of the above-mentioned crystal form A has an endothermic peak at 152.19 ⁇ 3°C and 216.79 ⁇ 3°C, and an exothermic peak at 161.50 ⁇ 3°C. .
  • the DSC chart of the above-mentioned crystal form A is shown in FIG. 2.
  • the present invention provides the B crystal form of the compound of formula (I), and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2 ⁇ angles: 5.13 ⁇ 0.20°, 19.14 ⁇ 0.20° and 21.18 ⁇ 0.20°.
  • the X-ray powder diffraction pattern of the above-mentioned crystal form B has characteristic diffraction peaks at the following 2 ⁇ angles: 5.13 ⁇ 0.20°, 7.34 ⁇ 0.20°, 10.14 ⁇ 0.20°, 10.56 ⁇ 0.20°, 11.72 ⁇ 0.20 °, 16.67 ⁇ 0.20°, 19.14 ⁇ 0.20° and 21.18 ⁇ 0.20°.
  • the X-ray powder diffraction pattern of the above-mentioned crystal form B has characteristic diffraction peaks at the following 2 ⁇ angles: 5.13 ⁇ 0.20°, 7.34 ⁇ 0.20°, 10.14 ⁇ 0.20°, 10.56 ⁇ 0.20°, 11.72 ⁇ 0.20 °, 16.67 ⁇ 0.20°, 19.14 ⁇ 0.20°, 21.18 ⁇ 0.20° and 21.78 ⁇ 0.20°.
  • the XRPD pattern of the above-mentioned crystal form B is shown in FIG. 3.
  • the XRPD pattern analysis data of the above-mentioned crystal form B is shown in Table 2.
  • the differential scanning calorimetry curve of the above-mentioned crystal form B has an endothermic peak at 193.99 ⁇ 3°C and 216.93 ⁇ 3°C; there is an exothermic peak at 200.10 ⁇ 3°C. .
  • the DSC chart of the above-mentioned crystal form B is shown in FIG. 4.
  • thermogravimetric analysis curve (TGA) of the above-mentioned crystal form B has a weight loss of 0.535% at 120 ⁇ 3°C.
  • the TGA pattern of the above-mentioned crystal form B is shown in FIG. 5.
  • the present invention provides crystal form C of the compound of formula (I), and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2 ⁇ angles: 8.92 ⁇ 0.20°, 18.66 ⁇ 0.20° and 20.26 ⁇ 0.20°.
  • the X-ray powder diffraction pattern of the above crystal form C has characteristic diffraction peaks at the following 2 ⁇ angles: 5.76 ⁇ 0.20°, 8.92 ⁇ 0.20°, 11.50 ⁇ 0.20°, 16.35 ⁇ 0.20°, 18.66 ⁇ 0.20 °, 19.17 ⁇ 0.20°, 20.26 ⁇ 0.20° and 24.79 ⁇ 0.20°.
  • the XRPD pattern of the above-mentioned crystal form C is shown in FIG. 6.
  • the XRPD pattern analysis data of the above-mentioned crystal form C is shown in Table 3.
  • the differential scanning calorimetry curve of the above-mentioned crystal form C has an endothermic peak starting point at 215.48°C.
  • the DSC chart of the above-mentioned crystal form C is shown in FIG. 7.
  • the present invention provides the D crystal form of the compound of formula (I), and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2 ⁇ angles: 7.12 ⁇ 0.20°, 20.54 ⁇ 0.20° and 21.42 ⁇ 0.20°.
  • the X-ray powder diffraction pattern of the above-mentioned crystal form D has characteristic diffraction peaks at the following 2 ⁇ angles: 7.12 ⁇ 0.20°, 12.45 ⁇ 0.20°, 14.64 ⁇ 0.20°, 18.31 ⁇ 0.20°, 20.54 ⁇ 0.20 °, 21.42 ⁇ 0.20° and 28.72 ⁇ 0.20°.
  • the X-ray powder diffraction pattern of the above crystal form D has characteristic diffraction peaks at the following 2 ⁇ angles: 7.12 ⁇ 0.20°, 10.28 ⁇ 0.20°, 12.45 ⁇ 0.20°, 14.64 ⁇ 0.20°, 17.50 ⁇ 0.20 °, 18.31 ⁇ 0.20°, 20.54 ⁇ 0.20°, 21.42 ⁇ 0.20° and 28.72 ⁇ 0.20°.
  • the XRPD pattern of the above-mentioned crystal form D is shown in FIG. 8.
  • the XRPD pattern analysis data of the above-mentioned crystal form D is shown in Table 4.
  • the present invention also provides the application of the above crystal form A, B crystal form, C crystal form and D crystal form in the preparation of drugs for treating JAK1 and/or TYK2 related diseases.
  • the above application is characterized in that the drug is a drug for the treatment of rheumatoid arthritis.
  • the compound of formula (I) of the present invention has good drug efficacy for in vivo administration, and its various crystal forms are stable, are less affected by light, heat and humidity, have high solubility, and have broad prospects for preparing medicines.
  • the intermediate compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by combining them with other chemical synthesis methods, and those skilled in the art.
  • Well-known equivalent alternatives, preferred implementations include but are not limited to the embodiments of the present invention.
  • the solvent used in the present invention is commercially available.
  • the present invention uses the following abbreviations: DCM stands for dichloromethane; DMF stands for N,N-dimethylformamide; DMSO stands for dimethyl sulfoxide; EtOH stands for ethanol; MeOH stands for methanol; TFA stands for trifluoroacetic acid; TsOH stands for P-toluenesulfonic acid; mp represents melting point; EtSO 3 H represents ethanesulfonic acid; MeSO 3 H represents methanesulfonic acid; ATP represents adenosine triphosphate; HEPES represents 4-hydroxyethylpiperazine ethanesulfonic acid; EGTA represents ethylene glycol bis(2 -Aminoethyl ether) tetraacetic acid; MgCl 2 stands for magnesium dichloride; MnCl 2 stands for manganese dichloride; DTT stands for dithiothreitol; DCC stands for dicycl
  • Light tube voltage 40kV
  • light tube current 40mA
  • DSC Differential Calorimetry Scanner
  • thermogravimetric analyzer (TGA) method of the present invention is thermogravimetric analyzer
  • Figure 1 XRPD pattern of crystal form A.
  • Figure 3 XRPD pattern of Form B.
  • Figure 8 XRPD pattern of crystal form D.
  • Step 1 At -78°C, LiHMDS (1M, 51.2 mL) was added dropwise to a THF (150 mL) solution in which compound 1-1 (10.2 g, 42.6 mmol) was dissolved. After the reaction solution was stirred at -78°C for 1 hour, 1,1,1-trifluoro-N-phenyl-N-(trifluoromethylsulfonyl)methanesulfonamide (16.7g, 46.9mmol) in THF( 150mL) solution was added to the reaction solution, and then stirred at 15°C for 12 hours.
  • reaction was quenched with 250 mL saturated ammonium chloride, diluted with 200 mL water, and then extracted with ethyl acetate (200 mL*3). The organic phases were combined, washed with saturated brine, dried over sodium sulfate, filtered and concentrated to obtain compound 1-2. The crude product was directly used in the next reaction without purification.
  • Step 2 Add potassium acetate (12.7g, 129.3mmol) and Pd( dppf) Cl 2 .CH 2 Cl 2 (3.5 g, 4.3 mmol), replaced with nitrogen 3 times and kept stirring at 70° C. for 3 hours in a nitrogen atmosphere.
  • the reaction solution was dispersed in a mixture of 300 mL of water and 400 mL of ethyl acetate.
  • the organic phase was separated, washed with saturated brine, dried over sodium sulfate, filtered and concentrated to obtain a crude product.
  • the crude product was purified by silica gel chromatography to obtain compound 1-3.
  • Step 3 In a nitrogen atmosphere, compound 1-3 (3.5g, 10.0mmol) and N-(5-bromo-[1,2,4]triazolo[1,5-a]pyridine-2 -Base) cyclopropane carboxamide (2.6g, 9.1mmol) in dioxane (60mL) and water (15mL) was added potassium carbonate (3.8g, 27.3mmol) and Pd(dppf)Cl 2 .CH 2 Cl 2 (744 mg, 911.0 ⁇ mol). The reaction solution was stirred at 90°C for 3 hours. The reaction solution was concentrated, and the obtained crude product was separated and purified by column chromatography to obtain compound 1-4. LCMS (ESI) m/z: 424.3 [M+H] + .
  • Step 4 To a dichloromethane (10 mL) solution in which compound 1-4 (3.5 g, 8.2 mmol) was dissolved was added hydrochloric acid/ethyl acetate (4M, 30 mL), and the reaction solution was stirred at 25° C. for 0.5 hours. The solid precipitated, filtered and dried to obtain compound 1-5 (3.3g hydrochloride, crude product), which was directly used in the next reaction without purification.
  • Step 5 In a nitrogen atmosphere, Pd/C (1 g, 10%) was added to a methanol (100 mL) solution in which compound 1-5 (3.0 g, 8.34 mmol, hydrochloride) was dissolved. This suspension was replaced with hydrogen three times, and then stirred under a hydrogen atmosphere (30 psi) at 30°C for 12 hours. The reaction solution was filtered and concentrated to obtain compound 1-6 (3g hydrochloride, crude product), which was directly used in the next reaction without purification.
  • Step 6 Dissolve compound 1-6 (0.87g, 2.40mmol, hydrochloride) in N,N-dimethylformamide (10mL), add HOBt (487mg, 3.6mmol,) and EDCI (691mg, 3.6 mmol), and then (1S)-2,2-difluorocyclopropylcarboxylic acid (323 mg, 2.6 mmol) and diisopropylethylamine (621 mg, 4.8 mmol) were added, and the reaction solution was reacted at 15°C for 12 hours. The reaction solution was concentrated under reduced pressure, and the residue was subjected to preparative HPLC (neutral system) to obtain the compound of formula (I).
  • the crystal form A of the compound of formula (I) was heated to 170°C, the XRPD (shown in Figure 6) was tested, the crystal form changed, and the new crystal form obtained was the crystal form C of the compound of formula (I).
  • DSC such as Shown in Figure 7).
  • sample B crystal form into a sample bottle, and then add 1.0 mL of different solvents to each [pure water, SGF (simulated gastric juice), FaSSIF (simulated intestinal fluid in fasting state), FeSSIF (simulated intestinal fluid in fed state) )], shake well. Place on a constant temperature shaker and shake at 37°C. After shaking for 24 hours, centrifugal separation, the obtained supernatant was tested for its solubility. After diluting the supernatant (diluent ACN/H 2 O (1/1)) to a certain multiple (the compound has low solubility, the supernatant is diluted twice except for SGF and 10 times for SGF), and its concentration is determined by HPLC.
  • SGF simulated gastric juice
  • FaSSIF simulated intestinal fluid in fasting state
  • FeSSIF simulated intestinal fluid in fed state
  • Diluent acetonitrile: water 1:1.
  • Mobile phase A 0.1% TFA aqueous solution, for example: Pipette 1 mL of TFA into 1 L of pure water, mix well, and ultrasonically degas.
  • Mobile phase B 100% acetonitrile.
  • STD solution preparation use crystal form B as the reference substance. Weigh about 5 mg of the reference substance in a glass bottle, dissolve it with 10 mL of diluent, sonicate for about 10 minutes to fully dissolve the sample, cool to room temperature and shake. Prepare two copies in parallel and label them as STD1 and STD2. Dilute the corresponding STD1 by 10, 100, 1000, and 2000 times with a diluent, and make a standard curve for testing.
  • sample solution After diluting the supernatant (diluent ACN/H 2 O (1/1)) a certain multiple (the compound has low solubility, the supernatant is diluted twice except for SGF, and 10 times for SGF), shake Evenly, place in a 1.5mL liquid phase vial to be tested, and determine its concentration by HPLC.
  • JAK2, JAK3 and TYK2 dilution 20mM 3-(N-morpholine) propanesulfonic acid (MOPS), 1mM EDTA, 0.01% Brij-35.5% glycerol, 0.1% ⁇ -mercaptoethanol, 1mg/mL BSA; JAK1 dilution: 20mM TRIS, 0.2mM EDTA, 0.1% ⁇ -mercaptoethanol, 0.01% Brij-35.5% glycerol. All compounds were prepared into 100% DMSO solution and reached 50 times the final measured concentration. The test compound was diluted by a 3-fold concentration gradient, and the final concentration was 9 concentrations from 10 ⁇ M to 0.001 ⁇ M. The content of DMSO in the detection reaction was 2%. The working stock solution of this compound is added as the first component of the reaction to the measurement well, and then the remaining components are added according to the protocol detailed in the measurement below.
  • MOPS 3-(N-morpholine) propanesulfonic acid
  • JAK1(h) was incubated with 20mM Tris/HCl pH7.5, 0.2mM EDTA, 500 ⁇ M MGEEPLYWSFPAKKK, 10mM magnesium acetate and [ ⁇ - 33 P]-ATP (activity and concentration as required). The reaction was started by adding Mg/ATP mixture. After incubating at room temperature for 40 minutes, the reaction was terminated by adding 0.5% phosphoric acid. Then 10 ⁇ L of the reactant was spotted on the P30 filter pad and washed three times with 0.425% phosphoric acid and once with methanol within 4 minutes, dried, and scintillated counted.
  • JAK2(h) was incubated with 8mM MOPS pH 7.0, 0.2mM EDTA, 100 ⁇ M KTFCGTPEYLAPEVRREPRILSEEEQEMFRDFDYIADWC, 10mM magnesium acetate and [ ⁇ - 33 P]-ATP (activity and concentration as required).
  • the reaction was started by adding Mg/ATP mixture. After incubating at room temperature for 40 minutes, the reaction was terminated by adding 0.5% phosphoric acid. Then 10 ⁇ L of the reactant was spotted on the P30 filter pad and washed three times with 0.425% phosphoric acid and once with methanol within 4 minutes, dried, and scintillated counted.
  • JAK3(h) was incubated with 8mM MOPS pH 7.0, 0.2mM EDTA, 500 ⁇ M GGEEEEYFELVKKKK, 10mM magnesium acetate and [ ⁇ - 33 P]-ATP (activity and concentration as required). The reaction was started by adding Mg/ATP mixture. After incubating at room temperature for 40 minutes, the reaction was terminated by adding 0.5% phosphoric acid. Then 10 ⁇ L of the reactant was spotted on the P30 filter pad and washed three times with 0.425% phosphoric acid and once with methanol within 4 minutes, dried, and scintillated counted.
  • TYK2(h) was incubated with 8mM MOPS pH 7.0, 0.2mM EDTA, 250 ⁇ M GGMEDIYFEFMGGKKK, 10mM magnesium acetate and [ ⁇ - 33 P]-ATP (activity and concentration as required). The reaction was started by adding Mg/ATP mixture. After incubating at room temperature for 40 minutes, the reaction was terminated by adding 0.5% phosphoric acid. Then 10 ⁇ L of the reactant was spotted on the P30 filter pad and washed three times with 0.425% phosphoric acid and once with methanol within 4 minutes, dried, and scintillated counted.
  • the IC 50 result is obtained by analyzing the XLFIT5 (205 formula) of IDBS, see Table 6 for details.
  • the compound of formula (I) of the present invention exhibits good selective inhibition of JAK1 and/or TYK2 in the in vitro activity test of 4 kinase subtypes JAK1, JAK2, JAk3 and TYK2.
  • the clear solution obtained by dissolving the test compound was injected into the tail vein and intragastrically administered to male mice (C57BL/6) or rats (SD) (overnight fasting, 7-8 weeks old).
  • the intravenous group (2mg/kg) at 0.117, 0.333, 1, 2, 4, 7 and 24 hours
  • the intragastric group (15 mg/kg) at 0.25, 0.5, 1, 2, 4, 8 At and 24 hours
  • blood was collected from the mandibular vein and centrifuged to obtain plasma.
  • the LC-MS/MS method was used to determine the blood drug concentration
  • the WinNonlin TM Version 6.3 pharmacokinetic software was used to calculate the relevant pharmacokinetic parameters by the non-compartmental model linear logarithmic ladder method.
  • the test results are as follows:
  • AUC 0-inf the area under the plasma concentration-time curve from time 0 to extrapolated to infinity
  • Bioavailability Bioavailability.
  • the compound of formula (I) of the present invention has good oral bioavailability in mice, and higher exposure is beneficial to produce good in vivo efficacy.
  • Rheumatoid arthritis is a type of multiple autoimmune diseases with a global incidence of about 1%. It is caused by autoimmune reactions that cause inflammation, damage, and deformities in the joints. In severe cases, it can cause systemic and systemic diseases. Inflammatory response.
  • the development of drugs for the treatment of RA can help relieve the symptoms of rheumatoid arthritis and improve the quality of life of patients.
  • Collagen-induced mouse arthritis model is an animal model that is often used to evaluate the efficacy of drugs in the treatment of RA. Its pathogenesis and symptoms are significantly related to RA disease. The model activates B cells by injecting type II collagen. The reactivity of T cells to bone collagen.
  • the activated B cells and T cells enter the joints and cause joint damage, thereby triggering a series of similar human rheumatoid arthritis symptom.
  • collagen-induced arthritis in mice is often used to evaluate its effectiveness.
  • the purpose of this experiment is to investigate the therapeutic effect of the compound of formula (I) and the reference compound Filgotinib on collagen-induced arthritis in mice, so as to provide preclinical pharmacodynamic information for subsequent clinical studies.
  • acetic acid Dilute 2N acetic acid to 100mM, filter with 0.22 micron membrane, and store at 4°C.
  • Bovine type II collagen solution Dissolve bovine type II collagen (CII) in 100 mM acetic acid solution and store at 4°C overnight. The final concentration of collagen is 8mg/ml.
  • Preparation of emulsion Mix the CII solution stored overnight with an equal volume of complete Freund's adjuvant, use a high-speed homogenizer, and homogenize on ice at 30,000 revolutions per minute for approximately 60 minutes, until the solution forms a stable emulsion.
  • mice were randomly assigned to different treatment groups.
  • the day of the first immunization was recorded as day 0, and the subsequent days were marked in order.
  • mice with a moderate degree of disease were selected and re-randomized into 5 treatment groups according to their body weight and score, with 8 mice in each group.
  • Dexamethasone (Dexamethasone, Dex.) was used as a reference drug to measure whether the model was successfully established, with a dose of 0.3 mg/kg (a commonly used dose in the CIA model); in addition, the test compound and The relative dose design of the reference compound Filgotinib is shown in Table 8-1:
  • the first group is normal mice without any treatment; the second group is given a blank group containing only a vehicle; the third group is given a dose of dexamethasone 0.3mg /kg; the doses of the sixth group, the seventh group and the eighth group are 15mg/kg and 15mg/kg respectively. It was administered twice a day for a total of 14 days.
  • PO oral; bid: twice a day; qd: once a day.
  • Clinical scoring After boosting immunity, observe the incidence of mice every day. When the mouse begins to develop symptoms (clinical symptoms of arthritis), according to the different degree of the disease (redness, joint deformation) according to the standard of 0-4 points, the highest score for each limb is 4 points, each animal is the highest The score is 16 points.
  • the scoring standards are shown in Table 8-2. Score at least three times a week.
  • the experimental data was expressed by Mean ⁇ SEM, and the area under the curve (AUC) was expressed by One-way ANOVA, and p ⁇ 0.05 was considered as a significant difference.
  • the average clinical scores of the compound of formula (I) and Filgotinib at this dose are reduced to 1.5 and 5.6 points (see Table 8-3 for values) ; It can be seen that the compound of formula (I) at 15mg/kg can effectively alleviate collagen-induced arthritis. Dexamethasone 0.3mg/kg (G3 group) treatment can significantly inhibit the clinical score of collagen-induced arthritis. From the 27th day, the clinical score is maintained at about 0.3 and on the 31st day (the clinical score drops to 0, see table 8-3 value).
  • Inhibition rate (average of the area under the curve of the blank group-average of the area under the curve of the administration group)/average of the area under the curve of the blank group
  • Incidence rate number of animals in each group/total number of animals in each group*100%
  • Adjuvant-induced Arthritis (AIA) rat model is one of the animal models commonly used in rheumatoid arthritis disease research and new drug development. Its pathogenesis and clinical symptoms are similar to those of human rheumatoid arthritis. similar.
  • the model injected Mycobacterium tuberculosis on the footpad to induce immune cells and antibodies with bone and joint damage, which caused a systemic response, which was manifested as joint swelling, osteolysis, synovial damage and other symptoms similar to human rheumatoid arthritis.
  • the purpose of this experiment is to evaluate the therapeutic effect of the compound of formula (I) on adjuvant-induced arthritis rat model, with dexamethasone and Filgotinib as reference compounds.
  • HE hematoxylin-eosin staining
  • Adjuvant preparation Weigh 100 mg of Mycobacterium tuberculosis H37Ra, grind for about 5 minutes, add 3 mL of paraffin oil to dissolve the powder, transfer to a brown dispensing bottle, and then wash the mortar twice with 3 mL and 4 mL of paraffin oil, and transfer all to In the brown dispensing bottle, the final concentration is 10 mg/mL. Ultrasonic break, ultrasonic for about 30 minutes in a mixture of ice and water.
  • the prepared adjuvant is shaken and mixed, and drawn with a 1 mL glass syringe (20G needle), and then replaced with a 25G needle to eliminate air bubbles.
  • Rats were anesthetized with isoflurane, and the syringe was turned upside down before immunizing each rat to thoroughly mix Mycobacterium tuberculosis. After anesthesia, 0.1 mL of adjuvant was injected subcutaneously into the sole of the left foot of the rat. Rats in the normal group were injected with 0.1 mL of paraffin oil subcutaneously in the soles of the feet. The day of adjuvant injection is day 0.
  • mice On the 13th day, all animals showed symptoms of arthritis such as erythema or swelling on the feet, and they were stratified and randomly grouped according to score, foot volume and weight.
  • the grouping situation is shown in Table 9-1. Seventy rats were divided into 7 groups, each with 10 rats, and the normal group with 5 rats. According to Table 9-1, the dosage of each group is as follows.
  • the volume of intragastric administration is 5mL/kg.
  • the compound was administered twice a day for a total of 14 days.
  • Weight Weigh three times a week from day 13 to day 27.
  • Foot volume measured once before immunization, and measured three times a week from day 13 to day 27.
  • Scoring Score three times a week from the 13th day to the 27th day. According to the different degree of lesions (redness, joint deformation) according to the standard of 0-4 points, the highest score for each limb is 4 points, and the highest score for each animal is 12 points (except for the left hind limb on the injection side). The scoring standards are shown in Table 9-2.
  • the experimental data were expressed by Mean ⁇ SEM, and the weight, clinical score, and pathology score were expressed by One-way ANOVA, and p ⁇ 0.05 was considered as a significant difference.
  • the compound of formula (I) at the four doses of 1, 3, 10, and 30 mg/kg significantly inhibited the clinical score of arthritic rats (compared with the solvent control group, The p values were all ⁇ 0.0001), and the clinical scores of arthritic rats were reduced to 5.4, 3.9, 3.2 and 2.7, respectively, in a dose-dependent manner (compared with the high-dose group and the low-dose group, p ⁇ 0.0001).
  • the effect of 30mg/kg of the compound of formula (I) is the most obvious (starting from the 17th day, there is a very significant difference compared with the solvent control group, p ⁇ 0.0001).
  • the average arthritis clinical score of this group decreased from a peak of 6.0 points on the 13th day to 2.7 points on the 27th day of the experimental end point (Table 9-4).
  • the BID score of the reference compound Filgotinib 30mg/kg dropped to 5.1 points on the 27th day of the experimental end point, which was significantly lower than the solvent control group (p ⁇ 0.001) but significantly higher than the 30mg/kg BID of the compound of formula (I) (p ⁇ 0.001).
  • the improvement effect of the compound of formula (I) on the clinical score of arthritis is significantly better than the effect of Filgotinib at the same dose.
  • This experiment evaluated the effect of compound of formula (I) on foot volume improvement in a rat arthritis (AIA) model, with dexamethasone and Filgotinib as references.
  • the average foot volume of animals in the solvent control group increased steadily from 1.9 mL on day 13 to 2.9 mL at the end of the experiment on day 27, marking the successful establishment of the AIA model (Table 9-5).
  • the compound of formula (I) at doses of 1, 3, 10 and 30 mg/kg can significantly inhibit the increase in foot volume of arthritic rats (compared with the solvent control group, p values are all ⁇ 0.0001).
  • the mean foot volume of arthritic rats decreased to 1.91 mL, 1.59 mL, and 1.26 mL and 1.21 mL, respectively, in a dose-dependent manner (compared with the high-dose group and the low-dose group, p ⁇ 0.0001).
  • the reference compound Filgotinib 30mg/kg BID decreased to 1.91 points on the 27th day of the experimental end point, which was significantly lower than the solvent control group (p ⁇ 0.0001) but significantly higher than the formula (I) compound 30mg/kg BID (p ⁇ 0/0001) ).
  • the effect of the compound of formula (I) on the foot volume of rats is significantly better than that of Filgotinib at the same dose.
  • the positive control dexamethasone treatment group also inhibited the increase in average foot volume very well. After the administration, the foot volume steadily decreased to the end of the experiment, which was stable at 1.21 mL. From day 17, it was extremely compared with the solvent control group. Significant difference, p ⁇ 0.0001 (Table 9-5)
  • the p values were 0.014, ⁇ 0.0001 and ⁇ 0.0001, and the inhibition rates were 29.4%, 72.5% and 90%.
  • the reference compound Filgotinib 30mg/kg total pathological score was 15.2 ⁇ 0.49, the inhibition rate was 5%, and there was no significant difference compared with the solvent group.
  • the total pathological score of the compound of formula (I) at the same dose (30mg/kg) was significantly lower than that of Filgotinib (p ⁇ 0.0001).
  • the control compound dexamethasone at a dose of 0.3 mg/kg extremely significantly reduced the pathological score of arthritic rats to 4.4 ⁇ 0.8, with a p value of ⁇ 0.0001, and an inhibition rate of 72.5% (Table 9-7).
  • the rats in the solvent control group developed clinical symptoms of arthritis and continued to worsen.
  • the compound of formula (I) (1, 3, 10, 30 mg/kg), Filgotinib (30 mg/kg) and dexamethasone (0.3 mg/kg) showed that the adjuvant-induced arthritis has Obvious inhibitory effect is manifested by delayed onset time, clinical symptoms and pathological changes significantly reduced, and the therapeutic effect of the compound of formula (I) on the adjuvant-induced arthritis model is dose-dependent.
  • the above experimental results show that the compound of formula (I) has a significant therapeutic effect on adjuvant-induced arthritis in rats and the effect is better than Filgotinib.

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Abstract

一种作为JAK抑制剂的晶型，以及在制备治疗JAK1或/和TYK2相关疾病的药物中的应用。

Description

JAK抑制剂的晶型及其应用
本申请要求申请日为2020年2月21日的中国专利申请CN202010110530.7的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及一种作为JAK抑制剂的晶型，以及在制备治疗JAK1或/和TYK2相关疾病的药物中的应用。
背景技术
JAK激酶是一类胞内非受体酪氨酸激酶家族，该激酶家族有4个成员:JAK1，JAK2，JAK3和TYK2(Robert Roskoski Jr.(2016))。JAK1、JAK2和TYK2在人体各组织细胞中均有表达，JAK3主要表达于各造血组织细胞中，主要存在于骨髓细胞、胸腺细胞、NK细胞及活化的B淋巴细胞、T淋巴细胞中。从功能获得性表达或突变分析来看，JAK1、JAK3更多的与免疫调节有关，JAK2则与红细胞和血小板的生成直接相关。从功能缺失分析来看，JAK1、JAK2功能缺失会造成老鼠胚胎致死，在人体中尚未发现JAK1、JAK2功能缺失相关的疾病，间接表明了JAK1、JAK2生理功能的重要性。JAK3功能缺失会造成严重的综合免疫缺陷。TYK2的功能研究较少，有报道其会引起与内在免疫相关的缺陷。(James D.Clark,(2014))。
JAKs的下游是信号转导与转录激活因子家族(STAT),JAK-STAT通路将来自多种细胞因子、生长因子和激素的细胞外信号传导到细胞核，并且负责数千个蛋白质编码基因的表达,当细胞因子与其受体结合时，JAK家族成员自磷酸化和/或彼此转磷酸化，随后STATs磷酸化，然后迁移至细胞核内以调节转录。JAK STAT细胞内信号转导适用于干扰素、大多数白细胞介素以及多种细胞因子和内分泌因子，例如EPO、TPO、GH、OSM、LIF、CNTF、GM CSF和PRL(Vainchenker W.等人(2008))。不同的JAK家族成员选择性地结合在不同的细胞因子受体上，赋予信号传导特异性，从而发挥不同的生理学作用，这种选择性的作用方式使得JAK抑制剂可以相对特异性地应用于疾病治疗。IL-2或IL-4受体连同共同的γ链与JAK1和JAK3结合，而具有相同β链的I型受体与JAK2结合；使用gp130(糖蛋白130)的I型受体和由杂二聚体细胞因子激活的I型受体优先结合JAK1/2和TYK2；由激素样细胞因子激活的I型受体结合并激活JAK2激酶；干扰素的Ⅱ型受体结合JAK1和TYK2，而IL-10细胞因子家族的受体与JAK1/2和TYK2结合。上述细胞因子及其受体与 JAK家族成员的各种特异结合引发不同的生理学作用，为不同疾病的治疗提供可能。
JAK-STAT信号通路参与着细胞的增殖、分化、凋亡以及免疫调节等许多重要的生物学过程。现有临床数据表明：JAK2长时间抑制会抑制细胞因子如G-CSF、TPO和EPO，从而影响造血干细胞增殖、分化；JAK3抑制会下调NK细胞数量并增加感染的机率。因此，作为JAK抑制剂可能会不同程度影响白细胞、红细胞以及淋巴细胞的数量及功能。然而，对细胞因子作用范围窄的选择性JAK1或/和TYK2抑制剂理论上可以保持功效并提高安全性(Daniella M.Schwartz,等人(2017))。
US2009220688公开了Filgotinib，是Galapagos公司处于临床三期用于类风湿性关节炎治疗的药物，
Figure PCTCN2021077228-appb-000001
发明内容
本发明提供了式(Ⅰ)化合物的A晶型，其X射线粉末衍射(XRPD)图谱在下列2θ角处具有特征衍射峰：6.91±0.20°、12.21±0.20°和19.06±0.20°。
Figure PCTCN2021077228-appb-000002
本发明的一些方案中，上述A晶型的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：6.91±0.20°、12.21±0.20°、13.69±0.20°、19.06±0.20°、19.86±0.20°、20.59±0.20°、22.06±0.20°和27.52±0.20°。
本发明的一些方案中，上述A晶型的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：6.91±0.20°、10.34±0.20°、12.21±0.20°、13.69±0.20°、18.11±0.20°、19.06±0.20°、19.86±0.20°、20.59±0.20°、22.06±0.20°和27.52±0.20°。
本发明的一些方案中，上述A晶型的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：6.91±0.20°、10.34±0.20°、12.21±0.20°、13.69±0.20°、17.44±0.20°、18.11±0.20°、19.06±0.20°、19.86±0.20°、20.59±0.20°、22.06±0.20°、24.46±0.20°和27.52±0.20°。
本发明的一些方案中，上述A晶型，其XRPD图谱如附图1所示。
本发明的一些方案中，上述A晶型的XRPD图谱解析数据如表1所示。
表1：式(Ⅰ)化合物的A晶型XRPD图谱解析数据
Figure PCTCN2021077228-appb-000003
本发明的一些方案中，上述A晶型，其差示扫描量热曲线在152.19±3℃和216.79±3℃各有一个吸热峰的峰值；在161.50±3℃有一个放热峰的峰值。
本发明的一些方案中，上述A晶型，其DSC图谱如附图2所示。
本发明提供了式(Ⅰ)化合物的B晶型，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：5.13±0.20°、19.14±0.20°和21.18±0.20°。
本发明的一些方案中，上述B晶型的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：5.13±0.20°、7.34±0.20°、10.14±0.20°、10.56±0.20°、11.72±0.20°、16.67±0.20°、19.14±0.20°和21.18±0.20°。
本发明的一些方案中，上述B晶型的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：5.13±0.20°、7.34±0.20°、10.14±0.20°、10.56±0.20°、11.72±0.20°、16.67±0.20°、19.14±0.20°、21.18±0.20°和21.78±0.20°。
本发明的一些方案中，上述B晶型，其XRPD图谱如附图3所示。
本发明的一些方案中，上述B晶型的XRPD图谱解析数据如表2所示。
表2：式(Ⅰ)化合物的B晶型XRPD图谱解析数据
Figure PCTCN2021077228-appb-000004
Figure PCTCN2021077228-appb-000005
本发明的一些方案中，上述B晶型，其差示扫描量热曲线在193.99±3℃和216.93±3℃各有一个吸热峰的峰值；在200.10±3℃有一个放热峰的峰值。
本发明的一些方案中，上述B晶型，其DSC图谱如附图4所示。
本发明的一些方案中，上述B晶型的热重分析曲线(TGA)在120±3℃时失重达0.535％。
本发明的一些方案中，上述B晶型的TGA图谱如图5所示。
本发明提供了式(Ⅰ)化合物的C晶型，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：8.92±0.20°、18.66±0.20°和20.26±0.20°。
本发明的一些方案中，上述C晶型的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：5.76±0.20°、8.92±0.20°、11.50±0.20°、16.35±0.20°、18.66±0.20°、19.17±0.20°、20.26±0.20°和24.79±0.20°。
本发明的一些方案中，上述C晶型，其XRPD图谱如附图6所示。
本发明的一些方案中，上述C晶型的XRPD图谱解析数据如表3所示。
表3：式(Ⅰ)化合物的C晶型XRPD图谱解析数据
Figure PCTCN2021077228-appb-000006
本发明的一些方案中，上述C晶型，其差示扫描量热曲线在215.48℃有一个吸热峰的起始点。
本发明的一些方案中，上述C晶型，其DSC图谱如附图7所示。
本发明提供了式(Ⅰ)化合物的D晶型，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：7.12±0.20°、20.54±0.20°和21.42±0.20°。
本发明的一些方案中，上述D晶型的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：7.12±0.20°、12.45±0.20°、14.64±0.20°、18.31±0.20°、20.54±0.20°、21.42±0.20°和28.72±0.20°。
本发明的一些方案中，上述D晶型的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：7.12±0.20°、10.28±0.20°、12.45±0.20°、14.64±0.20°、17.50±0.20°、18.31±0.20°、20.54±0.20°、21.42±0.20°和28.72±0.20°。
本发明的一些方案中，上述D晶型，其XRPD图谱如附图8所示。
本发明的一些方案中，上述D晶型的XRPD图谱解析数据如表4所示。
表4：式(Ⅰ)化合物的D晶型XRPD图谱解析数据
Figure PCTCN2021077228-appb-000007
本发明还提供了上述A晶型、B晶型、C晶型和D晶型在制备治疗JAK1和/或TYK2相关疾病的药物中的应用。
本发明的一些方案中，上述的应用，其特征在于，所述药物是用于类风湿性关节炎治疗的药物。
技术效果
本发明式(Ⅰ)化合物具有良好的体内给药药效且其各晶型稳定、受光热湿度影响小、溶解性高，成药前景广阔。
定义和说明
除非另有说明，本文所用的下列术语和短语旨在含有下列含义。一个特定的短语或术语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。当本文出现商品名时，旨在指代其对应的商品或其活性成分。
本发明的中间体化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。
本发明具体实施方式的化学反应是在合适的溶剂中完成的，所述的溶剂须适合于本发明的化学变化及其所需的试剂和物料。为了获得本发明的化合物，有时需要本领域技术人员在已有实施方式的基础上对合成步骤或者反应流程进行修改或选择。
下面会通过实施例具体描述本发明，这些实施例并不意味着对本发明的任何限制。
本发明所使用的所有溶剂是市售的，无需进一步纯化即可使用。
本发明所使用的溶剂可经市售获得。本发明采用下述缩略词：DCM代表二氯甲烷；DMF代表N，N-二甲基甲酰胺；DMSO代表二甲亚砜；EtOH代表乙醇；MeOH代表甲醇；TFA代表三氟乙酸；TsOH代表对甲苯磺酸；mp代表熔点；EtSO 3H代表乙磺酸；MeSO 3H代表甲磺酸；ATP代表三磷酸腺苷；HEPES代表4-羟乙基哌嗪乙磺酸；EGTA代表乙二醇双(2-氨基乙基醚)四乙酸；MgCl 2代表二氯化镁；MnCl 2代表二氯化锰；DTT代表二硫苏糖醇；DCC代表二环己基碳二亚胺；DMAP代表4-二甲氨基吡啶；EA代表乙酸乙酯；LiHMDS代表六甲基二硅基胺基锂；Pd(dppf)Cl 2.CH 2Cl 2代表[1,1'-双(二苯基膦基)二茂铁]二氯化钯的二氯甲烷络合物；EDCI代表碳化二亚胺；HOBt代表1-羟基苯并三唑。
XRPD、DSC、TGA的具体方法(包括设备型号以及各参数)
本发明粉末X-射线衍射(X-ray powder diffractometer,XRPD)方法
大约10～20mg样品用于XRPD检测
详细的XRPD参数如下：
光管：Cu,Cu:K-Alpha
Figure PCTCN2021077228-appb-000008
光管电压：40kV，光管电流：40mA
发散狭缝：0.60mm
探测器狭缝：10.50mm
防散射狭缝：7.10mm
扫描范围：3-40deg
扫描速率：10deg/min
样品盘转速：15rpm/0rpm
本发明差示量热扫描仪(DSC)方法
取样品(0.5～1mg)置于DSC铝锅内进行测试，在50mL/min N 2条件下，以10℃/min的升温速率，加热样品从30℃到250℃。
本发明热重分析仪(TGA)方法
取样品(2～5mg)置于TGA铂金锅内进行测试，在25mL/min N2条件下，以10℃/min的升温速率，加热样品从室温到300℃或失重20％。
本发明液相色谱分析方法(HPLC)
含量测试分析HPLC方法
Figure PCTCN2021077228-appb-000009
附图说明
图1：A晶型的XRPD图谱。
图2：A晶型的DSC图谱。
图3：B晶型的XRPD图谱。
图4：B晶型的DSC图谱。
图5：B晶型的TGA图谱。
图6：C晶型的XRPD图谱。
图7：C晶型的DSC图谱。
图8：D晶型的XRPD图谱。
具体实施方式
为了更好的理解本申请的内容，下面结合具体实施例来做进一步的说明但具体的实施方式并不是对本申请的内容所做的限制。
实施例1：式(Ⅰ)化合物的制备
Figure PCTCN2021077228-appb-000010
步骤1:在-78℃下，向溶有化合物1-1(10.2g,42.6mmol)的THF(150mL)溶液中滴加LiHMDS(1M,51.2mL)。该反应液在-78℃下搅拌1小时后将1,1,1-三氟-N-苯基-N-(三氟甲基磺酰基)甲磺酰胺(16.7g,46.9mmol)的THF(150mL)溶液加入到该反应液中,然后在15℃下搅拌12小时。用250mL饱和氯化铵淬灭反应，用200mL水稀释，然后用乙酸乙酯(200mL*3)萃取。合并有机相，经饱和食盐水洗涤，硫酸钠干燥，过滤浓缩得到化合物1-2。粗品未经纯化直接用于下一步反应。
1H NMR(400MHz,CDCl 3)δ5.63(br s,1H),3.50-3.65(m,4H),2.34(br s,4H),1.88(br t,J＝5.90Hz,2H),1.37(s,9H)。
步骤2：向溶有化合物1-2(16g,43.1mmol)和联硼酸频那醇酯(12.0g,47.4mmol)的DMF(100mL)溶液中加入醋酸钾(12.7g,129.3mmol)和Pd(dppf)Cl 2.CH 2Cl 2(3.5g,4.3 mmol)，用氮气置换3次并保持在氮气氛围中70℃下搅拌3小时。将反应液分散在300mL水和400mL乙酸乙酯混合液中。将有机相分离并经饱和食盐水洗涤，硫酸钠干燥，过滤浓缩得到粗品。粗品经硅胶色谱柱法纯化得到化合物1-3。
1H NMR(400MHz,CDCl 3)δ6.46(br s,1H),3.71-3.53(m,4H),2.31(br d,J＝3.0Hz,2H),2.24-2.16(m,2H),1.74(t,J＝6.3Hz,2H),1.44(s,9H),1.26(s,12H)。
步骤3：在氮气氛围中，向溶有化合物1-3(3.5g,10.0mmol)和N-(5-溴-[1,2,4]三唑并[1,5-a]吡啶-2-基)环丙烷甲酰胺(2.6g,9.1mmol)的二氧六环(60mL)和水(15mL)的溶液中加入碳酸钾(3.8g,27.3mmol)和Pd(dppf)Cl 2.CH 2Cl 2(744mg,911.0μmol)。该反应液在90℃下搅拌3小时。将反应液浓缩，所得粗品经柱色谱分离纯化得到化合物1-4。LCMS(ESI)m/z:424.3[M+H] +。
步骤4：向溶有化合物1-4(3.5g,8.2mmol)的二氯甲烷(10mL)溶液中加入盐酸/乙酸乙酯(4M,30mL)，该反应液在25℃下搅拌0.5小时。固体析出，过滤并干燥，得到化合物1-5(3.3g盐酸盐，粗品)，未经纯化，直接用于下一步反应。
LCMS(ESI)m/z:324.1[M+H] +。
步骤5：在氮气氛围中，向溶有化合物1-5(3.0g,8.34mmol,盐酸盐)的甲醇(100mL)溶液中加入Pd/C(1g,10％)。该悬浊液用氢气置换3次，然后在氢气氛围(30psi)30℃下搅拌12小时。将反应液过滤，浓缩得到化合物1-6(3g盐酸盐，粗品)，未经纯化，直接用于下一步反应。
LCMS(ESI)m/z:326.2[M+H] +。
步骤6：将化合物1-6(0.87g,2.40mmol,盐酸盐)溶解在N,N-二甲基甲酰胺(10mL)中，加入HOBt(487mg,3.6mmol,)和EDCI(691mg,3.6mmol)，之后加入(1S)-2,2-二氟环丙基甲酸(323mg,2.6mmol)和二异丙基乙胺(621mg,4.8mmol)，反应液在15℃反应12小时。反应液减压浓缩，残余物经由制备型HPLC(中性体系)得到式(Ⅰ)化合物。
1H NMR(400MHz,CD 3OD)δ7.32-7.73(m,2H),6.95(br s,1H),3.62-4.22(m,4H),3.45(br s,1H),3.18-3.37(m,1H),2.61(br s,1H),1.45-2.27(m,10H),0.78-1.17(m,4H)。LCMS(ESI)m/z:430.0[M+H] +。
实施例2：各晶型的制备方法
称取50mg式(Ⅰ)化合物加入到2.0mL玻璃小瓶中，加入0.4mL甲醇和水的溶剂混合物(体积比1:1)，所得悬浊液，加入磁子后，将上述样品置于加热磁力搅拌器上(40℃)进行搅拌。搅拌100小时后混悬的样品离心后置于35℃真空干燥箱过夜。干燥后的样品 经检测XRPD(如图1所示)，判断为式(Ⅰ)化合物的A晶型，同时检测DSC(如图2所示)。
称取大约50mg式(Ⅰ)化合物加入到2.0mL玻璃小瓶中，加入0.4mL乙酸乙酯。加入磁子后，将上述样品置于加热磁力搅拌器上(40℃)进行搅拌。搅拌100小时后混悬的样品离心后置于35℃真空干燥箱过夜。干燥后的样品经检测XRPD(如图3所示)，判断为式(Ⅰ)化合物的B晶型，同时检测DSC(如图4所示)和TGA(如图5所示)。
式(Ⅰ)化合物的A晶型加热至170℃，经检测XRPD(图6所示)，晶型发生改变，得到的新晶型为式(Ⅰ)化合物的C晶型，同时检测DSC(如图7所示)。
称取大约50mg式(Ⅰ)化合物加入到2.0mL玻璃小瓶中，加入0.4mL乙醇和水的溶剂混合物(体积比1:1)所得悬浊液。加入磁子后，将上述样品置于加热磁力搅拌器上(40℃)进行搅拌。搅拌100小时后混悬的样品离心后置于35℃真空干燥箱过夜。干燥后的样品经检测XRPD(如图8所示)，判断为式(Ⅰ)化合物的D晶型。
实施例3：式(Ⅰ)化合物的B晶型的固体稳定性研究
准确称重B晶型约5mg置于干燥洁净的玻璃瓶中，摊成薄薄一层，作为正式供试样品，放置于影响因素试验条件下(60℃，92.5％RH)和加速条件下(40℃/75％RH和60℃/75％RH)，其样品为完全暴露放样，用铝箔纸盖上，扎上小孔。在5天，10天取样分析。光照(可见光1200000Lux，紫外200W)条件下放置的样品为室温完全暴露放样。
实验结果表明，在影响因素条件(高温-60℃、高湿-92.5％RH、光照)和加速条件(40℃/75％RH和60℃/75％RH)下晶型均未发生变化。
实施例4：式(Ⅰ)化合物的B晶型的生物媒介溶解度研究
1 B晶型的生物媒介溶解度实验
分别称取样品B晶型约2mg于样品瓶中，然后向其分别加入各1.0mL不同的溶媒【纯水，SGF(模拟胃液)，FaSSIF(禁食状态模拟肠液)，FeSSIF(进食状态模拟肠液)】，振荡摇匀。置于恒温振荡仪上于37℃条件下振荡。振荡24小时后离心分离，所得上清液测试其溶解度大小。将上清液稀释(稀释剂ACN/H 2O(1/1))一定倍数后(化合物溶解度较小，上清液除SGF均稀释两倍，SGF稀释10倍)，用HPLC测定其浓度。
2稀释剂及流动相的配制
稀释剂：乙腈：水1:1。流动相A：0.1％TFA水溶液，例：移取1mL的TFA于1L的纯水中，混合均匀，超声脱气。流动相B：100％乙腈。
3对照品和样品液的配制
STD溶液配制：将B晶型作为对照品。分别称取5mg左右的对照品于玻璃瓶中， 用10mL稀释剂使其溶解，超声10分钟左右使样品充分溶解，冷却至室温摇匀。平行配制两份，标记为相应的STD1，STD2。将对应的STD1用稀释剂稀释10、100、1000和2000倍，做标准曲线进行测试。
样品液的配制：将上清液稀释(稀释剂ACN/H 2O(1/1))一定倍数后(化合物溶解度较小，上清液除SGF均稀释两倍，SGF稀释10倍)，摇匀，置于1.5mL液相小瓶中待测，用HPLC测定其浓度。
4生物媒介溶解度结果
表5 B晶型的生物媒介溶解度结果
Figure PCTCN2021077228-appb-000011
实验结论：B晶型的在模拟的生物媒介溶解度良好，这一特性有利于获得良好的体内生物利用度。
生物活性测试
实验例1：JAK1,JAK2,JAK3,TYK2激酶体外活性测试
实验材料
重组人源JAK1、JAK2、JAK3、Tyk2蛋白酶、主要仪器及试剂均由英国的Eurofins公司提供
实验方法
JAK2,JAK3和TYK2稀释：20mM 3-(N-吗啉)丙磺酸(MOPS),1mM EDTA,0.01％Brij-35.5％甘油,0.1％β-巯基乙醇,1mg/mL BSA；JAK1稀释：20mM TRIS,0.2mM EDTA,0.1％β-巯基乙醇,0.01％Brij-35.5％甘油。将所有化合物制备成100％的DMSO溶液并达到最终测定浓度50倍。测试化合物进行3倍浓度梯度稀释，终浓度为10μM到0.001μM共9个浓度，DMSO在检测反应中的含量为2％。将该化合物的工作储备液作为反应的第一组分添加到测定孔中，然后按照下面测定详述的方案加入其余组分。
JAK1(h)酶反应
JAK1(h)与20mM Tris/HCl pH7.5,0.2mM EDTA，500μM MGEEPLYWSFPAKKK，10mM乙酸镁和[γ- 33P]-ATP(根据需要制定活性和浓度)一起孵育。添加Mg/ATP混合物开始反应，在室温下孵育40分钟后，加入0.5％浓度的磷酸终止反应。然后将10μL反应物点在P30滤垫上并于4分钟内用0.425％磷酸洗涤三次和甲醇洗涤一次，干燥、闪烁计数。
JAK2(h)酶反应
JAK2(h)与8mM MOPS pH 7.0,0.2mM EDTA,100μM KTFCGTPEYLAPEVRREPRILSEEEQEMFRDFDYIADWC,10mM乙酸镁和[γ- 33P]-ATP(根据需要制定活性和浓度)一起孵育。添加Mg/ATP混合物开始反应，在室温下孵育40分钟后，加入0.5％浓度的磷酸终止反应。然后将10μL反应物点在P30滤垫上并于4分钟内用0.425％磷酸洗涤三次和甲醇洗涤一次，干燥、闪烁计数。
JAK3(h)酶反应
JAK3(h)与8mM MOPS pH 7.0,0.2mM EDTA,500μM GGEEEEYFELVKKKK,10mM乙酸镁和[γ- 33P]-ATP(根据需要制定活性和浓度)一起孵育。添加Mg/ATP混合物开始反应，在室温下孵育40分钟后，加入0.5％浓度的磷酸终止反应。然后将10μL反应物点在P30滤垫上并于4分钟内用0.425％磷酸洗涤三次和甲醇洗涤一次，干燥、闪烁计数。
TYK2(h)酶反应
TYK2(h)与8mM MOPS pH 7.0,0.2mM EDTA,250μM GGMEDIYFEFMGGKKK,10mM乙酸镁和[γ- 33P]-ATP(根据需要制定活性和浓度)一起孵育。添加Mg/ATP混合物开始反应，在室温下孵育40分钟后，加入0.5％浓度的磷酸终止反应。然后将10μL反应物点在P30滤垫上并于4分钟内用0.425％磷酸洗涤三次和甲醇洗涤一次，干燥、闪烁计数。
数据分析
IC 50结果由IDBS公司的XLFIT5(205公式)进行分析得到，具体见表6。
表6.本发明化合物体外筛选试验结果
Figure PCTCN2021077228-appb-000012
结论：本发明的式(Ⅰ)化合物在激酶4个亚型JAK1、JAK2、JAk3和TYK2的体外活性测试中展现了对JAK1和/或TYK2的良好的选择性抑制。
实验例2：药代动力学(PK)试验
将试验化合物溶解后得到的澄清溶液分别经尾静脉注射和灌胃给予雄性小鼠(C57BL/6)或大鼠(SD)体内(过夜禁食，7～8周龄)。给予受试化合物后，静脉注射组(2mg/kg)在0.117,0.333,1,2,4,7和24小时，灌胃组(15mg/kg)在0.25,0.5,1,2,4,8和24小时，分别从下颌静脉采血并离心后获得血浆。采用LC-MS/MS法测定血药浓度，使用WinNonlin TMVersion 6.3药动学软件，以非房室模型线性对数梯形法计算相关药代 动力学参数。测试结果如下：
表7式(Ⅰ)化合物在小鼠中的PK测试结果
PK参数 结果
T 1/2(hr) 1.61
C max(nM) 5105
AUC 0-inf(nM.hr) 9917
Bioavailability(％) a 38.1％
注：T 1/2：半衰期；C max：达峰浓度；
AUC 0-inf：从0时间到外推至无穷大时的血浆浓度-时间曲线下面积；
Bioavailability：生物利用度。
结论：本发明的式(Ⅰ)化合物在小鼠中都有良好的口服生物利用度，较高的暴露量，有利于产生良好的体内药效。
实验例3：在胶原诱导的小鼠关节炎(CIA)中的体内药效研究
实验目的：
类风湿性关节炎(RA)是一类多发的自身免疫性疾病，全球发病率约为1％左右，是由于自身免疫反应而导致关节部位发炎、损伤以及畸形，严重时会引起全身性系统性的炎症反应。研发治疗RA的药物，有助于缓解类风湿性关节炎症状，改善患者的生存质量。胶原诱导的小鼠关节炎模型，是经常用于评价药物治疗RA药效的动物模型，其发病机理和症状与RA疾病均有比较明显的相关性。模型通过注射II型胶原蛋白从而激活B细胞，T细胞对骨胶原蛋白的反应性，被激活的B细胞和T细胞进入关节部位引起关节的损伤，从而引发一系列类似于人类风湿性关节炎的症状。在临床前评价药物治疗类风湿性关节炎候选化合物的过程中，胶原诱导的小鼠关节炎常常被用来评价其有效性。
本次实验目的是考察式(Ⅰ)化合物和参考化合物Filgotinib在胶原诱导的小鼠关节炎上的治疗效果，从而为之后的临床研究提供临床前药效学相关信息。
实验方法：
1.二型胶原/完全弗氏佐剂免疫
乙酸的配制：稀释2N的乙酸至100mM，用0.22微米滤膜过滤后，4℃保存。
牛二型胶原溶液：将牛二型胶原(CII)溶解于100mM的乙酸溶液中，并置于4℃过夜保存。胶原蛋白的终浓度为8mg/ml。
乳剂的制备：将过夜保存的CII溶液与等体积的完全弗氏佐剂混合，使用高速匀浆机，在冰上以30,000转每分钟匀浆大约60分钟，直至溶液形成稳定的乳剂。
2.关节炎的诱导：
将小鼠随机分配到不同的治疗组。第一次免疫当天记为第0天，随后的天数依序标注。
DBA/1小鼠经异氟烷麻醉后，在尾部皮下(距尾根部2-3厘米)注射50微升的制备好的胶原乳剂(包含200微克CII)。第21天，尾部同法注射相同体积胶原乳剂。正常组的小鼠无需免疫。
3.给药和剂量设计
第28天，当平均临床评分达到1分左右时，挑选发病程度适中的小鼠50只，按照体重和评分，重新随机分组到5个治疗组，每组8只小鼠。
地塞米松(Dexamethasone，Dex.)作为衡量模型成功建立与否的参照药物，采用0.3mg/kg剂量(CIA模型中普遍使用剂量)；另外根据本实验前期预实验的结果确定了受试化合物和参考化合物Filgotinib的相关剂量设计如表8-1所示：第一组为正常小鼠，不做任何处理；第二组给予仅含溶媒的空白组；第三组给予地塞米松剂量为0.3mg/kg；第六组、第七组和第八组剂量分别为15mg/kg，15mg/kg。每天给药两次，共持续14天。
表8-1.分组及剂量设计
Figure PCTCN2021077228-appb-000013
注：PO：口服；bid：每日两次；qd：每日一次。
4.关节炎发病指标测定
临床观察：从免疫前7天至免疫后第21天，每日观察DBA/1小鼠的基本健康状况及体重变化(一周记录一次)。第22天之后，每日观察小鼠健康状况，发病情况，及体重变化(一周至少记录三次)，直至实验结束。
临床评分：增强免疫后，每天观察小鼠发病情况。当小鼠开始发病之后(出现关节炎的临床症状)，根据病变的不同程度(红肿，关节变形)按照0-4分的标准进行评分，每个肢体的最高评分为4分，每只动物最高评分为16分。评分标准如表8-2。至少每周评 分三次。
表8-2.关节炎临床评分标准
Figure PCTCN2021077228-appb-000014
5.统计学处理
实验数据应用平均数±标准误表示(Mean±SEM)，曲线下面积(AUC)用单因素方差分析(One-way ANOVA)，p<0.05认为有显著性差异。
实验结果：
1.临床评分及发病率：
第一次免疫后第28天(第二次免疫后第7天)，小鼠开始出现关节炎临床症状。第28天开始给药。实验详细结果如表8-3所示：溶剂对照组的平均临床评分逐渐升高，至第41天达到5.8分，提示胶原诱导的关节炎模型的成功建立；式(Ⅰ)化合物和Filgotinib在同剂量15mg/kg均可显著降低实验终点(第41天)关节炎小鼠的临床评分，该剂量下式(Ⅰ)化合物和Filgotinib临床平均评分下降至1.5和5.6分(见表8-3数值)；可见式(Ⅰ)化合物在15mg/kg可有效的减轻胶原诱导的关节炎。地塞米松0.3mg/kg(G3组)治疗可显著抑制胶原诱导的关节炎临床评分，从第27天开始起临床评分维持在0.3分左右并在第31天时(临床评分降至0，见表8-3数值)。
表8-3*本发明的平均临床评分
Figure PCTCN2021077228-appb-000015
Figure PCTCN2021077228-appb-000016
*注：平均临床评分±标准误
通过分析每组每只动物的临床评分曲线，计算曲线下面积(AUC)，通过组间AUC平均值，计算各给药组相对于溶剂对照组的抑制率，详细结果如表8-4：式(Ⅰ)化合物和Filgotinib在同剂量15mg/kg下均能够降低关节炎动物的临床评分AUC，抑制率分别为59.9％和18.7％，其中地塞米松也可显著性降低关节炎动物的临床评分，抑制率为97.3％。
表8-4*发病曲线下面积
Figure PCTCN2021077228-appb-000017
*注：曲线下面积的数值是根据动物的临床用Graphpad
Figure PCTCN2021077228-appb-000018
软件拟合出来的,分别是每一组每一只小鼠给药期的发病曲线下面积。抑制率＝(空白组曲线下面积的平均值-给药组的曲线下面积的平均值)/空白组曲线下面积的平均值
各治疗因素还可影响胶原诱导的关节炎发生率。实验详细结果如表8-5所示：式(Ⅰ)化合物发病率在第29天时至63％并稳定至实验结束(具体数值见表8-5)；Filgotinib组发病率在初次给药后呈降低，后逐渐回升直至最后一次给药后升到100％。溶剂对照组在免疫后第34天关节炎发生率达到并维持在100％；阳性对照地塞米松0.3mg/kg组的发病率从给药后就开始降低并于第31天降低至0％。
表8-5*本发明发病率
Figure PCTCN2021077228-appb-000019
Figure PCTCN2021077228-appb-000020
*注：发病率＝每组发病的动物数量/每组的动物总数*100％
2.体重
实验详细结果如表8-6所示：和正常组比较，小鼠免疫造模后体重均有降低，各给药组体重均在第28天至第34天下降，随后体重又开始缓慢恢复；其中地塞米松组体重下降幅度最大，但和其他组相比不存在显著性差异；式(Ⅰ)化合物和Filgotinib之间也不存在显著性差异，且体重变化趋势基本相同(见表8-6具体数值)，提示该化合物对小鼠的体重没有太大的影响。
表8-6*本发明平均体重
Figure PCTCN2021077228-appb-000021
*注：平均体重±标准误
结论：在胶原诱导的小鼠关节炎(CIA)模型中，式(Ⅰ)化合物展现出良好的疾病治疗效果，且对小鼠的体重没有明显的影响，并且同等剂量下优于Filgotinib的小鼠体内药效。
实验例4：佐剂诱导的大鼠关节炎(AIA)中的体内药效研究
实验目的：
佐剂诱导的关节炎(Adjuvant induced Arthritis，AIA)大鼠模型是在类风湿性关节炎疾病研究和新药开发中常用的动物模型之一，其发病机理和临床症状均与人类风湿性关节炎疾病类似。模型通过足垫注射结核分枝杆菌诱导具有骨关节损伤功能的免疫细胞和抗体引起了系统性的反应，具体表现为关节肿胀，骨溶解，滑膜损伤等类似人类风湿性关节炎的症状。本次实验旨在评价式(Ⅰ)化合物在佐剂诱导的关节炎大鼠模型上的治疗效果，以地塞米松和Filgotinib为参照化合物。本次实验共8组，分别为正常组(Normal组)，溶剂对照组(Vehicle组)，式(Ⅰ)化合物1mg/kg BID，3mg/kg BID，10mg/kg BID和30mg/kg BID剂量组，阳性药地塞米松0.3mg/kg QD组以及参照化合物Filgotinib 30mg/kg BID剂量组。除正常组外，所有大鼠于第0天，在左足皮下注射弗氏完全佐剂诱导关节炎。按照实验方案，于第13天根据体重及评分分组，并开始给药，持续给药14天。实验过程中监测大鼠的体重、足体积(第13天后，每周测量三次)以及临床评分。实验终点采集大鼠右后足进行苏木精—伊红染色(hematoxylin-eosin staining，HE)染色病理评分分析。
实验方法
1.关节炎模型
佐剂配制：称取结核分枝杆菌H37Ra 100mg，研磨约5分钟，加入3mL石蜡油溶解粉末，转移到棕色配药瓶中，然后分别用3mL、4mL石蜡油清洗研钵2次，并全部转移到棕色配药瓶中，终浓度为10mg/mL。超声破碎，冰水混合物中超声约30分钟。
2.关节炎的诱导
将配置好的佐剂震荡混匀，用1mL的玻璃注射器(20G针头)抽取，再换成25G针头，排除气泡。采用异氟烷麻醉大鼠，免疫每只大鼠前上下颠倒注射器，充分混匀结核分枝杆菌。待麻醉后在大鼠左脚脚掌皮下注射0.1mL佐剂。正常组大鼠脚掌皮下注射石蜡油0.1mL。注射佐剂当天为第0天。
3.给药
在第13天，所有动物均显现足部红斑或红肿等关节炎症状，按评分、足体积和体重进行分层随机分组。分组情况见表9-1。将70只大鼠分成7组，每组10只，正常组5只。根据表9-1，每组的给药剂量如下表。灌胃给药体积为5mL/kg。化合物给药每天两次，共持续14天。
表9-1分组及剂量设计
Figure PCTCN2021077228-appb-000022
Figure PCTCN2021077228-appb-000023
4.关节炎发病指标测定
体重：从第13天到第27天每周称重三次。
足体积：免疫前测量一次，从第13天到第27天每周测量三次。
评分：从第13天到第27天每周评分三次。根据病变的不同程度(红肿，关节变形)按照0-4分的标准进行评分，每个肢体的最高评分为4分，每只动物最高评分为12分(注射侧左后肢除外)。评分标准如表9-2。
表9-2.关节炎临床评分标准
分值 临床症状
0 无红斑和红肿
1 近跗骨附近或踝关节或跖骨出现红斑或轻度红肿或有一个脚趾有红斑和红肿
2 踝关节和跖骨轻微红斑和肿胀，有两个或两个以上脚趾有红肿和红斑
3 踝、腕关节和跖骨中度红斑和肿胀
4 踝、腕关节，跖骨和脚趾全部严重红肿
5.病理分析
第27天，安乐死大鼠。采血完成后，取大鼠右后脚，用10％福尔马林溶液浸泡，用甲酸溶液脱钙，石蜡包埋，切片，HE染色，显微镜观察。从炎症细胞浸润、血管翳生成、软骨损伤和骨吸收等四个方面对关节的损伤程度进行评价，并按照0-4分的标准进行评分。各项评分标准如下(表9-3)：
表9-3.关节炎病理学评分标准
Figure PCTCN2021077228-appb-000024
Figure PCTCN2021077228-appb-000025
6.统计学处理
实验数据应用平均数±标准误(Mean±SEM)表示，体重、临床评分、病理学评分用单因素方差分析(One-way ANOVA)，p<0.05认为有显著性差异。
实验结果
1.临床评分
本实验评价了式(Ⅰ)化合物在大鼠关节炎(AIA)模型中对临床评分的改善作用，以地塞米松和Filgotinib为参照。佐剂免疫后第6天大鼠开始出现关节炎症状。第13天开始给药，溶剂对照组的平均临床评分逐渐升高，实验结果显示，溶剂对照组的平均临床评分于第24天达到峰值，并稳定在约8分左右，标志着AIA模型的成功建立(表9-4)。
实验终点(第27天)时，式(Ⅰ)化合物在1，3，10，30mg/kg四个剂量下，对关节炎大鼠临床评分均有极显著性抑制(与溶剂对照组相比，p值均<0.0001)，将关节炎大鼠临床评分分别降低至5.4，3.9，3.2和2.7，且呈剂量依赖性(高剂量组和低剂量组相比， p<0.0001)。其中，式(Ⅰ)化合物30mg/kg的效果最为明显(从17天开始，与溶剂对照组比较有极显著性差异，p<0.0001)。该组的平均关节炎临床评分由第13天的峰值6.0分，降至实验终点第27天时的2.7分(表9-4)。参照化合物Filgotinib 30mg/kg BID在实验终点第27天评分降至5.1分，显著低于溶剂对照组(p<0.001)但显著高于式(Ⅰ)化合物30mg/kg BID(p<0.001)。式(Ⅰ)化合物在关节炎临床评分上的改善作用显著优于同剂量下Filgotinib的作用。
阳性对照地塞米松治疗组的平均临床评分在第13天之后，达到最高值6.0分，给药之后，临床评分持续下降，降至实验终点第27天时的2.7分，从17天开始，与溶剂对照组比较，有极显著性差异(表9-4)。
2足体积
本实验评价了式(Ⅰ)化合物在大鼠关节炎(AIA)模型中对足体积的改善作用，以地塞米松和Filgotinib为参照。溶剂对照组动物的平均足体积从13天的1.9mL稳定升高至第27天实验终点时的2.9mL，标志着AIA模型的成功建立(表9-5)。实验终点时，式(Ⅰ)化合物在1，3，10和30mg/kg剂量下，均能极显著抑制关节炎大鼠足体积的升高(与溶剂对照组比较，p值均<0.0001)，关节炎大鼠足体积均值分别减小至1.91mL，1.59mL，和1.26mL和1.21mL，且呈剂量依赖性(高剂量组与低剂量组之间相比，p<0.0001)。参照化合物Filgotinib 30mg/kg BID在实验终点第27天足体积降至1.91分，显著低于溶剂对照组(p<0.0001)但显著高于式(Ⅰ)化合物30mg/kg BID(p<0/0001)。式(Ⅰ)化合物对大鼠足体积的改善作用明显优于同剂量下Filgotinib的作用。阳性对照地塞米松治疗组也很好的抑制了平均足体积的升高，在给药之后足体积稳步下降，至实验终点，稳定在1.21mL，第17天起，与溶剂对照组比较有极显著性差异，p<0.0001(表9-5)
3体重
与正常组比较，大鼠免疫造模后体重均有降低，第13天开始给药以后，各给药组体重相比溶剂对照组均缓慢持续上升，而阳性对照地塞米松组体重恢复的较慢，此结果提示大鼠对Filgotinib和式(Ⅰ)化合物耐受性良好。式(Ⅰ)化合物30mg/kg组体重上升最快，4个剂量升高体重的趋势呈剂量依赖关系(表9-6)。
4组织病理学检测结果
溶剂对照组的关节炎大鼠，病理评分总分为16±0.00，式(Ⅰ)化合物在1mg/kg剂量下，评分降低至13.3±0.44(与溶剂对照组相比，P＝0.09无统计学差异)，抑制率为16.9％；而3mg/kg，10mg/kg和30mg/kg剂量下可显著性降低关节炎大鼠的病理评分，分别降低至11.3±1.64，4.4±1.16和1.6±0.47，p值分别为0.014，<0.0001和<0.0001，抑制率29.4％， 72.5％和90％。参考化合物Filgotinib 30mg/kg病理总评分为15.2±0.49，抑制率为5％，与溶剂组相比未见显著性差异。式(Ⅰ)化合物在同剂量下(30mg/kg)病理总评分显著低于Filgotinib(p<0.0001)。对照化合物地塞米松0.3mg/kg剂量下极显著降低关节炎大鼠的病理评分至4.4±0.8，p值<0.0001，抑制率为72.5％(表9-7)。
Figure PCTCN2021077228-appb-000026
Figure PCTCN2021077228-appb-000027
Figure PCTCN2021077228-appb-000028
表9-7.病理评分
Figure PCTCN2021077228-appb-000029
结论：溶剂对照组的大鼠出现关节炎的临床症状，并持续加重。与溶剂对照组相比，式(Ⅰ)化合物(1，3，10，30mg/kg)，Filgotinib(30mg/kg)以及地塞米松(0.3mg/kg)显示出对佐剂诱导的关节炎有明显的抑制作用，表现为发病时间推迟、临床症状和病理变化明显减轻，式(Ⅰ)化合物对佐剂诱导的关节炎模型的治疗效果呈剂量依赖性。以上实验结果表明，式(Ⅰ)化合物对佐剂诱导的大鼠关节炎有着明显的治疗效果且效果优于Filgotinib。

Claims (26)

  1. 式(Ⅰ)化合物的A晶型，其X射线粉末衍射(XRPD)图谱在下列2θ角处具有特征衍射峰：6.91±0.20°、12.21±0.20°和19.06±0.20°。
    Figure PCTCN2021077228-appb-100001
  2. 根据权利要求1所述的A晶型，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：6.91±0.20°、12.21±0.20°、13.69±0.20°、19.06±0.20°、19.86±0.20°、20.59±0.20°、22.06±0.20°和27.52±0.20°。
  3. 根据权利要求2所述的A晶型，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：6.91±0.20°、10.34±0.20°、12.21±0.20°、13.69±0.20°、18.11±0.20°、19.06±0.20°、19.86±0.20°、20.59±0.20°、22.06±0.20°和27.52±0.20°。
  4. 根据权利要求3所述的A晶型，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：6.91±0.20°、10.34±0.20°、12.21±0.20°、13.69±0.20°、17.44±0.20°、18.11±0.20°、19.06±0.20°、19.86±0.20°、20.59±0.20°、22.06±0.20°、24.46±0.20°和27.52±0.20°。
  5. 根据权利要求4所述的A晶型，其XRPD图谱如附图1所示。
  6. 根据权利要求1-5任意一项所述的A晶型，其差示扫描量热曲线在152.19±3℃和216.79±3℃各有一个吸热峰的峰值；在161.50±3℃有一个放热峰的峰值。
  7. 根据权利要求6所述的A晶型，其DSC图谱如附图2所示。
  8. 式(Ⅰ)化合物的B晶型，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：5.13±0.20°、19.14±0.20°和21.18±0.20°。
  9. 根据权利要求8所述的B晶型，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：5.13±0.20°、7.34±0.20°、10.14±0.20°、10.56±0.20°、11.72±0.20°、16.67±0.20°、19.14±0.20°和21.18±0.20°。
  10. 根据权利要求9所述的B晶型，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：5.13±0.20°、7.34±0.20°、10.14±0.20°、10.56±0.20°、11.72±0.20°、16.67±0.20°、19.14±0.20°、21.18±0.20°和21.78±0.20°。
  11. 根据权利要求10所述的B晶型，其XRPD图谱如附图3所示。
  12. 根据权利要求8-11任意一项所述的B晶型，其差示扫描量热曲线在193.99±3℃和216.93±3℃各有一个吸热峰的峰值；在200.10±3℃有一个放热峰的峰值。
  13. 根据权利要求12所述的B晶型，其DSC图谱如附图4所示。
  14. 根据权利要求8-11任意一项所述的B晶型，其热重分析曲线(TGA)在120±3℃时失重达0.535％。
  15. 根据权利要求14所述的B晶型，其TGA图谱如图5所示。
  16. 式(Ⅰ)化合物的C晶型，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：8.92±0.20°、18.66±0.20°和20.26±0.20°。
  17. 根据权利要求14所述的C晶型，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：5.76±0.20°、8.92±0.20°、11.50±0.20°、16.35±0.20°、18.66±0.20°、19.17±0.20°、20.26±0.20°和24.79±0.20°。
  18. 根据权利要求17所述的C晶型，其XRPD图谱如附图6所示。
  19. 根据权利要求16-18任意一项所述的C晶型，其差示扫描量热曲线在215.48℃有一个吸热峰的起始点。
  20. 根据权利要求19所述的C晶型，其DSC图谱如附图7所示。
  21. 式(Ⅰ)化合物的D晶型，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：7.12±0.20°、20.54±0.20°和21.42±0.20°。
  22. 根据权利要求21所述的D晶型，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：7.12±0.20°、12.45±0.20°、14.64±0.20°、18.31±0.20°、20.54±0.20°、21.42±0.20°和28.72±0.20°。
  23. 根据权利要求22所述的D晶型，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：7.12±0.20°、10.28±0.20°、12.45±0.20°、14.64±0.20°、17.50±0.20°、18.31±0.20°、20.54±0.20°、21.42±0.20°和28.72±0.20°。
  24. 根据权利要求23所述的D晶型，其XRPD图谱如附图8所示。
  25. 根据权利要求1-7任意一项所述的A晶型、权利要求8-15任意一项所述的B晶型、权利要求16-20任意一项所述的C晶型和权利要求21-24任意一项所述的D晶型在制备治疗JAK1和/或TYK2相关疾病的药物中的应用。
  26. 根据权利要求25所述的应用，其特征在于，所述药物是用于类风湿性关节炎治疗的药物。
PCT/CN2021/077228 2020-02-21 2021-02-22 Jak抑制剂的晶型及其应用 WO2021164786A1 (zh)

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CA3166743A CA3166743A1 (en) 2020-02-21 2021-02-22 Crystalline form of jak inhibitor and application thereof
US17/796,613 US20230091250A1 (en) 2020-02-21 2021-02-22 Crystalline form of jak inhibitor and application thereof
KR1020227032610A KR20220157398A (ko) 2020-02-21 2021-02-22 Jak 억제제의 결정형 및 그의 적용
EP21757247.8A EP4108664A4 (en) 2020-02-21 2021-02-22 CRYSTALLINE FORM OF JAK INHIBITOR AND USE THEREOF
JP2022549366A JP2023514291A (ja) 2020-02-21 2021-02-22 Ｊａｋ阻害剤の結晶形およびその使用

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