WO2021058001A1 - N杂五元环化合物的晶型及其应用 - Google Patents

N杂五元环化合物的晶型及其应用 Download PDF

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WO2021058001A1
WO2021058001A1 PCT/CN2020/118426 CN2020118426W WO2021058001A1 WO 2021058001 A1 WO2021058001 A1 WO 2021058001A1 CN 2020118426 W CN2020118426 W CN 2020118426W WO 2021058001 A1 WO2021058001 A1 WO 2021058001A1
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compound
crystal
formula
application
present application
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PCT/CN2020/118426
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English (en)
French (fr)
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王普军
郭猛
胡明通
吴杰
敖汪伟
张寅生
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正大天晴药业集团股份有限公司
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Priority to US17/763,114 priority Critical patent/US20220332684A1/en
Priority to CN202080066483.9A priority patent/CN114466837A/zh
Publication of WO2021058001A1 publication Critical patent/WO2021058001A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/30—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
    • C07D207/34—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • 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

  • This application relates to the crystal form of the N-hetero five-membered ring compound, in particular to the crystal form of the compound of formula I, and also includes the application of the crystal form in the preparation of drugs for the prevention or treatment of diseases that benefit from the inhibition of capsid protein assembly.
  • chronic viral hepatitis B is incurable and can only be controlled. At present, it is mainly limited to two types of agents (interferon and nucleoside analog/viral polymerase inhibitors).
  • the low cure rate of HBV is partly due to the presence and persistence of covalently closed circular DNA (cccDNA) in the nucleus of infected liver cells.
  • the current treatment plan cannot eliminate the cccDNA in the reservoir, and some new HBV targets such as core inhibitors, such as viral capsid protein formation or assembly inhibitors, cccDNA inhibitors, and interferon-stimulated gene activators It is expected to bring hope to cure hepatitis B (Mayur Brahmania, et al. New therapeutic agents for chronic hepatitis B).
  • HBV capsid is assembled by core protein. Before reverse transcription, HBV reverse transcriptase and pgRNA need to be correctly encapsulated by the capsid protein. Therefore, blocking the assembly of the capsid protein or accelerating the degradation of the capsid protein will block the process of capsid protein assembly, thereby affecting virus replication.
  • the present application provides a crystalline composition, wherein the crystals of the compound of formula I account for more than 50% of the weight of the crystalline composition, preferably more than 80%, more preferably more than 90%, most preferably 95%. %the above.
  • the present application provides a pharmaceutical composition, which comprises a therapeutically effective amount of the crystal of the compound of formula I described in the present application or a crystal form composition thereof.
  • the application also provides the use of the crystal of the compound of formula I, the crystal form composition, or the pharmaceutical composition thereof in the preparation of a medicine for preventing or treating diseases that benefit from the inhibition of capsid protein assembly.
  • the application also provides the use of the crystal of the compound of formula I, the crystal composition, or the pharmaceutical composition thereof in the preparation of a medicine for preventing or treating hepatitis B virus infection.
  • this application also provides the use of the crystal of the compound of formula I, its crystal form composition, or its pharmaceutical composition in the prevention or treatment of diseases that benefit from the inhibition of capsid protein assembly.
  • the present application also provides a method for treating diseases that benefit from capsid protein assembly inhibition, which includes administering a therapeutically effective amount of the crystal of the compound of formula I described in the present application, and its crystal form to a mammal in need of such treatment.
  • Composition, or its pharmaceutical composition are examples of the present application.
  • the present application also provides a crystal of the compound of formula I described in the present application, a crystal form composition thereof, or a pharmaceutical composition thereof for the prevention or treatment of diseases that benefit from the inhibition of capsid protein assembly.
  • Figure 1 is the XRPD spectrum of the Form A crystal of the compound of formula I.
  • Figure 2 is a DSC spectrum of the Form A crystal of the compound of formula I.
  • this application provides the type A crystal of the compound of formula I above, and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2 ⁇ angles: 8.25 ⁇ 0.20°, 10.10 ⁇ 0.20°, 14.46 ⁇ 0.20°, 15.48 ⁇ 0.20 ° and 18.97 ⁇ 0.20°; in some schemes of this application, the X-ray powder diffraction pattern of the above-mentioned type A crystal has characteristic diffraction peaks at the following 2 ⁇ angles: 8.25 ⁇ 0.20°, 9.46 ⁇ 0.20°, 10.10 ⁇ 0.20°, 14.46 ⁇ 0.20°, 15.48 ⁇ 0.20°, 18.97 ⁇ 0.20°, 20.33 ⁇ 0.20° and 22.02 ⁇ 0.20°; in some schemes of this application, the X-ray powder diffraction pattern of the above-mentioned type A crystal has characteristic diffraction peaks at the following 2 ⁇ angles : 8.25 ⁇ 0.20°, 9.46 ⁇ 0.20°, 10.10 ⁇ 0.20°, 14.46 ⁇ 0.20°
  • XRPD X-ray powder diffraction
  • the differential scanning calorimetry (DSC) curve of the above-mentioned type A crystal has an endothermic peak at 227.34 ⁇ 5°C.
  • the present application provides a method for preparing type A crystals, the method comprising the following steps:
  • the compound of the above formula I is added to the solvent, and then the solid is separated.
  • the method for preparing type A crystals includes the following steps: adding the compound of formula I above into a solvent, crystallizing, and then separating the solid.
  • the volume-mass ratio of the solvent to the compound of formula I is 1 ml to 400 ml/g; in some embodiments, the volume-mass ratio of the solvent to the compound of formula I is 5 ml ⁇ 250ml/g; in some embodiments, the volume-mass ratio of the solvent to the compound of formula I is 10ml-220ml/g; in some embodiments, the volume-mass ratio of the solvent to the compound of formula I is 10ml/g, 20ml /g, 30ml/g, 40ml/g, 50ml/g, 60ml/g, 70ml/g, 80ml/g, 90ml/g, 100ml/g, 110ml/g, 120ml/g, 130ml/g, 140ml/g , 150ml/g, 160ml/g, 170ml/g, 180ml/g, 190ml/g,
  • the solvent in the method for preparing type A crystals, is selected from one or more mixtures of methanol, acetonitrile, tetrahydrofuran, or water. In some aspects of the application, in the method for preparing type A crystals, the solvent is selected from methanol, a mixture of acetonitrile and water, or a mixture of tetrahydrofuran and water.
  • the volume mass ratio of the methanol to the compound of formula I is 10-100 ml/g; in some embodiments of the present application, the volume mass ratio of the methanol to the compound of formula I is 10 ml/g g, 20ml/g, 30ml/g, 40ml/g, 50ml/g, 60ml/g, 70ml/g, 80ml/g, 90ml/g, 100ml/g or a range formed by any ratio; in some implementations of this application In the scheme, the volume mass ratio of the methanol to the compound of formula I is 10-50 ml/g; in some embodiments of the present application, the volume mass ratio of the methanol to the compound of formula I is 50 ml/g.
  • the volume ratio of acetonitrile to water is 1:1-10:1; in some embodiments of the present application, it is 1:1, 2:1, 3:1, 4: 1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 or a range formed by any ratio; in some embodiments of the present application, it is 2:1 to 5:1; In some embodiments of this application, it is 3.4:1.
  • the volume ratio of the tetrahydrofuran to water is 1:1 to 1:10; in some embodiments of the present application, it is 1:1, 1:2, 1:3, 1: 4. 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or a range formed by any ratio; in some embodiments of the application, it is 1:1 to 1:5; In some embodiments of this application, it is 1:5.
  • the method for separating solids is selected from filtration.
  • the compound of formula I in the method for preparing the above-mentioned type A crystal, is added to a solvent, stirred to dissolve, and optionally heated to dissolve the solution; in some specific embodiments, it is heated to 50 ⁇ 50. 80°C; in some specific embodiments, heat to 60°C.
  • the method for preparing the type A crystals described above optionally includes a process of cooling to room temperature and/or cooling and crystallization in an ice-water bath, and/or optionally adding water to cause crystallization.
  • XRPD is detected by Bruker D8ADVANCE X-ray powder diffractometer, light tube: Cu, k ⁇ , Light tube voltage: 40kV, light tube current: 40mA; divergence slit: 0.618mm; scanning range: 3-60deg; step diameter: 0.02deg; step length: 0.1 second.
  • the DSC is detected by the Mettler DSC Type 1 Differential Thermal Scanning Calorimeter, the temperature range is 50-300°C, and the heating rate is 10.00K/min.
  • the present application provides a crystalline composition, wherein the crystals of the compound of formula I described in the present application account for more than 50% of the weight of the crystalline composition, preferably more than 80%, more preferably more than 90%, most preferably It is more than 95%.
  • the present application provides a crystal composition comprising the type A crystal described in the present application, wherein the type A crystal accounts for more than 50% of the weight of the crystal composition, preferably more than 80%, more preferably 90%. % Or more, preferably 95% or more.
  • the present application provides a pharmaceutical composition comprising a therapeutically effective amount of the crystal of the compound of formula I described in the present application, or a crystal form composition thereof.
  • the pharmaceutical composition of the present application may or may not contain pharmaceutically acceptable excipients.
  • the pharmaceutical composition of the present application may further include one or more other therapeutic agents.
  • the application also provides the use of the crystal of the compound of formula I, the crystal form composition, or the pharmaceutical composition thereof in the preparation of a medicine for preventing or treating diseases that benefit from the inhibition of capsid protein assembly.
  • the application also provides the use of the crystal of the compound of formula I, the crystal composition, or the pharmaceutical composition thereof in the preparation of a medicine for preventing or treating hepatitis B virus infection.
  • this application also provides the use of the crystal of the compound of formula I, its crystal form composition, or its pharmaceutical composition in the prevention or treatment of diseases that benefit from the inhibition of capsid protein assembly.
  • the present application also provides a method for preventing or treating diseases that benefit from capsid protein assembly inhibition, including administering a therapeutically effective amount of the formula I described in the present application to a mammal in need of such treatment or prevention, preferably a human.
  • a mammal in need of such treatment or prevention, preferably a human.
  • the crystal of the compound, its crystal form composition, or its pharmaceutical composition is administered to a mammal in need of such treatment or prevention, preferably a human.
  • the present application also provides a crystal of the compound of formula I described in the present application, a crystal form composition thereof, or a pharmaceutical composition thereof for the prevention or treatment of diseases that benefit from the inhibition of capsid protein assembly.
  • the diseases that benefit from capsid protein assembly inhibition refer to diseases caused by hepatitis B virus (HBV) infection.
  • HBV hepatitis B virus
  • the diseases that benefit from the inhibition of capsid protein assembly refer to liver diseases caused by hepatitis B virus (HBV) infection.
  • HBV hepatitis B virus
  • the prevention or treatment of diseases that benefit from capsid protein assembly inhibition refers to the control, reduction or elimination of HBV to prevent, alleviate or cure liver diseases in infected patients.
  • the position of the peak or the relative intensity of the peak may be different due to factors such as the measuring instrument and the measuring method/condition.
  • the measurement error of the 2 ⁇ value may be ⁇ 0.2°. Therefore, when determining each crystal type, this error should be taken into account, and the error also belongs to the scope of this application.
  • the position of the endothermic peak of DSC may be different due to factors such as measuring instrument, measuring method/condition and so on.
  • there may be an error in the position of the endothermic peak which can be ⁇ 5°C or ⁇ 3°C. Therefore, when determining each crystal type, this error should be taken into account, and the error also belongs to the scope of this application.
  • pharmaceutically acceptable excipients refers to inert substances that are administered together with the active ingredients to facilitate the administration of the active ingredients, including but not limited to those acceptable for use in humans or animals approved by the State Food and Drug Administration (such as livestock) any glidant, sweetener, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersant, disintegrant, suspending agent, stabilizer , Isotonic agent, solvent or emulsifier.
  • auxiliary materials include calcium carbonate, calcium phosphate, various sugars and various starches, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.
  • pharmaceutical composition refers to a mixture of one or more of the compounds of the application or their salts and pharmaceutically acceptable excipients.
  • the purpose of the pharmaceutical composition is to facilitate the administration of the compound of the present application to the organism.
  • the pharmaceutical composition of the present application can be prepared by combining the compound of the present application with suitable pharmaceutically acceptable excipients, for example, can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, and powders. , Granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols.
  • Typical routes for administering the crystalline, crystalline composition or pharmaceutical composition of the present application include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, and intramuscular Intravenous, subcutaneous, and intravenous administration.
  • the pharmaceutical composition of the present application can be manufactured by methods well known in the art, such as conventional mixing method, dissolution method, granulation method, sugar-coated pill method, grinding method, emulsification method, freeze-drying method, etc.
  • the pharmaceutical composition is in oral form.
  • the pharmaceutical composition can be formulated by mixing the active compound with pharmaceutically acceptable excipients well known in the art. These auxiliary materials enable the compound of the present application to be formulated into tablets, pills, lozenges, sugar-coated agents, capsules, liquids, gels, slurries, suspensions, etc., for oral administration to patients.
  • the therapeutic dose of the compound of the present application may be determined based on, for example, the following: the specific purpose of the treatment, the manner of administration of the compound, the health and condition of the patient, and the judgment of the prescribing physician.
  • the ratio or concentration of the compound of the present application in the pharmaceutical composition may not be fixed, depending on various factors, including dosage, chemical properties (for example, hydrophobicity), and route of administration.
  • treatment means administering the compound or formulation described in this application to improve or eliminate a disease or one or more symptoms related to the disease, and includes:
  • prevention means administering the compound or preparation described in this application to prevent a disease or one or more symptoms related to the disease, and includes: preventing the occurrence of a disease or disease state in a mammal, especially when Such mammals are susceptible to the disease state, but have not been diagnosed as having the disease state.
  • the term "therapeutically effective amount” refers to a sufficient amount of a drug or agent that is non-toxic but can achieve the desired effect.
  • the determination of the effective amount varies from person to person, and depends on the age and general conditions of the recipient, as well as the specific active substance. The appropriate effective amount in a case can be determined by those skilled in the art according to routine experiments.
  • the therapeutically effective amount of the crystals described in the present application is from about 0.0001 to 20 mg/Kg body weight/day, for example, from 0.001 to 10 mg/Kg body weight/day.
  • the dosage frequency of the crystals described in the present application is determined by the needs of the individual patient, for example, once or twice a day, or more times a day.
  • the administration may be intermittent, for example, where the patient receives the daily dose of the crystals described in this application during a period of several days, and then during a period of several days or more, the patient does not receive the crystals described in this application. Daily dose.
  • DMF N,N-dimethylformamide
  • EA ethyl acetate
  • MeOH stands for methanol
  • DMSO dimethyl sulfoxide
  • HATU 2-(7-oxybenzotriazide Azole)-N,N,N',N'-tetramethylurea hexafluorophosphate
  • DIPEA stands for N,N-diisopropylethylamine.
  • the crystal of the present application has good pharmacological activity, and at the same time has good high humidity, high temperature or light stability, which proves its good pharmacological properties and high prospects for preparing medicines.
  • Step A In an ice bath, under N 2 protection, add 2-chloro-2-oxoacetate (40.8g) and zinc oxide (1.22g) to the reaction flask, and then add 2,4-dimethyl- Ethyl 1H-pyrrole-3-carboxylate (5g), after the addition was completed, the mixture was stirred in an ice bath for 10 minutes, the ice bath was removed, and the mixture was stirred at room temperature. At the end of the reaction, the reaction solution was slowly added dropwise to 200 mL of ice-water mixture, EA (200 mL) was added, and the layers were separated.
  • Step B Into the reaction flask, add 5-(2-ethoxy-2-oxoacetyl)-2,4-dimethyl-1H-pyrrole-3-carboxylic acid ethyl ester (3.5g), MeOH in turn (40 mL), a solution of sodium hydroxide (1.05 g) in water (20 mL) was added dropwise under an ice bath, and stirred at room temperature.
  • Step C At room temperature, add 2-(4-(ethoxycarbonyl)-3,5-dimethyl-1H-pyrrol-2-yl)-2-oxoacetic acid (1g) to the reaction flask in sequence , DMF (20mL), HATU (2.07g) and DIPEA (1.08g), after the addition is complete, stir at room temperature for 10 minutes, add (S)-1,1,1-trifluoropropan-2-amine hydrochloride (0.63 g).
  • Step D Add (S)-2,4-dimethyl-5-(2-oxo-2-((1,1,1-trifluoroprop-2-yl)amino)acetyl to the reaction flask ) Ethyl -1H-pyrrole-3-carboxylate (300 mg), MeOH (2 mL), and NaOH (72 mg) in water (1 mL) were added. After the addition, the reaction temperature was heated to 80° C. to react overnight. After the reaction is over, concentrate, add water (20mL) and EA (60mL), separate the water layer, wash the organic phase with water (30mL), separate the layers, combine the water phases, adjust the water phase with 2N hydrochloric acid to make the pH around 3.
  • Step E At room temperature, sequentially add (S)-2,4-dimethyl-5-(2-oxo-2-((1,1,1-trifluoroprop-2-yl) to the reaction flask (Amino)acetyl)-1H-pyrrole-3-carboxylic acid (230mg), DMF (5mL), HATU (428mg) and DIPEA (194mg), after the addition is complete, stir for 10 minutes, and then add 5-amino-2-fluorobenzyl Nitrile (123mg), heated to 40°C and stirred for 20 hours.
  • Type A crystals of the compound of formula I prepared in Example 2 place them in a dry and clean weighing bottle, spread them into a thin layer, and use them as a test sample.
  • the samples were placed under the test conditions of influencing factors (40°C, 60°C, 75%RH, 92.5%RH, high temperature and high humidity (40°C, 75%RH)), and the samples were fully exposed lofting. Take samples for analysis within 10 days.
  • the samples placed under the conditions of light visible light 1200000 Lux ⁇ hr, ultraviolet 216W ⁇ hr/m 2 ) are fully exposed at room temperature for lofting.
  • the chromatographic column adopts Waters XBridge Shield RP18 column (4.6mm ⁇ 150mm, 3.5 ⁇ m).
  • the moisture content is measured with a Mettler V20 moisture analyzer.
  • the DMSO-dissolved compound was diluted with a complete medium, a 2-fold gradient, a total of 10 concentrations, the compound was added, the fresh medium containing the compound was replaced every 72h, and the compound was treated for 6 days. After aspirating the supernatant, add 300 ⁇ L of lysis solution (10mM Tris-HCl, 1mM EDTA, 1% NP-40) to each well. After lysis at room temperature for 10 minutes, DNA is extracted, and the intracellular viral capsid is determined by a real-time fluorescent quantitative PCR instrument. For HBV DNA, the inhibition rate is calculated based on the Ct value, and the EC50 value is calculated by the four-parameter method. The results are shown in Table 3 and Table 4.
  • HepG2.2.15 cells (Wuhan Institute of Virology) in good condition in the exponential growth phase, add 5mL PBS to wash it again, and add 2mL pancreatin. Put it into a cell incubator for digestion, take it out from time to time and observe it under the microscope. When the cells just fall off, discard 1mL trypsin, leaving only the residual liquid, put it in a 37°C incubator for digestion for 8-15 minutes, take it out and observe under the microscope Cells (whether they are single round, no adhesion between cells), add 5mL MEM medium to resuspend the cells. Count using a cell counter, dilute the complete medium, and adjust the cell density to 2*10 5 cells/mL.
  • Sample pretreatment 50 ⁇ L incubation sample, add 300 ⁇ L ice acetonitrile precipitation containing internal standard, vortex for 5min, centrifuge (12000rpm, 4°C) for 10min. Aspirate 75 ⁇ L of the supernatant, add 75 ⁇ L of ultrapure water, dilute and mix well, and 1 ⁇ L of sample for analysis. The results are shown in Table 6.
  • Plasma sample preparation draw 495 ⁇ L of blank plasma of corresponding species (mouse, rat, dog, monkey and human) respectively, and add 5 ⁇ L of the corresponding test compound solution or positive control to obtain the plasma sample solution, and make the compound plasma drug concentration separately It is 1 ⁇ M, 10 ⁇ M (prepared with acetonitrile).
  • Sample pretreatment 50 ⁇ L plasma side sample, add 450 ⁇ L ice acetonitrile precipitation with internal standard, vortex for 5min, centrifuge (12000rpm, 4°C) for 10min. Aspirate 75 ⁇ L of the supernatant, add 75 ⁇ L of ultrapure water to dilute and mix, 1 ⁇ L for sample analysis; 50 ⁇ L of PBS side sample, add 250 ⁇ L of ice acetonitrile with internal standard precipitation, vortex for 5min, centrifuge (12000rpm, 4°C) 10min. Aspirate 75 ⁇ L of the supernatant, add 75 ⁇ L of ultrapure water to dilute and mix well, and 2 ⁇ L of sample for analysis. The results are shown in Table 7.
  • the 300 ⁇ L final incubation system contains 30 ⁇ L liver microsomes (protein concentration: 0.15 mg/mL, Corning), 30 ⁇ L NADPH+MgCl 2 , 3 ⁇ L substrate (prepared with acetonitrile), and 237 ⁇ L PBS buffer. Make 2 servings for each species, each serving 0.3mL. Prepare each tube with a total volume of 270 ⁇ L of substrate and enzyme mixing solution. After pre-incubating with NADPH for 5 minutes at 37°C, add 30 ⁇ L of NADPH+MgCl 2 mixed solution to react at 0, 10, 30, and 60 minutes. Take out 50 ⁇ L and terminate the reaction with 300 ⁇ L ice acetonitrile containing internal standard.
  • Sample pretreatment 50 ⁇ L of warmed sample, 300 ⁇ L of ice acetonitrile precipitation containing internal standard diazepam, vortex for 5min, centrifugation (12000rpm, 4°C) for 10min. Aspirate 75 ⁇ L of the supernatant to a 96-well plate and dilute and mix with 75 ⁇ L ultrapure water, inject 0.5 ⁇ L, and perform LC-MS/MS analysis. The results are shown in Table 8.
  • mice Take 6-8 week old male C57BL/6 mice (Shanghai Lingchang Biological Technology Co., Ltd.), and inject rAAV8-1.3HBV virus (Beijing Wujiahe, adr subtype) to C57BL in the tail vein at a dose of 1 ⁇ 10 11 vg /6 In mice.
  • rAAV8-1.3HBV virus Beijing Wujiahe, adr subtype
  • mice Combined with the quantitative detection results of serological HBeAg, HBsAg and HBV DNA, the selected mice have HBV DNA expression levels greater than 1 ⁇ 10 4 IU/mL, HBeAg greater than 1 ⁇ 10 3 NCU/mL and HBsAg greater than 1 ⁇ 10 3 ng/mL.
  • the mice were divided into groups, and set up a blank control group, a vehicle control group, and a test substance group. The mice in each group were given intragastric administration for 3 weeks, once a day, and the drug was stopped for 1 week. During the experiment, blood was collected from the orbit every other week, the serum was separated, and the DNA content was detected by fluorescence quantitative PCR. The results are shown in Table 9 and Table 10.
  • Table 9 Decreased levels of HBV DNA in serum (log10) (administration dose 10mpk)
  • Table 10 Decreased levels of HBV DNA in serum (log10) (administration dose 1mpk)
  • mice Take 6-8 week old male C57BL/6 mice (Shanghai Lingchang Biotechnology Co., Ltd.), and dissolve the purified recombinant plasmid pAAV/HBV1.2 (10 ⁇ g) in PBS.
  • the injection volume of each mouse is about its body weight. 10% of it was injected into mice through the tail vein within 3-8s.
  • blood was taken from the orbit for detection of serum HBV DNA, and the model mice were selected for uniform serum DNA and grouped.
  • a model control group, a vehicle control group, and a test substance group were set up. Each group of mice was administered intragastrically for 10 consecutive days, once a day, at a dose of 3 mg/kg.
  • mice serum was collected at 0, 4, 7, and 10 days after administration. On the 10th day, the mice were sacrificed and liver tissue samples were collected. Fluorescence quantitative PCR method was used to detect the copy number of HBV DNA in the mouse serum and liver. The results are shown in Table 11.
  • SD rats (Shanghai Xipuer-Bike), weighing 180-220g, were used for 3 to 5 days, and then randomly divided into groups, 3 rats in each group, and were given a series of compounds at a dose of 20 mg/kg.
  • test animals SD rats were fasted for 12 hours before the administration, and were given food 4 hours after the administration. They were free to drink water before and after the experiment and during the experiment.

Abstract

公开了N杂五元环化合物的晶型，具体涉及式I化合物的晶型，还包括所述晶型在制备预防或者治疗受益于衣壳蛋白装配抑制的疾病的药物中的应用。

Description

N杂五元环化合物的晶型及其应用
相关申请的交叉引用
本申请要求于2019年09月29日向中国国家知识产权局提交的第201910933542.7号中国专利申请的优先权和权益，所述申请公开的内容通过引用整体并入本文中。
技术领域
本申请涉及N杂五元环化合物的晶型，具体涉及式I化合物的晶型，还包括所述晶型在制备预防或者治疗受益于衣壳蛋白装配抑制的疾病的药物中的应用。
背景技术
当前，针对慢性乙型病毒性肝炎不可治愈只能控制，目前主要限于两类药剂(干扰素和核苷类似物/病毒聚合酶的抑制剂)。HBV的治愈率低部分是由于受感染肝细胞的细胞核中共价闭合环状DNA(cccDNA)的存在和持续性。目前治疗方案无法将储存库中的cccDNA清除掉，而一些HBV的新靶点如核心抑制剂(Core inhibitors，例如病毒的衣壳蛋白形成或装配抑制剂和cccDNA抑制剂及干扰素刺激基因激活剂等)有望给治愈乙肝带来希望(Mayur Brahmania,et al.New therapeutic agents for chronic hepatitis B)。HBV衣壳由核心蛋白装配而成，在逆转录以前，HBV逆转录酶、pgRNA需要被衣壳蛋白正确包裹。因此，阻断衣壳蛋白装配，或加快衣壳蛋白降解，都会阻断衣壳蛋白装配过程，从而影响病毒复制。
发明概述
一方面，本申请提供式I化合物的结晶，
Figure PCTCN2020118426-appb-000001
又一方面，本申请提供晶型组合物，其中，所述式I化合物的结晶占晶型组合物重量的50％以上，较好为80％以上，更好是90％以上，最好是95％以上。
又一方面，本申请提供一种药物组合物，该药物组合物包含治疗有效量的本申请所述式I化合物的结晶或其晶型组合物。
另一方面，本申请还提供本申请所述式I化合物的结晶、其晶型组合物、或者其药物组合物在制备预防或治疗受益于衣壳蛋白装配抑制的疾病的药物中的用途。
另一方面，本申请还提供本申请所述式I化合物的结晶、其晶型组合物、或者其药物组合物在制备预防或治疗乙型肝炎病毒感染的药物中的用途。
另一方面，本申请还提供本申请所述式I化合物的结晶、其晶型组合物、或者其药物组合物在预防或者治疗受益于衣壳蛋白装配抑制的疾病中的用途。
另一方面，本申请还提供一种治疗受益于衣壳蛋白装配抑制的疾病的方法，包括对需要该治疗的哺乳动物，给予治疗有效量的本申请所述式I化合物的结晶、其晶型组合物、或者其药物组合物。
另一方面，本申请还提供了预防或治疗受益于衣壳蛋白装配抑制的疾病的本申请所述式I化合物的结晶、其晶型组合物、或者其药物组合物。
附图说明
图1为式I化合物的A型结晶的XRPD谱图。
图2为式I化合物的A型结晶的DSC谱图。
发明详述
一方面，本申请提供式I化合物的结晶，
Figure PCTCN2020118426-appb-000002
再一方面，本申请提供上述式I化合物的A型结晶，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：8.25±0.20°、10.10±0.20°、14.46±0.20°、15.48±0.20°和18.97±0.20°；本申请的一些方案中，上述A型结晶的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：8.25±0.20°、9.46±0.20°、10.10±0.20°、14.46±0.20°、15.48±0.20°、18.97±0.20°、20.33±0.20°和22.02±0.20°；本申请的一些方案中，上述A型结晶的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：8.25±0.20°、9.46±0.20°、10.10±0.20°、14.46±0.20°、15.48±0.20°、18.97±0.20°、20.33±0.20°、20.80±0.20°、21.67±0.20°、22.02±0.20°、24.37±0.20°、26.37±0.20°和30.77±0.20°；本申请的一些方案中，上述A型结晶的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：8.25±0.20°、9.46±0.20°、10.10±0.20°、14.46±0.20°、15.48±0.20°、18.97±0.20°、20.33±0.20°、20.80±0.20°、21.67±0.20°、22.02±0.20°、22.88±0.20°、23.93±0.20°、24.37±0.20°、24.97±0.20°、26.37±0.20°、28.68±0.20°、和30.77±0.20°。
本申请的一些方案中，上述A型结晶的XRPD图谱中，衍射峰的峰位置及相对强度由下表1表示：
表1 A型结晶的XRPD数据
Figure PCTCN2020118426-appb-000003
Figure PCTCN2020118426-appb-000004
本申请的一些方案中，上述A型结晶的X射线粉末衍射(XRPD)图谱如图1所示。
本申请的一些方案中，上述A型结晶的差示扫描量热(DSC)曲线在227.34±5℃处有吸热峰。
本申请的一些方案中，上述A型结晶的DSC图谱如图2所示。
又一方面，本申请提供一种A型结晶的制备方法，所述方法包括如下步骤：
将上述式I化合物加入溶剂中，然后分离固体。
本申请的一些方案中，A型结晶的制备方法包括如下步骤：将上述式I化合物加入溶剂中，析晶，然后分离固体。
本申请的一些方案中，上述A型结晶的制备方法，其中溶剂与式I化合物的体积质量比为1ml～400ml/g；在一些实施方案中，其中溶剂与式I化合物的体积质量比为5ml～250ml/g；在一些实施方案中，其中溶剂与式I化合物的体积质量比为10ml～220ml/g；在一些实施方案中，其中溶剂与式I化合物的体积质量比为10ml/g、20ml/g、30ml/g、40ml/g、50ml/g、60ml/g、70ml/g、80ml/g、90ml/g、100ml/g、110ml/g、120ml/g、130ml/g、140ml/g、150ml/g、160ml/g、170ml/g、180ml/g、190ml/g、200ml/g、210ml/g、220ml/g或者任意比例形成的范围；在一些实施方案中，其中溶剂与式I化合物的体积质量比为10ml/g、50ml/g或者220ml/g。
本申请的一些方案中，上述A型结晶的制备方法，其中溶剂选自甲醇、乙腈、四氢呋喃或水中的一种或多种的混合物。本申请的一些方案中，上述A型结晶的制备方法，其中溶剂选自甲醇、乙腈与水的混合物、或四氢呋喃与水的混合物。
在本申请的一些实施方案中，所述甲醇与式I化合物的体积质量比为10～100ml/g；在本申请的一些实施方案中，所述甲醇与式I化合物的体积质量比为10ml/g、20ml/g、30ml/g、40ml/g、50ml/g、60ml/g、70ml/g、80ml/g、90ml/g、100ml/g或者任意比例形成的范围；在本申请的一些实施方案中，所述甲醇与式I化合物的体积质量比为10～50ml/g；在本申请的一些实施方案中，所述甲醇与式I化合物的体积质量比 为50ml/g。
在本申请的一些实施方案中，所述乙腈与水的体积比为1:1～10:1；在本申请的一些实施方案中，为1:1、2:1、3:1、4:1、5:1、6:1、7:1、8:1、9:1、10:1或者任意比例形成的范围；在本申请的一些实施方案中，为2:1～5:1；在本申请的一些实施方案中，为3.4:1。
在本申请的一些实施方案中，所述四氢呋喃与水的体积比为1:1～1:10；在本申请的一些实施方案中，为1:1、1:2、1:3、1:4、1:5、1:6、1:7、1:8、1:9、1:10或者任意比例形成的范围；在本申请的一些实施方案中，为1:1～1:5；在本申请的一些实施方案中，为1:5。
本申请的一些方案中，上述A型结晶的制备方法，其中分离固体的方式选自过滤。
本申请的一些方案中，上述A型结晶的制备方法，将上述式I化合物加入溶剂中，搅拌溶清，任选地通过加热使溶液溶清；在一些具体的实施方案中，加热至50～80℃；在一些具体的实施方案中，加热至60℃。
本申请的一些方案中，上述A型结晶的制备方法，任选地包括冷却至室温的过程和/或在冰水浴中进行冷却析晶，和/或任选地加入水使得析晶。
本申请中，XRPD采用Bruker D8ADVANCE X-射线粉末衍射仪检测，光管：Cu,kα,
Figure PCTCN2020118426-appb-000005
光管电压：40kV，光管电流：40mA；发散狭缝：0.618mm；扫描范围：3-60deg；步径：0.02deg；步长：0.1秒。
本申请中，DSC采用梅特勒DSC 1型差热扫描量热仪检测，温度范围：50-300℃，升温速率：10.00K/min。
又一方面，本申请提供晶型组合物，其中，本申请所述式I化合物的结晶占晶型组合物重量的50％以上，较好为80％以上，更好是90％以上，最好是95％以上。
又一方面，本申请提供包含本申请所述A型结晶的晶型组合物，其中，所述A型结晶占晶型组合物重量的50％以上，较好为80％以上，更好是90％以上，最好是95％以上。
又一方面，本申请提供一种药物组合物，该药物组合物包含治疗有效量的本申请所述式I化合物的结晶、或其晶型组合物。本申请的药物组合物中可含有或不含有药学上可接受的辅料。此外，本申请的药物组合物可进一步包括一种或多种其他治疗剂。
另一方面，本申请还提供本申请所述式I化合物的结晶、其晶型组合物、或者其药物组合物在制备预防或治疗受益于衣壳蛋白装配抑制的疾病的药物中的用途。
另一方面，本申请还提供本申请所述式I化合物的结晶、其晶型组合物、或者其药物组合物在制备预防或治疗乙型肝炎病毒感染的药物中的用途。
另一方面，本申请还提供本申请所述式I化合物的结晶、其晶型组合物、或者其药物组合物在预防或者治疗受益于衣壳蛋白装配抑制的疾病中的用途。
另一方面，本申请还提供一种预防或治疗受益于衣壳蛋白装配抑制的疾病的方法，包括对需要该治疗 或预防的哺乳动物，优选人类，给予治疗有效量的本申请所述式I化合物的结晶、其晶型组合物、或者其药物组合物。
另一方面，本申请还提供了预防或治疗受益于衣壳蛋白装配抑制的疾病的本申请所述式I化合物的结晶、其晶型组合物、或者其药物组合物。
在本申请的一些实施方式中，所述受益于衣壳蛋白装配抑制的疾病指乙型肝炎病毒(HBV)感染引起的疾病。
在本申请的一些实施方式中，所述受益于衣壳蛋白装配抑制的疾病指乙型肝炎病毒(HBV)感染引起的肝脏疾病。
在本申请的部分实施方式中，所述预防或治疗受益于衣壳蛋白装配抑制的疾病指控制、降低或清除HBV以预防、缓解或治愈受感染患者的肝脏疾病。
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。
需要说明的是，在粉末X-射线衍射光谱中，峰的位置或峰的相对强度可能会因为测定仪器、测定方法/条件等因素而产生差异。对任何特定的晶型，峰的位置可能存在误差，2θ值的测定误差可以为±0.2°。因此，在确定每种晶型时，应该将此误差考虑在内，在误差内也属于本申请的范围。
需要说明的是，对于同种晶型，DSC的吸热峰出现位置可能会因为测定仪器、测定方法/条件等因素而产生差异。对任何特定的晶型，吸热峰的位置可能存在误差，误差可以为±5℃，可以为±3℃。因此，在确定每种晶型时，应该将此误差考虑在内，在误差内也属于本申请的范围。
所述词语“包括(comprise)”或“包含(comprise)”及其英文变体例如comprises或comprising应理解为开放的、非排他性的意义，即“包括但不限于”。
术语“药学上可接受的辅料”是指与活性成份一同给药的、有利于活性成份给药的惰性物质，包括但不限于国家食品药品监督管理局许可的可接受的用于人或动物(例如家畜)的任何助流剂、增甜剂、稀释剂、防腐剂、染料/着色剂、矫味增强剂、表面活性剂、润湿剂、分散剂、崩解剂、助悬剂、稳定剂、等渗剂、溶剂或乳化剂。所述辅料的非限制性实例包括碳酸钙、磷酸钙、各种糖和各类淀粉、纤维素衍生物、明胶、植物油和聚乙二醇。
术语“药物组合物”是指一种或多种本申请的化合物或其盐与药学上可接受的辅料组成的混合物。药物组合物的目的是有利于对有机体给予本申请的化合物。
本申请的药物组合物可通过将本申请的化合物与适宜的药学上可接受的辅料组合而制备，例如可配制成固态、半固态、液态或气态制剂，如片剂、丸剂、胶囊剂、粉剂、颗粒剂、膏剂、乳剂、悬浮剂、栓剂、 注射剂、吸入剂、凝胶剂、微球及气溶胶等。
给予本申请所述结晶、晶型组合物或其药物组合物的典型途径包括但不限于口服、直肠、局部、吸入、肠胃外、舌下、***内、鼻内、眼内、腹膜内、肌内、皮下、静脉内给药。
本申请的药物组合物可以采用本领域众所周知的方法制造，如常规的混合法、溶解法、制粒法、制糖衣药丸法、磨细法、乳化法、冷冻干燥法等。
在一些实施方案中，药物组合物是口服形式。对于口服给药，可以通过将活性化合物与本领域熟知的药学上可接受的辅料混合，来配制该药物组合物。这些辅料能使本申请的化合物被配制成片剂、丸剂、锭剂、糖衣剂、胶囊剂、液体、凝胶剂、浆剂、悬浮剂等，用于对患者的口服给药。
本申请化合物的治疗剂量可根据例如以下而定：治疗的具体用途、给予化合物的方式、患者的健康和状态，以及签处方医师的判断。本申请化合物在药用组合物中的比例或浓度可不固定，取决于多种因素，它们包括剂量、化学特性(例如疏水性)和给药途径。
术语“治疗”意为将本申请所述化合物或制剂进行给药以改善或消除疾病或与所述疾病相关的一个或多个症状，且包括：
(i)抑制疾病或疾病状态，即遏制其发展；
(ii)缓解疾病或疾病状态，即使该疾病或疾病状态消退。
术语“预防”意为将本申请所述化合物或制剂进行给药以预防疾病或与所述疾病相关的一个或多个症状，且包括：预防疾病或疾病状态在哺乳动物中出现，特别是当这类哺乳动物易患有该疾病状态，但尚未被诊断为已患有该疾病状态时。
针对药物或药理学活性剂而言，术语“治疗有效量”是指无毒的但能达到预期效果的药物或药剂的足够用量。有效量的确定因人而异，取决于受体的年龄和一般情况，也取决于具体的活性物质，个案中合适的有效量可以由本领域技术人员根据常规试验确定。
本申请所述结晶的治疗有效量为从约0.0001到20mg/Kg体重/天，例如从0.001到10mg/Kg体重/天。
本申请所述结晶的剂量频率由患者个体的需求决定，例如，每天1次或2次，或每天更多次。给药可以是间歇性的，例如，其中在若干天的期间内，患者接受本申请所述结晶的每日剂量，接着在若干天或更多天的期间，患者不接受本申请所述结晶的每日剂量。
本申请所使用的所有溶剂是市售的，无需进一步纯化即可使用。
本申请采用下述缩略词：DMF代表N,N-二甲基甲酰胺；EA代表乙酸乙酯；MeOH代表甲醇；DMSO代表二甲亚砜；HATU代表2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐；DIPEA代表N,N-二异丙基乙胺。
技术效果
本申请的结晶具有良好的药理活性，同时具有良好的高湿、高温或光照稳定性，证明其良好的药学性 质和较高的成药前景。
具体实施方式
下面通过实施例对本申请进行详细描述，但并不意味着对本申请任何不利限制。本文已经详细地描述了本申请，其中也公开了其具体实施例方式，对本领域的技术人员而言，在不脱离本申请精神和范围的情况下针对本申请具体实施方式进行各种变化和改进将是显而易见的。
实施例1式I化合物的制备
Figure PCTCN2020118426-appb-000006
步骤A：冰浴中，N 2保护下，反应瓶中依次加入2-氯-2-氧代乙酸乙酯(40.8g)和氧化锌(1.22g)，然后加入2,4-二甲基-1H-吡咯-3-羧酸乙酯(5g)，加料完毕，混合物在冰浴下搅拌10分钟，撤去冰浴，室温搅拌。反应结束，将反应液缓慢滴加到200mL冰水混合物中，加入EA(200mL)，分层，有机相用无水硫酸钠干燥，浓缩，柱层析制备得到5-(2-乙氧基-2-氧代乙酰基)-2,4-二甲基-1H-吡咯-3-羧酸乙酯(4.5g)。MS(ESI+,[M+Na] +)m/z：290.07。
步骤B:反应瓶中，依次加入5-(2-乙氧基-2-氧代乙酰基)-2,4-二甲基-1H-吡咯-3-羧酸乙酯(3.5g)、MeOH(40mL)，冰浴下滴加氢氧化钠(1.05g)的水(20mL)溶液，室温下搅拌。反应结束，用2N盐酸水溶液调节水相pH为3-4，加入EA(100mL*2)萃取，有机相用水(30mL)洗涤，浓缩，得到2-(4-(乙氧基羰基)-3,5-二甲基-1H-吡咯-2-基)-2-氧代乙酸(2.7g)。MS(ESI-,[M-H] -)m/z：238.1.
步骤C:室温下，向反应瓶中，依次加入2-(4-(乙氧基羰基)-3,5-二甲基-1H-吡咯-2-基)-2-氧代乙酸(1g)、DMF(20mL)、HATU(2.07g)及DIPEA(1.08g)，加料完毕，室温下搅拌10分钟，加入(S)-1,1,1-三氟丙-2-胺盐酸盐(0.63g)。反应结束，将反应液倒入50mL水中，EA(50mL*3)萃取，有机相用饱和硫酸钠水溶液 (50mL*3)洗涤，无水硫酸钠干燥，过滤，收集滤液，浓缩、经柱层析纯化，得到(S)-2,4-二甲基-5-(2-氧代-2-((1,1,1-三氟丙-2-基)氨基)乙酰基)-1H-吡咯-3-羧酸乙酯(0.5g)。MS(ESI-,[M-H] -)m/z：333.4.
步骤D:反应瓶中，加入(S)-2,4-二甲基-5-(2-氧代-2-((1,1,1-三氟丙-2-基)氨基)乙酰基)-1H-吡咯-3-羧酸乙酯(300mg)、MeOH(2mL)、加入NaOH(72mg)的水(1mL)溶液，加毕，加热反应温度至80℃反应过夜。反应结束，浓缩，然后加入水(20mL)及EA(60mL)，分离水层，有机相加水(30mL)洗涤，分层，合并水相，用2N盐酸调节水相，使pH为3左右，然后加入EA(100mL*2)萃取，分层，浓缩有机相，得到(S)-2,4-二甲基-5-(2-氧代-2-((1,1,1-三氟丙-2-基)氨基)乙酰基)-1H-吡咯-3-羧酸(230mg)。MS(ESI-,[M-H] -)m/z：305.4.
步骤E:室温下，向反应瓶中依次加入(S)-2,4-二甲基-5-(2-氧代-2-((1,1,1-三氟丙-2-基)氨基)乙酰基)-1H-吡咯-3-羧酸(230mg)、DMF(5mL)、HATU(428mg)及DIPEA(194mg)，加料完毕，搅拌10分钟，再加入5-氨基-2-氟苄腈(123mg)，加热至40℃搅拌反应20小时。反应结束，加入水(20mL)及EA(60mL)，分层，无水硫酸钠干燥，过滤，收集滤液，旋干，拌样，柱层析纯化得到(S)-N-(3-氰基-4-氟苯基)-2,4-二甲基-5-(2-氧代-2-((1,1,1-三氟丙-2-基)氨基)乙酰基)-1H-吡咯-3-甲酰胺(180mg)。 1H NMR(500MHz,DMSO-d6)δ12.05(s,1H),10.20(s,1H),9.49(d,J＝9.0Hz,1H),8.20(dd,J＝6.0,2.5Hz,1H),7.98-7.91(m,1H),7.53(t,J＝9.0Hz,1H),4.78-4.67(m,1H),2.40(s,3H),2.32(s,3H),1.34(d,J＝7.0Hz,3H)。MS(ESI-,[M-H] -)m/z：423.0。
实施例2式I化合物的A型结晶的制备
在室温下，将500mg式I化合物加入至25ml无水甲醇中搅拌，升温至60℃溶清，降至室温，放入冰水浴中，有大量白色固体析出，过滤，滤饼用无水甲醇淋洗，并于70℃鼓风干燥8h，得到类白色的式I化合物的A型结晶固体300mg，样品用XRPD检测，如图1所示，DSC检测如图2所示。
实验例1结晶稳定性实验
1.1样品制备
称取实施例2制备得到的式I化合物的A型结晶500mg，置于干燥洁净的称量瓶中，摊成薄薄一层，作为供试样品。将样品放置于影响因素试验条件下(40℃，60℃，75％RH，92.5％RH，高温高湿(40℃，75％RH))，其样品为完全暴露放样。在10天取样分析。光照(可见光1200000Lux·hr，紫外216W·hr/m 2)条件下放置的样品为室温完全暴露放样。
1.2色谱条件
色谱柱采用Waters XBridge Shield RP18柱(4.6mm×150mm，3.5μm)。
水分含量采用梅特勒V20型水分测定仪测定。
1.3样品溶液的制备
取出样品约12.5mg，适量的稀释剂乙腈-水(70：30)溶解。作为检测有关物质的浓度。
表2 稳定性试验结果
Figure PCTCN2020118426-appb-000007
a有关物质是指总杂。
表2的结果说明本申请结晶的有关物质和水分在上述多个考察项目下保持稳定，证明该结晶具有良好的高湿、高温或光照稳定性。
实验例2体外活性研究
2.1体外细胞HBV DNA抑制活性
取处于指数生长期状态良好的HepG2.2.15(武汉病毒所)或HepAD38细胞一瓶，加入5mL PBS清洗一遍，加入3mL胰酶。室温消化5min，弃掉2mL胰酶后再放入细胞培养箱中消化10min，取出显微镜下观察(是否为单个圆形，细胞间无粘连)，加入10mL完全培养基终止消化。吹打成单细胞悬液后，取10μL细胞悬液使用细胞计数仪计数，完全培养基进行稀释，调整细胞密度至1*10 5个/mL。使用排枪接种于24孔板上(24孔板提前使用50μg/mL Collagen Ⅰ溶液包被)，1mL/孔，置恒温CO 2培养箱中培养48h。
使用完全培养基将DMSO溶解的化合物稀释，2倍梯度，共10个浓度，进行化合物加样，每72h更换含化合物的新鲜培养基，化合物处理细胞6天。吸去上清后，每孔加入300μL裂解液(10mM Tris-HCl，1mM EDTA，1％NP-40)，室温放置裂解10min后，提取DNA，用实时荧光定量PCR仪测定胞内病毒衣壳中HBV DNA，根据Ct值计算抑制率，四参数法计算EC50值。结果如表3及表4所示。
表3 HepAD38细胞中抗HBV活性实验结果
编号 EC50(nM)
式I化合物 24
表4 HepG2.2.15细胞中抗HBV活性实验结果
编号 EC50(nM)
式I化合物 17.4
2.2体外细胞毒性
取处于指数生长期状态良好的HepG2.2.15细胞(武汉病毒所)一瓶，加入5mL PBS清洗一遍，加入2mL胰酶。放入细胞培养箱中进行消化，不时取出显微镜下观察，待细胞刚脱落时，弃掉1mL胰酶，仅仅留下 残液，放入37℃培养箱中消化8-15min，取出在显微镜下观察细胞(是否为单个圆形，细胞间无粘连)，加入5mL MEM培养基进行细胞重悬。使用细胞计数仪计数，完全培养基进行稀释，调整细胞密度至2*10 5个/mL。使用排枪接种于96孔板上(96孔板提前使用50μg/mL Collagen Ⅰ溶液包被)，100μL/孔，置恒温CO 2培养箱中培养24h，给药处理，每隔3天，更换含化合物的新鲜培养基，对照孔加不含药物的DMSO浓度为0.5％的培养基，并设普通培养基的对照孔，给药处理6天后，加CCK-8，10μL/孔，1-2h后酶标仪450nm处检测其吸光值，计算抑制率，并计算CC50。结果如表5所示。
表5
细胞 CC50(μM) 编号
HepG2.2.15 >100 式I化合物
2.3 CYP450酶抑制研究
500μL最终的温孵体系中，含50μL人肝微粒体(蛋白浓度：0.2mg/mL,Corning)，1μL混合的CYP450特异性底物(CYP1A2、CYP 2B6、CYP 2C9、CYP2C19、CYP 2D6、CYP 3A4)，398μL PBS缓冲液(PH7.4)，1μL特异性阳性抑制剂(阳性对照组)或受试化合物(乙腈配制)，50μL NADPH+MgCl 2。每个CYP450亚型做2份，每份0.5mL。每管先配好总体积为450μL的底物和酶的混匀液及NADPH分别在37℃预温孵5min后，加入50μL NADPH+MgCl 2混合溶液反应，于30min取出50μL用含内标的冰乙腈300μL终止反应。另外平行做2份空白组各500μL，不加NADPH，作为阴性对照组。
样品前处理：50μL温孵样品，加入300μL含内标的冰乙腈沉淀，涡旋震荡5min后，离心(12000rpm，4℃)10min。吸取上清液75μL，加入75μL超纯水，稀释混匀，1μL进样分析。结果如表6所示。
表6
Figure PCTCN2020118426-appb-000008
2.4血浆蛋白结合试验
血浆样品配制：分别吸取495μL相应种属(小鼠、大鼠、犬、猴及人)的空白血浆，加入5μL相应受试化合物溶液或阳性对照，即得血浆样品溶液，使化合物血浆药物浓度分别为1μM、10μM(乙腈配制)。
预处理好的透析膜置于高通量平衡透析装置中，吸取100μL血浆样品溶液及PBS缓冲液，分别加到透析膜的两侧(样品侧及缓冲液侧)(n＝3)，用贴膜将平衡装置封好后，放入37℃温孵过夜(100rpm)，达到透析平衡后，分别从样品侧及缓冲液侧吸取50μL样品，用含内标的冰乙腈终止反应。
样品前处理：50μL血浆侧样品，加入450μL含内标的冰乙腈沉淀，涡旋震荡5min后，离心(12000rpm，4℃)10min。吸取上清液75μL，加入75μL超纯水稀释混匀，1μL进样分析；50μLPBS侧样品，加入250μL含内标的冰乙腈沉淀，涡旋震荡5min后，离心(12000rpm，4℃)10min。吸取上清液75μL，加入75μL超纯水稀释混匀，2μL进样分析。结果如表7所示。
表7
Figure PCTCN2020118426-appb-000009
实验例3体外肝微粒体稳定性
300μL最终的温孵体系中，含30μL肝微粒体(蛋白浓度：0.15mg/mL，Corning)，30μL NADPH+MgCl 2，3μL底物(乙腈配制)，237μL PBS缓冲液。每个种属做2份，每份0.3mL。每管先配好总体积为270μL的底物及酶的混匀液，和NADPH分别在37℃预温孵5min后，加入30μL NADPH+MgCl 2混合溶液反应，分别于0、10、30、60min取出50μL用含内标的冰乙腈300μL终止反应。
样品前处理：50μL温孵样品，加入300μL含内标***的冰乙腈沉淀，涡旋震荡5min后，离心(12000rpm，4℃)10min。吸取上清液75μL至96孔板中用75μL超纯水稀释混匀，进样0.5μL，进行LC-MS/MS分析。结果如表8所示。
表8 体外肝微粒体稳定性
Figure PCTCN2020118426-appb-000010
实验例4体内动物药效
4.1 AAV小鼠模型评价抗病毒效果
取6-8周龄雄性C57BL/6小鼠(上海灵畅生物科技有限公司)，按照1×10 11vg剂量，尾静脉注射rAAV8-1.3HBV病毒(北京五加和，adr亚型)至C57BL/6小鼠体内。注射病毒第2、4周，小鼠眼眶采血，分离血清，测定血清中HBeAg和HBsAg表达水平以及HBV DNA拷贝数，判断模型构建成功与否。结合血清学HBeAg、HBsAg和HBV DNA的定量检测结果，挑选出的小鼠各自HBV DNA表达水平都大于1×10 4IU/mL，HBeAg大于1×10 3NCU/mL和HBsAg大于1×10 3ng/mL。小鼠进行分组，设空白对照组、溶媒对照组、受试物组。每组小鼠以灌胃方式连续给药3周，每日1次，停药1周。实验过程中，隔周分别眼眶采血，分离血清，荧光定量PCR方法检测DNA含量。结果如表9和表10所示。
表9 血清中HBV DNA下降水平(log10)(给药剂量10mpk)
编号 第7天 第14天 第21天 第28天
式I化合物 2.67 3.80 4.30 3.50
表10 血清中HBV DNA下降水平(log10)(给药剂量1mpk)
编号 第7天 第14天 第21天 第28天
式I化合物 2.49 3.41 3.65 3.25
4.2 pAAV/HBV模型实验方法
取6-8周龄雄性C57BL/6小鼠(上海灵畅生物科技有限公司)，将纯化的重组质粒pAAV/HBVl.2(10μg)溶解在PBS中，每只小鼠注射体积约为其体重的10％，通过尾静脉在3-8s内注射到小鼠体内。注射质粒3天后眼眶取血检测血清HBV DNA，挑选出模型小鼠血清DNA均一的进行分组，设模型对照组、溶媒对照组、受试物组。每组小鼠以灌胃方式连续给药10天，每日1次，剂量为3mg/kg。分别于给药后的0、4、7、10天取小鼠血清，第10天处死小鼠取肝组织样本，荧光定量PCR方法检测小鼠血清和肝脏中HBV DNA拷贝数。结果如表11所示。
表11 血清中HBV DNA下降水平(log10)(给药剂量3mpk)
编号 第4天 第7天 第10天
式I化合物 2.95 4.13 2.71
实验例5体内药物代谢动力学
大鼠体内药物代谢动力学(PK)研究
SD大鼠(上海西普尔-必凯)，体重180～220g，适应3～5天后，随机分组，每组3只，按20mg/kg剂量分别灌胃系列化合物。
受试动物(SD大鼠)给药前禁食12h，给药后4h给食物，实验前后和实验过程中均自由饮水。
给药后，于0min、15min、30min、1h、2h、4h、6h、8h、10h、24h、30h、48h于眼眶取血0.2mL左右，EDTA-K2抗凝后，30min内于4℃，4000rpm条件下离心10min分离血浆。收集全部血浆后立即于-20℃保存待测。
吸取50μL待测血浆样品和标曲样品，加入500μL含内标(***20mg/mL)的乙腈溶液，振荡混匀5min，12000rpm离心10min，取上清75μL，加入75μL超纯水稀释，混匀，吸取1μL用于LC/MS/MS测定。结果如表12所示。
表12
Figure PCTCN2020118426-appb-000011
Figure PCTCN2020118426-appb-000012
NA表示未检测。

Claims (10)

  1. 式I化合物的结晶，
    Figure PCTCN2020118426-appb-100001
  2. 如权利要求1所述的式I化合物的结晶，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：8.25±0.20°、10.10±0.20°、14.46±0.20°、15.48±0.20°和18.97±0.20°；任选地，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：8.25±0.20°、9.46±0.20°、10.10±0.20°、14.46±0.20°、15.48±0.20°、18.97±0.20°、20.33±0.20°和22.02±0.20°；任选地，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：8.25±0.20°、9.46±0.20°、10.10±0.20°、14.46±0.20°、15.48±0.20°、18.97±0.20°、20.33±0.20°、20.80±0.20°、21.67±0.20°、22.02±0.20°、24.37±0.20°、26.37±0.20°和30.77±0.20°；任选地，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：8.25±0.20°、9.46±0.20°、10.10±0.20°、14.46±0.20°、15.48±0.20°、18.97±0.20°、20.33±0.20°、20.80±0.20°、21.67±0.20°、22.02±0.20°、22.88±0.20°、23.93±0.20°、24.37±0.20°、24.97±0.20°、26.37±0.20°、28.68±0.20°、和30.77±0.20°。
  3. 如权利要求2所述的式I化合物的结晶，其XRPD图谱如图1所示。
  4. 如权利要求2所述的式I化合物的结晶，其DSC曲线在227.34±5℃处有吸热峰。
  5. 晶型组合物，所述权利要求1-4任一项所述的式I化合物的结晶占晶型组合物重量的50％以上，较好为80％以上，更好是90％以上，最好是95％以上。
  6. 药物组合物，其包含治疗有效量的如权利要求1-4任一项所述的式I化合物的结晶、或权利要求5所述的晶型组合物。
  7. 如权利要求1-4任一项所述的式I化合物的结晶、权利要求5所述的晶型组合物、或者权利要求6所述的药物组合物在制备预防或治疗受益于衣壳蛋白装配抑制的疾病的药物中的用途。
  8. 如权利要求7所述的用途，其中所述受益于衣壳蛋白装配抑制的疾病指乙型肝炎病毒感染引起的疾病。
  9. 如权利要求1-4任一项所述的式I化合物的结晶、或者如权利要求5所述的晶型组合物、或者如权利要求6所述的药物组合物在制备预防或治疗乙型肝炎病毒感染的药物中的用途。
  10. 用于预防或治疗受益于衣壳蛋白装配抑制的疾病的权利要求1-4任一项所述的式I化合物的结晶、或者如权利要求5所述的晶型组合物、或者如权利要求6所述的药物组合物。
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