WO2020151746A1 - 1,2,3-三氮唑并[1,5-a]吡嗪类衍生物的晶型及其制备方法 - Google Patents

1,2,3-三氮唑并[1,5-a]吡嗪类衍生物的晶型及其制备方法 Download PDF

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WO2020151746A1
WO2020151746A1 PCT/CN2020/073802 CN2020073802W WO2020151746A1 WO 2020151746 A1 WO2020151746 A1 WO 2020151746A1 CN 2020073802 W CN2020073802 W CN 2020073802W WO 2020151746 A1 WO2020151746 A1 WO 2020151746A1
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crystal form
compound
ray powder
triazolo
angle
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PCT/CN2020/073802
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English (en)
French (fr)
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韩龙
邵启云
冯君
贺峰
马亚辉
赵苗苗
杜振兴
王捷
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Priority to CA3126175A priority Critical patent/CA3126175A1/en
Priority to US17/425,495 priority patent/US20220081446A1/en
Priority to AU2020210998A priority patent/AU2020210998A1/en
Priority to EP20745058.6A priority patent/EP3915993A1/en
Priority to JP2021542521A priority patent/JP2022523035A/ja
Priority to CN202080007547.8A priority patent/CN113227096B/zh
Priority to KR1020217026840A priority patent/KR20210120036A/ko
Priority to BR112021014327-0A priority patent/BR112021014327A2/pt
Priority to MX2021008929A priority patent/MX2021008929A/es
Publication of WO2020151746A1 publication Critical patent/WO2020151746A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • 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
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4985—Pyrazines or piperazines ortho- or peri-condensed with heterocyclic ring systems
    • 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
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • 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
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • 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
    • 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
    • A61P31/22—Antivirals for DNA viruses for herpes 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

  • the present disclosure provides the compound (S)-N 5 -(3,4-difluorophenyl)-6-methyl-N 3 -((R)-1,1,1-trifluoropropan-2-yl)
  • PCT/CN2018/097170 (application date July 26, 2018) describes a compound (S)-N 5 -(3,4-difluorophenyl)-6-methyl-N 3 -((R) -1,1,1-Trifluoroprop-2-yl)-6,7-dihydro-[1,2,3]triazolo[1,5-a]pyrazine-3,5(4H) -Diformamide, pharmacodynamic experiments show that the compound has a significant inhibitory effect on the normal assembly of HBV capsid protein, and its pharmacokinetic absorption is good, and the bioavailability is high. At the same time, the compound of the new structure has no or little effect on the inhibition of HepG2 cell proliferation in vitro, and shows better safety.
  • Polymorphism refers to the existence of two or more different spatial arrangements of solid substances, which have different physical and chemical properties. There may also be differences in the bioavailability of different crystal forms of the same drug due to the difference in arrangement.
  • the compound (S)-N 5 -(3,4-difluorophenyl)-6-methyl-N 3- ((R)-1,1,1-trifluoroprop-2-yl)-6,7-dihydro-[1,2,3]triazolo[1,5-a]pyrazine-3 The study of polymorphs of 5(4H)-dimethylformamide to obtain crystals with high purity and stable chemical properties is of great significance to the development of drugs suitable for industrial production and with good biological activity.
  • the present disclosure provides compound (S)-N 5 -(3,4-difluorophenyl)-6-methyl-N 3 -((R)-1,1,1-trifluoropropane- 2-yl)-6,7-dihydro-[1,2,3]triazolo[1,5-a]pyrazine-3,5(4H)-dimethylformamide, crystal form A, by diffraction
  • the X-ray powder diffraction pattern represented by angle 2 ⁇ has characteristic peaks at 13.197, 14.239, 15.839, 17.680, 19.080, 19.780, and 22.539.
  • the crystalline form A has an X-ray powder diffraction pattern represented by a diffraction angle of 2 ⁇ , with characteristic peaks at 13.197, 14.239, 15.320, 15.839, 17.680, 19.080, 19.780, 22.539, and 25.519 .
  • the crystal form A has an X-ray powder diffraction pattern represented by a diffraction angle of 2 ⁇ angles at 11.022, 13.197, 14.239, 15.320, 15.839, 17.680, 19.080, 19.780, 20.879, 22.539, 25.519 and There is a characteristic peak at 26.041.
  • the X-ray powder diffraction pattern of the crystal form A expressed by the diffraction angle 2 ⁇ is shown in FIG. 2.
  • the present disclosure also provides the preparation compound (S)-N 5 -(3,4-difluorophenyl)-6-methyl-N 3 -((R)-1,1,1-trifluoropropane-2- Yl)-6,7-dihydro-[1,2,3]triazolo[1,5-a]pyrazine-3,5(4H)-dimethylformamide crystal form
  • said Methods include:
  • solvent (I) The compound (S)-N 5 -(3,4-difluorophenyl)-6-methyl-N 3 -((R)-1,1,1-trifluoropropan-2-yl) -6,7-Dihydro-[1,2,3]triazolo[1,5-a]pyrazine-3,5(4H)-dimethylamide is added to solvent (I), stirred to dissolve or heated Dissolving, the solvent (I) is selected from at least one of ethyl acetate, dichloromethane, isopropanol, isopropyl ether, preferably isopropanol/isopropyl ether, ethyl acetate/n-hexane, or dichloro Methane/isopropyl ether,
  • the solvent (I) in this method is selected from a mixed solvent of isopropyl alcohol/isopropyl ether, and the volume ratio of isopropyl alcohol to isopropyl ether is 2:1 to 1:10, which may be 2: 1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 and any between any two values
  • the ratio is preferably 1:3, 1:4 or 1:5.
  • the solvent (I) in this method is selected from a mixed solvent of ethyl acetate/n-hexane, and the volume ratio of ethyl acetate to n-hexane is 2:1 to 1:10, which can be 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 and any ratio between any two values, Preferably, 1:3, 1:4 or 1:5.
  • the solvent (I) in this method is selected from a mixed solvent of dichloromethane/isopropyl ether, and the volume ratio of dichloromethane to isopropyl ether is 1:5 to 1:30, which can be 1. :5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17 , 1:18, 1:19, 1:20, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30 and any Any ratio between the two values, preferably 1:20, 1:22 or 1:25.
  • the volume (ml) used in the solvent (I) in this method is 1-20 times the weight (g) of the compound, which can be 1, 2, 3, 4, 5, 6, 7, 8, 9. , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 times, and any value between any two values.
  • the present disclosure also provides the compound (S)-N 5 -(3,4-difluorophenyl)-6-methyl-N 3 -((R)-1,1,1-trifluoropropane -2-yl)-6,7-dihydro-[1,2,3]triazolo[1,5-a]pyrazine-3,5(4H)-dimethylformamide in crystalline form B,
  • the X-ray powder diffraction pattern represented by the diffraction angle 2 ⁇ angle has characteristic peaks at 6.273, 12.680, 14.178, 15.475, 17.685, 19.045 and 22.450.
  • the crystalline form B has an X-ray powder diffraction pattern represented by a diffraction angle of 2 ⁇ , with characteristic peaks at 6.273, 11.687, 12.680, 14.178, 15.475, 17.198, 17.685, 19.045 and 22.450 .
  • the crystal form B has an X-ray powder diffraction pattern expressed by a diffraction angle of 2 ⁇ as shown in FIG. 5.
  • the present disclosure also provides the compound (S)-N 5 -(3,4-difluorophenyl)-6-methyl-N 3 -((R)-1,1,1-trifluoropropan-2-yl )-6,7-dihydro-[1,2,3]triazolo[1,5-a]pyrazine-3,5(4H)-dimethylformamide crystal form C, with diffraction angle 2 ⁇
  • the indicated X-ray powder diffraction pattern has characteristic peaks at 6.326, 10.317, 11.833, 12.826, 13.805, 20.529 and 23.773.
  • the crystalline form C has an X-ray powder diffraction pattern expressed by diffraction angle 2 ⁇ angles at 6.326, 10.317, 11.833, 12.826, 13.805, 15.499, 16.875, 18.546, 20.529 and 23.773. Characteristic peaks.
  • the crystal form C has an X-ray powder diffraction pattern represented by a diffraction angle of 2 ⁇ as shown in FIG. 6.
  • the present disclosure also provides the compound (S)-N 5 -(3,4-difluorophenyl)-6-methyl-N 3 -((R)-1,1,1-trifluoropropan-2-yl )-6,7-dihydro-[1,2,3]triazolo[1,5-a]pyrazine-3,5(4H)-dimethylformamide crystal form D, with diffraction angle 2 ⁇
  • the indicated X-ray powder diffraction pattern has characteristic peaks at 10.296, 12.415, 15.542, 18.521, 19.298, 23.004 and 25.956.
  • the crystalline form D has an X-ray powder diffraction pattern represented by a diffraction angle of 2 ⁇ , with characteristic peaks at 10.296, 12.415, 15.542, 16.660, 18.521, 19.298, 20.058, 23.004 and 25.956 .
  • the crystalline form D the X-ray powder diffraction pattern expressed by diffraction angle 2 ⁇ , has characteristic peaks at 10.296, 12.415, 15.542, 16.660, 18.521, 19.298, 20.058, 21.214, 22.039, 23.004 and 25.956 .
  • the crystal form D has an X-ray powder diffraction pattern represented by a diffraction angle of 2 ⁇ as shown in FIG. 7.
  • the present disclosure also provides a pharmaceutical composition prepared from any of the aforementioned crystal forms. Further, the pharmaceutical composition also contains pharmaceutically acceptable carriers, diluents or excipients.
  • the present disclosure also provides a pharmaceutical composition, which contains the crystal form of the aforementioned compound and is optionally selected from a pharmaceutically acceptable carrier, diluent or excipient.
  • the present disclosure also provides the use of the aforementioned crystalline compound or the aforementioned pharmaceutical composition in the preparation of a medicament for the prevention and/or treatment of viral infections.
  • the virus can be hepatitis B virus, influenza virus, herpes virus and One or more of HIV, and the disease may be one or more of hepatitis B, influenza, herpes and AIDS.
  • the present disclosure also provides the use of the aforementioned crystalline compound or the aforementioned pharmaceutical composition in the preparation of drugs for capsid protein inhibitors.
  • the present disclosure also provides a method for preventing and/or treating viral infectious diseases, which comprises administering a therapeutically effective dose of a compound of the aforementioned crystal form to a patient in need thereof.
  • the virus may be hepatitis B virus, influenza virus, One or more of herpes virus and HIV, and the disease may be one or more of hepatitis B, influenza, herpes and AIDS.
  • Deliquescence absorb enough water to form a liquid
  • moisture absorption moisture absorption is less than 15% but not less than 2%;
  • weight gain by moisture absorption is less than 2% but not less than 0.2%;
  • the crystalline form A of the present disclosure under the condition of 90% RH, the moisture absorption and weight gain are 0.21%, slightly hygroscopic, or no/almost no hygroscopicity.
  • the "X-ray powder diffraction pattern" described in the present disclosure is measured using Cu-K ⁇ radiation.
  • the preparation method of the crystal form described in the present disclosure also includes steps such as filtration, washing or drying.
  • the Bragg equation can be satisfied on a certain atomic plane with d lattice plane spacing, and this group of X-ray powder diffraction patterns can be measured.
  • the "2 ⁇ or 2 ⁇ angle" mentioned in the present disclosure refers to the diffraction angle, ⁇ is the Bragg angle, and the unit is ° or degree; the error range of each characteristic peak 2 ⁇ is ⁇ 0.20, which can be -0.20, -0.19, -0.18 , -0.17, -0.16, -0.15, -0.14, -0.13, -0.12, -0.11, -0.10, -0.09, -0.08, -0.07, -0.06, -0.05, -0.04, -0.03, -0.02,- 0.01, 0.00, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20.
  • interplanar spacing or interplanar spacing (d value) means that the spatial lattice selects three non-parallel unit vectors a, b, and c connecting two adjacent lattice points.
  • the lattice is divided into juxtaposed parallelepiped units, called interplanar spacing.
  • the spatial lattice is divided according to the determined parallelepiped unit lines to obtain a set of linear grids, called spatial lattices or lattices.
  • Lattice and lattice use geometric points and lines to reflect the periodicity of the crystal structure.
  • the interplanar spacing that is, the distance between two adjacent parallel crystal planes
  • the unit is Or angstrom.
  • the “differential scanning calorimetry or DSC” mentioned in the present disclosure refers to the measurement of the temperature difference and heat flow difference between the sample and the reference material during the temperature rise or constant temperature process of the sample to characterize all physical changes related to thermal effects and Chemical changes to obtain phase change information of the sample.
  • the drying temperature mentioned in the present disclosure is generally 20°C to 100°C, preferably 25°C to 70°C, and can be dried under normal pressure or under reduced pressure (vacuum drying). Preferably, drying is dried under reduced pressure.
  • the monitoring of the reaction progress in the examples adopts thin-layer chromatography (TLC), the developing reagent used in the reaction, the eluent system of column chromatography used in the purification of the compound, and the developing reagent system of thin-layer chromatography include: A: Dichloromethane/methanol system, B: n-hexane/ethyl acetate system, C: petroleum ether/ethyl acetate system, the volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of triethylamine and Adjust with alkaline or acidic reagents such as acetic acid. Test conditions of the instruments used in the experiments in this disclosure:
  • XRPD is X-ray powder diffraction detection: the measurement is carried out using BRUKER D8 X-ray diffractometer, the specific information collected: Cu anode (40kV, 40mA), Cu-K ⁇ 1 ray ( ), K ⁇ 2 rays ( ), K ⁇ rays ( ).
  • the step-by-step scanning method is adopted, the number of scanning steps is 3, the scanning range of each step is 19°, the starting degree is 5°, the ending degree is 48°, and the length of each step is 75s.
  • DSC is differential scanning calorimetry: METTLER TOLEDO DSC 3+ differential scanning calorimeter is used for the measurement, the heating rate is 10°C/min, the specific temperature range refers to the corresponding graph (25-300 or 25-350°C), nitrogen purge speed 50mL/min.
  • TGA thermogravimetric analysis: METTLER TOLEDO TGA 2 type thermogravimetric analyzer is used for detection, the heating rate is 10°C/min, the specific temperature range refers to the corresponding graph (25-300°C), and the nitrogen purge speed is 20mL/min.
  • DVS dynamic moisture adsorption: the detection adopts SMS DVS Advantage, at 25°C, the humidity change is 50%-95%-0%-95%-50%, and the step is 10% (the last step is 5%) (specific humidity range) Based on the corresponding map, most of the methods listed here are used), the judgment standard is dm/dt not greater than 0.02%.
  • HPLC determination uses Agilent 1200DAD high pressure liquid chromatograph (Sunfire C18 150 ⁇ 4.6mm column) and Waters 2695-2996 high pressure liquid chromatograph (Gimini C18 150 ⁇ 4.6mm column).
  • the structure of the compound is determined by nuclear magnetic resonance (NMR) or/and mass spectrometry (MS).
  • NMR nuclear magnetic resonance
  • MS mass spectrometry
  • ⁇ is given in units of 10-6 (ppm).
  • NMR was measured with Bruker AVANCE-400 nuclear magnetic instrument, and the solvent was deuterated dimethyl sulfoxide (DMSO-d 6 ), deuterated chloroform (CDCl 3 ), deuterated methanol (CD 3 OD), and the internal standard was four Methylsilane (TMS); FINNIGAN LCQAd (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advantage MAX) was used for MS determination.
  • DMSO-d 6 deuterated dimethyl sulfoxide
  • CDCl 3 deuterated chloroform
  • CD 3 OD deuterated methanol
  • TMS Methylsilane
  • ESI FINNIGAN LCQAd
  • Figure 1 Amorphous XRPD pattern of the compound.
  • Figure 2 XRPD spectrum of the crystalline form A of the compound.
  • Figure 3 XRPD pattern of crystal form A in Example 5.
  • Figure 4 Comparison of X-ray powder diffraction before and after DVS of crystalline form A of the compound.
  • Figure 5 XRPD pattern of crystalline form B of the compound.
  • Figure 6 XRPD pattern of crystalline form C of the compound.
  • Figure 7 XRPD pattern of crystalline form D of the compound.
  • the crude compound 1d (2.20 g, 6.79 mmol) was dissolved in 20 mL of N,N-dimethylformamide, sodium azide (574 mg, 8.83 mmol,) was added, and the reaction was carried out at 80°C for 12 hours.
  • the reaction solution was cooled to room temperature, 50mL ethyl acetate was added, washed with water (20mL ⁇ 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure.
  • the resulting residue was purified by silica gel column chromatography with eluent system A to obtain the compound 1e (1.30 g, yield: 57.9%).
  • Test Example 1 In vitro anti-HBV activity test (quantitative analysis of intracellular HBV DNA)
  • HepG2.2.15 cells are stable expression cell lines that integrate the HBV genome, which can be secreted out of the cell through replication, transcription, translation, and packaging into virus particles with HBV DNA.
  • quantitative PCR was used to quantitatively analyze the HBV DNA produced by HepG2.2.15 in vitro proliferation, and the activity of the compounds in the present disclosure to inhibit HBV DNA replication by inhibiting the assembly of HBV capsid protein was determined.
  • HepG2.2.15 cells were cultured in DMEM/high glucose medium (10% FBS, 400 ⁇ g/ml G418), and passaged every 3 days. On the day of the experiment, a cell suspension was prepared with fresh cell culture medium, and cultured at 40,000 cells/well in a 96-well plate (Corning, #3599), 5% carbon dioxide, at 37°C. On the second day, the compound was dissolved in pure DMSO to a concentration of 20 mM, and then prepared with DMSO to make the first concentration of 2 mM, and then diluted 4-fold to 8 concentrations. Set the control wells and add 90 ⁇ L DMSO. Dilute 200 times with DMEM/high glucose medium.
  • the extracted DNA was eluted with DNA elution buffer at 100 ⁇ L/well.
  • DNA elution buffer 100 ⁇ L/well.
  • the quantitative standard curve adopts the standard sample that comes with the kit, and the experiment is carried out in parallel. Perform quantitative conversion for each sample according to the standard curve.
  • Graphpad Prism software was used to calculate the EC 50 value of the compound according to the concentration of the compound and the corresponding DNA value. Emax is the compound's maximum inhibition of HBV DNA replication.
  • Test Example 2 Effect on the proliferation of HepG2 cells in vitro
  • the DVS test showed that under normal storage conditions (ie 25°C, 60% RH), the sample's moisture gain was about 0.07%; under accelerated test conditions (ie 70% RH), the moisture gain was about 0.10%; under extreme conditions At 90% RH, the weight gain by moisture absorption is about 0.21%. During the 0%-95%RH humidity change process, the desorption process of this sample is consistent with the adsorption process.
  • the crystal form was retested after DVS detection, and the crystal form did not change, as shown in Figure 4 (a is the XRPD pattern after DVS detection, and b is the XRPD pattern before DVS detection).
  • Example 5 Place the crystal form A (Example 5) sample open and flat, and examine the stability of the sample under heating (40°C, 60°C), light (4500 Lux), and high humidity (RH 75%, RH 90%) conditions.
  • the sampling period is 30 days.

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Abstract

本公开提供了1,2,3-三氮唑并[1,5-a]吡嗪类衍生物的晶型及其制备方法。具体而言，本公开提供了化合物(S)-N5-(3,4-二氟苯基)-6-甲基-N3-((R)-1,1,1-三氟丙-2-基)-6,7-二氢-[1,2,3]三氮唑并[1,5-a]吡嗪-3,5(4H)-二甲酰胺的晶型及其制备方法。制备得到的晶型具有良好的稳定性以及临床应用价值。

Description

1,2,3-三氮唑并[1,5-a]吡嗪类衍生物的晶型及其制备方法
本申请要求申请日为2019年1月25日的中国专利申请CN201910072048.6的优先权。本申请引用上述中国专利申请的全文。
技术领域
本公开提供了化合物(S)-N 5-(3,4-二氟苯基)-6-甲基-N 3-((R)-1,1,1-三氟丙-2-基)-6,7-二氢-[1,2,3]三氮唑并[1,5-a]吡嗪-3,5(4H)-二甲酰胺的晶型及其制备方法。
背景技术
PCT/CN2018/097170(申请日2018年7月26日)描述了一种化合物(S)-N 5-(3,4-二氟苯基)-6-甲基-N 3-((R)-1,1,1-三氟丙-2-基)-6,7-二氢-[1,2,3]三氮唑并[1,5-a]吡嗪-3,5(4H)-二甲酰胺，药效实验显示了该化合物对HBV衣壳蛋白正常装配具有明显的抑制作用，并且其药代吸收良好，生物利用度高。同时该新型结构的化合物对HepG2细胞体外增殖抑制没有影响或影响较小，表现出较好的安全性。
多晶型现象是指固态物质存在两种或两种以上不同的空间排列方式，从而具有不同物理、化学性质。同种药物不同晶型之间由于排列方式的不同，其生物利用度也可能会存在差别。同时，鉴于固体药物晶型及其稳定性对其在临床治疗中的重要性，深入进行化合物(S)-N 5-(3,4-二氟苯基)-6-甲基-N 3-((R)-1,1,1-三氟丙-2-基)-6,7-二氢-[1,2,3]三氮唑并[1,5-a]吡嗪-3,5(4H)-二甲酰胺的多晶型研究，获得纯度高且化学性质稳定的晶型，对开发适合工业生产且生物活性良好的药物具有重要意义。
发明内容
本公开(The disclosure)提供了化合物(S)-N 5-(3,4-二氟苯基)-6-甲基-N 3-((R)-1,1,1-三氟丙-2-基)-6,7-二氢-[1,2,3]三氮唑并[1,5-a]吡嗪-3,5(4H)-二甲酰胺的晶型A，以衍射角2θ角度表示的X-射线粉末衍射图谱，在13.197、14.239、15.839、17.680、19.080、19.780和22.539处有特征峰。
在可选实施方案中，所述的晶型A，以衍射角2θ角度表示的X-射线粉末衍射图谱，在13.197、14.239、15.320、15.839、17.680、19.080、19.780、22.539和25.519处有特征峰。
在一些实施方案中，所述的晶型A，以衍射角2θ角度表示的X-射线粉末衍射图谱， 在11.022、13.197、14.239、15.320、15.839、17.680、19.080、19.780、20.879、22.539、25.519和26.041处有特征峰。
在另一些实施方案中，所述的晶型A，以衍射角2θ角度表示的X-射线粉末衍射图谱如图2所示。
本公开还提供了制备化合物(S)-N 5-(3,4-二氟苯基)-6-甲基-N 3-((R)-1,1,1-三氟丙-2-基)-6,7-二氢-[1,2,3]三氮唑并[1,5-a]吡嗪-3,5(4H)-二甲酰胺的晶型A的方法，所述方法包括：
(a)将化合物(S)-N 5-(3,4-二氟苯基)-6-甲基-N 3-((R)-1,1,1-三氟丙-2-基)-6,7-二氢-[1,2,3]三氮唑并[1,5-a]吡嗪-3,5(4H)-二甲酰胺加入溶剂(I)中，搅拌溶解或加热溶解，所述溶剂(I)选自乙酸乙酯、二氯甲烷、异丙醇、异丙醚中的至少一种，优选异丙醇/异丙醚、乙酸乙酯/正己烷，或二氯甲烷/异丙醚，
(b)搅拌析晶。
在一些实施方案中，本法所述溶剂(I)选自异丙醇/异丙醚的混合溶剂，异丙醇与异丙醚的体积比为2:1～1:10，可以为2:1、1:1、1:2、1:3、1:4、1:5、1:6、1:7、1:8、1:9、1:10及任意两个数值之间的任意比值，优选1:3、1:4或1:5。
在一些实施方案中，本法所述溶剂(I)选自乙酸乙酯/正己烷的混合溶剂，乙酸乙酯与正己烷的体积比为2:1～1:10，可以为2:1、1:1、1:2、1:3、1:4、1:5、1:6、1:7、1:8、1:9、1:10及任意两个数值之间的任意比值，优选1:3、1:4或1:5。
在另一些实施方案中，本法所述溶剂(I)选自二氯甲烷/异丙醚的混合溶剂，二氯甲烷与异丙醚的体积比为1:5～1:30，可以为1:5、1:6、1:7、1:8、1:9、1:10、1:11、1:12、1:13、1:14、1:15、1:16、1:17、1:18、1:19、1:20、1:22、1:23、1:24、1:25、1:26、1:27、1:28、1:29、1:30及任意两个数值之间的任意比值，优选1:20、1:22或1:25。
在一些实施方案中，本法所述溶剂(I)所用体积(ml)为化合物重量(g)的1～20倍，可以为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20倍，及任意两个数值之间的任意值。
另一方面，本公开还提供了化合物(S)-N 5-(3,4-二氟苯基)-6-甲基-N 3-((R)-1,1,1-三氟丙-2-基)-6,7-二氢-[1,2,3]三氮唑并[1,5-a]吡嗪-3,5(4H)-二甲酰胺的晶型B，以衍射角2θ角度表示的X-射线粉末衍射图谱，在6.273、12.680、14.178、15.475、17.685、19.045和22.450处有特征峰。
在可选实施方案中，所述的晶型B，以衍射角2θ角度表示的X-射线粉末衍射图谱，在6.273、11.687、12.680、14.178、15.475、17.198、17.685、19.045和22.450处有特征峰。
在另一些实施方案中，所述的晶型B，以衍射角2θ角度表示的X-射线粉末衍射图谱如图5所示。
本公开还提供了化合物(S)-N 5-(3,4-二氟苯基)-6-甲基-N 3-((R)-1,1,1-三氟丙-2-基)-6,7-二氢-[1,2,3]三氮唑并[1,5-a]吡嗪-3,5(4H)-二甲酰胺的晶型C，以衍射角2θ角度表示的X-射线粉末衍射图谱，在6.326、10.317、11.833、12.826、13.805、20.529和23.773处有特征峰。
在可选实施方案中，所述的晶型C，以衍射角2θ角度表示的X-射线粉末衍射图谱，在6.326、10.317、11.833、12.826、13.805、15.499、16.875、18.546、20.529和23.773处有特征峰。
在另一些实施方案中，所述的晶型C，以衍射角2θ角度表示的X-射线粉末衍射图谱如图6所示。
本公开还提供了化合物(S)-N 5-(3,4-二氟苯基)-6-甲基-N 3-((R)-1,1,1-三氟丙-2-基)-6,7-二氢-[1,2,3]三氮唑并[1,5-a]吡嗪-3,5(4H)-二甲酰胺的晶型D，以衍射角2θ角度表示的X-射线粉末衍射图谱，在10.296、12.415、15.542、18.521、19.298、23.004和25.956处有特征峰。
在可选实施方案中，所述的晶型D，以衍射角2θ角度表示的X-射线粉末衍射图谱，在10.296、12.415、15.542、16.660、18.521、19.298、20.058、23.004和25.956处有特征峰。
优选地，所述的晶型D，以衍射角2θ角度表示的X-射线粉末衍射图谱，在10.296、12.415、15.542、16.660、18.521、19.298、20.058、21.214、22.039、23.004和25.956处有特征峰。
在另一些实施方案中，所述的晶型D，以衍射角2θ角度表示的X-射线粉末衍射图谱如图7所示。
本公开还提供了一种由前述晶型任意一种制备而成的药物组合物。进一步地，所述药物组合物中还含有自药学上可接受的载体、稀释剂或赋形剂。
本公开还提供了一种药物组合物，其含有前述化合物的晶型和任选自药学上可接受的载体、稀释剂或赋形剂。
本公开还提供了前述晶型的化合物或前述药物组合物在制备用于预防和/或治疗病毒性感染疾病的药物中的用途，所述病毒可以为乙型肝炎病毒、流感病毒、疱疹病毒和艾滋病毒中的一种或多种，所述疾病可以为乙型肝炎、流感、疱疹和艾滋病中的一种或多种。
本公开还提供了前述晶型的化合物或前述药物组合物在制备用于衣壳蛋白抑制剂的药物中的用途。
本公开还提供了一种预防和/或治疗病毒性感染疾病的方法，其包括向需要其的患者施用治疗有效剂量的前述晶型的化合物，所述病毒可以为乙型肝炎病毒、流感病毒、疱疹病毒和艾滋病毒中的一种或多种，所述疾病可以为乙型肝炎、流感、疱疹和艾滋病中的一种或多种。
依据《中国药典》2015年版四部中“9103药物引湿性指导原则”中引湿性特征描述与引湿性增重的界定，
潮解：吸收足量水分形成液体；
极具引湿性：引湿增重不小于15％；
有引湿性：引湿增重小于15％但不小于2％；
略有引湿性：引湿增重小于2％但不小于0.2％；
无或几乎无引湿性：引湿增重小于0.2％。
本公开所述(S)-N 5-(3,4-二氟苯基)-6-甲基-N 3-((R)-1,1,1-三氟丙-2-基)-6,7-二氢-[1,2,3]三氮唑并[1,5-a]吡嗪-3,5(4H)-二甲酰胺的晶型A在70％RH条件下，引湿增重0.1％，小于0.2％，无或几乎无引湿性。
进一步地，本公开晶型A在90％RH条件下，引湿增重0.21％，略有引湿性，或无/几乎无引湿性。
本公开所述的“X-射线粉末衍射图谱”为使用Cu-Kα辐射测量得到。
本公开所述晶型的制备方法中还包括过滤、洗涤或干燥等的步骤。
本公开所述的“X-射线粉末衍射图谱或XRPD”是指根据布拉格公式2d sinθ＝nλ(式中，λ为X射线的波长，
Figure PCTCN2020073802-appb-000001
衍射的级数n为任何正整数，一般取一级衍射峰，n＝1)，当X射线以掠射角θ(入射角的余角，又称为布拉格角)入射到晶体或部分晶体样品的某一具有d点阵平面间距的原子面上时，就能满足布拉格方程，从而测得了这组X射线粉末衍射图。
本公开中所述的“2θ或2θ角度”是指衍射角，θ为布拉格角，单位为°或度；每个特征峰2θ的误差范围为±0.20，可以为-0.20、-0.19、-0.18、-0.17、-0.16、-0.15、-0.14、-0.13、-0.12、-0.11、-0.10、-0.09、-0.08、-0.07、-0.06、-0.05、-0.04、-0.03、-0.02、-0.01、0.00、0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.10、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.20。
本公开中所述的“晶面间距或晶面间距(d值)”是指空间点阵选择3个不相平行的连 结相邻两个点阵点的单位矢量a，b，c，它们将点阵划分成并置的平行六面体单位，称为晶面间距。空间点阵按照确定的平行六面体单位连线划分，获得一套直线网格，称为空间格子或晶格。点阵和晶格是分别用几何的点和线反映晶体结构的周期性，不同的晶面，其面间距(即相邻的两个平行晶面之间的距离)各不相同；单位为
Figure PCTCN2020073802-appb-000002
或埃。
本公开中所述的“差示扫描量热分析或DSC”是指在样品升温或恒温过程中，测量样品与参考物之间的温度差、热流差，以表征所有与热效应有关的物理变化和化学变化，得到样品的相变信息。
本公开中所述干燥温度一般为20℃～100℃，优选25℃～70℃，可以常压干燥，也可以减压干燥(真空干燥)。优选地，干燥在减压下干燥。
本公开中所用化学试剂、生物试剂可通过商业途径获得获得。本公开中化合物B参照PCT/CN2018/097170(申请日2018年7月26日)中方法进行，并将其中相关药理药效及动物体内研究内容引入本公开中以示说明。
实施例中的反应进程的监测采用薄层色谱法(TLC)，反应所使用的展开剂，纯化化合物采用的柱层析的洗脱剂的体系和薄层色谱法的展开剂体系包括：A：二氯甲烷/甲醇体系，B：正己烷/乙酸乙酯体系，C：石油醚/乙酸乙酯体系，溶剂的体积比根据化合物的极性不同而进行调节，也可以加入少量的三乙胺和醋酸等碱性或酸性试剂进行调节。本公开中实验所用仪器的测试条件：
XRPD为X射线粉末衍射检测：测定使用BRUKER D8型X射线衍射仪进行，具体采集信息：Cu阳极(40kV，40mA)，Cu-Kα1射线(
Figure PCTCN2020073802-appb-000003
)，Kα2射线(
Figure PCTCN2020073802-appb-000004
)，Kβ射线(
Figure PCTCN2020073802-appb-000005
)。扫描范围(2q范围)：3～64°、扫描步长0.02、狭缝宽度(准直器)1.0mm。采用分步扫描法，扫描步数为3步，每步扫描范围19°，起始度数5°，终止度数48°，每步时长75s。
DSC为差示扫描量热：测定采用METTLER TOLEDO DSC 3+示差扫描量热仪，升温速率10℃/min，温度具体范围参照相应图谱(为25-300或25-350℃)，氮气吹扫速度50mL/min。
TGA为热重分析：检测采用METTLER TOLEDO TGA 2型热重分析仪，升温速率10℃/min，温度具体范围参照相应图谱(为25-300℃)，氮气吹扫速度20mL/min。
DVS为动态水分吸附：检测采用SMS DVS Advantage，在25℃，湿度变化为50％-95％-0％-95％-50％，步进为10％(最后一步为5％)(湿度具体范围以相应图谱为准，此处所列为大多使用方法)，判断标准为dm/dt不大于0.02％。
HPLC的测定使用安捷伦1200DAD高压液相色谱仪(Sunfire C18 150×4.6mm色谱柱)和Waters 2695-2996高压液相色谱仪(Gimini C18 150×4.6mm色谱柱)。
化合物的结构是通过核磁共振(NMR)或/和质谱(MS)来确定的。NMR位移(δ)以10-6(ppm)的单位给出。NMR的测定是用Bruker AVANCE-400核磁仪，测定溶剂为氘代二甲基亚砜(DMSO-d 6)、氘代氯仿(CDCl 3)、氘代甲醇(CD 3OD)，内标为四甲基硅烷(TMS)；MS的测定用FINNIGAN LCQAd(ESI)质谱仪(生产商：Thermo，型号：Finnigan LCQ advantage MAX)。
附图说明
图1：化合物的无定型的XRPD图谱。
图2：化合物的晶型A的XRPD谱图。
图3：实施例5中晶型A的XRPD图谱。
图4：化合物的晶型A的DVS前后X-射线粉末衍射对比图。
图5：化合物的晶型B的XRPD图谱。
图6：化合物的晶型C的XRPD图谱。
图7：化合物的晶型D的XRPD图谱。
具体实施方式
以下将结合实施例或实验例更详细地解释本公开，本公开中的实施例或实验例仅用于说明本公开中的技术方案，并非限定本公开的实质和范围。
实施例1：(S)-N 5-(3,4-二氟苯基)-6-甲基-N 3-((R)-1,1,1-三氟丙-2-基)-6,7-二氢-[1,2,3]三氮唑并[1,5-a]吡嗪-3,5(4H)-二甲酰胺的制备(化合物B)
Figure PCTCN2020073802-appb-000006
第一步
(S)-4-((1-羟丙基-2-基)(4-甲氧基苄基)氨基))丁基-2炔-1-基乙酸酯1c
将化合物1a(3.00g，15.00mmol，采用公知的方法“Bioorganic&Medicinal Chemistry Letters,2015,25(5),1086-1091”制备而得)溶解于60mL二氧六环中，再加4-氯丁基-2-炔-1-基乙酸酯1b(5.73g，39.00mmol，采用公知的方法“Journal of Medicine Chemistry,2014,57(9),3687-3706”制备)，加入三乙胺(4.7g，46.00mmol)，60℃搅拌反应12小时。反应液过滤，滤液减压浓缩，所得残余物用硅胶柱色谱法以洗脱剂体系A纯化，得化合物1c(2.20g，产率：42.2％)。
MS m/z(ESI):306.2[M+1]。
第二步
(S)-4-((1-氯丙基-2-基)(4-甲氧基苄基)氨基)丁基-2炔-1-基乙酸酯1d
将化合物1c(2.20g，7.20mmol)和吡啶(854mg，10.08mmol)溶解于30mL二氯甲烷中，冰浴下缓慢滴加入氯化亚砜(1.50g，12.60mmol)，缓慢升至室温，搅拌反应2小时。反应液中加入100mL二氯甲烷，用水洗涤(50mL×2)，无水硫酸钠干燥，过滤，滤液减压浓缩，得标题化合物1d(2.20g)，产物不经纯化直接用于下一步反应。
第三步
(S)-(5-(4-甲氧基苄基)-6-甲基-4,5,6,7-四氢-[1,2,3]三氮唑并[1,5-a]吡嗪-3-基)甲基乙酸
酯1e
将粗品化合物1d(2.20g，6.79mmol)溶解于20mL N,N-二甲基甲酰胺中，加入叠氮化钠(574mg，8.83mmol，)，80℃反应12小时。反应液冷却至室温，加入50mL乙酸乙酯，用水洗涤(20mL×2)，无水硫酸钠干燥，过滤，滤液减压浓缩所得残余物用硅胶柱色谱法以洗脱剂体系A纯化，得化合物1e(1.30g，产率：57.9％)。
MS m/z(ESI):331.1[M+1]。
第四步
(S)-(6-甲基-4,5,6,7-四氢-[1,2,3]三氮唑并[1,5-a]哌嗪-3-基)甲基乙酸酯三氟乙酸盐1f
将化合物1e(1.30g，2.33mmol)溶解于5mL三氟乙酸中，微波加热至100℃，反应5分钟。反应液冷却至室温，减压浓缩，得粗品标题化合物1f(1.28g)，产物不经纯化直接用于下一步反应。
第五步
(S)-(5-((3,4-二氟苯基)氨基甲酰基)-6-甲基-4,5,6,7-四氢-[1,2,3]三氮唑并[1,5-a]吡嗪-3-基)甲基乙酸酯1h
将粗品化合物1f(200mg，0.62mmol)、化合物1g(228mg，1.90mmol)和三乙胺(290mg，2.86mmol)溶解于10mL四氢呋喃中，冰水浴下，加入双(三氯甲基)碳酸酯(87mg，0.3mmol)，搅拌反应3小时。反应液减压浓缩，残余物用硅胶柱色谱法以洗脱剂体系C纯化，得化合物1h(90mg，产率：40.1％)。
MS m/z(ESI):366.1[M+1]。
第六步
(S)-N-(3,4-二氟苯基)-3-(羟甲基)-6-甲基-6,7-二氢-[1,2,3]三氮唑并[1,5-a]吡嗪-5(4H)-甲酰胺1i
将化合物1h(442mg，1.21mmol)溶解于6mL甲醇和水(V:V＝5:1)的混合溶剂中，加入氢氧化锂(253.86mg，6.05mmol)，搅拌1.5小时。反应液减压浓缩，所得残余物中加入30mL乙酸乙酯，水洗涤(2mL×2)，有机相减压浓缩，得粗品化合物1i(391mg)，产物不经纯化直接用于下一步反应。
MS m/z(ESI):324.1[M+1]。
第七步
(S)-N-(3,4-二氟苯基)-3-甲酰基-6-甲基-6,7-二氢-[1,2,3]三氮唑并[1,5-a]吡嗪-5(4H)-甲酰胺1j
将粗品化合物1i(350mg，1.1mmol)溶解于12mL二氯甲烷中，加入氯铬酸吡啶盐(583.41mg，2.71mmol)和硅胶(550mg，100目)，搅拌2小时。过滤，减压浓缩，所得残余物用薄层色谱法以展开剂体系A纯化，得化合物1j(60mg，产率：17％)。
MS m/z(ESI):322.1[M+1]。
第八步
(S)-((3,4-二氟苯基)氨基羰基)-6-甲基-4,5,6,7-二氢-[1,2,3]三氮唑并[1,5-a]吡嗪-3-甲酸1k
将化合物1j(60mg，0.19mmol)溶解于5mL乙腈和水混合溶剂中(V:V＝3:2)，加入氨基磺酸(36.26mg，0.37mmol)，将亚氯酸钠(33.78mg，0.37mmol)溶于2mL水中加入到反应体系，室温搅拌3小时。加入1mL饱和亚硫酸钠溶液，用1N盐酸调pH至2，乙酸乙酯萃取(10mL×3)，合并有机相，减压浓缩，得粗品化合物1k(30mg)，产物不经纯化直接用于下一步反应。
MS m/z(ESI):338.4[M+1]。
第九步
(S)-N 5-(3,4-二氟苯基)-6-甲基-N 3-((R)-1,1,1-三氟丙-2-基)-6,7-二氢-[1,2,3]三氮唑并 [1,5-a]吡嗪-3,5(4H)-二甲酰胺1
将粗品化合物1k(50mg，148.2μmol)、O-(7-氮杂苯并三唑-1-基)-N,N,N′,N′-四甲基脲六氟磷酸酯(52.32mg，222.4μmol)、N,N-二异丙基乙胺(76.64mg，593μmol)溶解于3mL N,N-二甲基甲酰胺中，反应10分钟，加入化合物1l(33.25mg，222.4μmol，采用专利申请“CN102875270A”公开的方法制备而得)，搅拌反应2小时。反应液减压浓缩，用高效液相色谱法纯化所得残余物(分离条件：色谱柱：Gilson GX-281，流动相：A-水(10mmol醋酸氨)，B-乙腈，流速：18mL/min)，得到20mg粗品，进行手性制备手性制备(分离条件：手性制备柱：岛津手性制备色谱LC-20AP，CHIRALPAK-AY Lux LC Column 250*21.2mm,5um；流动相：A-正己烷：B-乙醇(0.1％DEA)＝85：15，流速：20mL/min)，收集其相应组分，减压浓缩，得到产物1(5mg)。
MS m/z(ESI):433.1[M+1]；
手性HPLC分析:保留时间8.647分钟，手性纯度：99.8％(色谱柱：AY Phenomenex Lux Amylose-2 150*4.6mm,5um；流动相：正己烷/乙醇(0.1％DEA)＝85/15(v/v))。
经X-射线粉末衍射检测，该晶型为无定型，XRPD谱图如图1。
MS m/z(ESI):439.0[M+1]。
1H NMR(400MHz，CD 3OD)：7.50-7.48(m,1H),7.18-7.16(m,2H),5.43(d,1H),5.08-5.06(m,1H),4.89-4.87(m,1H),4.74(d,1H),4.60(d,1H),4.49-4.46(m,1H),1.49(d,3H),1.21(d,3H)。
测试例1：体外抗HBV活性测试(细胞内HBV DNA定量分析)
一、实验材料及仪器
1.QIAamp 96 DNA QIAcube HT Kit(Qiagen)
2.QIAcube HT plasticware(Qiagen)
3.乙型肝炎病毒核酸定量检测试剂盒(泰普生物)
4.DNA提取设备(QIAcube)(Qiagen)
5.QuantStudio 6 Fiex(ABI,ThermFisher)
6.酶标仪(BMG)
7.HepG2.2.15细胞(上海瑞鹿生物技术有限公司)
二、实验步骤
HepG2.2.15细胞是整合了HBV基因组的稳定表达细胞株，可通过复制、转录、翻译、并包装成带有HBV DNA的病毒颗粒分泌到细胞外。本研究采用定量PCR的方法对HepG2.2.15体外增殖产生的HBV DNA进行定量分析，测定本公开中化合物通过对HBV 衣壳蛋白装配抑制进而抑制HBV DNA复制的活性。
HepG2.2.15细胞在DMEM/高葡萄糖培养基(10％FBS,400μg/ml G418)中培养，每3天传代一次。实验当天以新鲜细胞培养基制取细胞悬液，以40,000细胞/孔96孔板(Corning,#3599)，5％二氧化碳，于37℃培养。第二天先将化合物溶解在纯DMSO中浓度为20mM，再用DMSO配制成首个浓度2mM，并以4倍依次稀释成8个浓度，设置对照的孔加入90μL DMSO。用含DMEM/高葡萄糖培养基稀释200倍。取出第一天接种的细胞培养板，用负压吸取装置吸出孔板内的培养基，再将配制好的含有各浓度的化合物培养基分别加入各孔中，以200μl/孔，于37℃培养72小时。第五天，用含有同样化合物的新鲜培养基对该培养细胞进行换液，方法等同第二天，再于37℃培养72小时。第八天，取出细胞培养板，以300g离心3分钟，收集培养上清液200μL/孔。采用Qiagen自动DNA提取设备进行细胞培养上清液内HBV DNA提取，具体方法参考试剂和仪器使用说明。最后以100μL/孔，用DNA洗脱缓冲液洗脱提取的DNA。采用泰普生物的乙型肝炎病毒核酸定量检测试剂盒对提取的DNA进行HBV DNA定量PCR分析，具体方法参考试剂盒说明。定量标准曲线采用试剂盒自带标准样品，平行进行实验。根据标准曲线对各个样品进行定量换算。最后用Graphpad Prism软件根据化合物各浓度与相应的DNA值计算化合物的EC 50值。Emax为化合物最大限度抑制HBV DNA复制的效应值。
本公开中化合物B通过对HBV衣壳蛋白装配抑制进而抑制HBV DNA复制体外活性通过以上的试验进行测定，测得的EC 50＝19nM，Emax＝100％，表明对HBV DNA复制具有明显的抑制作用。
测试例2：对HepG2细胞体外增殖的影响
一、实验材料及仪器
1.HepG2细胞(ATCC)
2.CellTiter-Glo TM细胞增殖试剂盒(Promega)
3.自动移液工作站(Bravo)：Agilent Technologies公司
4.酶标仪(VICTOR 3)：PerkinElmer公司
5.CO 2培养箱(Fisher Scientific)
6.离心机(Fisher Scientific)
二、实验步骤
取对数生长期的HepG2细胞，用胰蛋白酶消化制备细胞悬液，以6,000细胞/孔96孔板(底透白色96孔板，PerkinElmer)，5％二氧化碳，于37℃培养16-20小时。第二天，化合物溶解在纯DMSO中浓度为20mM，利用自动移液工作站(Bravo)对化合物进行梯度 稀释，3倍稀释，每个化合物设8个浓度点，对照的孔为DMSO；接着用EMEM(含10％FBS)培养基对DMSO配制的各浓度点化合物进行200倍稀释。取出第一天接种的细胞培养板，用负压吸取装置吸出孔板内的培养基，再将配制好的含有各浓度的化合物培养基分别加入各孔中，以100μL/孔，于37℃培养72小时。第五天，取出96孔细胞培养板，向各孔中加入新鲜配制的CellTiter Glo，以100μL/孔，放置5-10分钟，用白色封底膜(PerkinElmer)将该96孔板底部封膜，置于酶标仪中，用酶标仪测定Luminescence信号。用Graphpad Prism软件根据化合物各浓度与相应的增殖抑制信号值计算化合物的CC 50值，CC 50>100μM，表明对HepG2细胞体外增殖抑制没有影响或影响较小，表现出高的安全性。
实施例2：
将化合物B(200mg，0.46mmol)加入到异丙醇和异丙醚(V:V＝1:4)混合溶剂，加热搅拌溶清，搅拌析晶，过滤，干燥得产物(122mg，产率：61％)。经X-射线粉末衍射检测，XRPD谱图如图2，将其定义为晶型A。
实施例3：
将化合物B(200mg，0.46mmol)加入到6mL乙酸乙酯和正己烷(V:V＝1:4)混合溶剂中，加热搅拌溶清，搅拌析晶，过滤，收集滤饼，真空干燥得产物(116mg，收率：58％)。
经X-射线粉末衍射为晶型A。
实施例4：
将化合物B(100mg，0.23mmol)加入到21mL二氯甲烷和异丙醚(V:V＝1:20)混合溶液中，加热搅拌溶清，缓慢冷却至室温，缓慢挥发，析出固体，过滤，收集滤饼，干燥得到产物(15mg，收率：15％)。
经XRPD检测为晶型A。
实施例5：
将化合物B的晶型A(38g，87.9mmol)加入到乙醚(80mL)中，室温打浆搅拌16小时，过滤，滤饼乙醚淋洗(30mL×2)，收集滤饼，干燥得产物(36.3g，收率：95.5％)。
经X-射线粉末衍射，为晶型A，其XRPD谱图如图3，其特征峰位置如下表1所示，DSC谱图中在149.39℃、184.81℃处有吸热峰，热重分析(TGA)在40℃-175℃之间失重 0.85％，
表1
Figure PCTCN2020073802-appb-000007
DVS检测显示在正常存储条件下(即25℃、60％RH)，该样品吸湿增重约为0.07％；在加速实验条件(即70％RH)，吸湿增重约为0.10％；在极端条件下(90％RH)，吸湿增重约为0.21％。在0％-95％RH湿度变化过程中，该样品的解吸附过程与吸附过程一致。DVS检测后复测晶型，晶型未转变，见图4(a为DVS检测后XRPD图谱，b为DVS检测前XRPD图谱)。
实施例6：
将化合物B的晶型A(40mg，92.5μmol)加入到0.5mL乙醇和水(V:V＝2:3)混合溶液中，室温打浆搅拌216小时，过滤，收集滤饼，真空干燥，得到产物(26mg，收率：65％)。
经XRPD检测为晶型A。
实施例7：
将化合物B的晶型A(40mg，92.5μmol)加入到环己烷(0.5mL)中，室温打浆搅拌216小时，过滤，收集滤饼，真空干燥，得到产物(35mg，收率：87.5％)。
经XRPD检测为晶型A。
实施例8：
将化合物B的晶型A(40mg，92.5μmol)加入到0.6mL丙酮和正庚烷(V:V＝1:5)混合溶液中，室温打浆搅拌216小时，过滤，收集滤饼，真空干燥，得到产物(33mg，收率：82.5％)。
经XRPD检测为晶型A。
实施例9：
实验例1：影响因素考察
将晶型A(实施例5)样品敞口平摊放置，考察在加热(40℃、60℃)、光照(4500Lux)、高湿(RH 75％、RH 90％)条件下样品的稳定性，取样考察期为30天。
表2
Figure PCTCN2020073802-appb-000008
Figure PCTCN2020073802-appb-000009
注：NA为未检测
由表2的影响因素实验结果表明：在光照、高温40℃、高温60℃、高湿75％、高湿90％条件下，放置30天，晶型A的物理化学稳定性较好。
实验例2：长期/加速稳定性
将晶型A(实施例5)放置25℃，60％RH和40℃，75％RH条件考察其稳定性
表3
Figure PCTCN2020073802-appb-000010
长期加速稳定性实验结果显示：化合物B的晶型A在长期(25℃、60％RH)、加速(40℃、75％RH)条件下放置6个月的物理化学稳定性优异。
实施例10：
将化合物B的晶型A(9.4mg)加入到40uL乙腈溶液中，室温挥发得到产物。
经X-射线粉末衍射，其XRPD谱图如图5，定义为晶型B，其特征峰位置如下表4所示，
表4
Figure PCTCN2020073802-appb-000011
Figure PCTCN2020073802-appb-000012
实施例11：
将化合物B的晶型A(9.4mg)加入到40uL硝基甲烷溶液中，室温挥发得到产物。
经X-射线粉末衍射，其XRPD谱图如图6，定义为晶型C，其特征峰位置如下表5所示，
表5
Figure PCTCN2020073802-appb-000013
Figure PCTCN2020073802-appb-000014
实施例12：
将化合物B的晶型A(13.6mg)加入到250uL 1,2-二氯乙烷溶液中，室温挥发得到产物。
经X-射线粉末衍射，其XRPD谱图如图7，定义为晶型D，其特征峰位置如下表6所示，
表6
Figure PCTCN2020073802-appb-000015
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (10)

  1. 一种化合物(S)-N 5-(3,4-二氟苯基)-6-甲基-N 3-((R)-1,1,1-三氟丙-2-基)-6,7-二氢-[1,2,3]三氮唑并[1,5-a]吡嗪-3,5(4H)-二甲酰胺的晶型A，其特征在于，以衍射角2θ角度表示的X-射线粉末衍射图谱，在13.197、14.239、15.839、17.680、19.080、19.780和22.539处有特征峰。
  2. 根据权利要求1所述的晶型A，其特征在于，以衍射角2θ角度表示的X-射线粉末衍射图谱，在13.197、14.239、15.320、15.839、17.680、19.080、19.780、22.539和25.519处有特征峰。
  3. 根据权利要求1或2所述的晶型A，其特征在于，以衍射角2θ角度表示的X-射线粉末衍射图谱，在11.022、13.197、14.239、15.320、15.839、17.680、19.080、19.780、20.879、22.539、25.519和26.041处有特征峰。
  4. 根据权利要求1-3任意一项所述的晶型A，其特征在于，以衍射角2θ角度表示的X-射线粉末衍射图谱如图2所示。
  5. 一种制备权利要求1-4任意一项所述的晶型A的方法，其包括：
    (a)将化合物(S)-N 5-(3,4-二氟苯基)-6-甲基-N 3-((R)-1,1,1-三氟丙-2-基)-6,7-二氢-[1,2,3]三氮唑并[1,5-a]吡嗪-3,5(4H)-二甲酰胺加入溶剂(I)中，搅拌溶解或加热溶解，所述溶剂(I)选自乙酸乙酯、二氯甲烷、异丙醇、异丙醚中的至少一种，优选异丙醇/异丙醚、乙酸乙酯/正己烷，或二氯甲烷/异丙醚，
    (b)搅拌析晶。
  6. 根据权利要求1-4任一项所述的晶型A，其特征在于，其在20.0％RH-80％RH条件下，无或几乎无引湿性。
  7. 根据权利要求1-4任一项所述的晶型，其特征在于，所述2θ角误差范围为±0.20。
  8. 一种药物组合物，其含有权利要求1-4任一项所述的晶型和任选自药学上可接受的载体、稀释剂或赋形剂。
  9. 一种药物组合物，其由权利要求1-4任一项所述的晶型和任选自药学上可接受的载体、稀释剂或赋形剂制备。
  10. 一种根据权利要求1-4任一项所述的晶型，或根据权利要求8或9所述的组合物在制备用于预防和/或治疗病毒性感染疾病的药物中的用途，所述病毒优选乙型肝炎病毒、流感病毒、疱疹病毒和艾滋病毒中的一种或多种。
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