WO2021047466A1 - Crystal form of p53-mdm2 inhibitor and preparation method therefor - Google Patents

Crystal form of p53-mdm2 inhibitor and preparation method therefor Download PDF

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WO2021047466A1
WO2021047466A1 PCT/CN2020/113712 CN2020113712W WO2021047466A1 WO 2021047466 A1 WO2021047466 A1 WO 2021047466A1 CN 2020113712 W CN2020113712 W CN 2020113712W WO 2021047466 A1 WO2021047466 A1 WO 2021047466A1
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compound
formula
crystal form
solvent
angles
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PCT/CN2020/113712
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French (fr)
Chinese (zh)
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颜小兵
陈新海
施斌
杨文谦
董加强
潘龙冈
王铁林
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罗欣药业(上海)有限公司
山东罗欣药业集团股份有限公司
南京明德新药研发有限公司
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Priority to CN202080049349.8A priority Critical patent/CN114096541B/en
Publication of WO2021047466A1 publication Critical patent/WO2021047466A1/en

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    • 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
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems

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  • the present invention relates to a crystal form of a p53-MDM2 inhibitor and a preparation method thereof, and specifically discloses the crystal forms A, B, C and D of a compound of formula (I) and a preparation method thereof, and also includes that the crystal form is used in preparation For the application of drugs for the treatment of cancer, bacterial infections and viral infections.
  • p53 is a tumor suppressor and transcription factor that responds to cellular stress by activating the transcription of many genes involved in cell cycle arrest, apoptosis, aging, and DNA repair. Unlike normal cells where p53 activation is caused by uncommon causes, tumor cells are under constant cell stress from various damages including hypoxia and activation of pro-apoptotic oncogenes. Therefore, the inactivation of the p53 pathway in tumors has a strong selective advantage, and it has been proposed that elimination of p53 function may be a prerequisite for tumor survival. To support this view, three investigative research groups have used mouse models to prove that the lack of p53 function is a continuing requirement for the maintenance of established tumors. When investigating researchers restored the p53 function of the p53-inactivated tumor, the tumor regressed.
  • p53 In 50% of solid tumors and 10% of liquid tumors, p53 is inactivated by mutation and/or deletion. In cancer, other major members of the p53 pathway also undergo genetic or epigenetic changes. MDM2 is an oncoprotein that inhibits p53 function and it is activated by gene amplification with a reported incidence of up to 10%. MDM2 was then inhibited by another tumor suppressor, p14ARF. Changes downstream of p53 are thought to be responsible for at least partially inactivating the p53 pathway in p53WT tumors (p53 wild-type). To support this concept, some p53WT tumors appear to show reduced apoptotic function, but their ability to withstand cell cycle arrest is still intact.
  • MDM2 inhibits p53 activity through three mechanisms: 1) acts as an E3 ubiquitin ligase to promote p53 degradation; 2) binds to the p53 transcription activation domain and blocks the p53 transcription activation domain; and 3) exports p53 from the nucleus to the cytoplasm . All three mechanisms will be blocked by counteracting the MDM2-p53 interaction.
  • this treatment strategy can be applied to p53WT tumors, and studies using small molecule MDM2 inhibitors have shown that tumor growth is hopefully reduced in vitro and in vivo. Further, in patients with p53-inactivated tumors, the stabilization of wild-type p53 in normal tissues caused by MDM2 inhibition may allow selective protection of normal tissues from damage by mitotic toxins.
  • MDM2 means human MDM2 protein
  • p53 means human p53 protein. It should be noted that human MDM2 can also be referred to as HDM2 or hMDM2.
  • the X-ray powder diffraction pattern of the crystal form A of the compound of formula (I) has characteristic diffraction peaks at the following 2 ⁇ angles: 5.5 ⁇ 0.2°, 8.8 ⁇ 0.2°, 11.0 ⁇ 0.2°,
  • the X-ray powder diffraction pattern of the above crystal form A has characteristic diffraction peaks at the following 2 ⁇ angles: 3.5 ⁇ 0.2°, 5.5 ⁇ 0.2°, 8.8 ⁇ 0.2°, 11.0 ⁇ 0.2°, 13.6 ⁇ 0.2°, 22.1 ⁇ 0.2°, 26.3 ⁇ 0.2°, 26.9 ⁇ 0.2°.
  • the X-ray powder diffraction pattern of the above crystal form A has characteristic diffraction peaks at the following 2 ⁇ angles: 3.501, 5.546, 8.835, 10.714, 11.008, 13.279, 13.590, 15.645, 16.115, 16.494, 16.943 , 18.506, 19.508, 20.025, 20.438, 21.245, 22.053, 22.551, 23.473, 25.819, 26.253, 26.867, 27.812, 28.248.
  • 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 the starting point of the endothermic peak at 150.13 ⁇ 3°C.
  • the DSC spectrum of the above-mentioned crystal form A is shown in FIG. 2.
  • thermogravimetric analysis curve of the above-mentioned crystal form A has a weight loss of 6.07% at 150.00 ⁇ 3°C.
  • the TGA spectrum of the above-mentioned crystal form A is shown in FIG. 3.
  • the DVS spectrum of the above-mentioned crystal form A is shown in FIG. 4.
  • the method for preparing the crystal form of compound A of formula (I) includes:
  • the solvent is methanol.
  • the present invention also 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: 5.7 ⁇ 0.2°, 11.2 ⁇ 0.2°, 22.3 ⁇ 0.2°.
  • the X-ray powder diffraction pattern of the above-mentioned crystal form B has characteristic diffraction peaks at the following 2 ⁇ angles: 5.7 ⁇ 0.2°, 8.5 ⁇ 0.2°, 11.2 ⁇ 0.2°, 13.5 ⁇ 0.2°, 17.5 ⁇ 0.2°, 22.3 ⁇ 0.2°, 23.3 ⁇ 0.2°, 24.3 ⁇ 0.2°.
  • the X-ray powder diffraction pattern of the above-mentioned crystal form B has characteristic diffraction peaks at the following 2 ⁇ angles: 3.541, 5.662, 8.464, 9.885, 10.691, 11.187, 13.513, 16.750, 17.458, 19.727, 20.065 , 20.595, 22.347, 22.862, 23.296, 24.287, 27.104, 28.090, 28.820.
  • the XRPD pattern of the above-mentioned crystal form B is shown in FIG. 5.
  • 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 the starting point of the endothermic peak at 152.49 ⁇ 3°C and the peak of the exothermic peak at 168.89 ⁇ 3°C.
  • the DSC spectrum of the above-mentioned crystal form B is shown in FIG. 6.
  • thermogravimetric analysis curve of the above-mentioned crystal form B has a weight loss of 4.16% at 162.20 ⁇ 3°C.
  • the TGA pattern of the above-mentioned crystal form B is shown in FIG. 7.
  • the present invention also provides a method for preparing the crystal form of compound B of formula (I), including:
  • the solvent is ethanol.
  • the present invention also 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: 3.3 ⁇ 0.2°, 6.6 ⁇ 0.2°, 9.1 ⁇ 0.2°.
  • the X-ray powder diffraction pattern of the above-mentioned crystal form C has characteristic diffraction peaks at the following 2 ⁇ angles: 3.3 ⁇ 0.2°, 6.6 ⁇ 0.2°, 9.1 ⁇ 0.2°, 10.6 ⁇ 0.2°, 11.2 ⁇ 0.2°, 14.0 ⁇ 0.2°, 21.1 ⁇ 0.2°, 22.3 ⁇ 0.2°.
  • the X-ray powder diffraction pattern of the above-mentioned crystal form C has characteristic diffraction peaks at the following 2 ⁇ angles: 3.325°, 3.522°, 6.118°, 6.591°, 7.003°, 9.055°, 10.554°, 11.162°, 13.988°, 16.710°, 21.067°, 22.309°.
  • the XRPD pattern of the above-mentioned crystal form C is shown in FIG. 8.
  • 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 the starting point of the endothermic peak at 93.20 ⁇ 3°C and 145.53 ⁇ 3°C, respectively.
  • the DSC chart of the above-mentioned crystal form C is shown in FIG. 9.
  • thermogravimetric analysis curve of the above crystal form has a weight loss of 1.39% at 71.79 ⁇ 3°C, a weight loss of 6.88% at 117.98 ⁇ 3°C, and a weight loss of 7.67% at 170.72 ⁇ 3°C. .
  • the TGA pattern of the above-mentioned crystal form C is shown in FIG. 10.
  • the present invention also provides a method for preparing the crystal form of compound C of formula (I), including:
  • the solvent is tetrahydrofuran.
  • the present invention also 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: 12.3 ⁇ 0.2°, 15.6 ⁇ 0.2°, 16.2 ⁇ 0.2°.
  • the X-ray powder diffraction pattern of the above-mentioned crystal form D has characteristic diffraction peaks at the following 2 ⁇ angles: 5.2 ⁇ 0.2°, 12.3 ⁇ 0.2°, 15.6 ⁇ 0.2°, 16.2 ⁇ 0.2°, 19.2 ⁇ 0.2°, 23.2 ⁇ 0.2°, 24.8 ⁇ 0.2°, 25.5 ⁇ 0.2°.
  • the X-ray powder diffraction pattern of the above-mentioned crystal form D has characteristic diffraction peaks at the following 2 ⁇ angles: 5.169°, 11.857°, 12.333°, 14.556°, 15.583°, 16.216°, 19.174°, 20.043°, 20.810°, 23.157°, 24.419°, 24.816°, 25.465°, 26.452°, 27.378°.
  • the XRPD pattern of the above-mentioned crystal form D is shown in FIG. 11.
  • the XRPD pattern analysis data of the above-mentioned crystal form D is shown in Table 4:
  • the differential scanning calorimetry curve of the above-mentioned crystal form D has the starting point of the endothermic peak at 113.74 ⁇ 3°C.
  • the DSC spectrum of the above-mentioned crystal form D is shown in FIG. 12.
  • thermogravimetric analysis curve of the above-mentioned crystal form D has a weight loss of 0.20% at 120.00 ⁇ 3°C and a weight loss of 0.63% at 220.00 ⁇ 3°C.
  • the TGA spectrum of the above-mentioned crystal form D is shown in FIG. 13.
  • the present invention also provides a method for preparing the crystal form of compound D of formula (I), which includes:
  • the solvent is selected from water, tetrahydrofuran, and a mixed solvent of water and ethanol.
  • the present invention also provides a method for preparing the crystal form of compound D of formula (I), which includes:
  • the solvent is selected from ethyl acetate, isopropyl acetate, n-heptane, methyl tert-butyl ether, a mixed solvent of ethyl acetate and n-heptane, and a mixture of ethyl acetate and methyl tert-butyl ether Solvent.
  • the present invention also provides a method for preparing the crystal form of compound D of formula (I), which includes:
  • the solvent is selected from ethanol, isopropanol, tert-butanol and ethylene glycol.
  • the present invention also provides a method for preparing the crystal form of compound D of formula (I), which includes:
  • the solvent is selected from water, tetrahydrofuran and a mixed solvent of water and ethanol.
  • the present invention also provides the application of the above-mentioned crystal form or the crystal form obtained by the above-mentioned preparation method in the preparation of drugs for treating cancer, bacterial infections or viral infections.
  • the compound of the present invention does not contain water of crystallization and crystallization solvent, has good stability, has almost no hygroscopicity, and has a good medicine prospect.
  • 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 of those skilled in the art.
  • Well-known equivalent alternatives, preferred implementations include but are not limited to the embodiments of the present invention.
  • the present invention uses the following abbreviations: min stands for minutes; hr stands for hours; RH stands for relative humidity; rt stands for room temperature; rpm stands for rotation speed per minute; THF stands for tetrahydrofuran; DCM stands for dichloromethane; EtOAc or EA stands for ethyl acetate; PE stands for petroleum ether; MeOH stands for methanol; EtOH stands for ethanol; Acetone stands for acetone; DIEA stands for N,N-diisopropylethylamine; Na 2 SO 4 stands for sodium sulfate; T 3 P stands for 1-propyl phosphoric anhydride; NBS Represents N-bromosuccinimide; KHMDS represents bis(trimethylsilyl) potassium amide; Pd(dppf)Cl 2.
  • CH 2 Cl 2 represents [1,1'-bis(diphenylphosphine) two Ferrocene] dichloride palladium dichloromethane complex; SOCl 2 stands for thionyl chloride; LDA stands for TLC stands for thin layer chromatography separation; HPLC stands for high performance liquid phase separation; SFC stands for supercritical fluid chromatography separation.
  • Test method Approximately 10-20mg sample is used for XRPD detection.
  • Light tube voltage 40kV
  • light tube current 40mA
  • Test method Take a sample (about 1 mg) and place it in a DSC aluminum pan for testing. Heat the sample from 30°C to 300°C at a heating rate of 10°C/min under the condition of 50mL/min N 2.
  • TGA Thermal Gravimetric Analyzer
  • Test method Take a sample (2 ⁇ 5mg) and place it in a TGA platinum pot for testing. Under the condition of 25mL/min N 2 and at a heating rate of 10°C/min, heat the sample from 30°C (room temperature) to 300°C or weight loss 20%.
  • Test method Take 10-15 mg of sample and place it on the DVS sample pan for testing.
  • the classification criteria for moisture absorption evaluation are as follows:
  • Moisture absorption classification Moisture absorption and weight gain* deliquescence Absorb enough water to form a liquid Very hygroscopic
  • the weight gain by dampening is not less than 15% Hygroscopic Moisture gain is less than 15% but not less than Slightly hygroscopic Moisture gain is less than 2% but not less than No or almost no hygroscopicity Moisture absorption weight gain is less than 0.2%
  • HPLC High Performance Liquid Chromatograph
  • Fig. 1 is an XRPD spectrum of Cu-K ⁇ radiation of the crystal form of compound A of formula (I).
  • Figure 2 is a DSC spectrum of the crystal form of compound A of formula (I).
  • Figure 3 is a TGA spectrum of the crystal form of compound A of formula (I).
  • Figure 4 is a DVS spectrum of the crystal form of compound A of formula (I).
  • the square dotted line represents the desorption process curve, and the diamond-shaped dotted solid line represents the adsorption curve.
  • Fig. 5 is an XRPD spectrum of Cu-K ⁇ radiation of the crystal form of compound B of formula (I).
  • Fig. 6 is a DSC chart of the crystal form of compound B of formula (I).
  • Figure 7 is a TGA spectrum of the crystal form of compound B of formula (I).
  • Fig. 8 is an XRPD spectrum of Cu-K ⁇ radiation of the crystalline form C of compound of formula (I).
  • Figure 9 is a DSC spectrum of the crystal form of compound C of formula (I).
  • Figure 10 is a TGA spectrum of the crystal form of compound C of formula (I).
  • Fig. 11 is an XRPD spectrum of Cu-K ⁇ radiation of the crystal form D of compound of formula (I).
  • Fig. 12 is a DSC chart of the crystalline form D of compound of formula (I).
  • Figure 13 is a TGA spectrum of the crystal form D of compound of formula (I).
  • Figure 14 is a DVS spectrum of the crystal form D of compound of formula (I).
  • Fig. 15 is an XRPD superimposed spectrum of Cu-K ⁇ radiation of the crystal form of compound D of formula (I) under 40°C and 75% RH conditions (the lower curve is 0 days and the upper curve is 3 months).
  • Figure 16 is an ellipsoid diagram of the three-dimensional structure of the compound of formula (I).
  • Step B At 25°C, 2-propylamine (2.64kg, 44.58mol, 3.82L, 3.00eq) was added to compound 2 (2.50kg, 14.86mol, 1.00eq), and the reaction solution was heated to 85°C and stirred for 16 hours. After the reaction solution was cooled to room temperature, it was concentrated under reduced pressure to obtain compound 3 (2.4 kg, yield 88.63%).
  • Step C Add K 3 PO 4 (3.70 kg, 17.43 mol, 2.65 eq) and NBS (2.50 kg, 14.05mol, 2.13eq), stirred for 12 hours.
  • the reaction solution was filtered, 20L saturated Na 2 SO 3 solution was added to the filtrate, extracted with EA (10L*2), the organic phases were combined and washed with saturated brine (10L*2), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure After getting the crude product.
  • Step D Under nitrogen protection conditions at -70°C, LDA (2M, 150mL, 1.57eq) was slowly added dropwise to a solution of compound 4 (50g, 191.49mmol, 1eq) in THF (500mL). After stirring for 0.5 hours, 4 -Chlorobenzaldehyde (32.30g, 229.78mmol, 1.2eq) in THF (30mL) solution was slowly added to the reaction solution, and the addition was completed and stirred at -70°C for 1.5 hours.
  • Step E At 25°C, SOCl 2 (21.32g, 179.25mmol, 13.00mL, 6eq) was added to a solution of compound 5 (12g, 29.87mmol, 1eq) in DCM (120mL) and stirred for 1 hour. Water (80 mL) was slowly added to the reaction solution, the organic phase was separated, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 6 (9 g, yield 71.66%).
  • Step G At room temperature, add NaOH (5.76g, 144.01mmol, 5eq) to a mixed solution of compound 7 (15g, 28.80mmol, 1eq) in MeOH (30mL), H 2 O (40mL) and THF (110mL), Stir at room temperature for 2 hours.
  • the pH of the reaction solution was adjusted to about 5 with aqueous HCl (1M, 30 mL), and extracted with ethyl acetate (200 mL*2).
  • the organic phases were combined and washed with saturated brine (100 mL*2), dried over Na 2 SO 4 , filtered, and the filter cake was collected and dried to obtain compound 8 (14 g, yield 98.65%).
  • T 3 P 36.16g, 56.82mmol, 33.79mL, 50% purity, 2eq
  • pyridine 11.24g, 142.06mmol, 11.47
  • Step I Under the protection of nitrogen at -70°C, slowly add a KHMDS (1M, 4.50 mL, 2.14 eq) solution to a THF (15 mL) solution of compound 9 (1 g, 2.11 mmol, 1 eq). After the addition, the mixture was stirred at -70°C for 1 hour, and then CH 3 I (3.020 g, 21.28 mmol, 1.32 mL, 10.10 eq) was added, and the reaction solution was stirred for 1.5 hours.
  • Step J Add compound 10 (300mg, 613.80 ⁇ mol, 1eq) and 2,4-dimethoxy-pyrimidine-5-boronic acid (180.00mg, 978.48 ⁇ mol, 1.59eq) in dioxane ( 12mL) and water (4mL) mixed solvents were added K 3 PO 4 (270.00mg, 1.27mmol, 2.07eq) and Pd(dppf)Cl 2 .CH 2 Cl 2 (102.00mg, 124.90 ⁇ mol, 2.03e-1eq) ), the reaction solution was heated to 100°C and stirred for 12 hours.
  • K 3 PO 4 270.00mg, 1.27mmol, 2.07eq
  • Pd(dppf)Cl 2 .CH 2 Cl 2 102.00mg, 124.90 ⁇ mol, 2.03e-1eq
  • crystal form A absorbs 5.4% moisture at 80% humidity and has hygroscopicity; crystal form D absorbs moisture and increases weight by 1.3%, which is slightly hygroscopic.
  • Example 7 Solid stability test under high temperature and high humidity conditions of the crystal form of compound D of formula (I)
  • Test conditions Point in time Crystal form (XRPD) — 0 days Crystal Form D 40°C/75%RH 4 weeks Crystal Form D 60°C/75%RH 4 weeks Crystal Form D
  • Example 8 Long-term stability test of crystal form of compound D of formula (I) under high temperature and high humidity conditions
  • Preparation of compound crystal of formula (I) weigh 10 mg of compound of formula (I) into a single-necked flask, add 2 mL of absolute ethanol to heat to dissolve, and then leave to stand in the open, pick out suitable crystals for X-ray single crystal structure analysis.
  • the MDM2/p53 protein binding experiment adopts the TR-FRET method for detection.
  • the specific steps are as follows: Use an Echo pipette (Labcyte) to perform a 3.162-fold gradient dilution of the test compound, dilute each compound at 11 concentrations and transfer 250 nL to a 384-well plate, and set up two replicate wells for each compound concentration. Set the well with positive compound (100% inhibition) as a positive control, and set the well with only DMSO as a negative control.
  • buffer 125mM NaCl, 1mM DTT, 0.01% Gelatin (animal gelatin), 0.1% Pluronic f-127 (polyether), 1 ⁇ PBS
  • the diluted mixture contains 0.3nM Eu2+anti-GST antibody and 9nM XL665 anti-His antibody .
  • Read on Envision multifunctional microplate reader PerkinElmer (excitation light 340nm, emission light 665nm, 615nm).
  • Ratio Signal intensity at 665nm/Signal intensity at 615nm ⁇ 10000. Use Ratio to calculate the inhibition rate.
  • Example 11 Cell-level activity determination of the compound of formula (I)
  • the SJSA-1 cell proliferation experiment uses propidium iodide staining. Propidium iodide cannot pass through the cell membrane of living cells, but it can pass through the cell membrane of apoptotic cells to stain cells.
  • the specific steps are as follows: Isolate the SJSA-1 cells in the logarithmic growth phase (from the cell bank of WuXi AppTec Department of Biology) in the cell culture flask, and count them. SJSA-1 cells were diluted to 1 ⁇ 10 5 cells per milliliter with RPMI1640 cell culture medium supplemented with 10% FBS, 1% double antibody and 1% L-glutamine.
  • cell lysate 150 mM NaCl, 2 mM Tris pH 7.5, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, ddH 2 O
  • cell lysate 150 mM NaCl, 2 mM Tris pH 7.5, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, ddH 2 O
  • Inhibition rate (add compound well signal-negative control Signal)/(positive control signal-negative control signal)*100%.
  • IC 50 value The anti-proliferative activity (IC 50 value) of the compound of formula (I) on SJSA-1 cells is shown in Table 13 below:
  • the compound of formula (I) shows good activity in binding to the MDM2 protein target and inhibiting the growth of SJSA-1 tumor cells.
  • mice Using female Balb/c mice as the test animals, the LC/MS/MS method was used to determine the mice's tail vein injection and oral cassette dosing method (cassette dosing) to give the positive reference compound NVP-HDM201, the compound of formula (I) The concentration of the drug in the plasma at different times afterwards. Study the pharmacokinetic behavior of the compound of the present invention in mice, and evaluate its pharmacokinetic characteristics.
  • mice Six female Balb/c mice were given a caudal intravenous injection at a dose of 0.5 mg/kg after a one-night fast; the other three mice were administered orally at a dose of 2 mg/kg.
  • Blood was collected at 0.08, 0.25, 0.5, 1, 2, 4, 8, and 24 hours after administration, placed in a heparinized anticoagulation tube, centrifuged at 7000 rpm (5204g), 4°C, and separated plasma. Store at 80°C. Eat food 4 hours after administration.
  • the LCMS/MS method was used to determine the content of the test compound in mouse plasma after intravenous injection and oral administration. Plasma samples were analyzed after pretreatment with precipitated protein.
  • the compound of formula (I) Compared with NVP-HDM201, the compound of formula (I) has a longer half-life in vivo when the dose of intravenous injection in mice is 0.5mpk.
  • the oral administration dose is 2 mg/kg, the plasma exposure of the compound of formula (I) is significantly greater, the oral bioavailability is higher, and the pharmacokinetic properties are better.
  • Example 13 Two-way permeability evaluation experiment of MDR1-MDCK cells
  • MDR1-MDCK cells that permanently express human P-glycoprotein were planted on a 96-well Insert cell plate and cultured for 4-7 days to form a convergent monolayer of cells.
  • the test compound was diluted with HBSS buffer (pH 7.4) to a concentration of 2 ⁇ M, added to the top or basolateral side of the cell, incubated at 37°C, 5% CO 2 , and 95% relative humidity for 2.5 hours, and then took the dosing hole
  • the sample solutions in the donor wells and receiver wells are immediately mixed with the cold acetonitrile solution containing the internal standard.
  • the cells were lysed with a cold acetonitrile solution containing an internal standard to measure the accumulation of intracellular compounds.
  • the LCMS/MS method was used to analyze the concentration of the test compound in all samples (including the initial dosing solution, the supernatant of the dosing hole, the receiving solution, and the cell lysate).
  • concentration of the test compound is expressed by the ratio of its peak area to the peak area of the internal standard, and the permeability of the test compound from the two directions A ⁇ B and B ⁇ A.
  • human colon cancer Caco-2 cells were seeded on a 96-well Insert cell plate at a density of 1 ⁇ 10 5 cells/cm 2 and cultured for 4-5 days to form a convergent monolayer of cells.
  • the compound of formula (I) was diluted with HBSS buffer (pH 7.4) to a concentration of 2 ⁇ M, added to the top or basolateral of the cell, incubated at 37°C, 5% CO 2 and saturated humidity for 2.5 hours, and then took the dosing hole ( The sample solutions in the donor wells and receiver wells are immediately mixed with the cold acetonitrile solution containing the internal standard.
  • the cells were lysed with a cold acetonitrile solution containing an internal standard to measure the accumulation of intracellular compounds.
  • the LCMS/MS method was used to analyze the concentration of the compound of formula (I) in all samples (including the initial dosing solution, the supernatant of the dosing hole, the receiving solution, and the cell lysis solution).
  • concentration of the test compound is expressed by the ratio of its peak area to the peak area of the internal standard, and the permeability of the test compound from the two directions A ⁇ B and B ⁇ A.
  • Example 15 Evaluation of the efficacy of the compound of formula (I) in acute myeloid leukemia animals
  • 0.2mL (10 ⁇ 10 6 cells, containing 50% Matrigel) of MV4-11 tumor cells were inoculated subcutaneously on the right back of each mouse to form a transplanted tumor.
  • the volume was randomly divided into groups, with 8 in the negative control group, 8 in each group in the positive control group, and 8 in each group in the experimental group.
  • the experimental group was orally orally administered with different doses of the positive drug NVP-HDM201 (6mg/kg) and the compound of formula (I) (6mg/kg and 12mg/kg) once a day, and the negative control group was given the same amount of solvent at the same time.
  • T RTV RTV of the treatment group
  • C RTV RTV of the negative control group.
  • Efficacy evaluation standard T/C%>60% is invalid; T/C% ⁇ 60%, and statistically processed P ⁇ 0.05 is valid.
  • the calculation formula of tumor growth inhibition rate (TGI) is as follows:
  • TGI(%) ⁇ [(CV t -CV 0 )-(TV t -TV 0 )]/(CV t -CV 0 ) ⁇ 100%
  • CV t is the tumor volume at the end of the administration of the control group
  • CV 0 is the tumor volume of the control group before caged administration
  • TV t is the tumor volume at the end of the administration group
  • TV 0 is the administration group Tumor volume before medication.
  • the difference in tumor volume between the treatment group and the control group was tested by t-test.
  • the nude mice of each group were weighed twice a week to preliminarily evaluate the toxic and side effects of the drugs.
  • the efficacy results of each compound in this model are shown in Table 17 below.
  • the compound of formula (I) has a better anti-tumor effect in a mouse transplanted MV4-11 human acute myeloid leukemia model, and shows a good dose-effect relationship.

Abstract

Disclosed in the present invention are a crystal form of a p53-MDM2 inhibitor and a preparation method therefor, and in particular, disclosed are A, B, C, and D crystal forms of a compound represented by formula (I) and a preparation method therefor, as well as application of the crystal forms in preparation of medicines for treating cancers, bacterial infections, and viral infections.

Description

一种p53-MDM2抑制剂的晶型及其制备方法Crystal form of p53-MDM2 inhibitor and preparation method thereof
本申请要求申请日为2019年9月12日的中国专利申请CN201910864542.6的优先权。本申请引用上述中国专利申请的全文。This application claims the priority of the Chinese patent application CN201910864542.6 whose filing date is September 12, 2019. This application quotes the full text of the aforementioned Chinese patent application.
技术领域Technical field
本发明涉及一种p53-MDM2抑制剂的晶型及其制备方法，具体公开了式(I)化合物的A、B、C和D晶型及其制备方法，还包括所述晶型在制备用于治疗癌症、细菌感染、病毒感染药物上的应用。The present invention relates to a crystal form of a p53-MDM2 inhibitor and a preparation method thereof, and specifically discloses the crystal forms A, B, C and D of a compound of formula (I) and a preparation method thereof, and also includes that the crystal form is used in preparation For the application of drugs for the treatment of cancer, bacterial infections and viral infections.
背景技术Background technique
p53是通过激活细胞周期停滞、凋亡、衰老和DNA修复中所涉及的许多基因的转录而响应于细胞应激的肿瘤抑制剂和转录因子。与p53激活是由不常见原因引起的正常细胞不同，肿瘤细胞处于来自包括缺氧和促凋亡癌基因激活在内的各种损害的恒定细胞应激下。因而，对肿瘤中p53途径的灭活具有强的选择性优势，并且已提出消除p53功能可能是肿瘤存活的前提。为了支持这一观点，三个调查研究组已经使用小鼠模型证明p53功能的缺少是已建立肿瘤的维持的持续要求。当调查研究人员恢复p53灭活的肿瘤的p53功能时，该肿瘤消退。p53 is a tumor suppressor and transcription factor that responds to cellular stress by activating the transcription of many genes involved in cell cycle arrest, apoptosis, aging, and DNA repair. Unlike normal cells where p53 activation is caused by uncommon causes, tumor cells are under constant cell stress from various damages including hypoxia and activation of pro-apoptotic oncogenes. Therefore, the inactivation of the p53 pathway in tumors has a strong selective advantage, and it has been proposed that elimination of p53 function may be a prerequisite for tumor survival. To support this view, three investigative research groups have used mouse models to prove that the lack of p53 function is a continuing requirement for the maintenance of established tumors. When investigating researchers restored the p53 function of the p53-inactivated tumor, the tumor regressed.
在50％的实体瘤和10％的液体瘤中，p53通过突变和/或缺失来进行灭活。在癌症中，p53途径的其他主要成员也发生遗传或表观遗传改变。MDM2是一种癌蛋白质，它抑制p53功能并且它以报道高达10％的发生率被基因扩增激活。MDM2继而被另一种肿瘤抑制剂p14ARF抑制。p53下游的改变被认为可能负责至少部分地灭活p53WT肿瘤(p53野生型)中的p53途径。为了支持这一概念，一些p53WT肿瘤似乎显示出降低的凋亡功能，但它们经受细胞周期停滞的能力仍然是完整的。一种癌症治疗策略涉及使用结合MDM2并抵消它与p53的相互作用的小分子。MDM2通过三种机制抑制p53活性：1)用作E3泛素连接酶以促进p53降解；2)结合至p53转录激活结构域并阻断p53转录激活结构域；以及3)从细胞核向细胞质输出p53。这三种机制都将通过抵消MDM2-p53相互作用来进行阻断。特别地，这种治疗策略可以应用于p53WT肿瘤，并且利用小分子MDM2抑制剂进行的研究已经显示出肿瘤生长在体外和体内有希望地减小。进一步地，在患有p53-灭活的肿瘤的患者中，由MDM2抑制引起的正常组织中野生型p53的稳定化可能允许选择性地保护正常组织免受有丝分裂毒物的损害。In 50% of solid tumors and 10% of liquid tumors, p53 is inactivated by mutation and/or deletion. In cancer, other major members of the p53 pathway also undergo genetic or epigenetic changes. MDM2 is an oncoprotein that inhibits p53 function and it is activated by gene amplification with a reported incidence of up to 10%. MDM2 was then inhibited by another tumor suppressor, p14ARF. Changes downstream of p53 are thought to be responsible for at least partially inactivating the p53 pathway in p53WT tumors (p53 wild-type). To support this concept, some p53WT tumors appear to show reduced apoptotic function, but their ability to withstand cell cycle arrest is still intact. One cancer treatment strategy involves the use of small molecules that bind MDM2 and counteract its interaction with p53. MDM2 inhibits p53 activity through three mechanisms: 1) acts as an E3 ubiquitin ligase to promote p53 degradation; 2) binds to the p53 transcription activation domain and blocks the p53 transcription activation domain; and 3) exports p53 from the nucleus to the cytoplasm . All three mechanisms will be blocked by counteracting the MDM2-p53 interaction. In particular, this treatment strategy can be applied to p53WT tumors, and studies using small molecule MDM2 inhibitors have shown that tumor growth is hopefully reduced in vitro and in vivo. Further, in patients with p53-inactivated tumors, the stabilization of wild-type p53 in normal tissues caused by MDM2 inhibition may allow selective protection of normal tissues from damage by mitotic toxins.
如本文所使用的，MDM2意指人MDM2蛋白，并且p53意指人p53蛋白。应注意，人MDM2也可以称为HDM2或hMDM2。As used herein, MDM2 means human MDM2 protein, and p53 means human p53 protein. It should be noted that human MDM2 can also be referred to as HDM2 or hMDM2.
基于抑制p53和MDM2之间的相互作用来治疗肿瘤等疾病的研究已进行了多年，目前尚无该靶点药物上市，但已有多个分子进入不同的临床阶段。诺华公司开发的小分子p53-MDM2抑制剂NVP-HDM201目前已进入临床II期，用于治疗脂肪肉瘤和急性髓性白血病，其在专利WO2013111105中公开，结构如下：Research on the treatment of tumors and other diseases based on inhibiting the interaction between p53 and MDM2 has been conducted for many years. Currently, there is no such target drug on the market, but many molecules have entered different clinical stages. The small molecule p53-MDM2 inhibitor NVP-HDM201 developed by Novartis has now entered clinical phase II for the treatment of liposarcoma and acute myeloid leukemia. It is disclosed in patent WO2013111105, and its structure is as follows:
Figure PCTCN2020113712-appb-000001
Figure PCTCN2020113712-appb-000001
发明内容Summary of the invention
式(I)化合物的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：5.5±0.2°、8.8±0.2°、11.0±0.2°，The X-ray powder diffraction pattern of the crystal form A of the compound of formula (I) has characteristic diffraction peaks at the following 2θ angles: 5.5±0.2°, 8.8±0.2°, 11.0±0.2°,
Figure PCTCN2020113712-appb-000002
Figure PCTCN2020113712-appb-000002
在本发明的一些方案中，上述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：3.5±0.2°、5.5±0.2°、8.8±0.2°、11.0±0.2°、13.6±0.2°、22.1±0.2°、26.3±0.2°、26.9±0.2°。In some aspects of the present invention, the X-ray powder diffraction pattern of the above crystal form A has characteristic diffraction peaks at the following 2θ angles: 3.5±0.2°, 5.5±0.2°, 8.8±0.2°, 11.0±0.2°, 13.6 ±0.2°, 22.1±0.2°, 26.3±0.2°, 26.9±0.2°.
在本发明的一些方案中，上述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：3.501、5.546、8.835、10.714、11.008、13.279、13.590、15.645、16.115、16.494、16.943、18.506、19.508、20.025、20.438、21.245、22.053、22.551、23.473、25.819、26.253、26.867、27.812、28.248。In some aspects of the present invention, the X-ray powder diffraction pattern of the above crystal form A has characteristic diffraction peaks at the following 2θ angles: 3.501, 5.546, 8.835, 10.714, 11.008, 13.279, 13.590, 15.645, 16.115, 16.494, 16.943 , 18.506, 19.508, 20.025, 20.438, 21.245, 22.053, 22.551, 23.473, 25.819, 26.253, 26.867, 27.812, 28.248.
在本发明的一些方案中，上述A晶型，其XRPD图谱如图1所示。In some aspects of the present invention, the XRPD pattern of the above-mentioned crystal form A is shown in FIG. 1.
在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：In some aspects of the present invention, the XRPD pattern analysis data of the above-mentioned crystal form A is shown in Table 1:
表1 式(I)化合物A晶型的XRPD图谱解析数据Table 1 XRPD pattern analysis data of formula (I) compound A crystal form
Figure PCTCN2020113712-appb-000003
Figure PCTCN2020113712-appb-000003
在本发明的一些方案中，上述A晶型，其差示扫描量热曲线在150.13±3℃处具有吸热峰的起始点。In some aspects of the present invention, the differential scanning calorimetry curve of the above-mentioned crystal form A has the starting point of the endothermic peak at 150.13±3°C.
在本发明的一些方案中，上述A晶型，其DSC图谱如图2所示。In some aspects of the present invention, the DSC spectrum of the above-mentioned crystal form A is shown in FIG. 2.
在本发明的一些方案中，上述A晶型，其热重分析曲线在150.00±3℃时失重达6.07％。In some aspects of the present invention, the thermogravimetric analysis curve of the above-mentioned crystal form A has a weight loss of 6.07% at 150.00±3°C.
在本发明的一些方案中，上述A晶型，其TGA图谱如图3所示。In some aspects of the present invention, the TGA spectrum of the above-mentioned crystal form A is shown in FIG. 3.
在本发明的一些方案中，上述A晶型，其DVS图谱如图4所示。In some aspects of the present invention, the DVS spectrum of the above-mentioned crystal form A is shown in FIG. 4.
式(I)化合物A晶型的制备方法，包括：The method for preparing the crystal form of compound A of formula (I) includes:
(a)将式(I)化合物加入溶剂中，升温至60-70℃；(a) The compound of formula (I) is added to the solvent, and the temperature is increased to 60-70°C;
(b)室温下放置至析出固体；(b) Place it at room temperature until a solid precipitates;
(c)35-45℃下真空干燥45～50小时；(c) Vacuum drying at 35-45°C for 45-50 hours;
(d)75-85℃下真空干燥1～5小时；(d) Vacuum drying at 75-85°C for 1 to 5 hours;
其中，所述溶剂为甲醇。Wherein, the solvent is methanol.
本发明还提供式(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：5.7±0.2°、11.2±0.2°、22.3±0.2°。The present invention also 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 2θ angles: 5.7±0.2°, 11.2±0.2°, 22.3±0.2°.
在本发明的一些方案中，上述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：5.7±0.2°、8.5±0.2°、11.2±0.2°、13.5±0.2°、17.5±0.2°、22.3±0.2°、23.3±0.2°、24.3±0.2°。In some aspects of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form B has characteristic diffraction peaks at the following 2θ angles: 5.7±0.2°, 8.5±0.2°, 11.2±0.2°, 13.5±0.2°, 17.5 ±0.2°, 22.3±0.2°, 23.3±0.2°, 24.3±0.2°.
在本发明的一些方案中，上述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：3.541、5.662、8.464、9.885、10.691、11.187、13.513、16.750、17.458、19.727、20.065、20.595、22.347、22.862、23.296、24.287、27.104、28.090、28.820。In some aspects of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form B has characteristic diffraction peaks at the following 2θ angles: 3.541, 5.662, 8.464, 9.885, 10.691, 11.187, 13.513, 16.750, 17.458, 19.727, 20.065 , 20.595, 22.347, 22.862, 23.296, 24.287, 27.104, 28.090, 28.820.
在本发明的一些方案中，上述B晶型，其XRPD图谱如图5所示。In some aspects of the present invention, the XRPD pattern of the above-mentioned crystal form B is shown in FIG. 5.
在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：In some aspects of the present invention, the XRPD pattern analysis data of the above-mentioned crystal form B is shown in Table 2:
表2 式(I)化合物B晶型的XRPD图谱解析数据Table 2 XRPD pattern analysis data of formula (I) compound B crystal form
Figure PCTCN2020113712-appb-000004
Figure PCTCN2020113712-appb-000004
在本发明的一些方案中，上述B晶型，其差示扫描量热曲线在152.49±3℃处具有吸热峰的起始点，在168.89±3℃处具有放热峰的峰值。In some aspects of the present invention, the differential scanning calorimetry curve of the above-mentioned crystal form B has the starting point of the endothermic peak at 152.49±3°C and the peak of the exothermic peak at 168.89±3°C.
在本发明的一些方案中，上述B晶型，其DSC图谱如图6所示。In some aspects of the present invention, the DSC spectrum of the above-mentioned crystal form B is shown in FIG. 6.
在本发明的一些方案中，上述B晶型，其热重分析曲线在162.20±3℃时失重达4.16％。In some aspects of the present invention, the thermogravimetric analysis curve of the above-mentioned crystal form B has a weight loss of 4.16% at 162.20±3°C.
在本发明的一些方案中，上述B晶型，其TGA图谱如图7所示。In some aspects of the present invention, the TGA pattern of the above-mentioned crystal form B is shown in FIG. 7.
本发明还提供式(I)化合物B晶型的制备方法，包括：The present invention also provides a method for preparing the crystal form of compound B of formula (I), including:
(a)将式(I)化合物加入溶剂中成为悬浊液；(a) adding the compound of formula (I) to a solvent to become a suspension;
(b)将上述悬浊液置于40℃的恒温混匀仪上，700rpm的转速振摇3～5天；(b) Place the above suspension on a constant temperature mixer at 40°C and shake at 700 rpm for 3 to 5 days;
(c)离心，固体残渣于25-35℃真空干燥箱内干燥10～16小时；(c) Centrifuge, dry the solid residue in a vacuum drying oven at 25-35°C for 10-16 hours;
其中，所述溶剂为乙醇。Wherein, the solvent is ethanol.
本发明还提供式(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：3.3±0.2°、6.6±0.2°、9.1±0.2°。The present invention also provides crystal form C of the compound of formula (I), and its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 3.3±0.2°, 6.6±0.2°, 9.1±0.2°.
在本发明的一些方案中，上述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：3.3±0.2°、6.6±0.2°、9.1±0.2°、10.6±0.2°、11.2±0.2°、14.0±0.2°、21.1±0.2°、22.3±0.2°。In some aspects of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form C has characteristic diffraction peaks at the following 2θ angles: 3.3±0.2°, 6.6±0.2°, 9.1±0.2°, 10.6±0.2°, 11.2 ±0.2°, 14.0±0.2°, 21.1±0.2°, 22.3±0.2°.
在本发明的一些方案中，上述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：3.325°、3.522°、6.118°、6.591°、7.003°、9.055°、10.554°、11.162°、13.988°、16.710°、21.067°、22.309°。In some aspects of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form C has characteristic diffraction peaks at the following 2θ angles: 3.325°, 3.522°, 6.118°, 6.591°, 7.003°, 9.055°, 10.554°, 11.162°, 13.988°, 16.710°, 21.067°, 22.309°.
在本发明的一些方案中，上述C晶型，其XRPD图谱如图8所示。In some aspects of the present invention, the XRPD pattern of the above-mentioned crystal form C is shown in FIG. 8.
在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：In some aspects of the present invention, the XRPD pattern analysis data of the above-mentioned crystal form C is shown in Table 3:
表3 式(I)化合物C晶型的XRPD图谱解析数据Table 3 XRPD pattern analysis data of compound C crystal form of formula (I)
Figure PCTCN2020113712-appb-000005
Figure PCTCN2020113712-appb-000005
Figure PCTCN2020113712-appb-000006
Figure PCTCN2020113712-appb-000006
在本发明的一些方案中，上述C晶型，其差示扫描量热曲线分别在93.20±3℃和145.53±3℃处具有吸热峰的起始点。In some aspects of the present invention, the differential scanning calorimetry curve of the above-mentioned crystal form C has the starting point of the endothermic peak at 93.20±3°C and 145.53±3°C, respectively.
在本发明的一些方案中，上述C晶型，其DSC图谱如图9所示。In some aspects of the present invention, the DSC chart of the above-mentioned crystal form C is shown in FIG. 9.
在本发明的一些方案中，上述C晶型，其热重分析曲线在71.79±3℃时失重达1.39％，在117.98±3℃时失重达6.88％，在170.72±3℃时失重达7.67％。In some aspects of the present invention, the thermogravimetric analysis curve of the above crystal form has a weight loss of 1.39% at 71.79±3°C, a weight loss of 6.88% at 117.98±3°C, and a weight loss of 7.67% at 170.72±3°C. .
在本发明的一些方案中，上述C晶型，其TGA图谱如图10所示。In some aspects of the present invention, the TGA pattern of the above-mentioned crystal form C is shown in FIG. 10.
本发明还提供式(I)化合物C晶型的制备方法，包括：The present invention also provides a method for preparing the crystal form of compound C of formula (I), including:
(a)将式(I)化合物加入溶剂中成为悬浊液；(a) adding the compound of formula (I) to a solvent to become a suspension;
(b)将上述悬浊液置于40℃的恒温混匀仪上，700rpm的转速振摇3～5天；(b) Place the above suspension on a constant temperature mixer at 40°C and shake at 700 rpm for 3 to 5 days;
(c)离心，固体残渣于25-35℃真空干燥箱内干燥10～16小时；(c) Centrifuge, dry the solid residue in a vacuum drying oven at 25-35°C for 10-16 hours;
其中，所述溶剂为四氢呋喃。Wherein, the solvent is tetrahydrofuran.
本发明还提供式(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：12.3±0.2°、15.6±0.2°、16.2±0.2°。The present invention also 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 2θ angles: 12.3±0.2°, 15.6±0.2°, 16.2±0.2°.
在本发明的一些方案中，上述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：5.2±0.2°、12.3±0.2°、15.6±0.2°、16.2±0.2°、19.2±0.2°、23.2±0.2°、24.8±0.2°、25.5±0.2°。In some aspects of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form D has characteristic diffraction peaks at the following 2θ angles: 5.2±0.2°, 12.3±0.2°, 15.6±0.2°, 16.2±0.2°, 19.2 ±0.2°, 23.2±0.2°, 24.8±0.2°, 25.5±0.2°.
在本发明的一些方案中，上述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：5.169°、11.857°、12.333°、14.556°、15.583°、16.216°、19.174°、20.043°、20.810°、23.157°、24.419°、24.816°、25.465°、26.452°、27.378°。In some aspects of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form D has characteristic diffraction peaks at the following 2θ angles: 5.169°, 11.857°, 12.333°, 14.556°, 15.583°, 16.216°, 19.174°, 20.043°, 20.810°, 23.157°, 24.419°, 24.816°, 25.465°, 26.452°, 27.378°.
在本发明的一些方案中，上述D晶型，其XRPD图谱如图11所示。In some aspects of the present invention, the XRPD pattern of the above-mentioned crystal form D is shown in FIG. 11.
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：In some aspects of the present invention, the XRPD pattern analysis data of the above-mentioned crystal form D is shown in Table 4:
表4 式(I)化合物D晶型的XRPD图谱解析数据Table 4 XRPD pattern analysis data of formula (I) compound D crystal form
Figure PCTCN2020113712-appb-000007
Figure PCTCN2020113712-appb-000007
Figure PCTCN2020113712-appb-000008
Figure PCTCN2020113712-appb-000008
在本发明的一些方案中，上述D晶型，其差示扫描量热曲线在113.74±3℃处具有吸热峰的起始点。In some aspects of the present invention, the differential scanning calorimetry curve of the above-mentioned crystal form D has the starting point of the endothermic peak at 113.74±3°C.
在本发明的一些方案中，上述D晶型，其DSC图谱如图12所示。In some aspects of the present invention, the DSC spectrum of the above-mentioned crystal form D is shown in FIG. 12.
在本发明的一些方案中，上述D晶型，其热重分析曲线在120.00±3℃时失重达0.20％，在220.00±3℃时失重达0.63％。In some aspects of the present invention, the thermogravimetric analysis curve of the above-mentioned crystal form D has a weight loss of 0.20% at 120.00±3°C and a weight loss of 0.63% at 220.00±3°C.
在本发明的一些方案中，上述D晶型，其TGA图谱如图13所示。In some aspects of the present invention, the TGA spectrum of the above-mentioned crystal form D is shown in FIG. 13.
本发明还提供式(I)化合物D晶型的制备方法，包括：The present invention also provides a method for preparing the crystal form of compound D of formula (I), which includes:
(a)将式(I)化合物加入溶剂中成为悬浊液；(a) adding the compound of formula (I) to a solvent to become a suspension;
(b)将上述悬浊液置于40℃的恒温混匀仪上，700rpm的转速振摇1～2天；(b) Place the above suspension on a constant temperature mixer at 40°C and shake at 700 rpm for 1 to 2 days;
(c)离心，固体残渣于25-35℃真空干燥箱内干燥10～16小时；(c) Centrifuge, dry the solid residue in a vacuum drying oven at 25-35°C for 10-16 hours;
其中，所述溶剂选自为水、四氢呋喃和水与乙醇的混合溶剂。Wherein, the solvent is selected from water, tetrahydrofuran, and a mixed solvent of water and ethanol.
本发明还提供式(I)化合物D晶型的制备方法，包括：The present invention also provides a method for preparing the crystal form of compound D of formula (I), which includes:
(a)将式(I)化合物加入溶剂中，升温至75-85℃，搅拌0.5～1.5小时；(a) Add the compound of formula (I) to the solvent, raise the temperature to 75-85°C, and stir for 0.5 to 1.5 hours;
(b)冷却至室温，过滤，干燥；(b) Cool to room temperature, filter and dry;
其中，所述溶剂选自乙酸乙酯、乙酸异丙酯、正庚烷、甲基叔丁基醚、乙酸乙酯与正庚烷的混合溶剂和乙酸乙酯与甲基叔丁基醚的混合溶剂。Wherein, the solvent is selected from ethyl acetate, isopropyl acetate, n-heptane, methyl tert-butyl ether, a mixed solvent of ethyl acetate and n-heptane, and a mixture of ethyl acetate and methyl tert-butyl ether Solvent.
本发明还提供式(I)化合物D晶型的制备方法，包括：The present invention also provides a method for preparing the crystal form of compound D of formula (I), which includes:
(a)将式(I)化合物加入溶剂中，升温至75-85℃，搅拌1.5～2.5小时；(a) Add the compound of formula (I) to the solvent, raise the temperature to 75-85°C, and stir for 1.5-2.5 hours;
(b)加入水；(b) Add water;
(c)冷却至室温，过滤，干燥；(c) Cool to room temperature, filter and dry;
其中，所述溶剂选自乙醇、异丙醇、叔丁醇和乙二醇。Wherein, the solvent is selected from ethanol, isopropanol, tert-butanol and ethylene glycol.
本发明还提供式(I)化合物D晶型的制备方法，包括：The present invention also provides a method for preparing the crystal form of compound D of formula (I), which includes:
(a)将式(I)化合物加入溶剂中，升温至95-105℃，搅拌1.5～2.5小时；(a) Add the compound of formula (I) to the solvent, raise the temperature to 95-105°C, and stir for 1.5-2.5 hours;
(b)冷却至室温，过滤，洗涤，干燥；(b) Cool to room temperature, filter, wash, and dry;
其中，所述溶剂选自水、四氢呋喃和水与乙醇的混合溶剂。Wherein, the solvent is selected from water, tetrahydrofuran and a mixed solvent of water and ethanol.
本发明还提供上述晶型或上述制备方法得到的晶型在制备治疗癌症、细菌感染或病毒感染药物中的应用。The present invention also provides the application of the above-mentioned crystal form or the crystal form obtained by the above-mentioned preparation method in the preparation of drugs for treating cancer, bacterial infections or viral infections.
技术效果Technical effect
本发明化合物不含结晶水和结晶溶剂，稳定性较好，且几乎无引湿性，具有良好的成药前景。The compound of the present invention does not contain water of crystallization and crystallization solvent, has good stability, has almost no hygroscopicity, and has a good medicine prospect.
定义和说明Definition and description
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。Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings. A specific phrase or term should not be considered uncertain or unclear without a special definition, but should be understood in its ordinary meaning. When a trade name appears herein, it is intended to refer to its corresponding commodity or its active ingredient.
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。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 of those skilled in the art. Well-known equivalent alternatives, preferred implementations include but are not limited to the embodiments of the present invention.
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。The chemical reaction in the specific embodiment of the present invention is completed in a suitable solvent, and the solvent must be suitable for the chemical change of the present invention and the required reagents and materials. In order to obtain the compounds of the present invention, it is sometimes necessary for those skilled in the art to modify or select the synthesis steps or reaction schemes on the basis of the existing embodiments.
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。Hereinafter, the present invention will be specifically described through examples, and these examples are not meant to limit the present invention in any way.
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。All solvents used in the present invention are commercially available and can be used without further purification.
本发明采用下述缩略词：min代表分钟；hr代表小时；RH代表相对湿度；r.t.代表室温；rpm代表每分钟转速；THF代表四氢呋喃；DCM代表二氯甲烷；EtOAc或EA代表乙酸乙酯；PE代表石油醚；MeOH代表甲醇；EtOH代表乙醇；Acetone代表丙酮；DIEA代表N,N-二异丙基乙胺；Na 2SO 4代表硫酸钠；T 3P代表1-丙基磷酸酐；NBS代表N-溴代丁二酰亚胺；KHMDS代表双(三甲基硅烷基)氨基钾；Pd(dppf)Cl 2.CH 2Cl 2代表[1,1'-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物；SOCl 2代表氯化亚砜；LDA代表TLC代表薄层色谱分离；HPLC代表高效液相分离；SFC表示超临界流体色谱分离。 The present invention uses the following abbreviations: min stands for minutes; hr stands for hours; RH stands for relative humidity; rt stands for room temperature; rpm stands for rotation speed per minute; THF stands for tetrahydrofuran; DCM stands for dichloromethane; EtOAc or EA stands for ethyl acetate; PE stands for petroleum ether; MeOH stands for methanol; EtOH stands for ethanol; Acetone stands for acetone; DIEA stands for N,N-diisopropylethylamine; Na 2 SO 4 stands for sodium sulfate; T 3 P stands for 1-propyl phosphoric anhydride; NBS Represents N-bromosuccinimide; KHMDS represents bis(trimethylsilyl) potassium amide; Pd(dppf)Cl 2. CH 2 Cl 2 represents [1,1'-bis(diphenylphosphine) two Ferrocene] dichloride palladium dichloromethane complex; SOCl 2 stands for thionyl chloride; LDA stands for TLC stands for thin layer chromatography separation; HPLC stands for high performance liquid phase separation; SFC stands for supercritical fluid chromatography separation.
化合物依据本领域常规命名原则或者使用
Figure PCTCN2020113712-appb-000009
软件命名，市售化合物采用供应商目录名称。 Compounds are based on conventional naming principles in the field or used
Figure PCTCN2020113712-appb-000009
The software is named, and the commercially available compounds use the supplier catalog name.
本发明粉末X-射线衍射(X-ray powder diffractometer,XRPD)方法Powder X-ray diffraction (X-ray powder diffractometer, XRPD) method of the present invention
仪器型号：布鲁克D8 advance X-射线衍射仪Instrument model: Bruker D8 advanced X-ray diffractometer
测试方法：大约10～20mg样品用于XRPD检测。Test method: Approximately 10-20mg sample is used for XRPD detection.
详细的XRPD参数如下：The detailed XRPD parameters are as follows:
光管：Cu,kα,
Figure PCTCN2020113712-appb-000010
Light pipe: Cu, kα,
Figure PCTCN2020113712-appb-000010
光管电压：40kV，光管电流：40mALight tube voltage: 40kV, light tube current: 40mA
发散狭缝：0.60mmDivergence slit: 0.60mm
探测器狭缝：10.50mmDetector slit: 10.50mm
防散射狭缝：7.10mmAnti-scatter slit: 7.10mm
扫描范围：4-40degScanning range: 4-40deg
步径：0.02degStep diameter: 0.02deg
步长：0.12秒Step length: 0.12 seconds
样品盘转速：15rpmSample plate speed: 15rpm
本发明差热分析(Differential Scanning Calorimeter,DSC)方法The differential thermal analysis (Differential Scanning Calorimeter, DSC) method of the present invention
仪器型号：TA Q2000差示扫描量热仪Instrument model: TA Q2000 Differential Scanning Calorimeter
测试方法：取样品(约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300℃。 Test method: Take a sample (about 1 mg) and place it in a DSC aluminum pan for testing. Heat the sample from 30°C to 300°C at a heating rate of 10°C/min under the condition of 50mL/min N 2.
本发明热重分析(Thermal Gravimetric Analyzer,TGA)方法Thermal Gravimetric Analyzer (TGA) method of the present invention
仪器型号：TA Q5000热重分析仪Instrument model: TA Q5000 thermogravimetric analyzer
测试方法：取样品(2～5mg)置于TGA铂金锅内进行测试，在25mL/min N 2条件下，以10℃/min的升温速率，加热样品从30℃(室温)到300℃或失重20％。 Test method: Take a sample (2～5mg) and place it in a TGA platinum pot for testing. Under the condition of 25mL/min N 2 and at a heating rate of 10°C/min, heat the sample from 30°C (room temperature) to 300°C or weight loss 20%.
本发明动态蒸汽吸附分析(Dynamic Vapor Sorption,DVS)方法The dynamic vapor adsorption analysis (Dynamic Vapor Sorption, DVS) method of the present invention
仪器型号：SMS DVS Advantage动态蒸汽吸附分析仪(PDS-PF-DVS-01/02)Instrument model: SMS DVS Advantage dynamic vapor adsorption analyzer (PDS-PF-DVS-01/02)
测试方法：取样品10-15mg样品置于DVS样品盘进行检测。Test method: Take 10-15 mg of sample and place it on the DVS sample pan for testing.
温度：25℃Temperature: 25℃
平衡dm/dt：0.01％/min：(时间：10min最大180min)Balance dm/dt: 0.01%/min: (time: 10min maximum 180min)
干燥：0％RH，120minDrying: 0%RH, 120min
RH(％)测量梯度：10％RH (%) measurement gradient: 10%
RH(％)测量梯度范围：0％～90％～0％RH(%) measuring gradient range: 0%～90%～0%
引湿性评价分类标准如下：The classification criteria for moisture absorption evaluation are as follows:
表5table 5
引湿性分类Moisture absorption classification 吸湿增重*Moisture absorption and weight gain*
潮解deliquescence 吸收足量水分形成液体Absorb enough water to form a liquid
极具引湿性Very hygroscopic 引湿增重不小于15％The weight gain by dampening is not less than 15%
有引湿性Hygroscopic 引湿增重小于15％但不小于Moisture gain is less than 15% but not less than
略有引湿性Slightly hygroscopic 引湿增重小于2％但不小于Moisture gain is less than 2% but not less than
无或几乎无引湿性No or almost no hygroscopicity 引湿增重小于0.2％Moisture absorption weight gain is less than 0.2%
*表示在25℃/80％RH下的吸湿增重。* Indicates the moisture absorption and weight gain at 25°C/80%RH.
本发明高效液相色谱仪(High Performance Liquid Chromatograph,HPLC)方法High Performance Liquid Chromatograph (HPLC) method of the present invention
表6Table 6
Figure PCTCN2020113712-appb-000011
Figure PCTCN2020113712-appb-000011
Figure PCTCN2020113712-appb-000012
Figure PCTCN2020113712-appb-000012
恒温恒湿箱Humidity Chamber
生产厂家：BinderManufacturer: Binder
设备型号：KBF-240Device model: KBF-240
附图说明Description of the drawings
图1为式(I)化合物A晶型的Cu-Kα辐射的XRPD谱图。Fig. 1 is an XRPD spectrum of Cu-Kα radiation of the crystal form of compound A of formula (I).
图2为式(I)化合物A晶型的DSC谱图。Figure 2 is a DSC spectrum of the crystal form of compound A of formula (I).
图3为式(I)化合物A晶型的TGA谱图。Figure 3 is a TGA spectrum of the crystal form of compound A of formula (I).
图4为式(I)化合物A晶型的DVS谱图。正方形点标线表示解吸附过程曲线，菱形点标实线表示吸附曲线。Figure 4 is a DVS spectrum of the crystal form of compound A of formula (I). The square dotted line represents the desorption process curve, and the diamond-shaped dotted solid line represents the adsorption curve.
图5为式(I)化合物B晶型的Cu-Kα辐射的XRPD谱图。Fig. 5 is an XRPD spectrum of Cu-Kα radiation of the crystal form of compound B of formula (I).
图6为式(I)化合物B晶型的DSC谱图。Fig. 6 is a DSC chart of the crystal form of compound B of formula (I).
图7为式(I)化合物B晶型的TGA谱图。Figure 7 is a TGA spectrum of the crystal form of compound B of formula (I).
图8为式(I)化合物C晶型的Cu-Kα辐射的XRPD谱图。Fig. 8 is an XRPD spectrum of Cu-Kα radiation of the crystalline form C of compound of formula (I).
图9为式(I)化合物C晶型的DSC谱图。Figure 9 is a DSC spectrum of the crystal form of compound C of formula (I).
图10为式(I)化合物C晶型的TGA谱图。Figure 10 is a TGA spectrum of the crystal form of compound C of formula (I).
图11为式(I)化合物D晶型的Cu-Kα辐射的XRPD谱图。Fig. 11 is an XRPD spectrum of Cu-Kα radiation of the crystal form D of compound of formula (I).
图12为式(I)化合物D晶型的DSC谱图。Fig. 12 is a DSC chart of the crystalline form D of compound of formula (I).
图13为式(I)化合物D晶型的TGA谱图。Figure 13 is a TGA spectrum of the crystal form D of compound of formula (I).
图14为式(I)化合物D晶型的DVS谱图。Figure 14 is a DVS spectrum of the crystal form D of compound of formula (I).
图15为式(I)化合物D晶型在40度、75％RH条件下放样取样(下端曲线为0天，上端曲线为3个月)的Cu-Kα辐射的XRPD叠加谱图。Fig. 15 is an XRPD superimposed spectrum of Cu-Kα radiation of the crystal form of compound D of formula (I) under 40°C and 75% RH conditions (the lower curve is 0 days and the upper curve is 3 months).
图16为式(I)化合物的立体结构椭球图。Figure 16 is an ellipsoid diagram of the three-dimensional structure of the compound of formula (I).
具体实施方式detailed description
为了更好的理解本发明的内容，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发明的内容所做的限制。In order to better understand the content of the present invention, a further description will be given below in conjunction with specific embodiments, but the specific implementation manners are not a limitation on the content of the present invention.
实施例1：式(I)化合物的制备Example 1: Preparation of the compound of formula (I)
Figure PCTCN2020113712-appb-000013
Figure PCTCN2020113712-appb-000013
步骤A：在25℃下，向化合物1(2.00kg,17.68mol,1.94L,1.00eq)的EtOH(25.00L)溶液中逐滴加入N,N-二甲基甲酰胺二甲基缩醛(2.74kg,22.98mol,3.04L,1.30eq)，反应液搅拌16小时。反应液减压浓缩得到粗品。粗产物通过硅胶色谱柱(PE:EA＝1:0-2.5:1)纯化分离得到化合物2(2.5kg，收率84.07％)。Step A: Add N,N-dimethylformamide dimethyl acetal (25.00L) to the EtOH (25.00L) solution of compound 1 (2.00kg, 17.68mol, 1.94L, 1.00eq) at 25°C. 2.74kg, 22.98mol, 3.04L, 1.30eq), the reaction solution was stirred for 16 hours. The reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was purified and separated by silica gel chromatography column (PE:EA=1:0-2.5:1) to obtain compound 2 (2.5kg, yield 84.07%).
步骤B：在25℃下，向化合物2(2.50kg,14.86mol,1.00eq)加入2-丙胺(2.64kg, 44.58mol,3.82L,3.00eq)，反应液升温至85℃搅拌16小时。反应液冷却至室温后，减压浓缩得到化合物3(2.4kg，收率88.63％)。Step B: At 25°C, 2-propylamine (2.64kg, 44.58mol, 3.82L, 3.00eq) was added to compound 2 (2.50kg, 14.86mol, 1.00eq), and the reaction solution was heated to 85°C and stirred for 16 hours. After the reaction solution was cooled to room temperature, it was concentrated under reduced pressure to obtain compound 3 (2.4 kg, yield 88.63%).
步骤C：在25℃下，向化合物3(1.20kg,6.59mol,1.00eq)的THF(30.00L)溶液中加入K 3PO 4(3.70kg,17.43mol,2.65eq)和NBS(2.50kg,14.05mol,2.13eq)，搅拌12小时。反应液过滤，向滤液中加入20L饱和Na 2SO 3溶液，EA(10L*2)萃取，合并有机相并用饱和食盐水(10L*2)洗，无水硫酸钠干燥后过滤，滤液减压浓缩后得粗品。粗产物通过硅胶色谱柱(PE:EA＝1:0-1:1)纯化分离得到化合物4(1.03kg，收率59.85％)。 Step C: Add K 3 PO 4 (3.70 kg, 17.43 mol, 2.65 eq) and NBS (2.50 kg, 14.05mol, 2.13eq), stirred for 12 hours. The reaction solution was filtered, 20L saturated Na 2 SO 3 solution was added to the filtrate, extracted with EA (10L*2), the organic phases were combined and washed with saturated brine (10L*2), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure After getting the crude product. The crude product was purified and separated by silica gel chromatography column (PE:EA=1:0-1:1) to obtain compound 4 (1.03 kg, yield 59.85%).
步骤D：-70℃、氮气保护条件下，向化合物4(50g,191.49mmol,1eq)的THF(500mL)溶液中缓慢滴加LDA(2M,150mL,1.57eq)，搅拌0.5小时后，将4-氯苯甲醛(32.30g,229.78mmol,1.2eq)的THF(30mL)溶液缓慢加入反应液中，滴加完毕-70℃搅拌1.5小时。将NH 4Cl(200mL)加入反应液中，EA(300mL*2)萃取，合并有机相后水洗，饱和食盐水洗，无水硫酸钠干燥后过滤，滤液减压浓缩得到粗品。粗产物通过硅胶色谱柱(PE:EA＝10:1-3:1)纯化分离得到化合物5(12g,收率15.62％)。 Step D: Under nitrogen protection conditions at -70°C, LDA (2M, 150mL, 1.57eq) was slowly added dropwise to a solution of compound 4 (50g, 191.49mmol, 1eq) in THF (500mL). After stirring for 0.5 hours, 4 -Chlorobenzaldehyde (32.30g, 229.78mmol, 1.2eq) in THF (30mL) solution was slowly added to the reaction solution, and the addition was completed and stirred at -70°C for 1.5 hours. NH 4 Cl (200 mL) was added to the reaction solution, extracted with EA (300 mL*2), combined the organic phases, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified and separated by silica gel chromatography column (PE:EA=10:1-3:1) to obtain compound 5 (12g, yield 15.62%).
步骤E：在25℃下，向化合物5(12g,29.87mmol,1eq)的DCM(120mL)溶液中加入SOCl 2(21.32g,179.25mmol,13.00mL,6eq)，搅拌1小时。水(80mL)缓慢加入反应液中，将有机相分离出来，饱和食盐水洗，无水硫酸钠干燥，过滤后滤液减压浓缩得到化合物6(9g,收率71.66％)。 Step E: At 25°C, SOCl 2 (21.32g, 179.25mmol, 13.00mL, 6eq) was added to a solution of compound 5 (12g, 29.87mmol, 1eq) in DCM (120mL) and stirred for 1 hour. Water (80 mL) was slowly added to the reaction solution, the organic phase was separated, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 6 (9 g, yield 71.66%).
步骤F：室温下，向化合物6(10.1g,24.04mmol,1eq)和胡椒胺(3.30g,24.04mmol,1eq)的乙腈(120mL)溶液中加入DIEA(12.43g,96.16mmol,16.75mL,4eq)，反应液升温至80℃搅拌12小时。冷却后向反应液中加入盐酸水溶液(1M,50mL)并浓缩，残余物用乙酸乙酯(200mL*2)萃取。合并有机相，饱和食盐水(100mL*2)洗涤，Na 2SO 4干燥，过滤浓缩。残余物经柱层析(SiO 2,PE:EA＝10:1至3:1)纯化分离得到化合物7(11g，收率87.86％)。 Step F: At room temperature, add DIEA (12.43g, 96.16mmol, 16.75mL, 4eq) to the acetonitrile (120mL) solution of compound 6 (10.1g, 24.04mmol, 1eq) and piperamine (3.30g, 24.04mmol, 1eq) ), the reaction solution was heated to 80°C and stirred for 12 hours. After cooling, an aqueous hydrochloric acid solution (1M, 50 mL) was added to the reaction solution and concentrated, and the residue was extracted with ethyl acetate (200 mL*2). The organic phases were combined, washed with saturated brine (100 mL*2), dried over Na 2 SO 4 , filtered and concentrated. The residue was purified and separated by column chromatography (SiO 2 , PE:EA=10:1 to 3:1) to obtain compound 7 (11 g, yield 87.86%).
步骤G：室温下，向化合物7(15g,28.80mmol,1eq)的MeOH(30mL)、H 2O(40mL)和THF(110mL)的混合溶液中加入NaOH(5.76g,144.01mmol,5eq)，室温下搅拌2小时。反应液经HCl(1M,30mL)水溶液调节pH至5左右，乙酸乙酯(200mL*2)萃取。合并有机相并有饱和食盐水(100mL*2)洗涤，Na 2SO 4干燥，过滤，收集并干燥滤饼得到化合物8(14g，收率98.65％)。 Step G: At room temperature, add NaOH (5.76g, 144.01mmol, 5eq) to a mixed solution of compound 7 (15g, 28.80mmol, 1eq) in MeOH (30mL), H 2 O (40mL) and THF (110mL), Stir at room temperature for 2 hours. The pH of the reaction solution was adjusted to about 5 with aqueous HCl (1M, 30 mL), and extracted with ethyl acetate (200 mL*2). The organic phases were combined and washed with saturated brine (100 mL*2), dried over Na 2 SO 4 , filtered, and the filter cake was collected and dried to obtain compound 8 (14 g, yield 98.65%).
步骤H：室温下，向化合物8(14g,28.41mmol,1eq)的DCM(140mL)溶液中加入T 3P(36.16g,56.82mmol,33.79mL,50％纯度,2eq)和吡啶(11.24g,142.06mmol,11.47mL,5eq)，25℃搅拌1小时。向反应液中加入NH 4Cl(100mL)水溶液，DCM(150mL*2)萃 取。合并有机相，饱和食盐水(100mL*2)洗涤，Na 2SO 4干燥，过滤浓缩。残余物经(EA:PE＝1:5,40mL)打浆得到化合物9(12g，收率88.97％)。 Step H: At room temperature, to a solution of compound 8 (14g, 28.41mmol, 1eq) in DCM (140mL) was added T 3 P (36.16g, 56.82mmol, 33.79mL, 50% purity, 2eq) and pyridine (11.24g, 142.06mmol, 11.47mL, 5eq), stirred at 25°C for 1 hour. An aqueous NH 4 Cl (100 mL) solution was added to the reaction solution, and extraction was performed with DCM (150 mL*2). The organic phases were combined, washed with saturated brine (100 mL*2), dried over Na 2 SO 4 , filtered and concentrated. The residue was slurried (EA:PE=1:5, 40 mL) to obtain compound 9 (12 g, yield 88.97%).
步骤I：-70℃、氮气保护下，向化合物9(1g,2.11mmol,1eq)的THF(15mL)溶液中缓慢加入KHMDS(1M,4.50mL,2.14eq)溶液。加毕于-70℃搅拌1小时，随后加入CH 3I(3.020g,21.28mmol,1.32mL,10.10eq)，反应液继续搅拌1.5小时。向反应液中加入20mL饱和NH 4Cl(aq.)水溶液，乙酸乙酯(20mL*2)萃取，合并有机相，饱和食盐水(20mL*1)洗涤，Na 2SO 4干燥，过滤浓缩。残余物经柱层析(SiO 2,PE:EA＝1:0-3:1-1:1)纯化分离得到化合物10(450mg，收率42.58％)。 Step I: Under the protection of nitrogen at -70°C, slowly add a KHMDS (1M, 4.50 mL, 2.14 eq) solution to a THF (15 mL) solution of compound 9 (1 g, 2.11 mmol, 1 eq). After the addition, the mixture was stirred at -70°C for 1 hour, and then CH 3 I (3.020 g, 21.28 mmol, 1.32 mL, 10.10 eq) was added, and the reaction solution was stirred for 1.5 hours. 20 mL of saturated NH 4 Cl (aq.) aqueous solution was added to the reaction solution, extracted with ethyl acetate (20 mL*2), the organic phases were combined, washed with saturated brine (20 mL*1), dried over Na 2 SO 4 , filtered and concentrated. The residue was purified and separated by column chromatography (SiO 2 , PE:EA=1:0-3:1-1:1) to obtain compound 10 (450 mg, yield 42.58%).
步骤J:室温、氮气保护下，向化合物10(300mg,613.80μmol,1eq)和2,4-二甲氧基-嘧啶-5-硼酸(180.00mg,978.48μmol,1.59eq)的二恶烷(12mL)和水(4mL)的混合溶剂中分别加入K 3PO 4(270.00mg,1.27mmol,2.07eq)和Pd(dppf)Cl 2.CH 2Cl 2(102.00mg,124.90μmol,2.03e-1eq)，反应液升温至100℃搅拌12小时。待反应液冷却后过滤，滤液用水10mL)稀释，乙酸乙酯(20mL*2)萃取。合并有机相，饱和食盐水(10mL*1)洗涤，Na 2SO 4干燥，过滤浓缩。残余物经柱层析(SiO2,PE:EA-1:0-3:1-1:1)和制备型HPLC(柱:Luna C18 150mm*25mm 5μm；流动相:[水(0.225％甲酸)-乙腈]；乙腈％:57％-67％)纯化分离得到式(I)化合物(保留时间：1.291min，60mg，收率17.54％)。 Step J: Add compound 10 (300mg, 613.80μmol, 1eq) and 2,4-dimethoxy-pyrimidine-5-boronic acid (180.00mg, 978.48μmol, 1.59eq) in dioxane ( 12mL) and water (4mL) mixed solvents were added K 3 PO 4 (270.00mg, 1.27mmol, 2.07eq) and Pd(dppf)Cl 2 .CH 2 Cl 2 (102.00mg, 124.90μmol, 2.03e-1eq) ), the reaction solution was heated to 100°C and stirred for 12 hours. After the reaction solution was cooled, it was filtered, the filtrate was diluted with water (10 mL), and extracted with ethyl acetate (20 mL*2). The organic phases were combined, washed with saturated brine (10 mL*1), dried over Na 2 SO 4 , filtered and concentrated. The residue was subjected to column chromatography (SiO2, PE:EA-1:0-3:1-1:1) and preparative HPLC (column: Luna C18 150mm*25mm 5μm; mobile phase: [water (0.225% formic acid)- Acetonitrile]; Acetonitrile%: 57%-67%) was purified and separated to obtain the compound of formula (I) (retention time: 1.291 min, 60 mg, yield 17.54%).
1H NMR(400MHz,DMSO-d 6):δ＝8.49(s,1H),7.47(d,J＝8.8Hz,2H),7.27(d,J＝8.4Hz,2H),6.82(d,J＝8.2Hz,1H),6.40(d,J＝2.0Hz,1H),6.16(dd,J＝2.0,8.3Hz,1H),6.02(d,J＝0.8Hz,1H),6.01(s,1H),4.18-4.07(m,1H),3.99(s,3H),3.94(s,3H),1.97(s,3H),1.17(d,J＝6.8Hz,3H),0.62(d,J＝6.8Hz,3H). 1 H NMR (400MHz, DMSO-d 6 ): δ = 8.49 (s, 1H), 7.47 (d, J = 8.8 Hz, 2H), 7.27 (d, J = 8.4 Hz, 2H), 6.82 (d, J ＝8.2Hz,1H),6.40(d,J＝2.0Hz,1H), 6.16(dd,J＝2.0,8.3Hz,1H), 6.02(d,J＝0.8Hz,1H), 6.01(s,1H ), 4.18-4.07 (m, 1H), 3.99 (s, 3H), 3.94 (s, 3H), 1.97 (s, 3H), 1.17 (d, J = 6.8 Hz, 3H), 0.62 (d, J = 6.8Hz, 3H).
实施例2：式(I)化合物A晶型的制备Example 2: Preparation of Compound A of Formula (I)
取式(I)化合物100mg置于单口瓶中，加入500mL甲醇溶解后浓缩干燥除去化合物中残留乙腈。向析出的样品中加入300μL甲醇后，将样品液加热至65℃，溶液澄清，放置室温下，析出固体，40℃真空干燥48小时； 1HNMR观察有甲醇残留。后又在80℃真空干燥箱内干燥3小时，得到的固体经XRPD鉴定为A晶型。式(I)化合物A晶型的XRPD谱图见图1，XRPD谱图解析数据见表1。 Take 100 mg of the compound of formula (I) and place it in a single-necked flask, add 500 mL of methanol to dissolve, concentrate and dry to remove residual acetonitrile in the compound. After adding 300 μL of methanol to the precipitated sample, the sample solution was heated to 65° C., the solution was clear, placed at room temperature, a solid precipitated, and dried under vacuum at 40° C. for 48 hours; 1 HNMR observed that there was methanol residue. Then, it was dried in a vacuum drying oven at 80°C for 3 hours, and the obtained solid was identified as A crystal form by XRPD. The XRPD spectrum of the crystal form of compound A of formula (I) is shown in Figure 1, and the analytical data of the XRPD spectrum is shown in Table 1.
实施例3：式(I)化合物B晶型的制备Example 3: Preparation of Compound B of Formula (I)
取式(I)化合物50mg至液相小瓶中，分别加入适量的乙醇使其成悬浊液。然后将上述悬浊液样品置于40℃的恒温混匀仪上以700rpm的转速振摇4天。然后离心(8000rpm，3min)分离出固体残渣，在30℃真空干燥箱内干燥过夜，得到的固体经XRPD鉴定为B晶型。式(I)化合物B晶型的XRPD谱图见图5，XRPD谱图解析数据见表2。Take 50 mg of the compound of formula (I) into a liquid phase vial, and add an appropriate amount of ethanol to make a suspension. Then the above suspension sample was placed on a constant temperature mixer at 40° C. and shaken at 700 rpm for 4 days. Then, the solid residue was separated by centrifugation (8000rpm, 3min) and dried overnight in a vacuum drying oven at 30°C. The obtained solid was identified as crystal form B by XRPD. The XRPD spectrum of the crystal form of compound B of formula (I) is shown in Fig. 5, and the analytical data of the XRPD spectrum is shown in Table 2.
实施例4：式(I)化合物C晶型的制备Example 4: Preparation of Compound C of Formula (I)
取式(I)化合物50mg至液相小瓶中，分别加入适量的四氢呋喃使其成悬浊液。然后将上述悬浊液样品置于40℃的恒温混匀仪上以700rpm的转速振摇4天。然后离心(8000rpm，3min)分离出固体残渣，在30℃真空干燥箱内干燥过夜，得到的固体经XRPD鉴定为C晶型。式(I)化合物C晶型的XRPD谱图见图8，XRPD谱图解析数据见表3。Take 50 mg of the compound of formula (I) into a liquid phase vial, and add appropriate amounts of tetrahydrofuran to make a suspension. Then the above suspension sample was placed on a constant temperature mixer at 40° C. and shaken at 700 rpm for 4 days. Then, the solid residue was separated by centrifugation (8000rpm, 3min) and dried overnight in a vacuum drying oven at 30°C. The obtained solid was identified as crystal form C by XRPD. The XRPD spectrum of the crystal form of compound C of formula (I) is shown in Figure 8, and the analytical data of the XRPD spectrum is shown in Table 3.
实施例5：式(I)化合物D晶型的制备Example 5: Preparation of Compound D of Formula (I)
方法1：method 1:
取式(I)化合物500mg至液相小瓶中，加入水使其成悬浊液。然后将上述悬浊液样品置于40℃的恒温混匀仪上以700rpm的转速振摇1天。然后离心分离出固体，在30℃真空干燥箱内干燥过夜，得到的固体经XRPD鉴定为D晶型。式(I)化合物D晶型的XRPD谱图见图11，XRPD谱图解析数据见表4。Take 500 mg of the compound of formula (I) into a liquid phase vial, and add water to make a suspension. Then the above suspension sample was placed on a constant temperature mixer at 40° C. and shaken at 700 rpm for 1 day. Then the solid was separated by centrifugation and dried overnight in a vacuum drying oven at 30°C. The obtained solid was identified as crystal form D by XRPD. The XRPD spectrum of the crystal form of compound D of formula (I) is shown in Fig. 11, and the analytical data of the XRPD spectrum is shown in Table 4.
方法2:Method 2:
取式(I)化合物1.4g，加入乙酸乙酯14mL，加热至80℃搅拌1小时。缓慢冷却至室温，过滤得到滤饼，滤饼真空干燥，得到800mg白色固体。得到的固体经XRPD鉴定为D晶型。Take 1.4 g of the compound of formula (I), add 14 mL of ethyl acetate, and heat to 80° C. and stir for 1 hour. Slowly cooled to room temperature, filtered to obtain a filter cake, and the filter cake was vacuum dried to obtain 800 mg of white solid. The obtained solid was identified as crystal form D by XRPD.
方法3：Method 3:
取式(I)化合物1.0g，加入乙醇12mL，加热至80℃搅拌2小时。加入水18mL，缓慢冷却至室温，过滤，滤饼真空干燥，得到0.90g白色固体。得到的固体经XRPD鉴定为D晶型。Take 1.0 g of the compound of formula (I), add 12 mL of ethanol, and heat to 80° C. and stir for 2 hours. Add 18 mL of water, slowly cool to room temperature, filter, and dry the filter cake under vacuum to obtain 0.90 g of white solid. The obtained solid was identified as crystal form D by XRPD.
方法4：Method 4:
取式(I)化合物1.0g，加入10mL水，加热至100℃搅拌2小时。冷却至室温，过滤，滤饼用水(5mL*2)洗涤，滤饼真空干燥，得到0.90g白色固体。得到的固体经XRPD鉴定为D晶型。Take 1.0 g of the compound of formula (I), add 10 mL of water, and heat to 100° C. and stir for 2 hours. Cool to room temperature, filter, wash the filter cake with water (5 mL*2), and dry the filter cake under vacuum to obtain 0.90 g of white solid. The obtained solid was identified as crystal form D by XRPD.
实施例6：式(I)化合物A晶型和D晶型的吸湿性研究Example 6: Study on Hygroscopicity of Compound A and D of Formula (I)
实验材料：Experimental Materials:
SMS DVS Advantage动态蒸汽吸附仪SMS DVS Advantage Dynamic Vapor Sorption Apparatus
实验方法：experimental method:
分别取式(I)化合物A和D晶型适量置于DVS样品盘内进行DVS分析。Take appropriate amounts of the crystal forms of compound A and D of formula (I) and place them in the DVS sample pan for DVS analysis.
实验结果：Experimental results:
式(I)化合物A晶型的DVS谱图如图4所示，在25℃/80％RH下△W＝5.4％；式 (I)化合物D晶型的DVS谱图如图14所示，△W＝1.3％；。The DVS spectrum of the crystal form of compound A of formula (I) is shown in Figure 4, and △W=5.4% at 25°C/80%RH; the DVS spectrum of the crystal form of compound D of formula (I) is shown in Figure 14. △W=1.3%;.
实验结论：Experimental results:
式(I)化合物在25℃/80％RH下，A晶型在80％湿度下吸湿5.4％，有引湿性；D晶型吸湿增重1.3％，略有引湿性。For the compound of formula (I) at 25° C./80% RH, crystal form A absorbs 5.4% moisture at 80% humidity and has hygroscopicity; crystal form D absorbs moisture and increases weight by 1.3%, which is slightly hygroscopic.
实施例7：式(I)化合物D晶型高温和高湿条件下的固体稳定性试验Example 7: Solid stability test under high temperature and high humidity conditions of the crystal form of compound D of formula (I)
实验目的：Purpose:
考察式(I)化合物D晶型在高温和高湿(40℃/75％RH,60℃/75％RH)条件下的稳定性。The stability of the crystal form of compound D of formula (I) under high temperature and high humidity (40℃/75%RH, 60℃/75%RH) conditions was investigated.
试验方法：experiment method:
取式(I)化合物D晶型大约10mg，准确称量，置于样品瓶中，摊成薄层，直接用铝箔纸包好瓶口并在铝箔纸上扎些小孔，垂直置于40℃/75％RH,60℃/75％RH的恒温恒湿箱中，每个时间点1份作为稳定性评价供试样品。对照的样品常温保存，待在考察时间点与加速供试样品一同分析。另外取一份样品约10mg用于考察样品的物理稳定性XRPD测试，样品瓶用铝箔纸包好并扎小孔，同样置于40℃/75％RH,60℃/75％RH的恒温恒湿箱中。试验期间在时间点为4周时取样分析。Take about 10 mg of compound D crystal form of formula (I), accurately weigh it, place it in a sample bottle, spread it into a thin layer, wrap the bottle mouth directly with aluminum foil paper and tie some small holes in the aluminum foil paper, and place it vertically at 40°C /75%RH, 60℃/75%RH constant temperature and humidity box, each time point 1 copy as the test sample for stability evaluation. The control sample is stored at room temperature and will be analyzed together with the accelerated test sample at the time of inspection. In addition, take a sample of about 10mg to examine the physical stability of the sample. XRPD test. The sample bottle is wrapped in aluminum foil and pierced with small holes. It is also placed at 40℃/75%RH, 60℃/75%RH at constant temperature and humidity. In the box. During the test, samples were taken for analysis at the time point of 4 weeks.
试验结果：见下表7、表8。Test results: see Table 7 and Table 8 below.
表7 式(I)化合物D晶型的固体稳定性试验Table 7 Solid stability test of compound D crystal form of formula (I)
试验条件Test conditions 时间点Point in time 晶型(XRPD)Crystal form (XRPD)
—— 0天0 days D晶型 Crystal Form D
40℃/75％RH40℃/75%RH 4周4 weeks D晶型 Crystal Form D
60℃/75％RH60℃/75%RH 4周4 weeks D晶型Crystal Form D
表8 式(I)化合物D晶型的固体稳定性试验HPLC分析结果Table 8 HPLC analysis results of solid stability test of crystal form D of compound of formula (I)
相对保留时间Relative retention time 0.540.54 0.710.71 0.860.86 0.970.97 1.091.09 1.421.42 纯度(％)purity(%) 含量(％)content(%)
0天0 days -- -- 0.110.11 0.150.15 1.061.06 0.110.11 98.5798.57 100100
40℃/75％RH，4周40℃/75%RH, 4 weeks 0.040.04 0.080.08 0.120.12 0.140.14 1.061.06 0.130.13 98.4398.43 103.45103.45
60℃/75％RH，4周60℃/75%RH, 4 weeks -- 0.080.08 0.120.12 0.140.14 1.071.07 0.230.23 98.3698.36 104.2104.2
实验结论：式(I)化合物D晶型在高温高湿条件下稳定。Experimental conclusion: The crystal form of compound D of formula (I) is stable under high temperature and high humidity conditions.
实施例8：式(I)化合物D晶型高温高湿条件下的长期晶型稳定性试验Example 8: Long-term stability test of crystal form of compound D of formula (I) under high temperature and high humidity conditions
考察式(I)化合物D晶型在40℃/75％RH条件下放置并在不同的时间点(0天，3个月)取样检测XRPD。XRPD结果见图15。The crystal form of compound D of formula (I) was placed under the condition of 40°C/75%RH and samples were taken at different time points (0 days, 3 months) to detect XRPD. The XRPD results are shown in Figure 15.
实验结论：式(I)化合物D晶型在长时间高温高湿条件下稳定。Experimental conclusion: The crystal form of compound D of formula (I) is stable under long-term high temperature and high humidity conditions.
实施例9：式(I)化合物立体构型确证Example 9: Confirmation of the stereo configuration of the compound of formula (I)
式(I)化合物晶体制备：称取式(I)化合物10mg置于单口瓶中，加入2mL无水乙醇加热溶解后敞口静置，挑出合适的晶体用于X-射线单晶结构解析。Preparation of compound crystal of formula (I): weigh 10 mg of compound of formula (I) into a single-necked flask, add 2 mL of absolute ethanol to heat to dissolve, and then leave to stand in the open, pick out suitable crystals for X-ray single crystal structure analysis.
实验结果：式(I)化合物的立体结构椭球图见图16，为S构型。式(I)化合物晶体结构数据和参数见表9、10、11和12。Experimental results: The ellipsoid diagram of the three-dimensional structure of the compound of formula (I) is shown in Figure 16, which is the S configuration. The crystal structure data and parameters of the compound of formula (I) are shown in Tables 9, 10, 11 and 12.
表9 式(I)化合物的晶体结构数据和测定参数Table 9 Crystal structure data and measurement parameters of the compound of formula (I)
Figure PCTCN2020113712-appb-000014
Figure PCTCN2020113712-appb-000014
表10 式(I)化合物晶体的原子坐标(×10 4)和等价各向同性移位参数
Figure PCTCN2020113712-appb-000015
 Table 10 Atomic coordinates (×10 4 ) and equivalent isotropic shift parameters of the crystals of the compound of formula (I)
Figure PCTCN2020113712-appb-000015
Figure PCTCN2020113712-appb-000016
Figure PCTCN2020113712-appb-000016
Figure PCTCN2020113712-appb-000017
Figure PCTCN2020113712-appb-000017
Figure PCTCN2020113712-appb-000018
Figure PCTCN2020113712-appb-000018
表11 式(I)化合物晶体的键长
Figure PCTCN2020113712-appb-000019
和键角[deg] Table 11 The bond length of the crystals of the compound of formula (I)
Figure PCTCN2020113712-appb-000019
And bond angle [deg]
Figure PCTCN2020113712-appb-000020
Figure PCTCN2020113712-appb-000020
Figure PCTCN2020113712-appb-000021
Figure PCTCN2020113712-appb-000021
Figure PCTCN2020113712-appb-000022
Figure PCTCN2020113712-appb-000022
表12 式(I)化合物晶体的扭转角度[deg]Table 12 The torsion angle of the crystal of the compound of formula (I) [deg]
Figure PCTCN2020113712-appb-000023
Figure PCTCN2020113712-appb-000023
Figure PCTCN2020113712-appb-000024
Figure PCTCN2020113712-appb-000024
实施例10：式(I)化合物酶水平活性测定Example 10: Determination of the enzyme level activity of the compound of formula (I)
本发明中应用MDM2/p53蛋白蛋白结合实验采用TR-FRET方法检测。具体步骤如下：用Echo移液器(Labcyte)对受试化合物进行3.162倍梯度稀释，每个化合物稀释11个浓度并分别转移250nL到384孔板中，每个化合物浓度设两复孔。设置加阳性化合物(100％抑制)的孔作为阳性对照，只加DMSO的孔作为阴性对照。用缓冲液(125mM NaCl，1mM DTT，0.01％Gelatin(动物明胶)，0.1％Pluronic f-127(聚醚)，1×PBS)将GST-MDM2蛋白(R&D-E3-202-050)稀释至0.625nM并加20μL到384孔板中。离心，震荡后将384孔板放入23℃温箱中孵育20min。用缓冲液将His-p53蛋白(R&D-SP-450-020)稀释至12.5nM并加20μL到384孔板中。离心，震荡后将384孔板放入23℃温箱中孵育60min。用缓冲液稀释Eu2+anti-GST抗体(Cisbio-61GSTKLB)和XL665 anti-His抗体(Cisbio-61HISXLB)，稀释得到的混合物中包含0.3nM的Eu2+anti-GST抗体和9nM的XL665 anti-His抗体。加10μL两种抗体的混合物到384孔板中。离心，震荡后将384孔板放入23℃温箱中孵育20h。在Envision多功能酶标仪(PerkinElmer)上读数(激发光340nm，发射光665nm、615nm)。Ratio＝665nm下信号强度/615nm下信号强度×10000，用Ratio值计算得到抑制率，公式如下：抑制率＝(加化合物孔Ratio-阴性对照Ratio)/(阳性对照Ratio-阴性对照Ratio)*100％，式(I)化合物的IC 50值示于下表13中。 In the present invention, the MDM2/p53 protein binding experiment adopts the TR-FRET method for detection. The specific steps are as follows: Use an Echo pipette (Labcyte) to perform a 3.162-fold gradient dilution of the test compound, dilute each compound at 11 concentrations and transfer 250 nL to a 384-well plate, and set up two replicate wells for each compound concentration. Set the well with positive compound (100% inhibition) as a positive control, and set the well with only DMSO as a negative control. Dilute GST-MDM2 protein (R&D-E3-202-050) to 0.625 with buffer (125mM NaCl, 1mM DTT, 0.01% Gelatin (animal gelatin), 0.1% Pluronic f-127 (polyether), 1×PBS) nM and add 20μL to a 384-well plate. After centrifugation and shaking, put the 384-well plate in a 23°C incubator and incubate for 20 min. Dilute His-p53 protein (R&D-SP-450-020) to 12.5 nM with buffer and add 20 μL to a 384-well plate. After centrifugation and shaking, put the 384-well plate in a 23°C incubator and incubate for 60 min. Dilute Eu2+anti-GST antibody (Cisbio-61GSTKLB) and XL665 anti-His antibody (Cisbio-61HISXLB) with buffer. The diluted mixture contains 0.3nM Eu2+anti-GST antibody and 9nM XL665 anti-His antibody . Add 10 μL of the mixture of the two antibodies to a 384-well plate. After centrifugation and shaking, place the 384-well plate in a 23°C incubator and incubate for 20 hours. Read on Envision multifunctional microplate reader (PerkinElmer) (excitation light 340nm, emission light 665nm, 615nm). Ratio=Signal intensity at 665nm/Signal intensity at 615nm×10000. Use Ratio to calculate the inhibition rate. The formula is as follows: Inhibition rate=(add compound well Ratio-negative control Ratio)/(positive control Ratio-negative control Ratio)*100 %, the IC 50 value of the compound of formula (I) is shown in Table 13 below.
实施例11：式(I)化合物细胞水平活性测定Example 11: Cell-level activity determination of the compound of formula (I)
SJSA-1细胞增殖实验采用碘化丙啶染色检测。碘化丙啶无法通过活细胞的细胞膜，却可以透过凋亡细胞的细胞膜，从而对细胞进行染色。具体步骤如下：分离细胞培养瓶中处于对数生长期的SJSA-1细胞(来自药明康德生物部细胞库)，计数。用添加了10％FBS、1％双抗和1％L-谷氨酰胺的RPMI1640细胞培养基将SJSA-1细胞稀释到1X10 5个细胞每毫升。向384孔板的最外围一圈孔中加入100μL PBS，向第二列孔中加入25μL RPMI1640细胞培养基作为阳性对照，向其它孔中加入25μL细胞悬液(2500个细胞每孔)。室温静置20min后，将细胞板放入细胞培养箱中过夜培养。第二天用Echo移液器(Labcyte)对受试化合物进行3.162倍梯度稀释，每个化合物稀释10个浓度并分别转移300nL到化合物板中，每个化合物浓度设两复孔。第24列不加化合物作为阴性对照。向化合物板除最外围一圈孔的所有孔中加30μL RPMI1640细胞培养基，离心，震荡。然后从化合物板中转移25μL化合物到细胞板中。将细胞板放入细胞培养箱中培养。72h后，向细胞板除最外围一圈孔的所有孔中加10μL 15μM YO-PRO-1(Invitrogen-Y3603)染料。离心，室温避光震荡20min后在Envision多功能酶标仪(PerkinElmer)上读数(激发光485nm，发射光535nm)。再向细胞板除最外围一圈孔的所有孔中加20μL细胞裂解液(150mM NaCl，2mM Tris pH 7.5，1mM EDTA，1mM EGTA，1％Triton X-100，ddH 2O)。离心，室温避光震荡20min后在Envision多功能酶标仪上读数。 The SJSA-1 cell proliferation experiment uses propidium iodide staining. Propidium iodide cannot pass through the cell membrane of living cells, but it can pass through the cell membrane of apoptotic cells to stain cells. The specific steps are as follows: Isolate the SJSA-1 cells in the logarithmic growth phase (from the cell bank of WuXi AppTec Department of Biology) in the cell culture flask, and count them. SJSA-1 cells were diluted to 1×10 5 cells per milliliter with RPMI1640 cell culture medium supplemented with 10% FBS, 1% double antibody and 1% L-glutamine. Add 100 μL of PBS to the outermost circle of the 384-well plate, add 25 μL of RPMI1640 cell culture medium to the second column of wells as a positive control, and add 25 μL of cell suspension (2500 cells per well) to the other wells. After standing at room temperature for 20 minutes, the cell plate was placed in a cell incubator for overnight culture. On the second day, the test compound was diluted 3.162 times with an Echo pipette (Labcyte), and each compound was diluted by 10 concentrations and transferred 300 nL to the compound plate. Two multiple wells were set up for each compound concentration. No compound was added in column 24 as a negative control. Add 30 μL of RPMI1640 cell culture medium to all the wells of the compound plate except the outermost circle of holes, centrifuge and shake. Then transfer 25 μL of compound from the compound plate to the cell plate. Put the cell plate into a cell incubator for culture. After 72 hours, add 10 μL of 15 μM YO-PRO-1 (Invitrogen-Y3603) dye to all the wells of the cell plate except the outermost circle of holes. After centrifugation and shaking at room temperature for 20 minutes, it was read on the Envision multifunctional microplate reader (PerkinElmer) (excitation light 485nm, emission light 535nm). Then add 20 μL of cell lysate (150 mM NaCl, 2 mM Tris pH 7.5, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, ddH 2 O) to all wells of the cell plate except for the outermost circle of wells. Centrifuge, shake at room temperature and avoid light for 20 minutes, and read on the Envision multi-function microplate reader.
用第二次读数得到的信号值减去第一次读数的信号值得到活细胞的信号值，按以下列公式计算药物对肿瘤细胞生长的抑制率：抑制率＝(加化合物孔信号-阴性对照信号)/(阳性对照信号-阴性对照信号)*100％。式(I)化合物对SJSA-1细胞的抗增殖活性(IC 50值)示于下表13中： Use the signal value of the second reading to subtract the signal value of the first reading to obtain the signal value of living cells. Calculate the inhibition rate of the drug on tumor cell growth according to the following formula: Inhibition rate = (add compound well signal-negative control Signal)/(positive control signal-negative control signal)*100%. The anti-proliferative activity (IC 50 value) of the compound of formula (I) on SJSA-1 cells is shown in Table 13 below:
表13 式(I)化合物体外筛选试验结果Table 13 In vitro screening test results of compounds of formula (I)
Figure PCTCN2020113712-appb-000025
Figure PCTCN2020113712-appb-000025
结论：式(I)化合物在与MDM2蛋白靶点的结合和抑制SJSA-1肿瘤细胞生长方面均表现出良好的活性。Conclusion: The compound of formula (I) shows good activity in binding to the MDM2 protein target and inhibiting the growth of SJSA-1 tumor cells.
实施例12：药代动力学研究Example 12: Pharmacokinetic study
以雌性Balb/c小鼠为受试动物，应用LC/MS/MS法测定了小鼠尾部静脉注射和口服盒式给药法(cassette dosing)给予阳性参照化合物NVP-HDM201、式(I)化合物后不同时刻血浆中的药物浓度。研究本发明的化合物在小鼠体内的药代动力学行为，评价其药代 动力学特征。Using female Balb/c mice as the test animals, the LC/MS/MS method was used to determine the mice's tail vein injection and oral cassette dosing method (cassette dosing) to give the positive reference compound NVP-HDM201, the compound of formula (I) The concentration of the drug in the plasma at different times afterwards. Study the pharmacokinetic behavior of the compound of the present invention in mice, and evaluate its pharmacokinetic characteristics.
实验药品：NVP-HDM201和式(I)化合物。Experimental drug: NVP-HDM201 and the compound of formula (I).
试验动物：Test animals:
健康幼年雌性Balb/c小鼠20-30g，总共6只。Healthy juvenile female Balb/c mice 20-30g, a total of 6 mice.
药物配制：Drug preparation:
称取适量样品，将NVP-HDM201和式(I)化合物，用5％DMSO/40％PEG400/55％水配制成0.2mg/mL的澄清溶液用于静脉注射，用0.5％MC水溶液配制成0.2mg/mL的混悬液用于口服组。Weigh an appropriate amount of sample, mix NVP-HDM201 and the compound of formula (I) with 5% DMSO/40% PEG400/55% water to prepare a clear solution of 0.2 mg/mL for intravenous injection, and prepare 0.2 with 0.5% MC aqueous solution. The mg/mL suspension was used in the oral group.
给药：Administration:
雌性Balb/c小鼠6只，禁食一夜后3只尾端静脉注射给药，剂量为0.5mg/kg；另外3只口服给药，剂量为2mg/kg。Six female Balb/c mice were given a caudal intravenous injection at a dose of 0.5 mg/kg after a one-night fast; the other three mice were administered orally at a dose of 2 mg/kg.
操作：operating:
于给药前及给药后0.08、0.25、0.5、1、2、4、8、24小时采血，置于肝素化抗凝试管中，7000rpm(5204g)、4℃下离心，分离血浆，于-80℃保存。给药后4小时进食。Blood was collected at 0.08, 0.25, 0.5, 1, 2, 4, 8, and 24 hours after administration, placed in a heparinized anticoagulation tube, centrifuged at 7000 rpm (5204g), 4°C, and separated plasma. Store at 80°C. Eat food 4 hours after administration.
用LCMS/MS法测定静脉注射和口服给药后小鼠血浆中的待测化合物含量。血浆样品经沉淀蛋白预处理后进行分析。The LCMS/MS method was used to determine the content of the test compound in mouse plasma after intravenous injection and oral administration. Plasma samples were analyzed after pretreatment with precipitated protein.
药代动力学参数结果：Pharmacokinetic parameter results:
表14 药代动力学研究结果Table 14 Pharmacokinetic study results
Figure PCTCN2020113712-appb-000026
Figure PCTCN2020113712-appb-000026
试验结论：Test Conclusions:
与NVP-HDM201比较，在小鼠静脉注射给药剂量为0.5mpk水平时，式(I)化合物体内半衰期更长。口服给药剂量为2mg/kg水平时，式(I)化合物血浆暴露量显著更大，口服生物利用度更高，具有更优的药代动力学性质。Compared with NVP-HDM201, the compound of formula (I) has a longer half-life in vivo when the dose of intravenous injection in mice is 0.5mpk. When the oral administration dose is 2 mg/kg, the plasma exposure of the compound of formula (I) is significantly greater, the oral bioavailability is higher, and the pharmacokinetic properties are better.
实施例13：MDR1-MDCK细胞双向渗透性评估实验Example 13: Two-way permeability evaluation experiment of MDR1-MDCK cells
实验目的：测定受试化合物在MDR1-MDCK细胞的渗透性Experimental purpose: To determine the permeability of the test compound in MDR1-MDCK cells
实验操作：取永久性表达人P-糖蛋白(P-glycoprotein)的MDR1-MDCK细胞种植在96孔Insert细胞板，培养4-7天后形成汇聚的单层细胞。受试化合物用HBSS缓冲液稀释(pH 7.4)至浓度为2μM，加于细胞顶端或基底外侧，于37℃,5％CO 2,95％相对湿度 的条件下孵育2.5小时后，取给药孔(donor wells)和接收孔(receiver wells)内的样品溶液立即与含有内标的冷乙腈溶液混合。并用含有内标的冷乙腈溶液裂解细胞来测量细胞内化合物的聚积量。采用LCMS/MS方法分析待测化合物在所有样品(包括起始给药液，给药孔上清液，接收液，细胞裂解液)中的浓度。待测化合物的浓度用其峰面积与内标峰面积之比来表示，检测化合物从A→B和B→A两个方向的渗透性。 Experimental operation: MDR1-MDCK cells that permanently express human P-glycoprotein (P-glycoprotein) were planted on a 96-well Insert cell plate and cultured for 4-7 days to form a convergent monolayer of cells. The test compound was diluted with HBSS buffer (pH 7.4) to a concentration of 2μM, added to the top or basolateral side of the cell, incubated at 37°C, 5% CO 2 , and 95% relative humidity for 2.5 hours, and then took the dosing hole The sample solutions in the donor wells and receiver wells are immediately mixed with the cold acetonitrile solution containing the internal standard. The cells were lysed with a cold acetonitrile solution containing an internal standard to measure the accumulation of intracellular compounds. The LCMS/MS method was used to analyze the concentration of the test compound in all samples (including the initial dosing solution, the supernatant of the dosing hole, the receiving solution, and the cell lysate). The concentration of the test compound is expressed by the ratio of its peak area to the peak area of the internal standard, and the permeability of the test compound from the two directions A→B and B→A.
表15 MDR1-MDCK细胞双向渗透性评估实验结果Table 15 Two-way permeability evaluation results of MDR1-MDCK cells
Figure PCTCN2020113712-appb-000027
Figure PCTCN2020113712-appb-000027
实验结论：式(I)化合物具有较好的渗透性。Experimental conclusion: The compound of formula (I) has better permeability.
实施例14：Caco-2细胞双向渗透性评估实验Example 14: Two-way permeability evaluation experiment of Caco-2 cells
实验目的：测定式(I)化合物在Caco-2细胞的渗透性Experimental purpose: To determine the permeability of the compound of formula (I) in Caco-2 cells
实验操作：将人结肠癌Caco-2细胞以1×10 5细胞/cm 2的密度种于96孔Insert细胞板，培养4-5天后形成汇聚的单层细胞。式(I)化合物用HBSS缓冲液稀释(pH 7.4)至浓度为2μM，加于细胞顶端或基底外侧，于37℃,5％CO 2和饱和湿度条件下孵育2.5小时后，取给药孔(donor wells)和接收孔(receiver wells)内的样品溶液立即与含有内标的冷乙腈溶液混合。并用含有内标的冷乙腈溶液裂解细胞来测量细胞内化合物的聚积量。采用LCMS/MS方法分析式(I)化合物在所有样品(包括起始给药液，给药孔上清液，接收液，细胞裂解液)中的浓度。待测化合物的浓度用其峰面积与内标峰面积之比来表示，检测化合物从A→B和B→A两个方向的渗透性。 Experimental operation: human colon cancer Caco-2 cells were seeded on a 96-well Insert cell plate at a density of 1×10 5 cells/cm 2 and cultured for 4-5 days to form a convergent monolayer of cells. The compound of formula (I) was diluted with HBSS buffer (pH 7.4) to a concentration of 2μM, added to the top or basolateral of the cell, incubated at 37°C, 5% CO 2 and saturated humidity for 2.5 hours, and then took the dosing hole ( The sample solutions in the donor wells and receiver wells are immediately mixed with the cold acetonitrile solution containing the internal standard. The cells were lysed with a cold acetonitrile solution containing an internal standard to measure the accumulation of intracellular compounds. The LCMS/MS method was used to analyze the concentration of the compound of formula (I) in all samples (including the initial dosing solution, the supernatant of the dosing hole, the receiving solution, and the cell lysis solution). The concentration of the test compound is expressed by the ratio of its peak area to the peak area of the internal standard, and the permeability of the test compound from the two directions A→B and B→A.
表16 Caco-2细胞双向渗透性评估实验结果Table 16 Two-way permeability evaluation results of Caco-2 cells
Figure PCTCN2020113712-appb-000028
Figure PCTCN2020113712-appb-000028
实验结论：式(I)化合物具有较好的渗透性。Experimental conclusion: The compound of formula (I) has better permeability.
实施例15：式(I)化合物在急性髓细胞白血病动物体内药效评价Example 15: Evaluation of the efficacy of the compound of formula (I) in acute myeloid leukemia animals
MV4-11肿瘤细胞按0.2mL(10×10 6个，含50％基质胶)分别皮下接种于每只小鼠的右后背，形成移植瘤，体积达到100～200mm 3时，将动物按瘤体积随机分组，阴性对照组8只，阳性对照组每组8只，实验组每组8只。实验组每天一次分别口服灌胃不同 剂量的阳性药NVP-HDM201(6mg/kg)和式(I)化合物(6mg/kg和12mg/kg)，阴性对照组同时给予等量的溶剂。每周两次用游标卡尺测量肿瘤长(A)、宽(B)，并由此计算肿瘤体积V＝A×B 2/2。相对肿瘤体积(RTV)的计算遵循：RTV＝V t/V 0，V t为给药结束时的肿瘤体积，V 0为分笼给药前测量所得肿瘤体积。抗肿瘤活性的药效学评价指标为相对肿瘤增殖率T/C(％)，计算公式为：T/C(％)＝T RTV/C RTV×100％。其中T RTV：治疗组RTV；C RTV：阴性对照组RTV。疗效评价标准：T/C％>60％为无效；T/C％≤60％，并经统计学处理P<0.05为有效。肿瘤生长抑制率(TGI)计算公式如下： 0.2mL (10×10 6 cells, containing 50% Matrigel) of MV4-11 tumor cells were inoculated subcutaneously on the right back of each mouse to form a transplanted tumor. When the volume reached 100-200mm 3 , the animals were pressed into the tumor The volume was randomly divided into groups, with 8 in the negative control group, 8 in each group in the positive control group, and 8 in each group in the experimental group. The experimental group was orally orally administered with different doses of the positive drug NVP-HDM201 (6mg/kg) and the compound of formula (I) (6mg/kg and 12mg/kg) once a day, and the negative control group was given the same amount of solvent at the same time. The length (A) and width (B) of the tumor were measured with a vernier caliper twice a week, and the tumor volume V=A×B 2 /2 was calculated from this. The calculation of the relative tumor volume (RTV) follows: RTV=V t /V 0 , V t is the tumor volume at the end of the administration, and V 0 is the tumor volume measured before the caged administration. The pharmacodynamic evaluation index of anti-tumor activity is the relative tumor proliferation rate T/C (%), and the calculation formula is: T/C (%) = T RTV /C RTV × 100%. Among them, T RTV: RTV of the treatment group; C RTV: RTV of the negative control group. Efficacy evaluation standard: T/C%>60% is invalid; T/C%≤60%, and statistically processed P<0.05 is valid. The calculation formula of tumor growth inhibition rate (TGI) is as follows:
TGI(％)＝{[(CV t-CV 0)-(TV t-TV 0)]/(CV t-CV 0)}×100％ TGI(%)={[(CV t -CV 0 )-(TV t -TV 0 )]/(CV t -CV 0 )}×100%
CV t为对照组给药结束时的肿瘤体积，CV 0为对照组分笼给药前的肿瘤体积，TV t为给药组给药结束时的肿瘤体积，TV 0为给药组分笼给药前的肿瘤体积。给药组及对照组的肿瘤体积的差异经t-检验。同时，每周两次称量各组裸鼠体重，以初步评价药物的毒副作用。各化合物在该模型中的药效结果如下表17中所示。 CV t is the tumor volume at the end of the administration of the control group, CV 0 is the tumor volume of the control group before caged administration, TV t is the tumor volume at the end of the administration group, TV 0 is the administration group Tumor volume before medication. The difference in tumor volume between the treatment group and the control group was tested by t-test. At the same time, the nude mice of each group were weighed twice a week to preliminarily evaluate the toxic and side effects of the drugs. The efficacy results of each compound in this model are shown in Table 17 below.
表17 式(I)化合物体内药效试验结果Table 17 In vivo efficacy test results of compounds of formula (I)
Figure PCTCN2020113712-appb-000029
Figure PCTCN2020113712-appb-000029
结论：式(I)化合物在小鼠移植MV4-11人急性髓细胞白血病模型中具有更好的抗肿瘤疗效，且显示具有良好的量效关系。Conclusion: The compound of formula (I) has a better anti-tumor effect in a mouse transplanted MV4-11 human acute myeloid leukemia model, and shows a good dose-effect relationship.

Claims (40)

  1. 式(I)化合物的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：5.5±0.2°、8.8±0.2°、11.0±0.2°，The X-ray powder diffraction pattern of the crystal form A of the compound of formula (I) has characteristic diffraction peaks at the following 2θ angles: 5.5±0.2°, 8.8±0.2°, 11.0±0.2°,
    Figure PCTCN2020113712-appb-100001
    Figure PCTCN2020113712-appb-100001
  2. 根据权利要求1所述的式(I)化合物的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：3.5±0.2°、5.5±0.2°、8.8±0.2°、11.0±0.2°、13.6±0.2°、22.1±0.2°、26.3±0.2°、26.9±0.2°。The crystal form A of the compound of formula (I) according to claim 1, whose X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 3.5±0.2°, 5.5±0.2°, 8.8±0.2°, 11.0± 0.2°, 13.6±0.2°, 22.1±0.2°, 26.3±0.2°, 26.9±0.2°.
  3. 根据权利要求2所述的式(I)化合物的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：3.501°、5.546°、8.835°、10.714°、11.008°、13.279°、13.590°、15.645°、16.115°、16.494°、16.943°、18.506°、19.508°、20.025°、20.438°、21.245°、22.053°、22.551°、23.473°、25.819°、26.253°、26.867°、27.812°、28.248°。The crystal form A of the compound of formula (I) according to claim 2, whose X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 3.501°, 5.546°, 8.835°, 10.714°, 11.008°, 13.279° , 13.590°, 15.645°, 16.115°, 16.494°, 16.943°, 18.506°, 19.508°, 20.025°, 20.438°, 21.245°, 22.053°, 22.551°, 23.473°, 25.819°, 26.253°, 26.867°, 27.812 °, 28.248 °.
  4. 根据权利要求3所述的式(I)化合物的A晶型，其XRPD图谱如图1所示。The crystal form A of the compound of formula (I) according to claim 3, and its XRPD pattern is shown in FIG. 1.
  5. 根据权利要求1～4任意一项所述的式(I)化合物的A晶型，其差示扫描量热曲线在150.13±3℃处具有吸热峰的起始点。The crystal form A of the compound of formula (I) according to any one of claims 1 to 4, whose differential scanning calorimetry curve has the starting point of the endothermic peak at 150.13±3°C.
  6. 根据权利要求5所述的式(I)化合物的A晶型，其DSC图谱如图2所示。The DSC spectrum of the crystal form A of the compound of formula (I) according to claim 5 is shown in FIG. 2.
  7. 根据权利要求1～4任意一项所述的式(I)化合物的A晶型，其热重分析曲线在150.00±3℃时失重达6.07％。According to the crystal form A of the compound of formula (I) according to any one of claims 1 to 4, its thermogravimetric analysis curve has a weight loss of 6.07% at 150.00±3°C.
  8. 根据权利要求7所述的式(I)化合物的A晶型，其TGA图谱如图3所示。The crystal form A of the compound of formula (I) according to claim 7, and its TGA pattern is shown in FIG. 3.
  9. 式(I)化合物A晶型的制备方法，其包括：The method for preparing the crystal form of compound A of formula (I), which includes:
    (a)将式(I)化合物加入溶剂中，升温至60-70℃；(a) The compound of formula (I) is added to the solvent, and the temperature is increased to 60-70°C;
    (b)室温下放置至析出固体；(b) Place it at room temperature until a solid precipitates;
    (c)35-45℃下真空干燥45～50小时；(c) Vacuum drying at 35-45°C for 45-50 hours;
    (d)75-85℃下真空干燥1～5小时；(d) Vacuum drying at 75-85°C for 1 to 5 hours;
    其中，所述溶剂为甲醇；Wherein, the solvent is methanol;
    Figure PCTCN2020113712-appb-100002
    Figure PCTCN2020113712-appb-100002
  10. 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：5.7±0.2°、11.2±0.2°、22.3±0.2°，Form B of the compound of formula (I), its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 5.7±0.2°, 11.2±0.2°, 22.3±0.2°,
    Figure PCTCN2020113712-appb-100003
    Figure PCTCN2020113712-appb-100003
  11. 根据权利要求10所述的式(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：5.7±0.2°、8.5±0.2°、11.2±0.2°、13.5±0.2°、17.5±0.2°、22.3±0.2°、23.3±0.2°、24.3±0.2°。The crystal form B of the compound of formula (I) according to claim 10, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 5.7±0.2°, 8.5±0.2°, 11.2±0.2°, 13.5± 0.2°, 17.5±0.2°, 22.3±0.2°, 23.3±0.2°, 24.3±0.2°.
  12. 根据权利要求11所述的式(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：3.541°、5.662°、8.464°、9.885°、10.691°、11.187°、13.513°、16.750°、17.458°、19.727°、20.065°、20.595°、22.347°、22.862°、23.296°、24.287°、27.104°、28.090°、28.820°。The crystal form B of the compound of formula (I) according to claim 11, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 3.541°, 5.662°, 8.464°, 9.885°, 10.691°, 11.187° , 13.513°, 16.750°, 17.458°, 19.727°, 20.065°, 20.595°, 22.347°, 22.862°, 23.296°, 24.287°, 27.104°, 28.090°, 28.820°.
  13. 根据权利要求12所述的式(I)化合物的B晶型，其XRPD图谱如图5所示。The XRPD pattern of the crystal form B of the compound of formula (I) according to claim 12 is shown in FIG. 5.
  14. 根据权利要求10～13任意一项所述的式(I)化合物的B晶型，其差示扫描量热曲线在152.49±3℃处具有吸热峰的起始点，在168.89±3℃处具有放热峰的峰值。The crystal form B of the compound of formula (I) according to any one of claims 10 to 13, whose differential scanning calorimetry curve has the starting point of the endothermic peak at 152.49±3°C and at 168.89±3°C The peak value of the exothermic peak.
  15. 根据权利要求14所述的式(I)化合物的B晶型，其DSC图谱如图6所示。The DSC chart of the crystal form B of the compound of formula (I) according to claim 14 is shown in FIG. 6.
  16. 根据权利要求10～13任意一项所述的式(I)化合物的B晶型，其热重分析曲线在162.20±3℃时失重达4.16％。According to the crystal form B of the compound of formula (I) according to any one of claims 10-13, its thermogravimetric analysis curve has a weight loss of 4.16% at 162.20±3°C.
  17. 根据权利要求16所述的式(I)化合物的B晶型，其TGA图谱如图7所示。The crystal form B of the compound of formula (I) according to claim 16, and its TGA pattern is shown in FIG. 7.
  18. 式(I)化合物B晶型的制备方法，其包括：The method for preparing the crystal form of compound B of formula (I), which includes:
    (a)将式(I)化合物加入溶剂中成为悬浊液；(a) adding the compound of formula (I) to a solvent to become a suspension;
    (b)将上述悬浊液置于40℃的恒温混匀仪上，700rpm的转速振摇3～5天；(b) Place the above suspension on a constant temperature mixer at 40°C and shake at 700 rpm for 3 to 5 days;
    (c)离心，固体残渣于25-35℃真空干燥箱内干燥10～16小时；(c) Centrifuge, dry the solid residue in a vacuum drying oven at 25-35°C for 10-16 hours;
    其中，所述溶剂为乙醇；Wherein, the solvent is ethanol;
    Figure PCTCN2020113712-appb-100004
    Figure PCTCN2020113712-appb-100004
  19. 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：3.3±0.2°、6.6±0.2°、9.1±0.2°，Form C of the compound of formula (I), its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 3.3±0.2°, 6.6±0.2°, 9.1±0.2°,
    Figure PCTCN2020113712-appb-100005
    Figure PCTCN2020113712-appb-100005
  20. 根据权利要求19所述的式(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：3.3±0.2°、6.6±0.2°、9.1±0.2°、10.6±0.2°、11.2±0.2°、14.0±0.2°、21.1±0.2°、22.3±0.2°。The crystal form C of the compound of formula (I) according to claim 19, whose X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 3.3±0.2°, 6.6±0.2°, 9.1±0.2°, 10.6± 0.2°, 11.2±0.2°, 14.0±0.2°, 21.1±0.2°, 22.3±0.2°.
  21. 根据权利要求20所述的式(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：3.325°、3.522°、6.118°、6.591°、7.003°、9.055°、10.554°、11.162°、13.988°、16.710°、21.067°、22.309°。The crystal form C of the compound of formula (I) according to claim 20, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 3.325°, 3.522°, 6.118°, 6.591°, 7.003°, 9.055° , 10.554°, 11.162°, 13.988°, 16.710°, 21.067°, 22.309°.
  22. 根据权利要求21所述的式(I)化合物的C晶型，其XRPD图谱如图8所示。The crystal form C of the compound of formula (I) according to claim 21, and its XRPD pattern is shown in FIG. 8.
  23. 根据权利要求19～22任意一项所述的式(I)化合物的C晶型，其差示扫描量热曲线分别在93.20±3℃和145.53±3℃处具有吸热峰的起始点。The crystal form C of the compound of formula (I) according to any one of claims 19-22, whose differential scanning calorimetry curves have the starting points of endothermic peaks at 93.20±3°C and 145.53±3°C, respectively.
  24. 根据权利要求23所述的式(I)化合物的C晶型，其DSC图谱如图9所示。The DSC chart of the crystal form C of the compound of formula (I) according to claim 23 is shown in FIG. 9.
  25. 根据权利要求19～22任意一项所述的式(I)化合物的C晶型，其热重分析曲线在71.79±3℃时失重达1.39％，在117.98±3℃时失重达6.88％，在170.72±3℃时失重达7.67％。According to the crystal form C of the compound of formula (I) according to any one of claims 19-22, its thermogravimetric analysis curve has a weight loss of 1.39% at 71.79±3°C and a weight loss of 6.88% at 117.98±3°C. The weight loss reached 7.67% at 170.72±3℃.
  26. 根据权利要求25所述的式(I)化合物的C晶型，其TGA图谱如图10所示。The crystal form C of the compound of formula (I) according to claim 25, and its TGA pattern is shown in FIG. 10.
  27. 式(I)化合物C晶型的制备方法，其包括：The method for preparing the crystal form of compound C of formula (I) includes:
    (a)将式(I)化合物加入溶剂中成为悬浊液；(a) adding the compound of formula (I) to a solvent to become a suspension;
    (b)将上述悬浊液置于40℃的恒温混匀仪上，700rpm的转速振摇3～5天；(b) Place the above suspension on a constant temperature mixer at 40°C and shake at 700 rpm for 3 to 5 days;
    (c)离心，固体残渣于25-35℃真空干燥箱内干燥10～16小时；(c) Centrifuge, dry the solid residue in a vacuum drying oven at 25-35°C for 10-16 hours;
    其中，所述溶剂为四氢呋喃；Wherein, the solvent is tetrahydrofuran;
    Figure PCTCN2020113712-appb-100006
    Figure PCTCN2020113712-appb-100006
  28. 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：12.3±0.2°、15.6±0.2°、16.2±0.2°，The crystal form D of the compound of formula (I), its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 12.3±0.2°, 15.6±0.2°, 16.2±0.2°,
    Figure PCTCN2020113712-appb-100007
    Figure PCTCN2020113712-appb-100007
  29. 根据权利要求28所述的式(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：5.2±0.2°、12.3±0.2°、15.6±0.2°、16.2±0.2°、19.2±0.2°、23.2±0.2°、24.8±0.2°、25.5±0.2°。The crystal form D of the compound of formula (I) according to claim 28, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 5.2±0.2°, 12.3±0.2°, 15.6±0.2°, 16.2± 0.2°, 19.2±0.2°, 23.2±0.2°, 24.8±0.2°, 25.5±0.2°.
  30. 根据权利要求29所述的式(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：5.169°、11.857°、12.333°、14.556°、15.583°、16.216°、19.174°、20.043°、20.810°、23.157°、24.419°、24.816°、25.465°、26.452°、27.378°。The crystal form D of the compound of formula (I) according to claim 29, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 5.169°, 11.857°, 12.333°, 14.556°, 15.583°, 16.216° , 19.174°, 20.043°, 20.810°, 23.157°, 24.419°, 24.816°, 25.465°, 26.452°, 27.378°.
  31. 根据权利要求30所述的式(I)化合物的D晶型，其XRPD图谱如图11所示。The crystal form D of the compound of formula (I) according to claim 30, and its XRPD pattern is shown in FIG. 11.
  32. 根据权利要求28～31任意一项所述的式(I)化合物的D晶型，其差示扫描量热曲线在113.74±3℃处具有吸热峰的起始点。The crystal form D of the compound of formula (I) according to any one of claims 28 to 31, whose differential scanning calorimetry curve has the starting point of the endothermic peak at 113.74±3°C.
  33. 根据权利要求32所述的式(I)化合物的D晶型，其DSC图谱如图12所示。The DSC chart of the crystal form D of the compound of formula (I) according to claim 32 is shown in FIG. 12.
  34. 根据权利要求28～31任意一项所述的式(I)化合物的D晶型，其热重分析曲线在120.00±3℃时失重达0.20％，在220.00±3℃时失重达0.63％。According to the crystal form D of the compound of formula (I) according to any one of claims 28 to 31, the thermogravimetric analysis curve has a weight loss of 0.20% at 120.00±3°C and a weight loss of 0.63% at 220.00±3°C.
  35. 根据权利要求34所述的式(I)化合物的D晶型，其TGA图谱如图13所示。The crystal form D of the compound of formula (I) according to claim 34, and its TGA pattern is shown in FIG. 13.
  36. 式(I)化合物D晶型的制备方法，其包括：The method for preparing the crystal form of compound D of formula (I) includes:
    (a)将式(I)化合物加入溶剂中成为悬浊液；(a) adding the compound of formula (I) to a solvent to become a suspension;
    (b)将上述悬浊液置于40℃的恒温混匀仪上，700rpm的转速振摇1～2天；(b) Place the above suspension on a constant temperature mixer at 40°C and shake at 700 rpm for 1 to 2 days;
    (c)离心，固体残渣于25-35℃真空干燥箱内干燥10～16小时；(c) Centrifuge, dry the solid residue in a vacuum drying oven at 25-35°C for 10-16 hours;
    其中，所述溶剂选自为水、四氢呋喃和水与乙醇的混合溶剂；Wherein, the solvent is selected from water, tetrahydrofuran, and a mixed solvent of water and ethanol;
    Figure PCTCN2020113712-appb-100008
    Figure PCTCN2020113712-appb-100008
  37. 式(I)化合物D晶型的制备方法，其包括：The method for preparing the crystal form of compound D of formula (I) includes:
    (a)将式(I)化合物加入溶剂中，升温至75-85℃，搅拌0.5～1.5小时；(a) Add the compound of formula (I) to the solvent, raise the temperature to 75-85°C, and stir for 0.5 to 1.5 hours;
    (b)冷却至室温，过滤，干燥；(b) Cool to room temperature, filter and dry;
    其中，所述溶剂选自乙酸乙酯、乙酸异丙酯、正庚烷、甲基叔丁基醚、乙酸乙酯与正庚烷的混合溶剂和乙酸乙酯与甲基叔丁基醚的混合溶剂；Wherein, the solvent is selected from ethyl acetate, isopropyl acetate, n-heptane, methyl tert-butyl ether, a mixed solvent of ethyl acetate and n-heptane, and a mixture of ethyl acetate and methyl tert-butyl ether Solvent
    Figure PCTCN2020113712-appb-100009
    Figure PCTCN2020113712-appb-100009
  38. 式(I)化合物D晶型的制备方法，其包括：The method for preparing the crystal form of compound D of formula (I) includes:
    (a)将式(I)化合物加入溶剂中，升温至75-85℃，搅拌1.5～2.5小时；(a) Add the compound of formula (I) to the solvent, raise the temperature to 75-85°C, and stir for 1.5-2.5 hours;
    (b)加入水；(b) Add water;
    (c)冷却至室温，过滤，干燥；(c) Cool to room temperature, filter and dry;
    其中，所述溶剂选自乙醇、异丙醇、叔丁醇和乙二醇；Wherein, the solvent is selected from ethanol, isopropanol, tert-butanol and ethylene glycol;
    Figure PCTCN2020113712-appb-100010
    Figure PCTCN2020113712-appb-100010
  39. 式(I)化合物D晶型的制备方法，其包括：The method for preparing the crystal form of compound D of formula (I) includes:
    (a)将式(I)化合物加入溶剂中，升温至95-105℃，搅拌1.5～2.5小时；(a) Add the compound of formula (I) to the solvent, raise the temperature to 95-105°C, and stir for 1.5-2.5 hours;
    (b)冷却至室温，过滤，洗涤，干燥；(b) Cool to room temperature, filter, wash, and dry;
    其中，所述溶剂选自水、四氢呋喃和水与乙醇的混合溶剂；Wherein, the solvent is selected from water, tetrahydrofuran and a mixed solvent of water and ethanol;
    Figure PCTCN2020113712-appb-100011
    Figure PCTCN2020113712-appb-100011
  40. 根据权利要求1-8、10-17、19～26和28～35任意一项所述的式(I)化合物的晶型或根据权利要求9、18、27和36～39任意一项所述的式(I)化合物晶型的制备方法得到的晶型在制备治疗癌症、细菌感染或病毒感染药物中的应用。The crystalline form of the compound of formula (I) according to any one of claims 1-8, 10-17, 19-26 and 28-35 or according to any one of claims 9, 18, 27 and 36-39 The application of the crystal form obtained by the method for preparing the crystal form of the compound of formula (I) in the preparation of medicines for the treatment of cancer, bacterial infections or viral infections.
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