WO2021047466A1 - Forme cristalline d'un inhibiteur de p53-mdm2 et son procédé de préparation - Google Patents

Forme cristalline d'un inhibiteur de p53-mdm2 et son procédé de préparation 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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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/zh
Publication of WO2021047466A1 publication Critical patent/WO2021047466A1/fr

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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

La présente invention concerne une forme cristalline d'un inhibiteur de p53-MDM2 et son procédé de préparation, et en particulier, l'invention concerne des formes cristallines A, B, C et D d'un composé représenté par la formule (I) et son procédé de préparation, ainsi que l'utilisation des formes cristallines dans la préparation de médicaments pour le traitement de cancers, d'infections bactériennes et d'infections virales. (I)
PCT/CN2020/113712 2019-09-12 2020-09-07 Forme cristalline d'un inhibiteur de p53-mdm2 et son procédé de préparation WO2021047466A1 (fr)

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WO2015084804A1 (fr) * 2013-12-03 2015-06-11 Novartis Ag Combinaison d'un inhibiteur de mdm2 et d'un inhibiteur de braf, et leur utilisation
WO2018161871A1 (fr) * 2017-03-06 2018-09-13 罗欣生物科技（上海）有限公司 Composé imidazopyridine utilisé en tant qu'inhibiteur de p53-mdm2
WO2019174576A1 (fr) * 2018-03-12 2019-09-19 罗欣药业(上海)有限公司 Composé imidaxopyrolone et son application

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WO2015084804A1 (fr) * 2013-12-03 2015-06-11 Novartis Ag Combinaison d'un inhibiteur de mdm2 et d'un inhibiteur de braf, et leur utilisation
WO2018161871A1 (fr) * 2017-03-06 2018-09-13 罗欣生物科技（上海）有限公司 Composé imidazopyridine utilisé en tant qu'inhibiteur de p53-mdm2
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