WO2019174589A1 - 一种Caspase抑制剂的结晶 - Google Patents

一种Caspase抑制剂的结晶 Download PDF

Info

Publication number
WO2019174589A1
WO2019174589A1 PCT/CN2019/077939 CN2019077939W WO2019174589A1 WO 2019174589 A1 WO2019174589 A1 WO 2019174589A1 CN 2019077939 W CN2019077939 W CN 2019077939W WO 2019174589 A1 WO2019174589 A1 WO 2019174589A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
formula
crystalline
ray powder
powder diffraction
Prior art date
Application number
PCT/CN2019/077939
Other languages
English (en)
French (fr)
Inventor
吴松亮
牟剑锋
贺海鹰
Original Assignee
正大天晴药业集团股份有限公司
南京明德新药研发有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 正大天晴药业集团股份有限公司, 南京明德新药研发有限公司 filed Critical 正大天晴药业集团股份有限公司
Priority to CA3093728A priority Critical patent/CA3093728A1/en
Priority to EP19768641.3A priority patent/EP3766873A4/en
Priority to US16/977,761 priority patent/US11091450B2/en
Priority to AU2019233195A priority patent/AU2019233195A1/en
Priority to CN201980016202.6A priority patent/CN111757871B/zh
Publication of WO2019174589A1 publication Critical patent/WO2019174589A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D261/00—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings
    • C07D261/02—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings
    • C07D261/06—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings having two or more double bonds between ring members or between ring members and non-ring members
    • C07D261/10—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings having two or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D261/18—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/13—Crystalline forms, e.g. polymorphs

Definitions

  • This application belongs to the field of medical chemistry and relates to the crystallization of a caspase inhibitor, and more particularly to (S)-3-((S)-2-(5-(2-chlorophenyl)isoxazol-3- Crystallization of formamide)propionyl)-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoic acid, as well as methods for preparing the same, pharmaceutical compositions and uses.
  • Necrotic cell death is one of the forms of cell death characterized by cell death due to pathological cell death caused by trauma or cell damage. Necrotic cell death is harmful to tissues, such as inflammation. In contrast, another physiological form of cell death occurs in an orderly, controlled manner. This ordered, controlled form of cell death is called apoptotic cell death.
  • apoptotic cell death Through a programmed approach to apoptotic cell death, organisms destroy unwanted cells (their activity and the presence of cells that are no longer needed) without damaging other tissues. Therefore, apoptotic cell death is an extremely important physiological process to maintain the normal development and dynamic balance of the organism.
  • caspase cyste aspartate-specific protease
  • Capase is a type of cysteine protease, and many important proteins in cells are its substrate. The process of apoptotic cell death involves the removal of fragments decomposed by the cells by caspase enzymes by other cells, or by macrophages or the like without causing inflammation or the like.
  • the application provides crystallization of a compound of formula I-A
  • the present application provides a crystalline composition of a compound of Formula I-A, wherein the crystal of the compound of Formula I-A above comprises more than 50% by weight, preferably more than 75% by weight of the crystalline composition. It is more than 90%, preferably more than 95%.
  • the application provides a pharmaceutical composition comprising a therapeutically effective amount of a crystal of a compound of Formula I-A above, or a crystalline composition of a compound of Formula I-A above; said pharmaceutical composition can comprise at least one A pharmaceutically acceptable carrier or other excipient.
  • the application provides crystallization of a compound of formula I-A above, a crystalline composition of a compound of formula I-A above, or the use of a pharmaceutical composition as described above for the manufacture of a medicament for treating a caspase receptor-related disorder in a mammal .
  • the application provides a method of treating a mammalian caspase receptor-associated disorder comprising administering to a mammal in need thereof a therapeutically effective amount of a crystal of a compound of Formula I-A above, a crystalline combination of a compound of Formula I-A above Or the above pharmaceutical composition.
  • the application provides crystallization of a compound of formula I-A above, a crystalline composition of a compound of formula I-A above, or a pharmaceutical composition as described above, for use in the treatment of a mammalian caspase receptor-related disorder.
  • One aspect of the present application is to provide crystallization of a compound of Formula I-A.
  • the crystal may be in the form of an unsolvated form or a form of a solvate such as a hydrate.
  • the compound of the formula I-A has high crystal stability, low hygroscopicity, good in vivo metabolism level, long half-life, and good inhibitory activity against Caspase enzyme, physical property, safety and metabolic stability. Aspects have better properties and are of higher value as drugs.
  • the crystallization of the compound of Formula I-A of the present application is Crystalline I of the compound of Formula I-A, characterized in that the X-ray powder diffraction spectrum is represented by 2 ⁇ values at about 14.0°, 16.3°, 23.0°, and 25.7.
  • the X-ray powder diffraction spectrum has diffraction peaks at about 9.6°, 14.0°, 14.5°, 15.0°, 16.3°, 23.0°, 25.1°, and 25.7° with 2 ⁇ values;
  • the X-ray powder diffraction spectrum is expressed at 2, 4, 7.4, 9.6, 14.0, 14.5, 15.0, 16.3, 17.1, 20.9, 21.7, 22.5, 23.0
  • the peak position and intensity of the characteristic peak of the X-ray powder diffraction spectrum of the crystal I of the compound of the formula I-A are shown in Table 1:
  • the crystalline I of the compound of Formula I-A, the X-ray powder diffraction pattern thereof is shown in FIG.
  • the crystalline I of the compound of Formula I-A, the starting point of the absorption peak in the differential scanning calorimetry (DSC) measurement chart is at about 120 ° C and 153 ° C.
  • the crystalline I of the compound of Formula I-A is shown in FIG.
  • the crystal I of the compound of Formula I-A, the thermogravimetric analysis (TGA) pattern thereof is shown in FIG.
  • the crystallization of the compound of Formula I-A of the present application is Crystalline II of the compound of Formula I-A, characterized in that the X-ray powder diffraction spectrum is expressed by the 2 ⁇ value at about 8.5°, 14.2°, 15.8°, 17.1. There are diffraction peaks at ° and 25.5°; typically, the X-ray powder diffraction spectrum is expressed by 2 ⁇ values at about 5.7°, 8.5°, 14.2°, 15.3°, 15.8°, 17.1°, 22.9°, 25.5°, 30.8°.
  • a diffraction peak at 33.3° is expressed by 2 ⁇ values at about 5.7°, 8.5°, 14.2°, 15.3°, 15.8°, 17.1°, 20.5°, 20.9°, 22.9°.
  • the peak positions and intensities of the X-ray powder diffraction spectrum characteristic peaks of the crystal II of the compound of the formula I-A are shown in Table 2:
  • the crystal II of the compound of Formula I-A has an X-ray powder diffraction pattern as shown in FIG.
  • the crystalline II of the compound of Formula I-A, the starting point of the absorption peak in the differential scanning calorimetry (DSC) measurement chart is at about 147 °C.
  • the crystalline II of the compound of Formula I-A is shown in FIG.
  • thermogravimetric analysis (TGA) thereof is shown in FIG.
  • the crystallization of the compound of Formula I-A of the present application is the crystalline IV of the compound of Formula I-A, characterized in that the X-ray powder diffraction spectrum is expressed by the 2 ⁇ value at about 11.2°, 15.1°, 15.6°, 16.7. There are diffraction peaks at ° and 25.6°; typically, X-ray powder diffraction spectra are diffracted at about 5.6°, 11.2°, 12.9°, 15.1°, 15.6°, 16.7°, 22.7°, and 25.6° with 2 ⁇ values.
  • the X-ray powder diffraction spectrum is expressed by the 2 ⁇ values at about 5.6°, 7.6°, 8.6°, 9.1°, 11.2°, 12.9°, 14.0°, 15.1°, 15.6°, 16.4°, 16.7°.
  • the peak position and intensity of the characteristic peak of the X-ray powder diffraction spectrum of the crystalline IV of the I-A compound are shown in Table 3:
  • the crystalline IV of the compound of Formula I-A, the X-ray powder diffraction pattern thereof is shown in FIG.
  • the crystalline IV of the compound of Formula I-A, the starting point of the absorption peak in the differential scanning calorimetry (DSC) measurement chart is at about 167 °C.
  • the crystalline IV of the compound of Formula I-A, the differential scanning calorimetry (DSC) measurement chart is shown in FIG.
  • thermogravimetric analysis (TGA) thereof is shown in FIG.
  • the crystallization of the compound of Formula I-A of the present application is the crystallization V of the compound of Formula I-A, characterized in that the X-ray powder diffraction spectrum is expressed by the 2 ⁇ value at about 6.9°, 8.3°, 13.9°, 15.7. There are diffraction peaks at °, 16.9°, 25.3°, and 32.9°; typically, X-ray powder diffraction spectra are expressed by 2 ⁇ values at about 6.9°, 8.0°, 8.3°, 13.9°, 14.5°, 15.1°, 15.7°. There are diffraction peaks at 16.9°, 19.2°, 22.8°, 25.3°, and 32.9°.
  • the peak positions and intensities of the X-ray powder diffraction spectrum characteristic peaks of the crystal V of the compound of the formula I-A are shown in Table 4:
  • the crystal V of the compound of Formula I-A has an X-ray powder diffraction pattern as shown in FIG.
  • the crystallization V of the compound of Formula I-A, the starting point of the absorption peak in the differential scanning calorimetry (DSC) measurement chart is at about 144 ° C and 169 ° C.
  • the crystalline V of the compound of Formula I-A, the differential scanning calorimetry (DSC) measurement chart is shown in FIG.
  • the crystal V of the compound of Formula I-A, the thermogravimetric analysis (TGA) pattern thereof is shown in FIG.
  • the crystal of the compound of Formula I-A of the present application is crystalline VII of the compound of Formula I-A, characterized in that the X-ray powder diffraction spectrum is expressed by the 2 ⁇ value at about 6.9°, 7.6°, 8.3°, 9.6. There are diffraction peaks at °, 13.9°, 15.2°, 16.4°, and 16.8°; typically, the X-ray powder diffraction spectrum is expressed by 2 ⁇ values at about 6.9°, 7.6°, 8.3°, 9.6°, 12.4°, 12.7°. There are diffraction peaks at 13.9, 14.6, 15.2, 16.4, 16.8, 19.2, 20.5, 21.9, 22.3, 23.1, 24.8, 25.6, 30.5, 30.9 and 32.1.
  • the peak position and intensity of the characteristic peak of the X-ray powder diffraction spectrum of the crystal VII of the compound of the formula I-A are shown in Table 5:
  • the crystalline VII of the compound of Formula I-A has an X-ray powder diffraction pattern as shown in FIG.
  • the crystalline VII of the compound of Formula I-A, the starting point of the absorption peak in the differential scanning calorimetry (DSC) measurement chart is at about 172 °C.
  • crystalline VII of the compound of Formula I-A which has a differential scanning calorimetry (DSC) measurement chart, is shown in FIG.
  • thermogravimetric analysis (TGA) pattern of crystalline VII of the compound of Formula I-A is shown in FIG.
  • the crystal of the compound of Formula I-A of the present application is crystalline VIII of the compound of Formula I-A, characterized in that the X-ray powder diffraction spectrum is expressed by the 2 ⁇ value at about 7.0°, 8.1°, 14.0°, 16.2. There are diffraction peaks at ° and 19.3°; typically, the X-ray powder diffraction spectrum is expressed by 2 ⁇ values at about 7.0°, 8.1°, 14.0°, 14.6°, 16.2°, 16.6°, 17.6°, 19.3°, 22.9°.
  • diffraction peaks at 25.4° and 26.6° There are diffraction peaks at 25.4° and 26.6°; more typically, the X-ray powder diffraction spectrum is expressed by 2 ⁇ values at about 7.0°, 8.1°, 8.4°, 13.3°, 14.0°, 14.6°, 15.8°, 16.2°. , 16.6°, 17.0°, 17.6°, 19.3°, 20.6°, 21.5°, 22.9°, 24.6°, 25.4°, 26.6°, 28.2°, 29.4°, 30.2°, 30.8°, 32.1°, 34.4° and 38.4 There is a diffraction peak at °.
  • the peak positions and intensities of the X-ray powder diffraction spectrum characteristic peaks of the crystal VIII of the compound of the formula I-A are shown in Table 6:
  • the crystalline VIII of the compound of Formula I-A has an X-ray powder diffraction pattern as shown in FIG.
  • the crystalline VIII of the compound of Formula I-A, the starting point of the absorption peak in the differential scanning calorimetry (DSC) measurement chart is at about 152 ° C and 171 ° C.
  • the crystalline VIII of the compound of Formula I-A which has a differential scanning calorimetry (DSC) measurement chart, is shown in FIG.
  • the crystal of the compound of Formula I-A of the present application is crystalline IX of the compound of Formula I-A, characterized in that the X-ray powder diffraction spectrum is expressed by the 2 ⁇ value at about 4.6°, 9.4°, 13.8°, 16.1. There are diffraction peaks at °, 16.9° and 25.6°; typically, the X-ray powder diffraction spectrum is expressed by 2 ⁇ values at about 4.6°, 7.2°, 9.0°, 9.4°, 13.8°, 14.3°, 14.8°, 16.1°.
  • the peak positions and intensities of the X-ray powder diffraction spectrum characteristic peaks of the crystal IX of the compound of the formula I-A are shown in Table 7:
  • the crystalline IX of the compound of Formula I-A, the X-ray powder diffraction pattern thereof is shown in FIG.
  • the crystalline IX of the compound of Formula I-A, the starting point of the absorption peak in the differential scanning calorimetry (DSC) measurement chart is at about 170 °C.
  • the crystalline IX of the compound of Formula I-A, the differential scanning calorimetry (DSC) measurement chart is shown in FIG.
  • the present application provides a method for preparing a crystal of the compound of the formula I-A, comprising: (1) adding a crude compound of the formula I-A to a solvent, sonicating, and separating the solid; (2) adding and step (1) The same solvent, stirred, filtered; (3) the filter cake is added to the same solvent as in step (1), and filtered to obtain crystal I of the compound of formula I-A; wherein, in the above steps (1), (2), (3)
  • the solvent is selected from the group consisting of methanol, ethanol, isopropanol, acetone, acetonitrile, tetrahydrofuran, ethylene glycol, propylene glycol, water or a mixed solvent of water and the above solvent.
  • the solvent in the above steps (1), (2), (3) is selected from the group consisting of acetonitrile or water.
  • the present application provides a process for the preparation of a crystalline form IV of a compound of Formula I-A, comprising:
  • the solvent described in the above step (1) is selected from the group consisting of a mixed solvent of acetone and water.
  • the volume ratio of acetone to water in the mixed solvent of acetone and water is 1:2.
  • the molar ratio of the crystal I of the compound of the formula I-A or the compound of the formula I-A in the above step (1) to the solvent is 0.01 to 0.1 mmol: 1 mL; preferably 0.02 to 0.08 mmol: 1 mL; There is a choice of 0.03 to 0.07 mmol: 1 mL.
  • the agitation described in the above step (2) is carried out at 20 to 50 ° C; preferably 30 to 50 ° C; more preferably 40 to 50 ° C.
  • the stirring time described in the above step (2) is from 12 to 48 hours; preferably from 16 to 48 hours.
  • the crude compound of formula I-A of the present application contains an enantiomer of a compound of formula I-B, which comprises from 0.1% to 15% by weight based on the total weight of the crude compound of formula I-A, wherein formula I-B
  • the compound structure is as follows:
  • Crystallization of the compound of formula I-A of the present application During the preparation, the enantiomer compound of formula I-B can also be converted to its crystalline form, the crystal of the compound of formula I-B being from 0.1% to 15% by weight based on the total weight of the crystal. .
  • the present application provides a crystalline composition of a compound of Formula I-A, wherein the crystal of the compound of Formula I-A above comprises more than 50% by weight, preferably more than 75% by weight of the crystalline composition. It is more than 90%, preferably more than 95%.
  • the crystalline composition may also contain minor amounts of other crystalline or amorphous forms of the compound of Formula I-A or crystals of the compound of Formula I-B.
  • the application provides a pharmaceutical composition comprising a therapeutically effective amount of a crystal of a compound of Formula I-A above, or a crystalline composition of a compound of Formula I-A above; said pharmaceutical composition can comprise at least one A pharmaceutically acceptable carrier or other excipient.
  • the application provides crystallization of a compound of formula I-A above, a crystalline composition of a compound of formula I-A above, or the use of a pharmaceutical composition as described above for the manufacture of a medicament for treating a caspase receptor-related disorder in a mammal .
  • the application provides a method of treating a mammalian caspase receptor-associated disorder comprising administering to a mammal in need thereof a therapeutically effective amount of a crystal of a compound of Formula I-A above, a crystalline combination of a compound of Formula I-A above Or the above pharmaceutical composition.
  • the application provides crystallization of a compound of formula I-A above, a crystalline composition of a compound of formula I-A above, or a pharmaceutical composition as described above, for use in the treatment of a mammalian caspase receptor-related disorder.
  • the mammal is a human.
  • the pharmaceutical composition can be formulated into a certain dosage form, and the administration route is preferably oral administration, parenteral administration (including subcutaneous, intramuscular, and intravenous), rectal administration, and the like.
  • parenteral administration including subcutaneous, intramuscular, and intravenous
  • rectal administration and the like.
  • dosage forms suitable for oral administration include tablets, capsules, granules, powders, pills, powders, troches, syrups or suspensions
  • suitable forms for parenteral administration include aqueous or non-aqueous injections.
  • Solutions or emulsions; dosage forms suitable for rectal administration include suppositories using hydrophilic or hydrophobic carriers.
  • the above dosage forms may also be formulated into a dosage form suitable for rapid release, delayed release or modified release of the active ingredient, as desired.
  • the mammalian caspase receptor-associated disorder is selected from the group consisting of non-alcoholic fatty liver disease, hepatitis, or liver fibrosis.
  • the X-ray powder diffraction spectrum of the sample was measured under the following conditions: Instrument: Bruker D8 ADVANCE X-ray diffractometer; Target: Cu: K ⁇ ; Wavelength 2 ⁇ angle range: 4 to 40°; scanning speed 10°/min; sample rotation speed: 15 rpm; Cu target tube pressure and tube flow: 40 KV, 40 mA.
  • the DSC spectrum was measured under the following conditions: Instrument: TA Q2000 Differential Scanning Calorimeter; Temperature Range: 25-300 ° C; Heating Rate: 10 ° C/min.
  • thermogravimetric analysis was carried out under the following conditions: Instrument: TA Q5000 thermogravimetric analyzer; temperature range: 25-300 ° C; heating rate: 10 ° C/min.
  • the diffraction spectrum obtained from the crystalline compound is often characteristic for a specific crystal, wherein the relative intensity of the band (especially at a low angle) may be due to crystallization conditions.
  • the dominant orientation effect due to the difference in particle size and other measurement conditions varies. Therefore, the relative intensities of the diffraction peaks are not characteristic for the crystals to be targeted.
  • the position of the peak can be shifted due to changes in temperature during sample analysis, sample movement, or calibration of the instrument, etc., and the measurement error of the 2 ⁇ value is sometimes about ⁇ 0.2°. Therefore, this error should be taken into account when determining each crystal structure.
  • the peak positions of the XRD spectrum have similarities as a whole, and the relative intensity error may be large.
  • DSC measures the transition temperature when crystallization absorbs or releases heat due to changes in its crystal structure or crystal melting.
  • the thermal transition temperature and melting point error is typically within about 5 ° C, usually within about 3 ° C, or within about 2 ° C, when we say one
  • DSC provides an auxiliary method for identifying different crystals. Different crystalline forms can be identified based on their different transition temperature characteristics. It should be noted that for mixtures, the DSC peak or melting point may vary over a larger range.
  • the melting temperature is related to the rate of temperature increase due to decomposition during the melting of the substance.
  • “Mammal” includes humans and domestic animals such as laboratory mammals and domestic pets (e.g., cats, dogs, pigs, sheep, cattle, sheep, goats, horses, rabbits), and non-domestic mammals, such as wild mammals.
  • pharmaceutical composition refers to a formulation of a compound of the present application and a medium generally accepted in the art for delivery of a biologically active compound to a mammal, such as a human.
  • the medium includes all pharmaceutically acceptable carriers for its use.
  • the pharmaceutical composition facilitates administration of the compound to the organism.
  • terapéuticaally effective amount refers to a sufficient amount of a drug or agent that is non-toxic but that achieves the desired effect. The determination of the effective amount will vary from person to person, depending on the age and general condition of the recipient, and also on the particular active substance, and a suitable effective amount in a case can be determined by one skilled in the art based on routine experimentation.
  • &quot pharmaceutically acceptable carrier" refers to those carriers which are administered with the active ingredient which are not irritating to the organism and which do not impair the biological activity and properties of the active compound.
  • pharmaceutically acceptable carrier&quot refers to those carriers which are administered with the active ingredient which are not irritating to the organism and which do not impair the biological activity and properties of the active compound.
  • room temperature means 20 to 25 °C.
  • Figure 1 is an X-ray powder diffraction pattern (XRPD) of the crystal I of the compound of the formula I-A of Example 2.
  • DSC differential scanning calorimetry
  • thermogravimetric analysis (TGA) chart of the crystal I of the compound of the formula I-A of Example 2.
  • Figure 4 is an X-ray powder diffraction pattern (XRPD) of Crystalline II of the compound of Formula I-A of Example 3.
  • Figure 5 is a differential scanning calorimetry (DSC) chart of Crystalline II of the compound of Formula I-A of Example 3.
  • FIG. 6 is a thermogravimetric analysis (TGA) diagram of Crystalline II of the compound of Formula I-A of Example 3.
  • Figure 7 is an X-ray powder diffraction pattern (XRPD) of the crystalline IV of the compound of Formula I-A of Example 5.
  • Figure 8 is a differential scanning calorimetry (DSC) chart of the crystalline IV of the compound of Formula I-A of Example 5.
  • Figure 9 is a thermogravimetric analysis (TGA) diagram of the crystalline IV of the compound of Formula I-A of Example 5.
  • Figure 10 is an X-ray powder diffraction pattern (XRPD) of the crystalline V of the compound of Formula I-A of Example 7.
  • Figure 11 is a differential scanning calorimetry (DSC) chart of the crystallization V of the compound of Formula I-A of Example 7.
  • Figure 12 is a thermogravimetric analysis (TGA) diagram of the crystalline V of the compound of Formula I-A of Example 7.
  • Figure 13 is an X-ray powder diffraction pattern (XRPD) of the crystalline VII of the compound of Formula I-A of Example 8.
  • Figure 14 is a differential scanning calorimetry (DSC) chart of the crystalline VII of the compound of Formula I-A of Example 8.
  • FIG. 15 is a thermogravimetric analysis (TGA) diagram of the crystalline VII of the compound of Formula I-A of Example 8.
  • Figure 16 is an X-ray powder diffraction pattern (XRPD) of Crystalline VIII of the compound of Formula I-A of Example 9.
  • Figure 17 is a differential scanning calorimetry (DSC) chart of the crystalline VIII of the compound of Formula I-A of Example 9.
  • Figure 18 is an X-ray powder diffraction pattern (XRPD) of Crystalline IX of the compound of Formula I-A of Example 10.
  • Figure 19 is a differential scanning calorimetry (DSC) chart of Crystalline IX of the compound of Formula I-A of Example 10.
  • t-BuOK for potassium t-butoxide
  • EtOAc for ethyl acetate
  • NaOH for sodium hydroxide
  • LiOH ⁇ H 2 O for lithium hydroxide monohydrate
  • DMF for N, N-dimethyl Base carboxamide
  • HCl stands for hydrogen chloride
  • T 3 P stands for propyl phosphoric anhydride
  • DIPEA stands for N,N-diisopropylethylamine
  • Boc stands for t-butoxycarbonyl
  • DEA stands for diethanolamine
  • SFC stands for supercritical fluid chromatography
  • DTT represents dithiothreitol
  • ddH 2 O represents deionized water
  • TFA represents trifluoroacetic acid.
  • Chiral SFC purity analysis method column signal: Chiralpak AS-3 100 ⁇ 4.6 mm ID, 3um; mobile phase: (A: CO 2 ; B: ethanol (0.05% DEA)); gradient: 4.5 minutes, 5% ⁇ 40% of B, followed by 40% of B for 2 minutes, and finally 5% of B for 1 minute; flow rate: 2.8 mL/min; column temperature: 40 °C.
  • Example 2 The crude compound of the formula I-A (347 g) obtained in Example 1 was added to a mixed solution of 2.8 L of acetone and 5.6 L of water, and the mixture was stirred at 40 to 50 ° C for 16 to 48 hours. Filtration to obtain crystalline IV of the compound of formula I-A (308 g, chiral SFC purity: 90.34%, chirality test showing 9.66% of compound of formula I-B), chiral SFC purity analysis method and analytical method of example 2 the same.
  • Example 2 50.01 mg of the compound I of the compound of the formula I-A prepared in Example 2 was weighed into a 4 mL glass vial, and 2 mL of ethanol-water (1:1) was added to make a suspension. After the magnetizer was added, it was placed on a magnetic heating stirrer and stirred at 8 ° C for one day. After centrifugation, the solid sample was taken and dried in a vacuum oven at room temperature overnight to obtain a crystal V of the compound of the formula I-A.
  • Example 2 50.01 mg of the compound I of the compound of the formula I-A prepared in Example 2 was weighed into a 4 mL glass vial, and 2 mL of ethanol-water (1:1) was added to make a suspension. After adding the magnetons, the mixture was stirred at 8 ° C for 6 days in a magnetic heating stirrer. After centrifugation, the solid sample was taken and dried in a vacuum oven at room temperature overnight to obtain a crystal VIII of the compound of the formula I-A.
  • Example 2 50.15 mg of the compound I of the compound of the formula I-A prepared in Example 2 was weighed into a 4 mL glass vial, and 3 mL of acetonitrile was added to make a suspension. After adding the magnetons, they were placed on a magnetic heating stirrer and stirred at 8 ° C for six days. After centrifugation, the solid sample was taken and dried in a vacuum oven at room temperature overnight to obtain crystal IX of the compound of formula I-A.
  • PDA detector was configured using an Agilent 1260 high performance liquid analyzer with a DAD detector or a Waters 2695 high performance liquid chromatography column: Waters Xselect CSH C18 (4.6 mm ⁇ 150 mm, 3.5 ⁇ m), column temperature: 40 ° C, flow rate : 1.0 mL / min, detection wavelength: 215 nm, injection volume: 10 uL, sample concentration: 0.5 mg / ml, diluent: methanol, using the mobile phase gradient in Table 9 for analysis.
  • This experiment used BioVision's Caspase Inhibitor Screening Kit to test the inhibitory activity of the test compound against Caspase.
  • each caspase enzymatic experiment uses the reagents in its corresponding kit. 550 ⁇ l of each 2X reaction for each enzyme The buffer was dissolved and stored at -80 °C.
  • the compound of formula I-A was diluted to a test concentration of 200* in DMSO, diluted to 2* test concentration with ddH 2 O, and added to a 384-well assay plate at 125 ⁇ l per well. Test compound and control compound were tested at 6 concentration points with test concentrations ranging from 1000 nM to 0.32 nM. To the control wells, 1% DMSO containing ddH 2 O was added to the control wells, and a high concentration of control compound (final concentration: 5 ⁇ M) was added to the 100% inhibition control wells.
  • the fluorogenic substrate of the caspase enzyme was diluted 5-fold with 2X reaction buffer containing 10 mM DTT, and then added to a 384-well experimental plate at 6.25 ⁇ l per well.
  • the total reaction volume was 25 ⁇ l, the final concentration of the substrate was 50 ⁇ M, and the final concentration of DMSO was 0.5%. After the substrate was added, the 384-well experimental plate was incubated at 37 ° C for 30 minutes.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Virology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

本申请涉及一种caspase抑制剂的结晶，更具体而言涉及(S)-3-((S)-2-(5-(2-氯苯基)异噁唑-3-甲酰胺基)丙酰胺基)-4-氧代-5-(2,3,5,6-四氟苯氧基)戊酸的结晶，其制备方法、其结晶组合物、药物组合物及其用途。本申请式Ⅰ-A化合物的结晶稳定性高、吸湿性小，在物理性质、安全性及代谢稳定性方面具备优势，有较高的成药价值。

Description

一种Caspase抑制剂的结晶
相关申请的引用
本申请要求于2018年03月13日向中华人民共和国国家知识产权局提交的第201810206043.3号中国专利申请和2018年07月20日向中华人民共和国国家知识产权局提交的第201810803581.0号中国专利申请的权益，在此将其全部内容以援引的方式整体并入本文中。
技术领域
本申请属于医药化学领域，涉及一种caspase抑制剂的结晶，更具体而言涉及(S)-3-((S)-2-(5-(2-氯苯基)异噁唑-3-甲酰胺基)丙酰胺基)-4-氧代-5-(2,3,5,6-四氟苯氧基)戊酸的结晶，以及其制备方法、药物组合物和用途。
背景技术
哺乳动物细胞数量的控制在一定程度上由细胞繁殖和死亡之间的平衡决定。坏死性细胞死亡是细胞死亡形式之一，其特点在于细胞的死亡是由于外伤或细胞损坏所导致的病理性细胞死亡。坏死性细胞死亡对组织有危害，如导致发炎等。相反，另外一种细胞死亡的生理形式却是以有序、控制的方式进行。这种有序、控制的细胞死亡形式称为凋亡性细胞死亡。通过凋亡性细胞死亡这种程序化的方式，生物体在不损害其他组织的情况下，消灭多余的细胞(其活动和存在已无需要的细胞)。因此，凋亡性细胞死亡是维持生物体正常发育及动态平衡的极为重要的生理过程。
有许多因素可以引起凋亡性细胞死亡。其中，最重要的因素是一类称为caspase的蛋白酶(cysteine aspartate-specific protease，已知有14种caspase蛋白酶)。Capase是一类半胱氨酸蛋白酶，细胞中的许多重要的蛋白质是它的作用底物。凋亡性细胞死亡的过程包括细胞在caspase酶作用下分解的碎片被其他的细胞吸收，或者被巨噬细胞等在不引发炎症等的情况下消灭。
发明概述
一方面本申请提供了式Ⅰ-A化合物的结晶
Figure PCTCN2019077939-appb-000001
另一方面，本申请提供了一种式Ⅰ-A化合物的结晶组合物，其中上述式Ⅰ-A化合物的结晶占所述结晶组合物重量的50％以上，较好是75％以上，更好是90％以上，最好是95％以上。
另一方面，本申请提供一种药物组合物，其包含治疗有效量的上述式Ⅰ-A化合物的结晶，或上述式Ⅰ-A化合物的结晶组合物；所述药物组合物可以包含至少一种药学上可接受的载体或其他赋形剂。
另一方面，本申请提供了上述式Ⅰ-A化合物的结晶，上述式Ⅰ-A化合物的结晶组合物，或上述药物组合物在制备用于治疗哺乳动物caspase受体相关病症的药物中的用途。
另一方面，本申请提供了治疗哺乳动物caspase受体相关病症的方法，其包括向有需要的哺乳动物给予治疗有效量的上述式Ⅰ-A化合物的结晶、上述式Ⅰ-A化合物的结晶组合物或上述药物组合物。
另一方面，本申请提供了用于治疗哺乳动物caspase受体相关病症的上述式Ⅰ-A化合物的结晶、上述式Ⅰ-A化合物的结晶组合物或上述药物组合物。
发明内容
结构如式Ⅰ-A所示的化合物，其化学名称为：(S)-3-((S)-2-(5-(2-氯苯基)异噁唑-3-甲酰胺基)丙酰胺基)-4-氧代-5-(2,3,5,6-四氟苯氧基)戊酸，
Figure PCTCN2019077939-appb-000002
本申请的一个方面在于提供式Ⅰ-A化合物的结晶。
所述结晶可以是非溶剂合物的形式，也可以是溶剂合物的形式，例如水合物。
所述式Ⅰ-A化合物的结晶稳定性高、吸湿性小，具有较好的体内代谢水平、较长的半衰期，且对Caspase酶的抑制活性较好，在物理性质、安全性及代谢稳定性方面都有较好的性质，作为药物的价值较高。
在一些实施方案中，本申请式Ⅰ-A化合物的结晶为式Ⅰ-A化合物的结晶Ⅰ，其特征是X-射线粉末衍射光谱用2θ值表示在约14.0°、16.3°、23.0°和25.7°处有衍射峰；典型地，X-射线粉末衍射光谱用2θ值表示在约9.6°、14.0°、14.5°、15.0°、16.3°、23.0°、25.1°和25.7°处有衍射峰；更典型地，X-射线粉末衍射光谱用2θ值表示在约4.7°、7.4°、9.6°、14.0°、14.5°、15.0°、16.3°、17.1°、20.9°、21.7°、22.5°、23.0°、24.5°、25.1°、25.7°、28.1°、32.0°和35.2°处有衍射峰；更典型地，X-射线粉末衍射光谱用2θ值表示在约4.7°、7.4°、7.9°、9.6°、14.0°、14.5°、15.0°、16.3°、17.1°、19.8°、20.4°、20.9°、21.7°、22.5°、23.0°、24.5°、25.1°、25.7°、28.1°、30.0°、32.0°、34.1°、35.2°和37.6°处有衍射峰。
作为本申请的一个实施方案，式Ⅰ-A化合物的结晶Ⅰ的X-射线粉末衍射光谱特征峰的峰位置及强度如表1所示：
表1：结晶Ⅰ的XRPD图谱表征数据
编号 衍射角2θ(°) 相对强度(％) 编号 衍射角2θ(°) 相对强度(％)
1 4.710 37.9 13 21.723 25.4
2 7.376 35.4 14 22.472 22.7
3 7.861 19.0 15 22.969 71.2
4 9.587 38.3 16 24.525 23.4
5 14.027 100 17 25.140 48.3
6 14.500 44.9 18 25.731 88.4
7 14.993 39.5 19 28.081 39.2
8 16.258 72.1 20 30.037 17.5
9 17.107 33.8 21 32.047 24.7
10 19.790 21.5 22 34.146 13.8
11 20.440 18.1 23 35.223 22.7
12 20.876 29.7 24 37.612 17.9
本申请的一个实施方案中，式Ⅰ-A化合物的结晶Ⅰ，其X-射线粉末衍射图谱如图1所示。
本申请的一个实施方案中，式Ⅰ-A化合物的结晶Ⅰ，其差示扫描量热(DSC)测量图中吸收峰的起始点在约120℃和153℃处。
本申请的一个实施方案中，式Ⅰ-A化合物的结晶Ⅰ，其差示扫描量热(DSC)测量图如图2所示。
本申请的一个实施方案中，式Ⅰ-A化合物的结晶Ⅰ，其热重分析(TGA)图如图3所示。
在一些实施方案中，本申请式Ⅰ-A化合物的结晶为式Ⅰ-A化合物的结晶Ⅱ，其特征是X-射线粉末衍射光谱用2θ值表示在约8.5°、14.2°、15.8°、17.1°和25.5°处有衍射峰；典型地，X-射线粉末衍射光谱用2θ值表示在约5.7°、8.5°、14.2°、15.3°、15.8°、17.1°、22.9°、25.5°、30.8°和33.3°处有衍射峰；更典型地，X-射线粉末衍射光谱用2θ值表示在约5.7°、8.5°、14.2°、15.3°、15.8°、17.1°、20.5°、20.9°、22.9°、23.3°、24.1°、25.1°、25.5°、26.2°、26.7°、28.0°、29.4°、30.8°、33.3°、35.6°和37.1°处有衍射峰。
作为本申请的一个实施方案，式Ⅰ-A化合物的结晶Ⅱ的X-射线粉末衍射光谱特征峰的峰位置及强度如表2所示：
表2：结晶Ⅱ的XRPD图谱表征数据
编号 衍射角2θ(°) 相对强度(％) 编号 衍射角2θ(°) 相对强度(％)
1 5.695 9.0 12 25.060 8.7
2 8.463 40.2 13 25.475 43.5
3 14.243 56.3 14 26.191 4.4
4 15.309 10.6 15 26.740 8.1
5 15.824 100 16 28.044 4.3
6 17.068 81.5 17 29.422 7.5
7 20.459 4.8 18 30.805 13.0
8 20.915 5.4 19 33.313 13.3
9 22.929 14.3 20 35.620 4.0
10 23.340 7.6 21 37.062 4.8
11 24.134 6.3      
本申请的一个实施方案中，式Ⅰ-A化合物的结晶Ⅱ，其X-射线粉末衍射图谱如图4所示。
本申请的一个实施方案中，式Ⅰ-A化合物的结晶Ⅱ，其差示扫描量热(DSC)测量图中吸收峰的起始点在约147℃处。
本申请的一个实施方案中，式Ⅰ-A化合物的结晶Ⅱ，其差示扫描量热(DSC)测量图如图5所示。
本申请的一个实施方案中，式Ⅰ-A化合物的结晶Ⅱ，其热重分析(TGA)图如图6所示。
在一些实施方案中，本申请式Ⅰ-A化合物的结晶为式Ⅰ-A化合物的结晶Ⅳ，其特征是X-射线粉末衍射光谱用2θ值表示在约11.2°、15.1°、15.6°、16.7°和25.6°处有衍射峰；典型地，X-射线粉末衍射光谱用2θ值表示在约5.6°、11.2°、12.9°、15.1°、15.6°、16.7°、22.7°和25.6°处有衍射峰；更典型地，X-射线粉末衍射光谱用2θ值表示在约5.6°、7.6°、8.6°、9.1°、11.2°、12.9°、14.0°、15.1°、15.6°、16.4°、16.7°、19.3°、22.7°、25.6°、27.2°、30.7°、31.5°、33.7°和34.7°处有衍射峰；更典型地，X-射线粉末衍射光谱用2θ值表示在约5.6°、7.6°、8.6°、9.1°、11.2°、12.0°、12.9°、14.0°、15.1°、15.6°、16.4°、16.7°、19.3°、22.7°、23.5°、25.1°、25.6°、27.2°、27.8°、29.1°、30.7°、31.5°、33.7°、34.7°、36.6°、37.0°和38.2°处有衍射峰。
作为本申请的一个实施方案，Ⅰ-A化合物的结晶Ⅳ的X-射线粉末衍射光谱特征峰的峰位置及强度如表3所示：
表3：结晶Ⅳ的XRPD图谱表征数据
编号 衍射角2θ(°) 相对强度(％) 编号 衍射角2θ(°) 相对强度(％)
1 5.599 29.7 15 23.485 7.1
2 7.573 15.8 16 25.097 9.6
3 8.582 13.1 17 25.555 100
4 9.051 11.0 18 27.195 12.7
5 11.163 70.1 19 27.824 7.0
6 12.035 6.7 20 29.144 5.4
7 12.865 36.1 21 30.746 15.6
8 13.966 10.2 22 31.514 17.9
9 15.094 98.7 23 33.746 11.4
10 15.645 99.5 24 34.674 11.0
11 16.416 16.2 25 36.550 8.5
12 16.731 92.5 26 36.964 7.3
13 19.320 29.8 27 38.224 6.3
14 22.672 47.8      
本申请的一个实施方案中，式Ⅰ-A化合物的结晶Ⅳ，其X-射线粉末衍射图谱如图7所示。
本申请的一个实施方案中，式Ⅰ-A化合物的结晶Ⅳ，其差示扫描量热(DSC)测量图中吸收峰的起始点在约167℃处。
本申请的一个实施方案中，式Ⅰ-A化合物的结晶Ⅳ，其差示扫描量热(DSC)测量图如图8所示。
本申请的一个实施方案中，式Ⅰ-A化合物的结晶Ⅳ，其热重分析(TGA)图如图9所示。
在一些实施方案中，本申请式Ⅰ-A化合物的结晶为式Ⅰ-A化合物的结晶Ⅴ，其特征是X-射线粉末衍射光谱用2θ值表示在约6.9°、8.3°、13.9°、15.7°、16.9°、25.3°和32.9°处有衍射峰；典型地，X-射线粉末衍射光谱用2θ值表示在约6.9°、8.0°、8.3°、13.9°、14.5°、15.1°、15.7°、16.9°、19.2°、22.8°、25.3°和32.9°处有衍射峰。
作为本申请的一个实施方案，式Ⅰ-A化合物的结晶Ⅴ的X-射线粉末衍射光谱特征峰的峰位置及强度如表4所示：
表4：结晶Ⅴ的XRPD图谱表征数据
编号 衍射角2θ(°) 相对强度(％) 编号 衍射角2θ(°) 相对强度(％)
1 6.898 45.4 7 15.666 73.2
2 7.988 11.7 8 16.889 40.9
3 8.323 62.5 9 19.218 8.2
4 13.89 43.9 10 22.848 11.3
5 14.470 7.1 11 25.297 25.3
6 15.111 10.3 12 32.923 100
本申请的一个实施方案中，式Ⅰ-A化合物的结晶Ⅴ，其X-射线粉末衍射图谱如图10所示。
本申请的一个实施方案中，式Ⅰ-A化合物的结晶Ⅴ，其差示扫描量热(DSC)测量图中吸收峰的起始点在约144℃和169℃处。
本申请的一个实施方案中，式Ⅰ-A化合物的结晶Ⅴ，其差示扫描量热(DSC)测量图如图11所示。
本申请的一个实施方案中，式Ⅰ-A化合物的结晶Ⅴ，其热重分析(TGA)图如图12所示。
在一些实施方案中，本申请式Ⅰ-A化合物的结晶为式Ⅰ-A化合物的结晶Ⅶ，其特征是X-射线粉末衍射光谱用2θ值表示在约6.9°、7.6°、8.3°、9.6°、13.9°、15.2°、16.4°和16.8°处有衍射峰；典型地，X-射线粉末衍射光谱用2θ值表示在约6.9°、7.6°、8.3°、9.6°、12.4°、12.7°、13.9°、14.6°、15.2°、16.4°、16.8°、19.2°、20.5°、21.9°、22.3°、23.1°、24.8°、25.6°、30.5°、30.9°和32.1°处有衍射峰。
作为本申请的一个实施方案，式Ⅰ-A化合物的结晶Ⅶ的X-射线粉末衍射光谱特征峰的峰位置及强度如表5所示：
表5：结晶Ⅶ的XRPD图谱表征数据
编号 衍射角2θ(°) 相对强度(％) 编号 衍射角2θ(°) 相对强度(％)
1 6.923 17.5 12 19.238 12.3
2 7.592 20.4 13 20.496 6.0
3 8.296 13.3 14 21.946 10.1
4 9.571 27.7 15 22.297 8.8
5 12.425 15.5 16 23.087 6.4
6 12.682 10.0 17 24.805 8.8
7 13.866 20.0 18 25.595 8.0
8 14.594 11.4 19 30.546 3.6
9 15.172 68.9 20 30.907 5.6
10 16.437 19.0 21 32.105 4.3
11 16.847 100      
本申请的一个实施方案中，式Ⅰ-A化合物的结晶Ⅶ，其X-射线粉末衍射图谱如图13所示。
本申请的一个实施方案中，式Ⅰ-A化合物的结晶Ⅶ，其差示扫描量热(DSC)测量图中吸收峰的起始点在约172℃处。
本申请的一个实施方案中，式Ⅰ-A化合物的结晶Ⅶ，其差示扫描量热(DSC)测量图如图14所示。
本申请的一个实施方案中，式Ⅰ-A化合物的结晶Ⅶ，其热重分析(TGA)图如图15所示。
在一些实施方案中，本申请式Ⅰ-A化合物的结晶为式Ⅰ-A化合物的结晶Ⅷ，其特征是X-射线粉末衍射光谱用2θ值表示在约7.0°、8.1°、14.0°、16.2°和19.3°处有衍射峰；典型地，X-射线粉末衍射光谱用2θ值表示在约7.0°、8.1°、14.0°、14.6°、16.2°、16.6°、17.6°、19.3°、22.9°、25.4°和26.6°处有衍射峰；更典型地，X-射线粉末衍射光谱用2θ值表示在约7.0°、8.1°、8.4°、13.3°、14.0°、14.6°、15.8°、16.2°、16.6°、17.0°、17.6°、19.3°、20.6°、21.5°、22.9°、24.6°、25.4°、26.6°、28.2°、29.4°、30.2°、30.8°、32.1°、34.4°和38.4°处有衍射峰。
作为本申请的一个实施方案，式Ⅰ-A化合物的结晶Ⅷ的X-射线粉末衍射光谱特征峰的峰位置及强度如表6所示：
表6：结晶Ⅷ的XRPD图谱表征数据
编号 衍射角2θ(°) 相对强度(％) 编号 衍射角2θ(° 相对强度(％)
1 7.019 54.0 14 21.451 7.6
2 8.089 34.6 15 22.870 13.6
3 8.423 9.2 16 24.608 9.0
4 13.291 8.8 17 25.378 13.3
5 14.026 100 18 26.597 23.3
6 14.597 21.4 19 28.197 8.1
7 15.789 8.4 20 29.401 5.0
8 16.178 39.9 21 30.168 4.2
9 16.554 10.0 22 30.786 4.0
10 17.025 8.6 23 32.067 8.2
11 17.642 26.5 24 34.359 3.6
12 19.335 38.4 25 38.443 4.1
13 20.565 7.4      
本申请的一个实施方案中，式Ⅰ-A化合物的结晶Ⅷ，其X-射线粉末衍射图谱如图16所示。
本申请的一个实施方案中，式Ⅰ-A化合物的结晶Ⅷ，其差示扫描量热(DSC)测量图中吸收峰的起始点在约152℃和171℃处。
本申请的一个实施方案中，式Ⅰ-A化合物的结晶Ⅷ，其差示扫描量热(DSC)测量图如图17所示。
在一些实施方案中，本申请式Ⅰ-A化合物的结晶为式Ⅰ-A化合物的结晶Ⅸ，其特征是X-射线粉末衍射光谱用2θ值表示在约4.6°、9.4°、13.8°、16.1°、16.9°和25.6°处有衍射峰；典型地，X-射线粉末衍射光谱用2θ值表示在约4.6°、7.2°、9.0°、9.4°、13.8°、14.3°、14.8°、16.1°、16.9°和25.6°处有衍射峰；更典型地，X-射线粉末衍射光谱用2θ值表示在约4.6°、7.2°、9.0°、9.4°、12.9°、13.8°、14.3°、14.8°、16.1°、16.9°、17.7°、21.5°、22.2°、23.2°、25.6°和31.8°处有衍射峰。
作为本申请的一个实施方案，式Ⅰ-A化合物的结晶Ⅸ的X-射线粉末衍射光谱特征峰的峰位置及强度如表7所示：
表7：结晶Ⅸ的XRPD图谱表征数据
编号 衍射角2θ(°) 相对强度(％) 编号 衍射角2θ(°) 相对强度(％)
1 4.551 100 9 16.099 61.3
2 7.157 22.3 10 16.931 44.1
3 9.037 15.1 11 17.658 15.4
4 9.393 49.7 12 21.509 11.9
5 12.923 9.9 13 22.238 14.4
6 13.829 66.8 14 23.164 14.4
7 14.304 28.2 15 25.593 52.5
8 14.781 27.9 16 31.812 8.5
本申请的一个实施方案中，式Ⅰ-A化合物的结晶Ⅸ，其X-射线粉末衍射图谱如图18所示。
本申请的一个实施方案中，式Ⅰ-A化合物的结晶Ⅸ，其差示扫描量热(DSC)测量图中吸收峰的起始点在约170℃处。
本申请的一个实施方案中，式Ⅰ-A化合物的结晶Ⅸ，其差示扫描量热(DSC)测量图如图19所示。
另一方面，本申请提供一种式Ⅰ-A化合物结晶Ⅰ的制备方法，包括：(1)将式Ⅰ-A化合物粗品加入溶剂中，超声，析出固体；(2)加入与步骤(1)相同的溶剂，搅拌，过滤；(3)滤饼加入步骤(1)相同的溶剂搅拌，过滤，得到式Ⅰ-A化合物结晶Ⅰ；其中，上述步骤(1)、(2)、(3)中的溶剂选自甲醇、乙醇、异丙醇、丙酮、乙腈、四氢呋喃、乙二醇、丙二醇、水或水与上述溶剂的混合溶剂。
本申请的一个实施方案中，上述步骤(1)、(2)、(3)中的溶剂选自乙腈或水。
另一方面，本申请提供一种式Ⅰ-A化合物结晶Ⅳ的制备方法，包括：
(1)式Ⅰ-A化合物或式Ⅰ-A化合物结晶Ⅰ于溶剂中，使其成混悬液或溶液；
(2)上述混悬液或溶液于磁力搅拌器中加热搅拌，离心或过滤得到式Ⅰ-A化合物结晶Ⅳ；
其中，上述步骤(1)中所述的溶剂选自丙酮与水的混合溶剂。
本申请的一个实施方案中，上述丙酮与水的混合溶剂中，丙酮与水的体积比为1：2。
本申请的一个实施方案中，上述步骤(1)中式Ⅰ-A化合物或式Ⅰ-A化合物的结晶Ⅰ与溶剂的摩尔体积比为0.01～0.1mmol：1mL；优选0.02～0.08mmol：1mL；更有选0.03～0.07mmol：1mL。
本申请的一个实施方案中，上述步骤(2)中所述的搅拌在20～50℃下进行；优选30～50℃；更优选40～50℃。
本申请的一个实施方案中，上述步骤(2)中所述的搅拌时间为12～48小时；优选16～48小时。
本申请的式Ⅰ-A化合物粗品中，含有对映异构体式Ⅰ-B化合物，所述式Ⅰ-B化合物占式Ⅰ-A化合物粗品总重量的0.1％～15％，其中式Ⅰ-B化合物结构式如下所示：
Figure PCTCN2019077939-appb-000003
本申请的式Ⅰ-A化合物结晶在制备过程中，对映异构体式Ⅰ-B化合物也可转化成其结晶形式，所述式Ⅰ-B化合物的结晶占结晶总重量的0.1％～15％。
另一方面，本申请提供了一种式Ⅰ-A化合物的结晶组合物，其中上述式Ⅰ-A化合物的结晶占所述结晶组合物重量的50％以上，较好是75％以上，更好是90％以上，最好是95％以上。所述结晶组合物中，还可能含有少量的式Ⅰ-A化合物其它结晶或非结晶形式或式Ⅰ-B化合物的结晶。
另一方面，本申请提供一种药物组合物，其包含治疗有效量的上述式Ⅰ-A化合物的结晶，或上述式Ⅰ-A化合物的结晶组合物；所述药物组合物可以包含至少一种药学上可接受的载体或其他赋形剂。
另一方面，本申请提供了上述式Ⅰ-A化合物的结晶，上述式Ⅰ-A化合物的结晶组合物，或上述药物组合物在制备用于治疗哺乳动物caspase受体相关病症的药物中的用途。
另一方面，本申请提供了治疗哺乳动物caspase受体相关病症的方法，其包括向有需要的哺乳动物给予治疗有效量的上述式Ⅰ-A化合物的结晶、上述式Ⅰ-A化合物的结晶组合物或上述药物组合物。
另一方面，本申请提供了用于治疗哺乳动物caspase受体相关病症的上述式Ⅰ-A化合物的结晶、上述式Ⅰ-A化合物的结晶组合物或上述药物组合物。
在本申请的一些实施方案中，所述哺乳动物为人类。
本申请中，药物组合物可制成一定的剂型，给药途径优选经口服给药、肠胃外给药(包括皮下、肌肉内和静脉内)、直肠给药等。例如，适合经口给药的剂型包括片剂、胶囊剂、颗粒剂、散剂、丸剂、粉剂、锭剂、糖浆剂或混悬剂；适合肠胃外给药的剂型包括水性或非水性的注射用溶液或乳液；适合直肠给药的剂型包括使用亲水性或疏水性载体的栓剂。根据需要，上述剂型还可制成适于活性成分的快速释放、延迟释放或调节释放的剂型。
本申请的一些实施方案中，所述的哺乳动物caspase受体相关病症选自非酒精型脂肪肝病、肝炎或肝纤维化。
本申请中，样品的X-射线粉末衍射光谱在下述条件下测定：仪器：Bruker D8 ADVANCE X射线衍射仪；靶：Cu:Kα；波长
Figure PCTCN2019077939-appb-000004
2θ角范围：4～40°；扫描速度10°/min；样品旋转速度:15rpm；Cu靶管压及管流：40KV，40mA。
本申请中，DSC光谱在下述条件下测定：仪器：TA Q2000差示扫描量热仪；温度范围：25～300℃；升温速率：10℃/min。
本申请中，TGA热重分析在下述条件下测定：仪器：TA Q5000热重分析仪；温度范围：25～300℃；升温速率：10℃/min。
需要说明的是，在X-射线粉末衍射光谱中，由结晶化合物得到的衍射谱图对于特定的结晶往往是特征性的，其中谱带(尤其是在低角度)的相对强度可能会因为结晶条件、粒径和其它测定条件的差异而产生的优势取向效果而变化。因此，衍射峰的相对强度对所针对的结晶并非是特征性的，判断是否与已知的结晶相同时，更应该注意的是峰的相对位置而不是它们的相对强度。此外，对任何给定的结晶而言，峰的位置可能存在轻微误差，这在结晶学领域中也是公知的。例如，由于分析样品时温度的变化、样品移动、或仪器的标定等，峰的位置可以移动，2θ值的测定误差有时约为±0.2°。因此，在确定每种结晶结构时，应该将此误差考虑在内。在XRD图谱中通常用2θ角或晶面距d表示峰位置，两者之间具有简单的换算关系：d＝λ/2sinθ，其中d代表晶面距，λ代表入射X射线的波长，θ为衍射角。对于同种化合物的同种结晶，其XRD谱的峰位置在整体上具有相似性，相对强度误差可能较大。还应指出的是，在混合物的鉴定中，由于含量下降等因素会造成部分衍射线的缺失，此时，无需依赖高纯试样中观察到的全部谱带，甚至一条谱带也可能对给定的结晶是特征性的。
DSC测定当结晶由于其结晶结构发生变化或结晶熔融而吸收或释放热时的转变温度。对于同种化合物的同种结晶，在连续的分析中，热转变温度和熔点误差典型的在约5℃之内，通常在约3℃之内，或在约2℃之内，当我们说一个化合物具有一给定的DSC峰或熔点时，这是指该DSC峰或熔点±5℃。DSC提供了一种辨别不同结晶的辅助方法。不同的结晶形态可根据其不同的转变温度特征而加以识别。需要指出的是 对于混合物而言，其DSC峰或熔点可能会在更大的范围内变动。此外，由于在物质熔化的过程中伴有分解，因此熔化温度与升温速率相关。
定义和说明
当用于本申请的说明书与权利要求中时，除非有相反的指定，否则下列术语具有所指示的意义：
“哺乳动物”包括人和家畜如实验室哺乳动物与家庭宠物(例如猫、狗、猪、羊、牛、绵羊、山羊、马、家兔)，及非驯养哺乳动物，如野生哺乳动物等。
术语“药物组合物”是指本申请化合物与本领域中通常接受的用于传递生物活性化合物至哺乳动物例如人的介质的制剂。所述介质包括所有供其使用的药物可接受的载体。药物组合物有利于化合物向生物体的给药。
术语“治疗有效量”是指无毒的但能达到预期效果的药物或药剂的足够用量。有效量的确定因人而异，取决于受体的年龄和一般情况，也取决于具体的活性物质，个案中合适的有效量可以由本领域技术人员根据常规试验确定。
本申请中，“药学上可接受的载体”是指与活性成份一同给药的、对有机体无明显刺激作用，而且不会损害该活性化合物的生物活性及性能的那些载体。关于载体的其他信息，可以参考Remington:The Science and Practice of Pharmacy,21st Ed.,Lippincott,Williams&Wilkins(2005)，该文献的内容通过引用的方式并入本文。
本申请中，“室温”是指20～25℃。
附图说明
图1为实施例2式Ⅰ-A化合物结晶Ⅰ的X-射线粉末衍射图(XRPD)。
图2为实施例2式Ⅰ-A化合物结晶Ⅰ的差示扫描量热(DSC)图。
图3为实施例2式Ⅰ-A化合物结晶Ⅰ的热重分析(TGA)图。
图4为实施例3式Ⅰ-A化合物结晶Ⅱ的X-射线粉末衍射图(XRPD)。
图5为实施例3式Ⅰ-A化合物结晶Ⅱ的差示扫描量热(DSC)图。
图6为实施例3式Ⅰ-A化合物结晶Ⅱ的热重分析(TGA)图。
图7为实施例5式Ⅰ-A化合物结晶Ⅳ的X-射线粉末衍射图(XRPD)。
图8为实施例5式Ⅰ-A化合物结晶Ⅳ的差示扫描量热(DSC)图。
图9为实施例5式Ⅰ-A化合物结晶Ⅳ的热重分析(TGA)图。
图10为实施例7式Ⅰ-A化合物结晶Ⅴ的X-射线粉末衍射图(XRPD)。
图11为实施例7式Ⅰ-A化合物结晶Ⅴ的差示扫描量热(DSC)图。
图12为实施例7式Ⅰ-A化合物结晶Ⅴ的热重分析(TGA)图。
图13为实施例8式Ⅰ-A化合物结晶Ⅶ的X-射线粉末衍射图(XRPD)。
图14为实施例8式Ⅰ-A化合物结晶Ⅶ的差示扫描量热(DSC)图。
图15为实施例8式Ⅰ-A化合物结晶Ⅶ的热重分析(TGA)图。
图16为实施例9式Ⅰ-A化合物结晶Ⅷ的X-射线粉末衍射图(XRPD)。
图17为实施例9式Ⅰ-A化合物结晶Ⅷ的差示扫描量热(DSC)图。
图18为实施例10式Ⅰ-A化合物结晶Ⅸ的X-射线粉末衍射图(XRPD)。
图19为实施例10式Ⅰ-A化合物结晶Ⅸ的差示扫描量热(DSC)图。
具体实施方案
下面的具体实施例，其目的是使本领域的技术人员能更清楚地理解和实施本申请。它们不应该被认为是对本申请范围的限制，而只是本申请的示例性说明和典型代表。
凡涉及易氧化或易水解的原料的所有操作都在氮气保护下进行。除非另有说明，本申请使用的原料都是市场上直接买到未经进一步纯化直接使用的。本申请使用的溶剂都是市场上直接买到未经特殊处理直接使用的。化合物经手工或者
Figure PCTCN2019077939-appb-000005
软件命名，市售化合物采用供应商目录名称。
本申请采用下述缩略词：t-BuOK代表叔丁醇钾；EtOAc代表乙酸乙酯；NaOH代表氢氧化钠；LiOH·H 2O代表一水合氢氧化锂；DMF代表N,N-二甲基甲酰胺；HCl代表氯化氢；T 3P代表丙基磷酸酐；DIPEA代表N,N-二异丙基乙胺；Boc代表叔丁氧羰基；DEA代表二乙醇胺；SFC代表超临界流体色谱；DMSO代表二甲基亚砜；DTT代表二硫苏糖醇；ddH 2O代表去离子水；TFA代表三氟乙酸。
实施例1：式Ⅰ-A化合物的制备
流程1：
Figure PCTCN2019077939-appb-000006
步骤1:化合物3的合成
将化合物2(122.22g)和t-BuOK(145.17g)溶于四氢呋喃(1500mL)，在0℃下向该混合溶液中缓慢滴加化合物1(80g)和四氢呋喃(500mL)的混合液。滴加完后，反应液在20℃下搅拌2小时。反应结束后，往反应液中加入1M盐酸水溶液，调pH至2-3。静置分层，水相用EtOAc(3L*2)萃取。合并有机相，用饱和食盐水(3L*3)洗涤，无水硫酸钠干燥，减压浓缩得到化合物3。粗品未经纯化直接用于下一步。
1H NMR(400MHz,CDCl 3)δ＝7.69-7.61(m,1H),7.50-7.37(m,3H),7.01-6.86(m,1H),3.94(d,J＝2.5Hz,3H)。
步骤2:化合物4的合成
将化合物3(446g)溶于甲醇(2000mL)，接着加入乙酸(222.60g)和盐酸羟胺(193.19g)。反应液在80℃下搅拌6小时。将反应体系冷却至室温，加水(2000mL)有固体析出。过滤，滤饼用水洗涤(1000mL*3)，得到化合物4。粗品未经纯化直接用于下一步。
1H NMR(400MHz,CDCl 3)δ＝8.01-7.95(m,1H),7.56-7.50(m,1H),7.45-7.38(m,2H),7.36(d,J＝1.3Hz,1H),4.03(d,J＝0.8Hz,3H)。
步骤3:化合物5的合成
将化合物4(300g)溶于甲醇(2000mL)，然后将NaOH(4M,631.21mL)加入到浑浊液中，溶液慢慢变澄清。将混合物在20℃下搅拌1小时，有白色固体生成。往反应混合液中加入1M盐酸水溶液，调pH至2-3。不断搅拌，原先的白色块状固体转化为白色粉状固体。通过布氏漏斗抽滤。滤饼用水洗涤(1000mL*3)。将滤饼用乙酸乙酯(5L)溶解，无水硫酸钠干燥，减压浓缩得化合物5。粗品未经纯化直接用于下一步。
1H NMR(400MHz,DMSO-d 6)δ＝7.98-7.85(m,1H),7.71-7.44(m,3H),7.34-7.22(m,1H)。
步骤4:化合物7的合成
将化合物5(145.78g)，T 3P(478.70g,纯度50％)，三乙胺(152.24g)溶于乙酸乙酯(350mL)，加入化合物6(70g)。反应液在25℃下搅拌19小时。将反应体系冷却至室温，加入乙酸乙酯(200mL)和水(600mL)稀释，分液后收集有机相，水相用乙酸乙酯(300mL*3)萃取。合并有机相，依次用0.5M的氢氧化钠溶液(500mL*5)和饱和食盐水(500mL*2)洗涤，经无水硫酸钠干燥，减压浓缩得到化合物7。
MS(ESI)m/z:308.9[M+H] +。
步骤5:化合物8的合成
将化合物7(149g)溶于四氢呋喃(250mL)和乙醇(250mL)的混合溶剂中，加入LiOH·H 2O(101.27g)和水(250mL)。反应液在15℃下搅拌3小时。将2M盐酸水溶液滴加到反应体系中，调pH至2-3，有固体析出，用砂芯漏斗减压抽滤。滤饼用乙酸乙酯(2L)溶解，然后用饱和食盐水洗涤(2000mL*2)，经无水硫酸钠干燥，减压浓缩得化合物8。
MS(ESI)m/z:294.9[M+H] +。
流程2：
Figure PCTCN2019077939-appb-000007
步骤6:化合物11的合成
将化合物9(17.5g)和4-甲基吗啉(8.76g)溶于四氢呋喃(200mL)中，溶液在冰浴下冷却至0℃，往其中滴加化合物10(11.09g)，并保持温度在0℃下搅拌1小时。将反应混合物过滤，滤液进经过冰浴预冷的三口烧瓶中，并用四氢呋喃(冰浴预冷，25mL)洗涤滤饼。保持温度在0℃，将CH 2N 2-乙醚溶液(200mL)在氮气保护下加入到合并的滤液中，得到的反应液在0℃下继续搅拌30分钟并升温至15℃再搅拌2小时。然后反应混合物冷却到0℃，慢慢加入HBr(35％乙酸溶液；18.77g,81.18mmol,纯度35％)，该混合物在0℃搅拌15分钟，升温至15℃继续搅拌45分钟。反应完毕后，往反应液中加入乙酸乙酯(300mL)和水(250mL)，分液。有机相依次用水(250mL)、饱和碳酸氢钠溶液(250mL)和饱和食盐水(250mL)洗涤，经无水硫酸钠干燥，过滤后浓缩得到化合物11。
MS(ESI)m/z:301.9[M-Boc+H] +。
步骤7:化合物13的合成
将化合物11(28.06g)溶于DMF(120mL)中，在-5℃下加入化合物12(11.64g)、碘化钾(1.16g)和碳酸氢钠(11.78g)。反应液在-5℃下搅拌61小时。往反应液中加入水(250mL)淬灭反应，混合液用乙酸乙酯(250mL*2)萃取。有机相合并后依次用饱和氯化铵(200mL)、水(200mL)和饱和食盐水(200mL)洗涤。有机相经无水硫酸钠干燥，过滤后浓缩得到粗品。粗品经石油醚/乙酸乙酯(10/1)打浆得到化合物13。
1H NMR(400MHz,DMSO-d 6)δ＝7.51-7.63(m,2H),7.28-7.40(m,5H),5.26(br d,J＝2.5Hz,2H),5.09(s,2H),4.46(q,J＝7.3Hz,1H),2.86(brdd,J＝6.0,16.6Hz,1H),2.65(dd,J＝7.5,16.6Hz,1H),1.38(s,9H)。
步骤8:化合物14的合成
将化合物13(15.70g)溶于乙酸乙酯(34mL)中，体系在冰浴下冷却，加入HCl/乙酸乙酯(4M,57mL)。反应液在0℃下搅拌1小时。反应液直接浓缩，得到化合物14。
1H NMR(400MHz,DMSO-d 6)δ＝8.67(br s,3H),7.63(tt,J＝7.4,10.8Hz,1H),7.30-7.42(m,5H),5.38(s,2H),5.13(d,J＝1.5Hz,2H),4.50(br t,J＝5.3Hz,1H),3.19(d,J＝5.8Hz,2H)。
步骤9:化合物15的合成
将化合物8(7.03g)溶于乙酸乙酯(185mL)，体系在冰浴下冷却至0℃，加入化合物14(7.71g)，接着加入T 3P/乙酸乙酯(50％,29.10g)，最后加入DIPEA(9.46g)。反应液在20℃搅拌1.5小时。往反应液中加入水(155mL)和乙酸乙酯(155mL)，分液。有机相用饱和食盐水(155mL)洗涤2次，洗涤时有固体析出，过滤后再分液。有机相经无水硫酸钠干燥，过滤后浓缩得到粗品。滤出的固体与粗品合并，依次用正庚烷/乙酸乙酯(2/1；210mL，20℃搅拌1小时)、正庚烷/乙酸乙酯(2/1；210mL，20℃搅拌13小时)、正庚烷/乙酸乙酯(1/1；100mL，25℃搅拌4小时)、正庚烷/乙酸乙酯(1/1；150mL，25℃搅拌14小时)打浆，过滤后得到化合物15。
MS(ESI)m/z:662.0[M+H] +。
步骤10:化合物Ⅰ-A的合成
将化合物15(10.02g)溶于四氢呋喃(400mL)，体系在冰浴下冷却，在0℃下缓慢滴加LiOH·H 2O(761.50mg)的水(200mL)溶液，滴加过程持续40分钟。滴加完毕，反应液在0℃下继续搅拌5分钟。用1M HCl调节反应液pH至1左右，加入乙酸乙酯(400mL)萃取。有机相用饱和食盐水(300mL)洗涤，经无水硫酸钠干燥，过滤后浓缩得到式Ⅰ-A化合物粗品。
MS(ESI)m/z:571.9[M+H] +。
1H NMR(400MHz,DMSO-d 6)δ＝9.04(br d,J＝6.5Hz,1H),8.67(br s,1H),8.01(dd,J＝2.0,7.0Hz,1H),7.72-7.80(m,1H),7.54-7.69(m,3H),7.40-7.45(m,1H),5.19(br s,2H),4.64-4.80(m,1H),4.48-4.59(m,1H),2.79-2.88(m,1H),2.72(br s,1H),1.42(d,J＝7.0Hz,3H)。
实施例2:式Ⅰ-A化合物结晶Ⅰ的制备
将实施例1中制备得到的式Ⅰ-A化合物粗品8.10g加入乙腈(300mL)，超声，有白色固体析出，加入乙腈(100mL)，室温搅拌5小时，过滤，得到4.20g白色固体。往该固体中加入乙腈(40mL)，在25℃下搅拌12小时，过滤，抽干溶剂后得到白色固体(3.20g,5.60mmol,手性SFC纯度：93.7％，包含6.3％的式Ⅰ-B化合物)，即为式Ⅰ-A化合物的结晶Ⅰ。
手性SFC纯度分析方法：柱子信号：Chiralpak AS-3 100×4.6mm I.D.,3um；流动相：(A:CO 2；B:乙醇(0.05％DEA))；梯度：4.5分钟内，5％～40％的B，随后40％的B保持2分钟，最后5％的B保持1分钟；流速：2.8mL/min；柱温：40℃。
实施例3：式Ⅰ-A化合物结晶Ⅱ的制备
称取30.03mg实施例2制备得到的式Ⅰ-A化合物结晶Ⅰ加入到1.5mL玻璃小瓶中，加入1.0mL乙醇-水(1:1)使其成悬浊液。加入磁子后，将上述悬浊液样品置于磁力加热搅拌器上于40℃进行搅拌两天。离心后取固体样品置于30℃真空干燥箱中干燥过夜，得到式Ⅰ-A化合物结晶Ⅱ。
实施例4：式Ⅰ-A化合物结晶Ⅱ的制备
称取29.97mg实施例2制备得到的式Ⅰ-A化合物结晶Ⅰ加入到1.5mL玻璃小瓶中，加入1.0mL乙醇使其成悬浊液。加入磁子后，将上述悬浊液样品置于磁力加热搅拌器上于40℃进行搅拌两天，溶液成澄清液。锡箔纸封口，扎孔，放入通风橱挥发，待5天左右有固体析出，取固体样品置于30℃真空干燥箱中干燥过夜，得到式Ⅰ-A化合物结晶Ⅱ。
实施例5：式Ⅰ-A化合物结晶Ⅳ的制备
称取29.86mg实施例2制备得到的式Ⅰ-A化合物结晶Ⅰ加入到1.5mL玻璃小瓶中，加入1.5mL丙酮-水(1:2)使其成悬浊液。加入磁子后，将上述悬浊液样品置于磁力加热搅拌器上于40℃进行搅拌两天。离心后取固体样品置于30℃真空干燥箱中干燥过夜，得到式Ⅰ-A化合物结晶Ⅳ。
实施例6：式Ⅰ-A化合物结晶Ⅳ的制备
将实施例1制备得到的式Ⅰ-A化合物粗品(347g)加入到2.8L丙酮和5.6L水的混合溶液中，混合液在40～50℃下搅拌16～48小时。过滤，得到式Ⅰ-A化合物结晶Ⅳ(308g，手性SFC纯度：90.34％，手性测试显示含有9.66％的式Ⅰ-B化合物)，手性SFC纯度分析方法与实施例2中的分析方法相同。
实施例7：式Ⅰ-A化合物结晶Ⅴ的制备
称取50.01mg实施例2制备得到的式Ⅰ-A化合物结晶Ⅰ加入到4mL玻璃小瓶中，加入2mL乙醇-水(1:1)使其成悬浊液。加入磁子后置于磁力加热搅拌器上于8℃进行搅拌一天，离心后取固体样品置于室温真空干燥箱中干燥过夜，得到式Ⅰ-A化合物结晶Ⅴ。
实施例8：式Ⅰ-A化合物结晶Ⅶ的制备
称取43.13mg实施例2制备得到的式Ⅰ-A化合物结晶Ⅰ加入到8mL玻璃小瓶中，加入6mL丙酮-水(1:2)使其成悬浊液。加入磁子后置于磁力加热搅拌器于8℃进行搅拌一天，离心后取固体样品置于室温真空干燥箱中干燥过夜，得到式Ⅰ-A化合物结晶Ⅶ。
实施例9：式Ⅰ-A化合物结晶Ⅷ的制备
称取50.01mg实施例2制备得到的式Ⅰ-A化合物结晶Ⅰ加入到4mL玻璃小瓶中，加入2mL乙醇-水(1:1)使其成悬浊液。加入磁子后置于磁力加热搅拌器于8℃进行搅拌六天，离心后取固体样品置于室温真空干燥箱中干燥过夜，得到式Ⅰ-A化合物结晶Ⅷ。
实施例10：式Ⅰ-A化合物结晶Ⅸ的制备
称取50.15mg实施例2制备得到的式Ⅰ-A化合物结晶Ⅰ加入到4mL玻璃小瓶中，加入3mL乙腈使其成悬浊液。加入磁子后置于磁力加热搅拌器上于8℃进行搅拌六天，离心后取固体样品置于室温真空干燥箱中干燥过夜，得到式Ⅰ-A化合物结晶Ⅸ。
实施例11：式Ⅰ-A化合物结晶Ⅸ的制备
称取43.13mg实施例2制备得到的式Ⅰ-A化合物结晶Ⅰ加入到8mL玻璃小瓶中，加入6mL丙酮-水(1:2)使其成悬浊液。加入磁子后置于磁力加热搅拌器上于8℃进行搅拌六天，离心后取固体样品置于室温真空干燥箱中干燥过夜，得到式Ⅰ-A化合物结晶Ⅸ。
实验例1：式Ⅰ-A化合物结晶Ⅳ的溶解度测试
分别移取2mL pH媒介溶液加入到4mL的玻璃瓶中，然后加入8mg式Ⅰ-A化合物结晶Ⅳ。磁力搅拌加热，温度保持37℃，分别于4小时和24小时后，取1mL样品快速离心，上清液测定其pH值并用稀释剂稀释，然后用HPLC测定其浓度。pH溶解度结果见表8。
表8.式Ⅰ-A化合物结晶Ⅳ在不同pH媒介溶液中的溶解度
Figure PCTCN2019077939-appb-000008
Figure PCTCN2019077939-appb-000009
实验例2：式Ⅰ-A化合物结晶Ⅳ稳定性试验
取8mg式Ⅰ-A化合物结晶Ⅳ，准确称量置于样品瓶中，铺成薄薄一层。0天的样品用瓶盖密封并用封口膜进一步密闭后保存于-20℃的冰箱内。分别在60℃、92.5％RH、25℃/60％RH、40℃/75％RH和60℃/75％RH以及光照条件下测试样品稳定性。
分析方法：使用Agilent 1260高效液相仪配置DAD检测器或Waters 2695高效液相仪配置PDA检测器，色谱柱：Waters Xselect CSH C18(4.6mm×150mm，3.5μm)，柱温：40℃，流速：1.0mL/分钟，检测波长：215nm，进样体积：10uL，配制样品浓度：0.5mg/ml，稀释剂：甲醇，采用表9中的流动相梯度进行分析。
高温高湿稳定性实验结果如表10所示，结果表明：式Ⅰ-A化合物结晶Ⅳ在高温高湿条件下稳定，产生的总杂质在0.5％以下，成药性好。
不同温度、湿度以及光照条件下的稳定性实验结果如表11所示，结果表明：式Ⅰ-A化合物结晶Ⅳ吸湿性小，高温下和光照条件下稳定性好，成药性较好。
表9.流动相梯度
梯度：时间(min) 流动相A：0.05％TFA/水(％) 流动相B：甲醇(％)
0.00 60 40
12.00 50 50
52.00 10 90
55.01 60 40
62.00 60 40
表10.结晶IV在高温高湿条件下的固体稳定性试验
Figure PCTCN2019077939-appb-000010
表11.结晶IV在不同温度和湿度及光照条件下稳定性试验结果
Figure PCTCN2019077939-appb-000011
实验例3：体外Caspase酶的抑制活性
实验目的：
本实验使用BioVision的Caspase抑制剂筛选试剂盒测试受试化合物对Caspase酶的抑制活性。
实验材料：
1)试剂盒：
Caspase-1抑制剂筛选试剂盒(BioVision#K151-100)
Caspase-3抑制剂筛选试剂盒(BioVision#K153-100)
Caspase-8抑制剂筛选试剂盒(BioVision#K158-100)
其中，每个caspase酶学实验使用其对应的试剂盒内的试剂。每个酶分别用550μl对应的2X反应 缓冲液溶解分装储存于-80℃。
2)黑色384孔板(PerkinElmer#6007279)
3)仪器：多功能酶标仪Molecular Devices(型号SpectraMax M2e)
实验方法：
1)将式Ⅰ-A化合物用DMSO倍比稀释至200*测试浓度，用ddH 2O稀释到2*测试浓度后以每孔125μl加入384孔实验板中。测试化合物和对照化合物测试6个浓度点，测试浓度范围为：1000nM-0.32nM。0％抑制对照孔中加入含1％DMSO的ddH 2O，100％抑制对照孔中加入高浓度对照化合物(终浓度为：5μM)。
2)配制含10mM DTT的2X反应缓冲液。将caspase酶储存液用含10mM DTT的2X反应缓冲液稀释5倍后以每孔6.25μl加入到384孔实验板中。混匀后将酶和化合物于37℃孵育30分钟。
3)将caspase酶的荧光底物用含10mM DTT的2X反应缓冲液稀释5倍后以每孔6.25μl加入到384孔实验板中。反应总体积为25μl,底物终浓度为50μM,DMSO终浓度为0.5％。加入底物后，384孔实验板置于37℃孵育30分钟。
4)使用多功能酶标仪测定荧光强度(激发光波长400nm，发射光波长为505nm)。荧光强度用于计算化合物对Caspase酶的抑制作用。使用GraphPad Prism软件对化合物抑制曲线进行拟合并计算IC 50值。实验结果：
供试化合物的实验结果见表12。
表12.供试化合物酶学活性测试结果
化合物编号 Caspase-1 Caspase-3 Caspase-8
式Ⅰ-A化合物 4.6nM 13.0nM 10.3nM

Claims (32)

  1. 式Ⅰ-A所示化合物的结晶
    Figure PCTCN2019077939-appb-100001
  2. 权利要求1的结晶，所述结晶为式Ⅰ-A所示化合物的结晶Ⅳ，其特征是X-射线粉末衍射光谱用2θ值表示在约11.2°、15.1°、15.6°、16.7°和25.6°处有衍射峰。
  3. 权利要求2所述的式Ⅰ-A化合物结晶Ⅳ，其特征是X-射线粉末衍射光谱用2θ值表示在约5.6°、11.2°、12.9°、15.1°、15.6°、16.7°、22.7°和25.6°处有衍射峰。
  4. 权利要求3所述的式Ⅰ-A化合物结晶Ⅳ，其特征是X-射线粉末衍射光谱用2θ值表示在约5.6°、7.6°、8.6°、9.1°、11.2°、12.9°、14.0°、15.1°、15.6°、16.4°、16.7°、19.3°、22.7°、25.6°、27.2°、30.7°、31.5°、33.7°和34.7°处有衍射峰。
  5. 权利要求4所述的式Ⅰ-A化合物结晶Ⅳ，其特征是X-射线粉末衍射光谱用2θ值表示在约5.6°、7.6°、8.6°、9.1°、11.2°、12.0°、12.9°、14.0°、15.1°、15.6°、16.4°、16.7°、19.3°、22.7°、23.5°、25.1°、25.6°、27.2°、27.8°、29.1°、30.7°、31.5°、33.7°、34.7°、36.6°、37.0°和38.2°处有衍射峰。
  6. 权利要求2-5任一项所述的式Ⅰ-A化合物结晶Ⅳ，其特征是差示扫描量热(DSC)测量图中吸收峰的起始点在约167℃处。
  7. 权利要求1的结晶，所述结晶为式Ⅰ-A化合物结晶Ⅰ，其特征是X-射线粉末衍射光谱用2θ值表示在约14.0°、16.3°、23.0°和25.7°处有衍射峰。
  8. 权利要求7所述的式Ⅰ-A化合物结晶Ⅰ，其特征是X-射线粉末衍射光谱用2θ值表示在约9.6°、14.0°、14.5°、15.0°、16.3°、23.0°、25.1°和25.7°处有衍射峰。
  9. 权利要求8所述的式Ⅰ-A化合物结晶Ⅰ，其特征是X-射线粉末衍射光谱用2θ值表示在约4.7°、7.4°、9.6°、14.0°、14.5°、15.0°、16.3°、17.1°、20.9°、21.7°、22.5°、23.0°、24.5°、25.1°、25.7°、28.1°、32.0°和35.2°处有衍射峰。
  10. 权利要求9所述的式Ⅰ-A化合物结晶Ⅰ，其特征是X-射线粉末衍射光谱用2θ值表示在约4.7°、7.4°、7.9°、9.6°、14.0°、14.5°、15.0°、16.3°、17.1°、19.8°、20.4°、20.9°、21.7°、22.5°、23.0°、24.5°、25.1°、25.7°、28.1°、30.0°、32.0°、34.1°、35.2°和37.6°处有衍射峰。
  11. 权利要求7-10任一项所述的式Ⅰ-A化合物结晶Ⅰ，其特征是差示扫描量热(DSC)测量图中吸收峰的起始点在约120℃和153℃处。
  12. 权利要求1的结晶，所述结晶为式Ⅰ-A化合物结晶Ⅱ，其特征是X-射线粉末衍射光谱用2θ值表示在约8.5°、14.2°、15.8°、17.1°和25.5°处有衍射峰。
  13. 权利要求12所述的式Ⅰ-A化合物结晶Ⅱ，其特征是X-射线粉末衍射光谱用2θ值表示在约5.7°、8.5°、14.2°、15.3°、15.8°、17.1°、22.9°、25.5°、30.8°和33.3°处有衍射峰。
  14. 权利要求13所述的式Ⅰ-A化合物结晶Ⅱ，其特征是X-射线粉末衍射光谱用2θ值表示在约5.7°、8.5°、14.2°、15.3°、15.8°、17.1°、20.5°、20.9°、22.9°、23.3°、24.1°、25.1°、25.5°、26.2°、26.7°、28.0°、29.4°、30.8°、33.3°、35.6°和37.1°处有衍射峰。
  15. 权利要求12-14任一项所述的式Ⅰ-A化合物结晶Ⅱ，其特征是差示扫描量热(DSC)测量图中吸收峰的起始点在约147℃处。
  16. 权利要求1的结晶，所述结晶为式Ⅰ-A化合物结晶Ⅴ，其特征是X-射线粉末衍射光谱用2θ值表示在约6.9°、8.3°、13.9°、15.7°、16.9°、25.3°和32.9°处有衍射峰。
  17. 权利要求16所述的式Ⅰ-A化合物结晶Ⅴ，其特征是X-射线粉末衍射光谱用2θ值表示在约6.9°、8.0°、8.3°、13.9°、14.5°、15.1°、15.7°、16.9°、19.2°、22.8°、25.3°和32.9°处有衍射峰。
  18. 权利要求16-17任一项所述的式Ⅰ-A化合物结晶Ⅴ，其特征是差示扫描量热(DSC)测量图中吸收峰的起始点在约144℃和169℃处。
  19. 权利要求1的结晶，所述结晶为式Ⅰ-A化合物结晶Ⅶ，其特征是X-射线粉末衍射光谱用2θ值表示在约6.9°、7.6°、8.3°、9.6°、13.9°、15.2°、16.4°和16.8处有衍射峰。
  20. 权利要求19所述的式Ⅰ-A化合物结晶Ⅶ，其特征是X-射线粉末衍射光谱用2θ值表示在约6.9°、7.6°、8.3°、9.6°、12.4°、12.7°、13.9°、14.6°、15.2°、16.4°、16.8°、19.2°、20.5°、21.9°、22.3°、23.1°、24.8°、25.6°、30.5°、30.9°和32.1°处有衍射峰。
  21. 权利要求19-20任一项所述的式Ⅰ-A化合物结晶Ⅶ，其特征是差示扫描量热(DSC)测量图中吸收峰的起始点在约172℃处有峰。
  22. 权利要求1的结晶，所述结晶为式Ⅰ-A化合物结晶Ⅷ，其特征是X-射线粉末衍射光谱用2θ值表示在约7.0°、8.1°、14.0°、16.2°和19.3°处有衍射峰。
  23. 权利要求22所述的式Ⅰ-A化合物结晶Ⅷ，其特征是X-射线粉末衍射光谱用2θ值表示在约7.0°、8.1°、14.0°、14.6°、16.2°、16.6°、17.6°、19.3°、22.9°、25.4°和26.6°处有衍射峰。
  24. 权利要求23所述的式Ⅰ-A化合物结晶Ⅷ，其特征是X-射线粉末衍射光谱用2θ值表示在约7.0°、8.1°、8.4°、13.3°、14.0°、14.6°、15.8°、16.2°、16.6°、17.0°、17.6°、19.3°、20.6°、21.5°、22.9°、24.6°、25.4°、26.6°、28.2°、29.4°、30.2°、30.8°、32.1°、34.4°和38.4°处有衍射峰。
  25. 权利要求22-24任一项所述的式Ⅰ-A化合物结晶Ⅷ，其特征是差示扫描量热(DSC)测量图中吸收峰的起始点在约152℃和171℃处。
  26. 权利要求1的结晶，所述结晶为式Ⅰ-A化合物结晶Ⅸ，其特征是X-射线粉末衍射光谱用2θ值表示在约4.6°、9.4°、13.8°、16.1°、16.9°和25.6°处有衍射峰。
  27. 权利要求26所述的式Ⅰ-A化合物结晶Ⅸ，其特征是X-射线粉末衍射光谱用2θ值表示在约4.6°、7.2°、9.0°、9.4°、13.8°、14.3°、14.8°、16.1°、16.9°和25.6°处有衍射峰。
  28. 权利要求27所述的式Ⅰ-A化合物结晶Ⅸ，其特征是X-射线粉末衍射光谱用2θ值表示在约4.6°、7.2°、9.0°、9.4°、12.9°、13.8°、14.3°、14.8°、16.1°、16.9°、17.7°、21.5°、22.2°、23.2°、25.6°和31.8°处有衍射峰。
  29. 权利要求26-28任一项所述的式Ⅰ-A化合物结晶Ⅸ，其特征是差示扫描量热(DSC)测量图中吸收峰的起始点在约170℃处。
  30. 结晶组合物，其中权利要求1所述的式Ⅰ-A化合物结晶占所述结晶组合物重量的50％以上，较好是75％以上，更好是90％以上，最好是95％以上。
  31. 药物组合物，其包含权利要求1所述的式Ⅰ-A化合物结晶或权利要求30所述的结晶组合物。
  32. 权利要求1所述的式Ⅰ-A化合物结晶、权利要求30所述的结晶组合物，或权利要求31所述的药物组合物在制备用于治疗哺乳动物caspase受体相关病症的药物中的用途。
PCT/CN2019/077939 2018-03-13 2019-03-13 一种Caspase抑制剂的结晶 WO2019174589A1 (zh)

Priority Applications (5)

Application Number Priority Date Filing Date Title
CA3093728A CA3093728A1 (en) 2018-03-13 2019-03-13 A crystal of a caspase inhibitor
EP19768641.3A EP3766873A4 (en) 2018-03-13 2019-03-13 PROCESS FOR PREPARING A CASPASE INHIBITOR
US16/977,761 US11091450B2 (en) 2018-03-13 2019-03-13 Crystal of a caspase inhibitor
AU2019233195A AU2019233195A1 (en) 2018-03-13 2019-03-13 A crystal of a caspase inhibitor
CN201980016202.6A CN111757871B (zh) 2018-03-13 2019-03-13 一种Caspase抑制剂的结晶

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN201810206043 2018-03-13
CN201810206043.3 2018-03-13
CN201810803581.0 2018-07-20
CN201810803581 2018-07-20

Publications (1)

Publication Number Publication Date
WO2019174589A1 true WO2019174589A1 (zh) 2019-09-19

Family

ID=67908560

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/077939 WO2019174589A1 (zh) 2018-03-13 2019-03-13 一种Caspase抑制剂的结晶

Country Status (6)

Country Link
US (1) US11091450B2 (zh)
EP (1) EP3766873A4 (zh)
CN (1) CN111757871B (zh)
AU (1) AU2019233195A1 (zh)
CA (1) CA3093728A1 (zh)
WO (1) WO2019174589A1 (zh)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1313846A (zh) * 1998-07-02 2001-09-19 伊邓药品公司 作为ICE/ced－3族半胱氨酸蛋白酶抑制剂的C端修饰的草氨酰二肽
CN1345332A (zh) * 1999-03-02 2002-04-17 伊邓药品公司 用作ice/ced－3族半胱氨酸蛋白酶抑制剂的c－端修饰的(n－取代)－2－吲哚基二肽
WO2018133870A1 (zh) * 2017-01-23 2018-07-26 正大天晴药业集团股份有限公司 作为Caspase抑制剂的联环化合物

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999047154A1 (en) * 1998-03-16 1999-09-23 Cytovia, Inc. Dipeptide caspase inhibitors and the use thereof
WO2017053864A1 (en) * 2015-09-23 2017-03-30 Intracellular Technologies, Llc Cysteine protease inhibitors
TW201739734A (zh) * 2016-05-11 2017-11-16 Chia Tai Tianqing Pharmaceutical Group Co Ltd Caspase抑制劑及其藥物組合物、用途和治療方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1313846A (zh) * 1998-07-02 2001-09-19 伊邓药品公司 作为ICE/ced－3族半胱氨酸蛋白酶抑制剂的C端修饰的草氨酰二肽
CN1345332A (zh) * 1999-03-02 2002-04-17 伊邓药品公司 用作ice/ced－3族半胱氨酸蛋白酶抑制剂的c－端修饰的(n－取代)－2－吲哚基二肽
WO2018133870A1 (zh) * 2017-01-23 2018-07-26 正大天晴药业集团股份有限公司 作为Caspase抑制剂的联环化合物

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
"Remington: The Science and Practice of Pharmacy", 2005, LIPPINCOTT, WILLIAMS & WILKINS
BARR ET AL., BIO/TECHNOLOGY, vol. 12, 1994, pages 487 - 497
See also references of EP3766873A4

Also Published As

Publication number Publication date
CN111757871B (zh) 2022-04-12
EP3766873A1 (en) 2021-01-20
EP3766873A4 (en) 2021-12-15
CA3093728A1 (en) 2019-09-19
AU2019233195A1 (en) 2020-09-24
US11091450B2 (en) 2021-08-17
US20210002235A1 (en) 2021-01-07
CN111757871A (zh) 2020-10-09

Similar Documents

Publication Publication Date Title
ES2386974T3 (es) Formas cristalinas de la 4-metil-N-[3-(4-metil-imidazol-1-il)-5-trifluoro-metil-fenil]-3-(4-piridin-3-il-pirimidin-2-il-amino)-benzamida
JP5524343B2 (ja) ベンゾジアゼピンブロモドメイン阻害剤
TWI598346B (zh) Crystallographic polymorphism of 4- [5- (pyridin-4-yl) -1H-1,2,4-triazol-3-yl] pyridine-2-carbonitriles and process for producing the same
US9012641B2 (en) Crystalline forms of Prasugrel salts
JP2015522037A (ja) ベムラフェニブコリン塩の固体形態
WO2023040513A1 (zh) Amg510化合物的晶型及其制备方法和用途
WO2016189486A1 (en) An improved process for preparation of apremilast and novel polymorphs thereof
WO2019174589A1 (zh) 一种Caspase抑制剂的结晶
US7655800B2 (en) Crystalline 1H-imidazo[4,5-b]pyridin-5-amine, 7-[5-[(cyclohexylmethylamino)-methyl]-1H-indol-2-yl]-2-methyl, sulfate (1:1), trihydrate and its pharmaceutical uses
CN116710169A (zh) 整合素抑制剂及其用途
US10561667B2 (en) Orbit azine-fumarate, hydrate, crystal form and preparation method therefor
EP4324829A1 (en) Sulfate of 5-[(1,1-dioxido-4-thiomorpholinyl)methyl]-2-phenyl-n-(tetrahydro-2h-pyran-4-yl)-1h-indole-7-amine, and novel crystal form thereof
JP2021533111A (ja) Ｌｔａ４ｈ阻害剤の結晶形態
WO2017076358A1 (zh) 咪唑基联苯基化合物盐的新晶型及其制备方法
CN106432244B (zh) 一种hedgehog信号通路抑制剂的结晶形式及其制备方法
WO2019001325A1 (zh) 雷西纳得的晶型xv及其制备方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19768641

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 3093728

Country of ref document: CA

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2019233195

Country of ref document: AU

Date of ref document: 20190313

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2019768641

Country of ref document: EP

Effective date: 20201013