WO2024007997A1 - Crystal form of pyridone polycyclic derivative and preparation method therefor - Google Patents

Crystal form of pyridone polycyclic derivative and preparation method therefor Download PDF

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WO2024007997A1
WO2024007997A1 PCT/CN2023/104923 CN2023104923W WO2024007997A1 WO 2024007997 A1 WO2024007997 A1 WO 2024007997A1 CN 2023104923 W CN2023104923 W CN 2023104923W WO 2024007997 A1 WO2024007997 A1 WO 2024007997A1
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compound
crystal form
solvent
formula
present
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PCT/CN2023/104923
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French (fr)
Chinese (zh)
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陈新海
秦东辉
王晶晶
郝恩慧
余继路
黎健
陈曙辉
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辉诺生物医药科技（杭州）有限公司
南京明德新药研发有限公司
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Publication of WO2024007997A1 publication Critical patent/WO2024007997A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/503—Pyridazines; Hydrogenated pyridazines spiro-condensed
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D421/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having selenium, tellurium, or halogen atoms as ring hetero atoms
    • C07D421/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having selenium, tellurium, or halogen atoms as ring hetero atoms containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains three hetero rings
    • C07D471/14—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains three hetero rings
    • C07D498/14—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D513/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00
    • C07D513/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains three hetero rings
    • C07D513/14—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F11/00—Compounds containing elements of Groups 6 or 16 of the Periodic Table

Definitions

  • the invention relates to a crystal form of a pyridone polycyclic derivative and a preparation method thereof.
  • Influenza virus also known as influenza virus (IFV)
  • IVF influenza virus
  • IVF influenza virus
  • Influenza viruses can cause very high morbidity and mortality, and influenza A viruses in particular can cause global pandemics.
  • Influenza A virus is a single negative-strand RNA virus with a genome divided into 8 segments encoding 8 proteins.
  • the 5' end and 3' end of the influenza virus genome fragment are highly conserved, and the sequences of the two ends are complementary to form a handle loop structure, which plays an important role in initiating viral RNA replication.
  • the proteins encoded by each gene segment of the virus are different in size and play different roles in the life cycle of the influenza virus. The basic functions of several major proteins are introduced below.
  • the HA of influenza virus is the ligand for influenza virus to recognize host receptors. It binds to virus-specific receptors on the cell surface and mediates the fusion of the virus outer membrane with the intracellular body membrane to release the virus nucleocapsid into the cytoplasm.
  • the receptors of influenza viruses are specific, and the receptors of influenza A viruses are sialoglycoproteins.
  • the NA protein of influenza virus can remove sialic acid on the surface of virus particles during the replication process, so that virus particles cannot continue to aggregate on the surface of host cells, which is beneficial to the release of virions and further infects more host cells.
  • the role of the M2 protein of influenza virus The role of the M2 protein of influenza virus:
  • the HA protein of influenza virus binds to sialic acid, and the influenza virus is endocytosed by the host cell.
  • the acidity and alkalinity in the phagocytic vacuole plays a crucial role in virus uncoating.
  • the ion channel of the M2 protein on the virus membrane can gradually reduce the pH value of the phagocytic vacuole.
  • the M2 protein is a transmembrane ion channel found only in influenza A viruses, with part of it extending to the surface of the virus's outer membrane.
  • influenza virus proteins also uses the translation mechanism of the host cell.
  • the virus can even pause the translation of host proteins and accelerate the synthesis of its own proteins.
  • Polyadenylation of host cell mRNA is completed by specific adenylation enzymes.
  • the adenylate tail of viral mRNA is formed by the continuous transcription of 5-7 uracils on the negative-strand vRNA. of.
  • the capping of each viral messenger RNA (mRNA) is completed in a similar way: PA and PB2 proteins grab the 5' capping primer of the host pre-mRNA transcript and then initiate viral mRNA synthesis.
  • cap is mainly accomplished by the virus's RNA-dependent RNA polymerase (RdRp), whose PA subunit has RNA endonuclease activity and is responsible for cutting host mRNA.
  • RdRp virus's RNA-dependent RNA polymerase
  • the viral mRNA exits the nucleus, enters the cytoplasm, and is translated like the host cell's mRNA.
  • the nuclear export of the viral vRNA fragment is mediated by the viral M1 protein and NS2 protein.
  • M1 protein when M1 protein can interact with vRNA and NP protein, it also interacts with nuclear export protein NS2; thus, nuclear export protein NS2 mediates M1-RNP to exit the nucleus and enter the cytoplasm of the host cell in the form of nuclear protein.
  • Influenza incurs direct costs due to lost productivity and related medical resources, as well as indirect costs of preventive measures.
  • influenza causes cumulative annual losses of approximately $10 billion, and future influenza pandemics are estimated to cause hundreds of billions of dollars in direct and indirect costs.
  • the cost of prevention is also very high. Governments around the world have spent billions of dollars preparing and planning for a possible H5N1 avian influenza pandemic, with costs associated with purchasing drugs and vaccines, as well as developing disaster drills and strategies to increase border controls.
  • influenza Current treatment options for influenza include vaccination and chemotherapy and chemoprophylaxis with antiviral drugs. Flu vaccination is often recommended for high-risk groups, such as children and older adults, or people with asthma, diabetes, or heart disease, but even vaccination cannot completely prevent getting the flu. Vaccines are prepared anew each season for specific influenza strains, but it is impossible to cover all strains of the virus that are actively infecting people around the world that season. In addition, because influenza viruses undergo a certain degree of antigenic drift, if more than one virus infects a single cell, the eight separate vRNA segments in the genome will mix or recombine, resulting in rapid changes in viral genetics. Antigenic shift and allows the virus to infect new host species and rapidly overcome protective immunity.
  • Antiviral drugs can also be used to treat influenza.
  • neuraminidase inhibitors such as oseltamivir (Tamiflu) are effective against influenza A viruses.
  • Tamiflu oseltamivir
  • the results are obvious, but clinical observations have found that virus strains resistant to this type of neuraminidase inhibitors have emerged.
  • anti-influenza virus drugs with new mechanisms of action, which can support the use of single drugs to treat influenza A, or can be used in combination with anti-influenza virus drugs with other mechanisms of action that are already on the market for the treatment of influenza A. Influenza prevention and treatment.
  • the invention provides crystal form A of the compound of formula (I),
  • the X-ray powder diffraction pattern of Cu K ⁇ radiation of the A crystal form has characteristic diffraction peaks at the following 2 ⁇ angles: 4.56 ⁇ 0.20°, 10.68 ⁇ 0.20°, 16.90 ⁇ 0.20°.
  • the X-ray powder diffraction pattern of Cu K ⁇ radiation of the above-mentioned A crystal form has characteristic diffraction peaks at the following 2 ⁇ angles: 4.56 ⁇ 0.20°, 10.68 ⁇ 0.20°, 16.90 ⁇ 0.20°, 19.42 ⁇ 0.20° , 20.72 ⁇ 0.20°.
  • the X-ray powder diffraction pattern of Cu K ⁇ radiation of the above-mentioned A crystal form has characteristic diffraction peaks at the following 2 ⁇ angles: 4.56 ⁇ 0.20°, 9.08 ⁇ 0.20°, 10.68 ⁇ 0.20°, 16.90 ⁇ 0.20° , 19.42 ⁇ 0.20°, 19.94 ⁇ 0.20°, 20.72 ⁇ 0.20°, 29.12 ⁇ 0.20°.
  • the X-ray powder diffraction pattern of Cu K ⁇ radiation of the above-mentioned A crystal form has characteristic diffraction peaks at the following 2 ⁇ angles: 4.56 ⁇ 0.20°, 9.08 ⁇ 0.20°, 10.68 ⁇ 0.20°, 11.80 ⁇ 0.20° , 16.90 ⁇ 0.20°, 17.84 ⁇ 0.20°, 19.42 ⁇ 0.20°, 19.94 ⁇ 0.20°, 20.72 ⁇ 0.20°, 22.66 ⁇ 0.20°, 24.30 ⁇ 0.20°, 29.12 ⁇ 0.20°.
  • the X-ray powder diffraction pattern of Cu K ⁇ radiation of the above-mentioned A crystal form has characteristic diffraction peaks at the following 2 ⁇ angles: 4.56 ⁇ 0.20°, 9.08 ⁇ 0.20°, 10.68 ⁇ 0.20°, 11.80 ⁇ 0.20° , 14.52 ⁇ 0.20°, 16.90 ⁇ 0.20°, 17.84 ⁇ 0.20°, 19.42 ⁇ 0.20°, 19.94 ⁇ 0.20°, 20.72 ⁇ 0.20°, 22.66 ⁇ 0.20°, 24.30 ⁇ 0.20°, 26.26 ⁇ 0.20°, 28.24 ⁇ 0.20° , 29.12 ⁇ 0.20°, 29.98 ⁇ 0.20°.
  • the X-ray powder diffraction pattern of Cu K ⁇ radiation of the above-mentioned A crystal form has characteristic diffraction peaks at the following 2 ⁇ angles: 4.56 ⁇ 0.20°, 9.08 ⁇ 0.20°, 10.68 ⁇ 0.20°, and/or 11.80 ⁇ 0.20°, and/or 14.52 ⁇ 0.20°, and/or 16.56 ⁇ 0.20°, and/or 16.90 ⁇ 0.20°, and/or 17.84 ⁇ 0.20°, and/or 19.42 ⁇ 0.20°, and/or 19.94 ⁇ 0.20 °, and/or 20.30 ⁇ 0.20°, and/or 20.72 ⁇ 0.20°, and/or 22.66 ⁇ 0.20°, and/or 24.30 ⁇ 0.20°, and/or 26.26 ⁇ 0.20°, and/or 28.24 ⁇ 0.20°, and/or 29.12 ⁇ 0.20°, and/or 29.98 ⁇ 0.20°.
  • the X-ray powder diffraction pattern of Cu K ⁇ radiation of the above-mentioned A crystal form has characteristic diffraction peaks at the following 2 ⁇ angles: 4.56°, 9.08°, 10.68°, 11.80°, 13.94°, 14.52°, 14.92 °, 15.28°, 15.74°, 16.56°, 16.90°, 17.84°, 18.42°, 19.42°, 19.94°, 20.30°, 20.72°, 21.12°, 22.66°, 23.80°, 24.30°, 24.68°, 25.46°, 25.80°, 26.26°, 26.86°, 28.24°, 29.12°, 29.98°, 30.88°, 31.94°, 33.60°, 35.00°, 37.88°, 38.48°.
  • the XRPD pattern of the above-mentioned crystal form A is shown in Figure 1.
  • the XRPD pattern of the above-mentioned crystal form A is basically as shown in Figure 1.
  • the differential scanning calorimetry (DSC) curve of the above-mentioned crystal form A shows that it has the starting point of the endothermic peak at 194.2°C ⁇ 5°C and the starting point of the exothermic peak at 201.4°C ⁇ 5°C. starting point.
  • the differential scanning calorimetry (DSC) curve of the above-mentioned Form A shows an endothermic peak at 198.4°C ⁇ 3°C.
  • the DSC spectrum of the above-mentioned crystal form A is shown in Figure 2.
  • the DSC spectrum of the above-mentioned crystal form A is basically as shown in Figure 2.
  • the weight loss of the thermogravimetric analysis (TGA) curve of the above-mentioned crystal form A at 200.0°C ⁇ 3°C is 2.87%.
  • the TGA spectrum of the above-mentioned crystal form A is shown in Figure 3.
  • the TGA spectrum of the above-mentioned crystal form A is basically as shown in Figure 3.
  • the present invention also provides crystal form A of the compound of formula (I),
  • Its preparation method includes the following steps:
  • the compound Z is selected from the group consisting of compound of formula (I), crystal form of compound of formula (I) B, crystal form of compound of formula (I) C, crystal form of compound of formula (I) D and crystal form of compound of formula (I) E; preferred , compound Z is the crystal form of compound B of formula (I);
  • the solvent X is ethyl acetate
  • the solvent Y is selected from n-heptane, n-hexane and cyclohexane; preferably, the solvent Y is n-heptane;
  • the volume ratio of solvent X to solvent Y is 1:1 to 1:6; preferably, the volume ratio of solvent X to solvent Y is 1:3.
  • the invention also provides a method for preparing crystal form A of the compound of formula (I), which includes the following steps:
  • Compound Z is the crystal form of compound B of formula (I);
  • Solvent X is ethyl acetate
  • Solvent Y is selected from n-heptane, n-hexane and cyclohexane;
  • the volume ratio of solvent X to solvent Y is 1:1 to 1:6.
  • the above-mentioned compound Z is a compound of formula (I).
  • the above-mentioned compound Z is the crystal form of compound B of formula (I) or the crystal form of compound C of formula (I).
  • the above-mentioned compound Z is the crystal form B of the compound of formula (I).
  • the above step (b) is to continue stirring at 10 to 25°C for 5 to 12 hours after the addition.
  • the above solvent Y is n-heptane.
  • the volume ratio of the above solvent X and solvent Y is 1:3.
  • the mass ratio of the total volume of the above-mentioned solvent X and solvent Y to compound Z is 1:8 to 1:15.
  • the mass ratio of the total volume of the above solvent X and solvent Y to compound Z is 1:10.
  • the present invention also provides the B crystal form of the compound of formula (I),
  • the X-ray powder diffraction pattern of Cu K ⁇ radiation of the B crystal form has characteristic diffraction peaks at the following 2 ⁇ angles: 9.70 ⁇ 0.20°, 12.16 ⁇ 0.20°, 14.91 ⁇ 0.20°, 18.08 ⁇ 0.20°.
  • the X-ray powder diffraction pattern of Cu K ⁇ radiation of the above-mentioned B crystal form has characteristic diffraction peaks at the following 2 ⁇ angles: 8.98 ⁇ 0.20°, 9.70 ⁇ 0.20°, 12.16 ⁇ 0.20°, 14.91 ⁇ 0.20° , 18.08 ⁇ 0.20°, 18.54 ⁇ 0.20°, 19.07 ⁇ 0.20°, 21.58 ⁇ 0.20°.
  • the X-ray powder diffraction pattern of Cu K ⁇ radiation of the above-mentioned B crystal form has characteristic diffraction peaks at the following 2 ⁇ angles: 8.98 ⁇ 0.20°, 9.70 ⁇ 0.20°, 12.16 ⁇ 0.20°, 14.91 ⁇ 0.20° , 17.02 ⁇ 0.20°, 18.08 ⁇ 0.20°, 18.54 ⁇ 0.20°, 19.07 ⁇ 0.20°, 21.58 ⁇ 0.20°, 22.23 ⁇ 0.20°, 24.87 ⁇ 0.20°, 26.93 ⁇ 0.20°.
  • the X-ray powder diffraction pattern of Cu K ⁇ radiation of the above-mentioned B crystal form has characteristic diffraction peaks at the following 2 ⁇ angles: 8.98 ⁇ 0.20°, 9.70 ⁇ 0.20°, 12.16 ⁇ 0.20°, 14.19 ⁇ 0.20° , 14.91 ⁇ 0.20°, 17.02 ⁇ 0.20°, 18.08 ⁇ 0.20°, 18.54 ⁇ 0.20°, 19.07 ⁇ 0.20°, 21.58 ⁇ 0.20°, 22.23 ⁇ 0.20°, 22.66 ⁇ 0.20°, 23.81 ⁇ 0.20°, 24.87 ⁇ 0.20° , 26.93 ⁇ 0.20°, 28.74 ⁇ 0.20°.
  • the X-ray powder diffraction pattern of Cu K ⁇ radiation of the above-mentioned B crystal form has characteristic diffraction peaks at the following 2 ⁇ angles: 9.70 ⁇ 0.20°, 12.16 ⁇ 0.20°, 14.91 ⁇ 0.20°, and/or 8.98 ⁇ 0.20°, and/or 17.02 ⁇ 0.20°, and/or 18.08 ⁇ 0.20°, and/or 18.54 ⁇ 0.20°, and/or 19.07 ⁇ 0.20°, and/or 21.58 ⁇ 0.20°, and/or 22.23 ⁇ 0.20 °, and/or 22.66 ⁇ 0.20°, and/or 23.81 ⁇ 0.20°, and/or 24.87 ⁇ 0.20°, and/or 26.93 ⁇ 0.20°, and/or 28.74 ⁇ 0.20°, and/or 10.48 ⁇ 0.20°, and/or 25.38 ⁇ 0.20°, and/or 23.38 ⁇ 0.20°, and/or 20.12 ⁇ 0.20°, and/or 32.87 ⁇ 0.20°.
  • the X-ray powder diffraction pattern of Cu K ⁇ radiation of the above-mentioned B crystal form has characteristic diffraction peaks at the following 2 ⁇ angles: 7.09°, 7.92°, 8.98°, 9.70°, 10.48°, 12.16°, 13.57 °, 13.84°, 14.19°, 14.91°, 15.90°, 17.02°, 18.08°, 18.54°, 19.07°, 20.12°, 21.18°, 21.58°, 21.74°, 22.23°, 22.66°, 23.38°, 23.81°, 24.47°, 24.87°, 25.38°, 25.87°, 26.93°, 28.74°, 29.27°, 31.17°, 32.04°, 32.87°, 34.35°, 36.05°, 39.04°.
  • the XRPD pattern of the above-mentioned Form B is shown in Figure 4.
  • the XRPD pattern of the above crystal form B is basically as shown in Figure 4.
  • the differential scanning calorimetry (DSC) curve of the above-mentioned B crystal form shows an endothermic peak at 154.2°C ⁇ 3°C.
  • the DSC spectrum of the above crystal form B is shown in Figure 5.
  • the DSC spectrum of the above crystal form B is basically as shown in Figure 5.
  • thermogravimetric analysis (TGA) curve of the above-mentioned B crystal form loses 4.82% weight at 150.0°C ⁇ 3°C.
  • the TGA spectrum of the above-mentioned B crystal form is shown in Figure 6.
  • the TGA spectrum of the above crystal form B is basically as shown in Figure 6.
  • the present invention also provides the B crystal form of the compound of formula (I),
  • Its preparation method includes the following steps:
  • Compound Z is selected from compounds of formula (I);
  • Solvent M is toluene
  • Solvent N does not exist, or solvent N is selected from n-heptane, n-hexane and cyclohexane; preferably, solvent N is n-heptane;
  • the volume ratio of the solvent M and the solvent N is 1:0.5 ⁇ 1:3.
  • the invention also provides a method for preparing the B crystal form of the compound of formula (I), which includes the following steps:
  • Compound Z is selected from compounds of formula (I);
  • Solvent M is toluene
  • Solvent N is absent or is selected from n-heptane, n-hexane and cyclohexane.
  • the above step (b) is stirring at 50-60°C for 1 to 5 hours.
  • the above-mentioned solvent N is selected from n-heptane, n-hexane and cyclohexane, and the volume ratio of solvent M to solvent N is 1:0.5-1:3.
  • the above-mentioned solvent N is n-heptane.
  • the volume ratio of the above solvent M and solvent N is 1:1.
  • the mass ratio of the total volume of the above-mentioned solvent M and solvent N to compound Z is 1:15 to 1:30.
  • the mass ratio of the total volume of the above-mentioned solvent M and solvent N to compound Z is 1:20.
  • the present invention also provides the C crystal form of the hydrate of the compound of formula (I),
  • the X-ray powder diffraction pattern of Cu K ⁇ radiation of the C crystal form has characteristic diffraction peaks at the following 2 ⁇ angles: 5.85 ⁇ 0.20°, 11.70 ⁇ 0.20°, 17.59 ⁇ 0.20°.
  • the X-ray powder diffraction pattern of Cu K ⁇ radiation of the above-mentioned C crystal form has characteristic diffraction peaks at the following 2 ⁇ angles: 5.85°, 11.70°, 17.59°.
  • the X-ray powder diffraction pattern of the above-mentioned C crystal form is shown in Figure 7.
  • the X-ray powder diffraction pattern of the above-mentioned C crystal form is basically as shown in Figure 7.
  • thermogravimetric analysis (TGA) curve of the above-mentioned C crystal form loses 12.61% of weight at 150.0°C ⁇ 3°C.
  • the TGA spectrum of the above crystal form C is shown in Figure 8.
  • the TGA spectrum of the above crystal form C is basically as shown in Figure 8.
  • the A crystal form, B crystal form, and C crystal form of the compound of formula (I) of the present invention may be in the form of a nonsolvate or a solvate, such as a hydrate.
  • the present invention also provides the use of the A crystal form, B crystal form, C crystal form or the crystal form obtained by its preparation method in the preparation of drugs for treating influenza.
  • the present invention also provides the use of the A crystal form of the above compound or the crystal form obtained by its preparation method in the preparation of drugs for treating influenza.
  • the compound of the present invention shows positive effects in inhibition of influenza virus replication tests at the cellular level, excellent body weight protection in animal efficacy models, and early recovery time.
  • Plasma protein binding rate test The results show that the compound of the present invention has a moderate plasma protein binding rate in plasma, and the PK results show that it has good pharmacokinetic properties and good pharmaceutical properties.
  • the crystal form of the compound of the present invention has a simple preparation process, is stable, and is convenient for preparation, and has great application prospects in preventing and/or treating influenza.
  • 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, embodiments formed by combining them with other chemical synthesis methods, and those skilled in the art.
  • Well-known equivalents and preferred embodiments include, but are not limited to, the embodiments of the present invention.
  • the position of the peak or the relative intensity of the peak may vary due to factors such as the measuring instrument, measuring method/conditions, etc.
  • the error in the determination of the 2 ⁇ value can be ⁇ 0.2°. Therefore, this error should be taken into account when determining each crystal form, and it is within the scope of this application.
  • the expression "substantially as shown" in a specific XRPD pattern means that the position of the diffraction peak in the XRPD pattern is basically within the range of visual inspection or with the help of a selected diffraction peak list ( ⁇ 0.20°, 2 ⁇ ). Same as above.
  • a selected diffraction peak list ⁇ 0.20°, 2 ⁇ .
  • the structure of the compound of the present invention can be confirmed by conventional methods well known to those skilled in the art. If the present invention involves the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art.
  • SXRD single crystal X-ray diffraction
  • the light source is CuK ⁇ radiation
  • the scanning mode is: ⁇ / ⁇ scanning.
  • Shelxs97 can confirm the absolute configuration by analyzing the crystal structure.
  • ACN represents acetonitrile
  • DMSO represents dimethyl sulfoxide.
  • N 2 nitrogen; RH: relative humidity; mL: milliliter; L: liter; min: minutes; °C: degrees Celsius; ⁇ m: micrometers; mm: millimeters; ⁇ L: microliters; moL/L: moles per liter; mg: milligrams ;s: seconds; nm: nanometers; MPa: megapascals; lux: lux; ⁇ w/cm 2 : microwatts per square centimeter; h: hours; Kg: kilograms; nM: nanomoles, rpm: rotational speed; XRPD represents X-rays Powder diffraction; DSC stands for differential scanning calorimetry; TGA stands for thermogravimetric analysis; 1 H NMR stands for hydrogen nuclear magnetic resonance spectroscopy.
  • the compounds of the present invention are named according to the conventional naming principles in this field or used Software nomenclature, commercially available compounds using supplier catalog names, all solvents used in this invention are commercially available.
  • the hygroscopicity evaluation categories are as follows:
  • ⁇ W% represents the moisture absorption weight gain of the test product at 25 ⁇ 1°C and 80 ⁇ 2%RH.
  • Figure 1 is the XRPD spectrum of the crystal form A of the compound of formula (I).
  • Figure 2 is a DSC spectrum of the crystal form A of the compound of formula (I).
  • Figure 3 is a TGA spectrum of the crystalline form A of compound (I).
  • Figure 4 is the XRPD spectrum of the crystal form B of the compound of formula (I).
  • Figure 5 is a DSC spectrum of the crystal form B of the compound of formula (I).
  • Figure 6 is a TGA spectrum of the crystal form B of the compound of formula (I).
  • Figure 7 is the XRPD spectrum of the hydrate crystal form C of the compound of formula (I).
  • Figure 8 is a TGA spectrum of the hydrate C crystal form of the compound of formula (I).
  • Figure 9 is the DVS spectrum of the crystalline form A of the compound of formula (I).
  • Figure 10 is an ellipsoid diagram of a single crystal of the compound of formula (I).
  • the obtained crude product was added to dichloromethane (460 mL), and triethylamine (77.60g, 766.87mmol, 106.74mL) and N,O-dimethylhydroxylamine hydrochloride (37.40g, 383.43mmol) were added under stirring.
  • the reaction solution was heated at 20 Stir for 1 hour at °C. Water (100 mL) was added, the liquid was separated, and the aqueous phase was extracted with dichloromethane (50 mL ⁇ 2).
  • Dissolve compound 1-4 (15g, 74.95mmol) in dichloromethane (60mL) and methanol (60mL), control the temperature from 0 to 20°C, and add trimethylsilyldiazomethane (2M, 44.97mL) dropwise.
  • the reaction solution was stirred at 20°C for 2 hours, then acetic acid (3 mL) was added and stirred for 5 minutes.
  • the reaction solution was concentrated under reduced pressure, added with water (50 mL), extracted with dichloromethane (50 mL ⁇ 2), combined the organic phases, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure.
  • Crystals were collected and diffraction intensity data were collected using a single crystal X-ray diffractometer (SC-XRD) (D8-VENTURE).
  • SC-XRD single crystal X-ray diffractometer
  • the absolute configuration of the compound of formula (I) can be determined, and the molecular formula of the compound is C 26 H 22 F 2 N 4 O 7 Se.
  • the ellipsoid diagram of the three-dimensional structure of the compound of formula (I) is shown in Figure 10.
  • the crystal structure data of the single crystal of the compound of formula (I) are shown in Table 7.
  • ICH conditions the total visible light illumination reached 1,200,000 Lux ⁇ hrs, the total ultraviolet light When the illumination reaches 200W ⁇ hrs/m 2 ), place it in the open under visible light and ultraviolet light (samples in the shading control group are placed at the same time and wrapped in tin foil), and placed 1, 2 and 3 under 40°C/75%RH (open) conditions. months, placed at 25°C/60%RH (exposure) for 3 months.
  • XRPD testing was performed on all stability samples to detect changes in crystal form, and the results are shown in Table 8.
  • HPLC tests were conducted on all stability samples. The specific results are summarized in Table 8. The HPLC test instruments and analysis conditions are shown in Tables 9 and 10.
  • Compound of formula (I) is a prodrug, which can be converted into compound 1-15 in the body, and will also be partially degraded into compound 1-15 during storage.
  • the purity data here include compound of formula (I) and compound 1 -15.
  • the antiviral activity of the compound against influenza virus was evaluated by measuring the half effective concentration (EC 50 ) value of the compound. Cytopathy assay is widely used to measure the protective effect of compounds on virus-infected cells to reflect the antiviral activity of compounds.
  • MDCK cells were seeded into a black 384-well cell culture plate at a density of 2,000 cells per well, and then placed in a 37°C, 5% CO2 incubator overnight. Compounds were diluted by the Echo555 non-contact nanoliter sonic pipetting system and added to the cell wells (4-fold dilution, 8 test concentration points). Influenza virus A/PR/8/34 (H1N1) strain was then added to the cell culture wells at 1-2 90% tissue culture infectious dose (TCID90) per well, with a final DMSO concentration of 0.5% in the culture medium. Set up virus control wells (add DMSO and viruses, no compounds), cell control wells (add DMSO, no compounds and viruses) and medium control wells (only medium, no cells).
  • the cytotoxicity test and the antiviral activity test of the compounds were carried out in parallel. Except that no virus was added, other experimental conditions were consistent with the antiviral activity test.
  • the cell plate was placed in a 37°C, 5% CO2 incubator for 5 days. After 5 days of culture, cell viability was detected using cell viability detection kit CCK8. Raw data were used for compound antiviral activity and cytotoxicity calculations.
  • the antiviral activity and cytotoxicity of the compound were expressed by the inhibition rate (%) of the compound on the cellular viral effect caused by the virus respectively. Calculated as follows:
  • the compounds of the present invention exhibit positive effects in the assay of inhibiting influenza virus replication at the cellular level.
  • mice were infected with influenza A/PR/8/34 (H1N1) via intranasal instillation. They were treated with the compound starting 48 hours after infection and administered orally for 7 consecutive days, twice a day. The anti-influenza A virus H1N1 effect of the compound in this model was evaluated by observing the weight changes and survival rate of mice.
  • mice SPF grade BALB/c mice, 6-7 weeks old, female were used in the experiment. After the mice arrived in the BSL-2 animal room, they were allowed to adapt for at least 3 days before starting the experiment. The day of infection was set as day 0 of the experiment. Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (75 mg/kg, 10 mL/kg). After the animals entered deep anesthesia, they were infected with A/PR/8/34 (H1N1) virus via intranasal instillation. The infection volume was 50 ⁇ L. From day 2 to day 8, 5 mg/kg (administration volume: 10 mL/kg) of the test compound was administered orally twice a day. The first administration time is 48 hours after infection. The status of the mice was observed every day, and the weight and survival rate of the mice were recorded. On day 14, all surviving animals were euthanized.
  • Weight loss rate (weight on day 0 – weight on day N)/body weight on day 0*100%.
  • Table 12 The results are shown in Table 12: The compound of formula (I) can achieve a maximum weight loss rate of 7.03% in protected animals on the third day, and then begins to recover. By the end of the experiment, the mouse survival rate is 100%.
  • the compounds of the present invention show excellent body weight protection and early recovery time in animal pharmacodynamic models.
  • MDCK cells were seeded in a 96-well cell culture plate at a density of 15,000 cells per well and cultured overnight in a 37°C, 5% CO2 incubator.
  • the compound solution (3-fold serial dilution, 8 concentration points, triple wells) and Baloxavir-resistant A/PR/8/34 (H1N1) influenza virus strain were added the next day.
  • the final concentration of DMSO in the cell culture medium was 0.5%.
  • the cells were cultured in a 5% CO 2 , 37°C incubator for 5 days until the cytopathic effects in the virus-infected control wells without compounds reached 80–95%.
  • CCK8 was used to detect cell viability in each well. If the cell viability of the compound-containing wells is higher than that of the virus-infected control wells, that is, the CPE is weakened, it indicates that the compound has an inhibitory effect on the tested virus.
  • the antiviral activity of a compound is expressed by the inhibitory activity (%) of the compound against viral effects on cells caused by viruses. Calculated as follows:
  • EC 50 uses GraphPad Prism (version 5) software to perform nonlinear fitting analysis on the inhibitory activity and cell viability of the compound.
  • the method is "log(inhibitor)vs.response--Variable slope”.
  • the experimental results are shown in Table 13.
  • the compound of the present invention shows a positive effect in inhibiting the replication of Baloxavir-resistant A/PR/8/34 (H1N1) influenza virus strain at the cellular level.
  • mice were infected with the Baloxavir-resistant A/PR/8/34(H1N1)I38T strain of influenza A virus through intranasal instillation. They were treated with the compound starting 2 hours before infection and administered orally for 7 consecutive days, twice a day. .
  • the anti-influenza A virus H1N1 effect of the compound in this model was evaluated by observing the weight changes and survival rate of mice.
  • mice SPF grade BALB/c mice, 6-7 weeks old, female were used in the experiment. After the mice arrived in the BSL-2 animal room, they were allowed to adapt for at least 3 days before starting the experiment. The day of infection was set as day 0 of the experiment. After the mice were deeply anesthetized by intraperitoneal injection of Serta 50/xylazine hydrochloride, they were infected with Baloxavir-resistant A/PR/8/34(H1N1)I38T virus strain via intranasal instillation, with an infection volume of 50 ⁇ L. From day 2 to day 8, 15 mg/kg or 50 mg/kg (administration volume 10 mL/kg) of the test compound was administered orally twice a day. The first administration time is 2 hours before infection. The status of the mice was observed every day, and the weight and survival rate of the mice were recorded. On day 14, all surviving animals were euthanized.
  • Weight loss rate (weight on day 0 – weight on day N)/body weight on day 0*100%.
  • Table 14 The experimental results are shown in Table 14 below.
  • the compounds of the present invention show excellent body weight protection and early recovery time in animal pharmacodynamic models.
  • Experimental purpose Use equilibrium dialysis method to evaluate the protein binding rate of the compound of the present invention in the plasma of CD-1 mice, SD rats and humans.
  • Test plan Dilute the test compound into the plasma of the above five species with dialysis buffer, prepare a sample with a final concentration of 2 ⁇ M, then add the sample to a 96-well equilibrium dialysis device, and incubate with phosphate at 37°C. The buffer solution was dialyzed for 4 hours. The experiment used warfarin as a control compound. The concentrations of test compounds and warfarin in plasma and buffer were determined by LC-MS/MS.
  • H stands for human
  • R stands for rat
  • M stands for mouse
  • D stands for dog
  • C stands for cynomolgus monkey.
  • the compounds of the present invention have moderate plasma protein binding rates in the plasma of the five species, which indicates that the free drug concentration ratio of the test compound in the plasma of the above five species is moderate and has good pharmaceutical properties.
  • mice Male SD rats, 6-8 weeks old, weighing 200-300 grams;
  • Plasma: dichlorvos solution 40:1.
  • the dichlorvos solution is a 40mM dichlorvos acetonitrile/water (1:1) solution.
  • the solution is heated at 4°C and 3000g. Centrifuge for 10 minutes to aspirate the supernatant plasma, quickly place it in dry ice, and store it in a -80°C refrigerator for LC-MS/MS analysis.
  • the compound of the present invention has a high oral plasma exposure and has good pharmacokinetic properties.
  • mice male beagle dogs, ⁇ 6 months old, weighing 6-12 kg;
  • Injection administration i.v.
  • the dose is 1mpk, the concentration is 1mg/mL, the solvent is 10% DMAC+90% (20% HP- ⁇ -CD+water);
  • the compound of formula (I) is administered orally (po) , the dosage is 10mpk, the concentration is 2mg/mL, the solvent is 3% DMSO+10% solution HS+87% water);
  • the crystal form of compound A of formula (I) is administered orally (po), the dosage is 5mpk, the concentration is 1mg/ mL, the solvent is 3% DMSO+10% solution HS+87% water).
  • Plasma 0.8 mL of blood sample was collected from the saphenous vein puncture of the experimental animals at each time point, and the actual blood collection time was recorded. All blood samples were added into commercial EDTA-K2 anticoagulant tubes with a specification of 1.5 mL. After the blood sample is collected, DDV is added to the plasma matrix as a stabilizer.
  • the compound of the present invention has low clearance rate, long half-life, high oral plasma exposure, and good pharmacokinetic properties.

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Abstract

Disclosed in the present invention are a crystal form of a pyridone polycyclic derivative and a preparation method therefor, and particularly a crystal form of a compound as represented by formula (I) and a preparation method therefor.

Description

吡啶酮多并环类衍生物的晶型及其制备方法Crystal forms of pyridone polycyclic derivatives and preparation methods thereof
本发明主张如下优先权：The present invention claims the following priority:
CN202210812490.X，申请日：2022年07月05日。CN202210812490.X, application date: July 5, 2022.
技术领域Technical field
本发明涉及一种吡啶酮多并环类衍生物的晶型及其制备方法。The invention relates to a crystal form of a pyridone polycyclic derivative and a preparation method thereof.
背景技术Background technique
流行性感冒病毒，即流感病毒(influenza virus,IFV)，是一种能够导致人和动物患流行感冒的分节状单链反义RNA病毒。流感病毒可引起非常高的发病率和死亡率，尤其A型流感病毒还能够导致全球性的大流行。Influenza virus, also known as influenza virus (IFV), is a segmented single-stranded antisense RNA virus that can cause influenza in humans and animals. Influenza viruses can cause very high morbidity and mortality, and influenza A viruses in particular can cause global pandemics.
A型流感病毒为单负链RNA病毒，基因组分为8个片段，编码8个蛋白。流感病毒基因组片段5’末端和3’末端高度保守，该两个末端的序列互补而形成柄环状结构，该结构在启动病毒RNA复制时发挥重要作用。病毒各个基因片段编码的蛋白大小不同，而且在流感病毒的生命周期中发挥着不同的作用，先将几种主要的蛋白的基本功能介绍如下。流感病毒的HA是流感病毒识别宿主受体的配体，与细胞表面病毒特异性受体结合，介导病毒外膜与细胞内小体膜融合释放病毒核衣壳进入胞浆。流感病毒的受体具有特异性，A型流感病毒的受体为唾液酸糖蛋白。流感病毒的NA蛋白在复制过程中可除去病毒颗粒表面的唾液酸，使病毒颗粒不能继续在宿主细胞表面聚集，从而有利于病毒子的释放并进一步感染更多的宿主细胞。Influenza A virus is a single negative-strand RNA virus with a genome divided into 8 segments encoding 8 proteins. The 5' end and 3' end of the influenza virus genome fragment are highly conserved, and the sequences of the two ends are complementary to form a handle loop structure, which plays an important role in initiating viral RNA replication. The proteins encoded by each gene segment of the virus are different in size and play different roles in the life cycle of the influenza virus. The basic functions of several major proteins are introduced below. The HA of influenza virus is the ligand for influenza virus to recognize host receptors. It binds to virus-specific receptors on the cell surface and mediates the fusion of the virus outer membrane with the intracellular body membrane to release the virus nucleocapsid into the cytoplasm. The receptors of influenza viruses are specific, and the receptors of influenza A viruses are sialoglycoproteins. The NA protein of influenza virus can remove sialic acid on the surface of virus particles during the replication process, so that virus particles cannot continue to aggregate on the surface of host cells, which is beneficial to the release of virions and further infects more host cells.
流感病毒的M2蛋白的作用：流感病毒的HA蛋白和唾液酸结合，流感病毒被宿主细胞内吞。吞噬泡中的酸碱性对于病毒脱衣壳起着至关重要的作用，病毒膜上的M2蛋白的离子通道可以使吞噬泡的pH值逐步降低，当pH值降至5.0-6.0时，导致HA2蛋白的发生变构，位于HA2蛋白氨基末端的融合肽移位，进而激活融合过程，导致病毒的双层类脂膜与细胞膜融合，释放出病毒颗粒内部的RNPs到宿主细胞浆。M2蛋白是一个跨膜的离子通道，仅在A型流感病毒中被发现，它有一部分延伸至病毒外膜表面。The role of the M2 protein of influenza virus: The HA protein of influenza virus binds to sialic acid, and the influenza virus is endocytosed by the host cell. The acidity and alkalinity in the phagocytic vacuole plays a crucial role in virus uncoating. The ion channel of the M2 protein on the virus membrane can gradually reduce the pH value of the phagocytic vacuole. When the pH value drops to 5.0-6.0, HA2 Allosteric protein occurs, and the fusion peptide located at the amino terminus of the HA2 protein is displaced, thereby activating the fusion process, causing the double-layer lipid membrane of the virus to fuse with the cell membrane, and releasing the RNPs inside the virus particles into the host cell plasma. The M2 protein is a transmembrane ion channel found only in influenza A viruses, with part of it extending to the surface of the virus's outer membrane.
流感病毒蛋白的合成也是利用宿主细胞翻译机制，甚至病毒可以暂停宿主蛋白的翻译，加快自身蛋白的合成。宿主细胞mRNA的多聚腺苷酸化是通过特异的腺苷酸化酶完成的，与之不同的是，病毒mRNA的腺苷酸尾是由负链的vRNA上连续的5-7个尿嘧啶转录形成的。病毒各个信使RNA(mRNA)的加帽是以相似的方式完成的：PA和PB2蛋白攫取宿主pre-mRNA转录体的5’加帽引物，并进而启动病毒mRNA合成，这个过程被称为“cap snatching”，主要通过病毒的RNA依赖性RNA聚合酶(RdRp)来完成，其PA亚基具有RNA内切酶活性，负责切断宿主mRNA。在完成了多聚腺苷酸化过程和加帽过程，病毒的mRNA即出核，进入细胞质，并像宿主细胞的mRNA一样进行翻译，病毒vRNA片段的核输出是由病毒的M1蛋白和NS2蛋白介导的，M1蛋白可以与vRNA和NP蛋白相互作用时，同时也与核输出蛋白NS2作用；由此，核输出蛋白NS2介导M1-RNP以核蛋白形式出核进入宿主细胞的细胞质。The synthesis of influenza virus proteins also uses the translation mechanism of the host cell. The virus can even pause the translation of host proteins and accelerate the synthesis of its own proteins. Polyadenylation of host cell mRNA is completed by specific adenylation enzymes. In contrast, the adenylate tail of viral mRNA is formed by the continuous transcription of 5-7 uracils on the negative-strand vRNA. of. The capping of each viral messenger RNA (mRNA) is completed in a similar way: PA and PB2 proteins grab the 5' capping primer of the host pre-mRNA transcript and then initiate viral mRNA synthesis. This process is called "cap "snatching" is mainly accomplished by the virus's RNA-dependent RNA polymerase (RdRp), whose PA subunit has RNA endonuclease activity and is responsible for cutting host mRNA. After completing the polyadenylation process and capping process, the viral mRNA exits the nucleus, enters the cytoplasm, and is translated like the host cell's mRNA. The nuclear export of the viral vRNA fragment is mediated by the viral M1 protein and NS2 protein. According to the guidance, when M1 protein can interact with vRNA and NP protein, it also interacts with nuclear export protein NS2; thus, nuclear export protein NS2 mediates M1-RNP to exit the nucleus and enter the cytoplasm of the host cell in the form of nuclear protein.
流感会产生由于丧失生产力和相关医疗资源的直接成本以及预防措施的间接成本。在美国，流感累计每年大约造成100亿美元的损失，据估计未来的流感大流行可引起数千亿美元的直接和间接成本。预防成本也非常高，全球各国政府已花费数十亿美元为可能的H5N1禽流感大流行做准备和计划，成本和购买药物和疫苗，以及发展灾难演练和提高边境管制的策略相关。Influenza incurs direct costs due to lost productivity and related medical resources, as well as indirect costs of preventive measures. In the United States, influenza causes cumulative annual losses of approximately $10 billion, and future influenza pandemics are estimated to cause hundreds of billions of dollars in direct and indirect costs. The cost of prevention is also very high. Governments around the world have spent billions of dollars preparing and planning for a possible H5N1 avian influenza pandemic, with costs associated with purchasing drugs and vaccines, as well as developing disaster drills and strategies to increase border controls.
目前的流感治疗选择包括接种疫苗和用抗病毒药物进行化疗和化学预防。经常向高危群体，例如儿童和老年人，或有哮喘、糖尿病或心脏病的人推荐接种抗流感的流感疫苗，但是，即使接种疫苗也不能完全避免患流感。每个季节重新制备一些特定流感株的疫苗，但不可能涵盖该季节时全球主动感染人的各种病毒株。另外，由于流感病毒会发生一定程度的抗原漂移，如果超过一种病毒感染了单个细胞，则基因组中8个单独的vRNA片段发生混合或重配，所导致的病毒遗传学上的快速变化可产生抗原转变并使得病毒能感染新宿主物种并迅速克服保护性免疫。Current treatment options for influenza include vaccination and chemotherapy and chemoprophylaxis with antiviral drugs. Flu vaccination is often recommended for high-risk groups, such as children and older adults, or people with asthma, diabetes, or heart disease, but even vaccination cannot completely prevent getting the flu. Vaccines are prepared anew each season for specific influenza strains, but it is impossible to cover all strains of the virus that are actively infecting people around the world that season. In addition, because influenza viruses undergo a certain degree of antigenic drift, if more than one virus infects a single cell, the eight separate vRNA segments in the genome will mix or recombine, resulting in rapid changes in viral genetics. Antigenic shift and allows the virus to infect new host species and rapidly overcome protective immunity.
抗病毒药物也可以用于治疗流感，其中神经氨酸酶抑制剂，如奥司他韦(达菲)，对于甲型流感病毒效 果明显，但是经过临床观察发现，对于该类神经氨酸酶抑制剂已经出现了耐药的病毒株。在抗流感病毒领域，临床上亟需全新作用机制的抗流感病毒药物，能够支持单药使用治疗甲型流感，或者通过和已上市的其他作用机制的抗流感病毒药物联用，用于甲型流感的预防和治疗。Antiviral drugs can also be used to treat influenza. Among them, neuraminidase inhibitors, such as oseltamivir (Tamiflu), are effective against influenza A viruses. The results are obvious, but clinical observations have found that virus strains resistant to this type of neuraminidase inhibitors have emerged. In the field of anti-influenza viruses, there is an urgent clinical need for anti-influenza virus drugs with new mechanisms of action, which can support the use of single drugs to treat influenza A, or can be used in combination with anti-influenza virus drugs with other mechanisms of action that are already on the market for the treatment of influenza A. Influenza prevention and treatment.
发明内容Contents of the invention
本发明提供了式(I)化合物的A晶型，
The invention provides crystal form A of the compound of formula (I),
所述A晶型的Cu Kα辐射的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：4.56±0.20°，10.68±0.20°，16.90±0.20°。The X-ray powder diffraction pattern of Cu Kα radiation of the A crystal form has characteristic diffraction peaks at the following 2θ angles: 4.56±0.20°, 10.68±0.20°, 16.90±0.20°.
本发明的一些方案中，上述A晶型的Cu Kα辐射的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：4.56±0.20°，10.68±0.20°，16.90±0.20°，19.42±0.20°，20.72±0.20°。In some aspects of the present invention, the X-ray powder diffraction pattern of Cu Kα radiation of the above-mentioned A crystal form has characteristic diffraction peaks at the following 2θ angles: 4.56±0.20°, 10.68±0.20°, 16.90±0.20°, 19.42±0.20° , 20.72±0.20°.
本发明的一些方案中，上述A晶型的Cu Kα辐射的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：4.56±0.20°，9.08±0.20°，10.68±0.20°，16.90±0.20°，19.42±0.20°，19.94±0.20°，20.72±0.20°，29.12±0.20°。In some aspects of the present invention, the X-ray powder diffraction pattern of Cu Kα radiation of the above-mentioned A crystal form has characteristic diffraction peaks at the following 2θ angles: 4.56±0.20°, 9.08±0.20°, 10.68±0.20°, 16.90±0.20° , 19.42±0.20°, 19.94±0.20°, 20.72±0.20°, 29.12±0.20°.
本发明的一些方案中，上述A晶型的Cu Kα辐射的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：4.56±0.20°，9.08±0.20°，10.68±0.20°，11.80±0.20°，16.90±0.20°，17.84±0.20°，19.42±0.20°，19.94±0.20°，20.72±0.20°，22.66±0.20°，24.30±0.20°，29.12±0.20°。In some aspects of the present invention, the X-ray powder diffraction pattern of Cu Kα radiation of the above-mentioned A crystal form has characteristic diffraction peaks at the following 2θ angles: 4.56±0.20°, 9.08±0.20°, 10.68±0.20°, 11.80±0.20° , 16.90±0.20°, 17.84±0.20°, 19.42±0.20°, 19.94±0.20°, 20.72±0.20°, 22.66±0.20°, 24.30±0.20°, 29.12±0.20°.
本发明的一些方案中，上述A晶型的Cu Kα辐射的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：4.56±0.20°，9.08±0.20°，10.68±0.20°，11.80±0.20°，14.52±0.20°，16.90±0.20°，17.84±0.20°，19.42±0.20°，19.94±0.20°，20.72±0.20°，22.66±0.20°，24.30±0.20°，26.26±0.20°，28.24±0.20°，29.12±0.20°，29.98±0.20°。In some aspects of the present invention, the X-ray powder diffraction pattern of Cu Kα radiation of the above-mentioned A crystal form has characteristic diffraction peaks at the following 2θ angles: 4.56±0.20°, 9.08±0.20°, 10.68±0.20°, 11.80±0.20° , 14.52±0.20°, 16.90±0.20°, 17.84±0.20°, 19.42±0.20°, 19.94±0.20°, 20.72±0.20°, 22.66±0.20°, 24.30±0.20°, 26.26±0.20°, 28.24±0.20° , 29.12±0.20°, 29.98±0.20°.
本发明的一些方案中，上述A晶型的Cu Kα辐射的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：4.56±0.20°，9.08±0.20°，10.68±0.20°，和/或11.80±0.20°，和/或14.52±0.20°，和/或16.56±0.20°，和/或16.90±0.20°，和/或17.84±0.20°，和/或19.42±0.20°，和/或19.94±0.20°，和/或20.30±0.20°，和/或20.72±0.20°，和/或22.66±0.20°，和/或24.30±0.20°，和/或26.26±0.20°，和/或28.24±0.20°，和/或29.12±0.20°，和/或29.98±0.20°。In some aspects of the present invention, the X-ray powder diffraction pattern of Cu Kα radiation of the above-mentioned A crystal form has characteristic diffraction peaks at the following 2θ angles: 4.56±0.20°, 9.08±0.20°, 10.68±0.20°, and/or 11.80 ±0.20°, and/or 14.52±0.20°, and/or 16.56±0.20°, and/or 16.90±0.20°, and/or 17.84±0.20°, and/or 19.42±0.20°, and/or 19.94±0.20 °, and/or 20.30±0.20°, and/or 20.72±0.20°, and/or 22.66±0.20°, and/or 24.30±0.20°, and/or 26.26±0.20°, and/or 28.24±0.20°, and/or 29.12±0.20°, and/or 29.98±0.20°.
本发明的一些方案中，上述A晶型的Cu Kα辐射的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：4.56°，9.08°，10.68°，11.80°，13.94°，14.52°，14.92°，15.28°，15.74°，16.56°，16.90°，17.84°，18.42°，19.42°，19.94°，20.30°，20.72°，21.12°，22.66°，23.80°，24.30°，24.68°，25.46°，25.80°，26.26°，26.86°，28.24°，29.12°，29.98°，30.88°，31.94°，33.60°，35.00°，37.88°，38.48°。In some aspects of the present invention, the X-ray powder diffraction pattern of Cu Kα radiation of the above-mentioned A crystal form has characteristic diffraction peaks at the following 2θ angles: 4.56°, 9.08°, 10.68°, 11.80°, 13.94°, 14.52°, 14.92 °, 15.28°, 15.74°, 16.56°, 16.90°, 17.84°, 18.42°, 19.42°, 19.94°, 20.30°, 20.72°, 21.12°, 22.66°, 23.80°, 24.30°, 24.68°, 25.46°, 25.80°, 26.26°, 26.86°, 28.24°, 29.12°, 29.98°, 30.88°, 31.94°, 33.60°, 35.00°, 37.88°, 38.48°.
本发明的一些方案中，上述A晶型的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 Figure 1.
本发明的一些方案中，上述A晶型的XRPD图谱基本上如图1所示。In some aspects of the present invention, the XRPD pattern of the above-mentioned crystal form A is basically as shown in Figure 1.
本发明的一些方案中，上述A晶型的Cu Kα辐射的XRPD图谱中，衍射峰的峰位置及相对强度如表1所示：In some solutions of the present invention, in the XRPD pattern of Cu Kα radiation of the above-mentioned crystal form A, the peak position and relative intensity of the diffraction peak are as shown in Table 1:
表1式(I)化合物A晶型的XRPD衍射数据

Table 1 XRPD diffraction data of compound A of formula (I)

本发明的一些方案中，上述A晶型的差示扫描量热(DSC)曲线显示在194.2℃±5℃处具有吸热峰的起始点，在201.4℃±5℃处具有放热峰的起始点。In some solutions of the present invention, the differential scanning calorimetry (DSC) curve of the above-mentioned crystal form A shows that it has the starting point of the endothermic peak at 194.2℃±5℃ and the starting point of the exothermic peak at 201.4℃±5℃. starting point.
本发明的一些方案中，上述A晶型的差示扫描量热(DSC)曲线显示在198.4℃±3℃处具有吸热峰的峰值。In some aspects of the present invention, the differential scanning calorimetry (DSC) curve of the above-mentioned Form A shows an endothermic peak at 198.4°C ± 3°C.
本发明的一些方案中，上述A晶型的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 Figure 2.
本发明的一些方案中，上述A晶型的DSC图谱基本上如图2所示。In some aspects of the present invention, the DSC spectrum of the above-mentioned crystal form A is basically as shown in Figure 2.
本发明的一些方案中，上述A晶型的热重分析(TGA)曲线在200.0℃±3℃时失重为2.87％。In some aspects of the present invention, the weight loss of the thermogravimetric analysis (TGA) curve of the above-mentioned crystal form A at 200.0°C ± 3°C is 2.87%.
本发明的一些方案中，上述A晶型的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 Figure 3.
本发明的一些方案中，上述A晶型的TGA图谱基本上如图3所示。In some aspects of the present invention, the TGA spectrum of the above-mentioned crystal form A is basically as shown in Figure 3.
本发明还提供了式(I)化合物的A晶型，
The present invention also provides crystal form A of the compound of formula (I),
其制备方法包括如下步骤：Its preparation method includes the following steps:
(a)将化合物Z加入溶剂X和溶剂Y的混合溶剂中；(a) Add compound Z to the mixed solvent of solvent X and solvent Y;
(b)加完后在10～35℃继续搅拌5～24小时；(b) After the addition is completed, continue stirring at 10-35°C for 5-24 hours;
(c)过滤，滤饼在25～60℃下真空干燥1～24小时；(c) Filter, and vacuum dry the filter cake at 25-60°C for 1-24 hours;
其中，in,
所述化合物Z选自式(I)化合物、式(I)化合物B晶型、式(I)化合物C晶型、式(I)化合物D晶型和式(I)化合物E晶型；优选的，化合物Z为式(I)化合物B晶型；The compound Z is selected from the group consisting of compound of formula (I), crystal form of compound of formula (I) B, crystal form of compound of formula (I) C, crystal form of compound of formula (I) D and crystal form of compound of formula (I) E; preferred , compound Z is the crystal form of compound B of formula (I);
所述溶剂X为乙酸乙酯；The solvent X is ethyl acetate;
所述溶剂Y选自正庚烷、正己烷和环己烷；优选的，溶剂Y为正庚烷；The solvent Y is selected from n-heptane, n-hexane and cyclohexane; preferably, the solvent Y is n-heptane;
所述溶剂X和溶剂Y的体积比为1:1～1:6；优选的，溶剂X和溶剂Y的体积比为1:3。The volume ratio of solvent X to solvent Y is 1:1 to 1:6; preferably, the volume ratio of solvent X to solvent Y is 1:3.
本发明还提供了式(I)化合物的A晶型的制备方法，包括如下步骤：The invention also provides a method for preparing crystal form A of the compound of formula (I), which includes the following steps:
(a)将化合物Z加入溶剂X和溶剂Y的混合溶剂中；(a) Add compound Z to the mixed solvent of solvent X and solvent Y;
(b)加完后在10～35℃继续搅拌5～24小时；(b) After the addition is completed, continue stirring at 10-35°C for 5-24 hours;
(c)过滤，滤饼在25～60℃下真空干燥1～24小时；(c) Filter, and vacuum dry the filter cake at 25-60°C for 1-24 hours;
其中，in,
化合物Z为式(I)化合物B晶型；Compound Z is the crystal form of compound B of formula (I);
溶剂X为乙酸乙酯；Solvent X is ethyl acetate;
溶剂Y选自正庚烷、正己烷和环己烷；Solvent Y is selected from n-heptane, n-hexane and cyclohexane;
溶剂X和溶剂Y的体积比为1:1～1:6。The volume ratio of solvent X to solvent Y is 1:1 to 1:6.
本发明的一些方案中，上述化合物Z为式(I)化合物。In some aspects of the present invention, the above-mentioned compound Z is a compound of formula (I).
本发明的一些方案中，上述化合物Z为式(I)化合物B晶型或式(I)化合物C晶型。In some aspects of the present invention, the above-mentioned compound Z is the crystal form of compound B of formula (I) or the crystal form of compound C of formula (I).
本发明的一些方案中，上述化合物Z为式(I)化合物B晶型。In some aspects of the present invention, the above-mentioned compound Z is the crystal form B of the compound of formula (I).
本发明的一些方案中，上述步骤(b)为加完后在10～25℃继续搅拌5～12小时。In some solutions of the present invention, the above step (b) is to continue stirring at 10 to 25°C for 5 to 12 hours after the addition.
本发明的一些方案中，上述溶剂Y为正庚烷。In some aspects of the present invention, the above solvent Y is n-heptane.
本发明的一些方案中，上述溶剂X和溶剂Y的体积比为1:3。In some aspects of the present invention, the volume ratio of the above solvent X and solvent Y is 1:3.
本发明的一些方案中，上述溶剂X和溶剂Y的总体积与化合物Z的质量比为1:8～1:15。In some aspects of the present invention, the mass ratio of the total volume of the above-mentioned solvent X and solvent Y to compound Z is 1:8 to 1:15.
本发明的一些方案中，上述溶剂X和溶剂Y的总体积与化合物Z的质量比为1:10。In some aspects of the present invention, the mass ratio of the total volume of the above solvent X and solvent Y to compound Z is 1:10.
本发明还提供了式(I)化合物的B晶型，
The present invention also provides the B crystal form of the compound of formula (I),
所述B晶型的Cu Kα辐射的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：9.70±0.20°，12.16±0.20°，14.91±0.20°，18.08±0.20°。The X-ray powder diffraction pattern of Cu Kα radiation of the B crystal form has characteristic diffraction peaks at the following 2θ angles: 9.70±0.20°, 12.16±0.20°, 14.91±0.20°, 18.08±0.20°.
本发明的一些方案中，上述B晶型的Cu Kα辐射的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：8.98±0.20°，9.70±0.20°，12.16±0.20°，14.91±0.20°，18.08±0.20°，18.54±0.20°，19.07±0.20°，21.58±0.20°。In some aspects of the present invention, the X-ray powder diffraction pattern of Cu Kα radiation of the above-mentioned B crystal form has characteristic diffraction peaks at the following 2θ angles: 8.98±0.20°, 9.70±0.20°, 12.16±0.20°, 14.91±0.20° , 18.08±0.20°, 18.54±0.20°, 19.07±0.20°, 21.58±0.20°.
本发明的一些方案中，上述B晶型的Cu Kα辐射的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：8.98±0.20°，9.70±0.20°，12.16±0.20°，14.91±0.20°，17.02±0.20°，18.08±0.20°，18.54±0.20°，19.07±0.20°，21.58±0.20°，22.23±0.20°，24.87±0.20°，26.93±0.20°。In some aspects of the present invention, the X-ray powder diffraction pattern of Cu Kα radiation of the above-mentioned B crystal form has characteristic diffraction peaks at the following 2θ angles: 8.98±0.20°, 9.70±0.20°, 12.16±0.20°, 14.91±0.20° , 17.02±0.20°, 18.08±0.20°, 18.54±0.20°, 19.07±0.20°, 21.58±0.20°, 22.23±0.20°, 24.87±0.20°, 26.93±0.20°.
本发明的一些方案中，上述B晶型的Cu Kα辐射的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：8.98±0.20°，9.70±0.20°，12.16±0.20°，14.19±0.20°，14.91±0.20°，17.02±0.20°，18.08±0.20°，18.54±0.20°，19.07±0.20°，21.58±0.20°，22.23±0.20°，22.66±0.20°，23.81±0.20°，24.87±0.20°，26.93±0.20°，28.74±0.20°。In some aspects of the present invention, the X-ray powder diffraction pattern of Cu Kα radiation of the above-mentioned B crystal form has characteristic diffraction peaks at the following 2θ angles: 8.98±0.20°, 9.70±0.20°, 12.16±0.20°, 14.19±0.20° , 14.91±0.20°, 17.02±0.20°, 18.08±0.20°, 18.54±0.20°, 19.07±0.20°, 21.58±0.20°, 22.23±0.20°, 22.66±0.20°, 23.81±0.20°, 24.87±0.20° , 26.93±0.20°, 28.74±0.20°.
本发明的一些方案中，上述B晶型的Cu Kα辐射的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：9.70±0.20°，12.16±0.20°，14.91±0.20°，和/或8.98±0.20°，和/或17.02±0.20°，和/或18.08±0.20°，和/或18.54±0.20°，和/或19.07±0.20°，和/或21.58±0.20°，和/或22.23±0.20°，和/或22.66±0.20°，和/或23.81±0.20°，和/或24.87±0.20°，和/或26.93±0.20°，和/或28.74±0.20°，和/或10.48±0.20°，和/或25.38±0.20°，和/或23.38±0.20°，和/或20.12±0.20°，和/或32.87±0.20°。In some aspects of the present invention, the X-ray powder diffraction pattern of Cu Kα radiation of the above-mentioned B crystal form has characteristic diffraction peaks at the following 2θ angles: 9.70±0.20°, 12.16±0.20°, 14.91±0.20°, and/or 8.98 ±0.20°, and/or 17.02±0.20°, and/or 18.08±0.20°, and/or 18.54±0.20°, and/or 19.07±0.20°, and/or 21.58±0.20°, and/or 22.23±0.20 °, and/or 22.66±0.20°, and/or 23.81±0.20°, and/or 24.87±0.20°, and/or 26.93±0.20°, and/or 28.74±0.20°, and/or 10.48±0.20°, and/or 25.38±0.20°, and/or 23.38±0.20°, and/or 20.12±0.20°, and/or 32.87±0.20°.
本发明的一些方案中，上述B晶型的Cu Kα辐射的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：7.09°，7.92°，8.98°，9.70°，10.48°，12.16°，13.57°，13.84°，14.19°，14.91°，15.90°，17.02°，18.08°，18.54°，19.07°，20.12°，21.18°，21.58°，21.74°，22.23°，22.66°，23.38°，23.81°，24.47°，24.87°，25.38°，25.87°，26.93°，28.74°，29.27°，31.17°，32.04°，32.87°，34.35°，36.05°，39.04°。In some aspects of the present invention, the X-ray powder diffraction pattern of Cu Kα radiation of the above-mentioned B crystal form has characteristic diffraction peaks at the following 2θ angles: 7.09°, 7.92°, 8.98°, 9.70°, 10.48°, 12.16°, 13.57 °, 13.84°, 14.19°, 14.91°, 15.90°, 17.02°, 18.08°, 18.54°, 19.07°, 20.12°, 21.18°, 21.58°, 21.74°, 22.23°, 22.66°, 23.38°, 23.81°, 24.47°, 24.87°, 25.38°, 25.87°, 26.93°, 28.74°, 29.27°, 31.17°, 32.04°, 32.87°, 34.35°, 36.05°, 39.04°.
本发明的一些方案中，上述B晶型的XRPD图谱如图4所示。In some aspects of the present invention, the XRPD pattern of the above-mentioned Form B is shown in Figure 4.
本发明的一些方案中，上述B晶型的XRPD图谱基本上如图4所示。In some aspects of the present invention, the XRPD pattern of the above crystal form B is basically as shown in Figure 4.
本发明的一些方案中，上述B晶型的Cu Kα辐射的XRPD图谱中，衍射峰的峰位置及相对强度由表2所示：In some solutions of the present invention, in the XRPD pattern of Cu Kα radiation of the above-mentioned B crystal form, the peak position and relative intensity of the diffraction peak are shown in Table 2:
表2式(I)化合物B晶型的XRPD衍射数据

Table 2 XRPD diffraction data of compound B crystal form of formula (I)

本发明的一些方案中，上述B晶型的差示扫描量热(DSC)曲线显示在154.2℃±3℃处具有吸热峰的峰值。In some aspects of the present invention, the differential scanning calorimetry (DSC) curve of the above-mentioned B crystal form shows an endothermic peak at 154.2°C ± 3°C.
本发明的一些方案中，上述B晶型的DSC图谱如图5所示。In some aspects of the present invention, the DSC spectrum of the above crystal form B is shown in Figure 5.
本发明的一些方案中，上述B晶型的DSC图谱基本上如图5所示。In some aspects of the present invention, the DSC spectrum of the above crystal form B is basically as shown in Figure 5.
本发明的一些方案中，上述B晶型的热重分析(TGA)曲线在150.0℃±3℃时失重4.82％。In some aspects of the present invention, the thermogravimetric analysis (TGA) curve of the above-mentioned B crystal form loses 4.82% weight at 150.0°C ± 3°C.
本发明的一些方案中，上述B晶型的TGA图谱如图6所示。In some aspects of the present invention, the TGA spectrum of the above-mentioned B crystal form is shown in Figure 6.
本发明的一些方案中，上述B晶型的TGA图谱基本上如图6所示。In some aspects of the present invention, the TGA spectrum of the above crystal form B is basically as shown in Figure 6.
本发明还提供了式(I)化合物的B晶型，
The present invention also provides the B crystal form of the compound of formula (I),
其制备方法包括如下步骤：Its preparation method includes the following steps:
(a)将化合物Z加入溶剂M中；(a) Add compound Z to solvent M;
(b)在40-60℃下搅拌1～12小时；(b) Stir at 40-60°C for 1 to 12 hours;
(c)冷却至10-30℃，加入溶剂N，继续搅拌1～12小时；(c) Cool to 10-30°C, add solvent N, and continue stirring for 1 to 12 hours;
(d)过滤，滤饼在25～60℃下真空干燥1～24小时；(d) Filter, and vacuum dry the filter cake at 25-60°C for 1-24 hours;
其中，in,
化合物Z选自式(I)化合物；Compound Z is selected from compounds of formula (I);
溶剂M为甲苯；Solvent M is toluene;
溶剂N不存在，或者，溶剂N选自正庚烷、正己烷和环己烷；优选的，溶剂N为正庚烷；Solvent N does not exist, or solvent N is selected from n-heptane, n-hexane and cyclohexane; preferably, solvent N is n-heptane;
当溶剂N选自正庚烷、正己烷和环己烷时，溶剂M和溶剂N的体积比为1:0.5～1:3。When the solvent N is selected from n-heptane, n-hexane and cyclohexane, the volume ratio of the solvent M and the solvent N is 1:0.5~1:3.
本发明还提供了式(I)化合物的B晶型的制备方法，包括如下步骤：The invention also provides a method for preparing the B crystal form of the compound of formula (I), which includes the following steps:
(a)将化合物Z加入溶剂M中；(a) Add compound Z to solvent M;
(b)在40-60℃下搅拌1～12小时；(b) Stir at 40-60°C for 1 to 12 hours;
(c)冷却至10-30℃，加入溶剂N，继续搅拌1～12小时；(c) Cool to 10-30°C, add solvent N, and continue stirring for 1 to 12 hours;
(d)过滤，滤饼在25～60℃下真空干燥1～24小时；(d) Filter, and vacuum dry the filter cake at 25-60°C for 1-24 hours;
其中，in,
化合物Z选自式(I)化合物；Compound Z is selected from compounds of formula (I);
溶剂M为甲苯；Solvent M is toluene;
溶剂N不存在，或者，溶剂N选自正庚烷、正己烷和环己烷。Solvent N is absent or is selected from n-heptane, n-hexane and cyclohexane.
本发明的一些方案中，上述步骤(b)为在50-60℃下搅拌1～5小时。In some aspects of the present invention, the above step (b) is stirring at 50-60°C for 1 to 5 hours.
本发明的一些方案中，上述溶剂N选自正庚烷、正己烷和环己烷，溶剂M和溶剂N的体积比为1:0.5～1:3。In some aspects of the present invention, the above-mentioned solvent N is selected from n-heptane, n-hexane and cyclohexane, and the volume ratio of solvent M to solvent N is 1:0.5-1:3.
本发明的一些方案中，上述溶剂N为正庚烷。In some aspects of the present invention, the above-mentioned solvent N is n-heptane.
本发明的一些方案中，上述溶剂M和溶剂N的体积比为1:1。In some aspects of the present invention, the volume ratio of the above solvent M and solvent N is 1:1.
本发明的一些方案中，上述溶剂M和溶剂N的总体积与化合物Z的质量比为1:15～1:30。In some aspects of the present invention, the mass ratio of the total volume of the above-mentioned solvent M and solvent N to compound Z is 1:15 to 1:30.
本发明的一些方案中，上述溶剂M和溶剂N的总体积与化合物Z的质量比为1:20。In some aspects of the present invention, the mass ratio of the total volume of the above-mentioned solvent M and solvent N to compound Z is 1:20.
本发明还提供了式(I)化合物的水合物的C晶型，
The present invention also provides the C crystal form of the hydrate of the compound of formula (I),
所述C晶型的Cu Kα辐射的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：5.85±0.20°，11.70±0.20°，17.59±0.20°。The X-ray powder diffraction pattern of Cu Kα radiation of the C crystal form has characteristic diffraction peaks at the following 2θ angles: 5.85±0.20°, 11.70±0.20°, 17.59±0.20°.
本发明的一些方案中，上述C晶型的Cu Kα辐射的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：5.85°，11.70°，17.59°。In some aspects of the present invention, the X-ray powder diffraction pattern of Cu Kα radiation of the above-mentioned C crystal form has characteristic diffraction peaks at the following 2θ angles: 5.85°, 11.70°, 17.59°.
本发明的一些方案中，上述C晶型的X-射线粉末衍射图谱如图7所示。In some aspects of the present invention, the X-ray powder diffraction pattern of the above-mentioned C crystal form is shown in Figure 7.
本发明的一些方案中，上述C晶型的X-射线粉末衍射图谱基本上如图7所示。In some aspects of the present invention, the X-ray powder diffraction pattern of the above-mentioned C crystal form is basically as shown in Figure 7.
本发明的一些方案中，上述C晶型的Cu Kα辐射的XRPD图谱中，衍射峰的峰位置及相对强度由表3所示：In some solutions of the present invention, in the XRPD pattern of Cu Kα radiation of the above-mentioned C crystal form, the peak position and relative intensity of the diffraction peak are shown in Table 3:
表3式(I)化合物的水合物C晶型的XRPD衍射数据
Table 3 XRPD diffraction data of hydrate crystal form C of the compound of formula (I)
本发明的一些方案中，上述C晶型的热重分析(TGA)曲线在150.0℃±3℃时失重12.61％。In some aspects of the present invention, the thermogravimetric analysis (TGA) curve of the above-mentioned C crystal form loses 12.61% of weight at 150.0°C ± 3°C.
本发明的一些方案中，上述C晶型的TGA图谱如图8所示。In some aspects of the present invention, the TGA spectrum of the above crystal form C is shown in Figure 8.
本发明的一些方案中，上述C晶型的TGA图谱基本上如图8所示。In some aspects of the present invention, the TGA spectrum of the above crystal form C is basically as shown in Figure 8.
本发明所述式(I)化合物的A晶型、B晶型、C晶型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。The A crystal form, B crystal form, and C crystal form of the compound of formula (I) of the present invention may be in the form of a nonsolvate or a solvate, such as a hydrate.
本发明还提供了上述化合物的A晶型、B晶型、C晶型或通过其制备方法得到的晶型在制备治疗流感的药物中的应用。The present invention also provides the use of the A crystal form, B crystal form, C crystal form or the crystal form obtained by its preparation method in the preparation of drugs for treating influenza.
本发明还提供了上述化合物的A晶型或通过其制备方法得到的晶型在制备治疗流感的药物中的应用。技术效果The present invention also provides the use of the A crystal form of the above compound or the crystal form obtained by its preparation method in the preparation of drugs for treating influenza. Technical effect
本发明化合物作为一种RNA聚合酶抑制剂，在细胞水平抑制流感病毒复制试验中展示出积极效应，在动物体内药效模型中表现出优异的体重保护，并且恢复时间早，血浆蛋白结合率测试结果显示本发明化合物在血浆中具中等的血浆蛋白结合率，PK结果显示具有良好的药代动力学性质，具有良好的成药性质。本发明化合物的晶型制备工艺简单，并且晶型稳定，便于制剂，在预防和/或治疗流感中具有较大的应用前景。As an RNA polymerase inhibitor, the compound of the present invention shows positive effects in inhibition of influenza virus replication tests at the cellular level, excellent body weight protection in animal efficacy models, and early recovery time. Plasma protein binding rate test The results show that the compound of the present invention has a moderate plasma protein binding rate in plasma, and the PK results show that it has good pharmacokinetic properties and good pharmaceutical properties. The crystal form of the compound of the present invention has a simple preparation process, is stable, and is convenient for preparation, and has great application prospects in preventing and/or treating influenza.
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 particular phrase or term should not be considered uncertain or unclear in the absence of a specific definition, but should be understood in its ordinary meaning. When a trade name appears herein, it is intended to refer to its corresponding trade name 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, embodiments formed by combining them with other chemical synthesis methods, and those skilled in the art. Well-known equivalents and preferred embodiments 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 reactions of the specific embodiments of the present invention are completed in a suitable solvent, and the solvent must be suitable for the chemical changes of the present invention and the required reagents and materials. In order to obtain the compounds of the present invention, those skilled in the art sometimes need to modify or select the synthesis steps or reaction procedures based on the existing embodiments.
除非另有说明，本发明化合物的差示扫描量热曲线中，向上表示放热(Exo Up)。Unless otherwise stated, in the differential scanning calorimetry curves of the compounds of the present invention, upward indicates exotherm (Exo Up).
除非另有说明，在粉末X-射线衍射光谱(XRPD)中，峰的位置或峰的相对强度可能会因为测定仪器、测定方法/条件等因素而产生差异。对任何特定的晶型，峰的位置可能存在误差，2θ值的测定误差可以为±0.2°。因此，在确定每种晶型时，应该将此误差考虑在内，在误差内也属于本申请的范围。Unless otherwise stated, in powder X-ray diffraction spectrum (XRPD), the position of the peak or the relative intensity of the peak may vary due to factors such as the measuring instrument, measuring method/conditions, etc. For any particular crystal form, there may be errors in the position of the peak, and the error in the determination of the 2θ value can be ±0.2°. Therefore, this error should be taken into account when determining each crystal form, and it is within the scope of this application.
本发明中表述“基本上如……所示”的特定XRPD图谱是指XRPD图谱中衍射峰的位置在目视检查或借助于被选择的衍射峰列表(±0.20°，2θ)的范围内基本上相同。普通技术人员理解，强度可以随着样品而改变。In the present invention, the expression "substantially as shown" in a specific XRPD pattern means that the position of the diffraction peak in the XRPD pattern is basically within the range of visual inspection or with the help of a selected diffraction peak list (±0.20°, 2θ). Same as above. One of ordinary skill understands that the intensity may vary from sample to sample.
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。The present invention will be described in detail through examples below. These examples do not mean any limitation to the present invention.
本发明的化合物可以通过本领域技术人员所熟知的常规方法来确认结构，如果本发明涉及化合物的绝对构型，则该绝对构型可以通过本领域常规技术手段予以确证。例如单晶X射线衍射法(SXRD)，把培养出的单晶用Bruker D8venture衍射仪收集衍射强度数据，光源为CuKα辐射，扫描方式：φ/ω扫描，收集相关数据后，进一步采用直接法(Shelxs97)解析晶体结构，便可以确证绝对构型。The structure of the compound of the present invention can be confirmed by conventional methods well known to those skilled in the art. If the present invention involves the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art. For example, in single crystal X-ray diffraction (SXRD), the cultured single crystal is used to collect diffraction intensity data using a Bruker D8venture diffractometer. The light source is CuKα radiation, and the scanning mode is: φ/ω scanning. After collecting relevant data, the direct method is further used ( Shelxs97) can confirm the absolute configuration by analyzing the crystal structure.
本发明采用下述缩略词：ACN代表乙腈；DMSO代表二甲基亚砜。N2：氮气；RH：相对湿度；mL：毫升；L：升；min：分钟；℃：摄氏度；μm：微米；mm：毫米；μL：微升；moL/L：摩尔每升；mg：毫克；s：秒；nm：纳米；MPa：兆帕；lux：勒克斯；μw/cm2：微瓦每平方厘米；h:小时；Kg：千克；nM：纳摩尔，rpm：转速；XRPD代表X射线粉末衍射；DSC代表差示扫描量热分析；TGA代表热重分析；1H NMR代表核磁共振氢谱。The following abbreviations are used in the present invention: ACN represents acetonitrile; DMSO represents dimethyl sulfoxide. N 2 : nitrogen; RH: relative humidity; mL: milliliter; L: liter; min: minutes; ℃: degrees Celsius; μm: micrometers; mm: millimeters; μL: microliters; moL/L: moles per liter; mg: milligrams ;s: seconds; nm: nanometers; MPa: megapascals; lux: lux; μw/cm 2 : microwatts per square centimeter; h: hours; Kg: kilograms; nM: nanomoles, rpm: rotational speed; XRPD represents X-rays Powder diffraction; DSC stands for differential scanning calorimetry; TGA stands for thermogravimetric analysis; 1 H NMR stands for hydrogen nuclear magnetic resonance spectroscopy.
本发明化合物依据本领域常规命名原则或者使用软件命名，市售化合物采用供应商目录名称，本发明所使用的所有溶剂是市售可得的。The compounds of the present invention are named according to the conventional naming principles in this field or used Software nomenclature, commercially available compounds using supplier catalog names, all solvents used in this invention are commercially available.
本发明仪器及分析方法Instrument and analysis method of the present invention
1.1X-射线粉末衍射(X-ray powder diffractometer,XRPD)方法1.1X-ray powder diffraction (X-ray powder diffractometer, XRPD) method
大约10mg样品用于XRPD检测，详细的仪器信息和参数如表4所示：About 10 mg of sample was used for XRPD detection. Detailed instrument information and parameters are shown in Table 4:
表4 XRPD仪器信息和参数
Table 4 XRPD instrument information and parameters
1.2差式扫描量热法(Differential Scanning Calorimeter,DSC)和热重分析(Thermal Gravimetric Analyzer,TGA)1.2 Differential Scanning Calorimeter (DSC) and Thermal Gravimetric Analyzer (TGA)
详细的参数如下表5所示：The detailed parameters are shown in Table 5 below:
表5 DSC和TGA仪器参数
Table 5 DSC and TGA instrument parameters
1.3动态水分吸附分析(Dynamic Vapor Sorption,DVS)方法1.3 Dynamic Vapor Sorption (DVS) method
详细的仪器信息和参数如下表6所示：Detailed instrument information and parameters are shown in Table 6 below:
表6 DVS仪器参数
Table 6 DVS instrument parameters
引湿性评价分类如下：The hygroscopicity evaluation categories are as follows:
吸收足量水分形成液体：潮解；ΔW％≥15％：极具吸湿性；15％>ΔW％≥2％：有吸湿性；2％>ΔW％≥0.2％：略有吸湿性；ΔW％<0.2％：无或几乎无吸湿性。ΔW％表示受试品在25±1℃和80±2％RH下的吸湿增重。Absorb enough water to form a liquid: deliquescence; ΔW% ≥ 15%: extremely hygroscopic; 15% > ΔW% ≥ 2%: hygroscopic; 2% > ΔW% ≥ 0.2%: slightly hygroscopic; ΔW% < 0.2%: No or almost no hygroscopicity. ΔW% represents the moisture absorption weight gain of the test product at 25±1℃ and 80±2%RH.
附图说明Description of the drawings
图1为式(I)化合物A晶型的XRPD谱图。Figure 1 is the XRPD spectrum of the crystal form A of the compound 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 A of the compound 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 crystalline form A of compound (I).
图4为式(I)化合物B晶型的XRPD谱图。Figure 4 is the XRPD spectrum of the crystal form B of the compound of formula (I).
图5为式(I)化合物B晶型的DSC谱图。Figure 5 is a DSC spectrum of the crystal form B of the compound of formula (I).
图6为式(I)化合物B晶型的TGA谱图。Figure 6 is a TGA spectrum of the crystal form B of the compound of formula (I).
图7为式(I)化合物的水合物C晶型的XRPD谱图。Figure 7 is the XRPD spectrum of the hydrate crystal form C of the compound of formula (I).
图8为式(I)化合物的水合物C晶型的TGA谱图。Figure 8 is a TGA spectrum of the hydrate C crystal form of the compound of formula (I).
图9为式(I)化合物A晶型的DVS谱图。Figure 9 is the DVS spectrum of the crystalline form A of the compound of formula (I).
图10为式(I)化合物单晶椭球图。Figure 10 is an ellipsoid diagram of a single crystal of the compound of formula (I).
具体实施方式 Detailed ways
下面通过实施例对本发明进行详细描述，但并不意味着对本发明任何不利限制。本文已经详细地描述了本发明，其中也公开了其具体实施例方式，对本领域的技术人员而言，在不脱离本发明精神和范围的情况下针对本发明具体实施方式进行各种变化和改进将是显而易见的。The present invention is described in detail below through examples, which do not mean any adverse limitations to the present invention. The present invention has been described in detail herein, and its specific embodiments are also disclosed. For those skilled in the art, various changes and improvements can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention. will be obvious.
实施例1：式(I)化合物的制备
Example 1: Preparation of compounds of formula (I)
步骤1：化合物1-2的合成Step 1: Synthesis of Compound 1-2
将化合物1-1(66g,383.43mmol)溶于二氯甲烷(460mL)中，加入N,N-二甲基甲酰胺(280.27mg,3.83mmol,295.02μL)，向反应液中滴加草酰氯(73.00g,575.15mmol,50.35mL)，滴完后反应液在20℃下搅拌30分钟，然后减压浓缩。所得粗品加入二氯甲烷(460mL)，搅拌下加入三乙胺(77.60g,766.87mmol,106.74mL)和N,O-二甲基羟胺盐酸盐(37.40g,383.43mmol)，反应液在20℃下搅拌1小时。加入水(100mL)，分液，水相用二氯甲烷(50mL×2)萃取。合并有机相，分别用稀盐酸(0.2M，50mL)，饱和碳酸氢钠水溶液(50mL)和饱和食盐水(50mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，得化合物1-2。1H NMR(400MHz,氘代氯仿)δ7.02-7.05(m,2H),3.81(brs,3H),3.49(s,3H),2.28(s,3H)。Dissolve compound 1-1 (66g, 383.43mmol) in dichloromethane (460mL), add N,N-dimethylformamide (280.27mg, 3.83mmol, 295.02μL), and add oxalyl chloride dropwise to the reaction solution (73.00g, 575.15mmol, 50.35mL). After the dripping was completed, the reaction solution was stirred at 20°C for 30 minutes and then concentrated under reduced pressure. The obtained crude product was added to dichloromethane (460 mL), and triethylamine (77.60g, 766.87mmol, 106.74mL) and N,O-dimethylhydroxylamine hydrochloride (37.40g, 383.43mmol) were added under stirring. The reaction solution was heated at 20 Stir for 1 hour at ℃. Water (100 mL) was added, the liquid was separated, and the aqueous phase was extracted with dichloromethane (50 mL × 2). Combine the organic phases, wash with dilute hydrochloric acid (0.2M, 50mL), saturated aqueous sodium bicarbonate solution (50mL) and saturated brine (50mL), dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain compound 1-2 . 1 H NMR (400MHz, deuterated chloroform) δ7.02-7.05 (m, 2H), 3.81 (brs, 3H), 3.49 (s, 3H), 2.28 (s, 3H).
步骤2：化合物1-3的合成Step 2: Synthesis of Compounds 1-3
将化合物1-2(20g,92.94mmol)溶于四氢呋喃(200mL)中，在0℃下滴加甲基溴化镁(3M,37.18mL)，滴完后反应液升温至20℃搅拌2小时。反应液用1M盐酸淬灭，调节pH至7，用乙酸乙酯(100mL×2)萃取，合并有机相，分别用稀盐酸(0.2M，50mL)，饱和碳酸氢钠溶液(50mL)，饱和食盐水(50mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩得化合物1-3。1H NMR(400MHz,氘代氯仿)δ7.47-7.50(m,1H),7.03-7.07(m,1H),2.60(s,3H),2.47(s,3H)。Compound 1-2 (20g, 92.94mmol) was dissolved in tetrahydrofuran (200mL), methylmagnesium bromide (3M, 37.18mL) was added dropwise at 0°C. After the dripping was completed, the reaction solution was heated to 20°C and stirred for 2 hours. The reaction solution was quenched with 1M hydrochloric acid, adjusted to pH 7, extracted with ethyl acetate (100mL×2), the organic phases were combined, and diluted hydrochloric acid (0.2M, 50mL), saturated sodium bicarbonate solution (50mL), and saturated salt were used. Wash with water (50 mL), dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain compound 1-3. 1 H NMR (400MHz, deuterated chloroform) δ7.47-7.50(m,1H),7.03-7.07(m,1H),2.60(s,3H),2.47(s,3H).
步骤3：化合物1-4的合成Step 3: Synthesis of Compounds 1-4
将化合物1-3(15g,88.15mmol)溶于吡啶(90mL)中，加入二氧化硒(19.56g,176.31mmol)，反应液 在110℃下搅拌12小时。将反应液冷却至室温，过滤，减压浓缩。粗品中加入水(50mL)，用1M盐酸调pH至4，用乙酸乙酯(50mL×3)萃取，合并有机相，用饱和食盐水(50mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩得化合物1-4。1H NMR(400MHz,氘代甲醇)δ7.64-7.68(m,1H),7.30-7.32(m,1H),2.52(s,3H)。Compound 1-3 (15g, 88.15mmol) was dissolved in pyridine (90mL), selenium dioxide (19.56g, 176.31mmol) was added, and the reaction solution Stir at 110°C for 12 hours. The reaction solution was cooled to room temperature, filtered, and concentrated under reduced pressure. Add water (50 mL) to the crude product, adjust the pH to 4 with 1M hydrochloric acid, extract with ethyl acetate (50 mL × 3), combine the organic phases, wash with saturated brine (50 mL), dry over anhydrous sodium sulfate, filter, and reduce the filtrate to Concentrate under pressure to obtain compound 1-4. 1 H NMR (400MHz, deuterated methanol) δ7.64-7.68 (m, 1H), 7.30-7.32 (m, 1H), 2.52 (s, 3H).
步骤4：化合物1-5的合成Step 4: Synthesis of Compounds 1-5
将化合物1-4(15g,74.95mmol)溶于二氯甲烷(60mL)和甲醇(60mL)中，控温0～20℃，滴加三甲基硅基重氮甲烷(2M,44.97mL)，反应液在20℃下搅拌2小时，然后加入乙酸(3mL)，搅拌5分钟。将反应液减压浓缩，加入水(50mL)，用二氯甲烷(50mL×2)萃取，合并有机相，用饱和食盐水(30mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩。所得粗品经硅胶柱纯化(乙酸乙酯/石油醚，乙酸乙酯比例：0～20％)得化合物1-5。1H NMR(400MHz,氘代氯仿)δ7.51-7.55(m,1H),7.12-7.16(m,1H),3.98(s,3H),2.54(s,3H)。Dissolve compound 1-4 (15g, 74.95mmol) in dichloromethane (60mL) and methanol (60mL), control the temperature from 0 to 20°C, and add trimethylsilyldiazomethane (2M, 44.97mL) dropwise. The reaction solution was stirred at 20°C for 2 hours, then acetic acid (3 mL) was added and stirred for 5 minutes. The reaction solution was concentrated under reduced pressure, added with water (50 mL), extracted with dichloromethane (50 mL × 2), combined the organic phases, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was purified through silica gel column (ethyl acetate/petroleum ether, ethyl acetate ratio: 0-20%) to obtain compound 1-5. 1 H NMR (400MHz, deuterated chloroform) δ7.51-7.55(m,1H),7.12-7.16(m,1H),3.98(s,3H),2.54(s,3H).
步骤5：化合物1-6的合成Step 5: Synthesis of Compounds 1-6
将化合物1-5(5g,23.35mmol)溶于1,2-二氯乙烷(50mL)中，加入N-溴代丁二酰亚胺(8.31g,46.69mmol)和偶氮二异丁腈(383.37mg,2.33mmol)，反应液在80℃下搅拌12小时。将反应液冷却至室温，分别用饱和亚硫酸钠溶液(20mL)，水(20mL)和饱和食盐水(20mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩。所得粗品经硅胶柱纯化(乙酸乙酯/石油醚，乙酸乙酯比例：0～5％)得化合物1-6。1H NMR(400MHz,氘代氯仿)δ7.61-7.64(m,1H),7.26-7.31(m,1H),4.94(s,2H),3.99(s,3H)。Compound 1-5 (5g, 23.35mmol) was dissolved in 1,2-dichloroethane (50mL), and N-bromosuccinimide (8.31g, 46.69mmol) and azobisisobutyronitrile were added (383.37mg, 2.33mmol), the reaction solution was stirred at 80°C for 12 hours. The reaction solution was cooled to room temperature, washed with saturated sodium sulfite solution (20 mL), water (20 mL) and saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified through silica gel column (ethyl acetate/petroleum ether, ethyl acetate ratio: 0-5%) to obtain compound 1-6. 1 H NMR (400MHz, deuterated chloroform) δ7.61-7.64(m,1H),7.26-7.31(m,1H),4.94(s,2H),3.99(s,3H).
步骤6：化合物1-7的合成Step 6: Synthesis of Compounds 1-7
将磷酸二氢钠(11.52g,96.05mmol)溶于水(60mL)中，然后加入乙腈(30mL)，加入3-(3-吡啶基二硒基)吡啶(3.62g,11.53mmol)，分批加入锌粉(1.88g,28.82mmol)，反应液在20℃下搅拌30分钟。加入化合物1-6(5.63g,19.21mmol)，反应液在20℃下搅拌3小时。将反应液过滤，滤液用乙酸乙酯(30mL×2)萃取。合并有机相，用饱和食盐水(30mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩。所得粗品经硅胶柱纯化(乙酸乙酯/石油醚，乙酸乙酯比例：0～60％)得化合物1-7。MS(ESI)m/z:373.8[M+H]+。Dissolve sodium dihydrogen phosphate (11.52g, 96.05mmol) in water (60mL), then add acetonitrile (30mL), and add 3-(3-pyridyldiselenyl)pyridine (3.62g, 11.53mmol) in batches Zinc powder (1.88g, 28.82mmol) was added, and the reaction solution was stirred at 20°C for 30 minutes. Compound 1-6 (5.63g, 19.21mmol) was added, and the reaction solution was stirred at 20°C for 3 hours. The reaction liquid was filtered, and the filtrate was extracted with ethyl acetate (30 mL×2). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was purified through silica gel column (ethyl acetate/petroleum ether, ethyl acetate ratio: 0-60%) to obtain compound 1-7. MS(ESI)m/z:373.8[M+H] + .
步骤7：化合物1-8的合成Step 7: Synthesis of Compounds 1-8
将化合物1-7(4.2g,11.28mmol)溶于二氯甲烷(80mL)中，加入戴斯-马丁过碘烷(7.18g,16.93mmol)，反应液在20℃下搅拌12小时。反应液中加入饱和亚硫酸钠溶液(30mL)，搅拌5分钟。用二氯甲烷(30mL×2)萃取，合并有机相，用饱和食盐水(30mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩。所得粗品经硅胶柱纯化(乙酸乙酯/石油醚，乙酸乙酯比例：0～50％)得化合物1-8。MS(ESI)m/z:371.9[M+H]+。Compound 1-7 (4.2g, 11.28mmol) was dissolved in dichloromethane (80mL), Dess-Martin periodane (7.18g, 16.93mmol) was added, and the reaction solution was stirred at 20°C for 12 hours. Saturated sodium sulfite solution (30 mL) was added to the reaction solution, and stirred for 5 minutes. Extract with dichloromethane (30 mL×2), combine the organic phases, wash with saturated brine (30 mL), dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. The crude product was purified through silica gel column (ethyl acetate/petroleum ether, ethyl acetate ratio: 0-50%) to obtain compound 1-8. MS(ESI)m/z:371.9[M+H] + .
步骤8：化合物1-9的合成Step 8: Synthesis of Compounds 1-9
将化合物1-8(2.9g,7.83mmol)溶于四氢呋喃(16mL)中，加入氢氧化钠水溶液(626.67mg,15.67mmol，4mL)，反应液在20℃下搅拌1小时。减压浓缩除去大部分四氢呋喃，水相用1N盐酸调节pH至6，固体过滤，滤饼减压干燥得化合物1-9。MS(ESI)m/z:357.9[M+H]+。Compound 1-8 (2.9g, 7.83mmol) was dissolved in tetrahydrofuran (16mL), aqueous sodium hydroxide solution (626.67mg, 15.67mmol, 4mL) was added, and the reaction solution was stirred at 20°C for 1 hour. Concentrate under reduced pressure to remove most of the tetrahydrofuran, adjust the pH of the aqueous phase to 6 with 1N hydrochloric acid, filter the solid, and dry the filter cake under reduced pressure to obtain compound 1-9. MS(ESI)m/z:357.9[M+H] + .
步骤9：化合物1-10的合成Step 9: Synthesis of Compounds 1-10
将化合物1-9(2.3g,6.46mmol)溶于二甲基亚砜(23mL)中，分别加入过硫酸铵(2.95g,12.91mmol)，硝酸银(109.69mg,645.74μmol)和浓硫酸(633.34mg,6.46mmol)，反应液在50℃下搅拌3小时。向反应液中分别加入饱和碳酸氢钠水溶液(20mL)，水(10mL)和二氯甲烷(20mL)，搅拌5分钟，过滤，滤液分液，水相用二氯甲烷(10mL)萃取。合并有机相，用饱和食盐水(30mL×3)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩。所得粗品经硅胶柱纯化(乙酸乙酯/石油醚，乙酸乙酯比例：0～50％)得化合物1-10。MS(ESI)m/z:311.8[M+H]+。Compound 1-9 (2.3g, 6.46mmol) was dissolved in dimethyl sulfoxide (23mL), and ammonium persulfate (2.95g, 12.91mmol), silver nitrate (109.69mg, 645.74μmol) and concentrated sulfuric acid ( 633.34 mg, 6.46 mmol), the reaction solution was stirred at 50°C for 3 hours. Saturated sodium bicarbonate aqueous solution (20 mL), water (10 mL) and dichloromethane (20 mL) were added to the reaction solution, stirred for 5 minutes, filtered, the filtrate was separated, and the aqueous phase was extracted with dichloromethane (10 mL). The organic phases were combined, washed with saturated brine (30 mL × 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was purified through silica gel column (ethyl acetate/petroleum ether, ethyl acetate ratio: 0-50%) to obtain compound 1-10. MS(ESI)m/z:311.8[M+H] + .
步骤10：化合物1-11的合成Step 10: Synthesis of Compounds 1-11
将化合物1-10(390mg,1.26mmol)溶于异丙醇(8mL)中，加入硼氢化钠(95.14mg,2.51mmol)，反应液在20℃下搅拌1小时。加入1N盐酸调节pH至7，用乙酸乙酯(15mL×2)萃取，合并有机相，用 饱和食盐水(10mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩。所得粗品经硅胶柱纯化(乙酸乙酯/石油醚，乙酸乙酯比例：0～50％)得化合物1-11。MS(ESI)m/z:313.8[M+H]+。Compound 1-10 (390 mg, 1.26 mmol) was dissolved in isopropanol (8 mL), sodium borohydride (95.14 mg, 2.51 mmol) was added, and the reaction solution was stirred at 20°C for 1 hour. Add 1N hydrochloric acid to adjust the pH to 7, extract with ethyl acetate (15mL×2), combine the organic phases, and use Wash with saturated brine (10 mL), dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. The crude product was purified through silica gel column (ethyl acetate/petroleum ether, ethyl acetate ratio: 0-50%) to obtain compound 1-11. MS(ESI)m/z:313.8[M+H] + .
步骤11：化合物1-12的合成Step 11: Synthesis of Compounds 1-12
将化合物1-11(330mg,1.06mmol)溶于二氯甲烷(6mL)中，加入二氯亚砜(251.53mg,2.11mmol,153.37μL)，反应液在20℃下搅拌1小时。将反应液减压浓缩得化合物1-12，粗品直接用于下一步反应。Compound 1-11 (330 mg, 1.06 mmol) was dissolved in dichloromethane (6 mL), thionyl chloride (251.53 mg, 2.11 mmol, 153.37 μL) was added, and the reaction solution was stirred at 20°C for 1 hour. The reaction solution was concentrated under reduced pressure to obtain compound 1-12, and the crude product was directly used in the next reaction.
步骤12：化合物1-14的合成Step 12: Synthesis of Compounds 1-14
将化合物1-13(340mg,1.04mmol)溶于乙腈(6mL)中，加入化合物1-12(343.41mg,1.04mmol)和碳酸铯(676.86mg,2.08mmol)，反应液在60℃下搅拌12小时。将反应液冷却到室温，加入水(5mL)，用乙酸乙酯(5mL×3)萃取，合并有机相，用饱和食盐水(5mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩。所得粗品经硅胶柱纯化(甲醇/二氯甲烷，甲醇比例：0～5％)，所得化合物经手性分离(柱子型号:DAICEL CHIRALPAK AD(250mm*30mm,10μm)；流动相:[A：二氧化碳，B：0.1％氨水/乙醇]；梯度：流动相B保持40％)得到化合物1-14(分析方法：柱子型号:Chiralpak AD-3(50mm*4.6mm,3μm)；流动相:[A：二氧化碳，B：0.05％二乙胺/乙醇]；梯度：流动相B浓度在2分钟内从5％增加至40％，40％保持1.2分钟，然后5％保持0.8分钟，化合物1-14的保留时间1.831min，ee＝96.1％)。Compound 1-13 (340 mg, 1.04 mmol) was dissolved in acetonitrile (6 mL), compound 1-12 (343.41 mg, 1.04 mmol) and cesium carbonate (676.86 mg, 2.08 mmol) were added, and the reaction solution was stirred at 60°C for 12 Hour. The reaction solution was cooled to room temperature, water (5 mL) was added, extracted with ethyl acetate (5 mL × 3), the organic phases were combined, washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was purified by silica gel column (methanol/dichloromethane, methanol ratio: 0~5%), and the obtained compound was chiral separated (column model: DAICEL CHIRALPAK AD (250mm*30mm, 10μm); mobile phase: [A: carbon dioxide, B: 0.1% ammonia/ethanol]; gradient: mobile phase B maintained at 40%) to obtain compound 1-14 (analysis method: column model: Chiralpak AD-3 (50mm*4.6mm, 3μm); mobile phase: [A: carbon dioxide , B: 0.05% diethylamine/ethanol]; gradient: mobile phase B concentration increased from 5% to 40% in 2 minutes, 40% held for 1.2 minutes, then 5% held for 0.8 minutes, retention time of compounds 1-14 1.831min, ee=96.1%).
步骤13：化合物1-15的合成Step 13: Synthesis of Compounds 1-15
将化合物1-14(6mg,9.65μmol)加入到N,N-二甲基乙酰胺(1mL)中，加入氯化锂(2.05mg,48.27μmol)，反应液在80℃下搅拌12小时。将反应液冷却到室温，用乙腈(1mL)稀释。粗品反应液经制备型高效液相色谱分离纯化(柱子:Phenomenex Gemini-NX C18 75*30mm*3μm；流动相:[A：水(0.225％甲酸)；B：乙腈]；梯度：乙腈％:30％-53％)得到化合物1-15。MS(ESI)m/z:533.1[M+H]+；1H NMR(400MHz,氘代甲醇)δ7.98-8.09(m,1H),7.70(dd,J＝1.51,8.03Hz,1H),7.41(d,J＝7.53Hz,1H),7.18-7.31(m,2H),7.13(dd,J＝4.52,8.03Hz,1H),5.75-5.88(m,2H),5.40-5.53(m,1H),4.71(dd,J＝3.01,10.04Hz,1H),4.63(br s,1H),4.17(d,J＝12.55Hz,1H),4.07(dd,J＝3.01,11.04Hz,1H),3.77(dd,J＝3.01,11.54Hz,1H),3.66(t,J＝10.54Hz,1H),3.48(dt,J＝2.51,11.80Hz,1H),3.04-3.18(m,1H)。Compound 1-14 (6 mg, 9.65 μmol) was added to N,N-dimethylacetamide (1 mL), lithium chloride (2.05 mg, 48.27 μmol) was added, and the reaction solution was stirred at 80°C for 12 hours. The reaction solution was cooled to room temperature and diluted with acetonitrile (1 mL). The crude reaction solution was separated and purified by preparative high performance liquid chromatography (column: Phenomenex Gemini-NX C18 75*30mm*3μm; mobile phase: [A: water (0.225% formic acid); B: acetonitrile]; gradient: acetonitrile%: 30 %-53%) to obtain compound 1-15. MS (ESI) m/z: 533.1 [M+H] + ; 1 H NMR (400MHz, deuterated methanol) δ7.98-8.09 (m, 1H), 7.70 (dd, J = 1.51, 8.03Hz, 1H) ,7.41(d,J＝7.53Hz,1H),7.18-7.31(m,2H),7.13(dd,J＝4.52,8.03Hz,1H),5.75-5.88(m,2H),5.40-5.53(m ,1H),4.71(dd,J＝3.01,10.04Hz,1H),4.63(br s,1H),4.17(d,J＝12.55Hz,1H),4.07(dd,J＝3.01,11.04Hz,1H ),3.77(dd,J＝3.01,11.54Hz,1H),3.66(t,J＝10.54Hz,1H),3.48(dt,J＝2.51,11.80Hz,1H),3.04-3.18(m,1H) .
步骤14：式(I)化合物的合成Step 14: Synthesis of compounds of formula (I)
将化合物1-15(130.00mg,244.65μmol)加入到N,N-二甲基乙酰胺(2mL)中，然后加入氯甲基碳酸甲酯(45.70mg,366.98μmol)，碳酸钾(67.63mg,489.30μmol)和碘化钾(40.61mg,244.65μmol)，反应液在70℃下搅拌3小时。反应液冷却到室温，加入水(10mL)，用乙酸乙酯(10mL×2)萃取，有机相用饱和食盐水(10mL×4)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩。所得粗品经硅胶柱(二氯甲烷:甲醇＝1:0至20:1)纯化得式(I)化合物。MS(ESI)m/z:621.0[M+H]+；1H NMR(400MHz,氘代氯仿)δ8.03(dd,J＝1.00,4.52Hz,1H),7.44(dd,J＝1.51,8.03Hz,1H),6.99-7.16(m,3H),6.95(dd,J＝4.52,8.03Hz,1H),5.90(d,J＝6.53Hz,1H),5.74-5.85(m,1H),5.22-5.35(m,3H),4.60(dd,J＝2.01,13.55Hz,1H),4.50(dd,J＝3.01,10.04Hz,1H),4.03(d,J＝12.55Hz,1H),3.95(dd,J＝3.01,11.04Hz,1H),3.77-3.83(m,3H),3.73(dd,J＝3.01,12.05Hz,1H),3.54(t,J＝10.54Hz,1H),3.41(dt,J＝2.51,11.80Hz,1H),2.85-2.97(m,1H)。Compound 1-15 (130.00 mg, 244.65 μmol) was added to N,N-dimethylacetamide (2 mL), then chloromethyl methyl carbonate (45.70 mg, 366.98 μmol), potassium carbonate (67.63 mg, 489.30 μmol) and potassium iodide (40.61 mg, 244.65 μmol). The reaction solution was stirred at 70°C for 3 hours. The reaction solution was cooled to room temperature, water (10 mL) was added, and extracted with ethyl acetate (10 mL × 2). The organic phase was washed with saturated brine (10 mL × 4), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained crude product was purified through a silica gel column (dichloromethane:methanol=1:0 to 20:1) to obtain the compound of formula (I). MS (ESI) m/z: 621.0[M+H] + ; 1 H NMR (400MHz, deuterated chloroform) δ8.03 (dd, J＝1.00, 4.52Hz, 1H), 7.44 (dd, J＝1.51, 8.03Hz,1H),6.99-7.16(m,3H),6.95(dd,J＝4.52,8.03Hz,1H),5.90(d,J＝6.53Hz,1H),5.74-5.85(m,1H), 5.22-5.35(m,3H),4.60(dd,J＝2.01,13.55Hz,1H),4.50(dd,J＝3.01,10.04Hz,1H),4.03(d,J＝12.55Hz,1H),3.95 (dd,J＝3.01,11.04Hz,1H),3.77-3.83(m,3H),3.73(dd,J＝3.01,12.05Hz,1H),3.54(t,J＝10.54Hz,1H),3.41( dt,J＝2.51,11.80Hz,1H),2.85-2.97(m,1H).
实施例2：式(I)化合物B晶型的制备
Example 2: Preparation of crystal form B of compound of formula (I)
20℃下，向式(I)化合物(59g)中加入甲苯(590mL)并在50℃下搅拌1小时，然后冷却至20℃，滴加正庚烷(590mL)，继续搅拌1小时。过滤，滤饼在45℃下真空干燥2小时，得到式(I)化合物B晶型。XRPD谱图如图4所示，DSC谱图如图5所示，TGA图谱如图6所示。Toluene (590 mL) was added to the compound of formula (I) (59 g) at 20° C. and stirred at 50° C. for 1 hour. Then, it was cooled to 20° C., n-heptane (590 mL) was added dropwise, and stirring was continued for 1 hour. Filter, and the filter cake is vacuum dried at 45°C for 2 hours to obtain the crystal form B of compound of formula (I). The XRPD spectrum is shown in Figure 4, the DSC spectrum is shown in Figure 5, and the TGA spectrum is shown in Figure 6.
实施例3：式(I)化合物A晶型的制备
Example 3: Preparation of crystal form A of compound of formula (I)
将式(I)化合物B晶型(约55g)加入乙酸乙酯(125mL)和正庚烷(375mL)的混合溶剂中并在20℃下搅拌12小时，过滤，滤饼在45℃下真空干燥2小时，得到式(I)化合物A晶型。XRPD谱图如图1所示，DSC谱图如图2所示，TGA图谱如图3所示。Add crystal form B (about 55g) of compound of formula (I) to a mixed solvent of ethyl acetate (125mL) and n-heptane (375mL) and stir at 20°C for 12 hours. Filter and dry the filter cake under vacuum at 45°C for 2 hours. hours, the crystal form A of the compound of formula (I) is obtained. The XRPD spectrum is shown in Figure 1, the DSC spectrum is shown in Figure 2, and the TGA spectrum is shown in Figure 3.
实施例4：式(I)化合物的水合物的C晶型的制备
Example 4: Preparation of Form C of the Hydrate of the Compound of Formula (I)
称取式(I)化合物约15毫克加入到甲醇/甲基叔丁基醚(体积比为1:9，0.5mL)中，悬浊液在室温下搅拌8天。离心，收集固体得到式(I)化合物的水合物C晶型。XRPD谱图如图7所示，TGA图谱如图8所示。Weigh about 15 mg of the compound of formula (I) and add it to methanol/methyl tert-butyl ether (volume ratio: 1:9, 0.5 mL), and the suspension is stirred at room temperature for 8 days. Centrifuge and collect the solid to obtain the hydrate crystal form C of the compound of formula (I). The XRPD spectrum is shown in Figure 7, and the TGA spectrum is shown in Figure 8.
实施例5：式(I)化合物的单晶X射线衍射检测分析
Example 5: Single crystal X-ray diffraction detection and analysis of the compound of formula (I)
使用正己烷气相扩散法培养单晶。称取式(I)化合物约5mg，溶于异丙醇(0.3mL)，用滤膜(0.45μM)过滤，滤液装入2mL样品瓶中，样品瓶用铝箔纸封口，然后用注射器扎3-5个小孔，置入密闭的棕色瓶中，棕色瓶中加入20mL正己烷，约0.3cm高，密闭后在20～25℃下静置2天，析出四方的柱状晶体。收集晶体，用单晶X射线衍射仪(SC-XRD)(D8-VENTURE)收集衍射强度数据。单晶数据显示， 可以确定式(I)化合物的绝对构型，该化合物的分子式为C26H22F2N4O7Se。式(I)化合物的立体结构椭球图见附图10。式(I)化合物单晶的晶体结构数据见表7。Single crystals were grown using n-hexane vapor diffusion method. Weigh about 5 mg of the compound of formula (I), dissolve it in isopropyl alcohol (0.3 mL), filter it with a filter membrane (0.45 μM), put the filtrate into a 2 mL sample bottle, seal the sample bottle with aluminum foil, and then pierce it with a syringe for 3- Make 5 small holes and put them into a sealed brown bottle. Add 20mL n-hexane to the brown bottle, about 0.3cm high. After sealing, let it stand for 2 days at 20-25°C to precipitate square columnar crystals. Crystals were collected and diffraction intensity data were collected using a single crystal X-ray diffractometer (SC-XRD) (D8-VENTURE). Single crystal data display, The absolute configuration of the compound of formula (I) can be determined, and the molecular formula of the compound is C 26 H 22 F 2 N 4 O 7 Se. The ellipsoid diagram of the three-dimensional structure of the compound of formula (I) is shown in Figure 10. The crystal structure data of the single crystal of the compound of formula (I) are shown in Table 7.
表7式(I)化合物单晶的晶体数据
Table 7 Crystal data of compound single crystal of formula (I)
实施例6：式(I)化合物A晶型的固体稳定性试验Example 6: Solid stability test of crystal form A of compound of formula (I)
依据《原料药与制剂稳定性试验指导原则》(中国药典2020版四部通则9001)，为评估式(I)化合物A晶型的固体稳定性，对A晶型进行了影响因素(高温、高湿及光照)、40℃/75％RH及25℃/60％RH条件的稳定性考察。将A晶型分别在高温(60℃，敞口)、高湿(25℃/92.5％RH，敞口)条件下各放置30天，按照ICH条件(可见光总照度达到1200000Lux·hrs、紫外光总照度达到200W·hrs/m2)敞口放置在可见光及紫外光下(遮光对照组样品同时放置并用锡箔纸包裹)，在40℃/75％RH(敞口)条件下放置1、2和3个月，在25℃/60％RH(敞口)条件下放置3个月。对所有稳定性样品进行了XRPD测试，以检测晶型的变化，结果如表8所示。According to the "Guiding Principles for Stability Testing of Raw Materials and Preparations" (Chinese Pharmacopoeia 2020 Edition Four General Chapters 9001), in order to evaluate the solid stability of the crystalline form A of the compound of formula (I), the influencing factors (high temperature, high humidity) of the crystalline form A were tested. and light), 40℃/75%RH and 25℃/60%RH conditions. Crystal form A was placed under high temperature (60°C, exposed) and high humidity (25°C/92.5%RH, exposed) conditions for 30 days respectively. According to ICH conditions (the total visible light illumination reached 1,200,000 Lux·hrs, the total ultraviolet light When the illumination reaches 200W·hrs/m 2 ), place it in the open under visible light and ultraviolet light (samples in the shading control group are placed at the same time and wrapped in tin foil), and placed 1, 2 and 3 under 40℃/75%RH (open) conditions. months, placed at 25℃/60%RH (exposure) for 3 months. XRPD testing was performed on all stability samples to detect changes in crystal form, and the results are shown in Table 8.
准确称取该晶型约10mg置于干燥洁净的玻璃瓶中，摊成薄薄一层，敞口放置于影响因素试验条件下和加速条件下。光照(可见光1200000Lux·hrs，紫外200W·hrs/m2)条件下放置的样品采用透明玻璃瓶，完全暴露，用于XRPD检测的样品单独放置。Accurately weigh about 10 mg of the crystal form and place it in a dry and clean glass bottle, spread it into a thin layer, and place it exposed under the conditions of the influencing factors test and acceleration conditions. The samples placed under illumination (visible light 1200000 Lux·hrs, ultraviolet 200W·hrs/m 2 ) conditions are in transparent glass bottles, which are completely exposed, and the samples used for XRPD detection are placed separately.
样品到时间点取出后，盖好盖子，使用封口膜密封，置于-20℃冰箱保存。配样时，将样品从冰箱取出，恢复至室温，加入25mL稀释液(乙腈/水，1:1，v/v)，使样品溶解，得浓度约为0.5mg/mL溶液，使用液相进行进样分析，检测结果与0天的初始检测结果进行比较，试验结果见下表8所示。After the sample is taken out at the time point, cover it, seal it with a sealing film, and store it in a -20°C refrigerator. When preparing the sample, take the sample out of the refrigerator, return it to room temperature, add 25 mL of diluent (acetonitrile/water, 1:1, v/v) to dissolve the sample, and obtain a solution with a concentration of approximately 0.5 mg/mL. Use the liquid phase. Inject samples for analysis, and compare the test results with the initial test results on day 0. The test results are shown in Table 8 below.
0天标准溶液的配制：称取该晶型约25mg于50mL容量瓶中，使用50％乙腈溶解并定容至刻度。 Preparation of 0-day standard solution: Weigh about 25 mg of this crystal form into a 50 mL volumetric flask, dissolve it in 50% acetonitrile and dilute to the mark.
同时，对所有稳定性样品进行了HPLC测试，具体结果汇总于表8，HPLC测试仪器和分析条件见表9和表10所示。At the same time, HPLC tests were conducted on all stability samples. The specific results are summarized in Table 8. The HPLC test instruments and analysis conditions are shown in Tables 9 and 10.
表8式(I)化合物A晶型的固体稳定性试验结果
Table 8 Solid stability test results of compound A crystal form of formula (I)
注*：式(I)化合物为前药，其在体内可转化成化合物1-15，在保存过程也会部分降解成化合物1-15，此处纯度的数据包含式(I)化合物和化合物1-15。Note*: Compound of formula (I) is a prodrug, which can be converted into compound 1-15 in the body, and will also be partially degraded into compound 1-15 during storage. The purity data here include compound of formula (I) and compound 1 -15.
表9 HPLC仪器信息及分析方法

Table 9 HPLC instrument information and analysis methods

表10化合物1-15检测HPLC仪器信息及分析方法
Table 10 HPLC instrument information and analysis methods for detection of compounds 1-15
结论：式(I)化合物A晶型在所有稳定性(高温，高湿，光照)条件下，晶型稳定且具有较好的化学稳定性。Conclusion: The crystal form of compound A of formula (I) is stable and has good chemical stability under all stability conditions (high temperature, high humidity, light).
实施例7：式(I)化合物A晶型的吸湿性研究Example 7: Study on hygroscopicity of crystal form A of compound of formula (I)
实验材料：SMS DVS intrinsic plus动态水分吸附仪Experimental materials: SMS DVS intrinsic plus dynamic moisture adsorption instrument
实验方法：取式(I)化合物A晶型(10～20mg)置于DVS样品盘内进行测试。Experimental method: Take the crystal form (10~20 mg) of compound A of formula (I) and place it in a DVS sample tray for testing.
实验结果：式(I)化合物A晶型在25℃/80％RH下的吸湿增重ΔW％＝1.37％，DVS谱图如图9所示。实验结论：式(I)化合物A晶型在25℃/80％RH下的吸湿增重2％>ΔW％≥0.2％，略有吸湿性。Experimental results: The hygroscopic weight gain ΔW% of the crystal form of compound A of formula (I) at 25°C/80% RH = 1.37%, and the DVS spectrum is shown in Figure 9. Experimental conclusion: The hygroscopic weight gain of the crystal form of compound A of formula (I) at 25°C/80%RH is 2%>ΔW%≥0.2%, which is slightly hygroscopic.
生物测试数据：Biological test data:
实验例1：流感病毒细胞病变(CPE)实验Experimental Example 1: Influenza Virus Cytopathy (CPE) Experiment
通过测定化合物的半数有效浓度(EC50)值来评价化合物对流感病毒(Inflμenza virus,IFV)的抗病毒活性。细胞病变实验被广泛用于测定化合物对病毒感染细胞的保护作用来反映化合物的抗病毒活性。The antiviral activity of the compound against influenza virus (IFV) was evaluated by measuring the half effective concentration (EC 50 ) value of the compound. Cytopathy assay is widely used to measure the protective effect of compounds on virus-infected cells to reflect the antiviral activity of compounds.
流感病毒CPE实验Influenza virus CPE experiment
将MDCK细胞以2,000细胞每孔的密度种入黑色384孔细胞培养板中，随后置于37℃，5％CO2培养箱中培养过夜。化合物由Echo555非接触式纳升级声波移液系统进行稀释并加入到细胞孔内(4倍倍比稀释，8个测试浓度点)。流感病毒A/PR/8/34(H1N1)株随后以每孔1-2 90％组织培养感染剂量(TCID90)加入细胞培养孔中，培养基中DMSO终浓度为0.5％。设置病毒对照孔(加入DMSO和病毒，不加化合物)，细胞对照孔(加入DMSO，不加化合物和病毒)和培养基对照孔(只有培养基，不含细胞)。化合物的细胞毒性测定和抗病毒活性测定平行进行，除了不加病毒，其它的实验条件和抗病毒活性实验一致。细胞板置于37℃，5％CO2培养箱中培养5天。培养5天后使用细胞活力检测试剂盒CCK8检测细胞活性。原始数据用于化合物抗病毒活性和细胞毒性计算。 MDCK cells were seeded into a black 384-well cell culture plate at a density of 2,000 cells per well, and then placed in a 37°C, 5% CO2 incubator overnight. Compounds were diluted by the Echo555 non-contact nanoliter sonic pipetting system and added to the cell wells (4-fold dilution, 8 test concentration points). Influenza virus A/PR/8/34 (H1N1) strain was then added to the cell culture wells at 1-2 90% tissue culture infectious dose (TCID90) per well, with a final DMSO concentration of 0.5% in the culture medium. Set up virus control wells (add DMSO and viruses, no compounds), cell control wells (add DMSO, no compounds and viruses) and medium control wells (only medium, no cells). The cytotoxicity test and the antiviral activity test of the compounds were carried out in parallel. Except that no virus was added, other experimental conditions were consistent with the antiviral activity test. The cell plate was placed in a 37°C, 5% CO2 incubator for 5 days. After 5 days of culture, cell viability was detected using cell viability detection kit CCK8. Raw data were used for compound antiviral activity and cytotoxicity calculations.
化合物的抗病毒活性和细胞毒性由化合物分别对病毒引起的细胞病毒效应的抑制率(％)表示。计算公式如下：
The antiviral activity and cytotoxicity of the compound were expressed by the inhibition rate (%) of the compound on the cellular viral effect caused by the virus respectively. Calculated as follows:
使用GraphPad Prism软件对化合物的抑制率和细胞毒性进行非线性拟合分析，得到化合物的EC50值。实验结果见表11。Use GraphPad Prism software to perform nonlinear fitting analysis on the inhibition rate and cytotoxicity of the compound, and obtain the EC 50 value of the compound. The experimental results are shown in Table 11.
表11化合物对于流感病毒A/PR/8/34(H1N1)的抑制活性
Table 11 Inhibitory activity of compounds against influenza virus A/PR/8/34 (H1N1)
结论：本发明化合物在细胞水平抑制流感病毒复制试验中展示出积极效应。Conclusion: The compounds of the present invention exhibit positive effects in the assay of inhibiting influenza virus replication at the cellular level.
实验例2：体内药效研究Experimental Example 2: In vivo drug efficacy study
实验目的：评价化合物在甲型流感病毒H1N1小鼠感染模型中的药效Experimental purpose: To evaluate the efficacy of compounds in the influenza A virus H1N1 mouse infection model
实验方案：小鼠经滴鼻感染甲型流感病毒A/PR/8/34(H1N1)，感染后48小时开始用化合物处理，口服给药，连续7天，每天两次。通过观察小鼠体重变化及存活率，来评价化合物在该模型中的抗甲型流感病毒H1N1作用。Experimental protocol: Mice were infected with influenza A/PR/8/34 (H1N1) via intranasal instillation. They were treated with the compound starting 48 hours after infection and administered orally for 7 consecutive days, twice a day. The anti-influenza A virus H1N1 effect of the compound in this model was evaluated by observing the weight changes and survival rate of mice.
实验选用SPF级别的BALB/c小鼠，6-7周，雌性。小鼠到达BSL-2动物房后适应至少3天后开始实验。将感染当天设为实验第0天。小鼠经戊巴比妥钠腹腔注射麻醉(75mg/kg,10mL/kg)，待动物进入深麻状态后，经滴鼻感染A/PR/8/34(H1N1)病毒，感染体积为50μL。从第2天至第8天，每天口服给予5mg/kg(给药体积10mL/kg)待测化合物，每天两次。首次给药时间为感染后48小时。每天观察小鼠状态，并记录小鼠体重及存活率。在第14天时，将所有存活动物进行安乐死。SPF grade BALB/c mice, 6-7 weeks old, female were used in the experiment. After the mice arrived in the BSL-2 animal room, they were allowed to adapt for at least 3 days before starting the experiment. The day of infection was set as day 0 of the experiment. Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (75 mg/kg, 10 mL/kg). After the animals entered deep anesthesia, they were infected with A/PR/8/34 (H1N1) virus via intranasal instillation. The infection volume was 50 μL. From day 2 to day 8, 5 mg/kg (administration volume: 10 mL/kg) of the test compound was administered orally twice a day. The first administration time is 48 hours after infection. The status of the mice was observed every day, and the weight and survival rate of the mice were recorded. On day 14, all surviving animals were euthanized.
实验结果：Experimental results:
检测动物存活率及体重下降率，体重下降率＝(第0天体重–第N天体重)/第0天体重*100％。结果如表12所示：式(I)化合物在第3天可以实现保护动物体重最大下降率为7.03％，然后开始恢复，至实验结束小鼠存活率为100％。The animal survival rate and weight loss rate were detected. Weight loss rate = (weight on day 0 – weight on day N)/body weight on day 0*100%. The results are shown in Table 12: The compound of formula (I) can achieve a maximum weight loss rate of 7.03% in protected animals on the third day, and then begins to recover. By the end of the experiment, the mouse survival rate is 100%.
表12动物存活率及体重下降率结果
Table 12 Animal survival rate and weight loss rate results
结论：本发明化合物在动物体内药效模型中表现出优异的体重保护，并且恢复时间早。Conclusion: The compounds of the present invention show excellent body weight protection and early recovery time in animal pharmacodynamic models.
实验例3：对Baloxavir耐药A/PR/8/34(H1N1)I38T流感病毒株细胞病变(CPE)实验Experimental Example 3: Cytopathological Pathogenesis (CPE) Experiment on Baloxavir-Resistant A/PR/8/34(H1N1)I38T Influenza Virus Strain
实验目的：通过测定化合物的半数有效浓度(EC50)值来评价化合物对Baloxavir耐药A/PR/8/34(H1N1)I38T流感病毒株的抗病毒活性。Experimental purpose: To evaluate the antiviral activity of compounds against Baloxavir-resistant A/PR/8/34(H1N1)I38T influenza virus strain by measuring the half effective concentration (EC 50 ) value of the compound.
实验方案：MDCK细胞以每孔15,000个细胞的密度接种于96孔细胞培养板中，并于37℃，5％CO2培养箱中培养过夜。次日加入化合物溶液(3倍系列稀释、8个浓度点、三复孔)和Baloxavir耐药A/PR/8/34(H1N1)流感病毒株，细胞培养液中DMSO终浓度为0.5％。细胞在5％CO2、37℃培养箱中培养5天，直至无化合物的病毒感染对照孔内细胞病变达80–95％。然后用CCK8检测每孔细胞活力。如含化合物孔的细胞活力较病毒感染对照孔高，即CPE减弱，则表明化合物对所测病毒有抑制作用。Experimental protocol: MDCK cells were seeded in a 96-well cell culture plate at a density of 15,000 cells per well and cultured overnight in a 37°C, 5% CO2 incubator. The compound solution (3-fold serial dilution, 8 concentration points, triple wells) and Baloxavir-resistant A/PR/8/34 (H1N1) influenza virus strain were added the next day. The final concentration of DMSO in the cell culture medium was 0.5%. The cells were cultured in a 5% CO 2 , 37°C incubator for 5 days until the cytopathic effects in the virus-infected control wells without compounds reached 80–95%. Then CCK8 was used to detect cell viability in each well. If the cell viability of the compound-containing wells is higher than that of the virus-infected control wells, that is, the CPE is weakened, it indicates that the compound has an inhibitory effect on the tested virus.
实验结果：Experimental results:
化合物的抗病毒活性由化合物对病毒引起的细胞病毒效应的抑制活性(％)表示。计算公式如下：
The antiviral activity of a compound is expressed by the inhibitory activity (%) of the compound against viral effects on cells caused by viruses. Calculated as follows:
EC50使用GraphPad Prism(version 5)软件对化合物的抑制活性和细胞活率进行非线性拟合分析，拟合 方法为"log(inhibitor)vs.response--Variable slope"。实验结果见表13。EC 50 uses GraphPad Prism (version 5) software to perform nonlinear fitting analysis on the inhibitory activity and cell viability of the compound. The method is "log(inhibitor)vs.response--Variable slope". The experimental results are shown in Table 13.
表13本发明化合物对Baloxavir耐药A/PR/8/34(H1N1)I38T流感病毒株的抑制活性结果
Table 13 Inhibitory activity results of the compounds of the present invention against Baloxavir-resistant A/PR/8/34 (H1N1) I38T influenza virus strain
结论：本发明化合物在细胞水平抑制Baloxavir耐药A/PR/8/34(H1N1)流感病毒株复制试验中展示出积极效应。Conclusion: The compound of the present invention shows a positive effect in inhibiting the replication of Baloxavir-resistant A/PR/8/34 (H1N1) influenza virus strain at the cellular level.
实验例4：体内药效研究Experimental Example 4: In vivo drug efficacy study
实验目的：评价化合物在甲型流感病毒H1N1耐药株中小鼠感染模型中的药效Experimental purpose: To evaluate the efficacy of compounds in a mouse infection model with a drug-resistant strain of influenza A virus H1N1
实验方案：小鼠经滴鼻感染甲型流感病毒Baloxavir耐药A/PR/8/34(H1N1)I38T病毒株，感染前2小时开始用化合物处理，口服给药，连续7天，每天两次。通过观察小鼠体重变化及存活率，来评价化合物在该模型中的抗甲型流感病毒H1N1作用。Experimental protocol: Mice were infected with the Baloxavir-resistant A/PR/8/34(H1N1)I38T strain of influenza A virus through intranasal instillation. They were treated with the compound starting 2 hours before infection and administered orally for 7 consecutive days, twice a day. . The anti-influenza A virus H1N1 effect of the compound in this model was evaluated by observing the weight changes and survival rate of mice.
实验选用SPF级别的BALB/c小鼠，6-7周，雌性。小鼠到达BSL-2动物房后适应至少3天后开始实验。将感染当天设为实验第0天。小鼠经腹腔注射舒泰50/盐酸赛拉嗪深度麻醉后，经滴鼻感染Baloxavir耐药A/PR/8/34(H1N1)I38T病毒株，感染体积为50μL。从第2天至第8天，每天口服给予15mg/kg或50mg/kg(给药体积10mL/kg)待测化合物，每天两次。首次给药时间为感染前2小时。每天观察小鼠状态，并记录小鼠体重及存活率。在第14天时，将所有存活动物进行安乐死。SPF grade BALB/c mice, 6-7 weeks old, female were used in the experiment. After the mice arrived in the BSL-2 animal room, they were allowed to adapt for at least 3 days before starting the experiment. The day of infection was set as day 0 of the experiment. After the mice were deeply anesthetized by intraperitoneal injection of Serta 50/xylazine hydrochloride, they were infected with Baloxavir-resistant A/PR/8/34(H1N1)I38T virus strain via intranasal instillation, with an infection volume of 50 μL. From day 2 to day 8, 15 mg/kg or 50 mg/kg (administration volume 10 mL/kg) of the test compound was administered orally twice a day. The first administration time is 2 hours before infection. The status of the mice was observed every day, and the weight and survival rate of the mice were recorded. On day 14, all surviving animals were euthanized.
实验结果：Experimental results:
检测动物存活率及体重下降率，体重下降率＝(第0天体重–第N天体重)/第0天体重*100％。实验结果如下表14所示。式(I)化合物及式(I)化合物A晶型在给药剂量50mg/kg时小鼠体重几乎没有下降，至实验结束小鼠存活率为100％。The animal survival rate and weight loss rate were detected. Weight loss rate = (weight on day 0 – weight on day N)/body weight on day 0*100%. The experimental results are shown in Table 14 below. When the compound of formula (I) and the crystal form A of compound of formula (I) were administered at a dose of 50 mg/kg, the body weight of mice almost did not decrease, and the survival rate of mice was 100% by the end of the experiment.
表14动物存活率及体重下降率结果
Table 14 Animal survival rate and weight loss rate results
结论：本发明化合物在动物体内药效模型中表现出优异的体重保护，并且恢复时间早。Conclusion: The compounds of the present invention show excellent body weight protection and early recovery time in animal pharmacodynamic models.
实验例5化合物的血浆蛋白结合率测试Test of plasma protein binding rate of compounds in Experimental Example 5
实验目的：采用平衡透析法评估本发明化合物在CD-1小鼠、SD大鼠和人血浆中的蛋白结合率。Experimental purpose: Use equilibrium dialysis method to evaluate the protein binding rate of the compound of the present invention in the plasma of CD-1 mice, SD rats and humans.
试验方案：将受试化合物分别用透析缓冲液稀释到上述五个物种的血浆中，配制成终浓度为2μM的样品，然后将样品加入到96孔平衡透析装置中，在37℃下用磷酸盐缓冲溶液透析4小时。实验采用华法林(warfarin)作为对照化合物。血浆和缓冲液中受试化合物与warfarin的浓度用LC-MS/MS法进行测定。Test plan: Dilute the test compound into the plasma of the above five species with dialysis buffer, prepare a sample with a final concentration of 2 μM, then add the sample to a 96-well equilibrium dialysis device, and incubate with phosphate at 37°C. The buffer solution was dialyzed for 4 hours. The experiment used warfarin as a control compound. The concentrations of test compounds and warfarin in plasma and buffer were determined by LC-MS/MS.
实验结果：结果显示如表15。Experimental results: The results are shown in Table 15.
表15本发明化合物血浆蛋白结合率结果
Table 15 Results of plasma protein binding rates of compounds of the present invention
注：H代表人，R代表大鼠，M代表小鼠,D代表犬,C代表食蟹猴Note: H stands for human, R stands for rat, M stands for mouse, D stands for dog, and C stands for cynomolgus monkey.
结论：本发明化合物在五个种属血浆中均具中等的血浆蛋白结合率，预示在上述五个种属的血浆中，受试化合物的游离态药物浓度比例适中，具有良好的成药性质。Conclusion: The compounds of the present invention have moderate plasma protein binding rates in the plasma of the five species, which indicates that the free drug concentration ratio of the test compound in the plasma of the above five species is moderate and has good pharmaceutical properties.
实验例6：大鼠药代动力学研究试验Experimental Example 6: Rat pharmacokinetics research test
实验目的：考察本发明化合物单次静脉注射和灌胃给药后雄性SD大鼠体内血浆药代动力学。Experimental purpose: To investigate the plasma pharmacokinetics in male SD rats after a single intravenous injection and intragastric administration of the compound of the present invention.
实验动物：雄性SD大鼠，6-8周龄，体重200-300克； Experimental animals: male SD rats, 6-8 weeks old, weighing 200-300 grams;
实验过程：注射给药(i.v.)，剂量为1mpk，浓度为0.50mg/mL，溶媒为40％DMAC+40％PG+20％(20％HP-β-CD+水)；口服给药(po)，剂量为10mpk，浓度为1mg/mL，溶媒为3％DMSO+10％solutol HS+87％水)。Experimental process: Injection administration (i.v.), the dose is 1mpk, the concentration is 0.50mg/mL, the solvent is 40% DMAC+40% PG+20% (20% HP-β-CD+water); oral administration (po) , the dose is 10mpk, the concentration is 1mg/mL, the solvent is 3% DMSO+10% solution HS+87% water).
样品采集：实验动物每个时间点从隐静脉穿刺采集血液样本0.03mL，记录实际采血时间。所有血样均加入规格为1.5mL的商品化EDTA-K2抗凝管中。血样采集后，血浆基质当中添加DDV做稳定剂，其中，血浆：敌敌畏溶液＝40:1，敌敌畏溶液为40mM敌敌畏的乙腈/水(1:1)溶液，在半小时内，于4℃、3000g离心10分钟吸取上清血浆，迅速置于干冰中，于-80℃冰箱保存，用于LC-MS/MS分析。Sample collection: 0.03 mL of blood sample was collected from the saphenous vein puncture of the experimental animals at each time point, and the actual blood collection time was recorded. All blood samples were added into commercial EDTA-K2 anticoagulant tubes with a specification of 1.5 mL. After the blood sample is collected, DDV is added to the plasma matrix as a stabilizer. Plasma: dichlorvos solution = 40:1. The dichlorvos solution is a 40mM dichlorvos acetonitrile/water (1:1) solution. Within half an hour, the solution is heated at 4°C and 3000g. Centrifuge for 10 minutes to aspirate the supernatant plasma, quickly place it in dry ice, and store it in a -80°C refrigerator for LC-MS/MS analysis.
数据分析：采用Phoenix WinNonlin 6.3药动学软件的非房室模型处理血浆浓度，使用线性对数梯形法方法计算药动学参数：Cl(表观清除率)，T1/2(清除一半化合物所需时长)，Cmax(达峰浓度)，AUC0-last(0-末次取样时间内的浓度积分面积)，结果见表16。Data analysis: The non-compartmental model of Phoenix WinNonlin 6.3 pharmacokinetic software was used to process plasma concentration, and the linear logarithmic trapezoidal method was used to calculate pharmacokinetic parameters: Cl (apparent clearance), T 1/2 (the time required to clear half of the compound). time required), C max (peak concentration), AUC 0-last (0-concentration integrated area during the last sampling time), the results are shown in Table 16.
表16本发明化合物的大鼠PK结果
Table 16 Rat PK results of compounds of the present invention
实验结论：本发明化合物口服血浆暴露量较高，具有良好的药代动力学性质。Experimental conclusion: The compound of the present invention has a high oral plasma exposure and has good pharmacokinetic properties.
实验例7：比格犬药代动力学研究试验Experimental Example 7: Pharmacokinetics Research Test on Beagle Dogs
实验目的：考察本发明化合物单次静脉注射和灌胃给药后雄性比格犬体内血浆药代动力学。Experimental purpose: To investigate the plasma pharmacokinetics of the compound of the present invention in male beagle dogs after a single intravenous injection and intragastric administration.
实验动物：雄性比格犬，≥6月龄，体重6～12千克；Experimental animals: male beagle dogs, ≥6 months old, weighing 6-12 kg;
实验过程：注射给药(i.v.)，剂量为1mpk，浓度为1mg/mL，溶媒为10％DMAC+90％(20％HP-β-CD+水)；式(I)化合物口服给药(po)，剂量为10mpk，浓度为2mg/mL，溶媒为3％DMSO+10％solutol HS+87％水)；式(I)化合物A晶型口服给药(po)，剂量为5mpk，浓度为1mg/mL，溶媒为3％DMSO+10％solutol HS+87％水)。Experimental process: Injection administration (i.v.), the dose is 1mpk, the concentration is 1mg/mL, the solvent is 10% DMAC+90% (20% HP-β-CD+water); the compound of formula (I) is administered orally (po) , the dosage is 10mpk, the concentration is 2mg/mL, the solvent is 3% DMSO+10% solution HS+87% water); the crystal form of compound A of formula (I) is administered orally (po), the dosage is 5mpk, the concentration is 1mg/ mL, the solvent is 3% DMSO+10% solution HS+87% water).
样品采集：实验动物每个时间点从隐静脉穿刺采集血液样本0.8mL，记录实际采血时间。所有血样均加入规格为1.5mL的商品化EDTA-K2抗凝管中。血样采集后，血浆基质当中添加DDV做稳定剂，其中，血浆：敌敌畏溶液＝40:1，敌敌畏溶液为40mM敌敌畏的乙腈/水(1:1)溶液，在半小时内，于4℃、3000g离心10分钟吸取上清血浆，迅速置于干冰中，于-80℃冰箱保存，用于LC-MS/MS分析。Sample collection: 0.8 mL of blood sample was collected from the saphenous vein puncture of the experimental animals at each time point, and the actual blood collection time was recorded. All blood samples were added into commercial EDTA-K2 anticoagulant tubes with a specification of 1.5 mL. After the blood sample is collected, DDV is added to the plasma matrix as a stabilizer. Plasma: dichlorvos solution = 40:1. The dichlorvos solution is a 40mM dichlorvos acetonitrile/water (1:1) solution. Within half an hour, the solution is heated at 4°C and 3000g. Centrifuge for 10 minutes to aspirate the supernatant plasma, quickly place it in dry ice, and store it in a -80°C refrigerator for LC-MS/MS analysis.
数据分析：采用Phoenix WinNonlin 6.3药动学软件的非房室模型处理血浆浓度，使用线性对数梯形法方法计算药动学参数Cl,T1/2,Cmax,AUC0-last，结果见表17。Data analysis: The non-compartmental model of Phoenix WinNonlin 6.3 pharmacokinetic software was used to process plasma concentration, and the linear logarithmic trapezoidal method was used to calculate the pharmacokinetic parameters Cl, T 1/2 , C max , AUC 0-last . The results are shown in the table 17.
表17本发明化合物的比格犬PK结果
Table 17 Beagle PK results of compounds of the present invention
实验结论：本发明化合物清除率低，半衰期时间长，口服血浆暴露量高，具有良好的药代动力学性质。 Experimental conclusion: The compound of the present invention has low clearance rate, long half-life, high oral plasma exposure, and good pharmacokinetic properties.

Claims (15)

  1. 式(I)化合物的A晶型，
    Form A of the compound of formula (I),
    其特征在于，其Cu Kα辐射的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：4.56±0.20°，10.68±0.20°，16.90±0.20°。It is characterized in that its X-ray powder diffraction pattern of Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 4.56±0.20°, 10.68±0.20°, 16.90±0.20°.
  2. 根据权利要求1所述的A晶型，其Cu Kα辐射的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：4.56±0.20°，9.08±0.20°，10.68±0.20°，16.90±0.20°，19.42±0.20°，19.94±0.20°，20.72±0.20°，29.12±0.20°。According to the A crystal form of claim 1, the X-ray powder diffraction pattern of Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 4.56±0.20°, 9.08±0.20°, 10.68±0.20°, 16.90±0.20° , 19.42±0.20°, 19.94±0.20°, 20.72±0.20°, 29.12±0.20°.
  3. 根据权利要求2所述的A晶型，其Cu Kα辐射的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：4.56±0.20°，9.08±0.20°，10.68±0.20°，11.80±0.20°，16.90±0.20°，17.84±0.20°，19.42±0.20°，19.94±0.20°，20.72±0.20°，22.66±0.20°，24.30±0.20°，29.12±0.20°。According to the A crystal form of claim 2, the X-ray powder diffraction pattern of Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 4.56±0.20°, 9.08±0.20°, 10.68±0.20°, 11.80±0.20° , 16.90±0.20°, 17.84±0.20°, 19.42±0.20°, 19.94±0.20°, 20.72±0.20°, 22.66±0.20°, 24.30±0.20°, 29.12±0.20°.
  4. 根据权利要求3所述的A晶型，其Cu Kα辐射的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：4.56±0.20°，9.08±0.20°，10.68±0.20°，11.80±0.20°，14.52±0.20°，16.90±0.20°，17.84±0.20°，19.42±0.20°，19.94±0.20°，20.72±0.20°，22.66±0.20°，24.30±0.20°，26.26±0.20°，28.24±0.20°，29.12±0.20°，29.98±0.20°。According to the A crystal form of claim 3, the X-ray powder diffraction pattern of Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 4.56±0.20°, 9.08±0.20°, 10.68±0.20°, 11.80±0.20° , 14.52±0.20°, 16.90±0.20°, 17.84±0.20°, 19.42±0.20°, 19.94±0.20°, 20.72±0.20°, 22.66±0.20°, 24.30±0.20°, 26.26±0.20°, 28.24±0.20° , 29.12±0.20°, 29.98±0.20°.
  5. 根据权利要求4所述的A晶型，其Cu Kα辐射的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：4.56°，9.08°，10.68°，11.80°，13.94°，14.52°，14.92°，15.28°，15.74°，16.56°，16.90°，17.84°，18.42°，19.42°，19.94°，20.30°，20.72°，21.12°，22.66°，23.80°，24.30°，24.68°，25.46°，25.80°，26.26°，26.86°，28.24°，29.12°，29.98°，30.88°，31.94°，33.60°，35.00°，37.88°，38.48°。According to the A crystal form of claim 4, the X-ray powder diffraction pattern of Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 4.56°, 9.08°, 10.68°, 11.80°, 13.94°, 14.52°, 14.92 °, 15.28°, 15.74°, 16.56°, 16.90°, 17.84°, 18.42°, 19.42°, 19.94°, 20.30°, 20.72°, 21.12°, 22.66°, 23.80°, 24.30°, 24.68°, 25.46°, 25.80°, 26.26°, 26.86°, 28.24°, 29.12°, 29.98°, 30.88°, 31.94°, 33.60°, 35.00°, 37.88°, 38.48°.
  6. 根据权利要求1～5任意一项所述的A晶型，其特征在于，具备下列任意一项特征：The A crystal form according to any one of claims 1 to 5, characterized by having any one of the following characteristics:
    (1)其XRPD图谱基本上如图1所示；(1) Its XRPD pattern is basically as shown in Figure 1;
    (2)其差示扫描量热曲线在198.4℃±3℃处具有吸热峰的峰值；(2) Its differential scanning calorimetry curve has an endothermic peak at 198.4℃±3℃;
    (3)其DSC图谱基本上如图2所示；(3) Its DSC spectrum is basically as shown in Figure 2;
    (4)其热重分析曲线在200.0℃±3℃时失重为2.87％；(4) Its thermogravimetric analysis curve has a weight loss of 2.87% at 200.0℃±3℃;
    (5)其TGA图谱基本上如图3所示。(5) Its TGA spectrum is basically as shown in Figure 3.
  7. 权利要求1～5任意一项所述的式(I)化合物的A晶型的制备方法，
    The preparation method of crystal form A of the compound of formula (I) according to any one of claims 1 to 5,
    包括如下步骤：Includes the following steps:
    (a)将化合物Z加入溶剂X和溶剂Y的混合溶剂中；(a) Add compound Z to the mixed solvent of solvent X and solvent Y;
    (b)加完后在10～35℃继续搅拌5～24小时； (b) After the addition is completed, continue stirring at 10-35°C for 5-24 hours;
    (c)过滤，滤饼在25～60℃下真空干燥1～24小时；(c) Filter, and vacuum dry the filter cake at 25-60°C for 1-24 hours;
    其中，in,
    化合物Z为式(I)化合物B晶型；Compound Z is the crystal form of compound B of formula (I);
    溶剂X为乙酸乙酯；Solvent X is ethyl acetate;
    溶剂Y选自正庚烷、正己烷和环己烷；Solvent Y is selected from n-heptane, n-hexane and cyclohexane;
    溶剂X和溶剂Y的体积比为1:1～1:6。The volume ratio of solvent X to solvent Y is 1:1 to 1:6.
  8. 根据权利要求7所述的制备方法，其中，化合物Z为式(I)化合物B晶型，溶剂Y为正庚烷，溶剂X和溶剂Y的体积比为1:3。The preparation method according to claim 7, wherein compound Z is the crystal form of compound B of formula (I), solvent Y is n-heptane, and the volume ratio of solvent X and solvent Y is 1:3.
  9. 式(I)化合物的B晶型，
    Form B of the compound of formula (I),
    其特征在于，其Cu Kα辐射的X射线粉末衍射图谱在下列任意一组2θ角处具有特征衍射峰：It is characterized in that its X-ray powder diffraction pattern of Cu Kα radiation has characteristic diffraction peaks at any of the following sets of 2θ angles:
    (1)9.70±0.20°，12.16±0.20°，14.91±0.20°，18.08±0.20°；(1)9.70±0.20°, 12.16±0.20°, 14.91±0.20°, 18.08±0.20°;
    (2)8.98±0.20°，9.70±0.20°，12.16±0.20°，14.91±0.20°，18.08±0.20°，18.54±0.20°，19.07±0.20°，21.58±0.20°；(2)8.98±0.20°, 9.70±0.20°, 12.16±0.20°, 14.91±0.20°, 18.08±0.20°, 18.54±0.20°, 19.07±0.20°, 21.58±0.20°;
    (3)8.98±0.20°，9.70±0.20°，12.16±0.20°，14.91±0.20°，17.02±0.20°，18.08±0.20°，18.54±0.20°，19.07±0.20°，21.58±0.20°，22.23±0.20°，24.87±0.20°，26.93±0.20°。(3)8.98±0.20°, 9.70±0.20°, 12.16±0.20°, 14.91±0.20°, 17.02±0.20°, 18.08±0.20°, 18.54±0.20°, 19.07±0.20°, 21.58±0.20°, 22.23± 0.20°, 24.87±0.20°, 26.93±0.20°.
  10. 根据权利要求9所述的B晶型，其特征在于，具备下列任意一项特征：The B crystal form according to claim 9, characterized in that it has any one of the following characteristics:
    (1)其XRPD图谱基本上如图4所示；(1) Its XRPD pattern is basically as shown in Figure 4;
    (2)其差示扫描量热曲线在154.2℃±3℃处具有吸热峰的峰值；(2) Its differential scanning calorimetry curve has an endothermic peak at 154.2℃±3℃;
    (3)其DSC图谱基本上如图5所示；(3) Its DSC spectrum is basically as shown in Figure 5;
    (4)其热重分析曲线在150.0℃±3℃时失重4.82％；(4) Its thermogravimetric analysis curve loses 4.82% weight at 150.0℃±3℃;
    (5)其TGA图谱基本上如图6所示。(5) Its TGA spectrum is basically as shown in Figure 6.
  11. 权利要求9或10所述的式(I)化合物的B晶型的制备方法，
    The preparation method of the B crystal form of the compound of formula (I) according to claim 9 or 10,
    包括如下步骤：Includes the following steps:
    (a)将化合物Z加入溶剂M中；(a) Add compound Z to solvent M;
    (b)在40-60℃下搅拌1～12小时；(b) Stir at 40-60°C for 1 to 12 hours;
    (c)冷却至10-30℃，加入溶剂N，搅拌1～12小时； (c) Cool to 10-30°C, add solvent N, and stir for 1 to 12 hours;
    (d)过滤，滤饼在25～60℃下真空干燥1～24小时；(d) Filter, and vacuum dry the filter cake at 25-60°C for 1-24 hours;
    其中，in,
    化合物Z选自式(I)化合物；Compound Z is selected from compounds of formula (I);
    溶剂M为甲苯；Solvent M is toluene;
    溶剂N不存在，或者，溶剂N选自正庚烷、正己烷和环己烷。Solvent N is absent or is selected from n-heptane, n-hexane and cyclohexane.
  12. 根据权利要求11所述的制备方法，其中，溶剂N为正庚烷。The preparation method according to claim 11, wherein solvent N is n-heptane.
  13. 式(I)化合物的水合物的C晶型，
    Form C of the hydrate of the compound of formula (I),
    其特征在于，其Cu Kα辐射的X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：5.85±0.20°，11.70±0.20°，17.59±0.20°。It is characterized in that its X-ray powder diffraction pattern of Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 5.85±0.20°, 11.70±0.20°, 17.59±0.20°.
  14. 根据权利要求13所述的C晶型，其XRPD图谱基本上如图7所示。According to the C crystal form of claim 13, its XRPD pattern is basically as shown in Figure 7.
  15. 根据权利要求1～6任意一项所述的A晶型、权利要求9或10所述的B晶型、权利要求13或14所述的C晶型或根据权利要求7～8、11～12任意一项所述的方法制备得到的晶型在制备治疗流感的药物中的应用。 According to the A crystal form according to any one of claims 1 to 6, the B crystal form according to claims 9 or 10, the C crystal form according to claims 13 or 14, or according to claims 7 to 8, 11 to 12 The use of the crystal form prepared by any of the methods described in the preparation of drugs for treating influenza.
PCT/CN2023/104923 2022-07-05 2023-06-30 Crystal form of pyridone polycyclic derivative and preparation method therefor WO2024007997A1 (en)

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CN109721615A (en) * 2017-09-18 2019-05-07 广东东阳光药业有限公司 Inhibitors of influenza viruses replication and application thereof
CN113226327A (en) * 2019-07-11 2021-08-06 南京征祥医药有限公司 Compounds useful for the treatment of influenza virus infections
WO2022148434A1 (en) * 2021-01-08 2022-07-14 辉诺生物医药科技（杭州）有限公司 Pyridone multiple-membered ring derivatives and use thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109721615A (en) * 2017-09-18 2019-05-07 广东东阳光药业有限公司 Inhibitors of influenza viruses replication and application thereof
CN113226327A (en) * 2019-07-11 2021-08-06 南京征祥医药有限公司 Compounds useful for the treatment of influenza virus infections
WO2022148434A1 (en) * 2021-01-08 2022-07-14 辉诺生物医药科技（杭州）有限公司 Pyridone multiple-membered ring derivatives and use thereof

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