WO2024255837A1 - 一种enpp1抑制剂的晶型 - Google Patents

一种enpp1抑制剂的晶型 Download PDF

Info

Publication number
WO2024255837A1
WO2024255837A1 PCT/CN2024/099188 CN2024099188W WO2024255837A1 WO 2024255837 A1 WO2024255837 A1 WO 2024255837A1 CN 2024099188 W CN2024099188 W CN 2024099188W WO 2024255837 A1 WO2024255837 A1 WO 2024255837A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
formula
cancer
crystalline form
organic solvent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2024/099188
Other languages
English (en)
French (fr)
Inventor
周付刚
张启超
宋云龙
卢凯
贡振财
史凯
雷响
刘春磊
杨欣欣
寇红艳
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
CSPC Zhongqi Pharmaceutical Technology Shijiazhuang Co Ltd
Innovstone Therapeutics Ltd
Original Assignee
CSPC Zhongqi Pharmaceutical Technology Shijiazhuang Co Ltd
Innovstone Therapeutics Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by CSPC Zhongqi Pharmaceutical Technology Shijiazhuang Co Ltd, Innovstone Therapeutics Ltd filed Critical CSPC Zhongqi Pharmaceutical Technology Shijiazhuang Co Ltd
Priority to AU2024302003A priority Critical patent/AU2024302003A1/en
Priority to CN202480001155.9A priority patent/CN119497716A/zh
Priority to KR1020267001047A priority patent/KR20260023568A/ko
Priority to EP24822785.2A priority patent/EP4729519A1/en
Publication of WO2024255837A1 publication Critical patent/WO2024255837A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • 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
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5365—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07D471/02—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 two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/13—Crystalline forms, e.g. polymorphs

Definitions

  • the present invention belongs to the field of pharmaceutical chemistry, discloses a crystal form of an ENPP1 inhibitor, and also includes application of the crystal form in preparing drugs for treating diseases related to ENPP1.
  • ENPP1 is a type II transmembrane glycoprotein with nucleotide pyrophosphatase and phosphodiesterase activities. It belongs to the extracellular nucleotide pyrophosphatase/phosphodiesterase (Enpp) family, which consists of seven functionally distinct proteins (1-7).
  • the ENPP1 structure has two N-terminal SMB domains (SMB1 and SMB2), two linker regions (L1 and L2), a catalytic domain, and a nuclease-like domain.
  • ENPP1 is differentially expressed in immune cells, with low levels in NK cells, DCs, and macrophages, and high levels in neutrophils.
  • ENPP1 is also expressed in a small fraction of B cells, which may be involved in the regulation of T cell activity.
  • ENPP1 expression is elevated in M2 subtype macrophages, which play a role in tumor promotion.
  • ENPP1 expression is increased in astrocytic tumors, breast cancer, and head and neck tumors.
  • ENPP1 expression varies greatly between different tumor tissues.
  • ENPP1 is specifically highly expressed in some tumors and may be a driver of tumor immune escape and metastasis.
  • ENPP1 plays an important role in immune responses to various stimuli through the STING pathway, selectively activating the STING signaling pathway in tumor cells and other cells in the tumor microenvironment, achieving high selectivity.
  • ENPP1 can also catalyze the hydrolysis of ATP into PPi and AMP, promoting the production of adenosine, which has a strong immunosuppressive effect.
  • Inhibiting ENPP1 can relieve tumor immunosuppression by inhibiting the hydrolysis of ATP and reducing adenosine production.
  • STING agonists indiscriminately activate STING in cancer cells and host cells
  • ENPP1 limits the scope of STING activation to tumor tissues and tumor microenvironment, playing a role in enhancing anti-tumor immunity.
  • ENPP1 is selectively upregulated in metastatic and chromosomally unstable tumor cells. Systemic administration of ENPP1 inhibitors can interfere with the ability of diffuse tumor cells to evade immune surveillance, avoiding the technical difficulties of intratumoral administration of STING agonists.
  • ENPP1 inhibitors combined with radiotherapy have achieved good results in animal models, and have a certain synergistic effect when used in combination with various anti-tumor drugs such as PD-1 and other immune checkpoint inhibitors and PARP inhibitors.
  • various anti-tumor drugs such as PD-1 and other immune checkpoint inhibitors and PARP inhibitors.
  • ENPP1 inhibitors are in the preclinical research and development stage, but many pharmaceutical companies have published patents on ENPP1 inhibitors, such as WO2021061803A1, WO2021158829A1, WO2020190912A1, WO2019177971A1 and WO2019046778A1.
  • ENPP1 inhibitor small molecules have been disclosed in the prior art, there are currently no small molecule drugs on the market for clinical use, so it is still urgent to develop new compounds with potential for marketing and better efficacy and pharmacokinetic results.
  • the present invention designs a series of compounds with new structures shown in the general formula, and finds that compounds with such structures exhibit excellent effects and functions, which has positive significance for the development of ENPP1 inhibitors.
  • the present invention provides a compound of formula (I) in crystalline form
  • the compound represented by formula (I) in crystalline form is Form A, and its X-ray powder diffraction pattern using Cu-K ⁇ radiation has diffraction peaks ( ⁇ 0.2°) at the following 2 ⁇ angles: 24.9, 25.1, 25.7.
  • the crystalline form A of the compound of formula (I) above, using Cu-K ⁇ radiation has an X-ray powder diffraction pattern having diffraction peaks ( ⁇ 0.2°) at the following 2 ⁇ angles: 12.8, 14.0, 16.0, 18.5, 24.9, 25.1, 25.7.
  • the crystalline form A of the compound of formula (I) above, using Cu-K ⁇ radiation has an X-ray powder diffraction pattern having diffraction peaks ( ⁇ 0.2°) at the following 2 ⁇ angles: 12.8, 14.0, 16.0, 18.5, 24.9, 25.1, 25.7, 26.9, 28.5.
  • the crystalline form A of the compound of formula (I) above, using Cu-K ⁇ radiation has an X-ray powder diffraction pattern having diffraction peaks ( ⁇ 0.2°) at the following 2 ⁇ angles: 12.8, 14.0, 16.0, 18.5, 24.9, 25.1, 25.7, 26.9, 28.5, 29.8.
  • the crystalline form A of the compound of formula (I) above, using Cu-K ⁇ radiation has an X-ray powder diffraction pattern having diffraction peaks ( ⁇ 0.2°) at the following 2 ⁇ angles: 12.8, 14.0, 16.0, 17.0, 18.5, 20.5, 24.9, 25.1, 25.7, 26.9, 28.5, 29.8.
  • the XRPD spectrum of Form A of the compound of formula (I) above using Cu-K ⁇ radiation is shown in FIG. 1 or FIG. 7 .
  • the XRPD spectrum of Form A of the compound of formula (I) is analyzed using Cu-K ⁇ radiation as shown in Table 1 or Table 2.
  • the differential scanning calorimetry curve of the crystal form A of the compound of formula (I) has an endothermic peak at 233.23 ⁇ 4°C.
  • the differential scanning calorimetry curve of the crystal form A of the compound of formula (I) has an exothermic peak at 234.3 ⁇ 4°C.
  • the crystal form A of the compound of formula (I) has a differential scanning calorimetry (DSC) spectrum substantially as shown in FIG. 2 .
  • thermogravimetric analysis curve of the crystal form A of the compound of formula (I) above shows a weight loss of 13.50 ⁇ 1% between about 105-254°C, for example, a weight loss of 13.5111%.
  • thermogravimetric analysis curve of the crystal form A of the compound of formula (I) above shows a weight loss of 13.50 ⁇ 1% at 254.00 ⁇ 3°C, for example, a weight loss of 13.5111%.
  • the crystal form A of the compound of formula (I) has a thermogravimetric analysis (TGA) spectrum substantially as shown in FIG. 3 .
  • the present invention also provides a crystalline form B of the compound of formula (I), which has an X-ray powder diffraction pattern with diffraction peaks ( ⁇ 0.2°) at the following 2 ⁇ angles using Cu-K ⁇ radiation: 19.19, 27.18, 13.44, 22.55.
  • the crystalline form B of the compound of formula (I) above, using Cu-K ⁇ radiation has an X-ray powder diffraction pattern having diffraction peaks ( ⁇ 0.2°) at the following 2 ⁇ angles: 26.19, 20.76, 4.42, 22.84, 19.19, 27.18, 13.44, 22.55.
  • the XRPD spectrum of Form B of the compound of formula (I) above using Cu-K ⁇ radiation is shown in FIG4 .
  • the crystal form B of the compound of formula (I) above, using Cu-K ⁇ radiation has an XRPD pattern analysis.
  • the analysis is shown in Table 3.
  • the crystalline form B of the compound of formula (I) is a DMF solvate of the compound of formula (I).
  • the ratio of the compound of formula (I) to the DMF solvent is 1:1.
  • the present invention also provides a crystalline form C of the compound of formula (I), which has an X-ray powder diffraction pattern with diffraction peaks ( ⁇ 0.2°) at the following 2 ⁇ angles using Cu-K ⁇ radiation: 22.11, 22.55, 20.96, 26.63.
  • the crystalline form C of the compound of formula (I) above, using Cu-K ⁇ radiation has an X-ray powder diffraction pattern having diffraction peaks ( ⁇ 0.2°) at the following 2 ⁇ angles: 22.11, 22.55, 20.96, 26.63, 15.01, 25.68, 26.38, 28.54, 13.22, 18.68.
  • the XRPD spectrum of Form C of the compound of formula (I) above using Cu-K ⁇ radiation is shown in FIG5 .
  • the crystalline form C of the compound of formula (I) is a DMSO solvate of the compound of formula (I).
  • the present invention also provides a crystalline form D of the compound of formula (I), which has an X-ray powder diffraction pattern using Cu-K ⁇ radiation and has diffraction peaks ( ⁇ 0.2°) at the following 2 ⁇ angles: 22.71, 18.92, 22.96, 26.4, 24.44, 16.62, 13.15, 17.79, 27.12.
  • the crystalline form D of the compound of formula (I) above, using Cu-K ⁇ radiation has an X-ray powder diffraction pattern having diffraction peaks ( ⁇ 0.2°) at the following 2 ⁇ angles: 22.71, 18.92, 22.96, 26.4, 24.44, 16.62, 13.15, 17.79, 27.12, 24.17, 28.81, 19.95, 28.42, 24.85, 14.78, 17.36, 29.16.
  • the XRPD spectrum of Form D of the compound of formula (I) above using Cu-K ⁇ radiation is shown in FIG6 .
  • the XRPD spectrum analysis of the crystalline form D of the compound of formula (I) above using Cu-K ⁇ radiation is shown in Table 5.
  • the crystalline form D of the compound of formula (I) is an NMP solvate of the compound of formula (I).
  • the ratio of the compound of formula (I) to the NMP solvent is 1:1.
  • the present application provides a method for preparing the crystalline compound of formula (I) as described in the first aspect, comprising method 1:
  • the compound of formula I is added to organic solvent A to dissolve, and then the solution is added to organic solvent B to precipitate crystals.
  • the organic solvent A is selected from one or more of DMF (N,N-dimethylformamide), DMSO (dimethyl sulfoxide), and NMP (N-methylpyrrolidone);
  • the organic solvent B is selected from one or more of water, methanol, ethanol, MTBE, toluene, and dichloromethane.
  • the organic solvent A is selected from DMF, and the organic solvent B is selected from one or two of water and ethanol; or the organic solvent A is selected from DMSO, and the organic solvent B is selected from one or two of toluene and methanol.
  • the organic solvent A is selected from DMF
  • the organic solvent B is selected from one or two of MTBE and toluene.
  • the organic solvent A is selected from NMP
  • the organic solvent B is selected from one or more of water, dichloromethane, and MTBE.
  • the method for preparing the crystalline compound of formula (I) comprises method 2:
  • the compound of formula I is added to an organic solvent C to dissolve, and then an organic solvent D is added to precipitate crystals.
  • the organic solvent C is selected from one or more of DMF, DMSO, and NMP
  • the organic solvent D is selected from one or more of acetone, ethylene glycol methyl ether, water, dichloromethane, ethyl acetate, MTBE, toluene, tetrahydrofuran, dioxane, ethanol, trifluoroethanol, acetonitrile, ethylene glycol dimethyl ether, isopropanol, and butanone.
  • the organic solvent C is selected from DMF
  • the organic solvent D is selected from one or more of acetone, ethylene glycol methyl ether, water, and dichloromethane
  • the organic solvent C is selected from From DMSO
  • the organic solvent D is selected from one or more of ethanol, trifluoroethanol, ethylene glycol methyl ether, acetonitrile, dioxane, ethylene glycol dimethyl ether, butanone
  • the organic solvent C is selected from NMP
  • the organic solvent D is selected from one or more of water, acetonitrile, dichloromethane, ethylene glycol dimethyl ether.
  • the organic solvent C is selected from DMF
  • the organic solvent D is selected from one or more of ethyl acetate, MTBE, toluene, tetrahydrofuran, dioxane, and butanone.
  • the organic solvent C is selected from DMSO
  • the organic solvent D is selected from one or more of ethyl acetate, water, and toluene.
  • the organic solvent C is selected from NMP
  • the organic solvent D is selected from one or more of isopropanol, MTBE, toluene, tetrahydrofuran, and butanone.
  • the method for preparing the crystalline compound of formula (I) comprises method three:
  • the compound of formula I is added to organic solvent E for suspension, slurrying, and filtered to obtain crystals.
  • the organic solvent E is selected from one or more of methanol, ethanol, isopropanol, ethyl acetate, n-heptane, MTBE, ethylene glycol methyl ether, water, acetonitrile, toluene, dichloromethane, tetrahydrofuran, dioxane, ethylene glycol dimethyl ether, and butanone.
  • the organic solvent E is selected from DMF.
  • the organic solvent E is selected from DMSO.
  • the organic solvent E is selected from NMP.
  • the present application provides a crystalline composition comprising a crystalline compound of formula (I) as described in the first aspect.
  • the crystalline compound of formula (I) is selected from one or more of crystalline form A, crystalline form B, crystalline form C, and crystalline form D.
  • the crystalline form of the compound of formula (I) accounts for more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% or more than 99.5% of the weight of the crystalline composition.
  • the compound of formula (I) in crystalline form accounts for more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% or more than 99.5% by weight of the crystalline composition.
  • the crystalline composition comprises Form A of the compound of formula (I).
  • the crystalline form A accounts for more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% or more than 99.5% of the weight of the crystalline composition.
  • the crystalline form A accounts for more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% or more than 99.5% of the weight of the crystalline composition.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising a crystalline compound of formula (I) and an optional pharmaceutically acceptable carrier.
  • the crystalline compound of formula (I) is selected from one or more of crystalline form A, crystalline form B, crystalline form C, and crystalline form D.
  • the pharmaceutical composition comprises the crystalline form A of the compound represented by formula (I) and an optional pharmaceutically acceptable carrier.
  • the present application provides a pharmaceutical composition comprising the crystalline composition as described in the second aspect and an optional pharmaceutically acceptable carrier.
  • the present application provides the use of the compound of formula (I) in crystalline form as described in the first aspect, the crystalline composition as described in the second aspect, or the pharmaceutical composition of the third aspect as a drug or in the preparation of a drug.
  • the drug is used to prevent and/or treat ENPP1-mediated diseases.
  • the drug is used to prevent and/or treat cancer or tumor-related diseases, or cardiovascular diseases.
  • the pharmaceutical composition comprises another, two or more drugs for treating cancer or tumors.
  • the pharmaceutical composition is used in combination with another, two or more drugs or treatment methods for treating cancer or tumors.
  • the anti-tumor drug is a chemotherapy drug, a targeted therapy drug or an immunotherapy drug; preferably, the drug for preventing and/or treating cancer or tumors includes but is not limited to: cell signal transduction inhibitors, chlorambucil, melphalan, cyclophosphamide, ifosfamide, busulfan, carmustine, lomustine, streptozotocin, cisplatin, carboplatin, oxaliplatin, dacarbazine, temozolomide, procarbazine, methotrexate, fluorouracil, cytarabine, gemcitabine, mercaptopurine, fludarabine, vinblastine, vincristine, vinorelbine, paclitaxel, docetaxel, topotecan, irinotecan, etoposide, trabectedin, dactinomycin, doxorubicin, epirubicin, daconazole, daunorubic
  • afatinib alisertib, amuvatinib, apatinib, axitinib, bortezomib, bosutinib, brivanib, cabozantinib, cediranib, crenolanib, crizotinib, dabrafenib, dacomitinib, danusetib, dasatinib, dovitinib, erlotinib, foretinib, ganetespib, gefitinib, ibrutinib Icotinib, imatinib, iniparib, lapatinib, lenvatinib, linifanib, linsitinib, masitinib, momelotinib, motesanib, neratinib, nilotinib, niraparib, oprozomib, olaparib, pazopanib,
  • the drug is used to prevent and/or treat skin cancer, bladder cancer, ovarian cancer, breast cancer, gastric cancer, pancreatic cancer, prostate cancer, colon cancer, lung cancer, bone cancer, brain cancer, neuroblastoma, rectal cancer, colon cancer, familial adenomatous polyposis cancer, hereditary non-polyposis colorectal cancer, esophageal cancer, lip cancer, laryngeal cancer, hypopharyngeal cancer, tongue cancer, salivary gland cancer, adenocarcinoma, medullary thyroid cancer, papillary thyroid cancer, kidney cancer, renal parenchymal cancer, cervical cancer, uterine body cancer, endometrial cancer, choriocarcinoma, testicular cancer, urinary cancer, melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermal tumor, Hodgkin's lympho
  • the cardiovascular disease to be treated and/or prevented is the treatment and/or prevention of myocardial infarction (MI), Heart failure (HF), cardiac trauma, abnormal scarring of cardiac tissue, cardiomyopathy, cardiomyocyte death, enhanced cardiac repair, and release of pro-inflammatory molecules in cardiomyocytes.
  • MI myocardial infarction
  • HF Heart failure
  • cardiac trauma abnormal scarring of cardiac tissue
  • cardiomyopathy cardiomyocyte death
  • enhanced cardiac repair and release of pro-inflammatory molecules in cardiomyocytes.
  • the "compound of formula (I) in crystalline form” mentioned in the present application refers to the compound of formula (I) in crystalline form, including anhydrous and solvent-free form, hydrate form and solvate form of the compound of formula (I).
  • the crystalline form is preferably anhydrous and solvent-free form or hydrate form; more preferably anhydrous and solvent-free form.
  • solvate refers to an association formed by solvent molecules in stoichiometric or non-stoichiometric ratios and the compound represented by formula (I) of the present application, including an association containing both water molecules and one or more other solvent molecules, and an association containing only one or more other solvent molecules.
  • hydrate refers to an association formed by water molecules in a stoichiometric ratio or a non-stoichiometric ratio and the compound represented by formula (I) of the present application.
  • anhydrous and solvent-free form means that there are no water molecules or solvent molecules in the unit cell or crystal lattice, or the water molecules or solvent molecules coexist with the compound represented by formula (I) in a manner that is not bound by intermolecular forces, such as by adsorption.
  • crystalline composition refers to a solid form, which includes the crystal form A mentioned in the present application. Moreover, in addition to the crystal form of the present application, the crystalline composition may optionally include other crystal forms or other amorphous forms of the compound represented by formula (I) or its salt, or impurities other than these substances. It should be understood by those skilled in the art that the sum of the contents of each component in the crystalline composition should be 100%.
  • the "room temperature” is the room temperature conventionally known in the art, generally 10-30°C, preferably 25°C ⁇ 5°C.
  • the term “substantially” or “substantially as shown” refers to a substantially pure crystalline form, in which at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% of the peaks in the powder X-ray diffraction pattern appear in the given pattern. Further, when the content of a certain crystalline form in the product gradually decreases, some of the diffraction peaks attributed to the crystalline form in its X-ray powder diffraction pattern may become less due to the detection sensitivity of the instrument.
  • the term "substantially” or “substantially as shown” means that for the same crystalline form of the same compound, in consecutive analyses, the error of the thermal transition onset temperature, the peak temperature of the endothermic peak, the peak temperature of the exothermic peak, the melting point, etc. is typically within about 8°C, usually within about 5°C, and usually within about 3°C.
  • the error of the thermal transition onset temperature, the peak temperature of the endothermic peak, the peak temperature of the exothermic peak, the melting point, etc. is typically within about 8°C, usually within about 5°C, and usually within about 3°C.
  • the term “prevent” or “prevent” means that when used for a disease or condition (e.g., a viral disease), the compound or drug (e.g., the combination product claimed herein) can reduce the frequency of symptoms of the medical condition in a subject or delay its onset compared to a subject to which the compound or drug (e.g., the combination product claimed herein) is not administered.
  • a disease or condition e.g., a viral disease
  • the compound or drug e.g., the combination product claimed herein
  • the term "treat,” “treat,” “treat,” “treat,” or “treat” refers to alleviating, relieving, or ameliorating symptoms of a disease or condition, ameliorating underlying metabolically caused symptoms, inhibiting a disease or symptom, such as arresting the development of a disease or condition, alleviating a disease or condition, causing regression of a disease or condition, alleviating a condition caused by a disease or condition, or arresting symptoms of a disease or condition.
  • composition of the present application can be prepared by conventional methods in the art.
  • pharmaceutically acceptable carrier or “excipient” or “pharmaceutically acceptable excipient” or “pharmaceutically acceptable excipient” refers to an agent that has no significant irritation to organisms and will not impair the biological activity of the active compound.
  • pharmaceutically acceptable excipients includes: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, etc.
  • the intermediate compounds of the present application can be prepared by a variety of synthesis 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 equivalent substitution methods well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present application.
  • the compound of formula (I) of the present invention has good PK properties and oral bioavailability, its crystal form is stable, and its drug development prospects are good.
  • the compound of formula (I) exhibits good inhibitory activity against ENPP1; the compound of formula (I) has good pharmacokinetic indicators.
  • the crystalline form of the compound of formula (I) and the specific crystal form provided by the present invention have one or more of the following beneficial effects: (1) the crystalline form of the compound of formula (I) has good properties and is easy to weigh, transfer, separate, purify and store; (2) the crystalline form of the compound of formula (I) and the specific crystal form have good crystallinity; (3) the crystalline form of the compound of formula (I) and the specific crystal form have excellent operability, for example, they are easy to purify, filter and separate, especially the crystal form A, which is simple to prepare and has a high yield; (4) the preferred crystal form has good physical stability and chemical stability, especially the crystal form A, which has high purity and can be directly used as a raw material drug, and has good pharmaceutical prospects.
  • Step size 0.1s/step
  • Test conditions starting temperature 30°C, heating to 300°C at a heating rate of 10°C/min; purge gas flow rate: 50ml/min;
  • Drying gas flow rate 150 ml/min; Purge gas: N 2 ; Crucible: aluminum crucible.
  • Test conditions starting temperature 30°C, heating to 350°C at a heating rate of 10°C/min; balance protection gas flow rate:
  • reaction gas flow rate 50ml/min
  • purge gas N 2
  • crucible alumina crucible.
  • Test method 25°C0%RH-90%RH-0%RH; refer to Chinese Pharmacopoeia Part IV General Rules 0103
  • the preparation process of the biological medium is shown in the following table.
  • the crystalline sample is added to the biological medium and water and shaken at a constant temperature of 37°C for 24 hours. Samples are taken at 1 hour, 4 hours and 24 hours respectively.
  • the sampled solution is filtered with a 0.22 ⁇ m water filter membrane. Some samples with higher concentrations are appropriately diluted with diluents.
  • the signal peak area of the solution is measured by HPLC.
  • the concentration of the compound in the solution is calculated based on the peak area, the HPLC standard curve of the raw material and the dilution multiple.
  • samples at different time points are taken to test their pH values, and the remaining solids are tested by XRPD.
  • FaSSIF simulates the intestinal fluid in the small intestine of humans in a hungry state before a meal
  • FeSSIF simulates the intestinal fluid in the small intestine of humans in a full state after a meal
  • FaSSGF simulates the gastric fluid in an empty stomach when humans are hungry.
  • FIG1 is an XRPD spectrum of Form A of the compound of formula (I) prepared in Example 2.
  • FIG2 is a DSC spectrum of Form A of the compound of formula (I).
  • FIG3 is a TGA spectrum of Form A of the compound of formula (I).
  • FIG4 is an XRPD spectrum of Form B of the compound of formula (I) prepared in Example 4.
  • FIG5 is an XRPD spectrum of Form C of the compound of formula (I) prepared in Example 5.
  • FIG6 is an XRPD spectrum of Form D of the compound of formula (I) prepared in Example 6.
  • FIG. 7 is an XRPD spectrum of Form A of the compound of formula (I) prepared in Example 3.
  • Figure 8 shows the cardiac function indexes and body weight changes in the acute myocardial ischemia model of mice treated with the compound of formula (I), A) left ventricular ejection fraction of mice; B) left ventricular short axis shortening rate of mice; C) body weight changes of mice;
  • Figure 9 shows the changes in myocardial fibrosis in the acute myocardial ischemia model of mice treated with the compound of formula (I); D) Masson-stained pathological sections of mouse heart tissue; E) the degree of myocardial fibrosis in mice;
  • Figure 10 shows the cardiac function indexes and body weight changes in the acute myocardial ischemia model of rats treated with the compound of formula (I), a) left ventricular short axis shortening rate of rats; b) left ventricular ejection fraction of rats; c) body weight changes of rats;
  • Figure 11 shows the changes in myocardial fibrosis in the acute myocardial ischemia model of rats treated with the compound of formula (I); d) Masson-stained pathological sections of rat heart tissue; e) the degree of myocardial fibrosis in rats;
  • FIG. 12 is a comparison of XRPD patterns of Form A before and after the stability experiment.
  • Step 1 Preparation of ethyl 4-chloro-7-methoxy-1,8-naphthyridine-3-carboxylate
  • Step 2 Preparation of ethyl 4-((4-bromo-2,6-difluorobenzyl)amino)-7-methoxy-1,8-naphthyridine-3-carboxylate
  • Step 3 Preparation of (4-((4-bromo-2,6-difluorobenzyl)amino)-7-methoxy-1,8-naphthyridin-3-yl)methanol
  • Step 4 Preparation of 1-(4-bromo-2,6-difluorobenzyl)-8-methoxy-1,4-dihydro-2H-[1,3]oxazino[5,4-c][1,8]naphthylidene-2-one
  • Step 5 Preparation of 1-(2,6-difluoro-4-((4-methoxybenzyl)thio)benzyl)-8-methoxy-1,4-dihydro-2H-[1,3]oxazino[5,4-c][1,8]naphthylidene-2-one
  • Step 6 Preparation of 3,5-difluoro-4-((8-methoxy-2-oxo-2H-[1,3]oxazino[5,4-c][1,8]naphthyridin-1(4H)-yl)methyl)benzenesulfonamide
  • the compound of formula (I) (100 mg) prepared in Example 1 and dimethyl sulfoxide (0.3 mL) were added to the reaction flask in sequence, slowly heated to dissolve, and slowly added to water (1 mL) at 80° C. After slowly cooling to room temperature, stirred for one hour, filtered, and the filter cake was washed with an appropriate amount of water. The filter cake was dried under reduced pressure at 65° C. to obtain the crystal form A of the compound of formula (I).
  • the compound of formula (I) prepared in Example 1 (100 mg) was added to the reaction bottle, and then acetone (1 mL) was added, and the mixture was stirred for 24 hours at room temperature to slurry the suspension; the mixture was filtered, and the filter cake was dried under reduced pressure at 45° C. to obtain the crystalline form A of the compound of formula (I).
  • the crystal form did not change after the DVS test.
  • Example 2 20 mg of the Form A sample of the compound of formula (I) prepared in Example 2 was weighed into a vial and left open for 7 days under high temperature (60°C), long term (25°C/60% RH) and accelerated (40°C/75% RH) conditions. The samples were taken for purity detection and X-ray powder diffraction to investigate the stability of Example 2 (Form A) under different conditions. The results are shown in Table 6 and Figure 12.
  • Example 2 (Crystal Form A) can maintain chemical stability and crystal stability in the solid stability test.
  • Test Example 3 Stability test of the crystalline form A of the compound of formula (I) in biological solvents
  • Example 2 Crystal Form A
  • FaSSIF, FeSSIF and FaSSGF biological media
  • the remaining sample was subjected to X-ray powder diffraction after 24 hours. The results are shown in Table 7:
  • Example 2 (Crystal Form A) can maintain crystal stability in biological solvents.
  • ENPP1 is a transmembrane glycoprotein that can hydrolyze nucleotides and derivatives with nucleotide-5'-monophosphate structures.
  • ENPP1 can hydrolyze synthetic 5'-monophosphate p-nitrophenyl ester (TMP-pNP) into nucleotides'-monophosphate and p-nitrophenol, and p-nitrophenol is a color product.
  • TMP-pNP synthetic 5'-monophosphate p-nitrophenyl ester
  • the amount of p-nitrophenol product generated can be directly measured by its absorbance at 405nm, which is proportional to the enzyme activity.
  • % inhibition rate (OD high signal control - OD sample well) / (OD high signal control - OD low signal control) * 100
  • the IC50 value was calculated by four-parameter fitting, high signal control: DMSO group without inhibitor, low signal control: blank control group.
  • Test Example 5 Pharmacokinetic study of compound concentration in mice using LC-MS/MS
  • Test principle Use LC-MS/MS to measure the drug concentration of the target drug in plasma at different times and draw the pharmacokinetic curve of the target compound in vivo.
  • Test method Dissolve the test compound (the compound of formula (I) prepared in Example 1) in DMSO to prepare a stock solution with a final concentration of 20 mg/mL, and use a solvent containing 5% DMSO (Sigma-Aldrich, SHBJ2847), 45% PEG400 (Sigma-Aldrich, BCCC0015) and 50% dd H 2 O to dissolve the above compound to 1 mg/mL.
  • the mouse source is CD-1 male (JH Laboratory Animal Co.LTD), 9 mice are selected for each compound group, 10 mg/kg PO is administered, and blood is collected at 0.25hr, 0.5hr, 1hr, 2hr, 4hr, 8hr and 24hr, 3 samples are collected at each time, and 110 ⁇ L of whole blood (K2EDTA anticoagulation) is collected. The sample is immediately centrifuged at 2000g for 5 minutes at 4°, and the serum is collected and stored at -70°. The blood drug concentration was determined using a Triple-quadrupole MS system (SCIEX), including the preparation of standard curves and quality control and sample preparation.
  • SCIEX Triple-quadrupole MS system
  • Standard curve and quality control preparation dilute the working solution with MeOH:H 2 O (1:1), and add 3 ⁇ L of the standard curve and quality control working solution to 57 ⁇ L of blank plasma.
  • Sample preparation add 200 ⁇ L of internal standard solution (Propranolol, 40 ng/mL) to 30 ⁇ L of plasma sample, mix for 1 minute, centrifuge at 5800 rpm for 10 minutes, take 100 ⁇ L of supernatant and transfer to a new plate for injection analysis. Chromatographic conditions were optimized according to the sample, including the mobile phase composition, elution gradient conditions, flow rate and retention time, etc.
  • the chromatographic column was Waters BEH C18 (2.1 ⁇ 50 mm, 1.7 ⁇ m), and the injection volume was 1 ⁇ L.
  • the mass spectrometer used an electrospray ionization source (TuREo spray), and in the positive ion detection mode, the multi-channel reaction monitoring (MRM) mode was selected for secondary mass spectrometry analysis.
  • MRM multi-channel reaction monitoring
  • WinNonlin 8.2 software was used to calculate the pharmacokinetic parameters according to the non-compartmental model, including peak concentration Cmax, peak time Tmax, area under the drug-time curve AUC, and elimination half-life t1/2.
  • AUC was calculated using the linear up log down method.
  • test results show that the compound of formula (I) of the present invention has good in vivo pharmacokinetics and has drug-forming potential.
  • Test Example 6 Pharmacodynamic evaluation of the compound of formula (I) in the mouse pancreatic cancer Pan02 model
  • Pan02 cells were cultured in monolayer in vitro in DMEM medium with 10% fetal bovine serum, 0.01 mg/ml sinsulin, 1% penicillin/streptomycin/amphotericin B, and cultured in a 37°C 5% CO 2 incubator. When the cell saturation reached 80%-90% and the number reached the required level, the cells were collected, counted, and inoculated.
  • Tumor inoculation and animal grouping and administration 0.1 mL (5 ⁇ 10 6 cells) of Pan02 cells were subcutaneously inoculated on the right back of each mouse. Grouping and administration began when the average tumor volume reached about 100-200 mm 3. Weigh the animals before administration and measure the tumor volume. Randomly group the animals according to the tumor volume, with 8 mice in each group. The control group was given the solvent: 5% DMSO + 45% PEG400 + 50% H20 (1% CMC-Na). The experimental group was given a dose of 20 mg/kg, and was given oral gavage. The administration volume was 10 ⁇ l/g according to the mouse body weight, twice a day. The health status of the animals was observed daily. If the tumor volume exceeded 3,000 mm 3 , or there was a serious disease or pain, the body weight decreased by more than 20%, and continued to deteriorate, euthanasia was required.
  • Tumor growth inhibition rate Tumor diameter was measured with a vernier caliper twice a week.
  • the tumor inhibition efficacy of the compound TGI (%) [(1-(average tumor volume of the treatment group after administration - average tumor volume of the treatment group at the beginning of administration))/(average tumor volume of the solvent control group before treatment - average tumor volume of the solvent control group after treatment)] ⁇ 100%. Data analysis was performed using T test for comparison between the two groups, and p ⁇ 0.05 was considered to be significantly different.
  • the compound of formula (I) prepared in Example 1 of the present invention showed activity in the mouse pancreatic cancer Pan02 model.
  • the compound of formula (I) of the present invention was administered for 16 days, which could significantly inhibit the growth of tumors, with a TGI of 43.4%, which was significantly different from the control group (p ⁇ 0.05), and had no inhibition on the weight of mice, and had good safety.
  • the crystalline form of the compound of formula (I) (such as the crystalline form A of the compound of formula (I)) has basically the same efficacy.
  • Test Example 7 Pharmacodynamic evaluation of the compound in a mouse myocardial ischemia model
  • the aim of this study was to evaluate the efficacy of the compounds in a C57 BL/6 mouse model of myocardial ischemia.
  • mice After the animals arrived at the facility and adapted for a week, they were divided into 4 groups according to their body weight one day before surgery, with 6 animals in the sham operation group (Sham) and 8 animals in the model group (MI). The next day, they underwent myocardial ischemia surgery (the mice in the Sham group only underwent thoracotomy and threading without ligation). The model was established by ligating the left anterior descending branch of the coronary artery (LAD). After model establishment, they were treated with penicillin (20,000 units/mouse i.m.) for 3 days and disinfected to prevent infection.
  • penicillin 20,000 units/mouse i.m.
  • the corresponding compounds were administered to each group of animals 1 hour before surgery.
  • the control group (Vehicle) was given a solvent: 5% DMSO + 45% PEG400 + 50% H 2 O (1% CMC-Na), and the experimental groups were given 4 mg/kg and 20 mg/kg, respectively, and were given oral gavage twice a day for 28 consecutive days.
  • the body weight of the animals was monitored daily after administration, and cardiac ultrasound imaging indicators were measured on the 1st, 7th and 28th days.
  • the main indicators of echocardiography evaluation were left ventricular fractional shortening (FS) and left ventricular ejection fraction (LVEF). After the echocardiography measurement on the 28th day, the heart tissue was collected in tissue fixative for Masson trichrome staining pathological analysis.
  • FS left ventricular fractional shortening
  • LVEF left ventricular ejection fraction
  • the compound (I) of Example 1 of the present invention showed good cardiac function improvement activity in a mouse myocardial infarction model.
  • the compound of formula (I) shown in the invention has a protective effect on cardiac function after pre-administration one day before modeling.
  • 4mpk and 20mpk can increase the left ventricular ejection fraction LVEF of mice by 15% and 10% (Figure A) and the left ventricular short axis shortening rate FS by 7% and 5% ( Figure B).
  • the left ventricular ejection fraction LVEF of mice can be increased by 23.4% and 23.3% respectively, and the left ventricular short axis shortening rate FS can be increased by 12.2% and 12.3% respectively.
  • the compound of formula (I) of Example 1 shown in the present invention shows an improvement effect on myocardial fibrosis in mice with myocardial infarction.
  • Figure D is an electron microscope image of Masson-stained pathological sections of mouse heart tissue.
  • Figure E is a statistical result of the degree of fibrosis in mouse heart tissue.
  • the red myocardial fibers of mice are neatly arranged, and blue collagen fibers are rare.
  • myocardial cell necrosis, myocardial tissue structure disorder, and large areas of blue collagen fibers can be seen in the infarct area.
  • the myocardial tissue structure of mice in the group administered with the compound of formula (I) was improved to a certain extent, and the red myocardial cells and blue collagen in the infarct area were distributed alternately, and the collagen fibers were significantly reduced, indicating that the compound of formula (I) can reduce the degree of myocardial fibrosis after MI when administered for 28 days, and compared with the model solvent control group, the degree of fibrosis can be reduced by 8.8% and 9.6%, respectively (Figure E).
  • the crystalline form A of the compound of formula (I) has substantially the same efficacy.
  • Test Example 8 Pharmacodynamic evaluation of compounds in rat myocardial ischemia model
  • the purpose of this study is to evaluate the efficacy of the compound in the SD rat myocardial ischemia model.
  • the model was established by ligating the left anterior descending coronary artery (LAD). After the model was established, penicillin (80,000 units/rat i.m) was given for treatment for 3 days and disinfection was performed to prevent infection.
  • LAD left anterior descending coronary artery
  • mice were randomly divided into 4 groups according to the baseline body weight and cardiac ultrasound imaging indicators, with 4 animals in each sham operation group (Sham) and 6 animals in each myocardial infarction model group (MI), and drug administration began.
  • the control group was given a blank solvent Vehicle: 5% DMSO + 45% PEG400 + 50% H 2 O (1% CMC-Na), and the experimental groups were given oral gavage at doses of 30 mg/kg and 75 mg/kg, twice a day, for 28 consecutive days.
  • the body weight of the animals was monitored daily after administration, and cardiac ultrasound imaging indicators were measured on the 7th and 28th days.
  • the main indicators of echocardiography evaluation were left ventricular short axis shortening fraction (FS) and left ventricular ejection fraction (LVEF). After the echocardiography measurement on the 28th day, the heart tissue was collected in tissue fixative for Masson trichrome staining pathological analysis.
  • FS left ventricular short axis shortening fraction
  • LVEF left ventricular ejection fraction
  • the compound of formula (I) prepared in Example 1 of the present invention showed good cardiac function improvement activity in the rat myocardial infarction model ( Figure 7a, b).
  • the compound of formula (I) of the present invention can significantly improve the left ventricular ejection fraction LVEF and the left ventricular short axis shortening index FS of rats on the 7th day of administration.
  • the 30mpk and 75mpk groups increased the LVEF by 11.43% and 18.85% and the left ventricular short axis shortening rate FS by 9.73% and 12.62%, respectively.
  • the 30mpk and 75mpk dosage groups increased the LVEF by 18.75% and 23.8% and the left ventricular short axis shortening rate FS by 13.01% and 15.88%, respectively. Moreover, it showed a dose-dependent trend and had no significant effect on body weight (Figure c). Statistical significance was analyzed by t-test, and p ⁇ 0.05 was considered to be significantly different.
  • the compound of formula (I) prepared in Example 1 of the present invention showed an improvement effect on myocardial fibrosis in rats with myocardial infarction.
  • Figure d it is an electron microscope image of Masson-stained pathological sections of rat heart tissue.
  • Figure e is the statistical results of the degree of fibrosis in rat heart tissue.
  • the red myocardial fibers of rats were arranged neatly, and blue collagen fibers were rare.
  • MI model control group myocardial cell necrosis, myocardial tissue structure disorder, and large areas of blue collagen fibers were visible in the infarct area.
  • the myocardial tissue structure of rats in the group administered with the compound of formula (I) was improved to a certain extent, and the red myocardial cells and blue collagen in the infarct area were distributed alternately, and the collagen fibers were significantly reduced, indicating that the compound of formula (I) can reduce the degree of myocardial fibrosis after MI when administered for 28 days, and compared with the model solvent control group, the degree of fibrosis can be reduced by 5.2% and 6.5%, respectively ( Figure e).
  • the crystalline form A of the compound of formula (I) has basically the same efficacy.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

本申请提供了一种结晶形式的式(I)所示化合物、其具体晶型、包含其的药物组合物及其用途，结晶形式的式(I)所示化合物及其具体晶型具有良好的结晶度和化学纯度，且制备简便，收率高，具有良好的成药潜力

Description

一种ENPP1抑制剂的晶型
本申请要求2023年6月15日向中国国家知识产权局提交的，专利申请号为202310706966.6，发明名称为“一种ENPP1抑制剂的晶型”的在先申请的优先权。所述申请的全文通过引用的方式结合于本申请中。
技术领域
本发明属于药物化学领域，公开了一种ENPP1抑制剂的晶型，还包括所述晶型在制备治疗与ENPP1相关疾病的药物中的应用。
背景技术
ENPP1是一种具有核苷酸焦磷酸酶和磷酸二酯酶活性的II型跨膜糖蛋白，属于胞外核苷酸焦磷酸酶/磷酸二酯酶(Enpp)家族，该家族由7种功能不同的蛋白质组成(1-7)。ENPP1结构具有两个N端SMB结构域(SMB1和SMB2)、两个连接体区域(L1和L2)、一个催化结构域和一个核酸酶样结构域。ENPP1在免疫细胞中差异表达，在NK细胞、DC和巨噬细胞中低水平表达，在中性粒细胞中高水平表达。ENPP1也在一小部分B细胞中表达，这些细胞可能参与了T细胞活性的调节。ENPP1在M2亚型巨噬细胞中表达升高，而M2亚型巨噬细胞在肿瘤促进中起作用。ENPP1在星形细胞肿瘤、乳腺癌和头颈部肿瘤中的表达增加。ENPP1的表达在不同的肿瘤组织之间差异很大。ENPP1在部分肿瘤中特异性高表达，可能是肿瘤免疫逃逸和转移的驱动因素。
ENPP1通过STING途径在多种刺激的免疫应答中发挥重要作用，选择性地激活肿瘤细胞和肿瘤微环境中其他细胞的STING信号通路，实现高选择性。ENPP1还可催化ATP水解为PPi和AMP，促进腺苷的生成，而腺苷有很强的免疫抑制作用。抑制ENPP1可以通过抑制ATP的水解，减少腺苷生成，解除肿瘤免疫抑制。
STING激动剂无区分性地激活癌细胞和宿主细胞中的STING，而ENPP1则使STING激活的范围更加局限于肿瘤组织及肿瘤微环境，发挥增强抗肿瘤免疫力的作用。而且ENPP1在转移性和染色体不稳定的肿瘤细胞中选择性上调，ENPP1抑制剂全身给药能干扰扩散的肿瘤细胞对免疫监视的逃避能力，避开了STING激动剂瘤内给药的技术困难。
ENPP1抑制剂联合放疗在动物模型上取得较好的效果，与PD-1等免疫检查点抑制剂及PARP抑制剂等多种抗肿瘤药物联用也有一定协同作用。现阶段ENPP1抑制剂的开发均属于临床前研发阶段，但已有多家药企发表关于ENPP1抑制剂的专利，如WO2021061803A1、WO2021158829A1、WO2020190912A1、WO2019177971A1和WO2019046778A1。
尽管现有技术已公开了一些ENPP1抑制剂小分子，但目前暂未有上市临床的小分子药物，因此开发新的具有上市潜力的，具有更好药效、药代结果的化合物仍是迫切需要的。本发明设计了系列具有通式所示的新结构的化合物，并发现具有此类结构的化合物呈现出优异的效果和作用，对ENPP1抑制剂的开发具有积极意义。
发明内容
第一方面，本发明提供了结晶形式的式(I)化合物，
在本发明的一个优选方面，所述结晶形式的式(I)所示化合物，其为晶型A，使用Cu-Kα辐射，其X射线粉末衍射图谱在下列2θ角处具有衍射峰(±0.2°)：24.9，25.1，25.7。
在本发明的一些方案中，上述式(I)化合物的晶型A，使用Cu-Kα辐射，其X射线粉末衍射图谱在下列2θ角处具有衍射峰(±0.2°)：12.8，14.0，16.0，18.5，24.9，25.1，25.7。
在本发明的一些方案中，上述式(I)化合物的晶型A，使用Cu-Kα辐射，其X射线粉末衍射图谱在下列2θ角处具有衍射峰(±0.2°)：12.8，14.0，16.0，18.5，24.9，25.1，25.7，26.9，28.5。
在本发明的一些方案中，上述式(I)化合物的晶型A，使用Cu-Kα辐射，其X射线粉末衍射图谱在下列2θ角处具有衍射峰(±0.2°)：12.8，14.0，16.0，18.5，24.9，25.1，25.7，26.9，28.5，29.8。
在本发明的一些方案中，上述式(I)化合物的晶型A，使用Cu-Kα辐射，其X射线粉末衍射图谱在下列2θ角处具有衍射峰(±0.2°)：12.8，14.0，16.0，17.0，18.5，20.5，24.9，25.1，25.7，26.9，28.5，29.8。
在本发明的一些方案中，上述式(I)化合物的晶型A，使用Cu-Kα辐射，其XRPD图谱如图1或图7所示。
在本发明的一些方案中，上述式(I)化合物的晶型A，使用Cu-Kα辐射，其XRPD图谱解析如表1或表2所示。
在本发明的一些方案中，上述式(I)化合物的A晶型，其差示扫描量热曲线在233.23±4℃处具有吸热峰。
在本发明的一些方案中，上述式(I)化合物的A晶型，其差示扫描量热曲线在234.3±4℃处具有放热峰。
在本发明的一些方案中，上述式(I)化合物的A晶型，其具有基本上如图2所示的差示扫描量热(DSC)图谱。
在本发明的一些方案中，上述式(I)化合物的A晶型，其热重分析曲线在约105-254℃之间失重达13.50±1％，例如失重达13.5111％。
在本发明的一些方案中，上述式(I)化合物的A晶型，其热重分析曲线在254.00±3℃处失重达13.50±1％，例如失重达13.5111％。
在本发明的一些方案中，上述式(I)化合物的A晶型，其具有基本上如图3所示的热重分析(TGA)图谱。
本发明还提供了式(I)化合物的晶型B，使用Cu-Kα辐射，其X射线粉末衍射图谱在下列2θ角处具有衍射峰(±0.2°)：19.19，27.18，13.44，22.55。
在本发明的一些方案中，上述式(I)化合物的晶型B，使用Cu-Kα辐射，其X射线粉末衍射图谱在下列2θ角处具有衍射峰(±0.2°)：26.19，20.76，4.42，22.84，19.19，27.18，13.44，22.55。
在本发明的一些方案中，上述式(I)化合物的晶型B，使用Cu-Kα辐射，其XRPD图谱如图4所示。
在本发明的一些方案中，上述式(I)化合物的晶型B，使用Cu-Kα辐射，其XRPD图谱解 析如表3所示。
在本发明的一些方案中，上述式(I)化合物的晶型B为式(I)化合物的DMF溶剂合物。
在本发明的一些方案中，上述式(I)化合物的晶型B中式(I)化合物与DMF溶剂比例为1:1。
本发明还提供了式(I)化合物的晶型C，使用Cu-Kα辐射，其X射线粉末衍射图谱在下列2θ角处具有衍射峰(±0.2°)：22.11，22.55，20.96，26.63。
在本发明的一些方案中，上述式(I)化合物的晶型C，使用Cu-Kα辐射，其X射线粉末衍射图谱在下列2θ角处具有衍射峰(±0.2°)：22.11，22.55，20.96，26.63，15.01，25.68，26.38，28.54，13.22，18.68。
在本发明的一些方案中，上述式(I)化合物的晶型C，使用Cu-Kα辐射，其XRPD图谱如图5所示。
在本发明的一些方案中，上述式(I)化合物的晶型C，使用Cu-Kα辐射，其XRPD图谱解析如表4所示。
在本发明的一些方案中，上述式(I)化合物的晶型C为式(I)化合物的DMSO溶剂合物。
本发明还提供了结晶形式的式(I)化合物的晶型D，使用Cu-Kα辐射，其X射线粉末衍射图谱在下列2θ角处具有衍射峰(±0.2°)：22.71，18.92，22.96，26.4，24.44，16.62，13.15，17.79，27.12。
在本发明的一些方案中，上述式(I)化合物的晶型D，使用Cu-Kα辐射，其X射线粉末衍射图谱在下列2θ角处具有衍射峰(±0.2°)：22.71，18.92，22.96，26.4，24.44，16.62，13.15，17.79，27.12，24.17，28.81，19.95，28.42，24.85，14.78，17.36，29.16。
在本发明的一些方案中，上述式(I)化合物的晶型D，使用Cu-Kα辐射，其XRPD图谱如图6所示。
在本发明的一些方案中，上述式(I)化合物的晶型D，使用Cu-Kα辐射，其XRPD图谱解析如表5所示。
在本发明的一些方案中，上述式(I)化合物的晶型D为式(I)化合物的NMP溶剂合物。
在本发明的一些方案中，上述式(I)化合物的晶型D中式(I)化合物与NMP溶剂比例为1:1。
第二方面，本申请提供了如第一方面所述的结晶形式的式(I)化合物的制备方法，包括方法一：
将式I化合物加入有机溶剂A中溶解，再将溶液加入到有机溶剂B中，析出晶体。
所述有机溶剂A选自DMF(N,N-二甲基甲酰胺)，DMSO(二甲基亚砜)，NMP(N-甲基吡咯烷酮)中的一种或多种；所述有机溶剂B选自水，甲醇，乙醇，MTBE，甲苯，二氯甲烷中的一种或多种。
在本发明的一些方案中，当式(I)化合物为晶型A时，所述方法一中，有机溶剂A选自DMF，有机溶剂B选自水，乙醇中的一种或两种；或有机溶剂A选自DMSO，有机溶剂B选自甲苯，甲醇中的一种或两种。
在本发明的一些方案中，当式(I)化合物为晶型B时，所述方法一中，有机溶剂A选自DMF，有机溶剂B选自MTBE和甲苯中的一种或两种。
在本发明的一些方案中，当式(I)化合物为晶型D时，所述方法一中，有机溶剂A选自NMP，有机溶剂B选自水，二氯甲烷，MTBE的一种或多种。
所述的结晶形式的式(I)化合物的的制备方法，包括方法二：
将式I化合物加入有机溶剂C中溶解，再加入有机溶剂D，析出晶体。
所述有机溶剂C选自DMF，DMSO，NMP中的一种或多种，所述有机溶剂D选自丙酮，乙二醇甲醚，水，二氯甲烷，乙酸乙酯，MTBE，甲苯，四氢呋喃，二氧六环，乙醇，三氟乙醇，乙腈，乙二醇二甲醚，异丙醇，丁酮中的一种或多种。
在本发明的一些方案中，当式(I)化合物为晶型A时，所述方法一中，有机溶剂C选自DMF，有机溶剂D选自丙酮，乙二醇甲醚，水，二氯甲烷中的一种或多种；或有机溶剂C选 自DMSO，有机溶剂D选自乙醇，三氟乙醇，乙二醇甲醚，乙腈，二氧六环，乙二醇二甲醚，丁酮中的一种或多种；或有机溶剂C选自NMP，有机溶剂D选自水，乙腈，二氯甲烷，乙二醇二甲醚中的一种或多种。
在本发明的一些方案中，当式(I)化合物为晶型B时，所述方法二中，有机溶剂C选自DMF，有机溶剂D选自乙酸乙酯，MTBE，甲苯，四氢呋喃，二氧六环，丁酮中的一种或多种。
在本发明的一些方案中，当式(I)化合物为晶型C时，所述方法二中，有机溶剂C选自DMSO，有机溶剂D选自乙酸乙酯，水，甲苯中的一种或多种。
在本发明的一些方案中，当式(I)化合物为晶型D时，所述方法二中，有机溶剂C选自NMP，有机溶剂D选自异丙醇，MTBE，甲苯，四氢呋喃，丁酮中的一种或多种。
所述的结晶形式的式(I)化合物的制备方法，包括方法三：
将式I化合物加入有机溶剂E混悬，打浆，过滤后得到晶体。
所述有机溶剂E选自甲醇，乙醇，异丙醇，乙酸乙酯，正庚烷，MTBE，乙二醇甲醚，水，乙腈，甲苯，二氯甲烷，四氢呋喃，二氧六环，乙二醇二甲醚，丁酮，二甲基甲酰胺，二甲基亚砜，N甲基吡咯烷酮，丙酮中的一种或多种。
在本发明的一些方案中，当式(I)化合物为晶型A时，所述方法三中，有机溶剂E选自甲醇、乙醇、异丙醇、乙酸乙酯、正庚烷、MTBE、乙二醇甲醚、水、乙腈、甲苯、二氯甲烷、四氢呋喃、二氧六环、乙二醇二甲醚、丁酮中的一种或多种。
在本发明的一些方案中，当式(I)化合物为晶型B时，所述方法三中，有机溶剂E选自DMF。
在本发明的一些方案中，当式(I)化合物为晶型C时，所述方法三中，有机溶剂E选自DMSO。
在本发明的一些方案中，当式(I)化合物为晶型D时，所述方法三中，有机溶剂E选自NMP。
第三方面，本申请提供了一种结晶组合物，包含如第一方面所述的结晶形式的式(I)化合物。优选的，所述结晶组合物中，结晶形式的式(I)化合物选自晶型A、晶型B、晶型C、晶型D中的一种或多种。
本申请的一些方案中，所述结晶形式的式(I)化合物占所述结晶组合物重量的10％以上、20％以上、30％以上、40％以上、50％以上、60％以上、70％以上、80％以上、90％以上、91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上、99％以上或99.5％以上。
优选地，所述结晶形式的式(I)化合物占所述结晶组合物重量的50％以上、60％以上、70％以上、80％以上、90％以上、91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上、99％以上或99.5％以上。
本申请的一些方案中，所述结晶组合物，包含式(I)化合物的晶型A。
本申请的一些方案中，所述晶型A占所述结晶组合物重量的10％以上、20％以上、30％以上、40％以上、50％以上、60％以上、70％以上、80％以上、90％以上、91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上、99％以上或99.5％以上。
优选地，所述晶型A占所述结晶组合物重量的50％以上、60％以上、70％以上、80％以上、90％以上、91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上、99％以上或99.5％以上。
第三方面，本申请提供了一种药物组合物，包含结晶形式的式(I)所示化合物以及任选的药学上可接受的载体。优选的，所述药物组合物中，结晶形式的式(I)化合物选自晶型A、晶型B、晶型C、晶型D中的一种或多种。
本申请的一些方案中，所述药物组合物，包含式(I)所示化合物的晶型A以及任选的药学上可接受的载体。
第四方面，本申请提供了一种药物组合物，包含如第二方面所述的结晶组合物以及任选的药学上可接受的载体。
第五方面，本申请提供了如第一方面所述的结晶形式的式(I)所示化合物、如第二方面所述的结晶组合物、或第三方面的药物组合物作为药物或在制备药物中的应用。
本申请的一些方案中，所述药物用于预防和/或治疗ENPP1介导的疾病。
本申请的一些方案中，所述药物用于预防和/或治疗癌症或肿瘤相关疾病，或者心血管疾病。
本申请的一些方案中，所述药物组合物中包含另一种、两种或多种治疗癌症或肿瘤的药物。
本申请的一些方案中，所述的药物组合物与另一种、两种或多种治疗癌症或肿瘤的药物，或治疗手段联合使用。
优选的，所述抗肿瘤药物为化疗药物、靶向治疗药物或免疫治疗药物；优选的，所述用于预防和/或治疗癌症或肿瘤的药物包括但不限于：细胞信号转导抑制剂、苯丁酸氮芥、美法仑、环磷酰胺、异环磷酰胺、白消安、卡莫司汀、洛莫司汀、链脲佐菌素、顺铂、卡铂、奥沙利铂、达卡巴嗪、替莫唑胺、丙卡巴肼、甲氨蝶呤、氟尿嘧啶、阿糖胞苷、吉西他滨、巯基嘌呤、氟达拉滨、长春碱、长春新碱、长春瑞滨、紫杉醇、多西紫杉醇、拓扑替康、伊立替康、依托泊苷、曲贝替定、更生霉素、多柔比星、表柔比星、道诺霉素、米托蒽醌、博来霉素、丝裂霉素C、伊沙匹隆、他莫昔芬、氟他胺、戈那瑞林类似物、甲地孕酮、强的松、地塞米松、甲泼尼龙、沙利度胺、干扰素α、亚叶酸钙、西罗莫司、西罗莫司脂化物、依维莫司、阿法替尼、alisertib、amuvatinib、阿帕替尼、阿西替尼、硼替佐米、波舒替尼、布立尼布、卡博替尼、西地尼布、crenolanib、克卓替尼、达拉菲尼、达可替尼、达努塞替、达沙替尼、多维替尼、厄洛替尼、foretinib、ganetespib、吉非替尼、依鲁替尼、埃克替尼、伊马替尼、iniparib、拉帕替尼、乐伐替尼、linifanib、linsitinib、马赛替尼、momelotinib、莫替沙尼、来那替尼、尼罗替尼、尼拉帕尼、oprozomib、奥拉帕尼、帕唑帕尼、pictilisib、普纳替尼、奎扎替尼、瑞格菲尼、rigosertib、卢卡帕尼、鲁索利替尼、塞卡替尼、saridegib、索拉非尼、舒尼替尼、替拉替尼、tivantinib、替沃扎尼、托法替尼、曲美替尼、凡德他尼、维利帕尼、威罗菲尼、维莫德吉、volasertib、阿仑单抗、贝伐单抗、贝伦妥单抗维多汀、卡妥索单抗、西妥昔单抗、地诺单抗、吉妥珠单抗、伊匹单抗、尼妥珠单抗、奥法木单抗、帕尼单抗、利妥昔单抗、托西莫单抗、曲妥珠单抗、PI3K抑制剂、CSF1R抑制剂、A2A和/或A2B受体拮抗剂、IDO抑制剂、抗PD-1抗体、抗PD-L1抗体、LAG3抗体、TIM-3抗体、TIGIT抗体，CD47抗体，CLAUDIN 18.2抗体、抗CTLA-4抗体或其任意组合；优选的所述治疗手段为放疗或手术。
本申请的一些方案中，所述药物用于预防和/或治疗皮肤癌、膀胱癌、卵巢癌、乳腺癌、胃癌、胰腺癌、前列腺癌、结肠癌、肺癌、骨癌、脑癌、神经细胞瘤、直肠癌、结肠癌、家族性腺瘤性息肉性癌、遗传性非息肉性结直肠癌、食管癌、唇癌、喉癌、下咽癌、舌癌、唾液腺癌、腺癌、甲状腺髓样癌、乳头状甲状腺癌、肾癌、肾实质癌、宫颈癌、子宫体癌、子宫内膜癌、绒毛膜癌、睾丸癌、泌尿癌、黑素瘤、脑肿瘤诸如成胶质细胞瘤、星形细胞瘤、脑膜瘤、成神经管细胞瘤和外周神经外胚层肿瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤、伯基特淋巴瘤、白血病、急性淋巴性白血病(ALL)、慢性淋巴性白血病(CLL)、急性骨髓性白血病(AML)、慢性粒细胞白血病(CML)、成人T细胞白血病淋巴瘤、弥漫性大B细胞淋巴瘤(DLBCL)、肝细胞癌、胆囊癌、支气管癌、小细胞肺癌、非小细胞肺癌、多发性骨髓瘤、基底细胞瘤、畸胎瘤、成视网膜细胞瘤、脉络膜黑素瘤、精原细胞瘤、横纹肌肉瘤、颅咽管瘤、骨肉瘤、软骨肉瘤、肌肉瘤、脂肪肉瘤、纤维肉瘤、尤因肉瘤和浆细胞瘤。
本申请的一些方案中，所述治疗和/或预防的心血管疾病为治疗和/或预防心肌梗塞(心梗)、 心力衰竭(心衰)、心脏创伤、心脏组织异常瘢痕的形成、心肌病、心肌细胞死亡，增强心脏修复，心肌细胞中释放促炎分子。
定义和说明
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。
本申请提及的“结晶形式的式(I)所示化合物”是指呈结晶形态的式(I)所示化合物，包括式(I)所示化合物的无水且无溶剂形式、水合物形式和溶剂合物形式。所述结晶形式优选为无水且无溶剂形式或水合物形式；进一步优选为无水且无溶剂形式。
术语“溶剂化物”或“溶剂合物”是指化学计量比或非化学计量比的溶剂分子与本申请的式(I)所示化合物所形成的缔合物，包括同时含有水分子和一种或多种其它溶剂分子的缔合物，及仅含一种或多种其它溶剂分子的缔合物。
术语“水合物”是指化学计量比或非化学计量比的水分子与本申请的式(I)所示化合物所形成的缔合物。
所述“无水且无溶剂形式”是指晶胞或晶格中不含水分子或溶剂分子，或者水分子或溶剂分子以非分子间力结合的方式与式(I)所示化合物共存，例如吸附的方式。
术语“结晶组合物”指的是一种固体形式，其包含本申请提及的晶型A。而且，除了本申请的晶型以外，结晶组合物还可以任选地包含其它晶型或其它无定型形式的式(I)所示化合物或其盐，或者除了这些物质以外的杂质。本领域技术人员应当理解，结晶组合物中各成分的含量之和应当为100％。
所述“室温”为本领域常规意义上的室温温度，一般为10～30℃，优选25℃±5℃。
在X-射线粉末衍射图谱中，术语“基本上”或者“基本上如图所示”是指基本上纯净的某种晶型，其粉末X-射线衍射图谱中至少50％，或至少60％，或至少70％，或至少80％，或至少90％，或至少95％，或至少96％，或至少97％，或至少98％，或至少99％的峰出现在所给图谱中。进一步的，当产品中某种晶型的含量逐渐降低时，其X-射线粉末衍射图谱中的一些归属于该晶型的衍射峰可能会由于仪器的检测灵敏度的因素而变少。此外，对任何给定的晶型而言，峰的位置可能存在轻微误差，这在晶体学领域中也是公知的。例如，由于分析样品时温度的变化、样品移动或仪器的标定等，峰的位置可以移动，2θ值的测定误差有时约为±0.3°，通常约为±0.2°。因此，在确定每种晶型结构时，应该将此误差考虑在内，术语“基本上”或者“基本上如附图所示”也意图涵盖衍射峰位中的这样的差异性，是指±0.3°，优选±0.2°，进一步优选±0.1°。
在DSC图谱中，术语“基本上”或者“基本上如图所示”是指对于同种化合物的同种晶型，在连续的分析中，热转变起始温度、吸热峰峰值温度、放热峰峰值温度、熔点等的误差典型的在约8℃，通常在约5℃，通常约在3℃之内。当描述某个化合物具有某一给定的热转变起始温度、吸热峰峰值温度、放热峰峰值温度、熔点等时，指的是该温度±5℃。
在本文使用的术语“预防”是指当用于疾病或病症(例如病毒性疾病)时，与未施用化合物或药物(例如，本申请要求保护的组合产品)的受试者相比，所述化合物或药物能降低受试者体内的医学病症症状的频率或推迟其发病。
在本文中使用的术语“治疗”是指减轻、缓解或改善疾病或病症的症状，改善潜在的代谢引起的症状，抑制疾病或症状，例如阻止疾病或病症的发展、缓解疾病或病症、引起疾病或病症的消退、缓解疾病或病症引起的病况、或阻止疾病或病症的症状。
本申请的药物组合物可以采用本领域的常规方法制备得到。
在本申请的上下文中，术语“药学上可接受的载体”或“赋形剂”或“药学上可接受的辅料”或“药用可接受的辅料”是指对有机体无明显刺激作用，而且不会损害该活性化合物的生物活 性及性能的那些辅料。术语“药用可接受的辅料”包括：溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、黏合剂、崩解剂、填充剂、润滑剂、润湿剂、渗透压调节剂、稳定剂、助流剂、矫味剂、防腐剂、助悬剂、包衣材料、芳香剂、抗黏着剂、抗氧剂、螯合剂、渗透促进剂、pH值调节剂、缓冲剂、增塑剂、表面活性剂、发泡剂、消泡剂、增稠剂、包合剂、保湿剂、吸收剂、稀释剂、絮凝剂与反絮凝剂、助滤剂、释放阻滞剂等。本领域技术人员可根据实际需要选择具体的药用可接受的辅料。有关辅料的知识是本领域技术人员众所周知的，例如可以参考《药剂学》(崔福德主编，第5版，人民卫生出版社，2003)。
词语“包括(comprise)”或“包含(comprise)”及其英文变体例如comprises或comprising应理解为开放的、非排他性的意义，即“包括但不限于”。
在本申请的范围中，任一特征的各种选项可以与其它特征的各种选项相互组合，从而构成许多不同的实施方案。本申请意欲包括由所有技术特征的各种选项所组成的所有可能的实施方案。
本申请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申请的实施例。
本申请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申请的化学变化及其所需的试剂和物料。为了获得本申请的化合物，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。
下面会通过实施例具体描述本申请，这些实施例并不意味着对本申请的任何限制。
本申请所使用的所有试剂、溶剂是市售的，无需进一步纯化即可使用。
技术效果
本发明的式(I)化合物具有较好的PK性质及口服生物利用度，其晶型稳定好，成药前景良好。式(I)化合物对ENPP1展现出较好的抑制活性；式(I)化合物药代动力学指标良好。
本发明提供的结晶形式的式(I)所示化合物，以及具体晶型，它们具有以下一种或多种有益效果：(1)结晶形式的式(I)所示化合物具有良好的性状，便于称量、转移、分离、纯化和储存；(2)结晶形式的式(I)所示化合物及具体晶型具有良好的结晶度；(3)结晶形式的式(I)所示化合物及具体晶型可操作性能优良，例如，易于纯化、过滤和分离，特别是晶型A，其制备简便，收率高；(4)优选晶型具备良好的物理稳定性和化学稳定性，特别是晶型A，纯度高，可直接作为原料药使用，具有良好的药用前景。
1、仪器及分析方法
1.1.粉末X-射线衍射(X-ray powder diffractometer,XRPD)
仪器型号：D8 Advance
测试条件：中国药典四部通则0451
X-ray发生器：铜靶
管电压：40KV
散射狭缝：0.6mm
探测器狭缝：10.5mm
防散射狭缝：2.5°
扫描范围：3-50°
步径：0.1s/step
步长：0.02°
1.2.差热分析(Differential Scanning Calorimeter,DSC)
仪器型号：DSC 3
测试条件：起始温度30℃，以10℃/min的升温速率加热至300℃；吹扫气流速：50ml/min；
干燥气流速：150ml/min；吹扫气体：N2；坩埚：铝坩埚。
1.3.热重分析(Thermal Gravimetric Analyzer,TGA)
仪器型号：TGA 2
测试条件：起始温度30℃，以10℃/min的升温速率加热至350℃；天平保护气流速：
20ml/min；反应气流速：50ml/min；吹扫气体：N2；坩埚：氧化铝坩埚。
1.4.动态气体吸附仪(DVS)
仪器型号：DVS Intrinsic
测试方法：25℃0％RH-90％RH-0％RH；参考中国药典四部通则0103
平衡dm/dt：0.002
RH(％)测量梯度：10％一个台阶
RH(％)测量梯度范围：0％～90％
引湿性特征描述与引湿性增重的界定：
1.5水和生物介质中的晶型稳定性测试
生物介质的配制过程如下表所示。晶型样品加入生物介质和水中在37℃恒温震荡24h，分别于1h、4h和24h取样，将取样的溶液用0.22μm水系滤膜过滤，对部分浓度较高的样品用稀释剂进行适当稀释，用HPLC测量溶液的信号峰面积，最后根据峰面积、原料的HPLC标准曲线和稀释倍数计算溶液中化合物的浓度。此外，取不同时间点样品测试其pH值，对剩余固体进行XRPD测试。
备注：FaSSIF：模拟人类餐前饥饿状态下小肠内的肠液；FeSSIF：模拟人类餐后饱食状态下小肠内的肠液；FaSSGF：模拟人类饥饿状态下空胃时的胃液。
附图说明：
图1为实施例2制备的式(I)化合物晶型A的XRPD图谱。
图2为式(I)化合物的A晶型DSC图谱。
图3为式(I)化合物的A晶型TGA图谱。
图4为实施例4制备的式(I)化合物晶型B的XRPD图谱。
图5为实施例5制备的式(I)化合物晶型C的XRPD图谱。
图6为实施例6制备的式(I)化合物晶型D的XRPD图谱。
图7为实施例3制备的式(I)化合物的晶型A的XRPD图谱。
图8为式(I)化合物小鼠急性心肌缺血模型心脏功能指标及体重变化，A)小鼠左心室射血分数；B)小鼠左心室短轴缩短率；C)小鼠体重变化；
图9为式(I)化合物小鼠急性心肌缺血模型心肌纤维化变化，D)小鼠心脏组织Masson染色病理切片；E)小鼠心肌纤维化程度；
图10为式(I)化合物大鼠急性心肌缺血模型心脏功能指标及体重变化，a)大鼠左心室短轴缩短率；b)大鼠左心室射血分数；c)大鼠体重变化；
图11为式(I)化合物大鼠急性心肌缺血模型心肌纤维化变化，d)大鼠心脏组织Masson染色病理切片；e)大鼠心肌纤维化程度；
图12为晶型A稳定性实验前后XRPD图谱对比。
注：图示中出现的“化合物1”即式(I)化合物。
具体实施方式
为了更好的理解本发明的内容，下面结合具体实施例来做进一步的说明，但具体的实施方式并不是对本发明的内容所作的限制。
实施例1式(I)化合物的合成
第一步：4-氯-7-甲氧基-1,8-二氮杂萘-3-羧酸乙酯的制备
将2-(((6-甲氧基吡啶-2-基)氨基)亚甲基)丙二酸1,3-二乙基酯(12g,22.7mmol,1eq.)溶于三氯氧磷(60mL)，反应在氮气保护下110℃搅拌反应4小时。LCMS监测反应结束，将溶剂减压蒸馏除去，所得残渣用乙酸乙酯(60mL)溶清，加入水(20mL)迅速清洗，有机相用无水硫酸钠干燥，过滤，滤液旋干。得到的粗产品用快速色谱法分离纯化(Silica gel,DCM:EA＝1:1)得到目标化合物(1.5g,收率12％)。LCMS(ESI)[M+H]+＝266.0。
第二步：4-((4-溴-2,6-二氟苯甲基)氨基)-7-甲氧基-1,8-二氮杂萘-3-羧酸乙酯的制备
将4-氯-7-甲氧基-1,8-二氮杂萘-3-羧酸乙酯(3.0g,18.75mmol,1eq.)溶于乙腈(80mL)，然后加入碳酸钾(5.2g,37.5mmol,2.0eq.)，(4-溴-2,6-二氟苯基)甲胺(4.37g,19.69mmol,1.05eq.)，反应在氮气保护下在40℃进行20小时。LCMS监测反应完成，将反应液浓缩，用乙酸乙酯稀释，水洗后，有机相用无水硫酸钠干燥，过滤，滤液旋干。得到的粗产品用快速色谱法分离纯化(Silica gel,DCM:EA＝4:1)得到目标化合物(3.5g,收率69.2％)。LCMS(ESI)[M+H]+＝452.0。
第三步：(4-((4-溴-2,6-二氟苯甲基)氨基)-7-甲氧基-1,8-二氮杂萘-3-基)甲醇的制备
将4-((4-溴-2,6-二氟苯甲基)氨基)-7-甲氧基-1,8-二氮杂萘-3-羧酸乙酯(120mg,22.7mmol,1eq.)溶于乙醇(10mL)，缓慢加入硼氢化钠(134mg,5eq.)，反应在氮气保护下50℃搅拌反应16小时。LCMS监测反应结束，将溶剂旋干，所得残渣用饱和氯化钠溶液(5mL)淬灭反应，加入乙酸乙酯(10mL)萃取，有机相用无水硫酸钠干燥，过滤，滤液旋干。得到的粗产品用快 速色谱法分离纯化(Silica gel,DCM:MeOH＝2:1)得到目标化合物(120mg,收率38％)。LCMS(ESI)[M+H]+＝410.0；1H NMR(400MHz,CDCl3)δ8.43(s,1H),8.35(d,J＝9.2Hz,1H),7.09(d,J＝6.8Hz,2H),6.90(d,J＝9.2Hz,1H),4.75(s,2H),4.69(s,2H),4.12(s,3H).
第四步：1-(4-溴-2,6-二氟苯甲基)-8-甲氧基-1,4-二氢-2H-[1,3]噁嗪并[5,4-c][1,8]二氮杂萘-2-酮的制备
将(4-((4-溴-2,6-二氟苯甲基)氨基)-7-甲氧基-1,8-二氮杂萘-3-基)甲醇(300mg,0.73mmol,1eq.)溶于二氯甲烷(15mL)中，加入N,N-二异丙基乙胺(473mg,3.66mmol,5eq.)和三光气(651mg,2.19mmol,3eq.)，在0℃下反应2小时。
LCMS监测反应完全。将反应液用水洗涤，用无水硫酸钠干燥，过滤，滤液旋干。得到的粗产品用快速硅胶柱层析(Silica gel,MeOH:DCM＝5：95)分离纯化，得到目标化合物(250mg,收率78.51％)。LCMS(ESI)[M+H]+＝436.0.
第五步：1-(2,6-二氟-4-((4-甲氧苄基)硫基)苯甲基)-8-甲氧基-1,4-二氢-2H-[1,3]噁嗪并[5,4-c][1,8]二氮杂萘-2-酮的制备
将1-(4-溴-2,6-二氟苯甲基)-8-甲氧基-1,4-二氢-2H-[1,3]噁嗪并[5,4-c][1,8]二氮杂萘-2-酮(200mg,0.46mmol,1eq.)溶于二氧杂环己烷(10mL)中，加入(4-甲氧基苯基)甲硫醇(141mg,0.92mmol,2eq.)，[5-(二苯基膦基)-9,9-二甲基-9H-黄原-4-基]二苯基膦(53mg,0.09mmol,0.2eq.)，三(1,5-二苯基五-1,4-二烯-3-酮)二钯(84mg,0.09mmol,0.2eq.)和N,N-二异丙基乙胺(178mg,1.38mmol,3eq.)。在90℃下反应2小时，LCMS监测反应完全。将反应液浓缩旋干后，用快速硅胶柱层析(Silica gel,MeOH:DCM＝5：95)分离纯化，得到目标化合物(110mg,收率47.09％)。LCMS(ESI)[M+H]+＝510.1.
第六步：3,5-二氟-4-((8-甲氧基-2-氧代-2H-[1,3]噁嗪并[5,4-c][1,8]二氮杂萘-1(4H)-基)甲基)苯磺酰胺的制备
将1-(2,6-二氟-4-((4-甲氧苄基)硫基)苯甲基)-8-甲氧基-1,4-二氢-2H-[1,3]噁嗪并[5,4-c][1,8]二氮杂萘-2-酮(100mg,0.2mmol,1.0eq.)，乙酸(83mg,1.37mmol,7.0eq.)，水(50mg,2.75mmol,14.0eq.)溶于四氢呋喃(15mL)中，在0℃加入二氯海因(116mg,0.59mmol,3eq.)，混合液于25℃反应1小时。加入氨水(0.5mL)后于25℃反应10分钟。LCMS监测原料反应完全。反应液浓缩后经制备HPLC纯化得到目标化合物(14.6mg,收率17.05％)。LCMS(ESI)[M+H]+＝437.1；1HNMR(400MHz,MeOD-d4)δ8.71(s,1H),8.59(d,J＝9.2Hz,1H),7.46(d,J＝7.6Hz,2H),7.13(d,J＝9.6Hz,1H),5.57(s,2H),5.30(s,2H),4.12(s,3H).
实施例2式(I)化合物A晶型的制备
向反应瓶中依次加实施例1制备的式(I)化合物(100mg)和二甲基亚砜(0.3mL)，缓慢加热溶清，80℃下将其缓慢加入到水(1mL)中。缓慢降温至室温后搅拌一小时，过滤，用适量水洗滤饼，滤饼在65℃下减压干燥得式(I)化合物A晶型。
表1式(I)化合物的A晶型XRPD图谱解析数据
实施例3式(I)化合物A晶型的制备
向反应瓶中加入实施例1制备的式(I)化合物(100mg)，再加入丙酮(1mL)，室温后搅拌24小时混悬打浆；过滤，滤饼在45℃下减压干燥得式(I)化合物A晶型。
表2式(I)化合物的A晶型XRPD图谱解析数据
实施例4式(I)化合物B晶型的制备
称取260mg样品，室温下溶于10mL的DMF中，取0.8mL溶液，滴加甲基叔丁基醚约8mL。室温搅拌一定时间后将有固体析出的体系离心分离后，并将固体室温真空干燥得到晶型B。
表3式(I)化合物的B晶型XRPD图谱解析数据
实施例5式(I)化合物C晶型的制备
称取260mg样品，室温下溶于4mL的DMSO中，取0.3mL溶液，滴加甲苯约3mL。室温搅拌一定时间后将有固体析出的体系离心分离后，并将固体室温真空干燥得到晶型C。
表4式(I)化合物的C晶型XRPD图谱解析数据
实施例6式(I)化合物D晶型的制备
称取260mg样品，室温下溶于10mL的NMP中，取0.8mL溶液，滴加甲基叔丁基醚约8mL。室温搅拌一定时间后将有固体析出的体系离心分离后，并将固体室温真空干燥得到晶型D。
表5式(I)化合物的D晶型XRPD图谱解析数据
测试例1晶型的吸湿性研究
实验仪器：DVS Intrinsic
实验方法：取实施例2制备的式化合物(I)的晶型A 25-35mg置于DVS样品盘内进行测试。
实验结论：式化合物(I)的晶型A图谱在25℃，80％湿度时增重ΔW＝0.4461％，略有引湿性。DVS测试后晶型未发生变化。
测试例2：固体稳定性研究
称取实施例2制备的式化合物(I)的晶型A样品20mg于小瓶中，在高温(60℃)、长期(25℃/60％RH)和加速(40℃/75％RH)条件下分别敞口放置7天，取样品分别进行纯度检测和X-射线粉末衍射，考察实施例2(晶型A)在不同条件下的稳定性，结果见表6和图12。
表6固体稳定性实验结果
数据表明：实施例2(晶型A)在固体稳定性试验中均能保持化学稳定和晶型稳定。
测试例3：式(I)化合物晶型A在生物溶媒介质中的稳定性测试
取实施例2(晶型A)样品在3种生物介质(FaSSIF、FeSSIF和FaSSGF)和水中进行生物溶媒介质中的稳定性测试，取24h后剩余样品进行X-射线粉末衍射，结果见表7：
表7生物溶媒介质中的稳定性测试
数据表明：实施例2(晶型A)在生物溶媒介质中均能保持晶型稳定。
测试例4体外ENPP1酶学抑制测试
ENPP1是一种能够水解核苷酸和具有核苷酸-5’-单磷酸结构衍生物的跨膜糖蛋白。ENPPl能够将人工合成的5'-单磷酸对硝基苯基酯(TMP-pNP)水解成核苷酸'-单磷酸和对硝基苯酚，而对硝基苯酚为显色产物。对硝基苯酚产物的生成量可通过其在405nm处的吸光度直接测定，该与酶活性成正比。
实验步骤
用DMSO配制100x连续梯度稀释的化合物，1mM起始，4倍连续梯度稀释，10个浓度点。用Echo转移300nL梯度浓度的化合物至384孔检测板(终浓度为10μM起始，4倍连续梯度稀释，10个浓度点)，每孔中先加入15μL用实验缓冲液(250mM NaCl，50mM Tris，pH 9.5)配制的0.2ng/μL hENPP1酶(2x终浓度)。然后加入15μL用实验缓冲液配制的400μM TMP-pNP(2x终浓度)，37℃孵育0.5小时后，酶标仪OD405nm读值。公式计算百分抑制率：％抑制率＝(OD高信号对照-OD样品孔)/(OD高信号对照-OD低信号对照)*100，进行四参数拟合计算IC50值，高信号对照：无抑制剂DMSO组，低信号对照：空白对照组。实施例1制备的式(Ⅰ)化合物的数据在表8中示出。
表8 ENPP1酶抑制活性
测试例5：应用LC-MS/MS测定小鼠中化合物浓度的药代动力学试验
测试原理：使用LC-MS/MS测定目标药物在不同时间下血浆中的药物浓度，绘制出目标化合物在体内的药代动力曲线。
试验方法：DMSO溶解待测化合物(实施例1制备的式(I)化合物)，配制为终浓度20mg/mL的储备液，使用含5％DMSO(Sigma-Aldrich,SHBJ2847)，45％PEG400(Sigma-Aldrich,BCCC0015)及50％dd H2O的溶剂将上述化合物溶解为1mg/mL。小鼠来源为CD-1male(JH Laboratory Animal Co.LTD)，每组化合物取9只小鼠，10mg/kg PO给药，分别于0.25hr、0.5hr、1hr、2hr、4hr、8hr和24hr交叉采血，每个时间采样3个，采全血110μL(K2EDTA抗凝)，立刻将样本进行2000g 4°离心5分钟，收集血清，-70°保存。使用Triple-quadrupole MS system(SCIEX)进行血药浓度测定，包括标曲和质控制备和样品制备。标曲和质控配制：用MeOH:H2O(1:1)稀释配制工作液，在57μL空白血浆中加入上述3μL标曲和质控工作溶液。样本制备：取30μL血浆样品中加入200μL内标溶液(Propranolol，40ng/mL)，混匀1分钟，5800rpm离心10分钟，取100μL上清并转移至新板中进样分析。色谱条件根据样品优化各自的流动相组成、洗脱梯度条件、流速和保留时间等，色谱柱为Waters BEH C18(2.1×50mm,1.7μm)，进样体积1μL。质谱采用电喷雾离子源(TuREo spray)，在正离子检测模式下，选择多通道反应监测(MRM)模式进行二级质谱分析。根据药物浓度-时间数据，采用WinNonlin 8.2软件按非房室模型计算药动学参数，包括达峰浓度Cmax、达峰时间Tmax、药-时曲线下面积AUC、消除半衰期t1/2。AUC计算方式为线性梯形法(linear up log down)。
表9药代动力学参数如下：

试验结果表明本发明式(Ⅰ)化合物有良好的体内药代动力学，具有成药潜质。
测试例6：式(Ⅰ)化合物在小鼠胰腺癌Pan02模型中的药效学评价
1、实验目的：评价受试药在小鼠胰腺癌Pan02细胞皮下同种移植肿瘤模型上的体内药效。
2、试验方法：
2.1细胞培养：小鼠胰腺癌Pan02细胞体外单层培养，培养条件为DMEM培养基中加10％胎牛血清，0.01mg/ml sinsulin，1％青霉素/链霉素/两性霉素B，37℃ 5％CO2孵箱培养。当细胞饱和度为80％-90％，数量到达要求时，收取细胞，计数，接种。
2.2动物：C57BL/6小鼠，雌性，6-8周龄，体重18-22克。由上海西普尔-必凯实验动物有限公司或其他供应商提供。
2.3肿瘤接种及动物分组给药：将0.1mL(5×106个)Pan02细胞皮下接种于每只小鼠的右后背，肿瘤平均体积达到约100-200mm3时开始分组给药。给药前称重动物，测量瘤体积。根据瘤体积随机分组，每组8只，对照组给予溶媒：5％DMSO+45％PEG400+50％H20(1％CMC-Na)，实验组剂量20mg/kg，给予口服灌胃给药，给药容积根据小鼠体重10μl/g，每天两次。每日观察动物健康状况，若瘤体积超过3,000mm3，或有严重疾病，或疼痛，体重降低大于20％，持续恶化，须处以安乐死。
2.4肿瘤生长抑制率：每周两次用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5a×b2，a和b分别表示肿瘤的长径和短径。化合物的抑瘤疗效TGI(％)＝[(1-(处理组给药后平均瘤体积－该处理组开始给药时平均瘤体积))/(溶剂对照组处理前平均瘤体积－溶剂对照组处理后平均瘤体积)]×100％。数据分析采用T检验用于两组间比较，p<0.05认为有著性差异。
3、实验结果：
本发明实施例1制备的式(Ⅰ)化合物在小鼠胰腺癌Pan02模型显示出的活性。本发明的式(Ⅰ)化合物给药16天，能显著抑制肿瘤的生长，TGI为43.4％，与对照组有显著统计学差异(p＜0.05)，且对小鼠体重没有抑制，安全性较好。结晶形式的式(I)化合物(如式(I)化合物晶型A)具有基本相同的药效。
测试例7：化合物在小鼠心肌缺血模型中的药效学评价
1.实验目的
本研究目的是在C57 BL/6小鼠心肌缺血模型中评价化合物的药效。
2.试验方法
动物到达设施适应一周后，术前一天根据动物体重分成4组，假手术组(Sham)6只，模型组(MI)8只动物，第二天接受心肌缺血手术造模(Sham组小鼠仅开胸穿线，不进行结扎)，采用结扎左冠状动脉前降支(LAD)方法造模，造模后给予青霉素(20,000单位/小鼠i.m)治疗3天，消毒以防止感染。
术前1小时各组动物分别给予对应化合物。对照组(Vehicle)给予溶媒：5％DMSO+45％PEG400+50％H2O(1％CMC-Na)，实验组剂量分别为4mg/kg和20mg/kg，给予口服灌胃给药，每天两次，连续给药28天。给药后每日监测动物体重，第1天、第7天和第28天测量心脏超声影像学指标。心超评价主要指标为左室短轴缩短分数(FS)和左室射血分数(LVEF)。第28天心超测量结束后，采集心脏组织于组织固定液中用于Masson三色染色病理分析。
3.实验结果
本发明所示实施例1的化合物(I)在小鼠心梗模型中显示出较好的心脏功能改善活性。本发 明的式(I)化合物在造模前一天预给药后，对心脏功能具有保护作用。如图5所示，第1天，4mpk和20mpk与模型溶媒对照组比，能将小鼠左心室射血分数LVEF分别提高15％，10％(图A)和左心室短轴缩短率FS分别提高7％，5％(图B)。第7天，能将小鼠左心室射血分数LVEF分别提高23.4％，23.3％和左心室短轴缩短率FS分别提高12.2％，12.3％。第28天，模型溶媒对照组LVEF和FS进一步恶化，化合物(I)能进一步维持小鼠的左室LVEF和FS，且对体重无明显影响(图C)。统计显著性采用双因素方差分析/单因素方差分析/t检验分析，p<0.05认为有显著性差异。
本发明所示实施例1的式(I)化合物对心肌梗死小鼠心肌纤维化显示出改善作用。如图6所示，图D为小鼠心脏组织Masson染色病理切片的电镜图。图E为小鼠心脏组织纤维化程度统计结果。如图所示，假手术组，小鼠红色心肌纤维排列整齐，蓝色胶原纤维少见。MI模型对照组，心肌细胞坏死，心肌组织结构紊乱，梗死区可见大面积蓝色胶原纤维。式(I)化合物给药组小鼠心肌组织结构有一定程度改善，梗死区红色心肌细胞与蓝色胶原交互分布，胶原纤维明显减少，表明式(I)化合物在28天给药可减轻MI后心肌纤维化程度，且与模型溶媒对照组比，能将纤维化程度分别降低8.8％和9.6％(图E)。式(I)化合物晶型A具有基本相同的药效。
测试例8：化合物在大鼠心肌缺血模型中的药效学评价
1.实验目的：本研究目的是在SD大鼠心肌缺血模型中评价化合物的药效。
2.试验方法
动物到达设施适应一周后，采用结扎左冠状动脉前降支(LAD)方法造模，造模后给予青霉素(80,000单位/大鼠i.m)治疗3天，消毒以防止感染。
模型建立一天后，将成模的动物按基线体重和心脏超声影像学指标随机分为4组，假手术组(Sham)每组4只动物，心梗模型组(MI)每组6只动物，开始给药。对照组给予空白溶媒Vehicle：5％DMSO+45％PEG400+50％H2O(1％CMC-Na)，实验组剂量分别为30mg/kg和75mg/kg，给予口服灌胃给药，每天两次，连续给药28天，给药后每日监测动物体重，第7天和第28天测量心脏超声影像学指标。心超评价主要指标为左室短轴缩短分数(FS)和左室射血分数(LVEF)。第28天心超测量结束后，采集心脏组织于组织固定液中用于Masson三色染色病理分析。
3.实验结果
本发明实施例1制备的式(I)化合物在大鼠心梗模型显示出较好的心脏功能改善活性(图7a、b)。本发明的式(I)化合物，给药第7天，能显著提高大鼠左心室射血分数LVEF和左心室短轴缩短指数FS，30mpk和75mpk组将LVEF分别提高了11.43％，18.85％和左心室短轴缩短率FS分别提高了9.73％，12.62％。第28天，30mpk和75mpk剂量组能将LVEF分别提高了18.75％，23.8％和左心室短轴缩短率FS分别提高了13.01％，15.88％。而且呈现出剂量依赖性趋势且对体重无明显影响(图c)。统计显著性采用t检验分析，p<0.05认为有显著性差异。
本发明实施例1制备的式(I)化合物对心肌梗死大鼠心肌纤维化显示出改善作用。如图d为大鼠心脏组织Masson染色病理切片的电镜图。图e为大鼠心脏组织纤维化程度统计结果。如图8所示，假手术组，大鼠红色心肌纤维排列整齐，蓝色胶原纤维少见。MI模型对照组，心肌细胞坏死，心肌组织结构紊乱，梗死区可见大面积蓝色胶原纤维。式(I)化合物给药组大鼠心肌组织结构有一定程度改善，梗死区红色心肌细胞与蓝色胶原交互分布，胶原纤维明显减少，表明式(I)化合物在28天给药可减轻MI后心肌纤维化程度，且与模型溶媒对照组比，能将纤维化程度分别降低5.2％和6.5％(图e)。式(I)化合物晶型A具有基本相同的药效。

Claims (10)

  1. 结晶形式的式(I)所示化合物，
  2. 根据权利要求1所述的结晶形式的式(I)所示化合物，其特征在于，所述的结晶形式的式(I)所示化合物为晶型A，使用Cu-Kα辐射，其X-射线粉末衍射图谱包含在下列2θ角处的衍射峰(±0.2°)：24.9，25.1，25.7；
    或者，使用Cu-Kα辐射，其X-射线粉末衍射图谱包含在下列2θ角处的衍射峰(±0.2°)：12.8，14.0，16.0，18.5，24.9，25.1，25.7；
    或者，使用Cu-Kα辐射，其X-射线粉末衍射图谱包含在下列2θ角处的衍射峰(±0.2°)：12.8，14.0，16.0，18.5，24.9，25.1，25.7，26.9，28.5；
    或者，使用Cu-Kα辐射，其X-射线粉末衍射图谱包含在下列2θ角处的衍射峰(±0.2°)：12.8，14.0，16.0，18.5，24.9，25.1，25.7，26.9，28.5，29.8；
    或者，使用Cu-Kα辐射，其X-射线粉末衍射图谱包含在下列2θ角处的衍射峰(±0.2°)：12.8，14.0，16.0，17.0，18.5，20.5，24.9，25.1，25.7，26.9，28.5，29.8；
    或者，其具有基本上如图1或图7所示的X-射线粉末衍射图谱
    或者，使用Cu-Kα辐射，其XRPD图谱解析如表1或表2所示；
    或者，其差示扫描量热曲线在233.23±4℃处具有吸热峰；
    或者，其差示扫描量热曲线在234.3±4℃处具有放热峰；
    或者，其具有基本上如图2所示的差示扫描量热(DSC)图谱。
    或者，其热重分析曲线在约105-254℃之间失重达13.50±1％，例如失重达13.5111％。
    或者，其热重分析曲线在254.00±3℃处失重达13.50±1％，例如失重达13.5111％。
    或者，上述式(I)化合物的A晶型，其具有基本上如图3所示的热重分析(TGA)图谱。
    或者，所述的结晶形式的式(I)所示化合物为晶型B，使用Cu-Kα辐射，其X射线粉末衍射图谱在下列2θ角处具有衍射峰(±0.2°)：19.19，27.18，13.44，22.55；
    或者，所述晶型B，使用Cu-Kα辐射，其X射线粉末衍射图谱在下列2θ角处具有衍射峰(±0.2°)：26.19，20.76，4.42，22.84，19.19，27.18，13.44，22.55；
    或者，所述晶型B，使用Cu-Kα辐射，其XRPD图谱如图4所示；
    或者，所述晶型B，使用Cu-Kα辐射，其XRPD图谱解析如表3所示；
    或者，所述晶型B为式(I)化合物的DMF溶剂合物；
    或者，所述的结晶形式的式(I)所示化合物为晶型C，使用Cu-Kα辐射，其X射线粉末衍射图谱在下列2θ角处具有衍射峰(±0.2°)：22.11，22.55，20.96，26.63；
    或者，所述晶型C，使用Cu-Kα辐射，其X射线粉末衍射图谱在下列2θ角处具有衍射峰(±0.2°)：22.11，22.55，20.96，26.63，15.01，25.68，26.38，28.54，13.22，18.68；
    或者，所述晶型C，使用Cu-Kα辐射，其XRPD图谱如图5所示；
    或者，所述晶型C，使用Cu-Kα辐射，其XRPD图谱解析如表4所示；
    或者，所述晶型C为式(I)化合物的DMSO溶剂合物；
    或者，所述的结晶形式的式(I)所示化合物为晶型C，使用Cu-Kα辐射，其X射线粉末衍射图谱在下列2θ角处具有衍射峰(±0.2°)：22.71，18.92，22.96，26.4，24.44，16.62，13.15， 17.79，27.12；
    或者，所述晶型D，使用Cu-Kα辐射，其X射线粉末衍射图谱在下列2θ角处具有衍射峰(±0.2°)：22.71，18.92，22.96，26.4，24.44，16.62，13.15，17.79，27.12，24.17，28.81，19.95，28.42，24.85，14.78，17.36，29.16；
    或者，所述晶型D，使用Cu-Kα辐射，其XRPD图谱如图6所示；
    或者，所述晶型D，使用Cu-Kα辐射，其XRPD图谱解析如表5所示；
    或者，所述晶型D为式(I)化合物的NMP溶剂合物。
  3. 根据权利要求1或2所述结晶形式的式(I)化合物的制备方法，包括方法一：
    将式I化合物加入有机溶剂A中溶解，再将溶液加入到有机溶剂B中，析出晶体。
    所述有机溶剂A选自DMF(N,N-二甲基甲酰胺)，DMSO(二甲基亚砜)，NMP(N-甲基吡咯烷酮)中的一种或多种；所述有机溶剂B选自水，甲醇，乙醇，MTBE，甲苯，二氯甲烷中的一种或多种；
    在本发明的一些方案中，当式(I)化合物为晶型A时，所述方法一中，有机溶剂A选自DMF，有机溶剂B选自水，乙醇中的一种或两种；或有机溶剂A选自DMSO，有机溶剂B选自甲苯，甲醇中的一种或两种；
    在本发明的一些方案中，当式(I)化合物为晶型B时，所述方法一中，有机溶剂A选自DMF，有机溶剂B选自MTBE和甲苯中的一种或两种；
    在本发明的一些方案中，当式(I)化合物为晶型D时，所述方法一中，有机溶剂A选自NMP，有机溶剂B选自水，二氯甲烷，MTBE的一种或多种；
    或包括方法二：
    将式I化合物加入有机溶剂C中溶解，再加入有机溶剂D，析出晶体；
    所述有机溶剂C选自DMF，DMSO，NMP中的一种或多种，所述有机溶剂D选自丙酮，乙二醇甲醚，水，二氯甲烷，乙酸乙酯，MTBE，甲苯，四氢呋喃，二氧六环，乙醇，三氟乙醇，乙腈，乙二醇二甲醚，异丙醇，丁酮中的一种或多种；
    优选的，当式(I)化合物为晶型A时，所述方法一中，有机溶剂C选自DMF，有机溶剂D选自丙酮，乙二醇甲醚，水，二氯甲烷中的一种或多种；或有机溶剂C选自DMSO，有机溶剂D选自乙醇，三氟乙醇，乙二醇甲醚，乙腈，二氧六环，乙二醇二甲醚，丁酮中的一种或多种；或有机溶剂C选自NMP，有机溶剂D选自水、乙腈、二氯甲烷、乙二醇二甲醚中的一种或多种；
    优选的，当式(I)化合物为晶型B时，所述方法二中，有机溶剂C选自DMF，有机溶剂D选自乙酸乙酯，MTBE，甲苯，四氢呋喃，二氧六环，丁酮中的一种或多种；
    优选的，当式(I)化合物为晶型C时，所述方法二中，有机溶剂C选自DMSO，有机溶剂D选自乙酸乙酯，水，甲苯中的一种或多种；
    优选的，当式(I)化合物为晶型D时，所述方法二中，有机溶剂C选自NMP，有机溶剂D选自异丙醇，MTBE，甲苯，四氢呋喃，丁酮中的一种或多种；
    或包括方法三：
    将式I化合物加入有机溶剂E混悬，打浆，过滤后得到晶体；
    所述有机溶剂E选自甲醇，乙醇，异丙醇，乙酸乙酯，正庚烷，MTBE，乙二醇甲醚，水，乙腈，甲苯，二氯甲烷，四氢呋喃，二氧六环，乙二醇二甲醚，丁酮，二甲基甲酰胺，二甲基亚砜，N甲基吡咯烷酮，丙酮中的一种或多种；
    优选的，当式(I)化合物为晶型A时，所述方法三中，有机溶剂E选自甲醇、乙醇、异丙醇、乙酸乙酯、正庚烷、MTBE、乙二醇甲醚、水、乙腈、甲苯、二氯甲烷、四氢呋喃、二氧六环、乙二醇二甲醚、丁酮中的一种或多种；
    优选的，当式(I)化合物为晶型B时，所述方法三中，有机溶剂E选自DMF；
    优选的，当式(I)化合物为晶型C时，所述方法三中，有机溶剂E选自DMSO；
    优选的，当式(I)化合物为晶型D时，所述方法三中，有机溶剂E选自NMP。
  4. 一种结晶组合物，包含根据权利要求1-3中任一项所述的结晶形式的式(I)所示化合物；优选的，所述结晶组合物中，结晶形式的式(I)化合物选自晶型A、晶型B、晶型C、晶型D中的一种或多种；
    优选的，所述结晶形式的式(I)化合物占所述结晶组合物重量的10％以上、20％以上、30％以上、40％以上、50％以上、60％以上、70％以上、80％以上、90％以上、91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上、99％以上或99.5％以上；
    优选地，所述结晶形式的式(I)化合物占所述结晶组合物重量的50％以上、60％以上、70％以上、80％以上、90％以上、91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上、99％以上或99.5％以上。
  5. 一种药物组合物，包含权利要求1或2所述的结晶形式的式(I)所示化合物或如权利要求4所述的结晶组合物，以及任选的药学上可接受的载体。
  6. 根据权利要求5所述的药物组合物，其包含另一种、两种或多种治疗癌症或肿瘤的药物。
  7. 根据权利要求1或2所述的结晶形式的式(I)所示化合物、权利要求4所述的结晶组合物、权利要求5或6中所述的药物组合物作为药物或在制备药物中的应用，所述药物用于预防和/或治疗ENPP1介导的疾病。
  8. 根据权利要求1或2所述的结晶形式的式(I)所示化合物、权利要求4所述的结晶组合物、权利要求5或6中所述的药物组合物作为药物或在制备药物中的应用，所述药物用于预防和/或治疗癌症或肿瘤相关疾病，或者心血管疾病；
    优选的，权利要求1或2所述的结晶形式的式(I)所示化合物、权利要求4所述的结晶组合物、权利要求5或6中所述的药物组合物与另一种、两种或多种治疗癌症或肿瘤的药物，或治疗手段联合使用。
  9. 根据权利要求6所述的药物组合物或权利要求8所述的应用，其中，所述抗肿瘤药物为化疗药物、靶向治疗药物或免疫治疗药物；
    优选的，所述用于预防和/或治疗癌症或肿瘤的药物选自：细胞信号转导抑制剂、苯丁酸氮芥、美法仑、环磷酰胺、异环磷酰胺、白消安、卡莫司汀、洛莫司汀、链脲佐菌素、顺铂、卡铂、奥沙利铂、达卡巴嗪、替莫唑胺、丙卡巴肼、甲氨蝶呤、氟尿嘧啶、阿糖胞苷、吉西他滨、巯基嘌呤、氟达拉滨、长春碱、长春新碱、长春瑞滨、紫杉醇、多西紫杉醇、拓扑替康、伊立替康、依托泊苷、曲贝替定、更生霉素、多柔比星、表柔比星、道诺霉素、米托蒽醌、博来霉素、丝裂霉素C、伊沙匹隆、他莫昔芬、氟他胺、戈那瑞林类似物、甲地孕酮、强的松、地塞米松、甲泼尼龙、沙利度胺、干扰素α、亚叶酸钙、西罗莫司、西罗莫司脂化物、依维莫司、阿法替尼、alisertib、amuvatinib、阿帕替尼、阿西替尼、硼替佐米、波舒替尼、布立尼布、卡博替尼、西地尼布、crenolanib、克卓替尼、达拉菲尼、达可替尼、达努塞替、达沙替尼、多维替尼、厄洛替尼、foretinib、ganetespib、吉非替尼、依鲁替尼、埃克替尼、伊马替尼、iniparib、拉帕替尼、乐伐替尼、linifanib、linsitinib、马赛替尼、momelotinib、莫替沙尼、来那替尼、尼罗替尼、尼拉帕尼、oprozomib、奥拉帕尼、帕唑帕 尼、pictilisib、普纳替尼、奎扎替尼、瑞格菲尼、rigosertib、卢卡帕尼、鲁索利替尼、塞卡替尼、saridegib、索拉非尼、舒尼替尼、替拉替尼、tivantinib、替沃扎尼、托法替尼、曲美替尼、凡德他尼、维利帕尼、威罗菲尼、维莫德吉、volasertib、阿仑单抗、贝伐单抗、贝伦妥单抗维多汀、卡妥索单抗、西妥昔单抗、地诺单抗、吉妥珠单抗、伊匹单抗、尼妥珠单抗、奥法木单抗、帕尼单抗、利妥昔单抗、托西莫单抗、曲妥珠单抗、PI3K抑制剂、CSF1R抑制剂、A2A和/或A2B受体拮抗剂、IDO抑制剂、抗PD-1抗体、抗PD-L1抗体、LAG3抗体、TIM-3抗体、TIGIT抗体，CD47抗体，CLAUDIN 18.2抗体、抗CTLA-4抗体或其任意组合；优选的，所述治疗手段为放疗或手术。
  10. 根据权利要求8所述的应用，其特征在于，所述癌症或肿瘤选自皮肤癌、膀胱癌、卵巢癌、乳腺癌、胃癌、胰腺癌、前列腺癌、结肠癌、肺癌、骨癌、脑癌、神经细胞瘤、直肠癌、结肠癌、家族性腺瘤性息肉性癌、遗传性非息肉性结直肠癌、食管癌、唇癌、喉癌、下咽癌、舌癌、唾液腺癌、腺癌、甲状腺髓样癌、乳头状甲状腺癌、肾癌、肾实质癌、宫颈癌、子宫体癌、子宫内膜癌、绒毛膜癌、睾丸癌、泌尿癌、黑素瘤、脑肿瘤诸如成胶质细胞瘤、星形细胞瘤、脑膜瘤、成神经管细胞瘤和外周神经外胚层肿瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤、伯基特淋巴瘤、白血病、急性淋巴性白血病(ALL)、慢性淋巴性白血病(CLL)、急性骨髓性白血病(AML)、慢性粒细胞白血病(CML)、成人T细胞白血病淋巴瘤、弥漫性大B细胞淋巴瘤(DLBCL)、肝细胞癌、胆囊癌、支气管癌、小细胞肺癌、非小细胞肺癌、多发性骨髓瘤、基底细胞瘤、畸胎瘤、成视网膜细胞瘤、脉络膜黑素瘤、精原细胞瘤、横纹肌肉瘤、颅咽管瘤、骨肉瘤、软骨肉瘤、肌肉瘤、脂肪肉瘤、纤维肉瘤、尤因肉瘤和浆细胞瘤；
    所述心血管疾病选自心肌梗塞(心梗)、心力衰竭(心衰)、心脏创伤、心脏组织异常瘢痕的形成、心肌病、心肌细胞死亡，增强心脏修复，心肌细胞中释放促炎分子。
PCT/CN2024/099188 2023-06-15 2024-06-14 一种enpp1抑制剂的晶型 Ceased WO2024255837A1 (zh)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU2024302003A AU2024302003A1 (en) 2023-06-15 2024-06-14 Crystal form of enpp1 inhibitor
CN202480001155.9A CN119497716A (zh) 2023-06-15 2024-06-14 一种enpp1抑制剂的晶型
KR1020267001047A KR20260023568A (ko) 2023-06-15 2024-06-14 Enpp1 억제제의 결정형
EP24822785.2A EP4729519A1 (en) 2023-06-15 2024-06-14 Crystal form of enpp1 inhibitor

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202310706966 2023-06-15
CN202310706966.6 2023-06-15

Publications (1)

Publication Number Publication Date
WO2024255837A1 true WO2024255837A1 (zh) 2024-12-19

Family

ID=93851356

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2024/099188 Ceased WO2024255837A1 (zh) 2023-06-15 2024-06-14 一种enpp1抑制剂的晶型

Country Status (5)

Country Link
EP (1) EP4729519A1 (zh)
KR (1) KR20260023568A (zh)
CN (1) CN119497716A (zh)
AU (1) AU2024302003A1 (zh)
WO (1) WO2024255837A1 (zh)

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102015651A (zh) * 2008-03-03 2011-04-13 Irm责任有限公司 作为tlr活性调节剂的化合物和组合物
CN102712636A (zh) * 2009-09-02 2012-10-03 Irm责任有限公司 作为tlr活性调节剂的化合物和组合物
CN104884456A (zh) * 2012-09-12 2015-09-02 山东轩竹医药科技有限公司 PI3K和/或mTOR抑制剂
CN107530353A (zh) * 2015-03-04 2018-01-02 丹娜法伯癌症研究院 Melk的三环激酶抑制剂和使用方法
WO2019046778A1 (en) 2017-08-31 2019-03-07 Mavupharma, Inc. INHIBITORS OF ECTONUCLEOTIDE PYROPHOSPHATASE-PHOSPHODIESTERASE (ENPP-1) AND USES THEREOF
WO2019177971A1 (en) 2018-03-12 2019-09-19 Mavupharma, Inc. Ectonucleotide pyrophosphatase-phosphodiesterase 1 (enpp-1) inhibitors and uses thereof
WO2020190912A1 (en) 2019-03-19 2020-09-24 Stingray Therapeutics, Inc. Quinoline and quinazoline compounds and methods of use thereof
WO2021061803A1 (en) 2019-09-23 2021-04-01 Nanjing Zhengxiang Pharmaceuticals Co., Ltd. Phosphodiesterase inhibitors and use
WO2021158829A1 (en) 2020-02-04 2021-08-12 Stingray Therapeutics, Inc. Inhibitors of ectonucleotide pyrophosphatase/phosphodiesterase 1 (enpp1) and methods of use thereof
CN116262750A (zh) * 2021-12-15 2023-06-16 上海翊石医药科技有限公司 一种芳杂环类化合物及其制备方法和用途

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102015651A (zh) * 2008-03-03 2011-04-13 Irm责任有限公司 作为tlr活性调节剂的化合物和组合物
CN102712636A (zh) * 2009-09-02 2012-10-03 Irm责任有限公司 作为tlr活性调节剂的化合物和组合物
CN104884456A (zh) * 2012-09-12 2015-09-02 山东轩竹医药科技有限公司 PI3K和/或mTOR抑制剂
CN107530353A (zh) * 2015-03-04 2018-01-02 丹娜法伯癌症研究院 Melk的三环激酶抑制剂和使用方法
WO2019046778A1 (en) 2017-08-31 2019-03-07 Mavupharma, Inc. INHIBITORS OF ECTONUCLEOTIDE PYROPHOSPHATASE-PHOSPHODIESTERASE (ENPP-1) AND USES THEREOF
WO2019177971A1 (en) 2018-03-12 2019-09-19 Mavupharma, Inc. Ectonucleotide pyrophosphatase-phosphodiesterase 1 (enpp-1) inhibitors and uses thereof
WO2020190912A1 (en) 2019-03-19 2020-09-24 Stingray Therapeutics, Inc. Quinoline and quinazoline compounds and methods of use thereof
WO2021061803A1 (en) 2019-09-23 2021-04-01 Nanjing Zhengxiang Pharmaceuticals Co., Ltd. Phosphodiesterase inhibitors and use
CN115151253A (zh) * 2019-09-23 2022-10-04 南京征祥医药有限公司 磷酸二酯酶抑制剂及用途
WO2021158829A1 (en) 2020-02-04 2021-08-12 Stingray Therapeutics, Inc. Inhibitors of ectonucleotide pyrophosphatase/phosphodiesterase 1 (enpp1) and methods of use thereof
CN116262750A (zh) * 2021-12-15 2023-06-16 上海翊石医药科技有限公司 一种芳杂环类化合物及其制备方法和用途

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
"Pharmaceutics", 2003, THE PEOPLE'S HEALTH PRESS

Also Published As

Publication number Publication date
KR20260023568A (ko) 2026-02-20
CN119497716A (zh) 2025-02-21
EP4729519A1 (en) 2026-04-22
AU2024302003A1 (en) 2026-01-08

Similar Documents

Publication Publication Date Title
JP7128169B2 (ja) テトラヒドロ－ピリド［３，４－ｂ］インドールエストロゲン受容体モジュレーター及びその使用
CA3224105A1 (en) Nitrogen-containing heterocyclic compound, and preparation method therefor, intermediate thereof, and application thereof
WO2024040131A1 (en) Pyridopyrimidine kras inhibitors
KR102090453B1 (ko) 상피 성장 인자 수용체 키나제 억제제의 염
WO2025049274A2 (en) Tetracyclic derivatives, compositions and methods thereof
CN117247382A (zh) 吡啶并嘧啶酮化合物的晶型
EP3744723B1 (en) Substituted pyrazolo[1,5-a]pyrimidine macrocyclic compound
JP2021535909A (ja) 高活性ｓｔｉｎｇタンパク質アゴニスト化合物
JP2024516518A (ja) Ｓｏｓ１分解剤及びその製造方法並びに応用
EP4169917B1 (en) Quinazoline compounds as pi3k inhibitors for the treatment of cancer
JP2022516685A (ja) ホスファターゼ結合化合物およびそれらを使用する方法
WO2019029663A1 (zh) 1h-吡唑并[4,3-h]喹唑啉类化合物作为蛋白激酶抑制剂
EP2504331B1 (en) Compound, certain novel forms thereof, pharmaceutical compositions thereof and methods for preparation and use
CN110903283B (zh) 一种取代的喹唑啉类化合物、包含该化合物的药物组合物和该化合物的用途
TW202216712A (zh) 一種高活性的ｈｐｋ１激酶抑制劑
US20240350501A1 (en) SALT OF N-(2,6-DIETHYLPHENYL)-8-({4-[4-(DIMETHYLAMINO) PIPERIDIN-1-YL]-2-METHOXYPHENYL}AMINO)-1-METHYL-4,5-DIHYDRO-1H-PYRAZOLO[4,3-h]QUINAZOLINE-3-CARBOXAMIDE, ITS PREPARATION, AND FORMULATIONS CONTAINING IT
CN121909188A (zh) 一类nsd蛋白降解剂及其用途
US20220160713A1 (en) Selective inhibitor of protein arginine methyltransferase 5 (prmt5)
CN120607543A (zh) 胺基取代芳杂环类抑制剂及其制备方法和应用
EP3730492A1 (en) Substituted pyrazolo[1,5-a]pyrimidine compound and pharmaceutical composition and use thereof
TWI899051B (zh) 二氫唏衍生物
WO2019134573A1 (zh) 一种氘代二苯氨基嘧啶类化合物的制备方法及其晶型
AU2024302003A1 (en) Crystal form of enpp1 inhibitor
CA3245813A1 (en) POLYMORPHIC COMPOUNDS AND THEIR USES
JP2026503067A (ja) ２－メチル－２－（３－（ピペリジン－３－イル）フェノキシ）－２－メチルプロパンアミド骨格含有化合物

Legal Events

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

Ref document number: 24822785

Country of ref document: EP

Kind code of ref document: A1

WWP Wipo information: published in national office

Ref document number: 202480001155.9

Country of ref document: CN

WWE Wipo information: entry into national phase

Ref document number: AU2024302003

Country of ref document: AU

Ref document number: 828010

Country of ref document: NZ

WWE Wipo information: entry into national phase

Ref document number: 202517125903

Country of ref document: IN

Ref document number: 2025135585

Country of ref document: RU

ENP Entry into the national phase

Ref document number: 2025572859

Country of ref document: JP

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2501008579

Country of ref document: TH

WWP Wipo information: published in national office

Ref document number: 828010

Country of ref document: NZ

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112025027904

Country of ref document: BR

WWP Wipo information: published in national office

Ref document number: 202517125903

Country of ref document: IN

ENP Entry into the national phase

Ref document number: 2024302003

Country of ref document: AU

Date of ref document: 20240614

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 1020267001047

Country of ref document: KR

Free format text: ST27 STATUS EVENT CODE: A-0-1-A10-A15-NAP-PA0105 (AS PROVIDED BY THE NATIONAL OFFICE)

WWE Wipo information: entry into national phase

Ref document number: 1020267001047

Country of ref document: KR

WWP Wipo information: published in national office

Ref document number: 2025135585

Country of ref document: RU

WWE Wipo information: entry into national phase

Ref document number: 2024822785

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2024822785

Country of ref document: EP

Effective date: 20260115

ENP Entry into the national phase

Ref document number: 2024822785

Country of ref document: EP

Effective date: 20260115

WWE Wipo information: entry into national phase

Ref document number: 11202508313Y

Country of ref document: SG

WWP Wipo information: published in national office

Ref document number: 11202508313Y

Country of ref document: SG

ENP Entry into the national phase

Ref document number: 2024822785

Country of ref document: EP

Effective date: 20260115

ENP Entry into the national phase

Ref document number: 2024822785

Country of ref document: EP

Effective date: 20260115

WWP Wipo information: published in national office

Ref document number: 1020267001047

Country of ref document: KR

WWP Wipo information: published in national office

Ref document number: 2024822785

Country of ref document: EP