WO2024017229A1 - Use of pyrrolotriazine compound in preparation of anti-tumor drug - Google Patents

Use of pyrrolotriazine compound in preparation of anti-tumor drug Download PDF

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WO2024017229A1
WO2024017229A1 PCT/CN2023/107850 CN2023107850W WO2024017229A1 WO 2024017229 A1 WO2024017229 A1 WO 2024017229A1 CN 2023107850 W CN2023107850 W CN 2023107850W WO 2024017229 A1 WO2024017229 A1 WO 2024017229A1
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cancer
compound
acid
tumor
lymphoma
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PCT/CN2023/107850
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French (fr)
Chinese (zh)
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魏霞蔚
魏于全
姜宁
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成都嘉葆药银医药科技有限公司
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Publication of WO2024017229A1 publication Critical patent/WO2024017229A1/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
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • 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
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants

Definitions

  • the invention belongs to the field of medicine, and specifically relates to the application of a pyrrolotriazine compound in the preparation of anti-tumor drugs.
  • Mitogen-activated protein kinase interacting kinase (MAP kinase interacting kinase, MNK) is a serine/threonine protein kinase.
  • the MNK protein is encoded by two genes, MKNK1 and MKNK2.
  • MNK1a, MNK1b and MNK2a, MNK2b There are four subtypes of human MNK, MNK1a, MNK1b and MNK2a, MNK2b, which are expressed by the MNK1 and MNK2 genes respectively (Ahmed M Abdelaziz1, 2020).
  • All four isoforms contain a nuclear localization signal (NLS) and a sequence that binds to eIF4G at the N-terminus, allowing them to enter the nucleus and function, recognizing and binding to downstream eIF4E.
  • the C-termini of MNK1a and MNK2a subtypes have binding sites for MAPK and can be activated by upstream ERK and p38 phosphorylation (Jianling Xie, 2019).
  • the C-terminal nuclear export signal (NES) of MNK1a allows it to be widely present in the cytoplasm, while the other three isoforms mostly exist in the nucleus.
  • MNK protein can specifically phosphorylate Ser209 of eukaryotic translation initiation factor 4E (eIF4E), thereby indirectly regulating mRNA translation (Fukunaga R, 1997).
  • eukaryotic cell translation initiation factor eIF4E can strengthen the transcription of mRNA encoding and regulating cell cycle proteins and oncogenic proteins, thereby causing the upregulation of tumor-related protein expression (Truitt ML, 2015).
  • eIF4E, the skeleton protein eIF4G and the RNA helicase eIF4A form the eukaryotic initiation factor complex eIF4F. Since eIF4E is responsible for the binding of this complex to the 5' end cap structure of mRNA, it plays an irreplaceable regulatory role in the RNA translation process.
  • eIF4E In normal cells, the activity of eIF4E is limited and the transcription of these tumor-related mRNAs is inhibited, while in tumor cells or tissues, high expression or overactivation of eIF4E can cause the upregulation of the transcription levels of these mRNAs (Lama D, 2019).
  • MNK1/2 and eIF4E are the convergence points of the RAS-RAF-MEK-ERK and PI3K-AKT-mTOR signaling pathways and play an important role in signaling pathway conduction.
  • eIF4E, MNK1/2 and eIF4E signaling pathways can affect the synthesis of a variety of chemokines, cytokines and immune checkpoint proteins, thereby regulating immune responses (Gorentla, B.K. 2012).
  • VEGF/VEGFR pathway is the main signaling pathway for tumor angiogenesis.
  • vascular endothelial growth factor (VEGF) family members include VEGF-A, VEGF-B, VEGF-C, VEGF-D and PIGF.
  • VEGF also known as VEGF-A, is the most important stimulator of physiological and pathological angiogenesis.
  • the functions of VEGF include: maintaining the survival of endothelial cells, inducing endothelial cell proliferation and migration, recruiting bone marrow-derived hematopoietic progenitors or stem cells to induce blood vessel formation, and enhancing vascular permeability.
  • VEGF-A has two tyrosine kinase receptors, namely VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1).
  • VEGFR-2 is the main receptor for VEGF to exert its pro-angiogenic effects.
  • a variety of tumor cells and host cells such as platelets, myocytes, and stromal cells can produce VEGF.
  • Hypoxia is an important inducing factor for up-regulation of VEGF expression.
  • Other factors that promote the secretion of VEGF include: low pH; growth factors; inflammatory chemokines; and genetic mutations.
  • the invention provides the use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof in the preparation of anti-tumor drugs,
  • R 1 is H, F, Cl, Br or C 1-3 alkyl
  • R 2 and R 3 are each independently H or C 1-3 alkyl, wherein the C 1-3 alkyl is optionally replaced by 1, 2 or 3 substituents independently selected from F, Cl, Br or I replace;
  • R 2 and R 3 are joined together with the carbon atom to which they are attached to form cyclopentyl, cyclohexyl or piperidinyl, wherein said cyclopentyl, cyclohexyl and piperidinyl are optionally replaced by 1, 2 or 3 R replaced by a ;
  • Each R a is independently H, F, Cl, Br or C 1-3 alkyl
  • R 4 is H, F, Cl, Br or C 1-3 alkyl
  • R 5 and R 6 are each independently H, F, Cl, Br, I or C 1-3 alkyl
  • R 7 is pyrrolidinyl, wherein the pyrrolidinyl is optionally substituted by 1, 2 or 3 R b ;
  • Each R b is independently H, F, Cl, Br, I or C 1-3 alkyl, wherein the C 1-3 alkyl is optionally 1, 2 or 3 independently selected from F, Cl, Br or Substituted by the substituent of I;
  • n 1 or 2.
  • each of the above R a is independently H, F, Cl, Br, -CH 3 or -CH 2 CH 3 , and other variables are as defined in the present invention.
  • R 2 and R 3 are each independently H, -CH 3 or -CH 2 CH 3 , and other variables are as defined in the present invention.
  • R 2 and R 3 are connected together with the carbon atoms to which they are connected to form R a and other variables are as defined in the present invention.
  • R 2 and R 3 are connected together with the carbon atoms to which they are connected to form
  • Other variables are as defined in the present invention.
  • R 1 is C 1-3 alkyl, such as methyl.
  • R 2 and R 3 are joined together with the carbon atom to which they are connected to form
  • R 4 is C 1-3 alkyl, such as methyl.
  • R 5 and R 6 are each independently H or methyl; n is 2;
  • R 7 is substituted by 1, 2 or 3 H, F, Cl or methyl
  • the above compound has a structure represented by any of the formulas (I-1) to (I-4):
  • R 1 , R 4 , R 5 , R 6 , R 7 , R a and n are as defined in the present invention.
  • each of the above R b is independently H, F, Cl, Br, I, Other variables are as defined in the present invention.
  • R 7 is stated therein Optionally substituted by 1 or 2 R b , R b and other variables are as defined in the present invention.
  • R 7 is R b and other variables are as defined in the present invention.
  • R 4 is H or -CH 3 , and other variables are as defined in the present invention.
  • the above compound has a structure represented by any of the structural formulas of formula (I-5) to formula (I-9):
  • R 1 , R 5 , R 6 , R a and R b are as defined in the present invention.
  • R 1 is H, F, Cl or Other variables are as defined in the present invention.
  • R 5 and R 6 are each independently H or Other variables are as defined in the present invention. There are also some solutions of the present invention that are derived from any combination of the above variables.
  • the compound represented by formula (I) is selected from the following structures:
  • the pharmaceutically acceptable salt is a salt formed by a compound represented by formula (I) and an inorganic acid.
  • the inorganic acid includes, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, and bicarbonate, Phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydriodic acid, phosphorous acid, etc.; and organic acid salts, including acetic acid, propionic acid, isobutyric acid, maleic acid, Malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid and similar Acids; also include salts of amino acids (such as arginine, etc.), and salts of organic acids such
  • neoplastic includes “hyperproliferative disease,” which refers to excessive growth or proliferation compared to normal cells or unlysed cells, including dysplasia, neoplasia, noncontact suppression, or carcinogenesis.
  • Neoplastic refers to a variety of hyperproliferative diseases, including solid tumors and hematological tumors. These different forms of hyperproliferative diseases are known in the art and have established standards for diagnosis and classification.
  • Exemplary solid tumors include: breast cancer; prostate cancer; colon cancer; rectal cancer; liver cancer; kidney cancer; stomach cancer; bladder cancer; all forms of lung and bronchial cancer; bone marrow cancer; melanoma; neuroblastoma; parenchyma; pus Chest; choriomas; branching tumors; malignant carcinoid syndrome; carcinoid heart disease; cellular carcinoma (e.g., Walker carcinoma, basal cell carcinoma, basal squamous cell carcinoma, Brown-Pearce carcinoma, ductal carcinoma, myxoid carcinoma , non-small cell carcinoma, oat cell carcinoma, synovial cell carcinoma, bronchiolar carcinoma, bronchial carcinoma, squamous cell carcinoma and transitional cell carcinoma); plasmacytoma; reticuloendot
  • Exemplary hematological malignancies include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic eosinophilic leukemia (CEL), myelodysplastic syndrome (MDS), Hodgkin lymphoma, non-Hodgkin lymphoma (NHL) (such as follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), diffuse large B-cell lymphoma ( DLBCL), chronic lymphocytic leukemia (CLL), or multiple myeloma (MM).
  • ALL acute lymphoblastic leukemia
  • AML acute myeloid leukemia
  • CML chronic myelogenous leukemia
  • CEL chronic eosinophilic leukemia
  • MDS myelodysplastic syndrome
  • NHL non-Hodgkin lymphoma
  • NHL non-Hodgkin lympho
  • the tumor is selected from solid tumors or non-solid tumors, such as melanoma, kidney cancer, blood cancer, prostate cancer, colon cancer, rectal cancer, stomach cancer, esophageal cancer, bladder cancer, Head and neck cancer (such as head and neck squamous cell carcinoma), thyroid cancer, breast cancer (such as triple-negative breast cancer), ovarian cancer, cervical cancer, lung cancer (such as non-small cell lung cancer), urothelial cancer, pancreatic cancer, glioblastoma Cytoma, liver cancer, colon adenocarcinoma, astrocytoma, osteosarcoma, myeloma (eg, multiple myeloma), lymphoma (Hodgkin lymphoma, non-Hodgkin lymphoma (eg, follicular lymphoma) (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), diffuse large B-cell lympho
  • solid tumors or non-solid tumors
  • the tumor is selected from the group consisting of kidney cancer, prostate cancer, colon cancer, rectal cancer, gastric cancer, esophageal cancer, thyroid cancer, breast cancer, lung cancer (such as non-small cell lung cancer), ovarian cancer, glioblastoma Cell tumors, liver cancer, lymphoma ⁇ such as chronic lymphocytic leukemia (CLL) ⁇ , leukemia (such as acute myeloid leukemia).
  • lung cancer such as non-small cell lung cancer
  • ovarian cancer glioblastoma Cell tumors
  • liver cancer lymphoma ⁇ such as chronic lymphocytic leukemia (CLL) ⁇
  • leukemia such as acute myeloid leukemia
  • the tumor is selected from the group consisting of kidney cancer, colon cancer, rectal cancer, gastric cancer, lung cancer (such as non-small cell lung cancer), ovarian cancer, lymphoma, liver cancer, breast cancer, leukemia (such as acute myeloid leukemia) ).
  • the cells are colon cancer CT-26 cells, colon cancer HT-29 cells, colon cancer SW480 cells, lung cancer Calu-3 cells, lung cancer A549 cells, lung cancer H460 cells, and gastric cancer BGC-823 cells.
  • the compound represented by formula (I) or a pharmaceutically acceptable salt thereof has an inhibitory effect on VEGFR2, such as p-VEGFR2.
  • the compound represented by formula (I) or a pharmaceutically acceptable salt thereof has an inhibitory effect on both eIF4E and VEGFR2, for example, an inhibitory effect on p-eIF4E and p-VEGFR2.
  • the present invention also provides a pharmaceutical composition, including a compound represented by formula (I) or a pharmaceutically acceptable salt thereof and at least one second drug.
  • “Combination” refers to a compound represented by formula (I) and at least one second drug, each of which can be administered continuously, simultaneously or simultaneously (the compound represented by formula (I) can be administered first, or the second drug can be administered first). drug).
  • the mass ratio of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof to the second drug is 1:500 to 500:1, for example, 1 :50 to 50:1 or 1:10 to 10:1, examples are 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 10:3, 4:1, 25:6, 5:1, 25:1.
  • the mass ratio of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof to the second drug is 10:1 to 50:1, for example, 15:1 to 35:1, and examples are 20:1, 25:1, and 30:1.
  • the mass ratio of the compound represented by formula (I) or its pharmaceutically acceptable salt to the second drug is 1 :1 to 20:1, for example, 2:1 to 10:1, for example, 3:1, 4:1, 25:6, 5:1, 6:1, 8:1.
  • the mass ratio of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof to the second drug is 100:1 to 400:1, for example, 150:1 to 300:1, and examples are 200:1 and 250:1.
  • the second drug is selected from immune checkpoint inhibitors, chemotherapy drugs, or targeted drugs.
  • the immune checkpoint inhibitor targets include immunosuppressive signals involving PD-1, PD-L1, PD-L2, CTLA4, CD80, CD86, B7-H3, B7-H4, HVEM, BTLA, At least one of KIR, LAG3, GAL9, TIM3, 2B4, adenosine, A2aR, TGFP, BCL-2 and immunosuppressive cytokines (such as IL-10, IL-35).
  • the immunosuppressive inhibitor component can be a compound, antibody, antibody fragment or fusion polypeptide (eg Fc fusion, eg CTLA4-Fc), antisense molecule, ribozyme or RNAi molecule, or low molecular weight organic molecule.
  • the chemotherapy drug is selected from at least one of the following drugs: antimetabolites/anticancer agents, such as pyrimidine analogs (5-fluorouracil (5-fu), fluuridine, capecitabine azacitabine, gemcitabine, decitabine, azacitidine and cytarabine) and purine analogues, folate antagonists and related immunosuppressants (methotrexate, pemetrexed, mercaptopurine, thioguanine, Pentostatin and 2-chlorodeoxyadenosine (cladribine, fludarabine); coenzymes (tetrahydrofolate); antiproliferative/antimitotic agents, including natural products such as vinca alkaloids (vinblastine) , vincristine and vinorelbine), microtubule disruptors, such as taxanes (paclitaxel, docetaxel), vindesine, nocodazole, eflenox, epoth
  • the immune checkpoint inhibitor is selected from the group consisting of pembrolizumab, nivolumab, camrelizumab, toripalimab, cimepilimab, Valumab, avelumab, sintilimab, tislelizumab, cepalizumab, durvalumab, atezolizumab, envolizumab, sugalizumab, slulimumab, penpilimab, serelizumab, zimberizumab, jepunolizumab, spartalizumab, proglilimab, sugalizumab Cotinizumab, cosibelimab, AUNP-12, relevanizumab, retifalizumab, dotalizumab, cardonizumab, socazolizumab, adebre monoclonal antibody, ipilimum
  • the targeted drug is selected from the group consisting of B-Raf inhibitors (dabrafenib, sorafenib, vemurafenib and dabrafenib), MEK inhibitors (trametid nimetinib, selumetinib, binimetinib, PD-325901, cobimetinib, CI-1040 and PD035901), VEGF/VEGFR inhibitors (such as bevacizumab, ramucirumab, ramucirumab, Tizumab, aflibercept, conbercept, axitinib, cediranib, motesanib, lenvatinib, sunitinib, sorafenib, cabozantinib, Gorfenib, nintedanib, zopanib, osimertinib, fruquintinib, vandet
  • the second drug is preferably cisplatin, paclitaxel, doxorubicin or PD-1.
  • the present invention also provides the use of the pharmaceutical composition in preparing anti-tumor drugs.
  • the present invention provides the use of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or the pharmaceutical composition in the preparation of a drug for treating diseases related to the VEGFR2 signaling pathway.
  • the present invention provides the use of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or the pharmaceutical composition in the preparation of drugs for treating diseases related to eIF4E and VEGFR2 signaling pathways.
  • the present invention provides the use of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or the pharmaceutical composition in the preparation of a drug for regulating immunity.
  • the regulating immunity is preferably to enhance immunity.
  • the present invention also provides a method for treating tumor diseases, which method includes administering to a patient a therapeutically effective amount of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof.
  • the present invention also provides a method for joint treatment of tumor diseases, which method includes jointly administering a therapeutically effective amount of formula (I) to a tumor patient.
  • Combination treatments may be administered in any of a variety of ways, such as sequentially or simultaneously.
  • the method includes administering to the patient the pharmaceutical composition.
  • the tumor is selected from solid tumors or non-solid tumors, such as melanoma, kidney cancer, blood cancer, prostate cancer, colon cancer, rectal cancer, gastric cancer, esophageal cancer, bladder cancer, head and neck cancer (such as Head and neck squamous cell carcinoma), thyroid cancer, breast cancer (such as triple-negative breast cancer), ovarian cancer, cervical cancer, lung cancer (such as non-small cell lung cancer), urothelial cancer, pancreatic cancer, glioblastoma, liver cancer , colon adenocarcinoma, astrocytoma, colorectal cancer, osteosarcoma, myeloma (such as multiple myeloma), lymphoma (Hodgkin lymphoma, non-Hodgkin lymphoma (such as follicular lymphoma) (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), diffuse large B-cell lymphoma
  • the lung cells are lung cancer cells A549 and H460, and the colon cancer cells are SW480 and CT-26.
  • a method for treating diseases related to the VEGFR2 signaling pathway which method includes administering to a patient a therapeutically effective amount of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof or the pharmaceutical composition.
  • a method for treating diseases related to eIF4E and VEGFR2 signaling pathways which method includes administering to a patient a therapeutically effective amount of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof or the pharmaceutical composition.
  • the active compounds are combined or formulated with appropriate pharmaceutically acceptable carriers, diluents or excipients, and can be formulated into preparations in the form of solid, semi-solid, liquid or gas , such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres and aerosols.
  • Modes of administration include oral, intraperitoneal, transdermal, subcutaneous, intravenous or intramuscular injection, inhalation, topical, intralesional, infusion; liposome-mediated delivery; topical, intrathecal, gingival pocket, rectal, intrabronchial , nasal cavity, transmucosal, intestinal, eye or ear delivery, or any other method known in the art, can achieve the treatment of tumors;
  • the therapeutically effective amount or dose of the present invention will vary depending on several factors, including the route of administration chosen, the formulation of the composition, patient response, severity of condition, subject's weight, and the judgment of the prescribing physician, e.g. 1-200mg/kg, 40-150mg/kg, such as 50mg/kg. Dosage may be increased or decreased over time as individual patients require. In some cases, patients are initially given low doses and then increased to an effective dose that is tolerated by the patient. In addition, patients may be administered multiple doses over defined time periods, particularly in time increments (e.g., daily, weekly, biweekly, monthly, quarterly, biennially, or the like).
  • the research of the present invention has found that the compound represented by formula (I) has a dual-target inhibitory effect on eIF4E protein and/or VEGFR2 protein when used alone or in combination with other drugs, thereby having a good tumor inhibitory effect.
  • Figure 1 illustrates the dose-response curve of compound 12 corresponding to p-toluenesulfonate and Con-MNK on HCT116 cells.
  • Figure 2 illustrates the dose-response curve of compound 12 corresponding to p-toluenesulfonate and Con-MNK on human colon cancer HCT116 cells.
  • Figure 3 illustrates the body weight changes of tumor-bearing mice in the subcutaneous xenograft tumor model of human colon cancer HT-29 cells during the administration process.
  • Figure 4 illustrates the body weight changes of tumor-bearing mice in the subcutaneous xenograft tumor model of human colon cancer HT-29 cells during the administration process.
  • Figure 5 illustrates the tumor growth inhibition curve of human colon cancer HT-29 cell subcutaneous xenograft tumor model tumor-bearing mice after administration of drugs.
  • Figure 6 illustrates the mean plasma time-concentration plot in Balb/c nude mice after oral administration of 10, 30 and 100 mg/kg p-toluenesulfonate corresponding to compound 12.
  • Figure 7 illustrates the average plasma and tumor concentration profiles of Balb/c nude mice after oral administration of 10 mg/kg p-toluenesulfonate corresponding to Compound 12.
  • Figure 8 illustrates the average plasma and tumor concentration profiles of Balb/c nude mice after oral administration of 30 mg/kg p-toluenesulfonate corresponding to Compound 12.
  • Figure 9 illustrates the average plasma and tumor concentration profiles of Balb/c nude mice after oral administration of 100 mg/kg p-toluenesulfonate corresponding to Compound 12.
  • Figure 10 shows the protein expression of eIF4E and p-eIF4E in xenograft tumor HT-29 tissue after 2h/8h/24h administration.
  • Figure 11 illustrates the protein expression analysis of eIF4E and p-eIF4E in xenograft tumor HT-29 tissue after 2h/8h/24h administration.
  • Figure 12 shows the protein expression of VEGFR2 and p-VEGFR2 in xenograft tumor HT-29 tissue after 2h/8h/24h administration.
  • Figure 13 illustrates the protein expression analysis of p/t-VEGFR2 in xenograft tumor HT-29 tissue after 2h/8h/24h administration.
  • Figure 14 illustrates the effect of the combination of p-toluenesulfonate and 5-fu corresponding to Compound 12 on the tumor volume of human colon cancer SW480 cell Balb/c nude mouse transplanted tumor model.
  • Figure 15 illustrates the tumor growth curve of mouse colon cancer CT-26 cell subcutaneously transplanted tumor model in tumor-bearing mice after administration of drugs.
  • Figure 16 illustrates the inhibition of MNK1 kinase activity by test compounds.
  • Figure 17 illustrates the inhibition of MNK2 kinase activity by test compounds.
  • Figure 18 illustrates the effect of p-toluenesulfonate corresponding to Compound 12 on the tumor volume of the gastric cancer BGC-823 cell Balb/c nude mouse transplanted tumor model.
  • Figure 19 illustrates the effect of p-toluenesulfonate corresponding to Compound 12 on tumor volume in the lung cancer Calu-3 cell Balb/c nude mouse transplant tumor model.
  • Figure 20 illustrates the inhibition of HUVEC cells by the p-toluenesulfonate corresponding to Compound 12.
  • Figure 21 illustrates the effect of the combination of p-toluenesulfonate and cisplatin corresponding to compound 12 on the tumor volume of the lung cancer A549 cell Balb/c nude mouse transplant tumor model.
  • Figure 22 illustrates the effect of the combination of p-toluenesulfonate and paclitaxel corresponding to compound 12 on the tumor volume of the lung cancer H460 cell Balb/c nude mouse transplanted tumor model.
  • Figure 23 illustrates the tumor growth inhibition curve of human ovarian cancer ES2 cell subcutaneous xenograft tumor model tumor-bearing mice after administration of drugs.
  • Figure 24 illustrates the body weight changes of human ovarian cancer ES2 cell subcutaneous xenograft tumor model tumor-bearing mice during the administration process.
  • Figure 25 illustrates the effect of the combination of p-toluenesulfonate corresponding to Compound 12 and cisplatin or paclitaxel on the tumor volume of the human ovarian cancer ES2 cell subcutaneous xenograft model.
  • Figure 26 illustrates the tumor growth curves of p-toluenesulfonate corresponding to compound 12 used alone and in combination with doxorubicin on human DLBCL cell SU-DHL-6 xenograft tumors.
  • Figure 27 illustrates the body weight change curve of p-toluenesulfonate corresponding to compound 12 used alone and in combination with doxorubicin on human DLBCL cell SU-DHL-6 xenograft tumors
  • Figure 28 illustrates the tumor growth curve of p-toluenesulfonate corresponding to Compound 12 on MCL cell JeKo-1 cell xenograft tumors.
  • Figure 29 illustrates the tumor growth curve of compound 12 corresponding to p-toluenesulfonate and PD-1 antibody or PD-1 isotype antibody on the mouse LL2 subcutaneous tumor model.
  • Figure 30 illustrates the body weight change curve of p-toluenesulfonate corresponding to Compound 12 and PD-1 antibody or PD-1 isotype antibody on the mouse LL2 subcutaneous tumor model.
  • Figure 31 illustrates the tumor growth curve of the p-toluenesulfonate corresponding to compound 12 on the Hep G2 nude mouse transplanted tumor model.
  • Figure 32 illustrates the tumor growth curve of the p-toluenesulfonate corresponding to Compound 12 on the MDA-MB-231 nude mouse transplanted tumor model.
  • the compound of formula (I) above is a compound of the following formula:
  • the crude product was purified by high-performance liquid chromatography (hydrochloric acid conditions) to obtain hydrochloric acid of compound 5. Salt.
  • Methanol (5 mL) was added to the crude product, stirred at 15°C for 16 hours, filtered, and the filter cake was washed with methanol (2 mL ⁇ 2) and dried to obtain compound 11.
  • reaction solution is filtered, and the filtrate is concentrated under reduced pressure, diluted with dichloromethane (250 mL), washed with saturated aqueous potassium carbonate solution (75 mL ⁇ 1), the organic phase is collected, and the aqueous phase is extracted with dichloromethane (75 mL ⁇ 9) , the combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain colorless oily product 13b.
  • the crude product was purified by high performance liquid chromatography (hydrochloric acid system) to obtain the hydrochloride salt of compound 14.
  • the trifluoroacetate salt of compound 15c (200 mg, 0.554 mmol) was dissolved in tetrahydrofuran (3 mL) and methanol (3 mL), and di-tert-butyl dicarbonate (121 mg, 0.554 mmol) and triethylamine ( 224 mg, 2.21 mmol).
  • the reaction solution was stirred at 25°C for 1 hour.
  • the reaction solution was diluted with water (60mL), extracted with dichloromethane (60mL ⁇ 4), the organic phases were combined, washed with water (200mL ⁇ 1) and saturated brine (200mL ⁇ 1), dried over anhydrous sodium sulfate, and filtered.
  • the filtrate was concentrated under reduced pressure, and the crude product was purified by high-performance liquid chromatography (neutral conditions) to obtain compound 15d.
  • the hydrochloride of compound 15 (30 mg, 0.049 mmol) was dissolved in anhydrous methanol (3 mL), and aqueous formaldehyde solution (6.39 mg, 78.7 ⁇ mol, purity: 37%) and sodium cyanoborohydride (6.60 mg,0.105mmol).
  • the reaction solution was stirred at 10°C for 1 hour.
  • the reaction solution was quenched by adding water (20 mL), concentrated under reduced pressure to remove methanol, filtered, and washed with methanol (3 mL ⁇ 2).
  • the crude product was purified by high performance liquid chromatography (hydrochloric acid conditions) to obtain the hydrochloride salt of compound 16.
  • Cell culture Digest and isolate the cells in the T75 culture flask, resuspend in culture medium, count, adjust the cell concentration with culture medium to 2 ⁇ 10 5 cells/mL, add 100 ⁇ L of cell suspension to the 96-well plate, and incubate at room temperature. After leaving for 10 minutes, place it in a 37°C, 5% CO 2 incubator for overnight culture. Then place the cell plate back into the incubator for 3 hours. After 3 hours, remove the culture medium, add 50 ⁇ L 1X lysis buffer to each well, and rotate for 10 min at room temperature. Add 10 ⁇ L of lysis solution to the 384-well assay plate. Add 5 ⁇ L acceptor mix to each well, seal the plate with sealing film, and incubate at room temperature for 1 hour.
  • Cell culture Human colon cancer HT-29 cells (ATCC, product number: HTB-38) are cultured in vitro.
  • the culture conditions are McCoy's 5a medium plus 10% fetal bovine serum, 100U/mL penicillin and 100 ⁇ g/mL streptomycin. , cultured in 37°C 5% CO2 incubator. Passage was performed twice a week with routine digestion treatment with trypsin-EDTA. When the cell saturation is 80%-90% and the number reaches the required number, cells are collected, counted, and inoculated.
  • HT-29 cells were subcutaneously inoculated into the right back of each mouse. When the average tumor volume reached 154 mm 3 , random groups were divided according to tumor volume.
  • Tissuelyser LT uses the highest frequency to disrupt tissue for 5 minutes.
  • Electrophoresis 80 volts, 30 minutes, then 120 volts, 90 minutes.
  • Transfer Use iBlot2 transfer kit and transfer apparatus to transfer: for proteins with a molecular weight less than 100KDa, run the P3 program for 7 minutes; for proteins with a molecular weight greater than 100KDa, run the P3 program for 15 minutes.
  • Blocking Place the membrane in blocking solution (5% skim milk prepared with 1xTBST) for 1 hour at room temperature with shaking.
  • Chemiluminescence Add the HRP substrate in the West Femto Ultra-Sensitive Chemiluminescence Kit to the membrane.
  • AlphaviewSA software was used to perform relative quantification of the density intensity of the immunoblot chemiluminescent bands.
  • the average tumor volume in the vehicle control group was 1670mm 3 .
  • the average tumor volumes of the 10mg/kg, 30mg/kg, and 100mg/kg treatment dose groups of compound 12 corresponding to p-toluenesulfonate were 860mm 3 , 719mm 3 and 652mm 3 respectively, and the T/C were 51.72%, 43.56% and 38.89%, TGI are 53.46%, 62.69% and 67.16% respectively.
  • the p-toluenesulfonate corresponding to compound 12 showed a dose-dependent anti-tumor effect, and had a significant inhibitory effect on tumor growth at three concentrations, with statistical differences (p ⁇ 0.05 ).
  • Each dose group of the compound could significantly inhibit p-eIF4E protein expression at different time points.
  • Each dose group had a certain inhibitory effect on VEGFR2 and p-VEGFR2 protein expression 2h and 8h after administration.
  • 5-fu was formulated with DPBS to 3 mg/mL.
  • the specific preparation method is: weigh 30mg5-fu and add 10mL DPBS, vortex until clear, mark G2, G4-2, G5-2, G6-2, ready for use.
  • the corresponding compound of the present invention was formulated at 10 mg/mL.
  • the specific preparation method is: weigh 299.88mg compound and add 2.8mL NMP, vortex until clear, then add 2.8mL Solutol and vortex, then add 22.4mL water, vortex until uniform, marked as G3, G6-1 , prepared once a week.
  • the corresponding compound of the present invention was formulated at 1 mg/mL.
  • the specific preparation method is: weigh 14.99mg compound and add 1.4mL NMP, vortex until clear, then add 1.4mL Solutol and vortex, then add 11.2mL water, vortex until uniform, mark it as G4-1, every week Prepare once.
  • the corresponding compound of the present invention was formulated at 3 mg/mL.
  • the specific preparation method is: weigh 14.99 ⁇ 3mg compound and add 1.4mL NMP, vortex until clear, then add 1.4mL Solutol and vortex, then add 11.2mL water, vortex until uniform, mark it as G5-1. Prepare once a week.
  • Tumor volume was measured twice weekly. And calculate the tumor inhibition rate TGI (%).
  • TGI (%) [(1-average tumor volume at the end of treatment group administration-average tumor volume at the end of treatment group administration)/(average tumor volume at the end of treatment group for solvent control group-solvent control group group administration) average tumor volume)] ⁇ 100%.
  • TV 1 is the tumor volume on the day of group administration
  • TV n is the tumor volume on the day of measurement.
  • T/C% RTV t /RTV c ⁇ 100%
  • RTV t is the average relative tumor volume of the treatment group
  • RTV c is the average relative tumor volume of the vehicle control group.
  • Cell culture Mouse colon cancer CT-26 cells were cultured in vitro. The culture conditions were McCoy's 5a medium plus 10% fetal bovine serum, 100U/mL. Penicillin and 100 ⁇ g/mL streptomycin were cultured in a 37°C, 5% CO2 incubator. Passage was performed twice a week with routine digestion treatment with trypsin-EDTA. When the cell saturation is 80% to 90% and the number reaches the required number, cells are collected, counted, and inoculated.
  • CT-26 cells were subcutaneously inoculated into the right hind limb of each mouse. Group administration began when the tumor volume reached approximately 70 mm 3 on the 9th day after inoculation.
  • Tumor diameter was measured three times a week using vernier calipers.
  • the tumor inhibitory effect of the compound was evaluated by TGI (%) or tumor proliferation rate T/C (%).
  • TGI (%) reflects the tumor growth inhibition rate.
  • TGI (%) [(1-(average tumor volume at the end of treatment in a certain treatment group - average tumor volume at the beginning of treatment in the treatment group))/(average tumor volume at the end of treatment in the solvent control group - start of treatment in the solvent control group) average tumor volume)] ⁇ 100%.
  • T/C% TRTV/CRTV ⁇ 100% (TRTV: average RTV of the treatment group; CRTV: average RTV of the negative control group).
  • the relative tumor volume (RTV) of a single mouse is calculated based on the results of tumor measurement.
  • Tweight and Cweight represent the tumor weight of the drug administration group and the vehicle control group, respectively.
  • the p-toluenesulfonate monotherapy group corresponding to Compound 12 has a certain anti-tumor effect at a dose of 10-40mg/kg.
  • the tumor volume is 1057mm 3
  • T/C is 57.76%
  • TGI is 45.77%
  • p 0.051
  • There is a significant anti-tumor effect at the dose of 90mg/kg the tumor volume is 922mm 3
  • T/C is 47.26%
  • TGI is 53.20%
  • p 0.026.
  • the effect p 0.009, and it has better anti-tumor effect compared with the PD-1 monotherapy group.
  • Buffer 20mM Hepes (pH 7.5), 10mM MgCl 2 , 1mM EGTA, 0.02% Brij35, 0.02mg/ml BSA, 0.1mM Na 3 VO 4 , 2mM DTT, 1% DMSO
  • Kinase activity data are expressed as the percentage of remaining kinase activity in the test sample relative to the vehicle control (dimethyl sulfoxide).
  • BGC-823 cells were cultured in RPMI-1640 culture medium containing 10% fetal bovine serum (FBS) and 1% PS. BGC-823 cells in the logarithmic growth phase were collected, resuspended in HBSS to 5 ⁇ 10 6 /mL, and transplanted subcutaneously into BALB/c Nude mice under sterile conditions. Each mouse was inoculated with 5 ⁇ 10 5 /0.1mL. cell.
  • FBS fetal bovine serum
  • mice On the 10th day after vaccination, when the average tumor volume of the grouped mice reached 157.27 mm 3 , they were randomly divided into 3 groups according to tumor volume, with 6 mice in each group.
  • Tumor volume measurement Use vernier calipers to measure twice a week.
  • TGI% [1-T/C] ⁇ 100.
  • T/C% (delta T/T 0 ) ⁇ 100.
  • the relative tumor volume (RTV) is calculated based on the results of tumor measurement.
  • Tumor weight can be calculated by the relative tumor proliferation rate T/C (%).
  • T/C% Tweight/Cweight ⁇ 100% (Tweight: average tumor weight of the treatment group; Cweight: average tumor weight of the control group).
  • the p-toluenesulfonate (50mg/kg) corresponding to Compound 12 was The tumor growth inhibition rates TGI (%) of the drug group and the p-toluenesulfonate (150 mg/kg) administration group corresponding to compound 12 were 39.79% and 50.14%, respectively. Both the p-toluenesulfonate corresponding to compound 12 (50 mg/kg) and the p-toluenesulfonate corresponding to compound 12 (150 mg/kg) had statistically significant inhibitory effects on tumor growth (P ⁇ 0.05). At the same time, the anti-tumor efficacy of p-toluenesulfonate corresponding to compound 12 is positively correlated with the dosage. ( Figure 18)
  • Calu-3 cells were cultured in MEM medium containing 10% fetal bovine serum (FBS) and 1% PS. Calu-3 cells in the logarithmic growth phase were collected, resuspended in HBSS to 1 ⁇ 10 8 /mL, and transplanted subcutaneously into BALB/c Nude mice under sterile conditions. Each mouse was inoculated with 1 ⁇ 10 7 /0.2mL ( Contains 25% Matrigel) cells.
  • FBS fetal bovine serum
  • Tumor volume measurement Use vernier calipers to measure twice a week.
  • TGI% [1-T/C] ⁇ 100.
  • T/C% (delta T/T 0 ) ⁇ 100.
  • the relative tumor volume (RTV) is calculated based on the results of tumor measurement.
  • the tumor mass was taken and weighed, and photos were taken.
  • the p-toluenesulfonate (50mg/kg) group corresponding to compound 12 and the p-toluenesulfonate (150mg/kg) corresponding to compound 12 The tumor growth inhibition rates TGI (%) of the two groups were 45.74% and 69.42% respectively. According to the above result analysis, it can be seen that the p-toluenesulfonate (50mg/kg) group corresponding to compound 12 can inhibit tumor growth to a certain extent (P>0.05).
  • the p-toluenesulfonate (150mg/kg) group corresponding to compound 12 showed Statistically significant inhibitory effect on tumor growth (P ⁇ 0.05).
  • the anti-tumor efficacy of the corresponding compound of the present invention is positively correlated with the dosage.
  • the p-toluenesulfonate (50 mg/kg) group corresponding to Compound 12 was able to inhibit the subcutaneous transplantation of Calu-3 human lung cancer cells in mice to a certain extent. growth (P>0.05), and the p-toluenesulfonate (150mg/kg) group corresponding to compound 12 showed statistically significant inhibitory effects on tumor growth (P ⁇ 0.05).
  • IC50 For the determination of IC50, the data was analyzed using XLFit version 5.3 (ID Business Solutions), and the S-shaped dose-response curve (variable slope) was fitted by nonlinear regression analysis based on the mean value of each test concentration. When the top and/or bottom values exceed 10% or fall below -10%, the maximum and minimum limits of the curve may be redefined to 100 and 0, subject to meeting the QC criteria of R2.
  • HUVEC cell line Subculture in RPMI-1640 medium containing 10% fetal bovine serum, 100 u/ml penicillin and 100 mg/ml streptomycin at 37°C and 5% CO2 . Cells in the logarithmic growth phase, growing well, and with cell activity greater than 90% were selected for experiments.
  • HUVEC cells in logarithmic growth phase were used for experiments. Different concentrations of p-toluenesulfonate corresponding to Compound 12 were added to RPMI-1640 culture medium with 10% fetal calf serum, and the cell concentration was adjusted to 5 ⁇ 10 5 ml, each dose concentration is a group, placed in a 37°C, 5% CO 2 incubator for different times (48, 72h), incubated for 4h, and fully dissolved. Select the wavelength of 490nm and measure the absorbance (A) value of each well on an enzyme-linked immunoassay detector.
  • SPSS statistical software was used to conduct t test and one-way analysis of variance (ANOVA).
  • A549 cells were cultured in conventional cell culture medium. H460 cells in the logarithmic growth phase were collected, resuspended, and transplanted subcutaneously into mice under sterile conditions. Each mouse was inoculated with 2 ⁇ 10 7 mL cells.
  • mice were randomly divided into 4 groups according to tumor size, with 5 mice in each group.
  • Tumor volume measurement Use a vernier caliper to measure.
  • TGI% [1-T/C] ⁇ 100.
  • T/C% (delta T/T 0 ) ⁇ 100.
  • the relative tumor volume (RTV) is calculated based on the results of tumor measurement.
  • the tumor mass was taken and weighed, and photos were taken.
  • H460 cells were cultured in conventional cell culture medium. H460 cells in the logarithmic growth phase were collected, resuspended, and transplanted subcutaneously into mice under sterile conditions. Each mouse was inoculated with 1 ⁇ 10 7 mL cells.
  • mice were randomly divided into 4 groups according to tumor size, with 6 mice in each group.
  • Tumor volume measurement Use a vernier caliper to measure.
  • TGI% [1-T/C] ⁇ 100.
  • T/C% (delta T/T 0 ) ⁇ 100.
  • the relative tumor volume (RTV) is calculated based on the results of tumor measurement.
  • the tumor mass was taken and weighed, and photos were taken.
  • the TGIs of the p-toluenesulfonate (12 mg/kg + 50 mg/kg) group corresponding to paclitaxel + compound 12 were 30.17%, 66.24%, and 93.22%, respectively.
  • mice were randomly divided into 3 groups, with 5-8 mice in each group. Drug intervention was started on the 7th day after subcutaneous tumor inoculation.
  • the grouping and administration conditions were as follows:
  • Blank control group 100 ⁇ L normal saline was administered daily;
  • Solvent control group 100 ⁇ L of solvent was administered daily.
  • Drug experimental group The dose is 50 mg/kg, and about 100 ⁇ L of p-toluenesulfonate solution corresponding to Compound 12 is administered daily according to body weight.
  • volume formula long diameter * short diameter * short diameter / 2
  • the mice were sacrificed approximately 3 weeks after administration.
  • mice were randomly divided into 5 groups, with 5-8 mice in each group. Drug intervention was started on the 7th day after subcutaneous tumor inoculation.
  • the grouping and administration conditions were as follows:
  • Cisplatin (DDP) experimental group The drug dose is 2-4 mg/kg, and about 100 ⁇ L of DDP solution is injected intraperitoneally every week according to body weight;
  • Paclitaxel (PTX) experimental group 12 mg/kg, approximately 100 ⁇ L of PTX solution is injected intraperitoneally according to body weight every week;
  • the length, short diameter and body weight of the transplanted tumors in nude mice were measured every 1 day, and the tumor growth curve and body weight change curve were drawn respectively.
  • attention should be paid to the growth status of the mice such as skin color and temperature, whether there is weight loss, etc.
  • the mice were sacrificed approximately 3 weeks after administration.
  • nude mice The important organs of nude mice were fixed with 4% paraformaldehyde and reserved for subsequent paraffin embedding, sectioning and HE staining.
  • the p-toluenesulfonate corresponding to the MNK/VEGFR2 small molecule inhibitor compound 12 was used to treat the xenograft tumor model of human DLBCL cell SU-DHL-6 and MCL cell JeKo-1.
  • mice After tumor grafting, the patients were randomly divided into 5 groups: blank group, solvent group, p-toluenesulfonate single drug group corresponding to compound 12, doxorubicin single drug group and combination group. Among them, the p-toluenesulfonate corresponding to Compound 12 was intragastrically administered at 25 mg/kg daily, and doxorubicin was administered through the tail vein at 3.3 mg/kg, once every 10 days, a total of 2 times.
  • mice were sacrificed, and the subcutaneous tumors were carefully peeled off, photographed, weighed, and analyzed statistically. At the same time, fresh eye blood and important organs were collected.
  • mouse lung cancer cells LL2 were injected subcutaneously. Inject 100 ⁇ L of cell suspension containing 1X10 6 LL2 into the right back of female C57 mice. One week after inoculation (tumor volume is about 50-100mm 3 ), the mice were randomly divided into 4 groups, with 6 mice in each group.
  • the p-toluenesulfonate corresponding to compound 12 is administered orally daily at a dose of 50 mg/kg.
  • Single PD-1 antibody PD-1 antibody was administered intraperitoneally twice a week starting from the 5th day after tumor grafting, with a dose of 200 ⁇ g/animal.
  • PD-1 isotype control ISO Administration started on the 5th day after tumor grafting, twice a week, intraperitoneally, with a dose of 200 ⁇ g/animal.
  • Combination therapy After the start of treatment, the p-toluenesulfonate corresponding to Compound 12 was administered orally every day, and the PD-1 antibody was injected intraperitoneally twice a week, 200 ⁇ g/animal.
  • Cell culture Perform routine cell culture in 5% CO 2 , 37°C, and MEM culture medium containing 10% fetal bovine serum; digest and passage with 0.25% trypsin; according to cell growth, passage 2 to 3 times a week, passage ratio is 1:3 to 1:6.
  • Animal model preparation Collect Hep G2 cells in the logarithmic growth phase, count the cells and resuspend them in medium containing 50% serum-free MEM and 50% Matrigel. Adjust the cell concentration to 2.5 ⁇ 10 7 cells/mL; use a pipette Pipette the cells to disperse them evenly and then put them into a 50-mL centrifuge tube. Place the centrifuge tube in an ice box; use a 1-mL syringe to absorb the cell suspension and inject it into the subcutaneous axilla of the front right limb of the nude mouse. Each animal is inoculated with 200L. (5 ⁇ 10 6 cells/mouse) to establish a Hep G2 nude mouse transplanted tumor model.
  • the animal status and tumor growth were regularly observed, and the tumor diameter was measured using an electronic vernier caliper.
  • the data was directly entered into an Excel spreadsheet to calculate the tumor volume.
  • the tumor volume reaches 100-300mm 3
  • the day of grouping was regarded as the first day of the experiment (D1).
  • the tumor diameter was measured twice a week, the tumor volume was calculated, and the animal body weight was weighed and recorded.
  • the animal grouping and dosing schedule are shown in Table 2. Dosing will begin on the day of grouping and end the experiment after 3 weeks (or the tumor volume in the solvent control group reaches more than 2000 mm 3 , whichever comes first). The dosing volume is 10 mL ⁇ kg. -1 . On the last day of the experiment, the body weight and tumor diameter were measured, and the animals were euthanized by CO 2 inhalation. The tumor pieces were removed, weighed, and photographed.
  • MDA-MB-231 cells were cultured in L-15 culture medium containing 10% fetal bovine serum; according to the cell growth conditions, 0.25% pancreatic Enzyme digestion and passage, 1 to 2 times a week, with a passage ratio of 1:3 to 1:10.
  • Animal model preparation Collect MDA-MB-231 cells in the logarithmic growth phase, count the cells and resuspend them in serum-free L-15 culture medium. Adjust the cell concentration to 3 ⁇ 10 7 cells/mL; use a pipette to pipette the cells. Disperse it evenly and put it into a 50-mL centrifuge tube. Place the centrifuge tube in an ice box; use a 1-mL syringe to absorb the cell suspension and inject it into the subcutaneous skin of the axilla of the front right limb of nude mice. Each animal is inoculated with 100L (3.0 ⁇ 10 6 cells/mouse) to establish MDA-MB-231 nude mouse transplanted tumor model.
  • the animal status and tumor growth were regularly observed, and the tumor diameter was measured using an electronic vernier caliper.
  • the data was directly entered into an Excel spreadsheet to calculate the tumor volume.
  • the tumor volume reaches 100-300mm 3
  • the day of grouping was regarded as the first day of the experiment (D1).
  • the tumor diameter was measured twice a week, the tumor volume was calculated, and the animal body weight was weighed and recorded.
  • the animal grouping and dosing schedule are shown in Table 3. Dosing will begin on the day of grouping and end the experiment after 3 weeks (or the tumor volume in the solvent control group reaches more than 2000 mm 3 , whichever comes first). The dosing volume is 10 mL ⁇ kg. -1 . On the last day of the experiment, the body weight and tumor diameter were measured, and the animals were euthanized by CO 2 inhalation. The tumor pieces were removed, weighed, and photographed.

Abstract

The present invention relates to use of a compound represented by formula (I) in the preparation of an anti-tumor drug. The compound has an inhibitory effect on eIF4E protein and/or VEGFR2 protein, and has a good inhibitory effect on various solid tumors or non-solid tumors.

Description

一种吡咯并三嗪类化合物在制备抗肿瘤药物中的应用Application of a kind of pyrrolotriazine compound in the preparation of anti-tumor drugs
本发明要求享有于2022年7月19日向中国国家知识产权局提交的，专利申请号为2022108552809，名称为“一种吡咯并三嗪类化合物在制备抗肿瘤药物中的应用”的在先申请的优先权。该在先申请的全文通过引用的方式结合于本发明中。The present invention claims the benefit of a prior application submitted to the State Intellectual Property Office of China on July 19, 2022, with a patent application number of 2022108552809, titled "Application of a pyrrolotriazine compound in the preparation of anti-tumor drugs" priority. The entirety of this prior application is incorporated herein by reference.
技术领域Technical field
本发明属于医药领域，具体涉及一种吡咯并三嗪类化合物在制备抗肿瘤药物中的应用。The invention belongs to the field of medicine, and specifically relates to the application of a pyrrolotriazine compound in the preparation of anti-tumor drugs.
背景技术Background technique
有丝分裂原激活的蛋白激酶作用激酶(MAP kinase interacting kinase，MNK)，属于丝氨酸/苏氨酸蛋白激酶。MNK蛋白由两个基因MKNK1和MKNK2编码，人类的MNK有四种亚型，MNK1a，MNK1b和MNK2a，MNK2b，分别由MNK1和MNK2基因表达(Ahmed M Abdelaziz1，2020)。4种亚型在N端都含有细胞核定位信号序列(nuclear localization signal，NLS)和与eIF4G结合的序列，使其可以进入细胞核发挥作用，识别并结合下游eIF4E。MNK1a和MNK2a亚型C端具有与MAPK结合的位点，可以被上游ERK和p38磷酸化激活(Jianling Xie，2019)。MNK1a的C端的细胞核转出信号序列(nuclear export signal，NES)使其可以广泛存在于细胞质中，而其他3个亚型多数存在于细胞核中。MNK蛋白能够特异性的将真核细胞翻译起始因子4E(eukaryotic initiation factor 4E，eIF4E)的Ser209磷酸化，从而间接的调控mRNA翻译(Fukunaga R，1997)。Mitogen-activated protein kinase interacting kinase (MAP kinase interacting kinase, MNK) is a serine/threonine protein kinase. The MNK protein is encoded by two genes, MKNK1 and MKNK2. There are four subtypes of human MNK, MNK1a, MNK1b and MNK2a, MNK2b, which are expressed by the MNK1 and MNK2 genes respectively (Ahmed M Abdelaziz1, 2020). All four isoforms contain a nuclear localization signal (NLS) and a sequence that binds to eIF4G at the N-terminus, allowing them to enter the nucleus and function, recognizing and binding to downstream eIF4E. The C-termini of MNK1a and MNK2a subtypes have binding sites for MAPK and can be activated by upstream ERK and p38 phosphorylation (Jianling Xie, 2019). The C-terminal nuclear export signal (NES) of MNK1a allows it to be widely present in the cytoplasm, while the other three isoforms mostly exist in the nucleus. MNK protein can specifically phosphorylate Ser209 of eukaryotic translation initiation factor 4E (eIF4E), thereby indirectly regulating mRNA translation (Fukunaga R, 1997).
真核细胞翻译起始因子eIF4E作为一种重要的转录因子，能够加强编码调控细胞周期蛋白与致癌蛋白mRNA的转录从而引起肿瘤相关蛋白表达的上调(Truitt ML，2015)。eIF4E与骨架蛋白eIF4G和RNA解旋酶eIF4A组成了真核起始因子复合体eIF4F。由于eIF4E负责该复合体与mRNA的5’末端帽子结构结合，所以其在RNA翻译过程中发挥不可替代的调控作用。在正常细胞中，eIF4E的活性被限制，这些肿瘤相关mRNA的转录得到抑制，而在肿瘤细胞或组织中，eIF4E的高表达或过度活化会引起这些mRNA转录水平的上调(Lama D，2019)。As an important transcription factor, eukaryotic cell translation initiation factor eIF4E can strengthen the transcription of mRNA encoding and regulating cell cycle proteins and oncogenic proteins, thereby causing the upregulation of tumor-related protein expression (Truitt ML, 2015). eIF4E, the skeleton protein eIF4G and the RNA helicase eIF4A form the eukaryotic initiation factor complex eIF4F. Since eIF4E is responsible for the binding of this complex to the 5' end cap structure of mRNA, it plays an irreplaceable regulatory role in the RNA translation process. In normal cells, the activity of eIF4E is limited and the transcription of these tumor-related mRNAs is inhibited, while in tumor cells or tissues, high expression or overactivation of eIF4E can cause the upregulation of the transcription levels of these mRNAs (Lama D, 2019).
MNK1/2和eIF4E是RAS-RAF-MEK-ERK和PI3K-AKT-mTOR信号通路的汇聚点,在信号通路传导中起到重要作用。eIF4E作为一个重要的翻译限速因子，MNK1/2和eIF4E信号通路能够影响多种趋化因子，细胞因子以及免疫检查点蛋白的合成，进而调控免疫应答(Gorentla，B.K.2012)。MNK1/2 and eIF4E are the convergence points of the RAS-RAF-MEK-ERK and PI3K-AKT-mTOR signaling pathways and play an important role in signaling pathway conduction. As an important translation rate-limiting factor, eIF4E, MNK1/2 and eIF4E signaling pathways can affect the synthesis of a variety of chemokines, cytokines and immune checkpoint proteins, thereby regulating immune responses (Gorentla, B.K. 2012).
VEGF/VEGFR通路是肿瘤血管生成的主要信号通路。血管内皮生长因子(VascμLar endothelial growth factor,VEGF)家族成员包括VEGF-A、VEGF-B、VEGF-C、VEGF-D和PIGF。VEGF，也称VEGF-A，是生理和病理性血管生成最重要的刺激因子。VEGF作用有：维持内皮细胞的存活，诱导内皮细胞增殖和迁移，募集骨髓源性造血祖或干细胞诱导血管形成，增强血管通透性。VEGF-A有两种酪氨酸激酶受体，即VEGFR-1(Flt-1)和VEGFR-2(KDR/Flk-1)。VEGFR-2是VEGF发挥促血管生成效应的主要受体。多种肿瘤细胞和宿主细胞如血小板、肌细胞、基质细胞都能产生VEGF。缺氧是VEGF表达上调重要的诱导因素。促进VEGF的分泌的其它因素有：低pH值；生长因子；炎症趋化因子；基因突变。The VEGF/VEGFR pathway is the main signaling pathway for tumor angiogenesis. Vascular endothelial growth factor (VEGF) family members include VEGF-A, VEGF-B, VEGF-C, VEGF-D and PIGF. VEGF, also known as VEGF-A, is the most important stimulator of physiological and pathological angiogenesis. The functions of VEGF include: maintaining the survival of endothelial cells, inducing endothelial cell proliferation and migration, recruiting bone marrow-derived hematopoietic progenitors or stem cells to induce blood vessel formation, and enhancing vascular permeability. VEGF-A has two tyrosine kinase receptors, namely VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1). VEGFR-2 is the main receptor for VEGF to exert its pro-angiogenic effects. A variety of tumor cells and host cells such as platelets, myocytes, and stromal cells can produce VEGF. Hypoxia is an important inducing factor for up-regulation of VEGF expression. Other factors that promote the secretion of VEGF include: low pH; growth factors; inflammatory chemokines; and genetic mutations.
发明内容Contents of the invention
本发明提供了一种式(I)所示化合物或其药学上可接受的盐在制备抗肿瘤药物中的应用，
The invention provides the use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof in the preparation of anti-tumor drugs,
其中，R1为H、F、Cl、Br或C1-3烷基；Wherein, R 1 is H, F, Cl, Br or C 1-3 alkyl;
R2和R3各自独立地为H或C1-3烷基，其中所述C1-3烷基任选被1、2或3个独立选自F、Cl、Br或I的取代基所取代； R 2 and R 3 are each independently H or C 1-3 alkyl, wherein the C 1-3 alkyl is optionally replaced by 1, 2 or 3 substituents independently selected from F, Cl, Br or I replace;
或R2和R3与它们相连的碳原子连接在一起形成环戊基、环己基或哌啶基，其中所述环戊基、环己基和哌啶基任选被1、2或3个Ra所取代；Or R 2 and R 3 are joined together with the carbon atom to which they are attached to form cyclopentyl, cyclohexyl or piperidinyl, wherein said cyclopentyl, cyclohexyl and piperidinyl are optionally replaced by 1, 2 or 3 R replaced by a ;
各Ra独立地为H、F、Cl、Br或C1-3烷基；Each R a is independently H, F, Cl, Br or C 1-3 alkyl;
R4为H、F、Cl、Br或C1-3烷基；R 4 is H, F, Cl, Br or C 1-3 alkyl;
R5和R6各自独立地为H、F、Cl、Br、I或C1-3烷基；R 5 and R 6 are each independently H, F, Cl, Br, I or C 1-3 alkyl;
R7为吡咯烷基，其中所述吡咯烷基任选被1、2或3个Rb所取代；R 7 is pyrrolidinyl, wherein the pyrrolidinyl is optionally substituted by 1, 2 or 3 R b ;
各Rb独立地为H、F、Cl、Br、I或C1-3烷基，其中所述C1-3烷基任选被1、2或3个独立选自F、Cl、Br或I的取代基所取代；Each R b is independently H, F, Cl, Br, I or C 1-3 alkyl, wherein the C 1-3 alkyl is optionally 1, 2 or 3 independently selected from F, Cl, Br or Substituted by the substituent of I;
n为1或2。n is 1 or 2.
在本发明的一些方案中，上述各Ra独立地为H、F、Cl、Br、-CH3或-CH2CH3，其他变量如本发明所定义。In some aspects of the present invention, each of the above R a is independently H, F, Cl, Br, -CH 3 or -CH 2 CH 3 , and other variables are as defined in the present invention.
在本发明的一些方案中，上述R2和R3各自独立地为H、-CH3或-CH2CH3，其他变量如本发明所定义。In some aspects of the present invention, the above-mentioned R 2 and R 3 are each independently H, -CH 3 or -CH 2 CH 3 , and other variables are as defined in the present invention.
在本发明的一些方案中，上述R2和R3与它们相连的碳原子连接在一起形成 Ra及其他变量如本发明所定义。In some embodiments of the present invention, the above-mentioned R 2 and R 3 are connected together with the carbon atoms to which they are connected to form R a and other variables are as defined in the present invention.
在本发明的一些方案中，上述R2和R3与它们相连的碳原子连接在一起形成 其他变量如本发明所定义。In some embodiments of the present invention, the above-mentioned R 2 and R 3 are connected together with the carbon atoms to which they are connected to form Other variables are as defined in the present invention.
在本发明的一些方案中，上述结构单元为 R1、Ra及其他变量如本发明所定义。In some solutions of the present invention, the above structural units for R 1 , R a and other variables are as defined in the present invention.
在本发明的一些方案中，上述结构单元为 其他变量如本发明所定义。在本发明的一些方案中，R1为C1-3烷基，如甲基。In some solutions of the present invention, the above structural units for Other variables are as defined in the present invention. In some embodiments of the invention, R 1 is C 1-3 alkyl, such as methyl.
在本发明的一些方案中，R2和R3与它们相连的碳原子连接在一起形成 In some embodiments of the invention, R 2 and R 3 are joined together with the carbon atom to which they are connected to form
在本发明的一些方案中，R4为C1-3烷基，如甲基。In some embodiments of the invention, R 4 is C 1-3 alkyl, such as methyl.
在本发明的一些方案中，R5和R6各自独立地为H或甲基；n为2；In some embodiments of the invention, R 5 and R 6 are each independently H or methyl; n is 2;
在本发明的一些方案中，为 In some aspects of the invention, for
在本发明的一些方案中，R7为被1、2或3个H、F、Cl或甲基所取代的例如为在本发明的一些方案中，上述化合物具有式(I-1)～(I-4)任一结构式所示结构：
In some embodiments of the invention, R 7 is substituted by 1, 2 or 3 H, F, Cl or methyl For example In some aspects of the present invention, the above compound has a structure represented by any of the formulas (I-1) to (I-4):
其中，R1、R4、R5、R6、R7、Ra和n如本发明所定义。Among them, R 1 , R 4 , R 5 , R 6 , R 7 , R a and n are as defined in the present invention.
在本发明的一些方案中，上述各Rb独立地H、F、Cl、Br、I、其他变量如本发明所定义。In some aspects of the present invention, each of the above R b is independently H, F, Cl, Br, I, Other variables are as defined in the present invention.
在本发明的一些方案中，上述R7为其中所述任选被1或2个Rb所取代，Rb及其他变量如本发明所定义。In some aspects of the present invention, the above-mentioned R 7 is stated therein Optionally substituted by 1 or 2 R b , R b and other variables are as defined in the present invention.
在本发明的一些方案中，上述R7为Rb及其他变量如本发明所定义。In some aspects of the present invention, the above-mentioned R 7 is R b and other variables are as defined in the present invention.
在本发明的一些方案中，上述R7为其他变量如本发明所定义。In some aspects of the present invention, the above-mentioned R 7 is Other variables are as defined in the present invention.
在本发明的一些方案中，上述R4为H或-CH3，其他变量如本发明所定义。In some aspects of the present invention, the above-mentioned R 4 is H or -CH 3 , and other variables are as defined in the present invention.
在本发明的一些方案中，上述化合物具有式(I-5)～式(I-9)任一结构式所示结构：

In some aspects of the present invention, the above compound has a structure represented by any of the structural formulas of formula (I-5) to formula (I-9):

其中，R1、R5、R6、Ra和Rb如本发明所定义。Among them, R 1 , R 5 , R 6 , R a and R b are as defined in the present invention.
在本发明的一些方案中，上述R1为H、F、Cl或其他变量如本发明所定义。In some aspects of the invention, the above R 1 is H, F, Cl or Other variables are as defined in the present invention.
在本发明的一些方案中，上述R5和R6各自独立地为H或其他变量如本发明所定义。本发明还有一些方案是由上述变量任意组合而来。In some aspects of the present invention, the above R 5 and R 6 are each independently H or Other variables are as defined in the present invention. There are also some solutions of the present invention that are derived from any combination of the above variables.
根据本发明的实施方案，式(I)所示化合物选自以下结构：

According to an embodiment of the present invention, the compound represented by formula (I) is selected from the following structures:

根据本发明的实施方案，所述药学上可接受的盐为式(I)所示化合物与无机酸形成的盐，所述无机酸包括例如盐酸、氢溴酸、硝酸、碳酸，碳酸氢根，磷酸、磷酸一氢根、磷酸二氢根、硫酸、硫酸氢根、氢碘酸、亚磷酸等；以及有机酸盐，所述有机酸包括如乙酸、丙酸、异丁酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、酒石酸和甲磺酸等类似的酸；还包括氨基酸(如精氨酸等)的盐，以及如葡糖醛酸等有机酸的盐；优选为式(I)所示化合物的盐酸盐、对甲苯磺酸盐。According to an embodiment of the present invention, the pharmaceutically acceptable salt is a salt formed by a compound represented by formula (I) and an inorganic acid. The inorganic acid includes, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, and bicarbonate, Phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydriodic acid, phosphorous acid, etc.; and organic acid salts, including acetic acid, propionic acid, isobutyric acid, maleic acid, Malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid and similar Acids; also include salts of amino acids (such as arginine, etc.), and salts of organic acids such as glucuronic acid; preferably the hydrochloride and p-toluenesulfonate of the compound represented by formula (I).
如本文所述，“肿瘤”包含“高增殖性疾病”，“高增殖性疾病”指与正常细胞或未溶解的细胞相比过度生长或增殖，包括异型增生、肿瘤、非接触性抑制或癌变细胞、癌症、细胞癌、肉瘤、恶性细胞、癌前细胞，以及非肿瘤性或非恶性高增殖性疾病(例如，腺瘤、纤维瘤、脂肪瘤、平滑肌瘤、血管瘤、纤维化、再狭窄等)。As used herein, "neoplastic" includes "hyperproliferative disease," which refers to excessive growth or proliferation compared to normal cells or unlysed cells, including dysplasia, neoplasia, noncontact suppression, or carcinogenesis. Cells, cancers, cellular carcinomas, sarcomas, malignant cells, precancerous cells, and nonneoplastic or nonmalignant hyperproliferative diseases (e.g., adenomas, fibromas, lipomas, leiomyomas, hemangiomas, fibrosis, narrow, etc.).
“肿瘤”指各种高增殖性疾病，包括实体瘤和血液肿瘤，这些不同形式的高增值性疾病在本领域是已知的，并且建立了诊断和分类的标准。示例性实体瘤包括：乳腺癌；前列腺癌；结肠癌；直肠癌；肝癌；肾癌；胃癌；膀胱癌；所有形式的肺支气管癌；骨髓癌；黑色素瘤；神经母细胞瘤；状瘤；脓胸；绒毛膜瘤；分支瘤；恶性类癌综合征；类癌性心脏病；细胞癌(例如，Walker癌、基底细胞癌、基底鳞状细胞癌、Brown-Pearce癌、导管癌、粘液样癌、非小细胞癌、燕麦细胞癌、状细胞癌、细支气管癌、支气管癌、鳞状细胞癌和移行细胞癌)；浆细胞瘤；网状内皮综合征；软骨母细胞瘤；软骨病；软骨肉瘤；纤维瘤；巨细胞肿瘤；组织细胞瘤；脂肪瘤；脂肪肉瘤；间皮瘤；骨瘤；骨肉瘤；脊椎瘤；颅咽管瘤；生殖细胞瘤；错构瘤；间质瘤；中胚层肌肉瘤；神经母细胞瘤；骨髓瘤；牙龈瘤；畸胎瘤；胸腺瘤；滋养层肿瘤；膀胱腺瘤；胰岛细胞肿瘤；平滑肌肉瘤；横纹肌瘤；室管膜瘤；神经节性神经瘤；胶质瘤；髓母细胞瘤；脑膜瘤；神经性腺瘤；神经母细胞瘤；神经上皮瘤；神经纤维瘤；神经瘤；血管内皮瘤；血管瘤；血管肉瘤。示例性血液恶性肿瘤包括：急性淋巴细胞白血病(ALL)、急性髓系白血病(AML)、慢性粒细胞白血病(CML)、慢性嗜酸性粒细胞白血病(CEL)、骨髓增生异常综合征(MDS)、霍奇金淋巴瘤、非霍奇金淋巴瘤(NHL)(例如滤泡性淋巴瘤(FL)、套细胞淋巴瘤(MCL)、边缘区淋巴瘤(MZL)、弥漫性大B细胞淋巴瘤(DLBCL)、慢性淋巴细胞白血病(CLL))或多发性骨髓瘤(MM)。 "Neoplastic" refers to a variety of hyperproliferative diseases, including solid tumors and hematological tumors. These different forms of hyperproliferative diseases are known in the art and have established standards for diagnosis and classification. Exemplary solid tumors include: breast cancer; prostate cancer; colon cancer; rectal cancer; liver cancer; kidney cancer; stomach cancer; bladder cancer; all forms of lung and bronchial cancer; bone marrow cancer; melanoma; neuroblastoma; parenchyma; pus Chest; choriomas; branching tumors; malignant carcinoid syndrome; carcinoid heart disease; cellular carcinoma (e.g., Walker carcinoma, basal cell carcinoma, basal squamous cell carcinoma, Brown-Pearce carcinoma, ductal carcinoma, myxoid carcinoma , non-small cell carcinoma, oat cell carcinoma, synovial cell carcinoma, bronchiolar carcinoma, bronchial carcinoma, squamous cell carcinoma and transitional cell carcinoma); plasmacytoma; reticuloendothelial syndrome; chondroblastoma; rickets; chondrosis Sarcoma; Fibroma; Giant cell tumor; Histiocytoma; Lipoma; Liposarcoma; Mesothelioma; Osteoma; Osteosarcoma; Spinal tumor; Craniopharyngioma; Germ cell tumor; Hamartoma; Stromal tumor; Medium Germ sarcoma; neuroblastoma; myeloma; gingival tumor; teratoma; thymoma; trophoblastic tumor; bladder adenoma; islet cell tumor; leiomyosarcoma; rhabdomyomas; ependymoma; ganglioneuroma ; Glioma; Medulloblastoma; Meningioma; Neurogenic adenoma; Neuroblastoma; Neuroepithelioma; Neurofibroma; Neuroma; Hemangioendothelioma; Hemangioma; Angiosarcoma. Exemplary hematological malignancies include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic eosinophilic leukemia (CEL), myelodysplastic syndrome (MDS), Hodgkin lymphoma, non-Hodgkin lymphoma (NHL) (such as follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), diffuse large B-cell lymphoma ( DLBCL), chronic lymphocytic leukemia (CLL), or multiple myeloma (MM).
具体而言，根据本发明的实施方案，所述肿瘤选自实体瘤或非实体瘤，例如黑色素瘤、肾癌、血液癌、前列腺癌、结肠癌、直肠癌、胃癌、食道癌、膀胱癌、头颈癌(如头颈部鳞癌)、甲状腺癌、乳腺癌(如三阴性乳腺癌)、卵巢癌、宫颈癌、肺癌(如非小细胞肺癌)、尿路上皮癌、胰腺癌、胶质母细胞瘤、肝癌、结肠腺癌、星形细胞瘤、骨肉瘤、骨髓瘤(如多发性骨髓瘤)、淋巴瘤{霍奇金淋巴瘤、非霍奇金淋巴瘤(例如，滤泡性淋巴瘤(FL)、套细胞淋巴瘤(MCL)、边缘区淋巴瘤(MZL)、弥漫性大B细胞淋巴瘤(DLBCL)、慢性淋巴细胞白血病(CLL)、)或多发性骨髓瘤(MM))}、骨髓增生异常综合征、脑癌、中枢神经系统癌、恶性胶质瘤、白血病(如急性淋巴性白血病、急性髓性白血病)中的至少一种或其任意组合。Specifically, according to an embodiment of the present invention, the tumor is selected from solid tumors or non-solid tumors, such as melanoma, kidney cancer, blood cancer, prostate cancer, colon cancer, rectal cancer, stomach cancer, esophageal cancer, bladder cancer, Head and neck cancer (such as head and neck squamous cell carcinoma), thyroid cancer, breast cancer (such as triple-negative breast cancer), ovarian cancer, cervical cancer, lung cancer (such as non-small cell lung cancer), urothelial cancer, pancreatic cancer, glioblastoma Cytoma, liver cancer, colon adenocarcinoma, astrocytoma, osteosarcoma, myeloma (eg, multiple myeloma), lymphoma (Hodgkin lymphoma, non-Hodgkin lymphoma (eg, follicular lymphoma) (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL), or multiple myeloma (MM)} , myelodysplastic syndrome, brain cancer, central nervous system cancer, malignant glioma, at least one of leukemia (such as acute lymphoblastic leukemia, acute myeloid leukemia) or any combination thereof.
根据本发明的实施方案，所述肿瘤选自肾癌、前列腺癌、结肠癌、直肠癌、胃癌、食道癌、甲状腺癌、乳腺癌、肺癌(如非小细胞肺癌)、卵巢癌、胶质母细胞瘤、肝癌、淋巴瘤{如慢性淋巴细胞白血病(CLL)}、白血病(如急性髓性白血病)。According to an embodiment of the present invention, the tumor is selected from the group consisting of kidney cancer, prostate cancer, colon cancer, rectal cancer, gastric cancer, esophageal cancer, thyroid cancer, breast cancer, lung cancer (such as non-small cell lung cancer), ovarian cancer, glioblastoma Cell tumors, liver cancer, lymphoma {such as chronic lymphocytic leukemia (CLL)}, leukemia (such as acute myeloid leukemia).
根据本发明的实施方案，所述肿瘤选自肾癌、结肠癌、直肠癌、胃癌、肺癌(如非小细胞肺癌)、卵巢癌、淋巴瘤、肝癌、乳腺癌、白血病(如急性髓性白血病)。According to an embodiment of the present invention, the tumor is selected from the group consisting of kidney cancer, colon cancer, rectal cancer, gastric cancer, lung cancer (such as non-small cell lung cancer), ovarian cancer, lymphoma, liver cancer, breast cancer, leukemia (such as acute myeloid leukemia) ).
根据本发明的实施方案，所述细胞为结肠癌CT-26细胞、结肠癌HT-29细胞、结肠癌SW480细胞、肺癌Calu-3细胞、肺癌A549细胞、肺癌H460细胞、胃癌BGC-823细胞。According to an embodiment of the present invention, the cells are colon cancer CT-26 cells, colon cancer HT-29 cells, colon cancer SW480 cells, lung cancer Calu-3 cells, lung cancer A549 cells, lung cancer H460 cells, and gastric cancer BGC-823 cells.
根据本发明的实施方案，式(I)所示化合物或其药学上可接受的盐对VEGFR2，例如对p-VEGFR2具有抑制作用。According to an embodiment of the present invention, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof has an inhibitory effect on VEGFR2, such as p-VEGFR2.
根据本发明的实施方案，式(I)所示化合物或其药学上可接受的盐对eIF4E和VEGFR2同时具有抑制作用，例如对p-eIF4E和p-VEGFR2具有抑制作用。According to an embodiment of the present invention, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof has an inhibitory effect on both eIF4E and VEGFR2, for example, an inhibitory effect on p-eIF4E and p-VEGFR2.
本发明还提供一种药物组合物，包括式(I)所示化合物或其药学上可接受的盐和至少一种第二药物。“组合”是指包含式(I)所示化合物和至少一种第二药物，其中每一种都可以连续、同时或同时施用(可首先施用式(I)所示化合物，或者先施用第二药物)。The present invention also provides a pharmaceutical composition, including a compound represented by formula (I) or a pharmaceutically acceptable salt thereof and at least one second drug. "Combination" refers to a compound represented by formula (I) and at least one second drug, each of which can be administered continuously, simultaneously or simultaneously (the compound represented by formula (I) can be administered first, or the second drug can be administered first). drug).
根据本发明的实施方案，所述药物组合物中，式(I)所示化合物或其药学上可接受的盐与所述第二药物的质量比为1:500至500:1，例如为1:50至50:1或1:10至10:1，示例性为1:5、1:4、1:3、1:2、1:1、2:1、3:1、10:3、4:1、25:6、5:1、25:1。According to an embodiment of the present invention, in the pharmaceutical composition, the mass ratio of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof to the second drug is 1:500 to 500:1, for example, 1 :50 to 50:1 or 1:10 to 10:1, examples are 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 10:3, 4:1, 25:6, 5:1, 25:1.
根据本发明的实施方案，所述药物组合物中，当所述第二药物为顺铂时，式(I)所示化合物或其药学上可接受的盐与所述第二药物的质量比为10:1至50:1，例如为15:1至35:1，示例性为20:1、25:1、30:1。According to an embodiment of the present invention, in the pharmaceutical composition, when the second drug is cisplatin, the mass ratio of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof to the second drug is 10:1 to 50:1, for example, 15:1 to 35:1, and examples are 20:1, 25:1, and 30:1.
根据本发明的实施方案，所述药物组合物中，当所述第二药物为紫杉醇时，式(I)所示化合物或其药学上可接受的盐与所述第二药物的质量比为1:1至20:1，例如为2:1至10:1，示例性为3:1、4:1、25:6、5:1、6:1、8:1。According to an embodiment of the present invention, in the pharmaceutical composition, when the second drug is paclitaxel, the mass ratio of the compound represented by formula (I) or its pharmaceutically acceptable salt to the second drug is 1 :1 to 20:1, for example, 2:1 to 10:1, for example, 3:1, 4:1, 25:6, 5:1, 6:1, 8:1.
根据本发明的实施方案，所述药物组合物中，当所述第二药物为阿霉素时，式(I)所示化合物或其药学上可接受的盐与所述第二药物的质量比为1:1至20:1，例如为2:1至10:1，示例性为3:1、4:1、5:1、6:1、7:1、250:33、8:1、9:1。According to an embodiment of the present invention, in the pharmaceutical composition, when the second drug is doxorubicin, the mass ratio of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof to the second drug 1:1 to 20:1, for example, 2:1 to 10:1, for example, 3:1, 4:1, 5:1, 6:1, 7:1, 250:33, 8:1, 9:1.
根据本发明的实施方案，所述药物组合物中，当所述第二药物为PD-1时，式(I)所示化合物或其药学上可接受的盐与所述第二药物的质量比为100:1至400:1，例如为150:1至300:1，示例性为200:1、250:1。According to an embodiment of the present invention, in the pharmaceutical composition, when the second drug is PD-1, the mass ratio of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof to the second drug The ratio is 100:1 to 400:1, for example, 150:1 to 300:1, and examples are 200:1 and 250:1.
根据本发明的实施方案，所述第二药物选自免疫检查点抑制剂、化疗药物、或靶向药物。According to an embodiment of the present invention, the second drug is selected from immune checkpoint inhibitors, chemotherapy drugs, or targeted drugs.
根据本发明的实施方案，所述免疫检查点抑制剂靶标包括免疫抑制信号涉及PD-1、PD-L1、PD-L2、CTLA4、CD80、CD86、B7-H3、B7-H4、HVEM、BTLA、KIR、LAG3、GAL9、TIM3、2B4、腺苷、A2aR、TGFP、BCL-2和免疫抑制细胞因子(如IL-10、IL-35)的至少一种。免疫抑制抑制剂组分可以是化合物、抗体、抗体片段或融合多肽(例如Fc融合，例如CTLA4-Fc)、反义分子、核酶或RNAi分子，或低分子量有机分子。According to embodiments of the present invention, the immune checkpoint inhibitor targets include immunosuppressive signals involving PD-1, PD-L1, PD-L2, CTLA4, CD80, CD86, B7-H3, B7-H4, HVEM, BTLA, At least one of KIR, LAG3, GAL9, TIM3, 2B4, adenosine, A2aR, TGFP, BCL-2 and immunosuppressive cytokines (such as IL-10, IL-35). The immunosuppressive inhibitor component can be a compound, antibody, antibody fragment or fusion polypeptide (eg Fc fusion, eg CTLA4-Fc), antisense molecule, ribozyme or RNAi molecule, or low molecular weight organic molecule.
根据本发明的实施方案，所述化疗药物选自以下药物中的至少一种：抗代谢物/抗癌剂，如嘧啶类似物(5-氟尿嘧啶(5-fu)、氟尿苷、卡培他滨、吉西他滨、地西他滨、阿扎胞苷和阿糖胞苷)和嘌呤类似物、叶酸拮抗剂和相关免疫抑制剂(氨甲蝶呤、培美曲塞、巯基嘌呤、硫鸟嘌呤、喷司他丁和2-氯脱氧腺苷(克拉屈滨)、氟达拉滨)；辅酶(四氢叶酸)；抗增殖/抗有丝分裂剂，包括天然产物，如长春花生物碱(长春花碱、长春新碱和长春瑞滨)、微管干扰物，如紫杉烷(紫杉醇、多西他赛)，长春地辛、诺考达唑、依非洛类、埃博霉素、埃里布林；表鬼臼毒素类(依托泊苷、替尼泊苷)；DNA损伤剂(放线菌素、安吖啶、阿姆沙克林、蒽环类药物、博来霉素、白消安、喜树碱、苯丁酸氮芥、环磷酰胺、囊毒素、放线菌素D、柔红霉素、多柔比星、表柔比星、六甲基三聚氰胺奥沙利铂、异环磷酰胺、美法仑、氮芥、丝裂霉素、米托蒽醌、亚硝基脲、普卡霉素、丙卡巴嗪、紫杉醇、泰索帝、替莫唑胺、替尼泊苷、三亚乙基硫代磷酰胺和依托泊苷(VP 16))；DNA甲基转移酶抑制剂(氮杂胞苷)；抗生素、如放线菌素(放线菌素D)、柔红霉素、多柔比星(阿霉素、伊达比星、蒽环类药物(如道诺霉素)、米托蒽醌、博来霉素、普卡霉素(光神霉素)和 丝裂霉素；酶(L-天冬酰胺酶；抗血小板药物；抗增殖/抗有丝分裂烷化剂，如氮芥类物质(氮芥、环磷酰胺及其类似物、美法仑、苯丁酸氮芥)、乙烯亚胺和甲基三聚氰胺(六甲基三胺和噻替哌)、烷基磺酸盐(白消安)、亚硝基脲(卡莫司汀(BCNU)和类似物、链脲霉素)，三氮烯(达卡巴嗪(DTIC))；抗增殖/抗有丝分裂剂抗代谢物如叶酸类似物(氨甲蝶呤)；铂配位复合物(顺铂、卡铂、菲铂、洛铂、奈达铂、奥沙利铂)，丙卡巴肼、羟基脲、米托坦、氨鲁米特；激素、激素类似物(雌激素、他莫昔芬、戈舍瑞林、比卡鲁胺、尼鲁米特)和芳香化酶抑制剂(来曲唑、阿那曲唑)；抗凝血剂(肝素、合成肝素盐和其他凝血酶抑制剂)；纤维蛋白溶解剂(如组织纤溶酶原激活剂、链激酶和尿激酶)、阿司匹林、双嘧达莫、噻氯匹定、氯吡格雷、阿昔单抗；抗迁移剂；抗分泌剂(布雷非德菌素)；免疫抑制剂(环孢菌素、他克莫司(FK-506)，西罗莫司(雷帕霉素)、硫唑嘌呤、霉酚酸酯)；抗血管生成化合物(T P470，染料木黄酮、泊马度胺)、蛋白酶体抑制剂(伊沙佐米)；血管紧张素受体阻滞剂；一氧化氮供体；反义寡核苷酸；嵌合抗原受体；细胞周期抑制剂(夫拉平度、roscovitine、bryostatin-1)和分化诱导剂(维A酸)；拓扑异构酶抑制剂(阿霉素)，安吖啶、喜树碱、柔红霉素、放线菌素D、依尼泊苷、表柔比星、依托泊苷、伊达比星、伊立替康(CPT-11)和米托蒽醌、托泊替康)，皮质类固醇(可的松、地塞米松、氢化可的松、甲基泼尼松龙、泼尼松和泼尼松龙)；干扰素(聚乙二醇干扰素α-2b)；细胞凋亡蛋白(IAP)拮抗剂(比瑞那帕)；线粒体功能障碍诱导剂、毒素如霍乱毒素、蓖麻毒素、假单胞菌外毒素、百日咳博德特氏菌腺苷酸环化酶毒素或白喉毒素以及半胱天冬酶激活剂和染色质干扰物。According to an embodiment of the present invention, the chemotherapy drug is selected from at least one of the following drugs: antimetabolites/anticancer agents, such as pyrimidine analogs (5-fluorouracil (5-fu), fluuridine, capecitabine azacitabine, gemcitabine, decitabine, azacitidine and cytarabine) and purine analogues, folate antagonists and related immunosuppressants (methotrexate, pemetrexed, mercaptopurine, thioguanine, Pentostatin and 2-chlorodeoxyadenosine (cladribine, fludarabine); coenzymes (tetrahydrofolate); antiproliferative/antimitotic agents, including natural products such as vinca alkaloids (vinblastine) , vincristine and vinorelbine), microtubule disruptors, such as taxanes (paclitaxel, docetaxel), vindesine, nocodazole, eflenox, epothilone, erib Lin; epipodophyllotoxins (etoposide, teniposide); DNA damaging agents (actinomycin, amsacrine, amsacrine, anthracyclines, bleomycin, busulfan , camptothecin, chlorambucil, cyclophosphamide, cystotoxin, actinomycin D, daunorubicin, doxorubicin, epirubicin, hexamethylmelamine oxaliplatin, heterocycline Phosphamide, melphalan, nitrogen mustard, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, paclitaxel, Taxotere, temozolomide, teniposide, triethylene Thiophosphoramide and etoposide (VP 16)); DNA methyltransferase inhibitor (azacytidine); antibiotics such as actinomycin (actinomycin D), daunorubicin, doxorubicin Bicin (doxorubicin, idarubicin, anthracyclines (such as daunorubicin), mitoxantrone, bleomycin, plicamycin (mithramycin), and Mitomycins; enzymes (L-asparaginase); antiplatelet drugs; antiproliferative/antimitotic alkylating agents such as nitrogen mustards (nitrogen mustards, cyclophosphamide and its analogs, melphalan, phenbutin nitrogen mustard), ethyleneimines and methylmelamines (hexamethyltriamine and thiotepa), alkyl sulfonates (busulfan), nitrosoureas (carmustine (BCNU) and similar , streptozotocin), triazenes (dacarbazine (DTIC)); antiproliferative/antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin , phenanplatin, loplatin, nedaplatin, oxaliplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen, goserin lin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other thrombin inhibitors); fibrinolytics (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; anti-migratory agents; anti-secretory agents (Brfeldtella antibiotics); immunosuppressants (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic compounds (T P470 , genistein, pomalidomide), proteasome inhibitor (ixazomib); angiotensin receptor blocker; nitric oxide donor; antisense oligonucleotide; chimeric antigen receptor; Cell cycle inhibitors (flapinidine, roscovitine, bryostatin-1) and differentiation inducers (tretinoin); topoisomerase inhibitors (doxorubicin), acridine, camptothecin, daunorubicin, Actinomycin D, iniposide, epirubicin, etoposide, idarubicin, irinotecan (CPT-11) and mitoxantrone, topotecan), corticosteroids (CPT-11) pine, dexamethasone, hydrocortisone, methylprednisolone, prednisone and prednisolone); interferon (peginterferon alpha-2b); antagonizing apoptosis protein (IAP) agents (birenapa); mitochondrial dysfunction inducers, toxins such as cholera toxin, ricin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase toxin or diphtheria toxin, and caspase Winter enzyme activators and chromatin disruptors.
根据本发明的实施方案，所述免疫检查点抑制剂选自帕博利珠单抗、纳武尤利单抗、卡瑞丽珠单抗、特瑞普利单抗、西米普利单抗、杜瓦鲁单抗、阿维鲁单抗、信迪利单抗、替雷丽珠单抗、赛帕利单抗、度伐利尤单抗、阿替利珠单抗、恩沃利单抗、舒格利单抗、斯鲁利单抗、派安普利单抗、赛瑞单抗、津伯利单抗、吉普诺利单抗、斯巴达珠单抗、普罗格利单抗、普可替尼单抗、可西贝利单抗、AUNP-12、瑞拉单抗、瑞替凡利单抗、多塔利单抗、卡多尼单抗、索卡唑单抗、阿德布雷单抗、易普利单抗、曲美木单抗。According to an embodiment of the present invention, the immune checkpoint inhibitor is selected from the group consisting of pembrolizumab, nivolumab, camrelizumab, toripalimab, cimepilimab, Valumab, avelumab, sintilimab, tislelizumab, cepalizumab, durvalumab, atezolizumab, envolizumab, sugalizumab, slulimumab, penpilimab, serelizumab, zimberizumab, jepunolizumab, spartalizumab, proglilimab, sugalizumab Cotinizumab, cosibelimab, AUNP-12, relevanizumab, retifalizumab, dotalizumab, cardonizumab, socazolizumab, adebre monoclonal antibody, ipilimumab, tremelimumab.
根据本发明的实施方案，所述靶向药物选自B-Raf抑制剂(达拉非尼、索拉非尼、维莫拉非尼和达布拉非尼)、MEK抑制剂(曲美替尼、塞卢美替尼、比尼美替尼、PD-325901、考比美替尼、CI-1040和PD035901)、VEGF/VEGFR抑制剂(如贝伐珠单抗、雷莫芦单抗、雷珠单抗、阿柏西普、康柏西普、阿西替尼、西地尼布、莫特沙尼、仑伐替尼、舒尼替尼、索拉非尼、卡博替尼、瑞戈非尼、尼达尼布、怕唑帕尼、奥希替尼、呋喹替尼、凡德他尼、阿帕替尼、派加他尼、安罗替尼、利尼替尼、普利德辛)、EGFR抑制剂(如西妥西单抗、帕尼单抗、尼妥珠单抗、纳西妥单抗、吉非替尼、阿帕替尼、厄洛替尼、凡德替尼、埃克替尼、阿法替尼、奥希替尼、奥姆替尼、布加替尼、拉泽替尼)、酪氨酸激酶抑制剂(TKIs)、mTOR抑制剂(如雷帕霉素、Sirolimus、FK-50、CCI-779)、PARP抑制剂(尼拉帕尼、奥拉帕尼、拉唑帕尼、维利帕尼、他拉唑帕尼、氟唑帕尼、帕米帕尼)；粘附斑激酶(FAK)抑制剂(defactinib(VS-6063),VS-4718,VS-6062,GSK2256098)；生长因子信号转导激酶抑制剂(cediranib,galunisertib,rociletinib,vandetanib,afatinib，EGF816，AZD4547)；c-Met抑制剂(卡马替尼、克唑替尼、卡博替尼、沃利替尼、特泊替尼、替万替尼、谷美替尼、西曲替尼、INC280)；丝氨酸/苏氨酸激酶抑制剂；生长因子和其受体抑制剂(如ziv-aflibercept)；成纤维细胞生长因子受体(FGFR)抑制剂，如erdafitinib和pemigatinib)；组蛋白去乙酰化酶(HDAC)抑制剂(伏立诺他、罗米地辛、西达本胺、panobinostat，mocetinostat，abexinostat，belinostat，entinostat，resminostat，givinostat，quisinostat，SB939)；ALK抑制剂(赛瑞替尼、克唑替尼、阿来替尼、布加替尼、恩沙替尼、劳拉替尼、洛普替尼)；抗体(曲妥珠单抗、帕尼单抗、帕妥珠单抗、西妥昔单抗、阿达木单抗、戈利单抗、英夫利昔单抗、利妥昔单抗、奥瑞珠单抗、奥法单抗、奥法珠单抗、奥非珠单抗、阿仑珠单抗、阿昔单抗、阿特利珠单抗、达氯珠单抗、地诺单抗、依法珠单抗、依洛珠单抗、罗维珠单抗、鲁普珠单抗、乌斯替金单抗、维西珠单抗、吉珠单抗奥佐加米辛、布伦通昔布维多丁)中的至少一种。According to an embodiment of the present invention, the targeted drug is selected from the group consisting of B-Raf inhibitors (dabrafenib, sorafenib, vemurafenib and dabrafenib), MEK inhibitors (trametid nimetinib, selumetinib, binimetinib, PD-325901, cobimetinib, CI-1040 and PD035901), VEGF/VEGFR inhibitors (such as bevacizumab, ramucirumab, ramucirumab, Tizumab, aflibercept, conbercept, axitinib, cediranib, motesanib, lenvatinib, sunitinib, sorafenib, cabozantinib, Gorfenib, nintedanib, zopanib, osimertinib, fruquintinib, vandetanib, apatinib, pegaptanib, anlotinib, linitinib, pranax Leadsin), EGFR inhibitors (such as cetuximab, panitumumab, nimotuzumab, nacituzumab, gefitinib, apatinib, erlotinib, vandetinib , icotinib, afatinib, osimertinib, ommutinib, brigatinib, lazetinib), tyrosine kinase inhibitors (TKIs), mTOR inhibitors (such as rapamycin (Sirolimus, FK-50, CCI-779), PARP inhibitors (niraparib, olaparib, lazopanib, veliparib, talazopanib, fluzopanib, pami PAK); adherent plaque kinase (FAK) inhibitors (defactinib (VS-6063), VS-4718, VS-6062, GSK2256098); growth factor signaling kinase inhibitors (cediranib, galunisertib, rociletinib, vandetanib, afatinib , EGF816, AZD4547); c-Met inhibitors (capmatinib, crizotinib, cabozantinib, savolitinib, tepotinib, tivantinib, gumetinib, cetriti Ni, INC280); serine/threonine kinase inhibitors; growth factors and their receptor inhibitors (such as ziv-aflibercept); fibroblast growth factor receptor (FGFR) inhibitors, such as erdafitinib and pemigatinib); histones Desacetylase (HDAC) inhibitors (vorinostat, romidepsin, chidamide, panobinostat, mocetinostat, abexinostat, belinostat, entinostat, resminostat, givinostat, quisinostat, SB939); ALK inhibitors (Serrel crizotinib, alectinib, brigatinib, ensartinib, lorlatinib, lopotinib); antibodies (trastuzumab, panitumumab, pertuzumab monoclonal antibody, cetuximab, adalimumab, golimumab, infliximab, rituximab, ocrelizumab, ofatumumab, ofatumumab, orphetamine Tizumab, alemtuzumab, abciximab, atezolizumab, daclizumab, denosumab, efalizumab, evolizumab, rovizumab, Lupumab At least one of (tizumab, ustekinumab, velcilizumab, gemizumab ozogamicin, brentoncoxib, vedodin).
根据本发明的实施方案，所述第二药物优选为顺铂、紫杉醇、阿霉素或PD-1。According to an embodiment of the present invention, the second drug is preferably cisplatin, paclitaxel, doxorubicin or PD-1.
本发明还提供所述药物组合物在制备抗肿瘤药物中的应用。The present invention also provides the use of the pharmaceutical composition in preparing anti-tumor drugs.
本发明提供了式(I)所示化合物或其药学上可接受的盐或所述药物组合物在制备治疗VEGFR2信号通路相关疾病的药物中的用途。The present invention provides the use of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or the pharmaceutical composition in the preparation of a drug for treating diseases related to the VEGFR2 signaling pathway.
本发明提供了式(I)所示化合物或其药学上可接受的盐或所述药物组合物在制备治疗eIF4E和VEGFR2信号通路相关疾病的药物中的用途。The present invention provides the use of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or the pharmaceutical composition in the preparation of drugs for treating diseases related to eIF4E and VEGFR2 signaling pathways.
本发明提供了式(I)所示化合物或其药学上可接受的盐或所述药物组合物在制备调节免疫的药物中的应用，所述调节免疫优选为增强免疫。The present invention provides the use of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof or the pharmaceutical composition in the preparation of a drug for regulating immunity. The regulating immunity is preferably to enhance immunity.
本发明还提供一种治疗肿瘤疾病的方法，所述方法包括给与患者治疗有效量的式(I)所示化合物或其药学上可接受的盐。The present invention also provides a method for treating tumor diseases, which method includes administering to a patient a therapeutically effective amount of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof.
本发明还提供一种联合治疗肿瘤疾病的方法，所述方法包括联合给予肿瘤患者治疗有效量的式(I)所 示化合物或其药学上可接受的盐和至少一种第二药物。联合治疗可以以多种方式中的任何方式给予，如相继给予或同时给予。在一些实施方案中，所述方法包括给与患者所述药物组合物。The present invention also provides a method for joint treatment of tumor diseases, which method includes jointly administering a therapeutically effective amount of formula (I) to a tumor patient. The compound or a pharmaceutically acceptable salt thereof and at least one second drug. Combination treatments may be administered in any of a variety of ways, such as sequentially or simultaneously. In some embodiments, the method includes administering to the patient the pharmaceutical composition.
根据本发明的实施方案，所述肿瘤选自实体瘤或非实体瘤，例如黑色素瘤、肾癌、血液癌、前列腺癌、结肠癌、直肠癌、胃癌、食道癌、膀胱癌、头颈癌(如头颈部鳞癌)、甲状腺癌、乳腺癌(如三阴性乳腺癌)、卵巢癌、宫颈癌、肺癌(如非小细胞肺癌)、尿路上皮癌、胰腺癌、胶质母细胞瘤、肝癌、结肠腺癌、星形细胞瘤、大肠癌、骨肉瘤、骨髓瘤(如多发性骨髓瘤)、淋巴瘤(霍奇金淋巴瘤、非霍奇金淋巴瘤(例如，例如滤泡性淋巴瘤(FL)、套细胞淋巴瘤(MCL)、边缘区淋巴瘤(MZL)、弥漫性大B细胞淋巴瘤(DLBCL)、慢性淋巴细胞白血病(CLL))、多发性骨髓瘤(MM)、骨髓增生异常综合征、脑癌、中枢神经系统癌、恶性胶质瘤、白血病(如急性淋巴性白血病)中的至少一种或其任意组合。According to an embodiment of the invention, the tumor is selected from solid tumors or non-solid tumors, such as melanoma, kidney cancer, blood cancer, prostate cancer, colon cancer, rectal cancer, gastric cancer, esophageal cancer, bladder cancer, head and neck cancer (such as Head and neck squamous cell carcinoma), thyroid cancer, breast cancer (such as triple-negative breast cancer), ovarian cancer, cervical cancer, lung cancer (such as non-small cell lung cancer), urothelial cancer, pancreatic cancer, glioblastoma, liver cancer , colon adenocarcinoma, astrocytoma, colorectal cancer, osteosarcoma, myeloma (such as multiple myeloma), lymphoma (Hodgkin lymphoma, non-Hodgkin lymphoma (such as follicular lymphoma) (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL)), multiple myeloma (MM), myeloproliferation At least one of abnormal syndromes, brain cancer, central nervous system cancer, malignant glioma, leukemia (such as acute lymphoblastic leukemia) or any combination thereof.
根据本发明的实施方案，所述肺细胞为肺癌细胞A549和H460，所述结肠癌细胞为SW480和CT-26。According to an embodiment of the present invention, the lung cells are lung cancer cells A549 and H460, and the colon cancer cells are SW480 and CT-26.
一种治疗VEGFR2信号通路相关疾病的方法，所述方法包括给与患者治疗有效量的式(I)所示化合物或其药学上可接受的盐或所述药物组合物。A method for treating diseases related to the VEGFR2 signaling pathway, which method includes administering to a patient a therapeutically effective amount of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof or the pharmaceutical composition.
一种治疗eIF4E和VEGFR2信号通路相关疾病的方法，所述方法包括给与患者治疗有效量的式(I)所示化合物或其药学上可接受的盐或所述药物组合物。A method for treating diseases related to eIF4E and VEGFR2 signaling pathways, which method includes administering to a patient a therapeutically effective amount of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof or the pharmaceutical composition.
在制备本发明所述的药物时，将活性化合物与适当的药学上可接受的载体、稀释剂或赋形剂组合或配制来制备，并且可以配制成固体、半固体、液体或气体形式的制剂，例如片剂、胶囊剂、散剂、颗粒剂、软膏剂、溶液剂、栓剂、注射剂、吸入剂、凝胶、微球和气雾剂。给药方式包括是口服，腹膜内，透皮，皮下，静脉或肌肉注射，吸入，局部，病灶内，输注；脂质体介导的递送；局部、鞘内、牙龈袋、直肠、支气管内、鼻腔、经粘膜、肠、眼或耳输送，或本领域已知的任何其他方法，均能够达到对肿瘤的治疗；In preparing the medicaments of the present invention, the active compounds are combined or formulated with appropriate pharmaceutically acceptable carriers, diluents or excipients, and can be formulated into preparations in the form of solid, semi-solid, liquid or gas , such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres and aerosols. Modes of administration include oral, intraperitoneal, transdermal, subcutaneous, intravenous or intramuscular injection, inhalation, topical, intralesional, infusion; liposome-mediated delivery; topical, intrathecal, gingival pocket, rectal, intrabronchial , nasal cavity, transmucosal, intestinal, eye or ear delivery, or any other method known in the art, can achieve the treatment of tumors;
本发明所述治疗有效量或剂量将根据几个因素而变化，包括选择的给药途径，组合物的配方，患者反应，病情的严重程度，受试者的体重以及处方医生的判断，例如为1-200mg/kg，40-150mg/kg，如50mg/kg。剂量可以随着时间的推移增加或减少，如个别患者需要。在某些情况下，患者最初被给予低剂量，然后增加到患者可耐受的有效剂量。此外，患者可以在确定的时间段内给予多种剂量，特别是时间增量(如每日、每周、每两周、每月、每季度、每两年或类似)。The therapeutically effective amount or dose of the present invention will vary depending on several factors, including the route of administration chosen, the formulation of the composition, patient response, severity of condition, subject's weight, and the judgment of the prescribing physician, e.g. 1-200mg/kg, 40-150mg/kg, such as 50mg/kg. Dosage may be increased or decreased over time as individual patients require. In some cases, patients are initially given low doses and then increased to an effective dose that is tolerated by the patient. In addition, patients may be administered multiple doses over defined time periods, particularly in time increments (e.g., daily, weekly, biweekly, monthly, quarterly, biennially, or the like).
有益效果beneficial effects
本发明研究发现式(I)所示化合物单独使用或与其他药物联用均对eIF4E蛋白和/或VEGFR2蛋白具有双靶点抑制作用，从而具有较好的肿瘤抑制效果。The research of the present invention has found that the compound represented by formula (I) has a dual-target inhibitory effect on eIF4E protein and/or VEGFR2 protein when used alone or in combination with other drugs, thereby having a good tumor inhibitory effect.
附图说明Description of drawings
图1示意化合物12对应的对甲苯磺酸盐和Con-MNK对HCT116细胞的剂量量效曲线。Figure 1 illustrates the dose-response curve of compound 12 corresponding to p-toluenesulfonate and Con-MNK on HCT116 cells.
图2示意化合物12对应的对甲苯磺酸盐和Con-MNK对人结肠癌HCT116细胞的剂量量效曲线。Figure 2 illustrates the dose-response curve of compound 12 corresponding to p-toluenesulfonate and Con-MNK on human colon cancer HCT116 cells.
图3示意人结肠癌HT-29细胞皮下异种移植瘤模型荷瘤鼠在给药过程中的体重变化。Figure 3 illustrates the body weight changes of tumor-bearing mice in the subcutaneous xenograft tumor model of human colon cancer HT-29 cells during the administration process.
图4示意人结肠癌HT-29细胞皮下异种移植瘤模型荷瘤鼠在给药过程中的体重变化。Figure 4 illustrates the body weight changes of tumor-bearing mice in the subcutaneous xenograft tumor model of human colon cancer HT-29 cells during the administration process.
图5示意人结肠癌HT-29细胞皮下异种移植瘤模型荷瘤鼠在给予药物后的肿瘤生长抑制曲线。Figure 5 illustrates the tumor growth inhibition curve of human colon cancer HT-29 cell subcutaneous xenograft tumor model tumor-bearing mice after administration of drugs.
图6示意口服给予10、30和100mg/kg化合物12对应的对甲苯磺酸盐后Balb/c裸小鼠平均血浆时间-浓度图。Figure 6 illustrates the mean plasma time-concentration plot in Balb/c nude mice after oral administration of 10, 30 and 100 mg/kg p-toluenesulfonate corresponding to compound 12.
图7示意口服给予10mg/kg化合物12对应的对甲苯磺酸盐后Balb/c裸小鼠平均血浆和肿瘤浓度图。Figure 7 illustrates the average plasma and tumor concentration profiles of Balb/c nude mice after oral administration of 10 mg/kg p-toluenesulfonate corresponding to Compound 12.
图8示意口服给予30mg/kg化合物12对应的对甲苯磺酸盐后Balb/c裸小鼠平均血浆和肿瘤浓度图。Figure 8 illustrates the average plasma and tumor concentration profiles of Balb/c nude mice after oral administration of 30 mg/kg p-toluenesulfonate corresponding to Compound 12.
图9示意口服给予100mg/kg化合物12对应的对甲苯磺酸盐后Balb/c裸小鼠平均血浆和肿瘤浓度图。Figure 9 illustrates the average plasma and tumor concentration profiles of Balb/c nude mice after oral administration of 100 mg/kg p-toluenesulfonate corresponding to Compound 12.
图10示意异种移植肿瘤HT-29组织在给药2h/8h/24h后eIF4E和p-eIF4E的蛋白表达情况。Figure 10 shows the protein expression of eIF4E and p-eIF4E in xenograft tumor HT-29 tissue after 2h/8h/24h administration.
图11示意异种移植肿瘤HT-29组织在给药2h/8h/24h后eIF4E和p-eIF4E的蛋白表达分析。Figure 11 illustrates the protein expression analysis of eIF4E and p-eIF4E in xenograft tumor HT-29 tissue after 2h/8h/24h administration.
图12示意异种移植肿瘤HT-29组织在给药2h/8h/24h后VEGFR2和p-VEGFR2的蛋白表达情况。Figure 12 shows the protein expression of VEGFR2 and p-VEGFR2 in xenograft tumor HT-29 tissue after 2h/8h/24h administration.
图13示意异种移植肿瘤HT-29组织在给药2h/8h/24h后p/t-VEGFR2的蛋白表达分析。Figure 13 illustrates the protein expression analysis of p/t-VEGFR2 in xenograft tumor HT-29 tissue after 2h/8h/24h administration.
图14示意化合物12对应的对甲苯磺酸盐和5-fu联用对人结肠癌SW480细胞Balb/c nude小鼠移植瘤模型肿瘤体积的影响。Figure 14 illustrates the effect of the combination of p-toluenesulfonate and 5-fu corresponding to Compound 12 on the tumor volume of human colon cancer SW480 cell Balb/c nude mouse transplanted tumor model.
图15示意鼠结肠癌CT-26细胞皮下移植瘤模型荷瘤鼠在给予药物后的肿瘤生长曲线。Figure 15 illustrates the tumor growth curve of mouse colon cancer CT-26 cell subcutaneously transplanted tumor model in tumor-bearing mice after administration of drugs.
图16示意测试化合物对MNK1激酶的活性抑制。Figure 16 illustrates the inhibition of MNK1 kinase activity by test compounds.
图17示意测试化合物对MNK2激酶的活性抑制。Figure 17 illustrates the inhibition of MNK2 kinase activity by test compounds.
图18示意化合物12对应的对甲苯磺酸盐对胃癌BGC-823细胞Balb/c nude小鼠移植瘤模型肿瘤体积的影响。 Figure 18 illustrates the effect of p-toluenesulfonate corresponding to Compound 12 on the tumor volume of the gastric cancer BGC-823 cell Balb/c nude mouse transplanted tumor model.
图19示意化合物12对应的对甲苯磺酸盐对肺癌Calu-3细胞Balb/c nude小鼠移植瘤模型肿瘤体积的影响。Figure 19 illustrates the effect of p-toluenesulfonate corresponding to Compound 12 on tumor volume in the lung cancer Calu-3 cell Balb/c nude mouse transplant tumor model.
图20示意化合物12对应的对甲苯磺酸盐对HUVEC细胞的抑制。Figure 20 illustrates the inhibition of HUVEC cells by the p-toluenesulfonate corresponding to Compound 12.
图21示意化合物12对应的对甲苯磺酸盐和顺铂联用对肺癌A549细胞Balb/c nude小鼠移植瘤模型肿瘤体积的影响。Figure 21 illustrates the effect of the combination of p-toluenesulfonate and cisplatin corresponding to compound 12 on the tumor volume of the lung cancer A549 cell Balb/c nude mouse transplant tumor model.
图22示意化合物12对应的对甲苯磺酸盐和紫杉醇联用对肺癌H460细胞Balb/c nude小鼠移植瘤模型肿瘤体积的影响。Figure 22 illustrates the effect of the combination of p-toluenesulfonate and paclitaxel corresponding to compound 12 on the tumor volume of the lung cancer H460 cell Balb/c nude mouse transplanted tumor model.
图23示意人源性卵巢癌ES2细胞皮下异种移植瘤模型荷瘤鼠在给予药物后的肿瘤生长抑制曲线。Figure 23 illustrates the tumor growth inhibition curve of human ovarian cancer ES2 cell subcutaneous xenograft tumor model tumor-bearing mice after administration of drugs.
图24示意人源性卵巢癌ES2细胞皮下异种移植瘤模型荷瘤鼠在给药过程中的体重变化。Figure 24 illustrates the body weight changes of human ovarian cancer ES2 cell subcutaneous xenograft tumor model tumor-bearing mice during the administration process.
图25示意化合物12对应的对甲苯磺酸盐和顺铂或紫杉醇联用对人源性卵巢癌ES2细胞皮下异种移植瘤模型肿瘤体积的影响。Figure 25 illustrates the effect of the combination of p-toluenesulfonate corresponding to Compound 12 and cisplatin or paclitaxel on the tumor volume of the human ovarian cancer ES2 cell subcutaneous xenograft model.
图26示意化合物12对应的对甲苯磺酸盐单独用药以及和阿霉素联用对人DLBCL细胞SU-DHL-6异种移植瘤的肿瘤生长曲线。Figure 26 illustrates the tumor growth curves of p-toluenesulfonate corresponding to compound 12 used alone and in combination with doxorubicin on human DLBCL cell SU-DHL-6 xenograft tumors.
图27示意化合物12对应的对甲苯磺酸盐单独用药以及和阿霉素联用对人DLBCL细胞SU-DHL-6异种移植瘤的体重变化曲线Figure 27 illustrates the body weight change curve of p-toluenesulfonate corresponding to compound 12 used alone and in combination with doxorubicin on human DLBCL cell SU-DHL-6 xenograft tumors
图28示意化合物12对应的对甲苯磺酸盐对MCL细胞JeKo-1细胞异种移植瘤的肿瘤生长曲线。Figure 28 illustrates the tumor growth curve of p-toluenesulfonate corresponding to Compound 12 on MCL cell JeKo-1 cell xenograft tumors.
图29示意化合物12对应的对甲苯磺酸盐与PD-1抗体或PD-1同型抗体对小鼠LL2皮下瘤模型的肿瘤生长曲线。Figure 29 illustrates the tumor growth curve of compound 12 corresponding to p-toluenesulfonate and PD-1 antibody or PD-1 isotype antibody on the mouse LL2 subcutaneous tumor model.
图30示意化合物12对应的对甲苯磺酸盐与PD-1抗体或PD-1同型抗体对小鼠LL2皮下瘤模型的体重变化曲线Figure 30 illustrates the body weight change curve of p-toluenesulfonate corresponding to Compound 12 and PD-1 antibody or PD-1 isotype antibody on the mouse LL2 subcutaneous tumor model.
图31示意化合物12对应的对甲苯磺酸盐对Hep G2裸鼠移植瘤模型的肿瘤生长曲线。Figure 31 illustrates the tumor growth curve of the p-toluenesulfonate corresponding to compound 12 on the Hep G2 nude mouse transplanted tumor model.
图32示意化合物12对应的对甲苯磺酸盐对MDA-MB-231裸鼠移植瘤模型的肿瘤生长曲线。Figure 32 illustrates the tumor growth curve of the p-toluenesulfonate corresponding to Compound 12 on the MDA-MB-231 nude mouse transplanted tumor model.
具体实施方式Detailed ways
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following examples are only illustrative and explain the present invention and should not be construed as limiting the scope of the present invention. All technologies implemented based on the above contents of the present invention are covered by the scope of protection intended by the present invention.
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.
在本发明的一些方案中，上述式(I)化合物为下式化合物：

In some aspects of the invention, the compound of formula (I) above is a compound of the following formula:

化合物制备例：Compound preparation example:
实施例1
Example 1
合成路线：
synthetic route:
第一步first step
将化合物1a(100g,462mmol)溶于乙醇(500mL)中，在0℃下滴加浓硫酸(49.94g,509mmol,纯度：98％)，反应液在95℃下搅拌16小时。反应完成后，反应液减压浓缩除掉大部分乙醇，浓缩液加入水(300mL)，用乙酸乙酯(250mL×3)萃取，合并的有机相用饱和碳酸氢钠(300mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，得到化合物1b。Compound 1a (100g, 462mmol) was dissolved in ethanol (500mL), concentrated sulfuric acid (49.94g, 509mmol, purity: 98%) was added dropwise at 0°C, and the reaction solution was stirred at 95°C for 16 hours. After the reaction is completed, the reaction solution is concentrated under reduced pressure to remove most of the ethanol. Water (300 mL) is added to the concentrated solution, and extracted with ethyl acetate (250 mL × 3). The combined organic phase is washed with saturated sodium bicarbonate (300 mL) and anhydrous. Dry over sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain compound 1b.
1H NMR(400MHz,CDCl3)δ8.60(s,1H),7.78(s,1H),4.48-4.42(m,2H),2.58(s,3H),1.43(t,J＝7.2Hz,3H)。 1 H NMR (400MHz, CDCl 3 ) δ8.60 (s, 1H), 7.78 (s, 1H), 4.48-4.42 (m, 2H), 2.58 (s, 3H), 1.43 (t, J = 7.2Hz, 3H).
第二步Step 2
将化合物1b(10.0g,41.0mmol)溶于二氯甲烷(200mL)中，在0℃下搅拌加入三氟乙酸酐(17.2g,81.9mmol)和过氧化氢尿素(8.09g,86.0mmol)，反应液升至25℃下搅拌16小时。向反应液中加入水(200mL)，用二氯甲烷(100mL×3)萃取，合并有机相，依次用饱和碳酸氢钠(200mL×2)和饱和食盐水(500mL)洗涤，用无水硫酸钠干燥，过滤，减压浓缩，得到化合物1c。Compound 1b (10.0g, 41.0mmol) was dissolved in dichloromethane (200mL), and trifluoroacetic anhydride (17.2g, 81.9mmol) and urea hydrogen peroxide (8.09g, 86.0mmol) were added with stirring at 0°C. The reaction solution was heated to 25°C and stirred for 16 hours. Add water (200mL) to the reaction solution, extract with dichloromethane (100mL×3), combine the organic phases, wash with saturated sodium bicarbonate (200mL×2) and saturated brine (500mL), and wash with anhydrous sodium sulfate. Dry, filter and concentrate under reduced pressure to obtain compound 1c.
1H NMR(400MHz,CDCl3)δ8.23(s,1H),7.29(s,1H),4.52-4.45(m,2H),2.29(s,3H),1.41(t,J＝7.2Hz,3H)。 1 H NMR (400MHz, CDCl 3 ) δ8.23 (s, 1H), 7.29 (s, 1H), 4.52-4.45 (m, 2H), 2.29 (s, 3H), 1.41 (t, J = 7.2Hz, 3H).
第三步third step
将化合物1c(22.0g,84.6mmol)溶于N,N-二甲基甲酰胺(130mL)中，在0℃下搅拌加入三氟乙酸酐(35.5g,169mmol)，反应液在50℃下搅拌1小时。向反应液中加入水(200mL)，用乙酸乙酯(100mL×4)萃取，合并有机相，依次用饱和碳酸氢钠(200mL×3)和饱和食盐水(150mL×3)洗涤，用无水硫酸钠干燥，过滤，减压浓缩。粗品在石油醚/乙酸乙酯混合液(8/1,90mL)中室温下搅拌2h，过滤得到化合物1d。Compound 1c (22.0g, 84.6mmol) was dissolved in N,N-dimethylformamide (130mL), and trifluoroacetic anhydride (35.5g, 169mmol) was added with stirring at 0°C, and the reaction solution was stirred at 50°C. 1 hour. Add water (200mL) to the reaction solution, extract with ethyl acetate (100mL×4), combine the organic phases, wash with saturated sodium bicarbonate (200mL×3) and saturated brine (150mL×3), and wash with anhydrous Dry over sodium sulfate, filter, and concentrate under reduced pressure. The crude product was stirred in petroleum ether/ethyl acetate mixture (8/1, 90 mL) at room temperature for 2 h, and then filtered to obtain compound 1d.
1H NMR(400MHz,CDCl3)δ9.89(brs,1H),7.75(s,1H),4.46-4.41(m,2H),2.44(s,3H),1.43(t,J＝7.2Hz,3H)。 1 H NMR (400MHz, CDCl 3 ) δ9.89 (brs, 1H), 7.75 (s, 1H), 4.46-4.41 (m, 2H), 2.44 (s, 3H), 1.43 (t, J = 7.2Hz, 3H).
第四步the fourth step
将化合物1d(2.00g,7.69mmol)溶于乙醇(20mL)中，加入氨水(16.2g,115mmol,纯度：25％)，反应液在40℃下搅拌16小时。反应液减压浓缩，粗品在甲醇/二氯甲烷混合液(1/5,48mL)中室温下搅拌过夜。然后过滤，用二氯甲烷(5mL×2)洗涤，滤饼减压浓缩得到化合物1e。Compound 1d (2.00g, 7.69mmol) was dissolved in ethanol (20mL), ammonia water (16.2g, 115mmol, purity: 25%) was added, and the reaction solution was stirred at 40°C for 16 hours. The reaction solution was concentrated under reduced pressure, and the crude product was stirred in a methanol/dichloromethane mixture (1/5, 48 mL) at room temperature overnight. Then it was filtered, washed with dichloromethane (5 mL×2), and the filter cake was concentrated under reduced pressure to obtain compound 1e.
MS-ESI计算值[M+H]+231和233，实测值231和233。MS-ESI calculated values [M+H] + 231 and 233, measured values 231 and 233.
第五步the fifth step
将化合物1e(37.0g,67.3mmol)和环己酮(26.4g,269mmol)溶于二氧六环(400mL)中，搅拌下滴加浓硫酸(3.30g,33.6mmol,纯度：98％)，反应液在95℃下搅拌3小时。反应液减压浓缩，粗品在乙酸乙酯(100mL)中室温下搅拌3小时，过滤，将滤饼在饱和碳酸氢钠溶液(350mL)中室温下搅拌1小时，过滤，滤饼减压干燥得到化合物1f。MS-ESI计算值[M+H]+311和313，实测值311和313。Compound 1e (37.0g, 67.3mmol) and cyclohexanone (26.4g, 269mmol) were dissolved in dioxane (400mL), and concentrated sulfuric acid (3.30g, 33.6mmol, purity: 98%) was added dropwise with stirring. The reaction solution was stirred at 95°C for 3 hours. The reaction solution was concentrated under reduced pressure, and the crude product was stirred in ethyl acetate (100 mL) at room temperature for 3 hours, filtered, and the filter cake was stirred in saturated sodium bicarbonate solution (350 mL) at room temperature for 1 hour, filtered, and the filter cake was dried under reduced pressure to obtain Compound 1f. MS-ESI calculated values [M+H] + 311 and 313, measured values 311 and 313.
第六步Step 6
将化合物1g(50g,0.442mol)，1,8-二氮杂双环[5.4.0]十一碳-7-烯(DBU,67.3g,0.442mol)溶于四氢呋喃(500mL)中。将反应液加热至55℃，在此温度下，向反应液中加入乙醛(9.74g,0.221mol)。反应液在55℃下搅拌18小时。将反应液降至22℃，用乙酸(25mL)淬灭。反应液减压浓缩，剩余物溶于乙酸乙酯(1000mL)和稀盐酸(1000mL,1M)，分液后保留有机相，水相用乙酸乙酯萃取(300mL×3)，合并有机相，有机相依次用饱和碳酸氢钠溶液(100mL)和食盐水(200mL)洗涤，无水硫酸钠干燥，过滤，浓缩，粗产物经过柱层析法分离(4/1,石油醚/乙酸乙酯,Rf＝0.56)得到化合物1h。Compound 1g (50g, 0.442mol), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 67.3g, 0.442mol) was dissolved in tetrahydrofuran (500mL). The reaction liquid was heated to 55°C, and at this temperature, acetaldehyde (9.74g, 0.221mol) was added to the reaction liquid. The reaction solution was stirred at 55°C for 18 hours. The reaction solution was lowered to 22°C and quenched with acetic acid (25 mL). The reaction solution was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate (1000mL) and dilute hydrochloric acid (1000mL, 1M). After separation, the organic phase was retained. The aqueous phase was extracted with ethyl acetate (300mL×3). The organic phases were combined. The phase was washed successively with saturated sodium bicarbonate solution (100 mL) and brine (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated by column chromatography (4/1, petroleum ether/ethyl acetate, Rf = 0.56) to obtain compound 1h.
MS-ESI计算值[M+H]+226，实测值226。1H NMR(400MHz,CDCl3)δ9.29(brs,1H),7.48(d,J＝3.2Hz,1H),4.35(q,J＝7.2Hz,2H),4.29(q,J＝7.2Hz,2H),2.61(s,3H),1.38(t,J＝7.2Hz,3H),1.35(m,J＝7.2Hz,3H)。MS-ESI calculated value [M+H] + 226, measured value 226. 1 H NMR (400MHz, CDCl 3 ) δ9.29 (brs, 1H), 7.48 (d, J = 3.2Hz, 1H), 4.35 (q, J = 7.2Hz, 2H), 4.29 (q, J = 7.2Hz ,2H),2.61(s,3H),1.38(t,J＝7.2Hz,3H),1.35(m,J＝7.2Hz,3H).
第七步Step 7
将化合物1h(11.0g,48.8mmol)溶于N-甲基吡咯烷酮(60mL)中，向反应液中加入叔丁醇钾(6.03g,53.7mmol)。反应液在25℃下搅拌0.5小时后，加入化合物1i(9.78g,53.7mmol)的N-甲基吡咯烷酮(30mL)溶液。反应液继续搅拌20小时。将反应液用水(200mL)洗涤，乙酸乙酯萃取(200mL×3)，合并有机相，有机相用饱和食盐水(20mL×3)洗涤，无水硫酸钠干燥，过滤，浓缩，粗产物经过柱层析法分离(4/1,石油醚/乙酸乙酯,Rf＝0.55)得到化合物1j。Compound 1h (11.0g, 48.8mmol) was dissolved in N-methylpyrrolidone (60mL), and potassium tert-butoxide (6.03g, 53.7mmol) was added to the reaction solution. After the reaction solution was stirred at 25° C. for 0.5 hours, a solution of compound 1i (9.78 g, 53.7 mmol) in N-methylpyrrolidone (30 mL) was added. The reaction solution was continued to stir for 20 hours. The reaction solution was washed with water (200 mL), extracted with ethyl acetate (200 mL × 3), the organic phases were combined, washed with saturated brine (20 mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was passed through a column Chromatographic separation (4/1, petroleum ether/ethyl acetate, Rf=0.55) gave compound 1j.
MS-ESI计算值[M+H]+241，实测值241。1H NMR(400MHz,CDCl3)δ7.49(s,1H),4.35(q,J＝7.2Hz,2H),4.27(q,J＝7.2Hz,2H),2.57(s,3H),1.40(t,J＝7.2Hz,3H),1.34(t,J＝7.2Hz,3H)。MS-ESI calculated value [M+H] + 241, measured value 241. 1 H NMR (400MHz, CDCl 3 ) δ7.49 (s, 1H), 4.35 (q, J = 7.2Hz, 2H), 4.27 (q, J = 7.2Hz, 2H), 2.57 (s, 3H), 1.40 (t, J=7.2Hz, 3H), 1.34 (t, J=7.2Hz, 3H).
第八步Step 8
将化合物1j(10.2g,42.5mmol)溶于甲酰胺(120mL)，向反应液中加入磷酸(832mg,8.49mmol)。反应液在125℃下搅拌16小时。将反应液降至22℃，此时有大量白色固体析出。将混合物过滤，收集的滤饼加入到石油醚/乙酸乙酯混合溶液(1/1,100mL)中，混合物在30℃下搅拌0.5小时，过滤得化合物1k。Compound 1j (10.2g, 42.5mmol) was dissolved in formamide (120mL), and phosphoric acid (832mg, 8.49mmol) was added to the reaction solution. The reaction solution was stirred at 125°C for 16 hours. The reaction solution was lowered to 22°C. At this time, a large amount of white solid precipitated. The mixture was filtered, and the collected filter cake was added to petroleum ether/ethyl acetate mixed solution (1/1, 100 mL). The mixture was stirred at 30°C for 0.5 hours, and filtered to obtain compound 1k.
MS-ESI计算值[M+H]+222，实测值222。1H NMR(400MHz,DMSO-d6)δ7.90(s,1H),7.84(s,1H),4.23(q,J＝7.2Hz,2H),2.61(s,3H),1.28(t,J＝7.2Hz,3H)。MS-ESI calculated value [M+H] + 222, measured value 222. 1 H NMR (400MHz, DMSO-d 6 ) δ7.90 (s, 1H), 7.84 (s, 1H), 4.23 (q, J = 7.2Hz, 2H), 2.61 (s, 3H), 1.28 (t, J=7.2Hz,3H).
第九步Step 9
将化合物1k(4.00g,18.0mmol)溶于无水四氢呋喃(50mL)中。在25℃下,向反应液滴加甲基溴化镁(30.1ml,90.3mmol)，滴加完毕，反应升温至25℃，搅拌15小时。反应液用饱和氯化铵溶液(100mL)淬灭，乙酸乙酯(50mL×5)萃取，合并有机相，有机相用饱和食盐水(10mL×1)洗涤，无水硫酸钠干燥，过滤，减压浓缩，粗产 物经过薄层色谱层析法分离(2/1,石油醚/乙酸乙酯,Rf＝0.39)得到化合物1l。MS-ESI计算值[M+H]+208，实测值208。Compound 1k (4.00 g, 18.0 mmol) was dissolved in anhydrous tetrahydrofuran (50 mL). At 25°C, methylmagnesium bromide (30.1 ml, 90.3 mmol) was added dropwise to the reaction solution. After the dropwise addition was completed, the reaction was heated to 25°C and stirred for 15 hours. The reaction solution was quenched with saturated ammonium chloride solution (100mL), extracted with ethyl acetate (50mL×5), the organic phases were combined, washed with saturated brine (10mL×1), dried over anhydrous sodium sulfate, filtered, and reduced to Pressure concentration, crude production The material was separated by thin layer chromatography (2/1, petroleum ether/ethyl acetate, Rf = 0.39) to obtain compound 1l. MS-ESI calculated value [M+H] + 208, measured value 208.
1H NMR(400MHz,CDCl3)δ7.30(br s,1H),7.24(br s,1H),2.59(s,3H),1.54(s,6H)。 1 H NMR (400MHz, CDCl 3 ) δ7.30 (br s, 1H), 7.24 (br s, 1H), 2.59 (s, 3H), 1.54 (s, 6H).
第十步Step 10
将化合物1l(1.00g,4.83mmol)，双氧水(4.64ml,48.26mmol,含量：30％)溶于无水四氢呋喃(30mL)中。在0℃下，向反应液滴加冷的甲磺酸(3.44mL,48.26mmol)的水(10mL)溶液。反应液在0℃，搅拌1小时。反应液用10％亚硫酸钠水溶液(15mL)淬灭，直至淀粉碘化钾试纸显示阴性。溶液用乙酸乙酯(50mL×3)萃取，合并有机相，有机相用饱和食盐水(10mL×1)洗涤，无水硫酸钠干燥，过滤，减压浓缩，粗产物经过柱层析色谱法分离(2/1,石油醚/乙酸乙酯,Rf＝0.38)得到化合物1m。Compound 11 (1.00g, 4.83mmol) and hydrogen peroxide (4.64ml, 48.26mmol, content: 30%) were dissolved in anhydrous tetrahydrofuran (30mL). At 0°C, a cold solution of methanesulfonic acid (3.44 mL, 48.26 mmol) in water (10 mL) was added dropwise to the reaction liquid. The reaction solution was stirred at 0°C for 1 hour. The reaction solution was quenched with 10% sodium sulfite aqueous solution (15 mL) until the starch potassium iodide test paper showed negative. The solution was extracted with ethyl acetate (50mL×3), and the organic phases were combined. The organic phase was washed with saturated brine (10mL×1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was separated by column chromatography. (2/1, petroleum ether/ethyl acetate, Rf=0.38) gave compound 1m.
MS-ESI计算值[M+H]+166，实测值166。MS-ESI calculated value [M+H] + 166, measured value 166.
第十一步Step 11
将化合物1m(400mg,2.42mmol)溶于无水四氢呋喃(10mL)，向反应液中加入三乙胺(0.674mL,4.84mmol)，特戊酰氯(350mg,4.84mmol)。反应液在0℃下搅拌1小时，反应液用水(10mL)洗涤，乙酸乙酯萃取(10mL×5)，合并有机相，有机相用饱和食盐水(10mL×1)洗涤，无水硫酸钠干燥，过滤，减压浓缩，粗产物经过薄层色谱层析法分离(2/1,石油醚/乙酸乙酯,Rf＝0.63)得到化合物1n。Compound 1m (400 mg, 2.42 mmol) was dissolved in anhydrous tetrahydrofuran (10 mL), and triethylamine (0.674 mL, 4.84 mmol) and pivaloyl chloride (350 mg, 4.84 mmol) were added to the reaction solution. The reaction solution was stirred at 0°C for 1 hour. The reaction solution was washed with water (10 mL) and extracted with ethyl acetate (10 mL × 5). The organic phases were combined, washed with saturated brine (10 mL × 1), and dried over anhydrous sodium sulfate. , filtered, concentrated under reduced pressure, and the crude product was separated by thin layer chromatography (2/1, petroleum ether/ethyl acetate, Rf = 0.63) to obtain compound 1n.
MS-ESI计算值[M+H]+250，实测值250。1H NMR(400MHz,MeOH-d4)δ7.61(s,1H),7.47(s,1H),2.34(s,3H),1.38(s,9H)。MS-ESI calculated value [M+H] + 250, measured value 250. 1 H NMR (400MHz, MeOH-d 4 ) δ7.61 (s, 1H), 7.47 (s, 1H), 2.34 (s, 3H), 1.38 (s, 9H).
第十二步Step 12
将化合物1n(450mg,1.81mmol)溶于三氯氧磷(8.85mL)中。反应液在100℃下搅拌1小时。将反应液降至室温，倒入饱和碳酸氢铵溶液(300mL)中。混合物用二氯甲烷萃取(50mL×3)，合并有机相，有机相用饱和食盐水(20mL×1)洗涤，无水硫酸钠干燥，过滤，浓缩得到化合物1o。Compound 1n (450 mg, 1.81 mmol) was dissolved in phosphorus oxychloride (8.85 mL). The reaction solution was stirred at 100°C for 1 hour. The reaction solution was cooled to room temperature and poured into saturated ammonium bicarbonate solution (300 mL). The mixture was extracted with dichloromethane (50 mL × 3), and the organic phases were combined. The organic phase was washed with saturated brine (20 mL × 1), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 1o.
MS-ESI计算值[M+H]+268,实测值268。MS-ESI calculated value [M+H] + 268, measured value 268.
第十三步Step 13
将化合物1o(2.00g,7.47mmol)，2,4-二甲氧基苄胺(1.87g,11.21mmol)，三乙胺(2.27g,22.4mmol)溶于无水四氢呋喃(30mL)中。反应液在70℃下搅拌1小时。反应液减压浓缩得到粗品化合物1p。Compound 1o (2.00g, 7.47mmol), 2,4-dimethoxybenzylamine (1.87g, 11.21mmol), and triethylamine (2.27g, 22.4mmol) were dissolved in anhydrous tetrahydrofuran (30mL). The reaction solution was stirred at 70°C for 1 hour. The reaction solution was concentrated under reduced pressure to obtain crude compound 1p.
MS-ESI计算值[M+H]+399，实测值399。MS-ESI calculated value [M+H] + 399, measured value 399.
第十四步Step 14
将化合物1p(3.50g,8.78mmol)溶于甲醇(3mL)和四氢呋喃(20mL)中，向反应液中加入氢氧化钠(703mg,17.6mmol)的水(20mL)溶液。反应液在25℃下搅拌0.5小时。反应液浓缩除去有机溶剂，水相用稀盐酸水溶液(1M)调节至pH＝7，混合物用二氯甲烷萃取(50mL×3)，合并有机相，有机相用饱和食盐水(10mL×1)洗涤，无水硫酸钠干燥，过滤，减压浓缩，粗产物经过柱层析法分离(2/1,石油醚/乙酸乙酯,Rf＝0.32)得到化合物1q。Compound 1p (3.50g, 8.78mmol) was dissolved in methanol (3mL) and tetrahydrofuran (20mL), and a solution of sodium hydroxide (703mg, 17.6mmol) in water (20mL) was added to the reaction solution. The reaction solution was stirred at 25°C for 0.5 hours. The reaction solution was concentrated to remove the organic solvent. The aqueous phase was adjusted to pH=7 with dilute hydrochloric acid aqueous solution (1M). The mixture was extracted with dichloromethane (50mL×3). The organic phases were combined and washed with saturated brine (10mL×1). , dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was separated by column chromatography (2/1, petroleum ether/ethyl acetate, Rf = 0.32) to obtain compound 1q.
MS-ESI计算值[M+H]+315，实测值315。1H NMR(400MHz,MeOH-d4)δ7.67(s,1H),7.21(d,J＝8.4Hz,1H),7.07(s,1H),6.56(d,J＝2.4Hz,1H),6.47(dd,J＝2.4,8.4Hz,1H),4.66(s,2H),3.90(s,3H),3.79(s,3H),2.36(s,3H)。MS-ESI calculated value [M+H] + 315, measured value 315. 1 H NMR (400MHz, MeOH-d 4 ) δ7.67 (s, 1H), 7.21 (d, J = 8.4Hz, 1H), 7.07 (s, 1H), 6.56 (d, J = 2.4Hz, 1H) ,6.47(dd,J=2.4,8.4Hz,1H),4.66(s,2H),3.90(s,3H),3.79(s,3H),2.36(s,3H).
第十五步Step 15
将化合物1q(350mg,1.11mmol)溶于N,N-二甲基甲酰胺(10mL)中，加入氢氧化钠(89.1mg,2.23mmol)和1-(2-溴乙基)吡咯烷(238mg,1.34mmol)。反应液在50℃下搅拌2.5小时。反应液加入水(10mL)，乙酸乙酯(20mL×4)萃取，合并有机相，用饱和食盐水(50mL×3)洗涤，用无水硫酸钠干燥，过滤，减压浓缩，剩余物经柱层析法(10/1,二氯甲烷/甲醇,Rf＝0.28)分离得到化合物1r。Compound 1q (350 mg, 1.11 mmol) was dissolved in N,N-dimethylformamide (10 mL), and sodium hydroxide (89.1 mg, 2.23 mmol) and 1-(2-bromoethyl)pyrrolidine (238 mg) were added ,1.34mmol). The reaction solution was stirred at 50°C for 2.5 hours. Water (10 mL) was added to the reaction solution, extracted with ethyl acetate (20 mL × 4), the organic phases were combined, washed with saturated brine (50 mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was passed through a column Compound 1r was isolated by chromatography (10/1, dichloromethane/methanol, Rf=0.28).
MS-ESI计算值[M+H]+412，实测值412。MS-ESI calculated value [M+H] + 412, measured value 412.
第十六步Step 16
将化合物1r(190mg,462μmol)加入三氟乙酸(5mL)中，反应液在100℃下搅拌24小时。反应液直接减压浓缩。剩余物经高效液相色谱法(三氟乙酸条件)纯化得到化合物1s的三氟乙酸盐。Compound 1r (190 mg, 462 μmol) was added to trifluoroacetic acid (5 mL), and the reaction solution was stirred at 100°C for 24 hours. The reaction solution was directly concentrated under reduced pressure. The residue was purified by high performance liquid chromatography (trifluoroacetic acid conditions) to obtain the trifluoroacetate salt of compound 1s.
MS-ESI计算值[M+H]+262，实测值262。MS-ESI calculated value [M+H] + 262, measured value 262.
第十七步Step 17
向化合物1s的三氟乙酸盐(100mg,0.266mmol)的无水二氧六环(5mL)溶液中加入1f(91.2mg,0.293mmol)，甲烷磺酸(2-二环己基膦)-3,6-二甲氧基-2,4,6-三异丙基-1,1-联苯)(2-氨基-1,1-联苯-2-基)钯(II)(24.2mg,26.6μmol)和碳酸铯(217mg,0.666mmol)。反应液用氮气置换三次，在95℃下搅拌16小时。反应液降至室温，减压浓缩，剩余物经过高效液相色谱法(盐酸条件)纯化得化合物1的盐酸盐。To a solution of compound 1s as the trifluoroacetate salt (100 mg, 0.266 mmol) in anhydrous dioxane (5 mL) was added 1f (91.2 mg, 0.293 mmol), methanesulfonic acid (2-dicyclohexylphosphine)-3 ,6-dimethoxy-2,4,6-triisopropyl-1,1-biphenyl)(2-amino-1,1-biphenyl-2-yl)palladium(II)(24.2mg, 26.6 μmol) and cesium carbonate (217 mg, 0.666 mmol). The reaction liquid was replaced with nitrogen three times and stirred at 95°C for 16 hours. The reaction solution was lowered to room temperature, concentrated under reduced pressure, and the residue was purified by high-performance liquid chromatography (hydrochloric acid conditions) to obtain the hydrochloride of compound 1.
MS-ESI计算值[M+H]+492，实测值492。1H NMR(400MHz,DMSO-d6)δ10.23(s,1H),8.93(s,1H),8.67(s,1H),8.13(s,1H),7.82(s,1H),4.35-4.34(m,2H),3.62-3.61(m,4H),3.22-3.13(m,6H),2.97-2.93(m,2H),2.04-1.91(m,4H),1.75-1.67(m,6H),1.51-1.48(m,2H),1.29-1.23(m,2H)。 MS-ESI calculated value [M+H] + 492, measured value 492. 1 H NMR (400MHz, DMSO-d 6 ) δ10.23(s,1H),8.93(s,1H),8.67(s,1H),8.13(s,1H),7.82(s,1H),4.35- 4.34(m,2H),3.62-3.61(m,4H),3.22-3.13(m,6H),2.97-2.93(m,2H),2.04-1.91(m,4H),1.75-1.67(m,6H ),1.51-1.48(m,2H),1.29-1.23(m,2H).
实施例2
Example 2
合成路线：
synthetic route:
第一步first step
将化合物1e(500mg,2.16mmol)和N-叔丁氧羰基-4-哌啶酮(1.72g,8.66mmol)溶于二氧六环(10mL)中，搅拌下滴加浓硫酸(106mg,1.08mmol,纯度：98％)，反应液在95℃下搅拌16小时。反应液减压浓缩，粗品在乙酸乙酯(20mL)中室温下搅拌1小时，过滤，减压浓缩得到化合物2a。Compound 1e (500mg, 2.16mmol) and N-tert-butoxycarbonyl-4-piperidone (1.72g, 8.66mmol) were dissolved in dioxane (10mL), and concentrated sulfuric acid (106mg, 1.08 mmol, purity: 98%), the reaction solution was stirred at 95°C for 16 hours. The reaction solution was concentrated under reduced pressure, and the crude product was stirred in ethyl acetate (20 mL) at room temperature for 1 hour, filtered, and concentrated under reduced pressure to obtain compound 2a.
MS-ESI计算值[M+Na]+434和436，实测值434和436。MS-ESI calculated values [M+Na] + 434 and 436, measured values 434 and 436.
第二步Step 2
将化合物1s的三氟乙酸盐(100mg,266umol)溶于无水二氧六环(5mL)中，加入化合物2a(157mg,293umol)，三(二亚苄基丙酮)二钯(24.4mg,26.6umol)，4,5-双(二苯基磷)-9,9-二甲基氧杂蒽(30.8mg,53.3umol)和碳酸铯(304mg,0.932mmol)，置换氮气三次，反应液在110℃搅拌16小时。反应液直接减压浓缩，剩余物经柱层析法分离(10/1,二氯甲烷/甲醇，Rf＝0.35)纯化，洗脱剂浓缩后得到化合物2b。Dissolve the trifluoroacetate salt of compound 1s (100mg, 266umol) in anhydrous dioxane (5mL), add compound 2a (157mg, 293umol), tris(dibenzylideneacetone) dipalladium (24.4mg, 26.6umol), 4,5-bis(diphenylphosphorus)-9,9-dimethylxanthene (30.8mg, 53.3umol) and cesium carbonate (304mg, 0.932mmol), replace nitrogen three times, and the reaction solution is Stir at 110°C for 16 hours. The reaction solution was directly concentrated under reduced pressure, and the residue was separated and purified by column chromatography (10/1, dichloromethane/methanol, Rf=0.35). Compound 2b was obtained after concentration of the eluent.
MS-ESI计算值[M+H]+593，实测值593。MS-ESI calculated value [M+H] + 593, measured value 593.
第三步third step
将化合物2b(150mg,132umol)溶于三氟乙酸(2mL)，在25℃搅拌1小时，反应液直接减压浓缩，剩余物经过高效液相色谱法(盐酸条件)纯化得化合物2的盐酸盐。Compound 2b (150mg, 132umol) was dissolved in trifluoroacetic acid (2mL), stirred at 25°C for 1 hour, the reaction solution was directly concentrated under reduced pressure, and the residue was purified by high performance liquid chromatography (hydrochloric acid conditions) to obtain the hydrochloric acid of compound 2 Salt.
MS-ESI计算值[M+H]+493，实测值493。1H NMR(400MHz,DMSO-d6)δ11.09(s,1H),10.55(s,1H),9.60(s,1H),8.68(s,1H),8.13(s,1H),7.83(s,1H),4.40-4.38(m,2H),3.60-3.59(m,4H),3.47-3.37(m,4H),3.28-3.25(m,2H),3.12-3.08(m,2H),2.53-2.52(m,6H),2.02-1.89(m,4H),1.84-1.81(m,2H)。MS-ESI calculated value [M+H] + 493, measured value 493. 1 H NMR (400MHz, DMSO-d 6 ) δ11.09(s,1H),10.55(s,1H),9.60(s,1H),8.68(s,1H),8.13(s,1H),7.83( s,1H),4.40-4.38(m,2H),3.60-3.59(m,4H),3.47-3.37(m,4H),3.28-3.25(m,2H),3.12-3.08(m,2H), 2.53-2.52(m,6H),2.02-1.89(m,4H),1.84-1.81(m,2H).
实施例3
Example 3
合成路线：synthetic route:
化合物2的盐酸盐→化合物3的盐酸盐Hydrochloride of compound 2 → hydrochloride of compound 3
第一步first step
将化合物2的盐酸盐(40.0mg,75.6μmol)溶于甲醇(2mL)和二氯甲烷(2mL)中，随后加入甲醛水溶液(18.4mg,0.226mmol,纯度：37％)，乙酸(7.72mg,0.128mmol)和醋酸硼氢化钠(64.1mg,0.302mol)，反应液在25℃下搅拌16小时。反应液直接减压浓缩。剩余物经高效液相色谱法(盐酸条件)纯化得到化合物3的盐酸盐。The hydrochloride of compound 2 (40.0 mg, 75.6 μmol) was dissolved in methanol (2 mL) and dichloromethane (2 mL), and then formaldehyde aqueous solution (18.4 mg, 0.226 mmol, purity: 37%) and acetic acid (7.72 mg were added ,0.128mmol) and sodium acetate borohydride (64.1mg, 0.302mol). The reaction solution was stirred at 25°C for 16 hours. The reaction solution was directly concentrated under reduced pressure. The residue was purified by high performance liquid chromatography (hydrochloric acid conditions) to obtain the hydrochloride salt of compound 3.
MS-ESI计算值[M+H]+507，实测值507。1H NMR(400MHz,D2O)δ7.67(s,1H),7.51(s,1H),6.93(s,1H),3.87-3.86(m,2H),3.78-3.76(m,2H),3.68-3.66(m,2H),3.52-3.51(m,2H),3.31-3.30(m,4H),3.19-3.13(m,2H),2.95(s, 3H),2.17-2.13(m,2H),2.10(s,3H),1.99-1.90(m,4H),1.83(s,3H)。MS-ESI calculated value [M+H] + 507, measured value 507. 1 H NMR (400MHz, D 2 O) δ7.67(s,1H),7.51(s,1H),6.93(s,1H),3.87-3.86(m,2H),3.78-3.76(m,2H) ,3.68-3.66(m,2H),3.52-3.51(m,2H),3.31-3.30(m,4H),3.19-3.13(m,2H),2.95(s, 3H),2.17-2.13(m,2H),2.10(s,3H),1.99-1.90(m,4H),1.83(s,3H).
实施例4
Example 4
合成路线：
synthetic route:
第一步first step
参照实施例1第一步得到化合物4b。Compound 4b was obtained by referring to the first step of Example 1.
1H NMR(400MHz,CDCl3)δ8.81(d,J＝1.6Hz,1H),8.03(d,J＝8.4Hz,1H),7.98(dd,J＝1.6,8.4Hz,1H),4.48(q,J＝7.2Hz,2H),1.44(t,J＝7.2Hz,3H)。 1 H NMR (400MHz, CDCl 3 ) δ8.81(d,J＝1.6Hz,1H),8.03(d,J＝8.4Hz,1H),7.98(dd,J＝1.6,8.4Hz,1H),4.48 (q, J=7.2Hz, 2H), 1.44 (t, J=7.2Hz, 3H).
第二步Step 2
参照实施例1第二步得到化合物4c。Compound 4c was obtained by referring to the second step of Example 1.
1H NMR(400MHz,CDCl3)δ8.40(d,J＝1.6Hz,1H),7.51(d,J＝8.4Hz,1H),7.40(dd,J＝1.6,8.4Hz,1H),4.46(q,J＝7.2Hz,2H),1.41(t,J＝7.2Hz,3H)。 1 H NMR (400MHz, CDCl 3 ) δ8.40(d,J＝1.6Hz,1H),7.51(d,J＝8.4Hz,1H),7.40(dd,J＝1.6,8.4Hz,1H),4.46 (q, J=7.2Hz, 2H), 1.41 (t, J=7.2Hz, 3H).
第三步third step
参照实施例1第三步得到化合物4d。Compound 4d was obtained with reference to the third step of Example 1.
MS-ESI计算值[M+H]+246和248，实测值246和248。MS-ESI calculated values [M+H] + 246 and 248, measured values 246 and 248.
第四步the fourth step
参照实施例1第四步得到化合物4e。Compound 4e was obtained with reference to the fourth step of Example 1.
MS-ESI计算值[M+H]+217和219，实测值217和219。MS-ESI calculated values [M+H] + 217 and 219, measured values 217 and 219.
第五步the fifth step
参照实施例1第五步得到化合物4f。Compound 4f was obtained with reference to the fifth step of Example 1.
MS-ESI计算值[M+H]+297和299，实测值297和299。MS-ESI calculated values [M+H] + 297 and 299, measured values 297 and 299.
第六步Step 6
将化合物1s的三氟乙酸盐(40.0mg,0.107mmol)溶于无水二氧六环(2mL)中，加入化合物4f(35.2mg,0.117mmol)，三(二亚苄基丙酮)二钯(9.76mg,10.7μmol)，4,5-双(二苯基膦)-9,9-二甲基氧杂蒽(12.3mg,21.3μmol)和碳酸铯(121mg,0.373mmol)，置换氮气三次，反应液在110℃搅拌16小时。反应液直接减压浓缩，粗产物经过柱层析法(10/1,二氯甲烷/甲醇,Rf＝0.32)分离纯化，粗品经高效液相色谱法(盐酸盐)纯化得到化合物4的盐酸盐。The trifluoroacetate salt of compound 1s (40.0mg, 0.107mmol) was dissolved in anhydrous dioxane (2mL), and compound 4f (35.2mg, 0.117mmol) and tris(dibenzylideneacetone)dipalladium were added (9.76mg, 10.7μmol), 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (12.3mg, 21.3μmol) and cesium carbonate (121mg, 0.373mmol), replacing nitrogen three times , the reaction solution was stirred at 110°C for 16 hours. The reaction solution was directly concentrated under reduced pressure. The crude product was separated and purified by column chromatography (10/1, dichloromethane/methanol, Rf = 0.32). The crude product was purified by high-performance liquid chromatography (hydrochloride) to obtain the salt of compound 4. Acid.
MS-ESI计算值[M+H]+478，实测值478。1H NMR(400MHz,DMSO-d6)δ11.12(s,1H),10.38(s,1H),8.84(s,1H),8.10(s,1H),7.83(s,1H),6.97(s,1H),4.74-4.70(m,2H),3.60(s,4H),3.11-2.96(m,4H),2.66(s,3H),2.02-1.91(m,4H),1.76-1.67(m,4H),1.53-1.51(m,2H),1.30-1.24(m,2H)。MS-ESI calculated value [M+H] + 478, measured value 478. 1 H NMR (400MHz, DMSO-d 6 ) δ11.12(s,1H),10.38(s,1H),8.84(s,1H),8.10(s,1H),7.83(s,1H),6.97( s,1H),4.74-4.70(m,2H),3.60(s,4H),3.11-2.96(m,4H),2.66(s,3H),2.02-1.91(m,4H),1.76-1.67( m,4H),1.53-1.51(m,2H),1.30-1.24(m,2H).
实施例5
Example 5
合成路线：
synthetic route:
第一步first step
参照实施例1第一步得到化合物5b。Compound 5b was obtained by referring to the first step of Example 1.
1H NMR(400MHz,CDCl3)δ8.62(d,J＝0.8Hz,1H),7.77(dd,J＝1.6,9.2Hz,1H),4.49(q,J＝7.2Hz,2H),1.44(t,J＝7.2Hz,4H)。 1 H NMR (400MHz, CDCl 3 ) δ8.62 (d, J = 0.8Hz, 1H), 7.77 (dd, J = 1.6, 9.2Hz, 1H), 4.49 (q, J = 7.2Hz, 2H), 1.44 (t,J=7.2Hz,4H).
第二步Step 2
参照实施例1第二步得到化合物5c。Compound 5c was obtained with reference to the second step of Example 1.
MS-ESI计算值[M+H]+264和266，实测值264和266。MS-ESI calculated values [M+H] + 264 and 266, measured values 264 and 266.
第三步third step
参照实施例1第三步得到化合物5d。Compound 5d was obtained by referring to the third step of Example 1.
1H NMR(400MHz,CDCl3)δ7.84(d,J＝8.4Hz,1H),4.47(q,J＝7.2Hz,2H),1.43(t,J＝7.2Hz,3H)。 1 H NMR (400MHz, CDCl 3 ) δ7.84 (d, J = 8.4 Hz, 1H), 4.47 (q, J = 7.2 Hz, 2H), 1.43 (t, J = 7.2 Hz, 3H).
第四步the fourth step
参照实施例1第四步得到化合物5e。Compound 5e was obtained with reference to the fourth step of Example 1.
MS-ESI计算值[M+H]+235和237，实测值235和237。MS-ESI calculated values [M+H] + 235 and 237, measured values 235 and 237.
第五步the fifth step
参照实施例1第五步得到化合物5f。Compound 5f was obtained with reference to the fifth step of Example 1.
MS-ESI计算值[M+H]+315和317，实测值315和317。MS-ESI calculated values [M+H] + 315 and 317, measured values 315 and 317.
第六步Step 6
将化合物1s的三氟乙酸盐(40.0mg,0.107mmol)溶于无水二氧六环(2mL)中，加入化合物15f(38.9mg,0.117mmol)，三(二亚苄基丙酮)二钯(9.76mg,10.6μmol),4,5-双(二苯基膦)-9,9-二甲基氧杂蒽(12.3mg,21.3μmol)和碳酸铯(121mg,0.373mmol)，置换氮气三次，反应液在110℃搅拌16小时。反应液直接减压浓缩，粗产物经过柱层析法(10/1,二氯甲烷/甲醇,Rf＝0.32)分离纯化，粗品经高效液相色谱法(盐酸条件)纯化得到化合物5的盐酸盐。The trifluoroacetate salt of compound 1s (40.0mg, 0.107mmol) was dissolved in anhydrous dioxane (2mL), and compound 15f (38.9mg, 0.117mmol) and tris(dibenzylideneacetone)dipalladium were added (9.76mg, 10.6μmol), 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (12.3mg, 21.3μmol) and cesium carbonate (121mg, 0.373mmol), replacing nitrogen three times , the reaction solution was stirred at 110°C for 16 hours. The reaction solution was directly concentrated under reduced pressure. The crude product was separated and purified by column chromatography (10/1, dichloromethane/methanol, Rf = 0.32). The crude product was purified by high-performance liquid chromatography (hydrochloric acid conditions) to obtain hydrochloric acid of compound 5. Salt.
MS-ESI计算值[M+H]+496，实测值496。1H NMR(400MHz,DMSO-d6)δ10.27(s,1H),8.96(s,1H),8.82(d,J＝10.8Hz,1H),8.17(s,1H),7.88(s,1H),4.36-4.34(m,2H),3.62(s,4H),3.17-3.14(m,2H),2.92-2.89(m,2H),2.53(s,3H),2.07-2.02(m,2H),1.92-1.90(m,2H),1.79-1.75(m,2H),1.68-1.65(m,2H),1.61-1.58(m,3H),1.29-1.23(s,1H)。MS-ESI calculated value [M+H] + 496, measured value 496. 1 H NMR (400MHz, DMSO-d 6 ) δ10.27 (s, 1H), 8.96 (s, 1H), 8.82 (d, J = 10.8Hz, 1H), 8.17 (s, 1H), 7.88 (s, 1H),4.36-4.34(m,2H),3.62(s,4H),3.17-3.14(m,2H),2.92-2.89(m,2H),2.53(s,3H),2.07-2.02(m, 2H),1.92-1.90(m,2H),1.79-1.75(m,2H),1.68-1.65(m,2H),1.61-1.58(m,3H),1.29-1.23(s,1H).
实施例6
Example 6
合成路线：
synthetic route:
第一步first step
参照实施例1第一步得到化合物6b。Compound 6b was obtained by referring to the first step of Example 1.
MS-ESI计算值[M+H]+264和266，实测值264和266。MS-ESI calculated values [M+H] + 264 and 266, measured values 264 and 266.
第二步Step 2
参照实施例1第二步得到化合物6c。Compound 6c was obtained by referring to the second step of Example 1.
MS-ESI计算值[M+H]+280和282，实测值280和282。MS-ESI calculated values [M+H] + 280 and 282, measured values 280 and 282.
第三步third step
参照实施例1第三步得到化合物6d。Compound 6d was obtained with reference to the third step of Example 1.
MS-ESI计算值[M+H]+280和282，实测值280和282。MS-ESI calculated values [M+H] + 280 and 282, measured values 280 and 282.
第四步the fourth step
参照实施例1第四步得到化合物6e。Compound 6e was obtained by referring to the fourth step of Example 1.
MS-ESI计算值[M+H]+251和253，实测值251和253。MS-ESI calculated values [M+H] + 251 and 253, measured values 251 and 253.
第五步the fifth step
参照实施例1第五步得到化合物6f。Compound 6f was obtained by referring to the fifth step of Example 1.
MS-ESI计算值[M+H]+331和333，实测值331和333。MS-ESI calculated values [M+H] + 331 and 333, measured values 331 and 333.
第六步Step 6
参照实施例4第六步得到化合物6的盐酸盐。The hydrochloride salt of compound 6 was obtained with reference to the sixth step of Example 4.
MS-ESI计算值[M+H]+512，实测值512。1H NMR(400MHz,DMSO-d6)δ10.71(s,1H),10.45(s,1H),8.80(brs,1H),8.18(brs,1H),7.87(br s,1H),4.37(brs,2H),3.66-3.65(m,4H),3.12(brs,2H),2.91(brs,2H),2.55(s,3H),2.04-2.00(m,2H),1.93-1.87(m,2H),1.78-1.74(m,2H),1.65-1.58(m,5H),1.26-1.21(m,1H)。MS-ESI calculated value [M+H] + 512, measured value 512. 1 H NMR (400MHz, DMSO-d 6 ) δ10.71(s,1H),10.45(s,1H),8.80(brs,1H),8.18(brs,1H),7.87(br s,1H),4.37 (brs,2H),3.66-3.65(m,4H),3.12(brs,2H),2.91(brs,2H),2.55(s,3H),2.04-2.00(m,2H),1.93-1.87(m ,2H),1.78-1.74(m,2H),1.65-1.58(m,5H),1.26-1.21(m,1H).
实施例7
Example 7
合成路线：
synthetic route:
第一步first step
将化合物1e(500mg,1.97mmol)和环戊酮(664mg,7.89mmol)溶于无水二氧六环(6mL)中，向反应液中逐滴加入浓硫酸(98.7mg,0.986mmol,纯度：98％)，反应液在95℃下搅拌3小时。反应液减压浓缩除去部分二氧六 环(约3mL)，过滤。向收集的滤饼中加入正己烷(10mL)，室温下搅拌2小时，过滤，滤饼真空干燥2小时，得到化合物7a。Compound 1e (500mg, 1.97mmol) and cyclopentanone (664mg, 7.89mmol) were dissolved in anhydrous dioxane (6mL), and concentrated sulfuric acid (98.7mg, 0.986mmol) was added dropwise to the reaction solution. Purity: 98%), the reaction solution was stirred at 95°C for 3 hours. The reaction solution was concentrated under reduced pressure to remove part of the dioxane. ring (approximately 3 mL) and filtered. Add n-hexane (10 mL) to the collected filter cake, stir at room temperature for 2 hours, filter, and vacuum dry the filter cake for 2 hours to obtain compound 7a.
MS-ESI计算值[M+H]+297和299，实测值297和299。1H NMR(400MHz,DMSO-d6)δ10.16(s,1H),8.02(s,1H),2.71-2.78(m,2H),2.37(s,3H),1.91-1.93(m,2H),1.79-1.84(m,2H),1.63-1.67(m,2H)。MS-ESI calculated values [M+H] + 297 and 299, measured values 297 and 299. 1 H NMR (400MHz, DMSO-d 6 ) δ10.16(s,1H),8.02(s,1H),2.71-2.78(m,2H),2.37(s,3H),1.91-1.93(m,2H ),1.79-1.84(m,2H),1.63-1.67(m,2H).
第二步Step 2
参照实施例4第六步得到化合物7的盐酸盐。The hydrochloride salt of compound 7 was obtained with reference to the sixth step of Example 4.
MS-ESI计算值[M+H]+478，实测值478。1H NMR(400MHz,D2O)δ7.68(s,1H),7.49(s,1H),7.00(s,1H),4.02-4.01(m,2H),3.70-3.69(m,2H),3.57-3.56(m,2H),3.28(s,3H),3.18-3.17(m,2H),2.58-2.57(m,2H),2.14-2.13(m,3H),2.00-1.93(m,5H),1.84-1.79(m,4H),1.69-1.66(m,1H)。MS-ESI calculated value [M+H] + 478, measured value 478. 1 H NMR (400MHz, D 2 O) δ7.68(s,1H),7.49(s,1H),7.00(s,1H),4.02-4.01(m,2H),3.70-3.69(m,2H) ,3.57-3.56(m,2H),3.28(s,3H),3.18-3.17(m,2H),2.58-2.57(m,2H),2.14-2.13(m,3H),2.00-1.93(m, 5H),1.84-1.79(m,4H),1.69-1.66(m,1H).
实施例8
Example 8
合成路线：
synthetic route:
第一步first step
将化合物1e(500mg,1.97mmol)和丙酮(458mg,7.89mmol)溶于无水二氧六环(6mL)中，向反应液中逐滴加入浓硫酸(96.7mg,0.966mmol,纯度：98％)，反应液在95℃下搅拌6小时。反应液减压浓缩除去二氧六环(约3mL)，过滤。收集的滤饼用石油醚/乙酸乙酯混合溶液(10/1,8mL×2)洗涤，滤饼真空干燥2小时，得到化合物8a。Compound 1e (500 mg, 1.97 mmol) and acetone (458 mg, 7.89 mmol) were dissolved in anhydrous dioxane (6 mL), and concentrated sulfuric acid (96.7 mg, 0.966 mmol) was added dropwise to the reaction solution, purity: 98% ), the reaction solution was stirred at 95°C for 6 hours. The reaction solution was concentrated under reduced pressure to remove dioxane (about 3 mL) and filtered. The collected filter cake was washed with petroleum ether/ethyl acetate mixed solution (10/1, 8 mL × 2), and the filter cake was vacuum dried for 2 hours to obtain compound 8a.
MS-ESI计算值[M+H]+271和273，实测值271和273。1H NMR(400MHz,DMSO-d6)δ9.82(s,1H),8.01(s,1H),2.37(s,3H),1.74(s,6H)。MS-ESI calculated values [M+H] + 271 and 273, measured values 271 and 273. 1 H NMR (400MHz, DMSO-d 6 ) δ9.82 (s, 1H), 8.01 (s, 1H), 2.37 (s, 3H), 1.74 (s, 6H).
第二步Step 2
参照实施例4第六步得到化合物8的盐酸盐。Referring to the sixth step of Example 4, the hydrochloride salt of compound 8 was obtained.
MS-ESI计算值[M+H]+452，实测值452。1H NMR(400MHz,DMSO-d6)δ10.96(s,1H),9.71(s,1H),8.65(s,1H),8.12(s,1H),7.82(s,1H),4.38-4.37(m,2H),3.67-3.65(m,2H),3.60-3.59(m,2H),3.16-3.10(m,2H),2.47-2.46(m,6H),2.02-1.90(m,4H),1.80-1.76(m,6H)。MS-ESI calculated value [M+H] + 452, measured value 452. 1 H NMR (400MHz, DMSO-d 6 ) δ10.96(s,1H),9.71(s,1H),8.65(s,1H),8.12(s,1H),7.82(s,1H),4.38- 4.37(m,2H),3.67-3.65(m,2H),3.60-3.59(m,2H),3.16-3.10(m,2H),2.47-2.46(m,6H),2.02-1.90(m,4H ),1.80-1.76(m,6H).
实施例9
Example 9
合成路线：
synthetic route:
第一步first step
将化合物1e(500mg,1.97mmol)和化合物9a(1.06g,7.88mmol)溶于无水二氧六环(6mL)中，向反应液中逐滴加入浓硫酸(98.7mg,0.985mmol,纯度：98％)，反应液在95℃下搅拌1.5小时。反应液减压浓缩除去部分二氧六环(约3mL)，过滤。向收集的滤饼中加入正己烷(12mL)，室温下搅拌2小时，过滤，滤饼真空干燥2小时，得到化合物9b。Compound 1e (500mg, 1.97mmol) and compound 9a (1.06g, 7.88mmol) were dissolved in anhydrous dioxane (6mL), and concentrated sulfuric acid (98.7mg, 0.985mmol) was added dropwise to the reaction solution. Purity: 98%), the reaction solution was stirred at 95°C for 1.5 hours. The reaction solution was concentrated under reduced pressure to remove part of dioxane (about 3 mL), and filtered. Add n-hexane (12 mL) to the collected filter cake, stir at room temperature for 2 hours, filter, and vacuum dry the filter cake for 2 hours to obtain compound 9b.
MS-ESI计算值[M+H]+347和349，实测值347和349。1H NMR(400MHz,DMSO-d6)δ10.57(s,1H),8.05(s,1H),3.17-3.25(m,2H),2.39(s,3H),2.14-2.27(m,4H),1.61-1.64(m,2H)。MS-ESI calculated values [M+H] + 347 and 349, measured values 347 and 349. 1 H NMR (400MHz, DMSO-d 6 ) δ10.57(s,1H),8.05(s,1H),3.17-3.25(m,2H),2.39(s,3H),2.14-2.27(m,4H ),1.61-1.64(m,2H).
第二步Step 2
参照实施例4第六步得到化合物9的盐酸盐。Referring to the sixth step of Example 4, the hydrochloride salt of compound 9 was obtained.
MS-ESI计算值[M+H]+528，实测值528。1H NMR(400MHz,DMSO-d6)δ10.91(s,1H),10.46(s,1H),8.68(s,1H),8.13(s,1H),7.83(s,1H),4.38-4.37(m,2H),3.60-3.59(m,4H),3.27-3.10(m,4H),2.43-2.41(m,6H),2.18-2.16(m,4H),2.07-2.02(m,2H),1.90-1.88(m,2H),1.69-1.66(m,2H)。MS-ESI calculated value [M+H] + 528, measured value 528. 1 H NMR (400MHz, DMSO-d 6 ) δ10.91(s,1H),10.46(s,1H),8.68(s,1H),8.13(s,1H),7.83(s,1H),4.38- 4.37(m,2H),3.60-3.59(m,4H),3.27-3.10(m,4H),2.43-2.41(m,6H),2.18-2.16(m,4H),2.07-2.02(m,2H ),1.90-1.88(m,2H),1.69-1.66(m,2H).
实施例10
Example 10
合成路线：
synthetic route:
第一步first step
将化合物6e(1.50g,4.71mmol)和环戊酮(1.59g,18.9mmol)溶于二氧六环(10mL)中，搅拌下滴加浓硫酸(462mg,4.71mmol)，反应液在95℃下搅拌16小时。反应液加入水(10mL)，乙酸乙酯(15mL×4)萃取，合并有机相，用无水硫酸钠干燥，过滤，减压浓缩。剩余物经柱层析分离(1/1,石油醚/乙酸乙酯，Rf＝0.31)纯化得到化合物10a。Compound 6e (1.50g, 4.71mmol) and cyclopentanone (1.59g, 18.9mmol) were dissolved in dioxane (10mL), and concentrated sulfuric acid (462mg, 4.71mmol) was added dropwise with stirring. The reaction solution was heated at 95°C. Stir for 16 hours. Water (10 mL) was added to the reaction solution, and extracted with ethyl acetate (15 mL × 4). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (1/1, petroleum ether/ethyl acetate, Rf=0.31) to obtain compound 10a.
MS-ESI计算值[M+H]+317和319,实测值317和319。MS-ESI calculated values [M+H] + 317 and 319, measured values 317 and 319.
第二步Step 2
将化合物1s的三氟乙酸盐(230mg,613μmol)溶于无水二氧六环(5mL)中，加入化合物10a(249mg,674μmol)，三(二亚苄基丙酮)二钯(56.1mg,61.3μmol)，4,5-双(二苯基膦)-9,9-二甲基氧杂蒽(70.9mg,123μmol)和碳酸铯(699mg,2.14mmol)，置换氮气三次，反应液在110℃搅拌16小时。反应液直接减压浓缩，剩余物经柱层析法分离(10/1,二氯甲烷/甲醇，Rf＝0.35)纯化，洗脱剂浓缩后再经高效液相色谱法(盐酸体系)纯化得到化合物10的盐酸盐。The trifluoroacetate salt of compound 1s (230 mg, 613 μmol) was dissolved in anhydrous dioxane (5 mL), and compound 10a (249 mg, 674 μmol), tris(dibenzylideneacetone) dipalladium (56.1 mg, 61.3μmol), 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (70.9mg, 123μmol) and cesium carbonate (699mg, 2.14mmol), replace nitrogen three times, and the reaction solution is at 110 °C and stirred for 16 hours. The reaction solution was directly concentrated under reduced pressure, and the residue was separated and purified by column chromatography (10/1, dichloromethane/methanol, Rf=0.35). After the eluent was concentrated, it was purified by high-performance liquid chromatography (hydrochloric acid system) to obtain Compound 10 hydrochloride salt.
MS-ESI计算值[M+H]+498,实测值498。1H NMR(400MHz,DMSO-d6)δ10.68(s,1H),10.26(s,1H),8.79(s,1H),8.17(s,1H),7.86(s,1H),4.38-4.36(m,2H),3.61-3.60(m,4H),3.13-3.11(m,2H),2.83-2.78(m,2H),2.52(s,3H),2.03-1.83(m,8H),1.75-1.74(m,2H)。MS-ESI calculated value [M+H] + 498, measured value 498. 1 H NMR (400MHz, DMSO-d 6 ) δ10.68(s,1H),10.26(s,1H),8.79(s,1H),8.17(s,1H),7.86(s,1H),4.38- 4.36(m,2H),3.61-3.60(m,4H),3.13-3.11(m,2H),2.83-2.78(m,2H),2.52(s,3H),2.03-1.83(m,8H), 1.75-1.74(m,2H).
实施例11
Example 11
合成路线：
synthetic route:
第一步first step
将化合物11a盐酸盐(500mg,3.48mmol)溶于1,2-二溴乙烷(5mL)，然后加入N,N-二异丙基乙胺(900mg,6.97mmol),反应液在25℃下搅拌14小时。反应完成后,反应液用水(30mL)稀释，乙酸乙酯(20mL×4)萃取，有机相用无水硫酸钠干燥，过滤，滤液减压浓缩，经柱层析分离(3:1,石油醚/乙酸乙酯,Rf＝0.6)得到化合物11b。Compound 11a hydrochloride (500 mg, 3.48 mmol) was dissolved in 1,2-dibromoethane (5 mL), then N, N-diisopropylethylamine (900 mg, 6.97 mmol) was added, and the reaction solution was heated at 25°C Stir for 14 hours. After the reaction was completed, the reaction solution was diluted with water (30 mL), extracted with ethyl acetate (20 mL × 4), the organic phase was dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and separated by column chromatography (3:1, petroleum ether /ethyl acetate, Rf = 0.6) to obtain compound 11b.
1H NMR(400MHz,CDCl3)δ＝3.41(t,J＝7.0Hz,2H),2.99(t,J＝13.3Hz,2H),2.92(t,J＝7.0Hz,2H),2.83(t,J＝7.0Hz,2H),2.29(tt,J＝7.1,14.5Hz,2H)。 1 H NMR (400MHz, CDCl 3 ) δ = 3.41 (t, J = 7.0Hz, 2H), 2.99 (t, J = 13.3Hz, 2H), 2.92 (t, J = 7.0Hz, 2H), 2.83 (t ,J＝7.0Hz,2H),2.29(tt,J＝7.1,14.5Hz,2H).
第二步Step 2
将化合物11b(250mg,795μmol)溶于N,N-二甲基甲酰胺(4mL)中，然后加入化合物1q(187mg,875μmol)和氢氧化钠(63.6mg,1.59mmol)，并于50℃下搅拌0.5小时。反应完成后，向反应液中加入水(50mL)稀释，用乙酸乙酯萃取(30mL×3)，合并的有机相用无水硫酸钠干燥，过滤，滤液减压浓缩，经柱层析分离(1:1,石油醚/乙酸乙酯,Rf＝0.1)得到化合物11c。Compound 11b (250 mg, 795 μmol) was dissolved in N,N-dimethylformamide (4 mL), then compound 1q (187 mg, 875 μmol) and sodium hydroxide (63.6 mg, 1.59 mmol) were added, and the mixture was incubated at 50°C. Stir for 0.5 hours. After the reaction was completed, water (50 mL) was added to the reaction solution to dilute, and the mixture was extracted with ethyl acetate (30 mL × 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and separated by column chromatography ( 1:1, petroleum ether/ethyl acetate, Rf=0.1) to obtain compound 11c.
MS-ESI计算值[M+H]+448,实测值448。MS-ESI calculated value [M+H] + 448, measured value 448.
第三步third step
将化合物11c(300mg,670μmol)溶于三氟乙酸(3.0mL)中，反应液在100℃下搅拌1小时。反应完成后，将反应液浓缩，剩余物经过高效液相色谱法(盐酸体系)纯化得化合物11d的盐酸盐。Compound 11c (300 mg, 670 μmol) was dissolved in trifluoroacetic acid (3.0 mL), and the reaction solution was stirred at 100°C for 1 hour. After the reaction is completed, the reaction solution is concentrated, and the residue is purified by high performance liquid chromatography (hydrochloric acid system) to obtain the hydrochloride salt of compound 11d.
MS-ESI计算值[M+H]+298,实测值298。MS-ESI calculated value [M+H] + 298, measured value 298.
第四步the fourth step
将化合物11d的盐酸盐(108mg,323μmol)，化合物1f(111mg,356μmol)溶于无水二氧六环(2mL)中，然后加入碳酸铯(264mg,809μmol)和甲烷磺酸(2-二环己基膦)-3,6-二甲氧基-2,4,6-三异丙基-1,1-联苯)(2-氨基-1,1-联苯-2-基)钯(II)(29.3mg,32.4μmol)，反应液在氮气保护下105℃搅拌12小时。反应完成后，将反应液减压浓缩，经柱层析分离(10:1,二氯甲烷/甲醇,Rf＝0.3)得到粗品化合物。向粗品中加入甲醇(5mL)，15℃搅拌16小时，过滤，滤饼用甲醇洗涤(2mL×2)，干燥得到化合物11。The hydrochloride of compound 11d (108 mg, 323 μmol) and compound 1f (111 mg, 356 μmol) were dissolved in anhydrous dioxane (2 mL), and then cesium carbonate (264 mg, 809 μmol) and methane sulfonic acid (2-dioxane) were added. Cyclohexylphosphine)-3,6-dimethoxy-2,4,6-triisopropyl-1,1-biphenyl)(2-amino-1,1-biphenyl-2-yl)palladium ( II) (29.3 mg, 32.4 μmol), the reaction solution was stirred at 105°C for 12 hours under nitrogen protection. After the reaction was completed, the reaction solution was concentrated under reduced pressure and separated by column chromatography (10:1, dichloromethane/methanol, Rf=0.3) to obtain the crude compound. Methanol (5 mL) was added to the crude product, stirred at 15°C for 16 hours, filtered, and the filter cake was washed with methanol (2 mL × 2) and dried to obtain compound 11.
MS-ESI计算值[M+H]+528,实测值528。1H NMR(400MHz,DMSO-d6)δ＝10.21(s,1H),8.91(s,1H),8.66(s,1H),8.09(s,1H),7.72(s,1H),4.09(t,J＝5.6Hz,2H),3.06-2.92(m,4H),2.89-2.77(m,4H),2.49-2.46(m,6H),2.31-2.18(m,2H),1.82-1.72(m,2H),1.71-1.59(m,3H),1.54-1.42(m,2H),1.35-1.20(m,1H)。MS-ESI calculated value [M+H] + 528, measured value 528. 1 H NMR (400MHz, DMSO-d 6 ) δ = 10.21 (s, 1H), 8.91 (s, 1H), 8.66 (s, 1H), 8.09 (s, 1H), 7.72 (s, 1H), 4.09 ( t,J＝5.6Hz,2H),3.06-2.92(m,4H),2.89-2.77(m,4H),2.49-2.46(m,6H),2.31-2.18(m,2H),1.82-1.72( m,2H),1.71-1.59(m,3H),1.54-1.42(m,2H),1.35-1.20(m,1H).
实施例12
Example 12
合成路线：
synthetic route:
第一步first step
将化合物11d的三氟乙酸盐(90mg,219μmol)，化合物7a(72mg,241μmol)溶于无水二氧六环(2mL)中，然后加入碳酸铯(250mg,766μmol)和甲烷磺酸(2-二环己基膦)-3,6-二甲氧基-2,4,6-三异丙基-1,1-联苯)(2-氨基-1,1-联苯-2-基)钯(II)(20mg,21.9μmol)，反应液在氮气保护下105℃搅拌12小时。减压浓缩反应液，经柱层析分离(10:1,二氯甲烷/甲醇,Rf＝0.3)纯化得到粗品化合物。向粗品中加入甲醇和乙醇混合溶液(4/1,10mL)，20℃搅拌16小时，过滤，滤饼用甲醇洗涤(2mL×2)，水洗涤(2mL×2)，干燥得到化合物12。The trifluoroacetate salt of compound 11d (90 mg, 219 μmol) and compound 7a (72 mg, 241 μmol) were dissolved in anhydrous dioxane (2 mL), and then cesium carbonate (250 mg, 766 μmol) and methane sulfonic acid (2 -Dicyclohexylphosphine)-3,6-dimethoxy-2,4,6-triisopropyl-1,1-biphenyl)(2-amino-1,1-biphenyl-2-yl) Palladium (II) (20 mg, 21.9 μmol), the reaction solution was stirred at 105°C for 12 hours under nitrogen protection. The reaction solution was concentrated under reduced pressure and purified by column chromatography (10:1, dichloromethane/methanol, Rf=0.3) to obtain the crude compound. Add a mixed solution of methanol and ethanol (4/1, 10 mL) to the crude product, stir at 20°C for 16 hours, filter, and wash the filter cake with methanol (2 mL × 2), water (2 mL × 2), and dry to obtain compound 12.
MS-ESI计算值[M+H]+514,实测值514。1H NMR(400MHz,DMSO-d6)δ＝10.00(s,1H),8.84(s,1H),8.64(s,1H),8.08(s,1H),7.70(s,1H),4.08(t,J＝5.6Hz,2H),3.00(t,J＝13.5Hz,2H),2.91-2.78(m,6H),2.47(s,3H),2.46(s,3H),2.31-2.18(m,2H),2.04-1.92(m,2H),1.91-1.78(m,2H),1.76-1.62(m,2H)。MS-ESI calculated value [M+H] + 514, measured value 514. 1 H NMR (400MHz, DMSO-d 6 ) δ = 10.00 (s, 1H), 8.84 (s, 1H), 8.64 (s, 1H), 8.08 (s, 1H), 7.70 (s, 1H), 4.08 ( t,J＝5.6Hz,2H),3.00(t,J＝13.5Hz,2H),2.91-2.78(m,6H),2.47(s,3H),2.46(s,3H),2.31-2.18(m ,2H),2.04-1.92(m,2H),1.91-1.78(m,2H),1.76-1.62(m,2H).
将化合物12(2g，3.89μmol)与六氟异丙醇(40mL)搅拌混匀，向溶液中加入一水合对甲苯磺酸(814.89mg,4.28mmol)，反应液在40℃下搅拌3小时，将反应液滴加入异丙醇中(160mL)，过滤，滤饼真空干燥得到化合物12对应的对甲苯磺酸盐。1H NMR(400MHz,DMSO-d6)δ＝10.02(s,1H),8.86(br s,1H),8.64(s,1H),8.11(s,1H),7.78(s,1H),7.48(d,J＝8.0Hz,2H),7.10(d,J＝8.0Hz,2H),4.31(br d,J＝4.4Hz,2H),4.08-3.62(m,6H),2.89-2.78(m,2H),2.72-2.57(m,2H),2.51(br s,3H),2.46(s,3H),2.28(s,3H),1.95-1.97(m,2H),1.89-1.79(m,2H),1.73-1.64(m,2H)。MS-ESI计算值[M+H]+514,实测值514。Compound 12 (2g, 3.89μmol) and hexafluoroisopropanol (40mL) were stirred and mixed, p-toluenesulfonic acid monohydrate (814.89mg, 4.28mmol) was added to the solution, and the reaction solution was stirred at 40°C for 3 hours. The reaction liquid was added dropwise to isopropanol (160 mL), filtered, and the filter cake was dried under vacuum to obtain the p-toluenesulfonate corresponding to compound 12. 1 H NMR (400MHz, DMSO-d 6 ) δ = 10.02 (s, 1H), 8.86 (br s, 1H), 8.64 (s, 1H), 8.11 (s, 1H), 7.78 (s, 1H), 7.48 (d,J＝8.0Hz,2H),7.10(d,J＝8.0Hz,2H),4.31(br d,J＝4.4Hz,2H),4.08-3.62(m,6H),2.89-2.78(m ,2H),2.72-2.57(m,2H),2.51(br s,3H),2.46(s,3H),2.28(s,3H),1.95-1.97(m,2H),1.89-1.79(m, 2H),1.73-1.64(m,2H). MS-ESI calculated value [M+H] + 514, measured value 514.
实施例13
Example 13
合成路线：
synthetic route:
第一步first step
将化合物13a(2.00g,26.6mmol)和1,4-二溴丁烷(5.75g,26.6mmol)溶于乙腈(100mL)，然后加入碳酸钾(7.36g,53.26mmol)，反应液在80℃下搅拌12小时。反应完成后，过滤反应液，滤液减压浓缩，用二氯甲烷(250mL)稀释，用饱和碳酸钾水溶液洗涤(75mL×1)，收集有机相，水相用二氯甲烷萃取(75mL×9)，合并的有机相用无水硫酸钠干燥，过滤，滤液减压浓缩得到无色油状产品13b。Compound 13a (2.00g, 26.6mmol) and 1,4-dibromobutane (5.75g, 26.6mmol) were dissolved in acetonitrile (100mL), then potassium carbonate (7.36g, 53.26mmol) was added, and the reaction solution was heated at 80°C Stir for 12 hours. After the reaction is completed, the reaction solution is filtered, and the filtrate is concentrated under reduced pressure, diluted with dichloromethane (250 mL), washed with saturated aqueous potassium carbonate solution (75 mL × 1), the organic phase is collected, and the aqueous phase is extracted with dichloromethane (75 mL × 9) , the combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain colorless oily product 13b.
MS-ESI计算值[M+H]+130,实测值130。1H NMR(400MHz,CDCl3)δ＝3.61-3.55(m,1H),3.40-3.34(m,1H),2.99(brs,1H),2.69-2.62(m,1H),2.61-2.54(m,4H),1.84-1.70(m,4H),1.04(d,J＝6.5Hz,3H)。MS-ESI calculated value [M+H] + 130, measured value 130. 1 H NMR (400MHz, CDCl 3 )δ=3.61-3.55(m,1H),3.40-3.34(m,1H),2.99(brs,1H),2.69-2.62(m,1H),2.61-2.54(m ,4H),1.84-1.70(m,4H),1.04(d,J=6.5Hz,3H).
第二步Step 2
将化合物13b(2.95g,23.1mmol)溶于二氯甲烷(25mL)，冷却至0℃，然后在0℃下加入三苯基膦(9.07g,34.6mmol)和四溴化碳(9.94g,30.0mmol)，反应液在15℃下搅拌12小时。反应完成后，加水(150mL)稀释，用二氯甲烷萃取(100mL×3)，收集有机相。用饱和碳酸氢钠水溶液将水相值调至pH＝9，用乙酸乙酯萃取(100mL×3)。合并有机相，用无水硫酸钠干燥，过滤，滤液减压浓缩得到黄色油状产品13c。粗品直接用于下一步反应。Compound 13b (2.95g, 23.1mmol) was dissolved in dichloromethane (25mL), cooled to 0°C, and then triphenylphosphine (9.07g, 34.6mmol) and carbon tetrabromide (9.94g) were added at 0°C. 30.0 mmol), the reaction solution was stirred at 15°C for 12 hours. After the reaction is completed, add water (150 mL) to dilute, extract with dichloromethane (100 mL × 3), and collect the organic phase. Adjust the aqueous phase value to pH=9 with saturated sodium bicarbonate aqueous solution, and extract with ethyl acetate (100 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain product 13c as a yellow oil. The crude product was directly used in the next reaction.
1H NMR(400MHz,CDCl3)δ＝2.84-2.77(m,1H),2.65-2.59(m,1H),2.58-2.52(m,1H),2.51-2.48(m,2H),2.51-2.46(m,1H),1.76-1.71(m,3H),1.74-1.71(m,2H),1.66(d,J＝6.6Hz,3H)。 1 H NMR (400MHz, CDCl 3 ) δ = 2.84-2.77 (m, 1H), 2.65-2.59 (m, 1H), 2.58-2.52 (m, 1H), 2.51-2.48 (m, 2H), 2.51-2.46 (m,1H),1.76-1.71(m,3H),1.74-1.71(m,2H),1.66(d,J＝6.6Hz,3H).
第三步third step
将化合物1q(200mg,636μmol)溶于N,N-二甲基甲酰胺(4mL)中，然后加入化合物13c(134mg,700μmol)和氢氧化钠(50.9mg,1.27mmol)，并于50℃下搅拌2小时。反应完成后，减压浓缩反应液，向反应液中加入水(50mL)稀释，用乙酸乙酯萃取(30mL×3)，合并的有机相用饱和食盐水洗涤(40mL×1)，用无水硫酸钠干燥， 过滤，减压浓缩滤液得到黄色固体化合物13d。粗品直接用于下一步反应。Compound 1q (200 mg, 636 μmol) was dissolved in N,N-dimethylformamide (4 mL), then compound 13c (134 mg, 700 μmol) and sodium hydroxide (50.9 mg, 1.27 mmol) were added, and the mixture was incubated at 50°C. Stir for 2 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure, diluted with water (50 mL), and extracted with ethyl acetate (30 mL × 3). The combined organic phases were washed with saturated brine (40 mL × 1), and washed with anhydrous water. Dry over sodium sulfate, Filter, and concentrate the filtrate under reduced pressure to obtain compound 13d as a yellow solid. The crude product was directly used in the next reaction.
MS-ESI计算值[M+H]+426,实测值426。MS-ESI calculated value [M+H] + 426, measured value 426.
第四步the fourth step
将化合物13d(300mg,670μmol)溶于三氟乙酸(10mL)中，反应液在100℃下搅拌12小时。反应完成后，将反应液浓缩，剩余物经过高效液相色谱法(盐酸体系)纯化得化合物13e的盐酸盐。Compound 13d (300 mg, 670 μmol) was dissolved in trifluoroacetic acid (10 mL), and the reaction solution was stirred at 100°C for 12 hours. After the reaction is completed, the reaction solution is concentrated, and the residue is purified by high-performance liquid chromatography (hydrochloric acid system) to obtain the hydrochloride salt of compound 13e.
MS-ESI计算值[M+H]+276,实测值276。MS-ESI calculated value [M+H] + 276, measured value 276.
第五步the fifth step
将化合物13e的盐酸盐(76.0mg,244μmol)，化合物7a(72.4mg,244μmol)溶于无水二氧六环(3mL)中，然后加入碳酸铯(159mg,487μmol)和甲烷磺酸(2-二环己基膦)-3,6-二甲氧基-2,4,6-三异丙基-1,1-联苯)(2-氨基-1,1-联苯-2-基)钯(II)(22.1mg,24.4μmol)，反应液在氮气保护下105℃搅拌12小时。反应完成后，将反应液减压浓缩，加入水(50mL)稀释，用乙酸乙酯萃取(30mL×3)，合并的有机相用饱和食盐水洗涤(40mL×1)，用无水硫酸钠干燥，过滤，减压浓缩滤液，剩余物经过高效液相色谱(盐酸体系)纯化得到化合物13的盐酸盐。化合物13的盐酸盐用二氯甲烷(30mL)溶解后依次用饱和碳酸氢钠水溶液(20mL)和饱和食盐水(20mL)洗涤，有机相用无水硫酸钠干燥，过滤，减压浓缩滤液，剩余物经薄层层析分离纯化(10:1,二氯甲烷/甲醇,Rf＝0.3)得到化合物13。The hydrochloride of compound 13e (76.0 mg, 244 μmol) and compound 7a (72.4 mg, 244 μmol) were dissolved in anhydrous dioxane (3 mL), and then cesium carbonate (159 mg, 487 μmol) and methane sulfonic acid (2 -Dicyclohexylphosphine)-3,6-dimethoxy-2,4,6-triisopropyl-1,1-biphenyl)(2-amino-1,1-biphenyl-2-yl) Palladium (II) (22.1 mg, 24.4 μmol), the reaction solution was stirred at 105°C for 12 hours under nitrogen protection. After the reaction was completed, the reaction solution was concentrated under reduced pressure, diluted with water (50 mL), extracted with ethyl acetate (30 mL × 3), the combined organic phases were washed with saturated brine (40 mL × 1), and dried over anhydrous sodium sulfate. , filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by high-performance liquid chromatography (hydrochloric acid system) to obtain the hydrochloride of compound 13. The hydrochloride of compound 13 was dissolved in dichloromethane (30 mL) and washed with saturated aqueous sodium bicarbonate solution (20 mL) and saturated brine (20 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was separated and purified by thin layer chromatography (10:1, dichloromethane/methanol, Rf=0.3) to obtain compound 13.
MS-ESI计算值[M+H]+492,实测值492。1H NMR(400MHz,MeOH-d4)δ＝8.87-8.77(m,1H),8.08-8.00(m,1H),7.63-7.52(m,1H),4.71-4.53(m,1H),4.37-4.12(m,1H),3.67-3.37(m,3H),3.05-2.91(m,3H),2.65-2.52(m,6H),2.22-2.04(m,6H),1.97-1.76(m,5H),1.55-1.33(m,3H)。MS-ESI calculated value [M+H] + 492, measured value 492. 1 H NMR (400MHz, MeOH-d 4 ) δ = 8.87-8.77 (m, 1H), 8.08-8.00 (m, 1H), 7.63-7.52 (m, 1H), 4.71-4.53 (m, 1H), 4.37 -4.12(m,1H),3.67-3.37(m,3H),3.05-2.91(m,3H),2.65-2.52(m,6H),2.22-2.04(m,6H),1.97-1.76(m, 5H),1.55-1.33(m,3H).
实施例14
Example 14
合成路线：
synthetic route:
第一步first step
将化合物14a(2.00g,26.6mmol)和1,4-二溴丁烷(5.75g,26.6mmol)溶于乙腈(100mL)，然后加入碳酸钾(7.36g,53.26mmol)，反应液在80℃下搅拌12小时。过滤反应液，滤液减压浓缩，用二氯甲烷(250mL)稀释，用饱和碳酸钾水溶液洗涤(75mL×1)，收集有机相，水相用二氯甲烷萃取(75mL×9)，合并的有机相用无水硫酸钠干燥，过滤，滤液减压浓缩得到化合物14b。Compound 14a (2.00g, 26.6mmol) and 1,4-dibromobutane (5.75g, 26.6mmol) were dissolved in acetonitrile (100mL), then potassium carbonate (7.36g, 53.26mmol) was added, and the reaction solution was heated at 80°C Stir for 12 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure, diluted with dichloromethane (250 mL), and washed with saturated aqueous potassium carbonate solution (75 mL × 1). The organic phase was collected, and the aqueous phase was extracted with dichloromethane (75 mL × 9). The combined organic The phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 14b.
MS-ESI计算值[M+H]+130,实测值130。1H NMR(400MHz,CDCl3)δ＝3.63-3.58(m,1H),3.41-3.35(m,1H),2.90(brs,1H),2.73-2.66(m,1H),2.63-2.57(m,4H),1.82-1.74(m,4H),1.06(d,J＝6.5Hz,3H)。MS-ESI calculated value [M+H] + 130, measured value 130. 1 H NMR (400MHz, CDCl 3 ) δ = 3.63-3.58 (m, 1H), 3.41-3.35 (m, 1H), 2.90 (brs, 1H), 2.73-2.66 (m, 1H), 2.63-2.57 (m ,4H),1.82-1.74(m,4H),1.06(d,J=6.5Hz,3H).
第二步Step 2
将化合物14b(2.48g,19.2mmol)溶于二氯甲烷(25mL)，冷却至0℃，然后在0℃下加入三苯基膦(7.55g,28.8mmol)和四溴化碳(8.28g,25.0mmol)，反应液在15℃下搅拌12小时。向反应液中加水(150mL)稀释，用二氯甲烷萃取(100mL×3)，收集有机相。用饱和碳酸氢钠水溶液将水相调至pH＝9，用乙酸乙酯萃取(100mL×3)。合并有机相，用无水硫酸钠干燥，过滤，滤液减压浓缩得到化合物14c。粗品直接用于下一步反应。Compound 14b (2.48g, 19.2mmol) was dissolved in dichloromethane (25mL), cooled to 0°C, and then triphenylphosphine (7.55g, 28.8mmol) and carbon tetrabromide (8.28g) were added at 0°C. 25.0 mmol), the reaction solution was stirred at 15°C for 12 hours. The reaction solution was diluted with water (150 mL), extracted with dichloromethane (100 mL × 3), and the organic phase was collected. The aqueous phase was adjusted to pH=9 with saturated sodium bicarbonate aqueous solution, and extracted with ethyl acetate (100 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 14c. The crude product was directly used in the next reaction.
第三步third step
将化合物1q(250mg,795μmol)溶于N,N-二甲基甲酰胺(3mL)中，然后加入化合物14c(229mg,1.19mmol)和氢氧化钠(63.6mg,1.59mmol)，并于50℃下搅拌2小时。反应完成后，减压浓缩反应液，向反应液中加入水(30mL)稀释，用1M盐酸水溶液将反应液调至pH＝3，用二氯甲烷洗涤(50mL×3)。用1M氢氧化钠水溶液将反 应液调至pH＝11，用二氯甲烷萃取(60mL×3)，合并的有机相用饱和食盐水洗涤(150mL×1)，用无水硫酸钠干燥，过滤，减压浓缩滤液得到化合物14d。粗品直接用于下一步反应。Compound 1q (250 mg, 795 μmol) was dissolved in N,N-dimethylformamide (3 mL), then compound 14c (229 mg, 1.19 mmol) and sodium hydroxide (63.6 mg, 1.59 mmol) were added, and the mixture was heated at 50°C Stir for 2 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure, water (30 mL) was added to the reaction solution to dilute, the reaction solution was adjusted to pH=3 with 1M hydrochloric acid aqueous solution, and washed with dichloromethane (50 mL × 3). Use 1M aqueous sodium hydroxide solution to The reaction solution was adjusted to pH=11, and extracted with dichloromethane (60 mL × 3). The combined organic phases were washed with saturated brine (150 mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 14d. . The crude product was directly used in the next reaction.
MS-ESI计算值[M+H]+426,实测值426。MS-ESI calculated value [M+H] + 426, measured value 426.
第四步the fourth step
将化合物14d(430mg,1.01mmol)溶于三氟乙酸(15mL)中，反应液在100℃下搅拌3小时。反应完成后，将反应液浓缩，剩余物经过高效液相色谱纯化(盐酸体系)得到化合物14e的盐酸盐。Compound 14d (430 mg, 1.01 mmol) was dissolved in trifluoroacetic acid (15 mL), and the reaction solution was stirred at 100°C for 3 hours. After the reaction is completed, the reaction solution is concentrated, and the residue is purified by high performance liquid chromatography (hydrochloric acid system) to obtain the hydrochloride salt of compound 14e.
MS-ESI计算值[M+H]+276,实测值276。MS-ESI calculated value [M+H] + 276, measured value 276.
第五步the fifth step
将化合物14e的盐酸盐(80.0mg,206μmol)，化合物7a(61.2mg,206μmol)溶于无水二氧六环(6mL)中，然后加入碳酸铯(168mg,515μmol)和甲烷磺酸(2-二环己基膦)-3,6-二甲氧基-2,4,6-三异丙基-1,1-联苯)(2-氨基-1,1-联苯-2-基)钯(II)(18.7mg,20.6μmol)，反应液在氮气保护下95℃搅拌12小时。反应完成后，将反应液减压浓缩，柱层析分离纯化(10:1,二氯甲烷/甲醇,Rf＝0.25)，得到粗品。粗品经过高效液相色谱纯化(盐酸体系)得到化合物14的盐酸盐。The hydrochloride of compound 14e (80.0 mg, 206 μmol) and compound 7a (61.2 mg, 206 μmol) were dissolved in anhydrous dioxane (6 mL), and then cesium carbonate (168 mg, 515 μmol) and methane sulfonic acid (2 -Dicyclohexylphosphine)-3,6-dimethoxy-2,4,6-triisopropyl-1,1-biphenyl)(2-amino-1,1-biphenyl-2-yl) Palladium (II) (18.7 mg, 20.6 μmol), the reaction solution was stirred at 95°C for 12 hours under nitrogen protection. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and separated and purified by column chromatography (10:1, dichloromethane/methanol, Rf=0.25) to obtain a crude product. The crude product was purified by high performance liquid chromatography (hydrochloric acid system) to obtain the hydrochloride salt of compound 14.
MS-ESI计算值[M+H]+492,实测值492。1H NMR(400MHz,DMSO-d6)δ＝7.76-7.68(m,1H),7.53-7.45(m,1H),7.05-6.98(m,1H),4.12-4.02(m,1H),3.98-3.88(m,1H),3.77-3.59(m,3H),3.31-3.21(m,2H),2.68-2.53(m,2H),2.20-2.08(m,5H),2.06-1.93(m,4H),1.89-1.77(m,5H),1.73-1.62(m,2H),1.53-1.47(m,3H)。MS-ESI calculated value [M+H] + 492, measured value 492. 1 H NMR (400MHz, DMSO-d 6 ) δ = 7.76-7.68 (m, 1H), 7.53-7.45 (m, 1H), 7.05-6.98 (m, 1H), 4.12-4.02 (m, 1H), 3.98 -3.88(m,1H),3.77-3.59(m,3H),3.31-3.21(m,2H),2.68-2.53(m,2H),2.20-2.08(m,5H),2.06-1.93(m, 4H),1.89-1.77(m,5H),1.73-1.62(m,2H),1.53-1.47(m,3H).
实施例15
Example 15
合成路线：
synthetic route:
第一步first step
将化合物1q(357mg,1.14mmol)和化合物15a(300mg,1.14mmol)溶于N,N-二甲基甲酰胺(3mL)中，向反应液中加入氢氧化钠(136mg,3.41mmol)，反应液在50℃下搅拌0.5小时。反应液用水(60mL)稀释，乙酸乙酯(60mL×3)萃取，合并有机相，有机相用水(200mL×1)和饱和食盐水(200mL×1)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，粗品经柱层析法分离(2/1,石油醚/乙酸乙酯,Rf＝0.45)得到化合物15b。Compound 1q (357mg, 1.14mmol) and compound 15a (300mg, 1.14mmol) were dissolved in N,N-dimethylformamide (3mL), sodium hydroxide (136mg, 3.41mmol) was added to the reaction solution, and the reaction The solution was stirred at 50°C for 0.5 hours. The reaction solution was diluted with water (60mL), extracted with ethyl acetate (60mL×3), the organic phases were combined, washed with water (200mL×1) and saturated brine (200mL×1), dried over anhydrous sodium sulfate, filtered, and the filtrate Concentrate under reduced pressure, and the crude product is separated by column chromatography (2/1, petroleum ether/ethyl acetate, Rf=0.45) to obtain compound 15b.
MS-ESI计算值[M+H]+498，实测值498。MS-ESI calculated value [M+H] + 498, measured value 498.
第二步Step 2
将化合物15b(255mg,0.509mmol)溶于三氟乙酸(20mL)中。反应液在氮气保护下100℃下搅拌2小时。反应液减压浓缩，得到化合物15c的三氟乙酸盐。Compound 15b (255 mg, 0.509 mmol) was dissolved in trifluoroacetic acid (20 mL). The reaction solution was stirred at 100°C for 2 hours under nitrogen protection. The reaction solution was concentrated under reduced pressure to obtain the trifluoroacetate salt of compound 15c.
MS-ESI计算值[M+H]+248，实测值248。MS-ESI calculated value [M+H] + 248, measured value 248.
第三步 third step
将化合物15c的三氟乙酸盐(200mg,0.554mmol)溶于四氢呋喃(3mL)和甲醇(3mL)，向反应液中加入二叔丁基二碳酸酯(121mg,0.554mmol)和三乙胺(224mg,2.21mmol)。反应液在25℃下搅拌1小时。反应液用水(60mL)稀释，二氯甲烷(60mL×4)萃取，合并有机相，有机相依次用水(200mL×1)和饱和食盐水(200mL×1)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，粗品经高效液相色谱法(中性条件)纯化得到化合物15d。The trifluoroacetate salt of compound 15c (200 mg, 0.554 mmol) was dissolved in tetrahydrofuran (3 mL) and methanol (3 mL), and di-tert-butyl dicarbonate (121 mg, 0.554 mmol) and triethylamine ( 224 mg, 2.21 mmol). The reaction solution was stirred at 25°C for 1 hour. The reaction solution was diluted with water (60mL), extracted with dichloromethane (60mL×4), the organic phases were combined, washed with water (200mL×1) and saturated brine (200mL×1), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the crude product was purified by high-performance liquid chromatography (neutral conditions) to obtain compound 15d.
MS-ESI计算值[M+H]+348，实测值348。MS-ESI calculated value [M+H] + 348, measured value 348.
第四步the fourth step
将化合物15d(40mg,0.115mmol)和1f(40.5mg,0.125mmol)溶于无水二氧六环(3mL)中，氮气下，向混合液中加入碳酸铯(102mg,0.313mmol)和甲烷磺酸(2-二环己基膦)-3,6-二甲氧基-2,4,6-三异丙基-1,1-联苯)(2-氨基-1,1-联苯-2-基)钯(II)(9.45mg,10.4μmol)，反应液在100℃下搅拌12小时。反应液浓缩，粗产物经过柱层析法分离(20:1,二氯甲烷/甲醇,Rf＝0.35)得到化合物15e。Compounds 15d (40 mg, 0.115 mmol) and 1f (40.5 mg, 0.125 mmol) were dissolved in anhydrous dioxane (3 mL). Under nitrogen, cesium carbonate (102 mg, 0.313 mmol) and methane sulfonate were added to the mixture. Acid (2-dicyclohexylphosphine)-3,6-dimethoxy-2,4,6-triisopropyl-1,1-biphenyl) (2-amino-1,1-biphenyl-2 -base) palladium (II) (9.45 mg, 10.4 μmol), and the reaction solution was stirred at 100°C for 12 hours. The reaction solution was concentrated, and the crude product was separated by column chromatography (20:1, dichloromethane/methanol, Rf=0.35) to obtain compound 15e.
MS-ESI计算值[M+H]+578，实测值578。MS-ESI calculated value [M+H] + 578, measured value 578.
第五步the fifth step
将化合物15e(68.0mg,0.113mmol)溶于无水甲醇(3mL)中，加入盐酸/甲醇溶液(4.23mL,4M,16.9mmol)，反应液在25℃下搅拌1小时。反应液浓缩，剩余物在甲醇(30mL)中搅拌1小时，过滤，甲醇(10mL×2)洗涤。滤饼在甲醇(30mL)中搅拌1小时，过滤，甲醇(10mL×2)洗涤，收集滤饼，减压干燥得到化合物15的盐酸盐。Compound 15e (68.0 mg, 0.113 mmol) was dissolved in anhydrous methanol (3 mL), hydrochloric acid/methanol solution (4.23 mL, 4 M, 16.9 mmol) was added, and the reaction solution was stirred at 25°C for 1 hour. The reaction solution was concentrated, and the residue was stirred in methanol (30 mL) for 1 hour, filtered, and washed with methanol (10 mL × 2). The filter cake was stirred in methanol (30 mL) for 1 hour, filtered, and washed with methanol (10 mL × 2). The filter cake was collected and dried under reduced pressure to obtain the hydrochloride of compound 15.
MS-ESI计算值[M+H]+478，实测值478。1H NMR(400MHz,DMSO-d6)δ10.22(s,1H),8.99(brs,2H),8.90(s,1H),8.66(s,1H),8.10(s,1H),7.72(s,1H),4.09-3.96(m,2H),3.23-3.12(m,2H),3.07-2.92(m,3H),2.81-2.71(m,1H),2.53-2.51(m,6H),2.17-2.05(m,1H),1.82-1.58(m,7H),1.54-1.45(m,2H),1.34-1.21(m,1H)。MS-ESI calculated value [M+H] + 478, measured value 478. 1 H NMR (400MHz, DMSO-d 6 ) δ10.22(s,1H),8.99(brs,2H),8.90(s,1H),8.66(s,1H),8.10(s,1H),7.72( s,1H),4.09-3.96(m,2H),3.23-3.12(m,2H),3.07-2.92(m,3H),2.81-2.71(m,1H),2.53-2.51(m,6H), 2.17-2.05(m,1H),1.82-1.58(m,7H),1.54-1.45(m,2H),1.34-1.21(m,1H).
实施例16
Example 16
合成路线：synthetic route:
化合物15的盐酸盐→化合物16的盐酸盐Hydrochloride salt of compound 15 → hydrochloride salt of compound 16
将化合物15的盐酸盐(30mg,0.049mmol)溶于无水甲醇(3mL)中，向反应液中加入甲醛水溶液(6.39mg,78.7μmol,纯度：37％)和氰基硼氢化钠(6.60mg,0.105mmol)。反应液在10℃下搅拌1小时。反应液加入水(20mL)淬灭，减压浓缩除去甲醇，过滤，甲醇(3mL×2)洗涤。粗品经高效液相色谱法(盐酸条件)纯化得到化合物16的盐酸盐。The hydrochloride of compound 15 (30 mg, 0.049 mmol) was dissolved in anhydrous methanol (3 mL), and aqueous formaldehyde solution (6.39 mg, 78.7 μmol, purity: 37%) and sodium cyanoborohydride (6.60 mg,0.105mmol). The reaction solution was stirred at 10°C for 1 hour. The reaction solution was quenched by adding water (20 mL), concentrated under reduced pressure to remove methanol, filtered, and washed with methanol (3 mL × 2). The crude product was purified by high performance liquid chromatography (hydrochloric acid conditions) to obtain the hydrochloride salt of compound 16.
MS-ESI计算值[M+H]+492，实测值492。1H NMR(400MHz,DMSO-d6)δ10.65-10.43(m,1H),10.22(s,1H),8.91(s,1H),8.66(s,1H),8.10(s,1H),7.76-7.70(m,1H),4.14-3.99(m,2H),3.79-3.42(m,2H),3.16-2.92(m,4H),2.86-2.81(m,3H),2.53-2.51(m,6H),2.30-1.84(m,2H),1.81-1.58(m,6H),1.53-1.42(m,2H),1.34-1.22(m,1H)。MS-ESI calculated value [M+H] + 492, measured value 492. 1 H NMR (400MHz, DMSO-d 6 ) δ10.65-10.43(m,1H),10.22(s,1H),8.91(s,1H),8.66(s,1H),8.10(s,1H), 7.76-7.70(m,1H),4.14-3.99(m,2H),3.79-3.42(m,2H),3.16-2.92(m,4H),2.86-2.81(m,3H),2.53-2.51(m ,6H),2.30-1.84(m,2H),1.81-1.58(m,6H),1.53-1.42(m,2H),1.34-1.22(m,1H).
生物实施例1：Biological Example 1:
化合物12对应的对甲苯磺酸盐对HCT116细胞中p-eIF4E水平的抑制活性的研究Study on the inhibitory activity of p-toluenesulfonate corresponding to compound 12 on p-eIF4E levels in HCT116 cells
细胞培养：消化分离T75培养瓶中的细胞，用培养基重悬，计数，将细胞浓度用培养基调节至2×105个细胞/mL，向96孔板中加入100μL细胞悬液，室温静置10min后，置于37℃，5％CO2培养箱中培养过夜。然后将细胞板放回到培养箱中培养3小时。3小时后，去掉培养基，每孔加入50μL 1X lysis buffer，室温旋转裂解10min。取10μL裂解液加入到384孔assay板中。每孔加入5μL acceptor mix，用封板膜将板子密封，常温孵育1小时。每孔加入5μL donor mix，用封板膜将板子密封，常温孵育1小时。在EnVision上用标准AlphaLISA程序读取信号。受试物的配置：第二天将化合物进行3倍连续稀释，共10个点，取10μL加入到培养板中，化合物终浓度为0.051nM至1000nM。Cell culture: Digest and isolate the cells in the T75 culture flask, resuspend in culture medium, count, adjust the cell concentration with culture medium to 2×10 5 cells/mL, add 100 μL of cell suspension to the 96-well plate, and incubate at room temperature. After leaving for 10 minutes, place it in a 37°C, 5% CO 2 incubator for overnight culture. Then place the cell plate back into the incubator for 3 hours. After 3 hours, remove the culture medium, add 50 μL 1X lysis buffer to each well, and rotate for 10 min at room temperature. Add 10 μL of lysis solution to the 384-well assay plate. Add 5 μL acceptor mix to each well, seal the plate with sealing film, and incubate at room temperature for 1 hour. Add 5 μL donor mix to each well, seal the plate with sealing film, and incubate at room temperature for 1 hour. Signals were read using the standard AlphaLISA program on EnVision. Preparation of the test substance: On the next day, the compound was serially diluted 3 times, with a total of 10 points, and 10 μL was added to the culture plate. The final concentration of the compound was 0.051 nM to 1000 nM.
数据分析:data analysis:
根据所测得ble slope：Y＝Bottom+(Top-Bottom)/(1+10^((LogIC50-X)*HillSlope))According to the measured ble slope: Y=Bottom+(Top-Bottom)/(1+10^((LogIC50-X)*HillSlope))
实验结果：以Con-MNK为参照，结果显示对照化合物Con-MNK对p-eIF4E水平抑制的IC50值为11.11nM。化合物12对应的对甲苯磺酸盐对p-eIF4E的水平有明显的抑制，IC50为18.82nM。根据以上结果分析，化合物12对应的对甲苯磺酸盐能显著抑制HCT116细胞中p-eIF4E的水平，以化合物Con-MNK为参照，本品抑制活性与参照化合物相当，同样具有肿瘤抑制活性。(图1-图2)
Experimental results: Using Con-MNK as a reference, the results showed that the IC50 value of the control compound Con-MNK for inhibiting p-eIF4E levels was 11.11nM. The p-toluenesulfonate corresponding to compound 12 significantly inhibited the level of p-eIF4E, with an IC50 of 18.82nM. According to the above result analysis, the p-toluenesulfonate corresponding to Compound 12 can significantly inhibit the level of p-eIF4E in HCT116 cells. Taking Compound Con-MNK as a reference, the inhibitory activity of this product is equivalent to that of the reference compound and also has tumor inhibitory activity. (Figure 1-Figure 2)
生物实施例2：Biological Example 2:
化合物12对应的对甲苯磺酸盐对人结肠癌HT-29细胞皮下异种移植肿瘤BALB/c Nude小鼠模型的体内药效学研究In vivo pharmacodynamics study of p-toluenesulfonate corresponding to compound 12 on human colon cancer HT-29 cell subcutaneous xenograft tumor BALB/c Nude mouse model
细胞培养：人结肠癌HT-29细胞(ATCC，货号：HTB-38)体外贴壁培养，培养条件为McCoy's 5a培养基中加10％胎牛血清，100U/mL青霉素和100μg/mL链霉素，37℃ 5％CO2孵箱培养。一周两次用胰酶-EDTA进行常规消化处理传代。当细胞饱和度为80％-90％，数量到达要求时，收取细胞，计数，接种。Cell culture: Human colon cancer HT-29 cells (ATCC, product number: HTB-38) are cultured in vitro. The culture conditions are McCoy's 5a medium plus 10% fetal bovine serum, 100U/mL penicillin and 100μg/mL streptomycin. , cultured in 37℃ 5% CO2 incubator. Passage was performed twice a week with routine digestion treatment with trypsin-EDTA. When the cell saturation is 80%-90% and the number reaches the required number, cells are collected, counted, and inoculated.
细胞接种：将0.2mL(5×106个)HT-29细胞皮下接种于每只小鼠的右后背，肿瘤平均体积达到154mm3时，根据肿瘤体积进行随机分组。Cell inoculation: 0.2 mL (5 × 10 6 ) HT-29 cells were subcutaneously inoculated into the right back of each mouse. When the average tumor volume reached 154 mm 3 , random groups were divided according to tumor volume.
蛋白抽提及定量Protein extraction and quantification
1)-80℃冰箱中取出速冻组织样品。1) Take out the quick-frozen tissue samples from the -80°C refrigerator.
2)干冰上操作，剪取部分组织(约50-100mg)，放入2mL加有钢珠的离心管中，加500μL细胞裂解液RIPA(已新鲜加入1％的蛋白酶抑制剂和磷酸酶抑制剂)。2) Operate on dry ice, cut out part of the tissue (about 50-100mg), put it into a 2mL centrifuge tube with steel beads, and add 500μL cell lysis solution RIPA (1% protease inhibitor and phosphatase inhibitor have been freshly added) .
3)Tissuelyser LT使用最高频率破碎组织5分钟。3) Tissuelyser LT uses the highest frequency to disrupt tissue for 5 minutes.
4)组织裂解液置于冰上裂解30分钟。4) Place the tissue lysis solution on ice for 30 minutes.
5)12,000rpm at 4℃离心10分钟，取上清放入新的1.5mL离心管中。5) Centrifuge at 12,000rpm at 4℃ for 10 minutes, take the supernatant and put it into a new 1.5mL centrifuge tube.
6)用BCA定量试剂盒进行蛋白定量。6) Use BCA quantification kit for protein quantification.
7)根据定量结果，配置上样用的蛋白样品，统一样品蛋白浓度至1μg/μL，并加入LDS加样缓冲液(4X)和样品还原剂(10X)，100℃恒温加热样品，10分钟。7) According to the quantitative results, configure the protein sample for loading, unify the sample protein concentration to 1 μg/μL, add LDS loading buffer (4X) and sample reducing agent (10X), and heat the sample at a constant temperature of 100°C for 10 minutes.
8)蛋白印迹，或将已变性的样品-80℃冰箱保存。8) Western blot, or store the denatured samples in a -80°C refrigerator.
免疫印迹Western blot
1)上样样品解冻。1) Thaw the loaded sample.
2)上样：SDS-PAGE胶中，每孔上样10μL(上样量取决于各自需要)。2) Loading: In SDS-PAGE gel, load 10 μL of sample into each well (the loading amount depends on individual needs).
3)电泳：80伏，30分钟，后120伏，90分钟。3) Electrophoresis: 80 volts, 30 minutes, then 120 volts, 90 minutes.
4)转膜：利用iBlot2转膜套装及转膜仪转膜：蛋白分子量小于100KDa，P3程序运7分钟，蛋白分子量大于100KDa，P3程序运15分钟。4) Transfer: Use iBlot2 transfer kit and transfer apparatus to transfer: for proteins with a molecular weight less than 100KDa, run the P3 program for 7 minutes; for proteins with a molecular weight greater than 100KDa, run the P3 program for 15 minutes.
5)转膜结束后，按所需检测蛋白的分子量大小裁剪膜，1xTBST洗膜，3次，5分钟每次，室温，摇动。5) After the transfer, cut the membrane according to the molecular weight of the required detection protein, wash the membrane with 1xTBST, 3 times, 5 minutes each time, at room temperature, and shake.
6)封闭：膜置于封闭液(用1xTBST配置的5％的脱脂牛奶)中封闭，室温，摇动，1小时。6) Blocking: Place the membrane in blocking solution (5% skim milk prepared with 1xTBST) for 1 hour at room temperature with shaking.
7)1xTBST洗膜，3次，5分钟每次，室温，摇动。7) Wash the membrane with 1xTBST, 3 times, 5 minutes each time, at room temperature, and shake.
8)孵育一抗：加入合适稀释度的一抗(用1xTBST配置的5％的脱脂牛奶或牛血清白蛋白稀释)，4℃过夜，缓慢摇动。8) Incubate the primary antibody: Add the appropriate dilution of the primary antibody (diluted with 5% skim milk or bovine serum albumin configured with 1xTBST), overnight at 4°C, and shake slowly.
9)1xTBST洗膜，3次，10分钟每次，室温，摇动。9) Wash the membrane with 1xTBST, 3 times, 10 minutes each time, at room temperature, and shake.
10)孵育二抗：加入合适稀释度的二抗，室温，缓慢摇动，1小时。10) Incubate secondary antibody: Add secondary antibody of appropriate dilution, shake slowly at room temperature, for 1 hour.
11)1xTBST洗膜，3次，10分钟每次，室温，摇动。11) Wash the membrane with 1xTBST, 3 times, 10 minutes each time, at room temperature, and shake.
12)化学发光：膜上加入West Femto超敏感化学发光试剂盒中的HRP底物。12) Chemiluminescence: Add the HRP substrate in the West Femto Ultra-Sensitive Chemiluminescence Kit to the membrane.
13)ImageQuant LAS 4000机器上检测化学发光并拍照。13) Detect chemiluminescence and take pictures on ImageQuant LAS 4000 machine.
表达定量Expression quantification
运用AlphaviewSA软件对免疫印迹化学发光谱带的密度强度进行相对定量。AlphaviewSA software was used to perform relative quantification of the density intensity of the immunoblot chemiluminescent bands.
统计分析Statistical Analysis
统计分析基于试验结束时的数据运用SPSS软件进行分析。三组或多组间比较用one-way ANOVA进行 分析，各组别间方差有显著性差异(p<0.05)，应用Games-Howell法进行分析。p<0.05认为有显著性差异。Statistical analysis was performed using SPSS software based on the data at the end of the experiment. Comparisons between three or more groups were performed using one-way ANOVA After analysis, there was a significant difference in variance between each group (p<0.05), and the Games-Howell method was used for analysis. p<0.05 is considered to be a significant difference.
实验结果：在分组给药后第28天时，溶媒对照组的瘤体积均值为1670mm3。化合物12对应的对甲苯磺酸盐的10mg/kg，30mg/kg，100mg/kg治疗剂量组的平均肿瘤体积分别为860mm3、719mm3和652mm3，T/C分别为51.72％、43.56％和38.89％，TGI分别为53.46％、62.69％和67.16％。与溶媒对照组相比，化合物12对应的对甲苯磺酸盐呈现出剂量依赖性的抗肿瘤效果，在三个浓度下均有明显的抑制肿瘤生长的作用，且具有统计学差异(p<0.05)。化合物12对应的对甲苯磺酸盐与5-fu联用组均对人结肠癌HT-29细胞异种移植模型表现出显著的肿瘤生长抑制作用，且对动物体重没有明显影响。(图3-图5)Experimental results: On the 28th day after group administration, the average tumor volume in the vehicle control group was 1670mm 3 . The average tumor volumes of the 10mg/kg, 30mg/kg, and 100mg/kg treatment dose groups of compound 12 corresponding to p-toluenesulfonate were 860mm 3 , 719mm 3 and 652mm 3 respectively, and the T/C were 51.72%, 43.56% and 38.89%, TGI are 53.46%, 62.69% and 67.16% respectively. Compared with the vehicle control group, the p-toluenesulfonate corresponding to compound 12 showed a dose-dependent anti-tumor effect, and had a significant inhibitory effect on tumor growth at three concentrations, with statistical differences (p<0.05 ). The combination of p-toluenesulfonate corresponding to compound 12 and 5-fu showed significant tumor growth inhibition in the human colon cancer HT-29 cell xenograft model, and had no significant effect on the animal body weight. (Figure 3-Figure 5)
化合物各剂量组在不同时间点均可显著抑制p-eIF4E蛋白表达。各剂量组在给药2h和8h后对VEGFR2和p-VEGFR2蛋白表达均有一定抑制作用。(图6-图13)Each dose group of the compound could significantly inhibit p-eIF4E protein expression at different time points. Each dose group had a certain inhibitory effect on VEGFR2 and p-VEGFR2 protein expression 2h and 8h after administration. (Figure 6-Figure 13)
生物实施例3：Biological Example 3:
化合物12对应的对甲苯磺酸盐与5-fu联用在人结肠癌SW480细胞BALB/c裸小鼠皮下异种移植瘤模型的体内药效学研究In vivo pharmacodynamic study of the combination of p-toluenesulfonate corresponding to compound 12 and 5-fu in human colon cancer SW480 cell BALB/c nude mouse subcutaneous xenograft tumor model
化合物配制Compound formulation
5-fu用DPBS配制成3mg/mL。具体配置方法为：称取30mg5-fu加入10mL DPBS，涡旋振荡至澄清，标记为G2、G4-2、G5-2、G6-2，现用现配。5-fu was formulated with DPBS to 3 mg/mL. The specific preparation method is: weigh 30mg5-fu and add 10mL DPBS, vortex until clear, mark G2, G4-2, G5-2, G6-2, ready for use.
本发明对应的化合物用配制成10mg/mL。具体配置方法为：称取299.88mg化合物加入2.8mL NMP，涡旋振荡至澄清，之后加入2.8mL Solutol涡旋振荡，再加入22.4mL水，涡旋振荡均一即可，标记为G3、G6-1，每周配制一次。The corresponding compound of the present invention was formulated at 10 mg/mL. The specific preparation method is: weigh 299.88mg compound and add 2.8mL NMP, vortex until clear, then add 2.8mL Solutol and vortex, then add 22.4mL water, vortex until uniform, marked as G3, G6-1 , prepared once a week.
本发明对应的化合物用配制成1mg/mL。具体配置方法为：称取14.99mg化合物加入1.4mL NMP，涡旋振荡至澄清，之后加入1.4mLSolutol涡旋振荡，再加入11.2mL水，涡旋振荡均一即可，标记为G4-1，每周配制一次。The corresponding compound of the present invention was formulated at 1 mg/mL. The specific preparation method is: weigh 14.99mg compound and add 1.4mL NMP, vortex until clear, then add 1.4mL Solutol and vortex, then add 11.2mL water, vortex until uniform, mark it as G4-1, every week Prepare once.
本发明对应的化合物用配制成3mg/mL。具体配置方法为：称取14.99×3mg化合物加入1.4mL NMP，涡旋振荡至澄清，之后加入1.4mL Solutol涡旋振荡，再加入11.2mL水，涡旋振荡均一即可，标记为G5-1，每周配制一次。The corresponding compound of the present invention was formulated at 3 mg/mL. The specific preparation method is: weigh 14.99×3mg compound and add 1.4mL NMP, vortex until clear, then add 1.4mL Solutol and vortex, then add 11.2mL water, vortex until uniform, mark it as G5-1. Prepare once a week.
肿瘤体积测量Tumor volume measurement
每周测量2次肿瘤体积。并计算肿瘤抑制率TGI(％)。肿瘤体积(TV)的计算公式为：TV＝1/2×a×b2，其中，a、b分别表示肿瘤块的长径和短径。Tumor volume was measured twice weekly. And calculate the tumor inhibition rate TGI (%). The calculation formula of tumor volume (TV) is: TV=1/2×a×b2, where a and b respectively represent the long diameter and short diameter of the tumor mass.
肿瘤抑制率TGI(％)＝[(1-处理组给药结束时平均瘤体积-处理组分组给药时平均瘤体积)/(溶剂对照组治疗结束时平均瘤体积-溶剂对照组分组给药时平均瘤体积)]×100％。Tumor inhibition rate TGI (%) = [(1-average tumor volume at the end of treatment group administration-average tumor volume at the end of treatment group administration)/(average tumor volume at the end of treatment group for solvent control group-solvent control group group administration) average tumor volume)]×100%.
相对肿瘤体积计算公式为RTV＝TVn/TV1×100％The relative tumor volume calculation formula is RTV=TV n /TV 1 ×100%
其中，TV1为分组给药当天的肿瘤体积，而TVn为测量当天的肿瘤体积。Among them, TV 1 is the tumor volume on the day of group administration, and TV n is the tumor volume on the day of measurement.
相对肿瘤增殖率(T/C％)＝RTVt/RTVc×100％Relative tumor proliferation rate (T/C%) = RTV t /RTV c × 100%
其中RTVt为治疗组平均相对肿瘤体积，RTVc为溶媒对照组平均相对肿瘤体积。Among them, RTV t is the average relative tumor volume of the treatment group, and RTV c is the average relative tumor volume of the vehicle control group.
数据处理及统计分析Data processing and statistical analysis
所有数据均以平均值±SEM的形式表示。本实验采用分组后D32的数据使PRISM软件进行统计分析，两组间比较用T test进行分析，三组或多组间比较用one-way ANOVA进行分析。若方差齐，则采用turkey分析，若方差不齐，则采用Games-Howell法进行分析。All data are expressed as mean ± SEM. This experiment uses the grouped D32 data to perform statistical analysis with PRISM software. T test is used for comparison between two groups, and one-way ANOVA is used for comparison between three or more groups. If the variances are equal, turkey analysis is used; if the variances are uneven, the Games-Howell method is used.
实验结果：以5-fu单药为参照，给药32天后，溶媒对照组的平均肿瘤体积达984.61mm3，本化合物在10、30和100mg/kg剂量下和5-fu联用组的平均肿瘤体积分别为639.95mm3、539.01mm3和396.11mm3，TGI分别为39.15％、50.57％和66.92％，T/C分别为65.00％、54.74％和40.23％，p值分别为0.3692、0.0606和0.0140。上述结果表明化合物12对应的对甲苯磺酸盐和5-fu联用能显著的抑制肿瘤的生长，并有剂量依赖的趋势。参考化合物5-fu在30mg/kg剂量下的平均肿瘤体积为792.90mm3，T/C和TGI分别为80.53％和21.74％(p＝0.9195)，抑制肿瘤的生长效果不明显，但与剂量为10mg/kg的化合物12对应的对甲苯磺酸盐联用，抑瘤能力增强(TGI：21.74％与39.15％)。(图14)Experimental results: Taking 5-fu as a single drug as a reference, after 32 days of administration, the average tumor volume in the vehicle control group reached 984.61mm 3 , and the average tumor volume of this compound in combination with 5-fu at doses of 10, 30 and 100 mg/kg The tumor volumes were 639.95mm 3 , 539.01mm 3 and 396.11mm 3 respectively, the TGI were 39.15%, 50.57% and 66.92% respectively, the T/C were 65.00%, 54.74% and 40.23% respectively, and the p values were 0.3692, 0.0606 and 0.0606 respectively. 0.0140. The above results show that the combination of p-toluenesulfonate corresponding to compound 12 and 5-fu can significantly inhibit tumor growth in a dose-dependent manner. The average tumor volume of the reference compound 5-fu at the dose of 30 mg/kg was 792.90 mm 3 , and the T/C and TGI were 80.53% and 21.74% respectively (p=0.9195). The effect of inhibiting tumor growth was not obvious, but it was related to the dose of 5-fu. The combined use of 10 mg/kg of the corresponding p-toluenesulfonate of compound 12 enhanced the tumor inhibitory ability (TGI: 21.74% and 39.15%). (Figure 14)
生物实施例4：Biological Example 4:
化合物12对应的对甲苯磺酸盐在小鼠结肠癌CT-26细胞皮下移植肿瘤BALB/c小鼠模型的体内药效学研究Study on the in vivo pharmacodynamics of p-toluenesulfonate corresponding to compound 12 in BALB/c mouse model of subcutaneous transplantation of mouse colon cancer CT-26 cells
细胞培养：小鼠结肠癌CT-26细胞体外培养，培养条件为McCoy’s 5a培养基加10％胎牛血清，100U/mL 青霉素和100μg/mL链霉素，37℃，5％CO2培养箱培养。一周两次用胰酶-EDTA进行常规消化处理传代。当细胞饱和度为80％～90％，数量到达要求时，收取细胞，计数，接种。Cell culture: Mouse colon cancer CT-26 cells were cultured in vitro. The culture conditions were McCoy's 5a medium plus 10% fetal bovine serum, 100U/mL. Penicillin and 100 μg/mL streptomycin were cultured in a 37°C, 5% CO2 incubator. Passage was performed twice a week with routine digestion treatment with trypsin-EDTA. When the cell saturation is 80% to 90% and the number reaches the required number, cells are collected, counted, and inoculated.
细胞接种：将0.1mL(3×105个)CT-26细胞皮下接种于每只小鼠的右后肢，在接种后第9天肿瘤体积达到约70mm3时开始分组给药。Cell inoculation: 0.1 mL (3 × 10 5 ) CT-26 cells were subcutaneously inoculated into the right hind limb of each mouse. Group administration began when the tumor volume reached approximately 70 mm 3 on the 9th day after inoculation.
肿瘤体积测量：Tumor volume measurement:
每周三次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。Tumor diameter was measured three times a week using vernier calipers. The calculation formula of tumor volume is: V=0.5a×b2, where a and b represent the long and short diameters of the tumor respectively.
化合物的抑瘤疗效用TGI(％)或肿瘤增殖率T/C(％)评价。The tumor inhibitory effect of the compound was evaluated by TGI (%) or tumor proliferation rate T/C (%).
TGI(％)，反映肿瘤生长抑制率。TGI(％)＝[(1-(某处理组给药结束时平均瘤体积－该处理组开始给药时平均瘤体积))/(溶剂对照组治疗结束时平均瘤体积－溶剂对照组开始治疗时平均瘤体积)]×100％。TGI (%) reflects the tumor growth inhibition rate. TGI (%) = [(1-(average tumor volume at the end of treatment in a certain treatment group - average tumor volume at the beginning of treatment in the treatment group))/(average tumor volume at the end of treatment in the solvent control group - start of treatment in the solvent control group) average tumor volume)]×100%.
相对肿瘤增殖率T/C(％)：计算公式如下：T/C％＝TRTV/CRTV×100％(TRTV：治疗组平均RTV；CRTV：阴性对照组平均RTV)。根据肿瘤测量的结果计算出单只小鼠相对肿瘤体积(relative tumor volume，RTV)，计算公式为RTV＝Vt/V0，其中V0是分组单只小鼠给药时(即d0)测量所得肿瘤体积，Vt为单只小鼠某一次测量时的肿瘤体积，TRTV与CRTV取同一天数据，平均RTV值为同组小鼠的RTV值的平均值。Relative tumor proliferation rate T/C (%): The calculation formula is as follows: T/C% = TRTV/CRTV × 100% (TRTV: average RTV of the treatment group; CRTV: average RTV of the negative control group). The relative tumor volume (RTV) of a single mouse is calculated based on the results of tumor measurement. The calculation formula is RTV=Vt/V0, where V0 is the tumor volume measured when a single mouse is grouped into groups (i.e. d0). , Vt is the tumor volume of a single mouse at a certain time of measurement, TRTV and CRTV are taken on the same day, and the average RTV value is the average of the RTV values of mice in the same group.
在实验结束后将检测肿瘤重量，并计算T/Cweight百分比，Tweight和Cweight分别表示给药组和溶媒对照组的瘤重。After the experiment, the tumor weight will be measured and the T/Cweight percentage will be calculated. Tweight and Cweight represent the tumor weight of the drug administration group and the vehicle control group, respectively.
统计分析：Statistical Analysis:
统计分析基于试验结束时的数据运用SPSS软件进行分析。三组或多组间比较用T-test和one-way ANOVA进行分析。各组别间方差有显著性差异(p<0.05)，应用Games-Howell法进行分析。p<0.05认为有显著性差异。Statistical analysis was performed using SPSS software based on the data at the end of the experiment. Comparisons between three or more groups were analyzed using T-test and one-way ANOVA. There was a significant difference in variance between each group (p<0.05), and the Games-Howell method was used for analysis. p<0.05 is considered to be a significant difference.
实验结果：化合物12对应的对甲苯磺酸盐单药治疗组在10-40mg/kg剂量下有一定的抗肿瘤效果，肿瘤体积为1057mm3，T/C为57.76％，TGI为45.77％，p＝0.051；在90mg/kg剂量下有显著的抗肿瘤效果，肿瘤体积为922mm3，T/C为47.26％，TGI为53.20％，且与vehicle治疗组相比有显著性差异，p＝0.026。本发明对应的化合物(10-400mg/kg)与Anti-mPD-1(5mg/kg)联用组的肿瘤体积为738mm3，T/C＝36.87％，TGI＝63.31％，具有显著的抑瘤作用p＝0.009，且与PD-1单药治疗组相比有更好的抗肿瘤效果。(图15)Experimental results: The p-toluenesulfonate monotherapy group corresponding to Compound 12 has a certain anti-tumor effect at a dose of 10-40mg/kg. The tumor volume is 1057mm 3 , T/C is 57.76%, TGI is 45.77%, p =0.051; There is a significant anti-tumor effect at the dose of 90mg/kg, the tumor volume is 922mm 3 , T/C is 47.26%, TGI is 53.20%, and there is a significant difference compared with the vehicle treatment group, p=0.026. The tumor volume of the combination group of the compound corresponding to the present invention (10-400 mg/kg) and Anti-mPD-1 (5 mg/kg) is 738mm 3 , T/C=36.87%, TGI=63.31%, which has significant tumor inhibition. The effect p=0.009, and it has better anti-tumor effect compared with the PD-1 monotherapy group. (Figure 15)
生物实施例5：Biological Example 5:
化合物12对应的对甲苯磺酸盐对MNK1/MNK2的活性抑制作用Inhibitory effect of p-toluenesulfonate corresponding to compound 12 on the activity of MNK1/MNK2
反应条件：Reaction conditions:
缓冲液：20mM Hepes(pH 7.5),10mM MgCl2,1mM EGTA,0.02％Brij35,0.02mg/ml BSA,0.1mM Na3VO4,2mM DTT,1％DMSOBuffer: 20mM Hepes (pH 7.5), 10mM MgCl 2 , 1mM EGTA, 0.02% Brij35, 0.02mg/ml BSA, 0.1mM Na 3 VO 4 , 2mM DTT, 1% DMSO
*所需的辅助因子分别添加到每个激酶反应中。*Required cofactors are added individually to each kinase reaction.
反应操作步骤：Reaction steps:
1.在新鲜制备的反应缓冲液中准备相应的底物。1. Prepare the corresponding substrate in freshly prepared reaction buffer.
2.在上述底物溶液中加入需要的辅助因子。2. Add the required cofactors to the above substrate solution.
3.将相应的激酶加入到底物溶液中并轻轻混匀。3. Add the corresponding kinase to the substrate solution and mix gently.
4.利用声学技术仪器(Echo 550)将化合物的DMSO溶液加到激酶反应混合液中。4. Add the DMSO solution of the compound to the kinase reaction mixture using an acoustic technology instrument (Echo 550).
5.向反应混合液中加入33P-ATP(终比活性为0.01μCi/μ.)以引发反应。5. Add 33P-ATP (final specific activity: 0.01 μCi/μ.) to the reaction mixture to initiate the reaction.
6.在室温下孵化激酶反应120分钟。6. Incubate the kinase reaction for 120 minutes at room temperature.
7.将反应物点在P81离子交换纸上(Whatman#3698-915)。7. Spot the reactants on P81 ion exchange paper (Whatman #3698-915).
8.用0.75％磷酸充分清洗过滤器。8. Clean the filter thoroughly with 0.75% phosphoric acid.
9.测量留在滤纸上的放射性磷酸化底物。9. Measure the radioactive phosphorylated substrate remaining on the filter paper.
数据分析：data analysis:
激酶活性数据以测试样品中剩余激酶活性占溶媒对照组(二甲基亚砜)的百分比的形式表现。Kinase activity data are expressed as the percentage of remaining kinase activity in the test sample relative to the vehicle control (dimethyl sulfoxide).
使用Prism4软件(GraphPad)拟合曲线，计算IC50值。Prism4 software (GraphPad) was used to fit the curve and calculate the IC50 value.
实验结果：通过与对照药物星形孢菌素测试对比，化合物12对应的对甲苯磺酸盐对MNK1/2激酶的IC50值显著低于对照物。(图16和图17)Experimental results: Compared with the control drug staurosporine test, the IC50 value of p-toluenesulfonate corresponding to compound 12 for MNK1/2 kinase was significantly lower than the control substance. (Figure 16 and Figure 17)
生物实施例6：Biological Example 6:
化合物12对应的对甲苯磺酸盐对BGC-823人源胃癌CDX小鼠皮下移植瘤模型的药效学研究Study on the pharmacodynamics of p-toluenesulfonate corresponding to compound 12 on BGC-823 human gastric cancer CDX mouse subcutaneous transplant tumor model
肿瘤细胞接种 tumor cell inoculation
BGC-823细胞培养在含10％胎牛血清(FBS)和1％PS的RPMI-1640培养液中。收集对数生长期的BGC-823细胞，HBSS重悬至5×106/mL，无菌条件下，移植于BALB/c Nude小鼠皮下，每只小鼠接种5×105/0.1mL个细胞。BGC-823 cells were cultured in RPMI-1640 culture medium containing 10% fetal bovine serum (FBS) and 1% PS. BGC-823 cells in the logarithmic growth phase were collected, resuspended in HBSS to 5×10 6 /mL, and transplanted subcutaneously into BALB/c Nude mice under sterile conditions. Each mouse was inoculated with 5×10 5 /0.1mL. cell.
动物分组Animal grouping
在BGC-823模型药效实验中，在接种第10天当分组用小鼠肿瘤平均体积达到157.27mm3时，根据瘤体积大小随机分为3组，每组6只。In the BGC-823 model efficacy experiment, on the 10th day after vaccination, when the average tumor volume of the grouped mice reached 157.27 mm 3 , they were randomly divided into 3 groups according to tumor volume, with 6 mice in each group.
肿瘤体积测量：使用游标卡尺每周两次测量，肿瘤体积计算公式为V＝0.5a×b2，a，b分别代表肿瘤的长径和宽径；Tumor volume measurement: Use vernier calipers to measure twice a week. The tumor volume calculation formula is V=0.5a×b2, where a and b represent the long and wide diameters of the tumor respectively;
肿瘤生长抑制率TGI％＝[1-T/C]×100。当delta T>0时，T/C％＝(delta T/delta C)×100即T/C％＝(Ti-T0)/(Ci-C0)×100；当delta T<0时，T/C％＝(delta T/T0)×100。(delta T＝Ti-T0，Ti是给药组调查当天平均肿瘤体积，T0是给药组分组平均肿瘤体积，Ci是对照组调查当天平均肿瘤体积，C0是对照组分组平均肿瘤体积)。Tumor growth inhibition rate TGI%=[1-T/C]×100. When delta T>0, T/C%=(delta T/delta C)×100, that is, T/C%=(T i -T 0 )/(C i -C 0 )×100; when delta T<0 When, T/C%=(delta T/T 0 )×100. (delta T=T i -T 0 , T i is the average tumor volume of the administration group on the day of investigation, T 0 is the average tumor volume of the administration group, C i is the average tumor volume of the control group on the day of investigation, C 0 is the control group group mean tumor volume).
相对肿瘤增殖率T/C(％)：计算公式如下：T/C％＝TRTV/CRTV×100％(TRTV：治疗组RTV；CRTV：阴性对照组RTV)。根据肿瘤测量的结果计算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为RTV＝Vt/V0，其中V0是分组给药时(即d0)测量所得平均肿瘤体积，Vt为某一次测量时的平均肿瘤体积，TRTV与CRTV取同一天数据。Relative tumor proliferation rate T/C (%): The calculation formula is as follows: T/C% = TRTV/CRTV × 100% (TRTV: RTV of the treatment group; CRTV: RTV of the negative control group). The relative tumor volume (RTV) is calculated based on the results of tumor measurement. The calculation formula is RTV=V t /V 0 , where V 0 is the average tumor volume measured during group administration (i.e. d 0 ), V t It is the average tumor volume at a certain measurement time, and TRTV and CRTV take the data on the same day.
肿瘤瘤重可通过相对肿瘤增殖率T/C(％)计算，计算公式如下：T/C％＝Tweight/Cweight×100％(Tweight：治疗组平均肿瘤重量；Cweight：对照组平均肿瘤重量)。Tumor weight can be calculated by the relative tumor proliferation rate T/C (%). The calculation formula is as follows: T/C% = Tweight/Cweight × 100% (Tweight: average tumor weight of the treatment group; Cweight: average tumor weight of the control group).
统计分析Statistical Analysis
所有数据均采用Mean±SEM表示，用one-way ANOVA LSD(L)比较各组肿瘤体积和瘤重有无显著性差异。所有的数据均用Graphpad进行分析。*P<0.05即认为具有显著性差异。All data are expressed as Mean ± SEM, and one-way ANOVA LSD (L) was used to compare whether there were significant differences in tumor volume and tumor weight among each group. All data were analyzed using Graphpad. *P<0.05 is considered to have a significant difference.
实验结果：Vehicle组、化合物12对应的对甲苯磺酸盐(50mg/kg)组、化合物12对应的对甲苯磺酸盐(150mg/kg)组在BGC-823人源胃癌细胞小鼠皮下移植瘤上，荷瘤鼠的平均肿瘤体积分别为3059.77±444.29mm3、1904.8±342.27mm3、1603.17±148.52mm3，与Vehicle组相比较，化合物12对应的对甲苯磺酸盐(50mg/kg)给药组和化合物12对应的对甲苯磺酸盐(150mg/kg)给药组的肿瘤生长抑制率TGI(％)分别为39.79％和50.14％。化合物12对应的对甲苯磺酸盐(50mg/kg)和化合物12对应的对甲苯磺酸盐(150mg/kg)均具有统计学意义的抑制肿瘤生长的作用(P<0.05)。同时，化合物12对应的对甲苯磺酸盐的抗肿瘤药效与给药剂量呈正相关。(图18)Experimental results: Vehicle group, p-toluenesulfonate (50mg/kg) group corresponding to compound 12, and p-toluenesulfonate (150mg/kg) group corresponding to compound 12 subcutaneously transplanted tumors in BGC-823 human gastric cancer cell mice Above, the average tumor volumes of tumor-bearing mice were 3059.77±444.29mm 3 , 1904.8±342.27mm 3 , and 1603.17±148.52mm 3 respectively. Compared with the Vehicle group, the p-toluenesulfonate (50mg/kg) corresponding to Compound 12 was The tumor growth inhibition rates TGI (%) of the drug group and the p-toluenesulfonate (150 mg/kg) administration group corresponding to compound 12 were 39.79% and 50.14%, respectively. Both the p-toluenesulfonate corresponding to compound 12 (50 mg/kg) and the p-toluenesulfonate corresponding to compound 12 (150 mg/kg) had statistically significant inhibitory effects on tumor growth (P<0.05). At the same time, the anti-tumor efficacy of p-toluenesulfonate corresponding to compound 12 is positively correlated with the dosage. (Figure 18)
生物实施例7：Biological Example 7:
化合物12对应的对甲苯磺酸盐对Calu-3人源肺癌CDX小鼠皮下移植瘤模型的药效学研究Study on the pharmacodynamics of p-toluenesulfonate corresponding to compound 12 on Calu-3 human lung cancer CDX mouse subcutaneous transplant tumor model
肿瘤细胞接种tumor cell inoculation
Calu-3细胞培养在含含10％胎牛血清(FBS)和1％PS的MEM培养液中。收集对数生长期的Calu-3细胞，HBSS重悬至1×108/mL，无菌条件下，移植于BALB/c Nude小鼠皮下，每只小鼠接种1×107/0.2mL(包含25％Matrigel)个细胞。Calu-3 cells were cultured in MEM medium containing 10% fetal bovine serum (FBS) and 1% PS. Calu-3 cells in the logarithmic growth phase were collected, resuspended in HBSS to 1×10 8 /mL, and transplanted subcutaneously into BALB/c Nude mice under sterile conditions. Each mouse was inoculated with 1×10 7 /0.2mL ( Contains 25% Matrigel) cells.
动物分组Animal grouping
在Calu-3模型药效实验中，在接种第6天当分组用小鼠肿瘤平均体积达到144.93mm3时，根据瘤体积大小随机分为3组，每组6只。In the Calu-3 model efficacy experiment, on the 6th day after inoculation, when the average tumor volume of the grouped mice reached 144.93 mm 3 , they were randomly divided into 3 groups according to tumor volume, with 6 mice in each group.
肿瘤体积测量：使用游标卡尺每周两次测量，肿瘤体积计算公式为V＝0.5a×b2，a，b分别代表肿瘤的长径和宽径；Tumor volume measurement: Use vernier calipers to measure twice a week. The tumor volume calculation formula is V=0.5a×b2, where a and b represent the long and wide diameters of the tumor respectively;
肿瘤生长抑制率TGI％＝[1-T/C]×100。当delta T>0时，T/C％＝(delta T/delta C)×100即T/C％＝(Ti-T0)/(Ci-C0)×100；当delta T<0时，T/C％＝(delta T/T0)×100。(delta T＝Ti-T0，Ti是给药组调查当天平均肿瘤体积，T0是给药组分组平均肿瘤体积，Ci是对照组调查当天平均肿瘤体积，C0是对照组分组平均肿瘤体积)。Tumor growth inhibition rate TGI%=[1-T/C]×100. When delta T>0, T/C%=(delta T/delta C)×100, that is, T/C%=(T i -T 0 )/(C i -C 0 )×100; when delta T<0 When, T/C%=(delta T/T 0 )×100. (delta T=T i -T 0 , T i is the average tumor volume of the administration group on the day of investigation, T 0 is the average tumor volume of the administration group, C i is the average tumor volume of the control group on the day of investigation, C 0 is the control group group mean tumor volume).
相对肿瘤增殖率T/C(％)：计算公式如下：T/C％＝TRTV/CRTV×100％(TRTV：治疗组RTV；CRTV：阴性对照组RTV)。根据肿瘤测量的结果计算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为RTV＝Vt/V0，其中V0是分组给药时(即d0)测量所得平均肿瘤体积，Vt为某一次测量时的平均肿瘤体积，TRTV与CRTV取同一天数据。Relative tumor proliferation rate T/C (%): The calculation formula is as follows: T/C% = TRTV/CRTV × 100% (TRTV: RTV of the treatment group; CRTV: RTV of the negative control group). The relative tumor volume (RTV) is calculated based on the results of tumor measurement. The calculation formula is RTV=V t /V 0 , where V 0 is the average tumor volume measured during group administration (i.e. d 0 ), V t It is the average tumor volume at a certain measurement, and TRTV and CRTV take the data on the same day.
实验结束后，取肿瘤块称重，并拍照片。After the experiment, the tumor mass was taken and weighed, and photos were taken.
统计分析Statistical Analysis
所有数据均采用Mean±SEM表示，用one-way ANOVA LSD(L)比较各组肿瘤体积和瘤重有无显著性差异。所有的数据均用Graphpad进行分析。*P<0.05即认为具有显著性差异。 All data are expressed as Mean ± SEM, and one-way ANOVA LSD (L) was used to compare whether there were significant differences in tumor volume and tumor weight among each group. All data were analyzed using Graphpad. *P<0.05 is considered to have a significant difference.
实验结果：Vehicle组、化合物12对应的对甲苯磺酸盐(50mg/kg)组、化合物12对应的对甲苯磺酸盐(150mg/kg)组荷瘤鼠的平均肿瘤体积分别为1312.24±300.35mm3、779.88±161.66mm3、502.79±86.75mm3，与Vehicle组相比较，化合物12对应的对甲苯磺酸盐(50mg/kg)组和化合物12对应的对甲苯磺酸盐(150mg/kg)组的肿瘤生长抑制率TGI(％)分别为45.74％和69.42％。根据以上结果分析可知化合物12对应的对甲苯磺酸盐(50mg/kg)组能够一定程度地抑制肿瘤生长(P>0.05)，化合物12对应的对甲苯磺酸盐(150mg/kg)组表现出具有统计学意义的抑制肿瘤生长的作用(P<0.05)。同时，本发明对应的化合物的抗肿瘤药效与给药剂量呈正相关。经过治疗，在Calu-3人源肺癌细胞小鼠皮下移植瘤上，化合物12对应的对甲苯磺酸盐(50mg/kg)组能够一定程度地抑制Calu-3人源肺癌细胞小鼠皮下移植瘤的生长(P>0.05)，化合物12对应的对甲苯磺酸盐(150mg/kg)组则表现出具有统计学意义的抑制肿瘤生长的作用(P<0.05)。同时，化合物12对应的对甲苯磺酸盐的抗肿瘤药效与给药剂量呈正相关。另外在整个给药过程中，各组小鼠均未见体重下降以及其他明显异常现象，表明荷瘤小鼠对受试药物均能够耐受。(图19)Experimental results: The average tumor volume of tumor-bearing mice in the Vehicle group, the p-toluenesulfonate (50mg/kg) group corresponding to compound 12, and the p-toluenesulfonate (150mg/kg) group corresponding to compound 12 was 1312.24±300.35mm respectively. 3 , 779.88±161.66mm 3 , 502.79±86.75mm 3 , compared with the Vehicle group, the p-toluenesulfonate (50mg/kg) group corresponding to compound 12 and the p-toluenesulfonate (150mg/kg) corresponding to compound 12 The tumor growth inhibition rates TGI (%) of the two groups were 45.74% and 69.42% respectively. According to the above result analysis, it can be seen that the p-toluenesulfonate (50mg/kg) group corresponding to compound 12 can inhibit tumor growth to a certain extent (P>0.05). The p-toluenesulfonate (150mg/kg) group corresponding to compound 12 showed Statistically significant inhibitory effect on tumor growth (P<0.05). At the same time, the anti-tumor efficacy of the corresponding compound of the present invention is positively correlated with the dosage. After treatment, the p-toluenesulfonate (50 mg/kg) group corresponding to Compound 12 was able to inhibit the subcutaneous transplantation of Calu-3 human lung cancer cells in mice to a certain extent. growth (P>0.05), and the p-toluenesulfonate (150mg/kg) group corresponding to compound 12 showed statistically significant inhibitory effects on tumor growth (P<0.05). At the same time, the anti-tumor efficacy of p-toluenesulfonate corresponding to compound 12 is positively correlated with the dosage. In addition, during the entire administration process, no weight loss or other obvious abnormalities were seen in mice in each group, indicating that tumor-bearing mice were able to tolerate the tested drugs. (Figure 19)
生物实施例8:Biological Example 8:
化合物12对应的对甲苯磺酸盐对VEGFR1/VEGFR2/VEGFR3的活性抑制作用The inhibitory effect of p-toluenesulfonate corresponding to compound 12 on the activity of VEGFR1/VEGFR2/VEGFR3
化合物对每个选中激酶的检测均使用Eurofins标准的Kinase ProfilerTM实验流程，该流程遵循相关的标准操作规程。蛋白激酶(除了ATM(h)和DNA-PK(h))测试以辐射量进行检测；而脂质激酶，ATM(h)、ATR/ATRIP(h)和DNA-PK(h)的测试以进行检测。每个激酶的测试细节都列在Eurofins网站上或者随同的方案文件中。所有提供的化合物都使用100％二甲基亚砜溶液配制成50×实验终浓度。在适当的时候，将浓度更高的存储液用100％二甲基亚砜稀释成50×工作液。若提供的是化合物粉末，先通过100％二甲基亚砜溶液配成10mM的母液，再稀释成50×液体。Compounds were tested for each selected kinase using Eurofins' standard Kinase ProfilerTM experimental procedure, which followed relevant standard operating procedures. Tests for protein kinases (except ATM(h) and DNA-PK(h)) are based on the amount of radiation; tests for lipid kinases, ATM(h), ATR/ATRIP(h) and DNA-PK(h) are based on Perform testing. Test details for each kinase are listed on the Eurofins website or in the accompanying protocol document. All compounds provided were formulated in 100% dimethyl sulfoxide solution to 50× final experimental concentration. When appropriate, dilute the more concentrated stock solution with 100% dimethyl sulfoxide to create a 50× working solution. If the compound powder is provided, first prepare a 10mM stock solution with 100% dimethyl sulfoxide solution, and then dilute it into a 50× liquid.
取适量50×测试化合物的储存液到测试孔中，然后加入激酶和底物，混匀。加入一定浓度的ATP开始反应。在ATP加入前激酶和底物与化合物不需要预孵育。更详细的每个激酶的实验过程请参考网站或者随同的方案文件。Take an appropriate amount of 50× test compound stock solution into the test well, then add the kinase and substrate and mix well. Add a certain concentration of ATP to start the reaction. No preincubation of kinases, substrates, and compounds is required before adding ATP. For more detailed experimental procedures for each kinase, please refer to the website or accompanying protocol document.
统计分析Statistical Analysis
数据的处理使用定制的内部分析软件。结果表示为剩余酶活占DMSO对照组酶活的百分比。这个可以通过下面的公式计算得到：Data were processed using custom in-house analysis software. The results were expressed as the percentage of remaining enzyme activity to the enzyme activity of the DMSO control group. This can be calculated by the following formula:
(样品组的平均值-空白组的平均值)/对照组的平均值(average of the sample group - average of the blank group)/average of the control group
对于IC50的测定，数据使用XLFit 5.3版(ID Business Solutions)进行分析，S型的剂量效应曲线(可变斜率)基于每个测试浓度均值用非线性回归分析拟合得到。当顶端和/或者底部的数值超过10％或低于-10％，在满足R2的QC标准情况下，曲线的最大极限和最小极限可能被重新定义为100和0。For the determination of IC50, the data was analyzed using XLFit version 5.3 (ID Business Solutions), and the S-shaped dose-response curve (variable slope) was fitted by nonlinear regression analysis based on the mean value of each test concentration. When the top and/or bottom values exceed 10% or fall below -10%, the maximum and minimum limits of the curve may be redefined to 100 and 0, subject to meeting the QC criteria of R2.
实验结果：通过测试化合物12对应的对甲苯磺酸盐对激酶的抑制活性，本发明对应的化合物对VEGFR1激酶的IC50为18nM，对VEGFR2激酶的IC50为3nM，对VEGFR3激酶的IC50为2nM。(表1)Experimental results: By testing the inhibitory activity of p-toluenesulfonate corresponding to Compound 12 on kinases, the IC50 of the corresponding compound of the present invention against VEGFR1 kinase is 18nM, the IC50 against VEGFR2 kinase is 3nM, and the IC50 against VEGFR3 kinase is 2nM. (Table 1)
表1
Table 1
生物实施例9:Biological Example 9:
化合物12对应的对甲苯磺酸盐对HUVEC细胞的抑制活性的研究Study on the inhibitory activity of p-toluenesulfonate corresponding to compound 12 on HUVEC cells
细胞培养：HUVEC细胞株。在含有10％的胎牛血清、青霉素100u/ml及链霉素100mg/ml的RPMI-1640培养基中37℃、5％CO2条件下传代培养。取对数生长期、生长良好、细胞活性大于90％的细胞进行实验。Cell culture: HUVEC cell line. Subculture in RPMI-1640 medium containing 10% fetal bovine serum, 100 u/ml penicillin and 100 mg/ml streptomycin at 37°C and 5% CO2 . Cells in the logarithmic growth phase, growing well, and with cell activity greater than 90% were selected for experiments.
细胞生长活性检测：取对数生长期的HUVEC细胞进行实验，将不同浓度的化合物12对应的对甲苯磺酸盐加入10％胎牛血清的RPMI-1640培养基中，调细胞浓度为5×105ml，每种剂量浓度为一组，置37℃、5％CO2培养箱不同时间(48，72h)后，培养4h，充分溶解。选择490nm波长，酶联免疫检测仪上测定各孔吸光度(A)值。Cell growth activity detection: HUVEC cells in logarithmic growth phase were used for experiments. Different concentrations of p-toluenesulfonate corresponding to Compound 12 were added to RPMI-1640 culture medium with 10% fetal calf serum, and the cell concentration was adjusted to 5×10 5 ml, each dose concentration is a group, placed in a 37°C, 5% CO 2 incubator for different times (48, 72h), incubated for 4h, and fully dissolved. Select the wavelength of 490nm and measure the absorbance (A) value of each well on an enzyme-linked immunoassay detector.
统计分析：应用SPSS统计软件进行t检验、单因素方差分析(ANOVA)。Statistical analysis: SPSS statistical software was used to conduct t test and one-way analysis of variance (ANOVA).
实验结果：化合物12对应的对甲苯磺酸盐的对HUVEC细胞的抑制活性的研究结果显示本发明对应的化合物对HUVEC细胞有明显的抑制，48小时IC50为15.47μM，72小时IC50为10.63μM。(图20)Experimental results: The research results of the inhibitory activity of p-toluenesulfonate corresponding to Compound 12 on HUVEC cells showed that the corresponding compound of the present invention significantly inhibited HUVEC cells, with an IC50 of 15.47 μM at 48 hours and an IC50 of 10.63 μM at 72 hours. (Figure 20)
生物实施例10: Biological Example 10:
化合物12对应的对甲苯磺酸盐联合顺铂对小鼠皮下移植瘤A549模型的药效学研究Pharmacodynamic study of compound 12 corresponding to p-toluenesulfonate combined with cisplatin on mouse subcutaneous transplanted tumor A549 model
肿瘤细胞接种tumor cell inoculation
A549细胞培养在常规细胞培养液中。收集对数生长期的H460细胞，经重悬后，在无菌条件下移植于小鼠皮下，每只小鼠接种2×107mL个细胞。A549 cells were cultured in conventional cell culture medium. H460 cells in the logarithmic growth phase were collected, resuspended, and transplanted subcutaneously into mice under sterile conditions. Each mouse was inoculated with 2 × 10 7 mL cells.
动物分组Animal grouping
在A549模型药效实验中，小鼠根据瘤体积大小随机分为4组，每组5只。In the A549 model drug efficacy experiment, mice were randomly divided into 4 groups according to tumor size, with 5 mice in each group.
肿瘤体积测量：使用游标卡尺测量，肿瘤体积计算公式为V＝0.5a×b2，a，b分别代表肿瘤的长径和宽径；Tumor volume measurement: Use a vernier caliper to measure. The tumor volume calculation formula is V=0.5a×b2, where a and b represent the long and wide diameters of the tumor respectively;
肿瘤生长抑制率TGI％＝[1-T/C]×100。当delta T>0时，T/C％＝(delta T/delta C)×100即T/C％＝(Ti-T0)/(Ci-C0)×100；当delta T<0时，T/C％＝(delta T/T0)×100。(delta T＝Ti-T0，Ti是给药组调查当天平均肿瘤体积，T0是给药组分组平均肿瘤体积，Ci是对照组调查当天平均肿瘤体积，C0是对照组分组平均肿瘤体积)。Tumor growth inhibition rate TGI%=[1-T/C]×100. When delta T>0, T/C%=(delta T/delta C)×100, that is, T/C%=(T i -T 0 )/(C i -C 0 )×100; when delta T<0 When, T/C%=(delta T/T 0 )×100. (delta T=T i -T 0 , T i is the average tumor volume of the administration group on the day of investigation, T 0 is the average tumor volume of the administration group, C i is the average tumor volume of the control group on the day of investigation, C 0 is the control group group mean tumor volume).
相对肿瘤增殖率T/C(％)：计算公式如下：T/C％＝TRTV/CRTV×100％(TRTV：治疗组RTV；CRTV：阴性对照组RTV)。根据肿瘤测量的结果计算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为RTV＝Vt/V0，其中V0是分组给药时(即d0)测量所得平均肿瘤体积，Vt为某一次测量时的平均肿瘤体积，TRTV与CRTV取同一天数据。Relative tumor proliferation rate T/C (%): The calculation formula is as follows: T/C% = TRTV/CRTV × 100% (TRTV: RTV of the treatment group; CRTV: RTV of the negative control group). The relative tumor volume (RTV) is calculated based on the results of tumor measurement. The calculation formula is RTV=V t /V 0 , where V 0 is the average tumor volume measured during group administration (i.e. d 0 ), V t It is the average tumor volume at a certain measurement, and TRTV and CRTV take the data on the same day.
实验结束后，取肿瘤块称重，并拍照片。After the experiment, the tumor mass was taken and weighed, and photos were taken.
统计分析Statistical Analysis
所有数据均采用Mean±SEM表示，用one-way ANOVA LSD(L)比较各组肿瘤体积有无显著性差异。所有的数据均用Graphpad进行分析。P<0.05即认为具有显著性差异。All data are expressed as Mean ± SEM, and one-way ANOVA LSD (L) was used to compare whether there were significant differences in tumor volume among each group. All data were analyzed using Graphpad. P<0.05 was considered to be a significant difference.
实验结果：经过治疗，Vehicle组、顺铂(2mg/kg)组、化合物12对应的对甲苯磺酸盐(50mg/kg)组、顺铂+化合物12对应的对甲苯磺酸盐(2mg/kg+50mg/kg)组荷瘤鼠的平均肿瘤体积分别为747.076mm3、381.930mm3、291.653mm3、62.948mm3，顺铂(2mg/kg)组、化合物12对应的对甲苯磺酸盐(50mg/kg)组、顺铂+化合物12对应的对甲苯磺酸盐(2mg/kg+50mg/kg)组TGI分别为49.67％、72.31％、94.07％。根据以上结果分析可知，化合物12对应的对甲苯磺酸盐(50mg/kg)组、顺铂+化合物12对应的对甲苯磺酸盐(2mg/kg+50mg/kg)组均表现出具有统计学意义的抑制肿瘤生长的作用(P<0.01)，且顺铂+化合物12对应的对甲苯磺酸盐(2mg/kg+50mg/kg)联用组相比顺铂单剂量组以及化合物12对应的对甲苯磺酸盐单剂量组有更显著的抑瘤作用。(图21)Experimental results: After treatment, Vehicle group, cisplatin (2mg/kg) group, compound 12 corresponding p-toluenesulfonate (50mg/kg) group, cisplatin + compound 12 corresponding p-toluenesulfonate (2mg/kg) group The average tumor volumes of tumor-bearing mice in the +50mg/kg) group were 747.076mm 3 , 381.930mm 3 , 291.653mm 3 , and 62.948mm 3 respectively. In the cisplatin (2mg/kg) group, the corresponding p-toluenesulfonate ( The TGI of the cisplatin + compound 12 (2 mg/kg + 50 mg/kg) group and the p-toluenesulfonate (2 mg/kg + 50 mg/kg) group were 49.67%, 72.31%, and 94.07%, respectively. According to the analysis of the above results, it can be seen that the p-toluenesulfonate (50mg/kg) group corresponding to compound 12 and the p-toluenesulfonate (2mg/kg+50mg/kg) group corresponding to cisplatin + compound 12 showed statistically significant Significant inhibitory effect on tumor growth (P<0.01), and the combination group of cisplatin + p-toluenesulfonate (2mg/kg+50mg/kg) corresponding to compound 12 was compared with the cisplatin single-dose group and the corresponding group of compound 12. The single-dose p-toluenesulfonate group had a more significant anti-tumor effect. (Figure 21)
生物实施例11:Biological Example 11:
化合物12对应的对甲苯磺酸盐联合紫杉醇对小鼠皮下移植瘤H460模型的药效学研究Pharmacodynamic study of compound 12 corresponding to p-toluenesulfonate combined with paclitaxel on mouse subcutaneous transplanted tumor H460 model
肿瘤细胞接种tumor cell inoculation
H460细胞培养在常规细胞培养液中。收集对数生长期的H460细胞，经重悬后，在无菌条件下移植于小鼠皮下，每只小鼠接种1×107mL个细胞。H460 cells were cultured in conventional cell culture medium. H460 cells in the logarithmic growth phase were collected, resuspended, and transplanted subcutaneously into mice under sterile conditions. Each mouse was inoculated with 1×10 7 mL cells.
动物分组Animal grouping
在H460模型药效实验中，小鼠根据瘤体积大小随机分为4组，每组6只。In the H460 model drug efficacy experiment, mice were randomly divided into 4 groups according to tumor size, with 6 mice in each group.
肿瘤体积测量：使用游标卡尺测量，肿瘤体积计算公式为V＝0.5a×b2，a，b分别代表肿瘤的长径和宽径；Tumor volume measurement: Use a vernier caliper to measure. The tumor volume calculation formula is V=0.5a×b2, where a and b represent the long and wide diameters of the tumor respectively;
肿瘤生长抑制率TGI％＝[1-T/C]×100。当delta T>0时，T/C％＝(delta T/delta C)×100即T/C％＝(Ti-T0)/(Ci-C0)×100；当delta T<0时，T/C％＝(delta T/T0)×100。(delta T＝Ti-T0，Ti是给药组调查当天平均肿瘤体积，T0是给药组分组平均肿瘤体积，Ci是对照组调查当天平均肿瘤体积，C0是对照组分组平均肿瘤体积)。Tumor growth inhibition rate TGI%=[1-T/C]×100. When delta T>0, T/C%=(delta T/delta C)×100, that is, T/C%=(T i -T 0 )/(C i -C 0 )×100; when delta T<0 When, T/C%=(delta T/T 0 )×100. (delta T=T i -T 0 , T i is the average tumor volume of the administration group on the day of investigation, T 0 is the average tumor volume of the administration group, C i is the average tumor volume of the control group on the day of investigation, C 0 is the control group group mean tumor volume).
相对肿瘤增殖率T/C(％)：计算公式如下：T/C％＝TRTV/CRTV×100％(TRTV：治疗组RTV；CRTV：阴性对照组RTV)。根据肿瘤测量的结果计算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为RTV＝Vt/V0，其中V0是分组给药时(即d0)测量所得平均肿瘤体积，Vt为某一次测量时的平均肿瘤体积，TRTV与CRTV取同一天数据。Relative tumor proliferation rate T/C (%): The calculation formula is as follows: T/C% = TRTV/CRTV × 100% (TRTV: RTV of the treatment group; CRTV: RTV of the negative control group). The relative tumor volume (RTV) is calculated based on the results of tumor measurement. The calculation formula is RTV=V t /V 0 , where V 0 is the average tumor volume measured during group administration (i.e. d 0 ), V t It is the average tumor volume at a certain measurement, and TRTV and CRTV take the data on the same day.
实验结束后，取肿瘤块称重，并拍照片。After the experiment, the tumor mass was taken and weighed, and photos were taken.
统计分析Statistical Analysis
所有数据均采用Mean±SEM表示，用one-way ANOVA LSD(L)比较各组肿瘤体积有无显著性差异。所有的数据均用Graphpad进行分析。P<0.05即认为具有显著性差异。All data are expressed as Mean ± SEM, and one-way ANOVA LSD (L) was used to compare whether there were significant differences in tumor volume among each group. All data were analyzed using Graphpad. P<0.05 is considered to have a significant difference.
实验结果：经过治疗，Vehicle组、紫杉醇(12mg/kg)组、化合物12对应的对甲苯磺酸盐(50mg/kg)组、紫杉醇+化合物12对应的对甲苯磺酸盐(12mg/kg+50mg/kg)组荷瘤鼠的平均肿瘤体积分别为838.60mm3、591.21mm3、294.29mm3、75.29mm3，紫杉醇(12mg/kg)组、化合物12对应的对甲苯磺酸盐(50mg/kg)组、 紫杉醇+化合物12对应的对甲苯磺酸盐(12mg/kg+50mg/kg)组TGI分别为30.17％、66.24％、93.22％。根据以上结果分析可知，化合物12对应的对甲苯磺酸盐(50mg/kg)组、紫杉醇+化合物12对应的对甲苯磺酸盐(12mg/kg+50mg/kg)组均表现出具有统计学意义的抑制肿瘤生长的作用(P<0.001)。且紫杉醇+化合物12对应的对甲苯磺酸盐(12mg/kg+50mg/kg)联用组相比紫杉醇单剂量组以及化合物12对应的对甲苯磺酸盐单剂量组有更显著的抑瘤作用。(图22)Experimental results: After treatment, Vehicle group, paclitaxel (12mg/kg) group, compound 12 corresponding p-toluenesulfonate (50mg/kg) group, paclitaxel + compound 12 corresponding p-toluenesulfonate (12mg/kg+50mg) group /kg) group, the average tumor volumes of tumor-bearing mice were 838.60mm 3 , 591.21mm 3 , 294.29mm 3 , 75.29mm 3 respectively. The paclitaxel (12mg/kg) group and the p-toluenesulfonate corresponding to compound 12 (50mg/kg )Group, The TGIs of the p-toluenesulfonate (12 mg/kg + 50 mg/kg) group corresponding to paclitaxel + compound 12 were 30.17%, 66.24%, and 93.22%, respectively. According to the analysis of the above results, it can be seen that the p-toluenesulfonate (50mg/kg) group corresponding to compound 12 and the p-toluenesulfonate (12mg/kg+50mg/kg) group corresponding to paclitaxel + compound 12 showed statistically significant The effect of inhibiting tumor growth (P<0.001). And the combination group of paclitaxel + p-toluenesulfonate (12 mg/kg + 50 mg/kg) corresponding to compound 12 has a more significant anti-tumor effect than the single-dose group of paclitaxel and the single-dose group of p-toluenesulfonate corresponding to compound 12. . (Figure 22)
生物实施例12:卵巢癌实验方案与结果Biological Example 12: Ovarian Cancer Experimental Protocol and Results
1、人源性卵巢癌ES2皮下瘤模型1. Human ovarian cancer ES2 subcutaneous tumor model
将小鼠随机分为3组，每组5-8只。皮下瘤接种后第7天开始进行药物干预，分组及给药情况如下：The mice were randomly divided into 3 groups, with 5-8 mice in each group. Drug intervention was started on the 7th day after subcutaneous tumor inoculation. The grouping and administration conditions were as follows:
1)空白对照组：每日灌胃100μL生理盐水；1) Blank control group: 100 μL normal saline was administered daily;
2)溶剂对照组：每日灌胃100μL溶剂。2) Solvent control group: 100 μL of solvent was administered daily.
3)药物实验组：剂量为50mg/kg，每日根据体重灌胃约100μL的化合物12对应的对甲苯磺酸盐溶液。3) Drug experimental group: The dose is 50 mg/kg, and about 100 μL of p-toluenesulfonate solution corresponding to Compound 12 is administered daily according to body weight.
每隔1天测量裸鼠移植瘤的长、短径及其体重，分别绘制肿瘤生长曲线和体重变化曲线(体积公式＝长径*短径*短径/2)。另外，给药期间需注意观察小鼠生长状况，比如皮肤颜色和温度，有无体重减轻等。给药约3周后处死小鼠。The length, short diameter and body weight of the transplanted tumors in nude mice were measured every 1 day, and the tumor growth curve and weight change curve were drawn respectively (volume formula = long diameter * short diameter * short diameter / 2). In addition, during the administration period, attention should be paid to the growth status of the mice, such as skin color and temperature, whether there is weight loss, etc. The mice were sacrificed approximately 3 weeks after administration.
实验结果：与空白对照组和溶剂组相比，给药组肿瘤体积明显减小。小鼠体重无明显波动，提示药物没有明显毒副作用。(图23和图24)Experimental results: Compared with the blank control group and solvent group, the tumor volume in the drug administration group was significantly reduced. There was no significant fluctuation in the weight of the mice, indicating that the drug had no obvious toxic or side effects. (Figure 23 and Figure 24)
2、人源性卵巢癌ES2皮下瘤模型2. Human ovarian cancer ES2 subcutaneous tumor model
将小鼠随机分为5组，每组5-8只。皮下瘤接种后第7天开始进行药物干预，分组及给药情况如下：The mice were randomly divided into 5 groups, with 5-8 mice in each group. Drug intervention was started on the 7th day after subcutaneous tumor inoculation. The grouping and administration conditions were as follows:
a)溶剂对照组：每日灌胃100μL溶剂；a) Solvent control group: 100 μL of solvent was administered daily;
b)顺铂(DDP)实验组：药物剂量为2-4mg/kg，每周根据体重腹腔注射约100μL的DDP溶液；b) Cisplatin (DDP) experimental group: The drug dose is 2-4 mg/kg, and about 100 μL of DDP solution is injected intraperitoneally every week according to body weight;
c)紫杉醇(PTX)实验组：12mg/kg，每周根据体重腹腔注射约100μL的PTX溶液；c) Paclitaxel (PTX) experimental group: 12 mg/kg, approximately 100 μL of PTX solution is injected intraperitoneally according to body weight every week;
d)化合物12对应的对甲苯磺酸盐+DDP实验组：剂量为50mg/kg，每日根据体重灌胃约100μL的化合物12对应的对甲苯磺酸盐溶液+药物剂量为2-4mg/kg，每周根据体重腹腔注射约100μL的DDP溶液；d) Experimental group of p-toluenesulfonate + DDP corresponding to compound 12: The dose is 50 mg/kg. Approximately 100 μL of p-toluenesulfonate solution corresponding to compound 12 + drug dose is 2-4 mg/kg is administered daily according to body weight. , intraperitoneally inject approximately 100 μL of DDP solution according to body weight every week;
e)化合物12对应的对甲苯磺酸盐+PTX实验组：剂量为50mg/kg，每日根据体重灌胃约100μL的化合物12对应的对甲苯磺酸盐溶液+药物剂量为2-4mg/kg，每周根据体重腹腔注射约100μL的PTX溶液。e) Experimental group of p-toluenesulfonate + PTX corresponding to compound 12: The dose is 50 mg/kg. Approximately 100 μL of p-toluenesulfonate solution corresponding to compound 12 + drug dose is 2-4 mg/kg is administered daily according to body weight. , about 100 μL of PTX solution was injected intraperitoneally every week based on body weight.
每隔1天测量裸鼠移植瘤长、短径及其体重，分别绘制肿瘤生长曲线和体重变化曲线。另外，给药期间需注意观察小鼠生长状况，比如皮肤颜色和温度，有无体重减轻等。给药约3周后处死小鼠。The length, short diameter and body weight of the transplanted tumors in nude mice were measured every 1 day, and the tumor growth curve and body weight change curve were drawn respectively. In addition, during the administration period, attention should be paid to the growth status of the mice, such as skin color and temperature, whether there is weight loss, etc. The mice were sacrificed approximately 3 weeks after administration.
裸鼠处死相关实验：Experiments related to the execution of nude mice:
(1)将裸鼠搬至解剖房，取其眼球血，分装于EP管内以备血生化检测；(1) Move the nude mouse to the anatomy room, take blood from its eyeballs, and distribute it into EP tubes for blood biochemical testing;
(2)将所有裸鼠颈椎脱臼法处死；(2) All nude mice were killed by cervical dislocation;
(3)用组织剪剪开裸鼠近瘤处皮肤，用组织镊游离皮与瘤，剥离皮下瘤结节，称重并拍照；(3) Use tissue scissors to cut the skin of nude mice near the tumor, use tissue tweezers to separate the skin from the tumor, peel off the subcutaneous tumor nodules, weigh and take pictures;
(4)取下每只裸鼠重要脏器(心、肝、脾、肺、肾)；(4) Remove the important organs (heart, liver, spleen, lungs, kidneys) of each nude mouse;
(5)将裸鼠的肿瘤组织分为3份，分别用于石蜡包埋，低温冻存和流式分析。(5) Divide the tumor tissue of nude mice into three parts and use them for paraffin embedding, cryopreservation and flow analysis respectively.
裸鼠的重要脏器用4％多聚甲醛固定，留作后续石蜡包埋，切片和HE染色。The important organs of nude mice were fixed with 4% paraformaldehyde and reserved for subsequent paraffin embedding, sectioning and HE staining.
实验结果：与溶剂对照组相比，顺铂组和紫杉醇组治疗效果不明显，这与临床上透明细胞癌对化疗不敏感相一致；而联用化疗的这两个组的肿瘤明显更小。(图25)Experimental results: Compared with the solvent control group, the therapeutic effect of the cisplatin group and the paclitaxel group was not obvious, which is consistent with the clinical insensitivity of clear cell carcinoma to chemotherapy; the tumors of the two groups combined with chemotherapy were significantly smaller. (Figure 25)
生物实施例13:淋巴瘤实验方案与结果Biological Example 13: Lymphoma Experimental Protocol and Results
使用MNK/VEGFR2小分子抑制剂化合物12对应的对甲苯磺酸盐来治疗人DLBCL细胞SU-DHL-6和MCL细胞JeKo-1的异种移植瘤模型。The p-toluenesulfonate corresponding to the MNK/VEGFR2 small molecule inhibitor compound 12 was used to treat the xenograft tumor model of human DLBCL cell SU-DHL-6 and MCL cell JeKo-1.
1、人DLBCL细胞SU-DHL-6的异种移植瘤模型(联合治疗)1. Xenograft tumor model of human DLBCL cell SU-DHL-6 (combination therapy)
实验方案：接瘤后随机分为5组，空白组、溶剂组、化合物12对应的对甲苯磺酸盐单药组、阿霉素单药组以及联合组。其中化合物12对应的对甲苯磺酸盐每日灌胃25mg/kg，阿霉素尾静脉给药3.3mg/kg，每10天一次，总共2次。Experimental plan: After tumor grafting, the patients were randomly divided into 5 groups: blank group, solvent group, p-toluenesulfonate single drug group corresponding to compound 12, doxorubicin single drug group and combination group. Among them, the p-toluenesulfonate corresponding to Compound 12 was intragastrically administered at 25 mg/kg daily, and doxorubicin was administered through the tail vein at 3.3 mg/kg, once every 10 days, a total of 2 times.
实验结果：如图26和图27所示，化合物12对应的对甲苯磺酸盐、阿霉素单药均有治疗效果，而治疗效果最好的是联合组。以上体内外研究表明，化合物12对应的对甲苯磺酸盐联合阿霉素能产生协同抗肿瘤效果。Experimental results: As shown in Figure 26 and Figure 27, the single drugs corresponding to compound 12, p-toluenesulfonate and doxorubicin, both have therapeutic effects, and the best therapeutic effect is in the combination group. The above in vivo and in vitro studies show that the p-toluenesulfonate corresponding to compound 12 combined with doxorubicin can produce synergistic anti-tumor effects.
2、MCL细胞JeKo-1的皮下瘤模型2. Subcutaneous tumor model of MCL cell JeKo-1
实验方案：于接种15天后开始每日灌胃治疗，每隔3天测量肿瘤大小和小鼠体重(如图28所示)。总共治疗15天后处死小鼠，小心剥取皮下瘤，拍照、称重并做统计分析。同时留取新鲜眼球血和重要脏器。Experimental protocol: Start daily intragastric treatment 15 days after inoculation, and measure tumor size and mouse weight every 3 days (as shown in Figure 28). After a total of 15 days of treatment, the mice were sacrificed, and the subcutaneous tumors were carefully peeled off, photographed, weighed, and analyzed statistically. At the same time, fresh eye blood and important organs were collected.
实验结果：在治疗终点时，治疗组的肿瘤平均体积(259.80±56.51mm3)明显小于溶剂对照组 (825.58±141.46mm3)，其TGI达到73.62％。空白对照组和溶剂对照组在肿瘤体积上无明显差异。Experimental results: At the end of treatment, the average tumor volume of the treatment group (259.80±56.51mm 3 ) was significantly smaller than that of the solvent control group (825.58±141.46mm3), its TGI reaches 73.62%. There was no significant difference in tumor volume between the blank control group and the solvent control group.
以上结果表明化合物12对应的对甲苯磺酸盐对于非霍奇金淋巴瘤(NHL)体内模型具有良好的治疗效果。The above results indicate that the p-toluenesulfonate corresponding to Compound 12 has good therapeutic effect on the in vivo model of non-Hodgkin's lymphoma (NHL).
生物实施例14:肺癌实验方案与结果Biological Example 14: Lung Cancer Experimental Plan and Results
实验方案：Experimental program:
小鼠LL2皮下瘤模型的建立：小鼠肺癌细胞LL2，皮下注射。将100μL含1X106个LL2的细胞悬液注射至雌性C57小鼠右侧背部。接种一周后(肿瘤体积约50-100mm3)，将小鼠随机分为4组，每组6只。Establishment of mouse LL2 subcutaneous tumor model: mouse lung cancer cells LL2 were injected subcutaneously. Inject 100 μL of cell suspension containing 1X10 6 LL2 into the right back of female C57 mice. One week after inoculation (tumor volume is about 50-100mm 3 ), the mice were randomly divided into 4 groups, with 6 mice in each group.
分组设计group design
1:溶剂对照+PD-1同型抗体(V+ISO)1: Solvent control + PD-1 isotype antibody (V + ISO)
2:PD-1抗体单用2: PD-1 antibody alone
3:化合物12对应的对甲苯磺酸盐+PD-1同型抗体(MNK+ISO)3: p-toluenesulfonate+PD-1 isotype antibody corresponding to compound 12 (MNK+ISO)
4:化合物12对应的对甲苯磺酸盐+PD-1抗体(MNK+PD-1)4: p-toluenesulfonate+PD-1 antibody corresponding to compound 12 (MNK+PD-1)
药物剂量：Drug dosage:
单抑制剂:化合物12对应的对甲苯磺酸盐每天口服给药，剂量为50mg/kg。Single inhibitor: The p-toluenesulfonate corresponding to compound 12 is administered orally daily at a dose of 50 mg/kg.
单PD-1抗体:PD-1抗体自接瘤后第5天开始给药，每周2次，腹腔注射，剂量为200μg/只。Single PD-1 antibody: PD-1 antibody was administered intraperitoneally twice a week starting from the 5th day after tumor grafting, with a dose of 200 μg/animal.
PD-1同型对照ISO：自接瘤后第5天开始给药，每周2次，腹腔注射，剂量为200μg/只。PD-1 isotype control ISO: Administration started on the 5th day after tumor grafting, twice a week, intraperitoneally, with a dose of 200 μg/animal.
联合治疗:治疗开始后，化合物12对应的对甲苯磺酸盐每天口服给药，PD-1抗体每周两次腹腔注射，200μg/只。Combination therapy: After the start of treatment, the p-toluenesulfonate corresponding to Compound 12 was administered orally every day, and the PD-1 antibody was injected intraperitoneally twice a week, 200 μg/animal.
实验结果：联合组肿瘤平均体积低于PD-1抗体单药。小鼠体重无明显波动，提示药物没有明显毒副作用。(图29和图30)Experimental results: The average tumor volume in the combination group was lower than that of PD-1 antibody alone. There was no significant fluctuation in the weight of the mice, indicating that the drug had no obvious toxic or side effects. (Figure 29 and Figure 30)
生物实施例15:在Hep G2裸鼠移植瘤模型体内药效评价Biological Example 15: In vivo drug efficacy evaluation in Hep G2 nude mouse transplanted tumor model
细胞培养：在5％CO2、37℃以及含10％胎牛血清MEM培养液中进行常规细胞培养；以0.25％胰酶消化传代；根据细胞生长情况，每周传代2到3次，传代比例为1:3到1:6。Cell culture: Perform routine cell culture in 5% CO 2 , 37°C, and MEM culture medium containing 10% fetal bovine serum; digest and passage with 0.25% trypsin; according to cell growth, passage 2 to 3 times a week, passage ratio is 1:3 to 1:6.
动物模型制备：收取对数生长期Hep G2细胞，细胞计数后重悬于含50％无血清MEM培养基和50％基质胶中，调整细胞浓度至2.5×107细胞/mL；用移液器吹打细胞使其分散均匀后装入50-mL离心管中，将离心管置于冰盒中；用1-mL注射器吸取细胞悬液，注射到裸鼠前右肢腋窝皮下，每只动物接种200L(5×106细胞/只)，建立Hep G2裸鼠移植瘤模型。接种后定期观察动物状态及肿瘤生长情况，使用电子游标卡尺测量瘤径，数据直接输入Excel电子表格，计算肿瘤体积。待肿瘤体积达到100～300mm3，挑选健康状况良好、肿瘤体积相近的动物18只，根据肿瘤体积采用随机区组法分为3组(n＝6)，同时尽量保证每组平均体重保持一致。以分组当天为实验第一天(D1)，实验开始后每周测量2次瘤径，计算肿瘤体积，同时称量动物体重并记录。Animal model preparation: Collect Hep G2 cells in the logarithmic growth phase, count the cells and resuspend them in medium containing 50% serum-free MEM and 50% Matrigel. Adjust the cell concentration to 2.5×10 7 cells/mL; use a pipette Pipette the cells to disperse them evenly and then put them into a 50-mL centrifuge tube. Place the centrifuge tube in an ice box; use a 1-mL syringe to absorb the cell suspension and inject it into the subcutaneous axilla of the front right limb of the nude mouse. Each animal is inoculated with 200L. (5×10 6 cells/mouse) to establish a Hep G2 nude mouse transplanted tumor model. After inoculation, the animal status and tumor growth were regularly observed, and the tumor diameter was measured using an electronic vernier caliper. The data was directly entered into an Excel spreadsheet to calculate the tumor volume. When the tumor volume reaches 100-300mm 3 , 18 animals in good health and with similar tumor volumes are selected and divided into 3 groups (n=6) according to the tumor volume using random block method. At the same time, try to ensure that the average weight of each group is consistent. The day of grouping was regarded as the first day of the experiment (D1). After the start of the experiment, the tumor diameter was measured twice a week, the tumor volume was calculated, and the animal body weight was weighed and recorded.
肿瘤体积(TV)计算公式如下：The calculation formula of tumor volume (TV) is as follows:
TV(mm3)＝l×w2/2TV(mm3)＝l×w2/2
其中，l表示肿瘤长径(mm)；w表示肿瘤短径(mm)。Among them, l represents the long diameter of the tumor (mm); w represents the short diameter of the tumor (mm).
动物分组及给药方案见表2，于分组当天开始给药，3周后结束实验(或溶剂对照组肿瘤体积达到2000mm3以上，以先到指标为准)，给药体积均为10mL·kg-1。实验最后一天，称量体重、测量瘤径后采用CO2吸入法将动物安乐死，剥取瘤块称重、拍照。The animal grouping and dosing schedule are shown in Table 2. Dosing will begin on the day of grouping and end the experiment after 3 weeks (or the tumor volume in the solvent control group reaches more than 2000 mm 3 , whichever comes first). The dosing volume is 10 mL·kg. -1 . On the last day of the experiment, the body weight and tumor diameter were measured, and the animals were euthanized by CO 2 inhalation. The tumor pieces were removed, weighed, and photographed.
实验结果：与溶剂组相比，化合物12对应的对甲苯磺酸盐给药组肿瘤体积明显减小。(图31)Experimental results: Compared with the solvent group, the tumor volume in the p-toluenesulfonate administration group corresponding to Compound 12 was significantly reduced. (Figure 31)
表2.Hep G2裸鼠移植瘤模型药效实验给药方案
Table 2. Dosage regimen for drug efficacy experiments in Hep G2 nude mouse transplanted tumor model
生物实施例16:在MDA-MB-231裸鼠移植瘤模型体内药效评价 Biological Example 16: In vivo efficacy evaluation in MDA-MB-231 nude mouse transplanted tumor model
细胞培养：在100％空气、37℃、饱和湿度培养条件下，MDA-MB-231细胞在含10％胎牛血清L-15培养液中进行常规细胞培养；根据细胞生长情况，以0.25％胰酶消化传代，每周传代1到2次，传代比例为1:3到1:10。Cell culture: Under the conditions of 100% air, 37°C, and saturated humidity, MDA-MB-231 cells were cultured in L-15 culture medium containing 10% fetal bovine serum; according to the cell growth conditions, 0.25% pancreatic Enzyme digestion and passage, 1 to 2 times a week, with a passage ratio of 1:3 to 1:10.
动物模型制备：收取对数生长期MDA-MB-231细胞，细胞计数后重悬于无血清的L-15培养基中，调整细胞浓度至3×107细胞/mL；用移液器吹打细胞使其分散均匀后装入50-mL离心管中，将离心管置于冰盒中；用1-mL注射器吸取细胞悬液，注射到裸鼠前右肢腋窝皮下，每只动物接种100L(3.0×106细胞/只)，建立MDA-MB-231裸鼠移植瘤模型。接种后定期观察动物状态及肿瘤生长情况，使用电子游标卡尺测量瘤径，数据直接输入Excel电子表格，计算肿瘤体积。待肿瘤体积达到100～300mm3，挑选健康状况良好、肿瘤体积相近的动物20只左右，采用随机区组法分为3组(n＝6)。以分组当天为实验第一天(D1)，实验开始后每周测量2次瘤径，计算肿瘤体积，同时称量动物体重并记录。Animal model preparation: Collect MDA-MB-231 cells in the logarithmic growth phase, count the cells and resuspend them in serum-free L-15 culture medium. Adjust the cell concentration to 3×10 7 cells/mL; use a pipette to pipette the cells. Disperse it evenly and put it into a 50-mL centrifuge tube. Place the centrifuge tube in an ice box; use a 1-mL syringe to absorb the cell suspension and inject it into the subcutaneous skin of the axilla of the front right limb of nude mice. Each animal is inoculated with 100L (3.0 ×10 6 cells/mouse) to establish MDA-MB-231 nude mouse transplanted tumor model. After inoculation, the animal status and tumor growth were regularly observed, and the tumor diameter was measured using an electronic vernier caliper. The data was directly entered into an Excel spreadsheet to calculate the tumor volume. When the tumor volume reaches 100-300mm 3 , about 20 animals in good health and with similar tumor volumes are selected and divided into 3 groups (n=6) using the random block method. The day of grouping was regarded as the first day of the experiment (D1). After the start of the experiment, the tumor diameter was measured twice a week, the tumor volume was calculated, and the animal body weight was weighed and recorded.
动物分组及给药方案见表3，于分组当天开始给药，3周后结束实验(或溶剂对照组肿瘤体积达到2000mm3以上，以先到指标为准)，给药体积均为10mL·kg-1。实验最后一天，称量体重、测量瘤径后采用CO2吸入法将动物安乐死，剥取瘤块称重、拍照。The animal grouping and dosing schedule are shown in Table 3. Dosing will begin on the day of grouping and end the experiment after 3 weeks (or the tumor volume in the solvent control group reaches more than 2000 mm 3 , whichever comes first). The dosing volume is 10 mL·kg. -1 . On the last day of the experiment, the body weight and tumor diameter were measured, and the animals were euthanized by CO 2 inhalation. The tumor pieces were removed, weighed, and photographed.
实验结果：与和溶剂组相比，化合物12对应的对甲苯磺酸盐给药组肿瘤体积明显减小。(图32)Experimental results: Compared with the solvent group, the tumor volume in the p-toluenesulfonate administration group corresponding to compound 12 was significantly reduced. (Figure 32)
表3.MDA-MB-231裸鼠移植瘤模型药效实验给药方案
Table 3. Dosage regimen for drug efficacy experiment in MDA-MB-231 nude mouse transplanted tumor model
以上对本发明技术方案的实施方式进行了示例性的说明。应当理解，本发明的保护范围不拘囿于上述实施方式。凡在本发明的精神和原则之内，本领域技术人员所做的任何修改、等同替换、改进等，均应包含在本申请权利要求书的保护范围之内。 The above is an exemplary description of the implementation of the technical solution of the present invention. It should be understood that the protection scope of the present invention is not limited to the above-mentioned embodiments. Any modifications, equivalent substitutions, improvements, etc. made by those skilled in the art within the spirit and principles of the present invention shall be included in the protection scope of the claims of this application.

Claims (10)

  1. 一种式(I)所示化合物或其药学上可接受的盐在制备抗肿瘤药物中的应用，
    The use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof in the preparation of anti-tumor drugs,
    其中，R1为H、F、Cl、Br或C1-3烷基；Wherein, R 1 is H, F, Cl, Br or C 1-3 alkyl;
    R2和R3各自独立地为H或C1-3烷基，其中所述C1-3烷基任选被1、2或3个独立选自F、Cl、Br或I的取代基所取代；R 2 and R 3 are each independently H or C 1-3 alkyl, wherein the C 1-3 alkyl is optionally replaced by 1, 2 or 3 substituents independently selected from F, Cl, Br or I replace;
    或R2和R3与它们相连的碳原子连接在一起形成环戊基、环己基或哌啶基，其中所述环戊基、环己基和哌啶基任选被1、2或3个Ra所取代；Or R 2 and R 3 are joined together with the carbon atom to which they are attached to form cyclopentyl, cyclohexyl or piperidinyl, wherein said cyclopentyl, cyclohexyl and piperidinyl are optionally replaced by 1, 2 or 3 R replaced by a ;
    各Ra独立地为H、F、Cl、Br或C1-3烷基；Each R a is independently H, F, Cl, Br or C 1-3 alkyl;
    R4为H、F、Cl、Br或C1-3烷基；R 4 is H, F, Cl, Br or C 1-3 alkyl;
    R5和R6各自独立地为H、F、Cl、Br、I或C1-3烷基；R 5 and R 6 are each independently H, F, Cl, Br, I or C 1-3 alkyl;
    R7为吡咯烷基，其中所述吡咯烷基任选被1、2或3个Rb所取代；R 7 is pyrrolidinyl, wherein the pyrrolidinyl is optionally substituted by 1, 2 or 3 R b ;
    各Rb独立地为H、F、Cl、Br、I或C1-3烷基，其中所述C1-3烷基任选被1、2或3个独立选自F、Cl、Br或I的取代基所取代；Each R b is independently H, F, Cl, Br, I or C 1-3 alkyl, wherein the C 1-3 alkyl is optionally 1, 2 or 3 independently selected from F, Cl, Br or Substituted by the substituent of I;
    n为1或2。n is 1 or 2.
  2. 根据权利要求1所述的应用，其特征在于，所述Ra独立地为H、F、Cl、Br、-CH3或-CH2CH3；The application according to claim 1, wherein the R a is independently H, F, Cl, Br, -CH 3 or -CH 2 CH 3 ;
    优选地，所述R2和R3各自独立地为H、-CH3或-CH2CH3；Preferably, R 2 and R 3 are each independently H, -CH 3 or -CH 2 CH 3 ;
    优选地，所述R2和R3与它们相连的碳原子连接在一起形成 Preferably, the R 2 and R 3 are connected together with the carbon atoms to which they are connected to form
    优选地，所述R2和R3与它们相连的碳原子连接在一起形成 Preferably, the R 2 and R 3 are connected together with the carbon atoms to which they are connected to form
    优选地，所述为 Preferably, the for
    优选地，所述为 Preferably, the for
    优选地，式(I)所述化合物具有式(I-1)～(I-4)任一结构式所示结构：

    Preferably, the compound described in formula (I) has a structure represented by any one of formulas (I-1) to (I-4):

    其中，R1、R4、R5、R6、R7、Ra和n如以上或权利要求1所定义；Wherein, R 1 , R 4 , R 5 , R 6 , R 7 , R a and n are as defined above or in claim 1;
    优选地，所述各Rb独立地H、F、Cl、Br、I、 Preferably, each R b is independently H, F, Cl, Br, I,
    优选地，所述R7为其中所述任选被1或2个Rb所取代；Preferably, the R 7 is stated therein optionally replaced by 1 or 2 R b ;
    优选地，所述R7为 Preferably, the R 7 is
    优选地，所述R7为 Preferably, the R 7 is
    优选地，所述R4为H或-CH3；Preferably, the R 4 is H or -CH 3 ;
    优选地，式(I)所述化合物具有式(I-5)～式(I-9)任一结构式所示结构：
    Preferably, the compound described in formula (I) has a structure represented by any structural formula of formula (I-5) to formula (I-9):
    其中，R1、R5、R6、Ra和Rb如以上或权利要求1所定义；Wherein, R 1 , R 5 , R 6 , R a and R b are as defined above or in claim 1;
    优选地，所述R1为H、F、Cl或 Preferably, the R 1 is H, F, Cl or
    优选地，所述R5和R6各自独立地为H或 Preferably, R 5 and R 6 are each independently H or
    优选地，R1为C1-3烷基，如甲基；Preferably, R 1 is C 1-3 alkyl, such as methyl;
    优选地，R2和R3与它们相连的碳原子连接在一起形成 Preferably, R 2 and R 3 are joined together with the carbon atom to which they are attached to form
    优选地，R4为C1-3烷基，如甲基；Preferably, R 4 is C 1-3 alkyl, such as methyl;
    优选地，R5和R6各自独立地为H或甲基；n为2；Preferably, R 5 and R 6 are each independently H or methyl; n is 2;
    优选地，为 Preferably, for
    优选地，R7为被1、2或3个H、F、Cl或甲基所取代的例如为 Preferably, R 7 is substituted by 1, 2 or 3 H, F, Cl or methyl For example
    优选地，式(I)所示化合物选自以下结构：

    Preferably, the compound represented by formula (I) is selected from the following structures:

  3. 根据权利要求1或2所述的应用，其特征在于，所述药学上可接受的盐为式(I)所示化合物与无机酸形成的盐，所述无机酸包括例如盐酸、氢溴酸、硝酸、碳酸，碳酸氢根，磷酸、磷酸一氢根、磷酸二氢根、硫酸、硫酸氢根、氢碘酸、亚磷酸等；以及有机酸盐，所述有机酸包括如乙酸、丙酸、异丁酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、酒石酸和甲磺酸等类似的酸；还包括氨基酸(如精氨酸等)的盐，以及如葡糖醛酸等有机酸的盐；优选为式(I)所示化合物的盐酸盐、对甲苯磺酸盐。The application according to claim 1 or 2, characterized in that the pharmaceutically acceptable salt is a salt formed by a compound represented by formula (I) and an inorganic acid, and the inorganic acid includes, for example, hydrochloric acid, hydrobromic acid, Nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydriodic acid, phosphorous acid, etc.; and organic acid salts, the organic acids include such as acetic acid, propionic acid, Isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, Similar acids such as tartaric acid and methanesulfonic acid; also include salts of amino acids (such as arginine, etc.), and salts of organic acids such as glucuronic acid; preferably the hydrochloride salt of the compound represented by formula (I), Tosylate.
  4. 根据权利要求1-3任一项所述的应用，其特征在于，所述肿瘤选自实体瘤或非实体瘤，例如黑色素瘤、肾癌、血液癌、前列腺癌、结肠癌、直肠癌、胃癌、食道癌、膀胱癌、头颈癌(如头颈部鳞癌)、甲状腺癌、乳腺癌(如三阴性乳腺癌)、卵巢癌、宫颈癌、肺癌(如非小细胞肺癌)、尿路上皮癌、胰腺癌、胶质母细胞瘤、肝癌、结肠腺癌、星形细胞瘤、大肠癌、骨肉瘤、骨髓瘤(如多发性骨髓瘤)、淋巴瘤{霍奇金淋巴瘤、非霍奇金淋巴瘤(例如，滤泡性淋巴瘤(FL)、套细胞淋巴瘤(MCL)、边缘区淋巴瘤(MZL)、弥漫性大B细胞淋巴瘤(DLBCL)、慢性淋巴细胞白血病(CLL)、)或多发性骨髓瘤(MM))}、骨髓增生异常综合征、脑癌、中枢神经系统癌、恶性胶质瘤、白血病(如急性淋巴性白血病、急性髓性白血病)中的至少一种或其任意组合；The application according to any one of claims 1 to 3, characterized in that the tumor is selected from solid tumors or non-solid tumors, such as melanoma, kidney cancer, blood cancer, prostate cancer, colon cancer, rectal cancer, gastric cancer , esophageal cancer, bladder cancer, head and neck cancer (such as head and neck squamous cell carcinoma), thyroid cancer, breast cancer (such as triple-negative breast cancer), ovarian cancer, cervical cancer, lung cancer (such as non-small cell lung cancer), urothelial cancer , pancreatic cancer, glioblastoma, liver cancer, colon adenocarcinoma, astrocytoma, colorectal cancer, osteosarcoma, myeloma (such as multiple myeloma), lymphoma {Hodgkin lymphoma, non-Hodgkin lymphoma Lymphoma (eg, follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL), or multiple myeloma (MM)}, myelodysplastic syndrome, brain cancer, central nervous system cancer, malignant glioma, leukemia (such as acute lymphoblastic leukemia, acute myeloid leukemia) or at least one of them random combination;
    优选地，所述肿瘤选自肾癌、前列腺癌、结肠癌、直肠癌、胃癌、食道癌、甲状腺癌、乳腺癌、肺癌(如非小细胞肺癌)、卵巢癌、胶质母细胞瘤、肝癌、淋巴瘤{如慢性淋巴细胞白血病(CLL)}、白血病(如急性髓性白血病)；Preferably, the tumor is selected from the group consisting of kidney cancer, prostate cancer, colon cancer, rectal cancer, gastric cancer, esophageal cancer, thyroid cancer, breast cancer, lung cancer (such as non-small cell lung cancer), ovarian cancer, glioblastoma, liver cancer , lymphoma {such as chronic lymphocytic leukemia (CLL)}, leukemia (such as acute myeloid leukemia);
    优选地，所述肿瘤选自肾癌、结肠癌、直肠癌、胃癌、肺癌(如非小细胞肺癌)、卵巢癌、淋巴瘤、肝癌、乳腺癌、白血病(如急性髓性白血病)。Preferably, the tumor is selected from the group consisting of kidney cancer, colon cancer, rectal cancer, gastric cancer, lung cancer (such as non-small cell lung cancer), ovarian cancer, lymphoma, liver cancer, breast cancer, and leukemia (such as acute myeloid leukemia).
  5. 根据权利要求1-4任一项所述的应用，其特征在于，式(I)所示化合物或其药学上可接受的盐对VEGFR2，例如对p-VEGFR2具有抑制作用；The application according to any one of claims 1 to 4, characterized in that the compound represented by formula (I) or a pharmaceutically acceptable salt thereof has an inhibitory effect on VEGFR2, such as p-VEGFR2;
    优选地，式(I)所示化合物或其药学上可接受的盐对eIF4E和VEGFR2同时具有抑制作用，例如对p-eIF4E和p-VEGFR2具有抑制作用。Preferably, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof has an inhibitory effect on both eIF4E and VEGFR2, for example, an inhibitory effect on p-eIF4E and p-VEGFR2.
  6. 一种药物组合物，包括权利要求1-5中任一项所述式(I)所示化合物或其药学上可接受的盐和至少一种第二药物； A pharmaceutical composition comprising a compound represented by formula (I) according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof and at least one second drug;
    优选地，所述药物组合物中，式(I)所示化合物或其药学上可接受的盐与所述第二药物的质量比为1:100至100：1，例如为1:10至10:1；Preferably, in the pharmaceutical composition, the mass ratio of the compound represented by formula (I) or its pharmaceutically acceptable salt to the second drug is 1:100 to 100:1, for example, 1:10 to 10 :1;
    优选地，所述第二药物选自免疫检查点抑制剂、化疗药物或靶向药物；Preferably, the second drug is selected from immune checkpoint inhibitors, chemotherapy drugs or targeted drugs;
    优选地，所述免疫检查点抑制剂靶标包括免疫抑制信号涉及PD-1、PD-L1、PD-L2、CTLA4、CD80、CD86、CD244、B7-H3、B7-H4、HVEM、BTLA、KIR、LAG3、GAL9、TIM3、2B4、腺苷、A2aR、TGFP、BCL-2和免疫抑制细胞因子(如IL-10、IL-35)的至少一种。免疫抑制抑制剂组分可以是化合物、抗体、抗体片段或融合多肽(例如Fc融合，例如CTLA4-Fc)、反义分子、核酶或RNAi分子，或低分子量有机分子。Preferably, the immune checkpoint inhibitor targets include immunosuppressive signals involving PD-1, PD-L1, PD-L2, CTLA4, CD80, CD86, CD244, B7-H3, B7-H4, HVEM, BTLA, KIR, At least one of LAG3, GAL9, TIM3, 2B4, adenosine, A2aR, TGFP, BCL-2 and immunosuppressive cytokines (such as IL-10, IL-35). The immunosuppressive inhibitor component can be a compound, antibody, antibody fragment or fusion polypeptide (eg Fc fusion, eg CTLA4-Fc), antisense molecule, ribozyme or RNAi molecule, or low molecular weight organic molecule.
    优选地，所述化疗药物选自顺铂、卡铂、菲铂、洛铂、奈达铂、奥沙利铂、长春瑞滨、羟基喜树碱、吉西他滨、阿糖胞苷、阿扎胞苷、替加氟、甲氨蝶呤、紫杉醇、紫杉醇脂质体、白蛋白紫杉醇、多西他赛、培美曲塞、表柔比星、吡柔比星、伊达比星、丝裂霉素、米托蒽醌、氟嘧啶、氟尿嘧啶、5-氟尿嘧啶、卡培他滨、异环磷酰胺、氮烯咪胺、替吉奥、伊立替康、表阿霉素、亚叶酸钙、依托泊苷、甲酰四氢叶酸或其任意组合。Preferably, the chemotherapy drug is selected from the group consisting of cisplatin, carboplatin, phenanplatin, loplatin, nedaplatin, oxaliplatin, vinorelbine, hydroxycamptothecin, gemcitabine, cytarabine, and azacitidine , Tegafur, methotrexate, paclitaxel, paclitaxel liposome, albumin-paclitaxel, docetaxel, pemetrexed, epirubicin, pirarubicin, idarubicin, mitomycin , mitoxantrone, fluoropyrimidine, fluorouracil, 5-fluorouracil, capecitabine, ifosfamide, dacarbazine, tigio, irinotecan, epirubicin, leucovorin, etoposide , leucovorin or any combination thereof.
    优选地，所述免疫检查点抑制剂选自帕博利珠单抗、纳武尤利单抗、卡瑞丽珠单抗、特瑞普利单抗、西米普利单抗、杜瓦鲁单抗、阿维鲁单抗、信迪利单抗、替雷丽珠单抗、赛帕利单抗、度伐利尤单抗、阿替利珠单抗、恩沃利单抗、舒格利单抗、斯鲁利单抗、派安普利单抗、赛瑞单抗、津伯利单抗、吉普诺利单抗、斯巴达珠单抗、普罗格利单抗、普可替尼单抗、可西贝利单抗、AUNP-12、瑞拉单抗、瑞替凡利单抗、多塔利单抗、卡多尼单抗、索卡唑单抗、阿德布雷单抗、易普利单抗、曲美木单抗或其任意组合。Preferably, the immune checkpoint inhibitor is selected from pembrolizumab, nivolumab, camrelizumab, toripalimab, cimepilimab, durvalumab , avelumab, sintilimab, tislelizumab, cepalizumab, durvalumab, atezolizumab, envolizumab, sugalizumab anti, slulimumab, penpilimab, serelizumab, zimberizumab, jiprolimab, spartalizumab, proglimab, puxolitimab anti-, cosibelimab, AUNP-12, relamanumab, retifarimab, dotalizumab, cardonizumab, socazoliumab, adebrumab, easy Prilimumab, tremelimumab, or any combination thereof.
    优选地，所述靶向药物选自B-Raf抑制剂(达拉非尼、索拉非尼、维莫拉非尼和达布拉非尼)、MEK抑制剂(曲美替尼、塞卢美替尼、比尼美替尼、PD-325901、考比美替尼、CI-1040和PD035901)、VEGF/VEGFR抑制剂(如贝伐珠单抗、雷莫芦单抗、雷珠单抗、阿柏西普、康柏西普、阿西替尼、西地尼布、莫特沙尼、仑伐替尼、舒尼替尼、索拉非尼、卡博替尼、瑞戈非尼、尼达尼布、怕唑帕尼、奥希替尼、呋喹替尼、凡德他尼、阿帕替尼、派加他尼、安罗替尼、利尼替尼、普利德辛)、EGFR抑制剂(如西妥西单抗、帕尼单抗、尼妥珠单抗、纳西妥单抗、吉非替尼、阿帕替尼、厄洛替尼、凡德替尼、埃克替尼、阿法替尼、奥希替尼、奥姆替尼、布加替尼、拉泽替尼)、酪氨酸激酶抑制剂(TKIs)、mTOR抑制剂(如雷帕霉素、Sirolimus、FK-50、CCI-779)、PARP抑制剂(尼拉帕尼、奥拉帕尼、拉唑帕尼、维利帕尼、他拉唑帕尼、氟唑帕尼、帕米帕尼)；粘附斑激酶(FAK)抑制剂(defactinib(VS-6063),VS-4718,VS-6062,GSK2256098)；生长因子信号转导激酶抑制剂(cediranib,galunisertib,rociletinib,vandetanib,afatinib，EGF816，AZD4547)；c-Met抑制剂(卡马替尼、克唑替尼、卡博替尼、沃利替尼、特泊替尼、替万替尼、谷美替尼、西曲替尼、INC280)；丝氨酸/苏氨酸激酶抑制剂；生长因子和其受体抑制剂(如ziv-aflibercept)；成纤维细胞生长因子受体(FGFR)抑制剂，如erdafitinib和pemigatinib；组蛋白去乙酰化酶(HDAC)抑制剂(伏立诺他、罗米地辛、西达本胺、panobinostat，mocetinostat，abexinostat，belinostat，entinostat，resminostat，givinostat，quisinostat，SB939)；ALK抑制剂(赛瑞替尼、克唑替尼、阿来替尼、布加替尼、恩沙替尼、劳拉替尼、洛普替尼)；抗体(曲妥珠单抗、帕尼单抗、帕妥珠单抗、西妥昔单抗、阿达木单抗、戈利单抗、英夫利昔单抗、利妥昔单抗、奥瑞珠单抗、奥法单抗、奥法珠单抗、奥非珠单抗、阿仑珠单抗、阿昔单抗、阿特利珠单抗、达氯珠单抗、地诺单抗、依法珠单抗、依洛珠单抗、罗维珠单抗、鲁普珠单抗、乌斯替金单抗、维西珠单抗、吉珠单抗奥佐加米辛、布伦通昔布维多丁)或其任意组合；Preferably, the targeted drug is selected from the group consisting of B-Raf inhibitors (dabrafenib, sorafenib, vemurafenib and dabrafenib), MEK inhibitors (trametinib, selu Metinib, binimetinib, PD-325901, cobimetinib, CI-1040 and PD035901), VEGF/VEGFR inhibitors (such as bevacizumab, ramucirumab, ranibizumab, Aflibercept, conbercept, axitinib, cediranib, motesanib, lenvatinib, sunitinib, sorafenib, cabozantinib, regorafenib, Nintedanib, zopanib, osimertinib, fruquintinib, vandetanib, apatinib, pegaptanib, anlotinib, linitinib, pridersin) , EGFR inhibitors (such as cetuximab, panitumumab, nimotuzumab, nacituzumab, gefitinib, apatinib, erlotinib, vandetinib, icotinib ni, afatinib, osimertinib, ommutinib, brigatinib, lazetinib), tyrosine kinase inhibitors (TKIs), mTOR inhibitors (such as rapamycin, Sirolimus, FK-50, CCI-779), PARP inhibitors (niraparib, olaparib, razopanib, veliparib, talazopanib, fluzopanib, pamiparib); Adherent plaque kinase (FAK) inhibitors (defactinib (VS-6063), VS-4718, VS-6062, GSK2256098); growth factor signaling kinase inhibitors (cediranib, galunisertib, rociletinib, vandetanib, afatinib, EGF816, AZD4547 ); c-Met inhibitors (capmatinib, crizotinib, cabozantinib, savolitinib, tepotinib, tivantinib, gumetinib, cetrutinib, INC280) ; Serine/threonine kinase inhibitors; growth factors and their receptor inhibitors (such as ziv-aflibercept); fibroblast growth factor receptor (FGFR) inhibitors, such as erdafitinib and pemigatinib; histone deacetylase ( HDAC) inhibitors (vorinostat, romidepsin, chidamide, panobinostat, mocetinostat, abexinostat, belinostat, entinostat, resminostat, givinostat, quisinostat, SB939); ALK inhibitors (ceritinib, crizolin aletinib, brigatinib, ensartinib, lorlatinib, lopotinib); antibodies (trastuzumab, panitumumab, pertuzumab, cetol Xiximab, adalimumab, golimumab, infliximab, rituximab, ocrelizumab, ofatumumab, ofatumumab, ofatumumab, a Lentizumab, abciximab, atezolizumab, daclizumab, denosumab, efalizumab, evolizumab, rovizumab, lupilizumab, Stekinumab, velcilizumab, giltizumab ozogamicin, brentoncoxib vidotin) or any combination thereof;
    优选地，所述第二药物为顺铂、紫杉醇、阿霉素或PD-1。Preferably, the second drug is cisplatin, paclitaxel, doxorubicin or PD-1.
  7. 权利要求6所述药物组合物在制备抗肿瘤(黑色素瘤、肾癌、血液癌、前列腺癌、结肠癌、直肠癌、胃癌、食道癌、膀胱癌、头颈癌(如头颈部鳞癌)、甲状腺癌、乳腺癌(如三阴性乳腺癌)、卵巢癌、宫颈癌、肺癌(如非小细胞肺癌)、尿路上皮癌、胰腺癌、胶质母细胞瘤、肝癌、结肠腺癌、星形细胞瘤、大肠癌、骨肉瘤、骨髓瘤(如多发性骨髓瘤)、淋巴瘤{霍奇金淋巴瘤、非霍奇金淋巴瘤(例如，滤泡性淋巴瘤(FL)、套细胞淋巴瘤(MCL)、边缘区淋巴瘤(MZL)、弥漫性大B细胞淋巴瘤(DLBCL)、慢性淋巴细胞白血病(CLL)、或多发性骨髓瘤(MM))}、骨髓增生异常综合征、脑癌、中枢神经系统癌、恶性胶质瘤、白血病(如急性淋巴性白血病、急性髓性白血病)中的至少一种或其任意组合)药物中的应用；The pharmaceutical composition of claim 6 is used in the preparation of anti-tumor (melanoma, kidney cancer, blood cancer, prostate cancer, colon cancer, rectal cancer, gastric cancer, esophageal cancer, bladder cancer, head and neck cancer (such as head and neck squamous cell carcinoma), Thyroid cancer, breast cancer (such as triple negative breast cancer), ovarian cancer, cervical cancer, lung cancer (such as non-small cell lung cancer), urothelial cancer, pancreatic cancer, glioblastoma, liver cancer, colon adenocarcinoma, stellate cancer Cytoma, colorectal cancer, osteosarcoma, myeloma (eg, multiple myeloma), lymphoma {Hodgkin lymphoma, non-Hodgkin lymphoma (eg, follicular lymphoma (FL), mantle cell lymphoma) (MCL), marginal zone lymphoma (MZL), diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL), or multiple myeloma (MM)}, myelodysplastic syndrome, brain cancer , application in at least one of central nervous system cancer, malignant glioma, leukemia (such as acute lymphoblastic leukemia, acute myeloid leukemia) or any combination thereof);
    优选地，所述肿瘤选自肾癌、前列腺癌、结肠癌、直肠癌、胃癌、食道癌、甲状腺癌、乳腺癌、肺癌(如非小细胞肺癌)、卵巢癌、胶质母细胞瘤、肝癌、淋巴瘤{如慢性淋巴细胞白血病(CLL)}、白血病(如急性髓性白血病)；Preferably, the tumor is selected from the group consisting of kidney cancer, prostate cancer, colon cancer, rectal cancer, gastric cancer, esophageal cancer, thyroid cancer, breast cancer, lung cancer (such as non-small cell lung cancer), ovarian cancer, glioblastoma, liver cancer , lymphoma {such as chronic lymphocytic leukemia (CLL)}, leukemia (such as acute myeloid leukemia);
    优选地，所述肿瘤选自肾癌、结肠癌、直肠癌、胃癌、肺癌(如非小细胞肺癌)、卵巢癌、淋巴瘤、肝癌、乳腺癌、白血病(如急性髓性白血病)。Preferably, the tumor is selected from the group consisting of kidney cancer, colon cancer, rectal cancer, gastric cancer, lung cancer (such as non-small cell lung cancer), ovarian cancer, lymphoma, liver cancer, breast cancer, and leukemia (such as acute myeloid leukemia).
  8. 权利要求1-5任一项所述式(I)所示化合物或其药学上可接受的盐或所述药物组合物在制备治疗VEGFR2信号通路相关疾病的药物中的用途。 The use of the compound represented by formula (I) according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition in the preparation of a drug for treating diseases related to the VEGFR2 signaling pathway.
  9. 权利要求1-5任一项所述式(I)所示化合物或其药学上可接受的盐或所述药物组合物在制备治疗eIF4E和/或VEGFR2信号通路相关疾病的药物中的用途。The use of the compound represented by formula (I) according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition in the preparation of a drug for treating diseases related to eIF4E and/or VEGFR2 signaling pathways.
  10. 权利要求1-5任一项所述式(I)所示化合物或其药学上可接受的盐或所述药物组合物在制备调节免疫的药物中的应用，所述调节免疫优选为增强免疫。 The application of the compound represented by formula (I) according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition in the preparation of a drug for regulating immunity, and the regulating immunity is preferably to enhance immunity.
PCT/CN2023/107850 2022-07-19 2023-07-18 Use of pyrrolotriazine compound in preparation of anti-tumor drug WO2024017229A1 (en)

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WO2018218038A1 (en) * 2017-05-24 2018-11-29 Effector Therapeutics, Inc. Methods and compositions for cellular immunotherapy
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CN107750167A (en) * 2015-04-20 2018-03-02 效应物治疗公司 For treating cancer and the inhibitor of the immunologic test point conditioning agent of infection
WO2017087808A1 (en) * 2015-11-20 2017-05-26 Effector Therapeutics, Inc. Heterocyclic compounds that inhibit the kinase activity of mnk useful for treating various cancers
CN110719781A (en) * 2017-02-14 2020-01-21 效应治疗股份有限公司 Substituted piperidine MnK inhibitors and methods related thereto
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