WO2024000615A1 - 一种酪氨酸蛋白激酶抑制剂及其用途 - Google Patents

一种酪氨酸蛋白激酶抑制剂及其用途 Download PDF

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WO2024000615A1
WO2024000615A1 PCT/CN2022/104421 CN2022104421W WO2024000615A1 WO 2024000615 A1 WO2024000615 A1 WO 2024000615A1 CN 2022104421 W CN2022104421 W CN 2022104421W WO 2024000615 A1 WO2024000615 A1 WO 2024000615A1
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compound
receptor
alkyl
tyrosine
pharmaceutically acceptable
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French (fr)
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徐伟
吴曙光
吴诺萍
陈海昌
何绮琪
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广州百霆医药科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings

Definitions

  • the invention belongs to the field of medical technology and specifically relates to tyrosine protein kinase inhibitors, especially compounds that inhibit AXL, KDR and CSF-1R kinases; it also relates to the use of the inhibitors in the treatment of abnormalities in AXL, KDR and CSF-1R kinases. Use in medicines for tumors and related diseases.
  • PTK Protein tyrosine kinase
  • KDR vascular endothelial growth factor
  • Axl belongs to the TAM receptor tyrosine kinase family, which also includes Tyro3 and Mer (TAM) subfamilies.
  • Ligands that activate TAM receptors include growth arrest-specific gene 6 (Gas6), protein S, tubby, and tubby-like protein 1 (tulp1).
  • Gas6 growth arrest-specific gene 6
  • protein S protein S
  • tubby tubby-like protein 1
  • tulp1 tubby-like protein 1
  • Gas6 has subnanomolar affinity for Axl and is the only ligand that activates Axl; the binding affinity of Tyro and Mer to Gas6 is reduced, and protein S preferentially binds to Tyro and Mer.
  • TAM receptors have been shown to be overexpressed in many solid tumors such as breast, lung, brain and digestive tract tumors.
  • Axl For example, high expression of Axl has been detected in patients with acute leukemia; Axl induces tumor cell proliferation and survival, and induces Apoptosis and resistance to chemotherapy drugs; in addition, Axl also plays a role in mediating the migration and invasion of cancer cells.
  • CSF-1R colony-stimulating factor-1
  • CSF-1R colony-stimulating factor-1
  • CSF-1receptor colony growth factor receptor CSF-1R
  • Inhibition of CSF-1R may enhance the anti-tumor effects of other PTK inhibitors by enhancing tumor immune evasion. Studying the combined inhibition of CSF-1/CSF-1R and related signaling pathways is a new strategy for tumor treatment.
  • the present invention provides a new tyrosine protein kinase inhibitor that inhibits kinase activity and tumor cell proliferation in vitro, and exhibits excellent anti-tumor activity in animal models.
  • the present invention provides a compound represented by general formula (I), its isomer, its pharmaceutically acceptable salt or its deuterated product:
  • R 1 is C 1 -C 6 alkyl, R 2 is (D is an isotope of hydrogen); or R 2 is C 1 -C 6 alkyl, R 1 is (D is an isotope of hydrogen);
  • Y is selected from C 1 -C 10 alkylene, C 1 -C 10 alkyleneamine, C 1 -C 10 alkyleneoxy, C 1 -C 10 haloalkylene, piperidinylene, C 1 -C 10 alkylene piperidinyl, piperazinyl or C 1 -C 10 alkylene piperazinyl;
  • G 1 and G 2 are each independently selected from hydrogen, deuterium, halogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl or C 1 -C 6 alkoxy;
  • W is selected from the following groups:
  • R 1 is C 1 -C 3 alkyl
  • R 2 is Or R 2 is C 1 -C 3 alkyl
  • R 1 is
  • Y is selected from C 3 -C 6 alkylene, C 3 -C 6 alkyleneamine, C 3 -C 6 alkyleneoxy, C 3 -C 6 haloalkylene.
  • R 1 is C 1 -C 3 alkyl
  • R 2 is Y is selected from C 3 -C 5 alkylene or C 3 -C 5 haloalkylene.
  • G 1 and G 2 are each independently selected from hydrogen, deuterium, fluorine, chlorine, bromine or iodine.
  • W is selected from the following groups:
  • the invention also provides compounds including, but not limited to, the following:
  • the present invention provides a pharmaceutical composition, which includes a compound of the present invention, an isomer thereof, a pharmaceutically acceptable salt thereof or a deuterated product thereof, and a pharmaceutically acceptable excipient.
  • the compounds of the invention (including racemates, enantiomers, stereoisomers, deuterates) or their pharmaceutically acceptable salts, hydrates, solvates,
  • the prodrug and its pharmaceutically acceptable carrier or excipient are prepared into a pharmaceutical composition that is convenient for administration.
  • the administration route of the pharmaceutical composition of the present invention can be: (1) oral administration: such as tablets, capsules, etc.; (2) injection: such as intravenous injection, subcutaneous injection, intramuscular injection, eyeball injection, etc.; (3) intrarectally: For example, suppositories, gels, etc.; (4) Nostril inhalation: such as sprays, aerosols, etc.; (5) Administration with drug release systems such as liposomes, sustained release technology, controlled release technology, etc.
  • pharmaceutically acceptable salts refers to salts that maintain the biological activity of the compounds of the invention without exhibiting undesirable toxicological effects.
  • Illustrative examples thereof include, but are not limited to, acid addition salts formed with inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, hydrobromic acid, etc., as well as acid addition salts with acetic acid, malic acid, tartaric acid, oxalic acid, succinic acid, benzoic acid, tannic acid, Salts formed by organic acids such as alginic acid and polyglutamic acid; the compounds of the present invention can also be administered as pharmaceutically acceptable quaternary ammonium salts.
  • the present invention provides the compound, its isomer, its pharmaceutically acceptable salt or its deuterated product or the pharmaceutical composition prepared for the prevention or treatment of diseases caused by tyrosine protein kinase abnormalities. uses in medicines.
  • the tyrosine protein kinase is selected from one of receptor tyrosine kinase (AXL), vascular endothelial growth factor receptor 2 (KDR) and colony-stimulating factor-1 receptor (CSF-1R) or Various.
  • AXL receptor tyrosine kinase
  • KDR vascular endothelial growth factor receptor 2
  • CSF-1R colony-stimulating factor-1 receptor
  • the diseases caused by tyrosine protein kinase abnormalities include neoplastic diseases, such as solid tumors, such as gastric cancer, lung cancer, breast cancer, hematological tumors such as leukemia; and non-neoplastic diseases, such as inflammatory diseases, autologous immune diseases.
  • neoplastic diseases such as solid tumors, such as gastric cancer, lung cancer, breast cancer, hematological tumors such as leukemia
  • non-neoplastic diseases such as inflammatory diseases, autologous immune diseases.
  • the present invention provides the use of the compound, an isomer thereof, a pharmaceutically acceptable salt thereof or a deuterated product thereof, or the pharmaceutical composition in the preparation of a medicament for inhibiting tyrosine protein kinase.
  • the tyrosine protein kinase is selected from one of receptor tyrosine kinase (AXL), vascular endothelial growth factor receptor 2 (KDR) and colony-stimulating factor-1 receptor (CSF-1R) or Various.
  • AXL receptor tyrosine kinase
  • KDR vascular endothelial growth factor receptor 2
  • CSF-1R colony-stimulating factor-1 receptor
  • the present invention provides a method for preventing or treating diseases caused by tyrosine protein kinase abnormalities, which includes administering an effective amount of a compound of the present invention, an isomer thereof, or a pharmaceutically acceptable compound thereof to a subject in need thereof. salts or deuterated products thereof or the pharmaceutical composition.
  • the tyrosine protein kinase is selected from one of receptor tyrosine kinase (AXL), vascular endothelial growth factor receptor 2 (KDR) and colony-stimulating factor-1 receptor (CSF-1R) or Various.
  • AXL receptor tyrosine kinase
  • KDR vascular endothelial growth factor receptor 2
  • CSF-1R colony-stimulating factor-1 receptor
  • the diseases caused by tyrosine protein kinase abnormalities include neoplastic diseases, such as solid tumors, such as gastric cancer, lung cancer, breast cancer, hematological tumors such as leukemia; and non-neoplastic diseases, such as inflammatory diseases, autologous immune diseases.
  • neoplastic diseases such as solid tumors, such as gastric cancer, lung cancer, breast cancer, hematological tumors such as leukemia
  • non-neoplastic diseases such as inflammatory diseases, autologous immune diseases.
  • the tyrosine protein kinase inhibitor provided by the present invention can inhibit kinase activity and inhibit tumor cell proliferation in vitro, and exhibits excellent anti-tumor activity in animal models. Therefore, it can be used as a new inhibitor of PTK multi-target combination application. agent, which has unique and potential advantages in the treatment of tumors.
  • Figure 1 shows the anti-tumor effects of the compounds of Examples on human leukemia MOLM-13 cell transplanted tumors in nude mice.
  • Figure 2 shows the body weight changes of nude mice transplanted with tumors before and after administration.
  • AXL refers to receptor tyrosine kinases in the TAM (Tyro3, Axl, MerTK) family.
  • KDR refers to vascular endothelial growth factor receptor 2 (VEGFR2).
  • CSF-1R colony-stimulating factor-1 receptor
  • alkyl refers to straight-chain and branched aliphatic groups containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 carbon atoms, optionally substituted by one or more Substituted; the defined alkyl includes but is not limited to methyl, substituted methyl, ethyl, substituted ethyl, propyl, substituted propyl, isopropyl, substituted isopropyl, butyl, substituted butyl, iso Butyl, substituted isobutyl, pentyl, substituted pentyl, hexyl, substituted hexyl, etc.
  • alkylene refers to a -CH 2 - group located between and connecting two chemical groups; exemplary alkylene groups include, but are not limited to, methylene, ethylene, ethylene, Propyl and butylene etc.
  • haloalkyl refers to an alkyl chain in which one or more hydrogens are replaced by halogens.
  • halogen includes fluorine, chlorine, bromine and iodine.
  • alkoxy refers to -O alkyl, such as -OC 1 -C 6 alkyl.
  • cycloalkyl refers to a saturated or partially saturated cyclic group having 3, 4, 5 or 6 carbon atoms, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
  • heteroaryl refers to a monocyclic or bicyclic group composed of 5, 6, 7, 8, 9, or 10 ring atoms; among the atoms constituting the ring, in addition to carbon atoms, there are 1 or more For example, 1, 2, 3, 4, 5, 6, 7, 8, 9 heteroatoms selected from N, O and S.
  • aralkyl refers to an aryl group attached to an alkyl group, which may independently be optionally substituted.
  • heteroalkyl refers to an alkyl group in which one or more, for example, 1, 2, 3, 4, or 5 carbon atoms are substituted by O, S or N atoms.
  • aryl refers to a group containing 1, 2, or 3 aromatic rings, which may be optionally substituted; the aryl group includes but is not limited to phenyl, naphthyl, etc.
  • heterocyclyl refers to the ring atoms containing one or more (such as 1, 2, 3, 4, 5, 6, 7, 8, 9) nitrogen atoms and oxygen atoms in addition to carbon atoms. Or a 3, 4, 5, 6, 7, 8, 9, 10-membered ring group of sulfur atom, which can be a monocyclic, bicyclic, spirocyclic or bridged ring; the term “heterocyclylalkyl” refers to a group consisting of a sulfur atom and a sulfur atom. The alkyl group connected to the heterocyclyl group is then connected to other parts of the molecule.
  • the present invention provides compounds represented by general formula (I), isomers thereof, pharmaceutically acceptable salts or deuterated products thereof:
  • R 1 is C 1 -C 6 alkyl, R 2 is (D is an isotope of hydrogen); or R 2 is C 1 -C 6 alkyl, R 1 is (D is an isotope of hydrogen);
  • Y is selected from C 1 -C 10 alkylene, C 1 -C 10 alkyleneamine, C 1 -C 10 alkyleneoxy, C 1 -C 10 haloalkylene, piperidinylene, C 1 -C 10 alkylene piperidinyl, piperazinyl or C 1 -C 10 alkylene piperazinyl;
  • G 1 and G 2 are each independently selected from hydrogen, deuterium, halogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl or C 1 -C 6 alkoxy;
  • W is selected from the following groups:
  • R 1 is C 1 -C 3 alkyl
  • R 2 is Or R 2 is C 1 -C 3 alkyl
  • R 1 is
  • Y is selected from C 3 -C 6 alkylene, C 3 -C 6 alkyleneamine, C 3 -C 6 alkyleneoxy, C 3 -C 6 haloalkylene.
  • R 1 is C 1 -C 3 alkyl
  • R 2 is Y is selected from C 3 -C 5 alkylene or C 3 -C 5 haloalkylene.
  • G 1 and G 2 are each independently selected from hydrogen, deuterium, fluorine, chlorine, bromine or iodine.
  • W is selected from the following groups:
  • the invention also provides compounds including, but not limited to, the following:
  • Step 1 Compound 2, 7-(benzyloxy)-4-chloro-6-alkoxyquinoline, is used as the starting material, and reacts with nitrophenol compound 3 in DIPEA to form an ether to generate intermediate 4 ;
  • Step 2 Hydrolyze intermediate 4 in hydrochloric acid to release the benzyl group to generate intermediate 5;
  • Step 3 Intermediate 5 reacts with bromomethyl ester compound 6 under alkaline conditions to generate intermediate 7;
  • Step 4 After the nitro group of intermediate 7 is reduced to the amino group, intermediate 8 is generated;
  • Step 5 Condensation reaction is carried out between intermediate 8 and carboxylic acid compound 9 to generate amide intermediate 10;
  • Step 6 Intermediate 10 undergoes hydrolysis reaction to generate final product I.
  • step 1
  • Example 2 The synthesis of Example 2 was carried out with reference to the synthesis method of Example 1.
  • the intermediate raw material 5-bromopentanoic acid methyl ester was used instead of 4-bromobutyric acid methyl ester.
  • Example 3 The synthesis of Example 3 was carried out with reference to the synthesis method of Example 1. In the third step of the reaction, the intermediate raw material 6-bromocaproic acid methyl ester was used instead of 4-bromobutyric acid methyl ester.
  • Example 4 The synthesis of Example 4 was carried out with reference to the synthesis method of Example 1. In the first reaction step, the intermediate raw material 4-nitrophenol is used instead of 2-fluoro-4-nitrophenol.
  • Example 5 The synthesis of Example 5 was carried out with reference to the synthesis method of Example 1.
  • the intermediate raw material 4-nitrophenol is used to replace 2-fluoro-4-nitrophenol; in the third step of the reaction, the intermediate raw material 5-bromopentanoic acid methyl ester is used to replace 4-bromo-4-nitrophenol. Methyl butyrate.
  • Example 6 The synthesis of Example 6 was carried out with reference to the synthesis method of Example 1.
  • the intermediate raw material 4-nitrophenol is used to replace 2-fluoro-4-nitrophenol; in the third step of the reaction, the intermediate raw material 6-bromohexanoic acid methyl ester is used to replace 4-bromo-4-nitrophenol. Methyl butyrate.
  • Example 7 The synthesis of Example 7 was carried out with reference to the synthesis method of Example 1. In the reaction in step 5, the intermediate 1-(4-fluorophenyl)-2-oxopiperidine-3-carboxylic acid is used instead of 1-(4-fluorophenyl)-2-oxo-1,2- Dihydropyridine-3-carboxylic acid.
  • Example 8 The synthesis of Example 8 was carried out with reference to the synthesis method of Example 1.
  • In the third step of the reaction use the intermediate raw material 5-bromovalerate methyl ester instead of 4-bromobutyric acid methyl ester; in the fifth step of the reaction, use the intermediate 1-(4-fluorophenyl)-2- Oxypiperidine-3-carboxylic acid replaces 1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxylic acid.
  • Example 9 The synthesis of Example 9 was carried out with reference to the synthesis method of Example 1.
  • the intermediate raw material 6-bromocaproate methyl ester instead of 4-bromobutyric acid methyl ester;
  • the intermediate 1-(4-fluorophenyl)-2- Oxypiperidine-3-carboxylic acid replaces 1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxylic acid.
  • Example 10 The synthesis of Example 10 was carried out with reference to the synthesis method of Example 1.
  • 4-nitrophenol is used to replace 2-fluoro-4-nitrophenol;
  • the intermediate 1-(4-fluorophenyl)-2-oxopiperidine- 3-carboxylic acid replaces 1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxylic acid.
  • Example 11 The synthesis of Example 11 was carried out with reference to the synthesis method of Example 1.
  • 4-nitrophenol is used to replace 2-fluoro-4-nitrophenol
  • the intermediate raw material 5-bromopentanoic acid methyl ester is used to replace 4-bromobutyric acid methyl ester.
  • Example 12 The synthesis of Example 12 was carried out with reference to the synthesis method of Example 1.
  • 4-nitrophenol is used to replace 2-fluoro-4-nitrophenol
  • the intermediate raw material 6-bromocaproic acid methyl ester is used to replace 4-bromobutyric acid methyl ester.
  • Example 13 The synthesis of Example 13 was carried out with reference to the method of Example 1.
  • the intermediate raw material 5-bromovalerate methyl ester instead of 4-bromobutyric acid methyl ester; in the fifth step of the reaction, use the intermediate 1-(4-fluorophenyl)-2- Oxo-2,5-dihydro-1H-pyrrole-3-carboxylic acid replaces 1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxylic acid.
  • Example 14 The synthesis of Example 14 was carried out with reference to the synthesis method of Example 1.
  • the intermediate raw material 6-bromocaproate methyl ester instead of 4-bromobutyric acid methyl ester;
  • the intermediate 1-(4-fluorophenyl)-2- Oxo-2,5-dihydro-1H-pyrrole-3-carboxylic acid replaces 1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxylic acid.
  • Example 15 The synthesis of Example 15 was carried out with reference to the synthesis method of Example 1.
  • 4-nitrophenol is used to replace 2-fluoro-4-nitrophenol
  • the intermediate raw material 6-bromocaproic acid methyl ester is used to replace 4-bromobutyric acid methyl ester.
  • Ester; in step 5 reaction use the intermediate 1-(4-fluorophenyl)-2-oxo-2,5-dihydro-1H-pyrrole-3-carboxylic acid to replace 1-(4-fluorobenzene base)-2-oxo-1,2-dihydropyridine-3-carboxylic acid.
  • Example 16 The synthesis of Example 16 was carried out with reference to the synthesis method of Example 1. In step 5 of the reaction, the intermediate 1-(4-fluorophenyl)-2-oxopyrrolidine-3-carboxylic acid is used instead of 1-(4-fluorophenyl)-2-oxo-1,2- Dihydropyridine-3-carboxylic acid.
  • Example 17 The synthesis of Example 17 was carried out with reference to the method of Example 1.
  • the intermediate raw material 5-bromovalerate methyl ester is used instead of methyl 4-butyrate; in the reaction of step 5, the intermediate 1-(4-fluorophenyl)-2-oxo is used Pyrrolidine-3-carboxylic acid replaces 1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxylic acid.
  • Example 18 The synthesis of Example 18 was carried out with reference to the synthesis method of Example 1.
  • the intermediate raw material 6-bromohexanoic acid methyl ester is used instead of 4-butyric acid methyl ester;
  • the intermediate raw material 1-(4-fluorophenyl)-2-oxo Pyrrolidine-3-carboxylic acid replaces 1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxylic acid.
  • Example 19 The synthesis of Example 19 was carried out with reference to the synthesis method of Example 1.
  • 4-nitrophenol is used to replace 2-fluoro-4-nitrophenol;
  • the intermediate 1-(phenyl)-2-oxopyrrolidine-3-carboxylic is used Acid replacement for 1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxylic acid.
  • Example 20 The synthesis of Example 20 was carried out with reference to the synthesis method of Example 1.
  • 4-nitrophenol is used to replace 2-fluoro-4-nitrophenol
  • the intermediate raw material 5-bromopentanoic acid methyl ester is used to replace 4-bromobutyric acid methyl ester.
  • Example 21 The synthesis of Example 21 was carried out with reference to the synthesis method of Example 1.
  • 4-nitrophenol is used to replace 2-fluoro-4-nitrophenol
  • the intermediate raw material 6-bromocaproic acid methyl ester is used to replace 4-bromobutyric acid methyl ester.
  • biochemical activities of AXL, KDR and CSF-1R kinases were determined by phosphorylation of fluorescein-labeled tyrosine kinase substrates.
  • Reagents AXL, KDR and CSF-1R tyrosine kinase (purchased from Carna), KinEASE-TK kit and 96 microwell plate (purchased from Cisbio), ATP (purchased from Sigma), dithiothreitol (DTT) (purchased from Sigma), manganese chloride and magnesium chloride (purchased from Sigma) ), XL-184 (as a positive control) (purchased from Shanghai Hanxiang Biotechnology Co., Ltd.), full-wavelength multi-function microplate reader (TECAN M1000).
  • the three kinases were performed independently. 2 ⁇ L of AXL or KDR or CSF-1R kinase solution was added to the test wells of three 96-well microplates. Two wells were repeated for each concentration; 2 ⁇ L (1 ⁇ ) kinase was added to the control well. Buffer solution served as control. Add kinase buffer (4 ⁇ L), substrate solution (2 ⁇ L), and ATP solution (2 ⁇ L) to the experimental wells and control wells respectively.
  • Percent inhibition rate (F DMSO control - F sample ) / (F DMSO control - F negative control ) ⁇ 100%, add the value of DMSO as the solvent control group, without Kinase was added as a negative control group.
  • Tumor cells MOLM-13 acute leukemia cell line resistant to anti-vascular endothelial growth factor, and human gastric cancer cell MKN-45 cells were obtained from the Experimental Center of the School of Pharmacy, Southern Medical University.
  • Method Use the Luminometer luminescence method to determine the anti-proliferative activity of the compounds of the examples against MOLM-13 acute leukemia cell line.
  • Instrument Promega microplate detector (CellTiter-Glo Luminescent Cell Viability Assay, Promega).
  • Reagents and kits RPMI1640 medium, fetal calf serum, dimethyl sulfoxide, penicillin-streptomycin, Cell Titer-Gio detection kit. Cell culture flask, CO2 incubator, cell culture microplate (96-well plate).
  • Tumor cell culture MOLM-13 tumor cells frozen in liquid nitrogen are recovered and cultured in RPMI cell culture medium containing 10% fetal bovine serum and 10% penicillin-streptomycin; when the cells grow to the exponential growth phase , use a pipette to absorb the culture medium, gently pipet and collect the cells, and resuspend the cells in the culture medium; inoculate 5000-10000 cells into each well, and the cells are evenly distributed; the cells are maintained at a constant temperature of 37°C, 5% CO 2 and saturated humidity. Incubate continuously for 72 hours in a carbon dioxide incubator.
  • Example compounds and activity measurement The example compounds were dissolved in dimethyl sulfoxide to prepare 10 concentration gradients. Different concentrations of compounds were added to the test wells according to the experimental design and continued to be cultured for 72 hours. After the culture, take out the 96-well plate and place it at room temperature for 30 minutes, and then detect CTG. Add 100 ⁇ L CTG to the microwell, mix for 2 minutes, and leave at room temperature for 10 minutes. Then use a microplate luminometer to detect and record the luminescence signal value to observe the cell viability, and calculate the IC 50 value. The results are shown in Table 2.
  • Cell culture Human leukemia MOLM-13 cells were cultured in DMEM medium containing 10% fetal calf serum, 100 U/mL penicillin and 100 ⁇ g/mL streptomycin. The culture bottle was placed at 37°C, 5% CO 2 and saturated Culture in a humidified carbon dioxide incubator.
  • mice BALB/C-nu mice were purchased from Guangdong Yaokang Biotechnology Co., Ltd., 40 females, aged 28-34 days. License number: SCXK (Guangdong) 2020-0054, experimental unit use license number: SYXK (Guangdong) 2018-0131. 5 mice in one cage, animals were raised in the SPF animal room, and used for tumor cells after one week of testing Inoculation experiment.
  • MOLM-13 cell nude mouse transplanted tumor model When the number of MOLM-13 cells reaches the exponential growth phase, use a pipette to gently blow away the suspended cells in groups, collect them into a centrifuge tube, and centrifuge at 1000 r/min. After 5 minutes, collect the cells at the bottom of the centrifuge tube, resuspend the cells in serum-free medium, and adjust the cell concentration to 2 ⁇ 10 7 /mL. Use a 1mL syringe to draw the cell suspension, and inoculate it under the skin of the right front armpit of nude mice. Each nude mouse is inoculated with 0.1mL, and the number of cells inoculated is 2 ⁇ 10 6 /mouse.
  • the tumor cells After the tumor cells are inoculated, observe the tumor growth at the inoculated site of nude mice daily or regularly, and use a vernier caliper to measure the size of each animal's tumor. When the tumor reaches more than 100 mm3 , it can be selected for the experiment. Of the 40 nude mice inoculated with tumor cells, 30 met the inclusion criteria.
  • mice The experiment was randomly divided into 4 groups, including a solvent control group, a positive compound control group, and two dosage groups of 2.5 mg/kg and 5 mg/kg of the example compound.
  • Dissolve compound 3 with 50% PEG 400 and 50% distilled water (the total amount of PEG400 given to mice daily is controlled below 0.5%).
  • the drug is administered once a day, and each mouse is administered 0.1 mL by gavage, continuously. 7 days. Weigh the animals' weight every day, measure the tumor size of each animal every two days, and observe changes in the mice's diet, drinking water, body weight, hair and other basic conditions.
  • a is the long diameter of the tumor
  • b is the short diameter of the tumor.
  • TGI Tumor Growth Inhibition
  • BW is the body weight of nude mice (Body Weight)
  • the last day of BW is the body weight of nude mice at the end of the experiment
  • the first day of BW is the body weight of nude mice at the time of group administration.
  • the compound of Example 3 was continuously administered orally for 7 days to nude mice with MOLM-13 cell transplanted tumors, and showed a very significant anti-tumor effect with a rapid onset of effect.

Abstract

一种可作为酪氨酸蛋白激酶抑制剂的化合物，可抑制AXL、KDR和/或CSF-1R激酶，所述化合物还可用于制备治疗由AXL、KDR和/或CSF-1R异常引起的肿瘤及其相关疾病的药物。

Description

一种酪氨酸蛋白激酶抑制剂及其用途 技术领域
本发明属于医药技术领域，具体涉及酪氨酸蛋白激酶抑制剂，尤其是抑制AXL、KDR和CSF-1R激酶的化合物；还涉及所述抑制剂在治疗由AXL、KDR和CSF-1R激酶异常引起的肿瘤及其相关疾病的药物中的用途。
背景技术
蛋白质酪氨酸激酶(protein tyrosine kinase,PTK)是催化ATP的γ磷酸基转移到蛋白质底物酪氨酸上的关键激酶，通过磷酸化受体本身和下游信号蛋白上的酪氨酸将细胞外信号转导到细胞内，因此，PTK是细胞信号通路的重要组成部分。PTK能激活细胞内众多信号通路，导致细胞增殖、分化、迁移以及代谢的变化。
血管系统的发育依赖于PTK亚家族及其同源配体的协同作用。血管生成需要血管内皮生长因子(VEGF)以及与其协同发挥作用的受体KDR。KDR是一种参与血管生成的关键的信号蛋白。作为参与细胞信号转导过程的关键蛋白酪氨酸激酶，KDR的自磷酸化代表了促进血管生成的关键步骤。
Axl属于TAM受体酪氨酸激酶家族，其家族中还包含Tyro3、Mer(TAM)亚家族。激活TAM受体的配体有生长阻滞特异性基因6(Gas6)、蛋白S、tubby和tubby样蛋白1(tulp1)。Gas6对Axl具有亚纳摩尔的亲和力，是唯一激活Axl的配体；Tyro、Mer对Gas6的结合亲和力降低，而蛋白S优先与Tyro和Mer结合。TAM受体已被证明在许多实体肿瘤如乳腺、肺、脑以及消化道肿瘤等中有过表达，如在急性白血病患者检测到表达Axl的高表达；Axl诱导肿瘤细胞增殖与存活，并诱导对细胞凋亡以及化疗药物的耐受性；此外，Axl也在介导癌细胞的迁移和侵袭方面发挥作用。
CSF-1R(colony-stimulating factor-1，CSF-1R)是第一个被分离为纯蛋白的造血生长因子，通过骨髓祖细胞的集落生长因子受体CSF-1R(CSF-1receptor)发挥作用。对CSF-1R的抑制可能通过增强肿瘤免疫逃逸来增强其它PTK抑制剂的抗肿瘤作用，研究CSF-1/CSF-1R与相关信号通路的联合抑制是肿瘤治疗的新策略。
虽然单一靶点的PTK抑制剂通过其独特的靶点表现出各自的生物活性以及抗肿瘤作用，但越来越多的临床结果表明，疗效仍有一定局限性，因此开发新的PTK多靶点联合应用的抑制剂对肿瘤的治疗可能有着独特和潜在的优越性。
发明内容
为了解决上述技术问题，本发明提供一种新的酪氨酸蛋白激酶抑制剂，在体外抑制激酶活性和抑制肿瘤细胞增殖，以及在动物模型体内表现出优异的抗肿瘤活性。
第一方面，本发明提供通式(I)所示的化合物、其异构体、其药学上可接受的盐或其氘代物：
Figure PCTCN2022104421-appb-000001
其中，
R 1为C 1-C 6烷基，R 2为
Figure PCTCN2022104421-appb-000002
(D为氢的同位素)；或R 2为C 1-C 6烷基，R 1为
Figure PCTCN2022104421-appb-000003
(D为氢的同位素)；
Y选自C 1-C 10亚烷基、C 1-C 10亚烷基胺基、C 1-C 10亚烷基氧基、C 1-C 10卤代亚烷基、亚哌啶基、C 1-C 10亚烷基哌啶基、亚哌嗪基或C 1-C 10亚烷基哌嗪基；
G 1和G 2分别独立地选自氢、氘、卤素、C 1-C 6烷基、C 1-C 6卤代烷基或C 1-C 6烷氧基；
W选自如下基团：
Figure PCTCN2022104421-appb-000004
在本发明的一些优选实施方案中，通式(I)中，R 1为C 1-C 3烷基，R 2为
Figure PCTCN2022104421-appb-000005
Figure PCTCN2022104421-appb-000006
或R 2为C 1-C 3烷基，R 1为
Figure PCTCN2022104421-appb-000007
Y选自C 3-C 6亚烷基、C 3-C 6亚烷基胺基、C 3-C 6亚烷基氧基、C 3-C 6卤代亚烷基。
在本发明的一些优选实施方案中，通式(I)中，R 1为C 1-C 3烷基，R 2为
Figure PCTCN2022104421-appb-000008
Figure PCTCN2022104421-appb-000009
Y选自C 3-C 5亚烷基或C 3-C 5卤代亚烷基。
在本发明的一些优选实施方案中，通式(I)中，G 1和G 2分别独立地选自氢、氘、氟、氯、溴或碘。
在本发明的一些优选实施方案中，通式(I)中，W选自如下基团：
Figure PCTCN2022104421-appb-000010
在本发明的一些优选实施方案中，本发明还提供了包括，但不限于，以下所示的化合物：
Figure PCTCN2022104421-appb-000011
Figure PCTCN2022104421-appb-000012
第二方面，本发明提供一种药物组合物，其包括本发明的化合物、其异构体、其药学上可接受的盐或其氘代物，以及药学上可接受的赋形剂。
在本发明的一些优选实施方案中，本发明的化合物(包括消旋体、对映异构体、立体异构体、氘代物)或其药学上可接受的盐、水合物、溶剂合物、前药及其药学上可接受的载体或赋形剂制备成有利于给药的药物组合物。
本发明的药物组合物的给药途径可以为：(1)口服：例如片剂、胶囊等；(2)注射：例如静脉注射、皮下注射、肌肉注射、眼球注射等；(3)直肠内：例如栓剂、凝胶剂等；(4)鼻孔吸入： 例如喷雾剂、气雾剂等；(5)以脂质体、缓释技术、控释技术等药物释放系体系给药。
术语“药学上可接受的盐”是指能维持本发明化合物所具有的生物活性而不表现出不希望的毒理学效应的盐。其示例性实例包括但不限于与盐酸、硫酸、磷酸、硝酸、氢溴酸等无机酸形成的酸加成盐，以及与乙酸、苹果酸、酒石酸、草酸、琥珀酸、苯甲酸、鞣酸、藻酸、聚谷氨酸等有机酸形成的盐；本发明化合物还可以药学上可接受的季铵盐给药。
第三方面，本发明提供所述化合物、其异构体、其药学上可接受的盐或其氘代物或者所述药物组合物在制备用于预防或治疗由酪氨酸蛋白激酶异常引起的疾病的药物中的用途。
优选地，所述酪氨酸蛋白激酶选自受体酪氨酸激酶(AXL)、血管内皮生长因子受体2(KDR)和集落刺激因子-1受体(CSF-1R)中的一种或多种。
优选地，所述由酪氨酸蛋白激酶异常引起的疾病包括肿瘤性疾病，如实体瘤，诸如胃癌、肺癌、乳腺癌，血液系统肿瘤诸如白血病；以及非肿瘤性疾病，如炎症性疾病、自身免疫性疾病。
第四方面，本发明提供所述化合物、其异构体、其药学上可接受的盐或其氘代物或者所述药物组合物在制备用于抑制酪氨酸蛋白激酶的药物中的用途。
优选地，所述酪氨酸蛋白激酶选自受体酪氨酸激酶(AXL)、血管内皮生长因子受体2(KDR)和集落刺激因子-1受体(CSF-1R)中的一种或多种。
第五方面，本发明提供预防或治疗由酪氨酸蛋白激酶异常引起的疾病的方法，其包括向有需要的对象施用有效量的本发明所述化合物、其异构体、其药学上可接受的盐或其氘代物或者所述药物组合物。
优选地，所述酪氨酸蛋白激酶选自受体酪氨酸激酶(AXL)、血管内皮生长因子受体2(KDR)和集落刺激因子-1受体(CSF-1R)中的一种或多种。
优选地，所述由酪氨酸蛋白激酶异常引起的疾病包括肿瘤性疾病，如实体瘤，诸如胃癌、肺癌、乳腺癌，血液系统肿瘤诸如白血病；以及非肿瘤性疾病，如炎症性疾病、自身免疫性疾病。
本发明提供的酪氨酸蛋白激酶抑制剂，在体外能够抑制激酶活性和抑制肿瘤细胞增殖，以及在动物模型体内表现出优异的抗肿瘤活性，因此可作为新的PTK多靶点联合应用的抑制剂，对肿瘤的治疗发挥独特和潜在的优越性。
附图说明
图1示出实施例化合物对人白血病MOLM-13细胞裸鼠移植瘤的抗肿瘤作用。
图2示出肿瘤移植裸鼠给药前后的体重变化。
具体实施方式
以下实施例用于进一步说明本发明，而不是用来限制本发明的范围。
定义
本发明使用的术语及其技术的含义与所属领域的技术人员的理解相同。
术语“AXL”是指TAM(Tyro3、Axl、MerTK)家族中受体酪氨酸激酶。
术语“KDR”是指血管内皮生长因子受体2(VEGFR2)。
术语“CSF-1R”是指集落刺激因子-1受体(colony-stimulating factor-1 receptor，CSF-1R)。
术语“烷基”指含1、2、3、4、5、6、7、8、9、10个碳原子的直链和支链脂族基团，并任选被一个或两个以上取代基取代；所定义的烷基包括但不限于甲基，取代甲基，乙基，取代乙基，丙基，取代丙基，异丙基，取代异丙基，丁基，取代丁基，异丁基，取代异丁基，戊基，取代戊基，己基，取代己基等。
术语“亚烷基”指位于两个化学基团之间并连接这两个化学基团的-CH 2-基团；示例性的亚烷基包括但不限于亚甲基、亚乙基、亚丙基和亚丁基等。
术语“卤代烷基”指一个或两个以上的氢被卤素替代的烷基链。
术语“卤素”包括氟、氯、溴和碘。
术语“烷氧基”指-O烷基，例如-O-C 1-C 6烷基。
术语“环烷基”是指具有3、4、5、6个碳组成的饱和的或部分饱和的环状基团，包括但不限于环丙基、环丁基、环戊基和环己基。
术语“杂芳基”是指由5、6、7、8、9、10个环原子组成的单环或双环基团；在组成环的原子中，除碳原子外还含有1个或多个例如1、2、3、4、5、6、7、8、9个选自N、O和S中的杂原子。
术语“芳烷基”是指与烷基连接的芳基，可独立任选被取代。
术语“杂烷基”指烷基中一个或多个例如1、2、3、4、5个碳原子被O、S或N原子取代的烷基基团。
术语“芳基”是指含1、2、3个芳环组成的基团，可任选被取代；所述芳基包括但不限于苯基和萘基等。
术语“杂环基”指成环的原子中除碳原子外还含有一个或两个以上(例如1、2、3、4、5、6、7、8、9个)的氮原子、氧原子或硫原子的3、4、5、6、7、8、9、10元环的基团，可以是单环、双环、螺环或桥环；术语“杂环基烷基”是指通过与杂环基相连的烷基再与分子其他部分连接的基团。
化合物
本发明提供通式(I)所示的化合物、其异构体、其药学上可接受的盐或其氘代物：
Figure PCTCN2022104421-appb-000013
其中，
R 1为C 1-C 6烷基，R 2为
Figure PCTCN2022104421-appb-000014
(D为氢的同位素)；或R 2为C 1-C 6烷基，R 1为
Figure PCTCN2022104421-appb-000015
(D为氢的同位素)；
Y选自C 1-C 10亚烷基、C 1-C 10亚烷基胺基、C 1-C 10亚烷基氧基、C 1-C 10卤代亚烷基、亚哌啶基、C 1-C 10亚烷基哌啶基、亚哌嗪基或C 1-C 10亚烷基哌嗪基；
G 1和G 2分别独立地选自氢、氘、卤素、C 1-C 6烷基、C 1-C 6卤代烷基或C 1-C 6烷氧基；
W选自如下基团：
Figure PCTCN2022104421-appb-000016
在本发明的一些优选实施方案中，通式(I)中，R 1为C 1-C 3烷基，R 2为
Figure PCTCN2022104421-appb-000017
Figure PCTCN2022104421-appb-000018
或R 2为C 1-C 3烷基，R 1为
Figure PCTCN2022104421-appb-000019
Y选自C 3-C 6亚烷基、C 3-C 6亚烷基胺基、C 3-C 6亚烷基氧基、C 3-C 6卤代亚烷基。
在本发明的一些优选实施方案中，通式(I)中，R 1为C 1-C 3烷基，R 2为
Figure PCTCN2022104421-appb-000020
Figure PCTCN2022104421-appb-000021
Y选自C 3-C 5亚烷基或C 3-C 5卤代亚烷基。
在本发明的一些优选实施方案中，通式(I)中，G 1和G 2分别独立地选自氢、氘、氟、氯、溴或碘。
在本发明的一些优选实施方案中，通式(I)中，W选自如下基团：
Figure PCTCN2022104421-appb-000022
本发明还提供了包括，但不限于，以下所示的化合物：
Figure PCTCN2022104421-appb-000023
Figure PCTCN2022104421-appb-000024
实施方案与实施例
实施方案和实施例是对本发明的实施进行详细具体的描述，而不是限制本发明的范围。本发明中所述的化合物可由本领域技术人员所知的多种方法来制备，这些方法包括但不限于本实施例中采用的方法和其它的替代方法。在本发明设计思路的前提下，对本发明技术方案中所进行的修改或替换，都属于本发明的保护范围。
本发明通式(I)的化合物可通过以下通式路线的方法合成：
Figure PCTCN2022104421-appb-000025
主要的合成反应过程如下：
步骤1，以化合物2即7-(苄氧基)-4-氯-6-烷氧基喹啉为起始原料，与硝基苯酚类化合物3在DIPEA中发生成醚反应，生成中间体4；
步骤2，将中间体4在盐酸中水解脱下苄基生成中间体5；
步骤3，中间体5与溴代甲酯类化合物6在碱性条件下反应，生成中间体7；
步骤4，中间体7的硝基经还原氨基后，生成中间体8；
步骤5，中间体8与羧酸类化合物9进行缩合反应，生成酰胺类中间体10；
步骤6，中间体10经过水解反应，生成终产物I。
实施例1. 4-((4-(2-氟-4-(1-(4-氟苯基)-2-氧代-1,2-二氢吡啶-3-甲酰胺)苯氧基)-6-甲氧基喹啉-7-基)氧基)丁酸
Figure PCTCN2022104421-appb-000026
步骤1：
Figure PCTCN2022104421-appb-000027
将N,N-二异丙基乙胺(6.25mL，35.86mmol，2.15eq)加入7-苄氧基-4-氯-6-甲氧基喹啉(5.00g，16.68mmol，1.00eq)和2-氟-4-硝基苯酚(3.67g，23.35mmol，1.40eq)的氯苯(25mL)混和液中。140℃油浴，回流过夜。反应完成。冷却至室温。加入1M氢氧化钠水溶液(50mL)，析出固体。减压过滤，滤饼用水淋洗三遍，得到黄绿色滤饼。滤饼用乙醇搅洗四遍，得到7-(苄氧基)-4-(2-氟-4-硝基苯氧基)-6-甲氧基喹啉(4.52g，64.46％，棕色固体)。
步骤2：
Figure PCTCN2022104421-appb-000028
将7-(苄氧基)-4-(2-氟-4-硝基苯氧基)-6-甲氧基喹啉(5.80g，13.80mmol)和6M盐酸水溶液(60mL)混和，油浴120℃，回流两小时反应完成。冷却至室温。加入水稀释反应液。减压过滤，用水洗涤滤饼三次，烘干。得到4-(2-氟-4-硝基苯氧基)-6-甲氧基喹啉-7-醇(3.70g，81.20％，灰色固体)。
步骤3：
Figure PCTCN2022104421-appb-000029
将碳酸钾(1.85g，13.35mmol，3.00eq)和4-溴代丁酸甲酯(1.61g，8.90mmol，2.00eq)加入4-(2-氟-4-硝基苯氧基)-6-甲氧基喹啉-7-醇(1.47g，4.45mmol，1.00eq)的N,N-二甲基甲酰胺(15mL)溶液中，油浴90℃反应四个半小时。反应完成，冷却至室温。将反应液倒入水中，析出大量固体。减压过滤，滤饼用淋洗三遍，烘干。得到4-((4-(2-氟-4-硝基苯氧基)-6-甲氧基喹啉-7-基)氧基)丁酸甲酯(1.43g，收率74.9％，黄色固体)。
步骤4：
Figure PCTCN2022104421-appb-000030
将4-((4-(2-氟-4-硝基苯氧基)-6-甲氧基喹啉-7-基)氧基)丁酸甲酯(2.65g，6.22mmol，1.00eq)、甲酸铵(1.18g，18.65mmol，3.00eq)、10％钯碳(992.37mg)和甲醇(30mL)混和。室温(27℃)反应四小时。反应完成。硅藻土过滤，减压浓缩得到棕色固体。乙酸乙酯溶解后，用水洗三遍，饱和氯化钠洗两遍。无水硫酸钠干燥，过滤，减压浓缩。烘干。得到4-((4-(4-氨基-2-氟苯氧基)-6-甲氧基喹啉-7-基)氧基)丁酸甲酯(2.05g，收率82.2％，棕色固体)。
步骤5：
Figure PCTCN2022104421-appb-000031
将4-((4-(4-氨基-2-氟苯氧基)-6-甲氧基喹啉-7-基)氧基)丁酸甲酯(0.93g，2.33mmol，1.00eq)、1-(4-氟苯基)-2-氧代-1,2-二氢吡啶-3-羧酸(816.41mg，3.50mmol，1.50eq)、2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(1.77g，4.67mmol，2.00eq)、4-二甲氨基吡啶(142.57mg，1.17mmol，0.50eq)和N,N-二异丙基乙胺(1.63mL，9.34mmol，4.00eq)溶于N,N-二甲基甲酰胺(10mL)中。室温(20℃)反应过夜。反应完成。将反应液倒入水中，析出固体，减压过滤。用水洗涤滤饼三遍。柱层析纯化(DCM:MeOH＝30:1)，得到4-((4-(2-氟-4-(1-(4-氟苯基)-2-氧代-1,2-二氢吡啶-3-甲酰胺)苯氧基)-6-甲氧基喹啉-7-基)氧基)丁酸甲酯(1.19g，收率81.9％，棕色固体)。
步骤6：
Figure PCTCN2022104421-appb-000032
将4-((4-(2-氟-4-(1-(4-氟苯基)-2-氧代-1,2-二氢吡啶-3-甲酰胺)苯氧基)-6-甲氧基喹啉-7-基)氧基)丁酸甲酯(521mg，847.1μmol，1.00eq)溶于四氢呋喃(5mL)。加入氢氧化锂一水合物(142.12mg，3.39mmol，4.00eq)的水(5mL)溶液。室温反应一小时。反应完成。加入水稀释反应液，用4M盐酸水溶液调pH≈1，析出胶状物，过滤。柱层析(DCM:MeOH＝20:1)纯化，减压浓缩。先后用二氯甲烷和四氢呋喃搅洗，过滤，得到白色滤饼。烘干，得到4-((4-(2-氟-4-(1-(4-氟苯基)-2-氧代-1,2-二氢吡啶-3-甲酰胺)苯氧基)-6-甲氧基喹啉-7-基)氧基)丁酸(332.9mg，收率65.4％，白色固体)。
1H NMR(400MHz,DMSO)δ12.20(s,1H),8.80(d,J＝6.2Hz,1H),8.60(d,J＝7.1Hz,1H), 8.24(t,J＝8.8Hz,2H),7.68(s,2H),7.60-7.54(m,4H),7.32(t,J＝6.8Hz,2H),6.80(d,J＝7.1Hz,1H),6.72(t,J＝7.1Hz,1H),4.15(t,J＝6.2Hz,2H),3.90(s,3H),3.64–3.56(m,1H),2.21(t,J＝7.1Hz,2H),1.62–1.50(m,2H)MS:m/z(ESI+)602.7[M+H] +。
实施例2. 5-((4-(2-氟-4-(1-(4-氟苯基)-2-氧代-1,2-二氢吡啶-3-甲酰胺)苯氧基)-6-甲氧基喹啉-7-基)氧基)戊酸
Figure PCTCN2022104421-appb-000033
实施例2的合成参照实施例1的合成方法进行，第3步反应中，用中间体原料5-溴代戊酸甲酯替代4-溴代丁酸甲酯。
1H NMR(400MHz,DMSO)δ12.16(s,1H),8.74(d,J＝6.8Hz,1H),8.54(d,J＝7.0Hz,1H),8.14(t,J＝7.6Hz,2H),7.70(s,2H),7.68-7.59(m,4H),7.38(t,J＝7.1Hz,2H),6.84(d,J＝7.0Hz,1H),6.62(t,J＝6.9Hz,1H),4.19(t,J＝6.6Hz,2H),3.87(s,3H),3.60–3.52(m,1H),2.26(t,J＝7.1Hz,2H),1.72–1.69(m,2H),1.62–1.54(m,2H)MS:m/z(ESI+)616.4[M+H] +。
实施例3. 6-((4-(2-氟-4-(1-(4-氟苯基)-2-氧代-1,2-二氢吡啶-3-甲酰胺)苯氧基)-6-甲氧基喹啉-7-基)氧基)己酸
Figure PCTCN2022104421-appb-000034
实施例3的合成参照实施例1的合成方法进行，第3步反应中，用中间体原料6-溴代己酸甲酯替代4-溴代丁酸甲酯。
1H NMR(400MHz,DMSO)δ12.19(s,1H),8.81(d,J＝6.4Hz,1H),8.59(d,J＝7.1Hz,1H),8.14(t,J＝9.0Hz,2H),7.74(s,2H),7.632-7.561(m,4H),7.43(t,J＝8.6Hz,2H),6.97(d,J＝6.4Hz,1H),6.74(t,J＝6.9Hz,1H),4.22(t,J＝6.0Hz,2H),4.04(s,3H),3.65–3.55(m,1H),2.26(t,J＝7.1Hz,2H),1.93–1.81(m,2H),1.66–1.56(m,2H),1.54–1.42(m,2H)。MS:m/z(ESI+)630.8[M+H] +。
实施例4. 4-((4-(4-(1-(4-氟苯基)-2-氧代-1,2-二氢吡啶-3-甲酰胺)苯氧基)-6-甲氧基喹啉-7-基)氧基)丁酸
Figure PCTCN2022104421-appb-000035
实施例4的合成参照实施例1的合成方法进行。在第1步反应中，用中间体原料4-硝基苯酚替代2-氟-4-硝基苯酚。
1H NMR(400MHz,DMSO)δ12.19(s,1H),8.81(d,J＝6.4Hz,1H),8.59(d,J＝7.1Hz,1H),8.14(t,J＝9.0Hz,2H),7.74(s,2H),7.632-7.561(m,4H),7.43(t,J＝8.6Hz,2H),6.97(d,J＝6.4Hz,1H),6.74(t,J＝6.9Hz,1H),4.22(t,J＝6.0Hz,2H),4.04(s,3H),3.65–3.55(m,1H),2.26(t,J＝7.1Hz,2H),1.93–1.81(m,2H),1.66–1.56(m,2H),1.54–1.42(m,2H),MS:m/z(ESI+)583.57[M+H] +。
实施例5. 5-((4-(4-(1-(4-氟苯基)-2-氧代-1,2-二氢吡啶-3-甲酰胺)苯氧基)-6-甲氧基喹啉-7-基)氧基)戊酸
Figure PCTCN2022104421-appb-000036
实施例5的合成参照实施例1合成方法进行。在第1步反应中，用中间体原料4-硝基苯酚替代2-氟-4-硝基苯酚；第3步反应中，用中间体原料5-溴代戊酸甲酯替代4-溴代丁酸甲酯。
1H NMR(400MHz,DMSO)δ11.89(s,1H),8.74(d,J＝7.1Hz,1H),8.54(d,J＝6.8Hz,1H),8.18(t,J＝7.1Hz,2H),7.68(s,2H),7.58-7.42(m,4H),7.30(t,J＝7.0Hz,2H),6.83(t,J＝7.6Hz,2H),6.62(d,J＝6.8Hz,1H),3.98(t,J＝6.8Hz,2H),3.72(s,3H),3.48–3.32(m,1H),2.21(t,J＝7.2Hz,2H),1.58–1.50(m,2H)1.48–1.40(m,2H)MS:m/z(ESI+)598.5[M+H] +。
实施例6. 6-((4-(4-(1-(4-氟苯基)-2-氧代-1,2-二氢吡啶-3-甲酰胺)苯氧基)-6-甲氧基喹啉-7-基)氧基)己酸
Figure PCTCN2022104421-appb-000037
实施例6的合成参照实施例1合成方法进行。在第1步反应中，用中间体原料4-硝基苯 酚替代2-氟-4-硝基苯酚；第3步反应中，用中间体原料6-溴代己酸甲酯替代4-溴代丁酸甲酯。
1H NMR(400MHz,DMSO)δ11.89(s,1H),8.74(d,J＝7.1Hz,1H),8.54(d,J＝6.8Hz,1H),8.18(t,J＝7.1Hz,2H),7.68(s,2H),7.58-7.42(m,4H),7.30(t,J＝7.0Hz,2H),6.83(t,J＝7.6Hz,2H),6.62(d,J＝6.8Hz,1H),3.98(t,J＝6.8Hz,2H),3.72(s,3H),3.48–3.32(m,1H),2.21(t,J＝7.2Hz,2H),1.58–1.50(m,2H)1.48–1.40(m,2H)MS:m/z(ESI+)611.63[M+H] +。
实施例7. 4-((4-(2-氟-4-(1-(4-氟苯基)-2-氧哌啶-3-甲酰胺)苯氧基)-6-甲氧基喹啉-7-基)氧基)丁酸
Figure PCTCN2022104421-appb-000038
实施例7的合成参照实施例1的合成方法进行。在第5步反应中，用中间体1-(4-氟苯基)-2-氧哌啶-3-羧酸替代1-(4-氟苯基)-2-氧代-1,2-二氢吡啶-3-羧酸。
1H NMR(400MHz,DMSO)δ12.17(s,1H),8.50(d,J＝7.1Hz,1H),8.36(t,J＝8.2Hz,2H),7.68(s,2H),7.60-7.54(m,3H),7.32(t,J＝6.8Hz,2H),6.80(d,J＝7.1Hz,1H),4.20(t,J＝7.4Hz,2H),3.80(s,3H),3.72–3.42(m,5H),2.19(t,J＝8.2Hz,2H),1.94–1.70(m,3H)1.60–1.52(m,2H)MS:m/z(ESI+)606.4[M+H] +。
实施例8. 5-((4-(2-氟-4-(1-(4-氟苯基)-2-氧哌啶-3-甲酰胺)苯氧基)-6-甲氧基喹啉-7-基)氧基)戊酸
Figure PCTCN2022104421-appb-000039
实施例8的合成参照实施例1的合成方法进行。第3步反应中，用中间体原料5-溴代戊酸甲酯替代4-溴代丁酸甲酯；在第5步反应中，用中间体1-(4-氟苯基)-2-氧哌啶-3-羧酸替代1-(4-氟苯基)-2-氧代-1,2-二氢吡啶-3-羧酸。
1H NMR(400MHz,DMSO)δ12.22(s,1H),8.68(d,J＝6.8Hz,1H),8.28(t,J＝7.2Hz,2H),7.60(s,2H),7.70-7.58(m,3H),7.46(t,J＝7.1Hz,2H),6.72(d,J＝6.8Hz,1H),4.08(t,J＝7.1Hz,2H),3.60(s,3H),3.52–3.36(m,5H),2.18(t,J＝8.0Hz,2H),1.90–1.71(m,3H),1.70–1.62(m,2H)1.60–1.50(m,2H)1.42–1.36(m,2H)MS:m/z(ESI+)620.8[M+H] +。
实施例9. 6-((4-(2-氟-4-(1-(4-氟苯基)-2-氧哌啶-3-甲酰胺)苯氧基)-6-甲氧基喹啉-7-基)氧基)己酸
Figure PCTCN2022104421-appb-000040
实施例9的合成参照实施例1的合成方法进行。第3步反应中，用中间体原料6-溴代己酸甲酯替代4-溴代丁酸甲酯；在第5步反应中，用中间体1-(4-氟苯基)-2-氧哌啶-3-羧酸替代1-(4-氟苯基)-2-氧代-1,2-二氢吡啶-3-羧酸。
1H NMR(400MHz,DMSO)δ12.12(s,1H),8.54(d,J＝7.2Hz,1H),8.18(t,J＝6.8Hz,2H),7.66(s,2H),7.58-7.48(m,3H),7.38(t,J＝6.8Hz,2H),6.64(d,J＝6.8Hz,1H),4.02(t,J＝9.1Hz,2H),3.60(s,3H),3.50–3.38(m,5H),2.20(t,J＝8.0Hz,2H),2.06–1.81(m,3H),1.66–1.52(m,2H)1.48–1.30(m,2H)MS:m/z(ESI+)634.7[M+H] +。
实施例10. 4-((4-(4-(1-(4-氟苯基)-2-氧哌啶-3-甲酰胺)苯氧基)-6-甲氧基喹啉-7-基)氧基)丁酸
Figure PCTCN2022104421-appb-000041
实施例10的合成参照实施例1的合成方法进行。在第1步反应中，用4-硝基苯酚替代2-氟-4-硝基苯酚；在第5步反应中，用中间体1-(4-氟苯基)-2-氧哌啶-3-羧酸替代1-(4-氟苯基)-2-氧代-1,2-二氢吡啶-3-羧酸。
1H NMR(400MHz,DMSO)δ12.20(s,1H),8.52(d,J＝7.1Hz,1H),8.36(t,J＝6.8Hz,2H),7.68(s,2H),7.62-7.52(m,4H),7.30(t,J＝7.1Hz,2H),6.80(d,J＝6.8Hz,1H),4.18(t,J＝7.2Hz,2H),3.76(s,3H),3.70–3.40(m,5H),2.20(t,J＝9.0Hz,2H),1.94–1.70(m,3H)1.64–1.56(m,2H)MS:m/z(ESI+)588.6[M+H] +。
实施例11. 5-((4-(4-(1-(4-氟苯基)-2-氧哌啶-3-甲酰胺)苯氧基)-6-甲氧基喹啉-7-基)氧基)戊酸
Figure PCTCN2022104421-appb-000042
实施例11的合成参照实施例1合成方法进行。在第1步反应中，用4-硝基苯酚替代2-氟-4-硝基苯酚；第3步反应中，用中间体原料5-溴代戊酸甲酯替代4-溴代丁酸甲酯；在第5步反应中，用中间体1-(4-氟苯基)-2-氧哌啶-3-羧酸替代1-(4-氟苯基)-2-氧代-1,2-二氢吡啶-3-羧酸。
1H NMR(400MHz,DMSO)δ12.26(s,1H),8.74(d,J＝8.2Hz,1H),8.37(t,J＝7.1Hz,2H),7.72(s,2H),7.66-7.48(m,4H),7.36(t,J＝6.8Hz,2H),6.68(d,J＝7.1Hz,1H),4.02(t,J＝7.2Hz, 2H),3.54(s,3H),3.48–3.42(m,5H),2.28(t,J＝8.0Hz,2H),2.00–1.71(m,3H),1.66–1.60(m,2H)1.58–1.42(m,2H)MS:m/z(ESI+)602.5[M+H] +。
实施例12. 6-((4-(4-(1-(4-氟苯基)-2-氧哌啶-3-甲酰胺)苯氧基)-6-甲氧基喹啉-7-基)氧基)己酸
Figure PCTCN2022104421-appb-000043
实施例12的合成参照实施例1合成方法进行。在第1步反应中，用4-硝基苯酚替代2-氟-4-硝基苯酚；第3步反应中，用中间体原料6-溴代己酸甲酯替代4-溴代丁酸甲酯；在第5步反应中，用中间体1-(4-氟苯基)-2-氧哌啶-3-羧酸替代1-(4-氟苯基)-2-氧代-1,2-二氢吡啶-3-羧酸。
1H NMR(400MHz,DMSO)δ12.26(s,1H),8.74(d,J＝8.2Hz,1H),8.37(t,J＝7.1Hz,2H),7.72(s,2H),7.66-7.48(m,4H),7.36(t,J＝6.8Hz,2H),6.68(d,J＝7.1Hz,1H),4.02(t,J＝7.2Hz,2H),3.54(s,3H),3.48–3.42(m,5H),2.28(t,J＝8.0Hz,2H),2.00–1.71(m,3H),1.66–1.60(m,2H)1.58–1.42(m,2H)MS:m/z(ESI+)615.66[M+H] +。
实施例13. 5-((4-(2-氟-4-(1-(4-氟苯基)-2-氧代-2,5-二氢-1H-吡咯-3-甲酰胺)苯氧基)-6-甲氧基喹啉-7-基)氧基)戊酸
Figure PCTCN2022104421-appb-000044
实施例13的合成参照实施例1成方法进行。第3步反应中，用中间体原料5-溴代戊酸甲酯替代4-溴代丁酸甲酯；在第5步反应中，用中间体1-(4-氟苯基)-2-氧代-2,5-二氢-1H-吡咯-3-羧酸替代1-(4-氟苯基)-2-氧代-1,2-二氢吡啶-3-羧酸。
1H NMR(400MHz,DMSO)δ12.17(s,1H),8.70(d,J＝7.1Hz,1H),8.35(t,J＝7.2Hz,2H),7.70(s,2H),7.64-7.52(m,3H),7.32(t,J＝6.8Hz,2H),7.02(t,J＝2.4Hz,1H)6.64(d,J＝6.8Hz,1H),4.22(t,J＝1.6Hz,2H),4.08(t,J＝7.2Hz,2H),3.54(s,3H),3.50–3.36(m,5H),2.22(t,J＝8.0Hz,2H),MS:m/z(ESI+)604.6[M+H] +。
实施例14. 6-((4-(2-氟-4-(1-(4-氟苯基)-2-氧代-2,5-二氢-1H-吡咯-3-甲酰胺)苯氧基)-6-甲氧基喹啉-7-基)氧基)己酸
Figure PCTCN2022104421-appb-000045
实施例14的合成参照实施例1的合成方法进行。第3步反应中，用中间体原料6-溴代己酸甲酯替代4-溴代丁酸甲酯；在第5步反应中，用中间体1-(4-氟苯基)-2-氧代-2,5-二氢-1H-吡咯-3-羧酸替代1-(4-氟苯基)-2-氧代-1,2-二氢吡啶-3-羧酸。
1H NMR(400MHz,DMSO)δ12.21(s,1H),8.65(d,J＝6.4Hz,1H),8.24(t,J＝7.1Hz,2H),7.62(s,2H),7.56-7.46(m,3H),7.36(t,J＝7.4Hz,2H),7.14(t,J＝2.1Hz,1H)6.56(d,J＝6.8Hz,1H),4.30(t,J＝2.1Hz,2H),4.08(t,J＝6.8Hz,2H),3.64(s,3H),3.52–3.34(m,5H),2.28(t,J＝7.4Hz,2H),2.00–1.71(m,2H),MS:m/z(ESI+)618.6[M+H] +。
实施例15. 6-((4-(4-(1-(4-氟苯基)-2-氧代-2,5-二氢-1H-吡咯-3-甲酰胺)苯氧基)-6-甲氧基喹啉-7-基)氧基)己酸
Figure PCTCN2022104421-appb-000046
实施例15的合成参照实施例1的合成方法进行。在第1步反应中，用4-硝基苯酚替代2-氟-4-硝基苯酚；第3步反应中，用中间体原料6-溴代己酸甲酯替代4-溴代丁酸甲酯；在第5步反应中，用中间体1-(4-氟苯基)-2-氧代-2,5-二氢-1H-吡咯-3-羧酸替代1-(4-氟苯基)-2-氧代-1,2-二氢吡啶-3-羧酸。
1H NMR(400MHz,DMSO)δ12.16(s,1H),8.70(d,J＝7.1Hz,1H),8.45(t,J＝6.6Hz,2H),7.80(s,2H),7.74-7.62(m,4H),7.30(t,J＝7.1Hz,2H),7.12(t,J＝2.1Hz,1H)6.70(d,J＝7.8Hz,1H),4.22(t,J＝2.2Hz,2H),4.13(t,J＝9.0Hz,2H),3.60(s,3H),3.56–3.40(m,5H),2.22(t,J＝7.4Hz,2H),1.82–1.68(m,2H),MS:m/z(ESI+)600.5[M+H] +。
实施例16. 4-((4-(2-氟-4-(1-(4-氟苯基)-2-氧吡咯烷-3-甲酰胺)苯氧基)-6-甲氧基喹啉-7-基)氧基)丁酸
Figure PCTCN2022104421-appb-000047
实施例16的合成参照实施例1合成方法进行。在第5步反应中，用中间体1-(4-氟苯基)-2-氧吡咯烷-3-羧酸替代1-(4-氟苯基)-2-氧代-1,2-二氢吡啶-3-羧酸。
1H NMR(400MHz,DMSO)δ12.10(s,1H),8.63(d,J＝7.4Hz,1H),8.40(t,J＝6.8Hz,2H),7.50(s,2H),7.48-7.36(m,3H),7.24(t,J＝7.1Hz,2H),7.12(d,J＝4.2Hz,1H),4.30(t,J＝2.1Hz, 2H),4.20-4.12(m,2H),3.66(s,3H),3.58(t,J＝1.7Hz,1H)3.56–3.32(m,3H),,2.10–1.81(m,2H),MS:m/z(ESI+)592.6[M+H] +。
实施例-17. 5-((4-(2-氟-4-(1-(4-氟苯基)-2-氧吡咯烷-3-甲酰胺)苯氧基)-6-甲氧基喹啉-7-基)氧基)戊酸
Figure PCTCN2022104421-appb-000048
实施例17的合成参照实施例1成方法进行。第3步反应中，用中间体原料5-溴代戊酸甲酯替代4-代丁酸甲酯；在第5步反应中，用中间体1-(4-氟苯基)-2-氧吡咯烷-3-羧酸替代1-(4-氟苯基)-2-氧代-1,2-二氢吡啶-3-羧酸。
1H NMR(400MHz,DMSO)δ12.18(s,1H),8.70(d,J＝7.1Hz,1H),8.53(t,J＝7.1Hz,2H),7.54(s,2H),7.50-7.38(m,3H),7.22(t,J＝7.2Hz,2H),7.16(d,J＝3.6Hz,1H),4.34(t,J＝2.1Hz,2H),4.28-4.16(m,2H),3.72(s,3H),3.64(t,J＝1.7Hz,1H)3.60–3.38(m,3H),,2.08–1.80(m,2H),1.76–1.68(m,2H),MS:m/z(ESI+)606.7[M+H] +。
实施例18. 6-((4-(2-氟-4-(1-(4-氟苯基)-2-氧吡咯烷-3-甲酰胺)苯氧基)-6-甲氧基喹啉-7-基)氧基)己酸
Figure PCTCN2022104421-appb-000049
实施例18的合成参照实施例1合成方法进行。第3步反应中，用中间体原料6-溴代己酸甲酯替代4-代丁酸甲酯；在第5步反应中，用中间体1-(4-氟苯基)-2-氧吡咯烷-3-羧酸替代1-(4-氟苯基)-2-氧代-1,2-二氢吡啶-3-羧酸。
1H NMR(400MHz,DMSO)δ11.98(s,1H),8.64(d,J＝7.2Hz,1H),8.57(t,J＝6.8Hz,2H),7.66(s,2H),7.52-7.36(m,3H),7.18(t,J＝7.1Hz,2H),7.08(d,J＝4.2Hz,1H),4.52(t,J＝2.0Hz,2H),4.35-4.22(m,2H),3.86(s,3H),3.68(t,J＝2.1Hz,1H)3.64–3.40(m,3H),,2.32–2.08(m,2H),1.88–1.64(m,2H),1.50–1.44(m,2H)MS:m/z(ESI+)620.8[M+H] +。
实施例19. 4-((4-(4-(1-(4-氟苯基)-2-氧吡咯烷-3-甲酰胺)苯氧基)-6-甲氧基喹啉-7-基)氧基)丁酸
Figure PCTCN2022104421-appb-000050
实施例19的合成参照实施例1合成方法进行。在第1步反应中，用4-硝基苯酚替代2-氟-4-硝基苯酚；在第5步反应中，用中间体1-(苯基)-2-氧吡咯烷-3-羧酸替代1-(4-氟苯基)-2-氧代-1,2-二氢吡啶-3-羧酸。
1H NMR(400MHz,DMSO)δ11.86(s,1H),8.70(d,J＝7.2Hz,1H),8.48(t,J＝6.8Hz,2H),7.62(s,2H),7.58-7.44(m,4H),7.36(t,J＝6.8Hz,2H),7.22(d,J＝3.2Hz,1H),4.42(t,J＝1.7Hz,2H),4.34-4.38(m,2H),3.72(s,3H),3.64(t,J＝2.1Hz,1H)3.60–3.44(m,3H),,1.93–1.76(m,2H),MS:m/z(ESI+)573.58,[M+H] +。
实施例20. 5-((4-(4-(1-(4-氟苯基)-2-氧吡咯烷-3-甲酰胺)苯氧基)-6-甲氧基喹啉-7-基)氧基)戊酸
Figure PCTCN2022104421-appb-000051
实施例20的合成参照实施例1的合成方法进行。在第1步反应中，用4-硝基苯酚替代2-氟-4-硝基苯酚；第3步反应中，用中间体原料5-溴代戊酸甲酯替代4-溴代丁酸甲酯；在第5步反应中，用中间体1-苯基-2-氧吡咯烷-3-羧酸替代1-(4-氟苯基)-2-氧代-1,2-二氢吡啶-3-羧酸。
1H NMR(400MHz,DMSO)δ11.86(s,1H),8.70(d,J＝7.2Hz,1H),8.48(t,J＝6.8Hz,2H),7.62(s,2H),7.58-7.44(m,4H),7.36(t,J＝6.8Hz,2H),7.22(d,J＝3.2Hz,1H),4.42(t,J＝1.7Hz,2H),4.34-4.38(m,2H),3.72(s,3H),3.64(t,J＝2.1Hz,1H)3.60–3.44(m,3H),,1.93–1.76(m,2H),MS:m/z(ESI+)587.60[M+H] +。
实施例21. 6-((4-(4-(1-(4-氟苯基)-2-氧吡咯烷-3-甲酰胺)苯氧基)-6-甲氧基喹啉-7-基)氧基)己酸
Figure PCTCN2022104421-appb-000052
实施例21的合成参照实施例1的合成方法进行。在第1步反应中，用4-硝基苯酚替代2-氟-4-硝基苯酚；第3步反应中，用中间体原料6-溴代己酸甲酯替代4-溴代丁酸甲酯；第5步反应中，用中间体1-苯基-2-氧吡咯烷-3-羧酸替代1-(4-氟苯基)-2-氧代-1,2-二氢吡啶-3-羧酸。
1H NMR(400MHz,DMSO)δ11.72(s,1H),8.62(d,J＝7.2Hz,1H),8.40(t,J＝8.6Hz,2H),7.60(s,2H),7.44-7.32(m,4H),7.30(t,J＝7.1Hz,2H),7.04(d,J＝4.1Hz,1H),4.32(t,J＝2.1Hz,2H),4.26-4.12(m,2H),3.76(s,3H),3.60(t,J＝4.6Hz,1H)3.46–3.34(m,4H),2.38–2.26(m,2H),1.86–1.64(m,2H),1.52–1.38(m,2H)MS:m/z(ESI+)602.9[M+H] +。
实施例化合物生物活性评价
一.实施例化合物对AXL、KDR和CSF-1R激酶生化活性的抑制作用
用荧光素标记的酪氨酸激酶底物的磷酸化测定AXL、KDR和CSF-1R三种激酶的生化活性。
试剂：AXL、KDR和CSF-1R酪氨酸激酶(购自Carna公司)，
Figure PCTCN2022104421-appb-000053
KinEASE-TK试剂盒和96微孔板(购自Cisbio公司)，ATP(购自Sigma公司)，二硫苏糖醇(DTT)(购自Sigma公司)，氯化锰和氯化镁(购自Sigma公司)，XL-184(作为阳性对照)(购自上海瀚香生物科技有限公司)，全波长多功能酶标仪(TECAN
Figure PCTCN2022104421-appb-000054
M1000)。
方法：
根据试剂盒说明书，分别配AXL、KDR、CSF-1R酪氨酸激酶缓冲溶液(1×)，催化底物溶液(5×，5μM)，ATP溶液(500μM)，终止反应溶液(4×)。试验时三种激酶各自独立进行，分别往三块96微孔板的试验孔中加入AXL或KDR或CSF-1R激酶溶液2μL，各个浓度重复设置两孔；对照孔中加入2μL(1×)激酶缓冲溶液作为对照。实验孔和对照孔分别加入激酶缓冲液(4μL)，底物溶液(2μL)，ATP溶液(2μL)。上述反应混合物在37℃分别孵育0min、10min、20min、30min、40min、50min、60min，然后加入终止反应溶液10μL，再孵育1小时。用全波长多功能酶标仪在317nm为激发光波长下，检测665nm和620nm时的荧光信号强度，并根据665nm荧光信号强度/620nm荧光信号强度的比值，计算化合物产生50％抑制作用时的浓度(IC 50值)，结果见表1。AXL、KDR和CSF-1R激酶活性抑制率计算：百分抑制率＝(F DMSO对照-F 样品)/(F DMSO对照-F 阴性对照)×100％，加DMSO的值作为溶剂对照组，不加激酶的作为阴性对照组。
表1.实施例化合物对AXL、KDR和CSF-1R激酶生化活性的抑制作用(IC 50，nM)
实施例化合物 AXL活性(IC 50) KDR活性(IC 50) CSF-1R活性(IC 50)
1 11.62 9.11 11.57
2 4.41 6.34 9.11
3 5.01 7.12 8.35
4 20.15 15.70 17.32
5 15.79 20.91 25.48
6 20.44 17.23 24.68
7 30.57 22.62 39.52
8 50.34 34.73 40.29
9 6.27 7.09 10.18
10 51.15 47.32 57.02
11 67.93 100.72 87.32
12 52.11 36.82 61.47
13 6.32 8.37 9.56
14 15.22 10.63 12.75
15 10.19 17.20 15.68
16 25.31 34.09 55.47
17 23.38 30.07 60.51
18 7.23 9.15 11.92
19 27.94 40.11 30.08
20 16.62 19.38 20.25
21 40.97 27.19 30.93
XL-184 8.02 9.25 10.11
实验结果：
表1结果表明：实施例化合物能显著抑制AXL、KDR和CSF-1R激酶的生化活性。
二.实施例化合物对人白血病MOLM-13和人胃癌MKN-45细胞增殖的抑制作用
1.肿瘤细胞：对抗血管内皮生长因子耐药的MOLM-13急性白血病细胞株，人胃癌细胞MKN-45细胞获自南方医科大学药学院实验中心。
2.方法：用Luminometer发光法测定实施例化合物对MOLM-13急性白血病细胞株的抗增殖活性。
3.仪器：Promega微孔板检测仪(CellTiter-Glo Luminescent Cell Viability Assay，Promega)。
4.试剂和试剂盒：RPMI1640培养基，胎牛血清，二甲基亚砜，青霉素-链霉素，Cell Titer-Gio检测试剂盒。细胞培养瓶、CO 2培养箱，细胞培养微孔板(96孔板)。
5.肿瘤细胞培养：液氮冻存的MOLM-13肿瘤细胞复苏，用含10％胎牛血清，加10％青霉素-链霉素的RPMI细胞培养基培养细胞；当细胞生长至指数增长期时，用移液枪吸取培养基轻轻吹打并收集细胞，将细胞重悬于培养液中；每孔接种5000-10000个细胞，细胞均匀分布；细胞在恒温37℃、5％CO 2、饱和湿度的二氧化碳培养箱中连续培养72小时。
6.实施例化合物与活性测定：实施例化合物用二甲基亚砜溶解，配成10个浓度梯度，根据实验设计在测试孔中加入不同浓度的化合物继续培养72h。培养结束后取出96孔板在室温下放置30分钟，然后检测CTG。在微孔中加入100μL CTG，混匀2分钟，在室温下放置10分钟。然后用微孔板发光仪检测并记录发光信号值观察细胞活力，并计算IC 50值，结果见表2。
表2.实施例化合物对人白血病MOLM-13和人胃癌MKN-45细胞增殖的抑制作用
Figure PCTCN2022104421-appb-000055
Figure PCTCN2022104421-appb-000056
表2结果表明：实施例化合物能显著抑制人白血病MOLM-13细胞的增殖和人胃癌MKN-45细胞的增殖。
三.实施例化合物在体内对人白血病MOLM-13细胞的抗肿瘤作用
1.细胞培养：人白血病MOLM-13细胞在含10％的胎牛血清，100U/mL青霉素和100μg/mL链霉素的DMEM培养基中，培养瓶置于37℃、5％CO 2、饱和湿度的二氧化碳培养箱中培养。
2.实验动物：BALB/C-nu小鼠购自广东药康生物科技有限公司雌性，40只，日龄28-34天。许可证号：SCXK(粤)2020-0054，实验单位使用许可证编号：SYXK(粤)2018-0131。5只小鼠于一笼，动物在SPF动物房饲养，经一周检验后用于肿瘤细胞接种实验。
3.MOLM-13细胞裸鼠移植瘤模型：MOLM-13细胞数目达到指数生长期时，用移液枪轻轻吹散成团的悬浮细胞，并收集至离心管中，在1000r/min，离心5min，收集离心管底部的细胞，用无血清培养基重悬细胞，并调整细胞浓度为2×10 7/mL。用1mL注射器吸取细胞悬液，接种于裸鼠右前腋皮下，每只裸鼠接种0.1mL，接种的细胞数2×10 6/只。肿瘤细胞接种后，每日或定期观察裸鼠接种部位的肿瘤生长，并用游标卡尺测量每只动物肿瘤的大小，当肿瘤长体积达到100mm 3以上时，则可以入选进行实验。在接种肿瘤细胞的40只裸鼠中，有30只达到入选标准。
4.实验分组和给药：实验随机分为4组，包括溶剂对照组，阳性化合物对照组以及实施例化合物2.5mg/kg和5mg/kg两个剂量组。以50％PEG 400和50％蒸馏水溶解化合物3(小鼠每日给予的PEG400总量控在0.5％以下)，药物每日一次，每只小鼠每次0.1mL灌胃给药，连续给药7天。每天称量动物体重，每两天测量每只动物肿瘤大小，同时观察小鼠饮食，饮水、体重、毛发等基本情况变化。
5.化合物抗肿瘤作用的评价方法：给药结束后，荷瘤小鼠麻醉整体拍照，并取出肿瘤并 称重(瘤重以平均数±SD表示)，计算抑瘤率。
5-1.肿瘤体积(tumor volume，TV)的计算：
TV＝1/2×a×b 2
其中，a为肿瘤的长径，b为肿瘤的短径。
抑瘤率(Tumor Growth Inhibition，TGI)的计算：
Figure PCTCN2022104421-appb-000057
5-2.裸鼠给药前后体重变化百分比的计算：
Figure PCTCN2022104421-appb-000058
BW为裸鼠体重(Body Weight)，BW 最后一天为实验结束时的裸鼠体重，BW 第一天为分组给药时的裸鼠体重。
5-3.实验结果
如图1所示，实施例3的化合物在MOLM-13细胞移植瘤裸鼠连续灌胃给药7天，表现出非常显著的抗肿瘤作用，起效快。
如图2所示，比较了给药前后的体重变化，2.5mg/kg给药组在给药期间未观察到动物体重的下降；5mg/kg给药组的体重增加，显示化合物的低毒性。
对本申请的其他化合物同法检测，结果与实施例3的化合物类似。

Claims (10)

  1. 通式(I)所示的化合物、其异构体、其药学上可接受的盐或其氘代物：
    Figure PCTCN2022104421-appb-100001
    其中，
    R 1为C 1-C 6烷基，R 2为
    Figure PCTCN2022104421-appb-100002
    或R 2为C 1-C 6烷基，R 1为
    Figure PCTCN2022104421-appb-100003
    Y选自C 1-C 10亚烷基、C 1-C 10亚烷基胺基、C 1-C 10亚烷基氧基、C 1-C 10卤代亚烷基、亚哌啶基、C 1-C 10亚烷基哌啶基、亚哌嗪基或C 1-C 10亚烷基哌嗪基；
    G 1和G 2分别独立地选自氢、氘、卤素、C 1-C 6烷基、C 1-C 6卤代烷基或C 1-C 6烷氧基；
    W选自如下基团：
    Figure PCTCN2022104421-appb-100004
  2. 根据权利要求1所述的化合物，其中，R 1为C 1-C 3烷基，R 2为
    Figure PCTCN2022104421-appb-100005
    Figure PCTCN2022104421-appb-100006
    或R 2为C 1-C 3烷基，R 1为
    Figure PCTCN2022104421-appb-100007
    Y选自C 3-C 6亚烷基、C 3-C 6亚烷基胺基、C 3-C 6亚烷基氧基、C 3-C 6卤代亚烷基。
  3. 根据权利要求2所述的化合物，其中，R 1为C 1-C 3烷基，R 2为
    Figure PCTCN2022104421-appb-100008
    Figure PCTCN2022104421-appb-100009
    Y选自C 3-C 5亚烷基或C 3-C 5卤代亚烷基。
  4. 根据权利要求1所述的化合物，其中，G 1和G 2分别独立地选自氢、氘、氟、氯、溴或碘。
  5. 根据权利要求1所述的化合物，其中，W选自如下基团：
    Figure PCTCN2022104421-appb-100010
  6. 根据权利要求1-5任一项所述的化合物，其中，所述化合物选自：
    Figure PCTCN2022104421-appb-100011
    Figure PCTCN2022104421-appb-100012
  7. 药物组合物，其包括权利要求1-6任一项所述的化合物、其异构体、其药学上可接受的盐或其氘代物，以及药学上可接受的赋形剂。
  8. 权利要求1-6任一项所述的化合物、其异构体、其药学上可接受的盐或其氘代物或者权利要求7所述的药物组合物在制备用于预防或治疗由酪氨酸蛋白激酶异常引起的疾病的药物中的用途；
    优选地，所述酪氨酸蛋白激酶选自受体酪氨酸激酶(AXL)、血管内皮生长因子受体2(KDR)和集落刺激因子-1受体(CSF-1R)中的一种或多种；
    优选地，所述由酪氨酸蛋白激酶异常引起的疾病包括实体瘤如胃癌、肺癌、乳腺癌，血 液系统肿瘤，炎症性疾病和自身免疫性疾病。
  9. 权利要求1-6任一项所述的化合物、其异构体、其药学上可接受的盐或其氘代物或者权利要求7所述的药物组合物在制备用于抑制酪氨酸蛋白激酶的药物中的用途；
    优选地，所述酪氨酸蛋白激酶选自受体酪氨酸激酶(AXL)、血管内皮生长因子受体2(KDR)和集落刺激因子-1受体(CSF-1R)中的一种或多种。
  10. 预防或治疗由酪氨酸蛋白激酶异常引起的疾病的方法，其包括向有需要的对象施用有效量的权利要求1-6任一项所述的化合物、其异构体、其药学上可接受的盐或其氘代物或者权利要求7所述的药物组合物；
    优选地，所述酪氨酸蛋白激酶选自受体酪氨酸激酶(AXL)、血管内皮生长因子受体2(KDR)和集落刺激因子-1受体(CSF-1R)中的一种或多种；
    优选地，所述由酪氨酸蛋白激酶异常引起的疾病包括实体瘤如胃癌、肺癌、乳腺癌，血液系统肿瘤，炎症性疾病和自身免疫性疾病。
PCT/CN2022/104421 2022-06-29 2022-07-07 一种酪氨酸蛋白激酶抑制剂及其用途 WO2024000615A1 (zh)

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