WO2018137610A1 - Substituted 1-(isoxazole-3-yl)-3-(3-fluorine-4-phenyl)urea derivative, and preparation method therefor and use thereof - Google Patents

Substituted 1-(isoxazole-3-yl)-3-(3-fluorine-4-phenyl)urea derivative, and preparation method therefor and use thereof Download PDF

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WO2018137610A1
WO2018137610A1 PCT/CN2018/073793 CN2018073793W WO2018137610A1 WO 2018137610 A1 WO2018137610 A1 WO 2018137610A1 CN 2018073793 W CN2018073793 W CN 2018073793W WO 2018137610 A1 WO2018137610 A1 WO 2018137610A1
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phenyl
substituted
group
halogen
alkoxy
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杨胜勇
李琳丽
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四川大学
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    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
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    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
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    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
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    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
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    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • 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
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    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/04—Ortho-condensed systems
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Definitions

  • the invention belongs to the technical field of organic synthetic drugs, and in particular relates to substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivatives and preparation methods and uses thereof.
  • AML Acute myeloid leukemia
  • FLT3 FMS-like tyrosine kinase 3
  • FLT3 gene mutations of which FLT3 gene internal tandem repeat mutation (FLT3-ITD), accounting for about 23% of patients with primary AML, FLT3 tyrosine kinase domain single amino acid mutation (FLT3-TKD), accounting for approximately 7% of AML patients.
  • FLT3-ITD FLT3 gene internal tandem repeat mutation
  • FLT3-TKD FLT3 tyrosine kinase domain single amino acid mutation
  • FLT3 inhibitors have been reported, some of which have entered clinical trials, including: MLN518, KW-2449, TKI-258, sorafenib, TCS-359, punatinib (AP24534), DCC- 2036, SKLB-1028, AC220, etc.
  • AC220 is highly selective for FLT3, and the chemotherapy-resistant relapsing patient population, the rate of complete remission of the compound of up to 44 to 54% 1,2.
  • late molecular mechanism studies showed that after continuous treatment with AC220, FLT3 kinase produced a second mutation on the basis of the original mutation, which led to drug binding difficulties and drug resistance.
  • the present invention provides a substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative having the structural formula shown in Formula I:
  • X is carbon or nitrogen
  • Y is nitrogen, oxygen or sulfur
  • R 1 and R 2 are independently -H, -NO 2 , halogen, a phenyl group, a 5- to 12-membered saturated or unsaturated heterocyclic group; or a combination of R 1 and R 2 to form a ring, the ring being a substituted phenyl group, a substituted 5 to 12 membered saturated or unsaturated heterocyclic group
  • the 5-12 saturated or unsaturated heterocyclic group contains 1 to 2 hetero atoms; the hetero atom is nitrogen, oxygen or sulfur;
  • R 6 is a C 1 -C 6 alkyl group, a halogen, a C 1 -C 6 alkoxy group, Or phenyl.
  • X is carbon or nitrogen; Y is oxygen;
  • R 1 and R 2 are independently -H, -NO 2 , halogen, a phenyl group, a 5- to 12-membered saturated or unsaturated heterocyclic group; or a combination of R 1 and R 2 to form a ring, the ring being a substituted phenyl group, a substituted 5 to 12 membered saturated or unsaturated heterocyclic group
  • the 5-12 saturated or unsaturated heterocyclic group contains 1 to 2 hetero atoms; the hetero atom is nitrogen, oxygen or sulfur;
  • R 6 is a C 1 -C 6 alkyl group, a halogen, a C 1 -C 6 alkoxy group, Or phenyl.
  • X is carbon or nitrogen; Y is oxygen;
  • R 1 and R 2 are independently -H, -NO 2 , halogen, Or a 5- to 10-membered saturated or unsaturated heterocyclic group; or R 1 and R 2 are combined to form a ring, said ring being a substituted phenyl group, a substituted 5 to 10 membered saturated or unsaturated heterocyclic group;
  • the 5- to 10-membered saturated or unsaturated heterocyclic group has 1 to 2 hetero atoms; the hetero atom is nitrogen, oxygen or sulfur;
  • R 6 is a C 1 -C 6 alkyl group, a halogen, a C 1 -C 6 alkoxy group, Or phenyl.
  • X is carbon or nitrogen; Y is oxygen;
  • R 1 and R 2 are independently -H, -NO 2 , halogen, Or a 5- to 6-membered saturated heterocyclic group; or R 1 and R 2 are combined to form a ring, said ring being a substituted phenyl group, a substituted 5 to 10 membered unsaturated heterocyclic group; said 5 to 10 a mono-saturated or unsaturated heterocyclic group containing 1 to 2 hetero atoms; the hetero atom is nitrogen or oxygen;
  • R 6 is a C 1 -C 6 alkyl group, a halogen, a C 1 -C 6 alkoxy group, Or phenyl.
  • X is carbon or nitrogen; Y is oxygen;
  • R 1 and R 2 are independently -H, -NO 2 , halogen, Or a 5- to 6-membered saturated heterocyclic group; or R 1 and R 2 are combined to form a ring, said ring being a substituted phenyl group, a substituted 5- to 6-membered unsaturated heterocyclic group; said 5 to 6 a monosaturated heterocyclic group or a 5 to 10 membered unsaturated heterocyclic group having 1 to 2 hetero atoms; the hetero atom is nitrogen or oxygen;
  • R 6 is a C 1 -C 6 alkyl group, a halogen, a C 1 -C 6 alkoxy group, Or phenyl.
  • X is carbon or nitrogen; Y is oxygen;
  • R 1 and R 2 are independently -H, -NO 2 , halogen, Or a 5- to 6-membered saturated heterocyclic group; or R 1 and R 2 are combined to form a ring, said ring being a substituted phenyl group, a substituted 5- to 6-membered unsaturated heterocyclic group; said 5 to 6 a monosaturated heterocyclic group or a 5 to 10 membered unsaturated heterocyclic group having 1 to 2 hetero atoms; the hetero atom is nitrogen or oxygen;
  • R 6 is halogen, C 1 -C 6 alkoxy, Or phenyl.
  • X is carbon or nitrogen; Y is oxygen;
  • R 1 and R 2 are independently -H, -NO 2 , halogen, Or a 5- to 6-membered saturated heterocyclic group; or R 1 and R 2 are combined to form a ring, said ring being a substituted phenyl group, a substituted 5- to 6-membered unsaturated heterocyclic group; said 5 to 6 a monosaturated heterocyclic group or a 5- to 10-membered unsaturated heterocyclic group containing one nitrogen atom;
  • R 6 is a C 1 -C 6 alkoxy group, Or phenyl.
  • R 6 is a C 1 -C 6 alkyl group, a halogen, a C 1 -C 6 alkoxy group, Or phenyl.
  • R 6 is a C 1 -C 6 alkyl group, a halogen, a C 1 -C 6 alkoxy group, Or phenyl.
  • R 6 is a C 1 -C 6 alkyl group, a halogen, a C 1 -C 6 alkoxy group, Or phenyl.
  • R 6 is halogen, C 1 -C 6 alkoxy, Or phenyl.
  • R 6 is halogen, C 1 -C 6 alkoxy or
  • the present invention also provides a process for the preparation of the above substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative.
  • the raw material 4 and the pyrimidine derivative are dissolved in N,N-dimethylformamide, and then the cesium carbonate is added, and the reaction is carried out at 120 to 140 ° C for 4 to 6 hours, and then the reaction liquid is concentrated and purified by column chromatography to obtain Intermediate 11; the molar ratio of the starting material 4 and the pyrimidine derivative is 5:6;
  • the present invention also provides pharmaceutically acceptable salts and hydrates of the above substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivatives.
  • the present invention also provides pharmaceutically acceptable salts and hydrates of the above substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivatives.
  • the invention also provides prodrugs of the compounds of Formula I, Formula II and Formula III.
  • the prodrugs are derivatives of the compounds of the above formula I, formula II and formula III which may themselves have weak or even no activity, but after administration, under physiological conditions (for example by metabolism) , solvolysis or otherwise) is converted to the corresponding biologically active form.
  • the present invention also provides a composition which is pharmaceutically acceptable by the above substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative provided by the present invention. Prepared from auxiliary ingredients.
  • the present invention also provides the above substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative, a salt or hydrate thereof for preparing FMS-like tyrosine Use in kinase 3 inhibitors.
  • the above substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative, its salt or hydrate is tandemly repeated in tandem against the FLT3 tyrosine kinase Mutation of FLT3-ITD/F691L or FLT3-ITD/D835Y inhibitors in a single amino acid mutation based on the original internal tandem repeat mutation (FLT3-ITD) of FLT3 tyrosine kinase use.
  • the present invention also provides the above-mentioned substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative, a salt or a hydrate thereof for preparing a medicament for treating acute myeloid leukemia Use in.
  • the present invention also provides the above-mentioned substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative, a salt or a hydrate thereof for preparing an anti-autoimmune disease drug Use in.
  • the present invention also provides the above substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative, its salt or hydrate in the preparation of a neovascularization inhibitor the use of.
  • the present invention also provides the use of the above substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative, a salt or hydrate thereof for preparing an antitumor drug .
  • the compound of the invention effectively overcomes various drug resistances produced by the clinical drug AC220, and has a better therapeutic effect especially for the secondary mutation resistance FLT3-ITD/F691L and FLT3-ITD/D835Y.
  • the present invention provides a novel class of pyrazolopyrimidine derivatives which are predominantly substituted at the 4-position and which provide a simple, efficient and cost-effective process for the preparation of the pyrazolopyrimidine derivatives of the present invention.
  • the 1-(5-(tert-butyl)isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative of the invention has good inhibitory activity against various kinases, and is applicable to various entities.
  • Tumor, leukemia and autoimmune diseases have inhibitory effects, providing new effective options for the preparation of kinase inhibitors, preparation of anti-autoimmune diseases, preparation of neovascularization inhibitors and anti-tumor drugs. , has a good application prospects.
  • Figure 1 shows the results of in vitro apoptosis assay of compound SKLB707.
  • Figure 2 shows the results of in vitro detection of FLT3 signaling pathway by compound SKLB707.
  • Figure 2B shows the inhibitory effects of the compounds SKLB707 and AC220 on key proteins in the FLT3 signaling pathway.
  • Figure 3 shows the results of in vivo pharmacodynamic test of compound SKLB707. Different doses of SKLB707 completely abolished mouse subcutaneous tumors (Fig. 3A), and the body weight of the mice did not decrease (Fig. 3B), and the state of the mice did not change significantly.
  • the starting material 1 (4,4-dimethyl-3-oxopentanonitrile) (1.25 g, 10 mM) was weighed into a 250 mL round bottom flask containing 100 mL of water, then hydroxylamine hydrochloride (759 mg, 11 mM) and hydroxide were added. Sodium (440 mg, 11 mM), the reaction solution was stirred at room temperature for half an hour, and then an appropriate amount of sodium hydroxide was added to adjust the pH to 8-9, and then the temperature was raised to 50 ° C for 10 h. After the TCL detection reaction was completed, 100 mL of methyl t-butyl ether was added to the solution in three portions for extraction, and the organic layer was discarded.
  • reaction liquid was concentrated under reduced pressure, and water and a dilute hydrochloric acid solution were added to adjust the pH to 3-4, and then extracted with ethyl acetate (150 mL), and added to the aqueous layer three times, and the ethyl acetate layer was collected. Then, the ethyl acetate layer was washed three times with brine, and the ethyl acetate layer was dried over anhydrous sodium sulfate. Yield 71%), HPLC purity 95%.
  • Example 9 The synthesis method was the same as in Example 9, except that 4-chloro-6-methoxypyrido[3,4-d]pyrimidine in Example 9 was replaced with 4-chloro-6-ethylpyrido[3,4- d] Pyrimidine, the reaction treatment gave an off-white powder compound 11b, the reaction yield was 18.6%, and the HPLC purity was 96%.
  • Example 9 The synthesis method was the same as in Example 9, except that 4-chloro-6-methoxypyrido[3,4-d]pyrimidine in Example 9 was replaced with (2-methoxyethoxy)pyridine [3, 4-d]pyrimidine as a reactant, which was subjected to a reaction to give white powdery compound 11c, yield: 16.6%, HPLC purity 97%.
  • Example 9 The synthesis method was the same as in Example 9, except that 4-chloro-6-methoxypyrido[3,4-d]pyrimidine in Example 9 was replaced with 4-(2-((4-chloropyridin[3, 4-d]pyrimidin-6-yl)oxy)ethyl)morpholine was used as the reaction product, and the reaction was afforded to give a white solid solid compound 11d, yield: 14.8%, HPLC purity: 96%.
  • EtOAc EtOAc
  • Example 14 The synthesis method was the same as that in Example 14, and the 4,7-dichloroquinazoline in Example 14 was replaced with 4-chloro-6,7-dimethoxyquinazoline as a reaction product, and the reaction was carried out to obtain an off-white solid compound SKLB707. The yield was 78.34%.
  • Example 14 The synthesis method was the same as in Example 14, and the 4,7-dichloroquinazoline in Example 14 was replaced with 4-chloro-6,7-bis(2-methoxyethoxy)quinazoline as a reactant.
  • the reaction treatment gave Compound 13c as an off-white solid (yield: 48.66%).
  • the raw material 2d (4-chloro-6,7-dimethoxyquinoline, 500 mg, 1.0 mM) and the starting material 4 (4-nitro-2-fluorophenol, 704 mg, 2.0 mM) were weighed into a 100 mL round bottom flask. Add 10 mL of 2,6-lutidine to dissolve the mixture, then add the organic base 1,8-diazabicycloundec-7-ene, raise the temperature to 147 ° C for 8 h, then concentrate under reduced pressure.
  • 6-Acetoxy-4-chloro-7-methoxyquinazoline 100 mg, 1.0 mM
  • Intermediate 3 116 mg, 1.0 mM
  • potassium carbonate 157 mg, 2.0 mM
  • the analysis method finally gave 13 g of a yellow compound (yield: 62.5%).
  • the raw material 2k (7-benzyloxy-4-chloro-6-methoxyquinazoline, 1 g, 1.0 mM) was weighed into a 100 mL round bottom flask, dissolved in 20 mL of methanol, and then palladium on carbon (706 g, 2.0). mM), the temperature was raised to 70 ° C under nitrogen for 4 h, and the reaction mixture was suction filtered and concentrated under reduced pressure to give 6-acetoxy-4-chloro-7-methoxy quinazoline.
  • 6-Acetoxy-4-chloro-7-methoxyquinazoline 200 mg, 1.0 mM was weighed into a 100 mL round bottom flask, and a mixed solution of 10 mL of NaOH aqueous solution and dioxane (1:1) was added. The compound was dissolved, and the mixture was stirred at room temperature for 8 hours, and then the mixture was adjusted to pH 3-4, and the organic phase was concentrated to give a yellow solid 4-chloro-7-methoxyquinazoline-6-ol.
  • Example 23 The synthesis method was the same as in Example 23, and the 3,4-dihydro-7-methoxy-4-oxoquinazolin-6-ol acetate in Example 23 was replaced with 4-(2-(( 4-Chloro-7-methoxyquinolin-6-yl)oxy)ethyl)morpholine was used as the reaction product, and the reaction was afforded to give a pale white solid compound 13l.
  • Example 24 The synthesis method was the same as in Example 24, and the 7-benzyloxy-4-chloro-6-methoxyquinazoline in Example 23 was replaced with 4-(2-((4-chloro-6-methylquin).
  • Oxazolin-7-yl)oxy)ethyl)morpholine was used as the reactant, which was subjected to a reaction to give a pale white solid compound 13 m.
  • Example 23 The synthesis method was the same as in Example 23, and the 3,4-dihydro-7-methoxy-4-oxoquinazolin-6-ol acetate in Example 23 was replaced with 4-(3-(( 4-Chloro-7-methoxyquinolin-6-yl)oxy)propyl)morpholine was used as the reaction product, which was subjected to a reaction to give a pale white solid compound 13n.
  • Example 24 The synthesis method was the same as in Example 24, and the 7-benzyloxy-4-chloro-6-methoxyquinazoline in Example 23 was replaced with 4-(4-((4-chloro-6-methylquin).
  • Oxazolin-7-yl)oxy)butyl)morpholine is the reactant, which is reacted to give the compound 13o as an off-white solid.
  • Example 23 The synthesis method was the same as in Example 23, and the 3,4-dihydro-7-methoxy-4-oxoquinazolin-6-ol acetate in Example 23 was replaced with 3-((4-chloro). -7-Methoxyquinolin-6-yl)oxy)-N,N-dimethylpropan-1-amine was obtained as a reaction mixture.
  • Example 24 The synthesis method was the same as in Example 24, and the 7-benzyloxy-4-chloro-6-methoxyquinazoline in Example 23 was replaced with 4-((4-chloro-6-methylquinazoline- 7-yl)oxy)-N,N-dimethylbutan-1-amine is the reactant, which is reacted to give the compound 13r as an off-white solid.
  • Raw material 3a (4,6-dichloro-5-methoxypyrimidine, 168 mg, 1.2 mM) and starting material 4 (4-amino-2-fluorophenol, 100 mg, 1.0 mM) were weighed into a 100 mL round bottom flask and added.
  • Raw material 3b (4-chloro-N-methylmethylpyridine amide, 500 mg, 1.0 mM) and starting material 4 (4-amino-2-fluorophenol, 745 mg, 2.0 mM) were weighed into a 100 mL round bottom flask, and 10 mL was added. The N,N-dimethylformamide was dissolved in the mixture, then cesium carbonate (3820 mg, 4.0 mM) was added, and the temperature was raised to 130 ° C for 5 h, and then concentrated under reduced pressure to a mixture of 300-400 mm of crude silica gel, using DCM.
  • Raw material 3c (4-chloro-1H-pyrrolo[2,3-b]pyridine, 500 mg, 1.0 mM) and starting material 4 (4-nitro-2-fluorophenol, 516 mg, 1.2 mM) were weighed into a 100 mL round bottom. In the flask, 10 mL of 2,6-lutidine was added to dissolve the mixture, then 4-dimethylaminopyridine (401 mg, 1.0 mM) was added, and the temperature was raised to 147 ° C for 8 hours, and then concentrated under reduced pressure to add 300-400 mm.
  • T7 phage strain Escherichia coli, HEK-293 cells, streptavidin-coated magnetic beads, SeaBlock (Pierce, for termination of reaction), 1% BSA (bovine serum albumin), 0.05% Tween (spit Temperature) 20, 1 mM DTT (dithiothreitol), 0.17x PBS (phosphate buffer), 6 mM DTT, polystyrene 96-well plate, 1 x PBS, 0.5 ⁇ M non-biotinylated affinity ligand, 100% DMSO (dimethyl sulfoxide), the above materials were supplied by DiscoverRX (USA). AC220 and Ponatinib (Punatinib) were purchased from Selleck Chemicals (Shanghai, China).
  • the obtained phage is infected with HEK-293 cells, and the protein protein to be detected is produced by using the protein expression system in the host cell, and the kinase protein produced by the method has a DNA fragment, and in the subsequent experiments, the DNA content can be detected by qPCR technology.
  • the amount of kinase was determined indirectly.
  • the affinity to the kinase to be tested was selected and the streptavidin-coated magnetic beads were treated with biotin-labeled small molecule ligand for 30 min to produce an affinity resin for the kinase assay.
  • the treated magnetic beads were then incubated with excess biotin and rinsed with blocking buffer (SeaBlock, 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and reduce non-specific binding.
  • blocking buffer SeaBlock, 1% BSA, 0.05% Tween 20, 1 mM DTT
  • the test enzyme with the DNA fragment, the magnetic beads with the small molecule ligand and the specific concentration of the compound solution are added to the binding buffer (20% SeaBlock, 0.17x PBS, 0.05% Tween 20, 6mM DTT).
  • the binding reaction is carried out. All reactions were carried out in polystyrene 96-well plates and the final volume of the reaction solution was 0.135 ml.
  • the reaction system was shaken at room temperature for 1 h, and the affinity magnetic beads were washed with a washing buffer (1 x PBS, 0.05% Tween 20). The beads were then resuspended in elution buffer (1 x PBS, 0.05% Tween 20, 0.5 ⁇ M non-biotinylated affinity ligand) and shaken for 30 minutes at room temperature.
  • the kinase concentration in the eluate was measured by qPCR. Eleven test points for a 3-fold gradient dilution of each test compound were prepared in 100% DMSO and subsequently diluted to 1 x at the time of assay (final DMSO concentration was 1%).
  • the binding ability (kd value) of the compound of the present invention to the mutated FLT3 kinases (FLT3-ITD, FLT3 (D835Y), FLT3-ITD/F691L, and FLT3-ITD/D835V) was tested by the above test methods.
  • the Kd values of specific compounds are shown in Table 1. Among them, "-" means not tested.
  • test compound SKLB707 is significantly better than that of positive compound AC220 for FLT3-ITD/F691L and FLT3-ITD/D835V mutants; for FLT3-ITD The /D835V mutant, the activity of the test compound SKLB707 was also significantly better than the positive compound Ponatinib (Punatinib).
  • the purpose of this experiment was to test the potentiation inhibitory activity of the compound of the present invention against an AC220-resistant secondary mutant cell line using the MTT (tetramethylazozolium salt) colorimetric method.
  • RPMI-1640 medium was purchased from Gibco BRL (Invitrogen Corporation, USA), fetal bovine serum was purchased from Pan-Biotech (Germany), IL-3 (interleukin-3) was purchased from PeproTech, G418 (genetic mold) Originally purchased from Life Science. Tetramethylazozolium salt (MTT), sodium dodecyl sulfate (SDS) and DMSO (dimethyl sulfoxide) are products of Sigma (USA). The compound was formulated into a 10 mM stock solution in 100% DMSO, stored in a refrigerator at -20 ° C in the dark, and diluted to the desired concentration with a complete medium at the time of use.
  • MTT trimethylazozolium salt
  • SDS sodium dodecyl sulfate
  • DMSO dimethyl sulfoxide
  • Cell line and culture The cell line used in this experiment, mouse B lymphocyte Ba/F3 and acute myeloid leukemia cell MV4-11 were purchased from American ATCC (American type culture collection) and kept by our laboratory.
  • Ba/F3 cells were cultured in RPMI-1640 complete medium containing 10% fetal bovine serum, 100 U/mL penicillin, 100 ⁇ g/mL streptomycin, 10 ng/ml IL-3 at 5% CO 2 at 37 °C.
  • Ba/F3FLT3-ITD, Ba/F3FLT3-ITD/D835V and Ba/F3FLT3-ITD/F691L using RPMI-1640 with 10% fetal bovine serum, 100 U/mL penicillin, 100 ⁇ g/mL streptomycin and 500 ⁇ g/mL G418 culture medium at 5% CO 2, 37 °C.
  • a 100 [mu]L gradient of test compound was added to a 96-well plate at a DMSO concentration of 0.1% with 3 replicate wells per dose.
  • a cell suspension having a cell concentration of 2 ⁇ 10 5 /mL was adjusted with a complete cell culture medium, and seeded in a 96-well plate, 100 ⁇ l of a cell suspension per well.
  • a drug-free negative control group and an equal volume of a complete medium control group were cultured at 37 ° C under 5% CO 2 .
  • test compound not only has strong inhibitory activity against the AC220-sensitive cell line Ba/F3FLT3-ITD, but also the cell line with the AC220 resistance mutation Ba/F3Ba/F3FLT3-ITD/D835V and FLT3.
  • -ITD/F691L also has good inhibition ability, which can effectively overcome the clinical resistance mutation of AC220.
  • RPMI-1640 and IMDM media were purchased from Gibco BRL (Invitrogen Corporation, USA), fetal bovine serum was purchased from Pan-Biotech (Germany), and G418 was purchased from Life Science.
  • PBS was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.
  • the apoptosis detection kit was purchased from Shanghai Biyuntian Biotechnology Co., Ltd.
  • the compound was formulated into a 10 mM stock solution in 100% DMSO, stored in a refrigerator at -20 ° C in the dark, and diluted to the desired concentration with a complete medium at the time of use.
  • Cell lines and culture cell lines Ba/F3FLT3-ITD, Ba/F3FLT3-ITD/D835V and Ba/F3FLT3-ITD/F691L containing 10% fetal bovine serum, 100 U/mL penicillin, 100 ⁇ g/mL streptomycin, 500 ⁇ g/ ml G418 in complete RPMI-1640 medium at 5% CO 2, 37 °C conditions.
  • MV4-11 cells were cultured in IMDM complete medium containing 20% fetal bovine serum at 5% CO 2 at 37 °C.
  • the cells to be tested were seeded in a 6-well plate at a number of 2 ⁇ 10 5 /well per well, while different concentrations of the test compound SKLB707 solution were added, and the 6-well plate was placed in a cell culture incubator for 24 hours. Thereafter, the cell suspension was collected into a corresponding flow tube and washed twice with PBS, followed by resuspension of the cells by adding 500 ⁇ L of Binding Buffer (binding buffer). After the cells were resuspended, 5 ⁇ L of Annexin V-FITC (phospholipid binding protein-fluorescein isothiocyanate) solution and 5 ⁇ L of PI (propidium iodide) solution were sequentially added to the flow tube, and the mixture was mixed. Incubate for 5-10 min in the dark at room temperature and immediately test by flow cytometry.
  • Binding Buffer binding buffer
  • SKLB707 can induce significant apoptosis in cells.
  • SKLB707 can also induce significant apoptosis in cells, and increase the concentration of apoptotic cells.
  • SKLB707 induced apoptosis in MV4-11 cells at 1, 5, 10, 50 and 100 nM, respectively, at 38.2%, 65.2%, 70.3%, 73.2% and 81.25%; SKLB707 at 1, 5, 10, 50 and 100 nM
  • the proportions of Ba/F3FLT3-ITD cells induced apoptosis were 6.4%, 39.1%, 44.2%, 73.3% and 81.25%, respectively;
  • SKLB707 induced Ba/F3FLT3-ITD/F691L cells at 1, 5, 10, 50 and 100 nM
  • the proportion of death was 5.18%, 6.6%, 11.67%, 37.1% and 45.35%, respectively.
  • the proportion of SKLB707 induced apoptosis in Ba/F3FLT3-ITD/D835V cells at 1, 5, 10, 50 and 100 nM was 5.66%. 7.72%, 13.08%, 39.11% and 60.01%.
  • the purpose of this experiment was to examine the inhibition of the FLT3 signaling pathway by the inventive compound SKLB707 at the cellular level in vitro.
  • MV4-11 and the laboratory self-constructed cell lines Ba/F3FLT3-ITD, Ba/F3FLT3-ITD/D835V and Ba/F3FLT3-ITD/F691L were used to treat the cells for a certain period of time, and the total protein was extracted by Western blot.
  • the method detects the expression level of a signal related to the signal pathway of FLT3.
  • RPMI-1640 and IMDM medium were purchased from Gibco BRL (Invitrogen Corporation, USA), fetal bovine serum was purchased from Pan-Biotech (Germany), G418 was purchased from Life Science, and physiological saline was purchased from Sichuan Kelun.
  • RIPA lysate was purchased from Shanghai Biyuntian Biotechnology Co., Ltd.
  • Cocktail prote inhibitor
  • PMSF phenylmethylsulfonyl fluoride
  • the FLT3 antibody was purchased from Santa Cruz, the ⁇ -actin antibody was purchased from Beijing Zhongshan Jinqiao Company, and the remaining antibodies were purchased from Cell Signaling Technology.
  • HRP (horseradish peroxidase)-labeled secondary antibody was purchased from Beijing Zhongshan Jinqiao Co., Ltd.
  • PVDF membrane (polyvinylidene fluoride membrane) and developing substrate were purchased from Millipore.
  • the compound was formulated into a 10 mM stock solution in 100% DMSO, stored in a refrigerator at -20 ° C in the dark, and diluted to the desired concentration with a complete medium at the time of use.
  • Cell lines and culture cell lines Ba/F3FLT3-ITD, Ba/F3FLT3-ITD/D835V and Ba/F3FLT3-ITD/F691L containing 10% fetal bovine serum, 100 U/mL penicillin, 100 ⁇ g/mL streptomycin and 500 ⁇ g/
  • the RPMI-1640 complete medium of ml G418 was cultured at 5% CO 2 at 37 °C.
  • MV4-11 was cultured in IMDM complete medium containing 20% fetal bovine serum at 5% CO 2 at 37 °C.
  • the cells to be tested were seeded into a 6-well plate, and after appropriate cell density was amplified, test compounds of different concentrations were added, and cultured at 37 ° C, 5% CO 2 for 1 hour.
  • the cells were collected by centrifugation, the cells were washed twice with pre-cooled physiological saline, and the cells were simultaneously transferred to a 1.5 mL centrifuge tube, and the supernatant was aspirated and discarded, and an appropriate volume of RIPA lysate (containing 1% cocktail and 1) was added. %PMSF). Vortex to allow the cells to completely resuspend in the lysate and immediately lyse on ice. After about 15 min, cell disruption was performed using an ultrasonic disruption apparatus.
  • the treated cells were then centrifuged (13000 r/min, 15 min) in a low temperature high speed centrifuge to remove cell debris.
  • the protein was quantified by BCA (bisquinolinecarboxylic acid) method, and a standard curve was prepared by using BSA (bovine serum albumin) standard protein. The concentration of each group of proteins was calculated according to the standard curve, and the concentration of each group of protein samples was unified, and 5 ⁇ was added thereto.
  • the protein loading buffer was then placed in a 100 ° C dry thermostat for 10 min to denature the protein.
  • the processed protein samples were dispensed in the required amount and stored at -20 ° C until use. Protein samples avoid repeated freeze-thaw cycles.
  • the protein is separated.
  • the protein was transferred to the PVDF membrane by the trough wet transfer method, and the PVDF membrane was placed in TBS/T (trishydroxymethylaminomethane-hydrochloric acid/Tween containing 5% (m:v) skim milk powder.
  • TBS/T trishydroxymethylaminomethane-hydrochloric acid/Tween containing 5% (m:v) skim milk powder.
  • Block in the buffered saline solution for 2 h cut the protein bands of different molecular weights, dilute the primary antibody in the dilution ratio recommended by the corresponding antibody instructions and incubate the bands overnight at 4 ° C in the refrigerator.
  • each band was taken out, rinsed with TBS/T buffer, and then added with a 1:5000 dilution of HRP-labeled secondary antibody, incubated at 37 ° C for 1 h, followed by elution with TBS/T to remove excess antibody on the PVDF membrane.
  • the HRP substrate was evenly added dropwise and developed in a rapid gel imaging system.
  • Figure 2B shows the inhibitory effects of the compounds SKLB707 and AC220 on key proteins in the FLT3 signaling pathway.
  • pFLT3 represents phosphorylated FLT3
  • Total FLT3 represents total FLT3 level
  • pSTAT5 represents phosphorylated STAT5
  • Total STAT5 represents total STAT5 content
  • pErk1/2 represents phosphorylated Erk1/2
  • Total Erk1/2 represents total Erk1/2 content
  • ⁇ -actin indicates the content of ⁇ -actin.
  • SKLB707 can inhibit the autophosphorylation level of FLT3 receptor in a dose-dependent manner without affecting The expression level of total protein of FLT3.
  • phosphorylation of STAT5 and Erk1/2, downstream proteins of FLT3 kinase was also significantly inhibited.
  • the same dose of AC220 did not significantly inhibit the phosphorylation level of these proteins, indicating that after two mutations in FLT3 kinase, SKLB707 can still bind to its active region, overcoming AC220 resistance.
  • MV4-11 was subcutaneously inoculated into NOD SCID mice to construct a tumor xenograft model, and AC220 was used as a positive control to detect the inhibition of subcutaneous tumor growth by SKLB707.
  • IMDM medium was purchased from Gibco BRL (Invitrogen Corporation, USA) and fetal calf serum was purchased from Pan-Biotech (Germany).
  • PEG400 polyethylene glycol 400
  • DMSO was purchased from Sigma Aldrich (Shanghai) Trading Co., Ltd.
  • p-FLT3, p-STAT5, and p-ERK antibodies were purchased from Cell Signaling Technology.
  • MV4-11 was cultured in IMDM complete medium containing 20% fetal bovine serum at 5% CO 2 at 37 °C.
  • NOD SCID mice female 5-6 week old immunodeficient mice
  • the MV4-11 cells were expanded and the cells in the logarithmic growth cycle were collected.
  • the cells were resuspended in IMDM medium containing no serum and antibiotics, centrifuged and washed three times, and the final cell count was 1 ⁇ 10 8 cells/mL.
  • a subcutaneous tumor model of MV4-11 mice was constructed by inoculating 100 ⁇ L of the cell suspension to the back of the right side of NOD SCID mice. When the tumor grew to a volume of about 400 mm 3 , the mice were randomly divided into 5 groups of 6 animals each, and the gavage administration was started.
  • the five groups were the solvent control group, the SKLB707 1 mg/kg group, the SKLB707 3 mg/kg group, the SKLB707 10 mg/kg group, and the AC220 3 mg/kg group.
  • the 5% DMSO, 25% PEG400 (50% PEG400 with sterilized water) and 75% sterilized water were used as solvents to sequentially add the weighed compounds to prepare different concentrations of the drug stock solution. After being prepared, store in a refrigerator at 4 ° C for use. Oral gavage was administered once a day, tumor volume and mouse body weight were measured every three days, and the experiment was terminated after 21 days of administration.
  • SKLB707 10mg/kg group can completely eliminate tumors after 11 days of administration; SKLB707 1mg/kg group, SKLB707 3mg/kg group and AC220 3mg/kg group can completely eliminate tumors after 12 days of administration, indicating that SKLB707 inhibits tumor growth The effect is equivalent to AC220.
  • Mouse tumor xenograft model experiments show that SKLB707 has a good anti-tumor effect in vivo.

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Abstract

A substituted 1-(isoxazole-3-yl)-3-(3-fluorine-4-phenyl)urea derivative, and a preparation method therefor and a use thereof, relating to the technical field of organic synthesis of medicines. A substituted 1-(isoxazole-3-yl)-3-(3-fluorine-4-phenyl)urea derivative is provided, and the structural formula thereof is as represented by formula (I). Also provided is a preparation method for the substituted 1-( isoxazole-3-yl)-3-(3- fluorine-4-phenyl)urea derivative, and a use in preparing an FMS-fins-like tyrosine kinase 3 inhibitor. The present invention provides a novel effective choice for preparation of kinase inhibitors, preparation of medicines for treating autoimmune diseases, and preparation of angiogenesis inhibitors and antitumor medicines in the art, and has a very good application prospect.

Description

取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物及其制备方法和用途Substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative, preparation method and use thereof 技术领域Technical field
本发明属于有机合成药物技术领域，特别涉及取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物及其制备方法和用途。The invention belongs to the technical field of organic synthetic drugs, and in particular relates to substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivatives and preparation methods and uses thereof.
背景技术Background technique
急性髓性白血病(AML)是骨髓性白细胞异常增殖的一种血液肿瘤。近年来，随着分子生物学、疾病基因组学等的发展，人们发现了一些与AML发生发展密切相关的靶基因，为AML的有效治疗提供了新的选择。在这些靶基因中，FMS-样酪氨酸激酶3(FLT3)被证明是其中最重要的一种。FLT3在造血干细胞、树突细胞、B细胞和自然杀伤细胞前体细胞的增殖和分化的调节中起着重要作用。研究表明，约三分之一的AML患者发生了FLT3基因突变，其中FLT3基因内部串联重复突变(FLT3-ITD)，约占原发性AML患者的23％，FLT3酪氨酸激酶域单个氨基酸突变(FLT3-TKD)，约占AML患者的7％。另外，大量研究也表明FLT3突变与AML患者的预后直接相关。因此，FLT3被认为是AML治疗的有效靶点。研发FLT3抑制剂已成为当前的研究热点。Acute myeloid leukemia (AML) is a blood tumor that abnormally proliferates myeloid leukocytes. In recent years, with the development of molecular biology, disease genomics, etc., some target genes closely related to the development of AML have been discovered, which provides a new choice for the effective treatment of AML. Among these target genes, FMS-like tyrosine kinase 3 (FLT3) proved to be the most important one. FLT3 plays an important role in the regulation of proliferation and differentiation of hematopoietic stem cells, dendritic cells, B cells, and natural killer cell precursor cells. Studies have shown that about one-third of AML patients have FLT3 gene mutations, of which FLT3 gene internal tandem repeat mutation (FLT3-ITD), accounting for about 23% of patients with primary AML, FLT3 tyrosine kinase domain single amino acid mutation (FLT3-TKD), accounting for approximately 7% of AML patients. In addition, a large number of studies have also shown that FLT3 mutations are directly related to the prognosis of patients with AML. Therefore, FLT3 is considered to be an effective target for AML treatment. The development of FLT3 inhibitors has become a hot research topic.
目前已有多个FLT3抑制剂报道，其中部分抑制剂已进入临床试验，包括：MLN518、KW-2449、TKI-258、索拉非尼、TCS-359、普纳替尼(AP24534)、DCC-2036、SKLB-1028、AC220等。这些抑制剂中，AC220对FLT3具有高度选择性，在复发型和化疗耐药型病人群体中，该化合物的完全缓解率可达44％至54％ 1,2。但是后期分子机制研究显示，AC220连续治疗后，FLT3激酶在原有突变的基础上产生二次突变，导致药物结合困难和耐药的发生。主要突变位点集中在位于“activation loop”的第835位天冬氨酸(D835)和位于“gatekeeper”的第691位苯丙氨酸(F691) 3。这些点突变的出现，使得用于AML治疗的药物研发又遇到了新的挑战。因此，研发可克服FLT3耐药突变的新型高活性FLT3抑制剂已成为当前的一项迫切任务和研究热点之一。 Several FLT3 inhibitors have been reported, some of which have entered clinical trials, including: MLN518, KW-2449, TKI-258, sorafenib, TCS-359, punatinib (AP24534), DCC- 2036, SKLB-1028, AC220, etc. These inhibitors, AC220 is highly selective for FLT3, and the chemotherapy-resistant relapsing patient population, the rate of complete remission of the compound of up to 44 to 54% 1,2. However, late molecular mechanism studies showed that after continuous treatment with AC220, FLT3 kinase produced a second mutation on the basis of the original mutation, which led to drug binding difficulties and drug resistance. The major mutation sites are concentrated in the 835th aspartic acid (D835) located in the "activation loop" and the 691th phenylalanine (F691) 3 located in the "gatekeeper". The emergence of these point mutations has brought new challenges to drug development for AML treatment. Therefore, the development of new high-activity FLT3 inhibitors that can overcome FLT3 resistance mutations has become one of the current urgent tasks and research hotspots.
发明内容Summary of the invention
本发明提供了一种取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物，其结构式如式Ⅰ所 示：The present invention provides a substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative having the structural formula shown in Formula I:
Figure PCTCN2018073793-appb-000001
Figure PCTCN2018073793-appb-000001
其中，X为碳或氮；Y为氮、氧或硫；Wherein X is carbon or nitrogen; Y is nitrogen, oxygen or sulfur;
R 1、R 2独立地为-H、-NO 2、卤素、
Figure PCTCN2018073793-appb-000002
苯基、5～12元饱和或不饱和的杂环基；或者R 1和R 2组合成环，所述的环为取代的苯基、取代的5～12元饱和或不饱和的杂环基；所述5～12饱和或不饱和的杂环基含有1～2个杂原子；所述的杂原子为氮、氧或硫； R 1 and R 2 are independently -H, -NO 2 , halogen,
Figure PCTCN2018073793-appb-000002
a phenyl group, a 5- to 12-membered saturated or unsaturated heterocyclic group; or a combination of R 1 and R 2 to form a ring, the ring being a substituted phenyl group, a substituted 5 to 12 membered saturated or unsaturated heterocyclic group The 5-12 saturated or unsaturated heterocyclic group contains 1 to 2 hetero atoms; the hetero atom is nitrogen, oxygen or sulfur;
所述取代的苯基、取代的5～12元饱和或不饱和杂环基的取代基为-H、卤素、C 1～C 6烷基、C 1～C 6烷氧基、
Figure PCTCN2018073793-appb-000003
n＝1～4； The substituent of the substituted phenyl group or the substituted 5- to 12-membered saturated or unsaturated heterocyclic group is -H, halogen, C 1 -C 6 alkyl group, C 1 -C 6 alkoxy group,
Figure PCTCN2018073793-appb-000003
n=1～4;
R 6为C 1～C 6烷基、卤素、C 1～C 6烷氧基、
Figure PCTCN2018073793-appb-000004
或苯基。 R 6 is a C 1 -C 6 alkyl group, a halogen, a C 1 -C 6 alkoxy group,
Figure PCTCN2018073793-appb-000004
Or phenyl.
作为本发明优选的方案，X为碳或氮；Y为氧；As a preferred embodiment of the invention, X is carbon or nitrogen; Y is oxygen;
R 1、R 2独立地为-H、-NO 2、卤素、
Figure PCTCN2018073793-appb-000005
苯基、5～12元饱和或不饱和的杂环基；或者R 1和R 2组合成环，所述的环为取代的苯基、取代的5～12元饱和或不饱和的杂环基；所述5～12饱和或不饱和的杂环基含有1～2个杂原子；所述的杂原子为氮、氧或硫； R 1 and R 2 are independently -H, -NO 2 , halogen,
Figure PCTCN2018073793-appb-000005
a phenyl group, a 5- to 12-membered saturated or unsaturated heterocyclic group; or a combination of R 1 and R 2 to form a ring, the ring being a substituted phenyl group, a substituted 5 to 12 membered saturated or unsaturated heterocyclic group The 5-12 saturated or unsaturated heterocyclic group contains 1 to 2 hetero atoms; the hetero atom is nitrogen, oxygen or sulfur;
所述取代的苯基、取代的5～12元饱和或不饱和杂环基的取代基为-H、卤素、C 1～C 6烷基、C 1～C 6烷氧基、
Figure PCTCN2018073793-appb-000006
n＝1～4； The substituent of the substituted phenyl group or the substituted 5- to 12-membered saturated or unsaturated heterocyclic group is -H, halogen, C 1 -C 6 alkyl group, C 1 -C 6 alkoxy group,
Figure PCTCN2018073793-appb-000006
n=1～4;
R 6为C 1～C 6烷基、卤素、C 1～C 6烷氧基、
Figure PCTCN2018073793-appb-000007
或苯基。 R 6 is a C 1 -C 6 alkyl group, a halogen, a C 1 -C 6 alkoxy group,
Figure PCTCN2018073793-appb-000007
Or phenyl.
优选的，X为碳或氮；Y为氧；Preferably, X is carbon or nitrogen; Y is oxygen;
R 1、R 2独立地为-H、-NO 2、卤素、
Figure PCTCN2018073793-appb-000008
或5～10元饱和或不饱和的杂环基；或者R 1和R 2组合成环，所述的环为取代的苯基、取代的5～10元饱和或不饱和的杂环基；所述的5～10元饱和或不饱和的杂环基含有1～2个杂原子；所述的杂原子为氮、氧或硫； R 1 and R 2 are independently -H, -NO 2 , halogen,
Figure PCTCN2018073793-appb-000008
Or a 5- to 10-membered saturated or unsaturated heterocyclic group; or R 1 and R 2 are combined to form a ring, said ring being a substituted phenyl group, a substituted 5 to 10 membered saturated or unsaturated heterocyclic group; The 5- to 10-membered saturated or unsaturated heterocyclic group has 1 to 2 hetero atoms; the hetero atom is nitrogen, oxygen or sulfur;
所述取代的苯环、取代的5～10元饱和或不饱和的杂环基上的取代基为-H、卤素、C 1～C 4烷氧基、
Figure PCTCN2018073793-appb-000009
n＝1～4； The substituent on the substituted benzene ring, the substituted 5- to 10-membered saturated or unsaturated heterocyclic group is -H, halogen, C 1 -C 4 alkoxy,
Figure PCTCN2018073793-appb-000009
n=1～4;
R 6为C 1～C 6烷基、卤素、C 1～C 6烷氧基、
Figure PCTCN2018073793-appb-000010
或苯基。 R 6 is a C 1 -C 6 alkyl group, a halogen, a C 1 -C 6 alkoxy group,
Figure PCTCN2018073793-appb-000010
Or phenyl.
进一步优选的，X为碳或氮；Y为氧；Further preferably, X is carbon or nitrogen; Y is oxygen;
R 1、R 2独立地为-H、-NO 2、卤素、
Figure PCTCN2018073793-appb-000011
或5～6元饱和的杂环基；或者R 1和R 2组合成环，所述的环为取代的苯基、取代的5～10元不饱和的杂环基；所述的5～10元饱和或不饱和杂环基含有1～2个杂原子；所述的杂原子为氮或氧； R 1 and R 2 are independently -H, -NO 2 , halogen,
Figure PCTCN2018073793-appb-000011
Or a 5- to 6-membered saturated heterocyclic group; or R 1 and R 2 are combined to form a ring, said ring being a substituted phenyl group, a substituted 5 to 10 membered unsaturated heterocyclic group; said 5 to 10 a mono-saturated or unsaturated heterocyclic group containing 1 to 2 hetero atoms; the hetero atom is nitrogen or oxygen;
所述取代的苯基、取代的5～6元不饱和的杂环基上的取代基为-H、卤素、C 1～C 4烷氧基、
Figure PCTCN2018073793-appb-000012
n＝1～4； The substituent on the substituted phenyl group or the substituted 5- to 6-membered unsaturated heterocyclic group is -H, halogen, C 1 -C 4 alkoxy group,
Figure PCTCN2018073793-appb-000012
n=1～4;
R 6为C 1～C 6烷基、卤素、C 1～C 6烷氧基、
Figure PCTCN2018073793-appb-000013
或苯基。 R 6 is a C 1 -C 6 alkyl group, a halogen, a C 1 -C 6 alkoxy group,
Figure PCTCN2018073793-appb-000013
Or phenyl.
更进一步优选的，X为碳或氮；Y为氧；Still more preferably, X is carbon or nitrogen; Y is oxygen;
R 1、R 2独立地为-H、-NO 2、卤素、
Figure PCTCN2018073793-appb-000014
或5～6元饱和的杂环基；或者R 1和R 2组合成环，所述的环为取代的苯基、取代的5～6元不饱和的杂环基；所述的5～6元饱和杂环基或5～10元不饱和杂环基含有1～2个杂原子；所述的杂原子为氮或氧； R 1 and R 2 are independently -H, -NO 2 , halogen,
Figure PCTCN2018073793-appb-000014
Or a 5- to 6-membered saturated heterocyclic group; or R 1 and R 2 are combined to form a ring, said ring being a substituted phenyl group, a substituted 5- to 6-membered unsaturated heterocyclic group; said 5 to 6 a monosaturated heterocyclic group or a 5 to 10 membered unsaturated heterocyclic group having 1 to 2 hetero atoms; the hetero atom is nitrogen or oxygen;
所述取代的苯基、取代的5～10元不饱和的杂环基上的取代基为卤素、C 1～C 4烷氧基、
Figure PCTCN2018073793-appb-000015
n＝1～4； The substituent on the substituted phenyl group or the substituted 5- to 10-membered unsaturated heterocyclic group is a halogen, a C 1 -C 4 alkoxy group,
Figure PCTCN2018073793-appb-000015
n=1～4;
R 6为C 1～C 6烷基、卤素、C 1～C 6烷氧基、
Figure PCTCN2018073793-appb-000016
或苯基。 R 6 is a C 1 -C 6 alkyl group, a halogen, a C 1 -C 6 alkoxy group,
Figure PCTCN2018073793-appb-000016
Or phenyl.
再进一步优选的，X为碳或氮；Y为氧；Still further preferably, X is carbon or nitrogen; Y is oxygen;
R 1、R 2独立地为-H、-NO 2、卤素、
Figure PCTCN2018073793-appb-000017
或5～6元饱和的杂环基；或者R 1和R 2组合成环，所述的环为取代的苯基、取代的5～6元不饱和的杂环基；所述的5～6元饱和杂环基或5～10元不饱和杂环基含有1～2个杂原子；所述的杂原子为氮或氧； R 1 and R 2 are independently -H, -NO 2 , halogen,
Figure PCTCN2018073793-appb-000017
Or a 5- to 6-membered saturated heterocyclic group; or R 1 and R 2 are combined to form a ring, said ring being a substituted phenyl group, a substituted 5- to 6-membered unsaturated heterocyclic group; said 5 to 6 a monosaturated heterocyclic group or a 5 to 10 membered unsaturated heterocyclic group having 1 to 2 hetero atoms; the hetero atom is nitrogen or oxygen;
所述苯基、取代的5～10元不饱和的杂环基上的取代基为-H、卤素、C 1～C 4烷氧基、
Figure PCTCN2018073793-appb-000018
或
Figure PCTCN2018073793-appb-000019
n＝1～4； The substituent on the phenyl group, the substituted 5- to 10-membered unsaturated heterocyclic group is -H, a halogen, a C 1 -C 4 alkoxy group,
Figure PCTCN2018073793-appb-000018
or
Figure PCTCN2018073793-appb-000019
n=1～4;
R 6为卤素、C 1～C 6烷氧基、
Figure PCTCN2018073793-appb-000020
或苯基。 R 6 is halogen, C 1 -C 6 alkoxy,
Figure PCTCN2018073793-appb-000020
Or phenyl.
最优的，X为碳或氮；Y为氧；Optimally, X is carbon or nitrogen; Y is oxygen;
R 1、R 2独立地为-H、-NO 2、卤素、
Figure PCTCN2018073793-appb-000021
或5～6元饱和的杂环基；或者R 1和R 2组合成环，所述的环为取代的苯基、取代的5～6元不饱和的杂环基；所述的5～6元饱和杂环基或5～10 元不饱和杂环基含有1个氮原子； R 1 and R 2 are independently -H, -NO 2 , halogen,
Figure PCTCN2018073793-appb-000021
Or a 5- to 6-membered saturated heterocyclic group; or R 1 and R 2 are combined to form a ring, said ring being a substituted phenyl group, a substituted 5- to 6-membered unsaturated heterocyclic group; said 5 to 6 a monosaturated heterocyclic group or a 5- to 10-membered unsaturated heterocyclic group containing one nitrogen atom;
所述苯基、取代的5～10元不饱和的杂环基上的取代基为-H、-Cl、C 1～C 4烷氧基、
Figure PCTCN2018073793-appb-000022
或
Figure PCTCN2018073793-appb-000023
n＝1～4； The substituent on the phenyl group, the substituted 5- to 10-membered unsaturated heterocyclic group is -H, -Cl, C 1 -C 4 alkoxy,
Figure PCTCN2018073793-appb-000022
or
Figure PCTCN2018073793-appb-000023
n=1～4;
R 6为C 1～C 6烷氧基、
Figure PCTCN2018073793-appb-000024
或苯基。 R 6 is a C 1 -C 6 alkoxy group,
Figure PCTCN2018073793-appb-000024
Or phenyl.
上述取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物，当X为氮，Y为氧，R 1和R 2环合为取代的苯环时，其结构式如式Ⅱ所示： The above substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative, wherein X is nitrogen, Y is oxygen, and R 1 and R 2 are cyclized to be substituted When the benzene ring is used, its structural formula is as shown in formula II:
Figure PCTCN2018073793-appb-000025
Figure PCTCN2018073793-appb-000025
其中，R 3、R 4独立地为-H、卤素、C 1～C 4烷氧基、
Figure PCTCN2018073793-appb-000026
n＝1～4； Wherein R 3 and R 4 are independently -H, halogen, C 1 -C 4 alkoxy,
Figure PCTCN2018073793-appb-000026
n=1～4;
R 6为C 1～C 6烷基、卤素、C 1～C 6烷氧基、
Figure PCTCN2018073793-appb-000027
或苯基。 R 6 is a C 1 -C 6 alkyl group, a halogen, a C 1 -C 6 alkoxy group,
Figure PCTCN2018073793-appb-000027
Or phenyl.
作为本发明优选的方案，R 3、R 4独立地为-H、卤素、C 1～C 4烷氧基、
Figure PCTCN2018073793-appb-000028
n＝1～4；R 6为卤素、C 1～C 6烷氧基、
Figure PCTCN2018073793-appb-000029
或苯基。 As a preferred embodiment of the present invention, R 3 and R 4 are independently -H, halogen, C 1 -C 4 alkoxy,
Figure PCTCN2018073793-appb-000028
n=1～4; R 6 is halogen, C 1 -C 6 alkoxy,
Figure PCTCN2018073793-appb-000029
Or phenyl.
优选的，R 3、R 4独立地为-H、卤素、C 1～C 4烷氧基、
Figure PCTCN2018073793-appb-000030
n＝1～4；R 6为C 1～C 6烷氧基、
Figure PCTCN2018073793-appb-000031
或苯基。 Preferably, R 3 and R 4 are independently -H, halogen, C 1 -C 4 alkoxy,
Figure PCTCN2018073793-appb-000030
n=1 to 4; R 6 is a C 1 -C 6 alkoxy group,
Figure PCTCN2018073793-appb-000031
Or phenyl.
最优的，R 3、R 4独立地为-H、C 1～C 4烷氧基、
Figure PCTCN2018073793-appb-000032
n＝1～4；R 6为C 1～C 6烷氧基、
Figure PCTCN2018073793-appb-000033
或苯基。 Most preferably, R 3 and R 4 are independently -H, C 1 -C 4 alkoxy,
Figure PCTCN2018073793-appb-000032
n=1 to 4; R 6 is a C 1 -C 6 alkoxy group,
Figure PCTCN2018073793-appb-000033
Or phenyl.
上述取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物，当X为氮，Y为氧，R 1和R 2环合为含有1个氮原子的取代不饱和6元杂环时，其结构式如式Ⅲ所示： The above substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative, wherein X is nitrogen, Y is oxygen, and R 1 and R 2 are cyclized to contain 1 When a nitrogen atom is substituted with an unsaturated 6-membered heterocyclic ring, its structural formula is as shown in Formula III:
Figure PCTCN2018073793-appb-000034
Figure PCTCN2018073793-appb-000034
其中，R 5为-H、卤素、C 1～C 4烷氧基、
Figure PCTCN2018073793-appb-000035
n＝1～4； Wherein R 5 is -H, halogen, C 1 -C 4 alkoxy,
Figure PCTCN2018073793-appb-000035
n=1～4;
R 6为C 1～C 6烷基、卤素、C 1～C 6烷氧基、
Figure PCTCN2018073793-appb-000036
或苯基。 R 6 is a C 1 -C 6 alkyl group, a halogen, a C 1 -C 6 alkoxy group,
Figure PCTCN2018073793-appb-000036
Or phenyl.
作为本发明优选的方案，R 5为-H、卤素、C 1～C 4烷氧基或
Figure PCTCN2018073793-appb-000037
n＝1～4；R 6为C 1～ C 6烷基、卤素、C 1～C 6烷氧基、
Figure PCTCN2018073793-appb-000038
或苯基。 As a preferred embodiment of the present invention, R 5 is -H, halogen, C 1 -C 4 alkoxy or
Figure PCTCN2018073793-appb-000037
n=1 to 4; R 6 is a C 1 -C 6 alkyl group, a halogen, a C 1 -C 6 alkoxy group,
Figure PCTCN2018073793-appb-000038
Or phenyl.
优选的，R 5为-H、卤素、C 1～C 4烷氧基或
Figure PCTCN2018073793-appb-000039
n＝1～4；R 6为卤素、C 1～C 6烷氧基、
Figure PCTCN2018073793-appb-000040
或苯基。 Preferably, R 5 is -H, halogen, C 1 -C 4 alkoxy or
Figure PCTCN2018073793-appb-000039
n=1～4; R 6 is halogen, C 1 -C 6 alkoxy,
Figure PCTCN2018073793-appb-000040
Or phenyl.
最优的，R 5为-H、卤素、C 1～C 4烷氧基或
Figure PCTCN2018073793-appb-000041
n＝1～4；R 6为卤素、C 1～C 6烷氧基或
Figure PCTCN2018073793-appb-000042
Most preferably, R 5 is -H, halogen, C 1 -C 4 alkoxy or
Figure PCTCN2018073793-appb-000041
n=1～4; R 6 is halogen, C 1 -C 6 alkoxy or
Figure PCTCN2018073793-appb-000042
上述取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物，其结构式为：The above substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative having the structural formula:
Figure PCTCN2018073793-appb-000043
Figure PCTCN2018073793-appb-000043
本发明还提供了上述取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物的制备方法。The present invention also provides a process for the preparation of the above substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative.
路线一，式Ⅲ所示化合物的合成路线：Route 1, the route of the compound shown by formula III:
Figure PCTCN2018073793-appb-000044
Figure PCTCN2018073793-appb-000044
上述式Ⅲ所示化合物的合成步骤为：The synthetic steps of the compound of the above formula III are as follows:
a)将原料1、盐酸羟胺和氢氧化钠于常温下反应半小时，然后加入氢氧化钠调节pH至8～9，再升温至40～60℃反应8～12h；TCL检测反应完全后，反应液用甲基叔丁基醚萃取，弃去有机层，水层再用DCM(二氯甲烷)进行萃取，收集DCM层，用饱和食盐水洗涤、干燥、过滤之后，除去溶剂即得N'-羟基-4,4-二甲基-3-羰基戊脒；将N'-羟基-4,4-二甲基-3-羰基戊脒溶于水中，然后用浓盐酸调节溶液的pH至4～5，然后升高温度到40～60℃，反应2～4小时；反应完全后，将反应液冷却至室温，在0～5℃缓慢将溶液pH值调节至11～13，过滤出沉淀，低温烘干，得到中间体2；所述原料1、盐酸羟胺和氢氧化钠的摩尔比为10:11:11；a) The raw material 1, hydroxylamine hydrochloride and sodium hydroxide are reacted at room temperature for half an hour, then sodium hydroxide is added to adjust the pH to 8-9, and then the temperature is raised to 40-60 ° C for 8-12 hours; after the TCL detection reaction is complete, the reaction The liquid was extracted with methyl tert-butyl ether, the organic layer was discarded, and the aqueous layer was extracted with DCM (dichloromethane). The DCM layer was collected, washed with saturated brine, dried and filtered, and then solvent was obtained to obtain N'- Hydroxy-4,4-dimethyl-3-carbonylpentanyl; dissolving N'-hydroxy-4,4-dimethyl-3-carbonylpentanquinone in water, then adjusting the pH of the solution to 4~ with concentrated hydrochloric acid 5, then raise the temperature to 40 ~ 60 ° C, the reaction is 2 ~ 4 hours; after the reaction is complete, the reaction solution is cooled to room temperature, slowly adjust the pH of the solution to 11 ~ 13 at 0 ~ 5 ° C, filter out the precipitate, low temperature Drying to obtain intermediate 2; the molar ratio of the starting material 1, hydroxylamine hydrochloride and sodium hydroxide is 10:11:11;
b)在0～5℃，向三光气和乙酸乙酯中缓慢滴加中间体2的乙酸乙酯溶液，然后再在0～5℃缓慢滴加三乙胺，然后升温至60～80℃反应2～4小时；反应完毕后，将有机相除去，然后加入4-氨基-2-氟苯酚的乙酸乙酯溶液，然后反应液于60～80℃下加热反应4～6h；反应完毕后，将反应液中有机相除去后，加入碳酸钾的饱和水溶液萃，取并取有机层经柱层析提纯，得到中间体3；所述三光气、中间体2、三乙胺、4-氨基-2-氟苯酚的摩尔比为17.8:35.7:71.4:35；b) slowly add dropwise the ethyl acetate solution of the intermediate 2 to the triphosgene and ethyl acetate at 0 to 5 ° C, and then slowly add triethylamine at 0 to 5 ° C, and then raise the temperature to 60 to 80 ° C. 2~4 hours; after the reaction is completed, the organic phase is removed, then 4-amino-2-fluorophenol in ethyl acetate solution is added, and then the reaction solution is heated at 60-80 ° C for 4-6 hours; after the reaction is completed, After the organic phase in the reaction mixture is removed, a saturated aqueous solution of potassium carbonate is added to extract, and the organic layer is purified by column chromatography to obtain intermediate 3; the phosgene, intermediate 2, triethylamine, 4-amino-2. - the molar ratio of fluorophenol is 17.8:35.7:71.4:35;
c)将中间体4和BOC酸酐(二碳酸二叔丁酯)溶在二氧六环中，升温至80～110℃，反应6～10h；然后浓缩反应液，调节pH值为3～4，随后萃取，收集有机层，经洗涤、干燥、过滤，滤液除去有机相，得到中间体5；所述中间体4和BOC酸酐的摩尔比为15.6:18.7；c) dissolving intermediate 4 and BOC anhydride (di-tert-butyl dicarbonate) in dioxane, raising the temperature to 80-110 ° C, and reacting for 6-10 h; then concentrating the reaction solution to adjust the pH to 3-4. Subsequently, the organic layer is collected, washed, dried, filtered, and the organic phase is removed by the filtrate to obtain the intermediate 5; the molar ratio of the intermediate 4 and the BOC anhydride is 15.6:18.7;
d)将中间体5、TMEDA(四甲基乙二胺)、正丁基锂在四氢呋喃溶液中，无氧条件下于-78℃下保持2h，随后在冰水浴条件下保持10min，然后再次降温至-78℃以下开始缓慢通入CO 2气体，3～4h后缓慢升温至20～30℃，并充分搅拌后加入水淬灭；然后浓缩反应液，将pH值调节为2～3，然后萃取，合并有机层，经柱层析提纯，得到中间体6；所述正丁基锂为催化量； d) Intermediate 5, TMEDA (tetramethylethylenediamine), n-butyllithium in tetrahydrofuran solution, maintained at -78 ° C for 2 h under anaerobic conditions, then kept in ice water bath for 10 min, then cooled again Slowly introduce CO 2 gas to below -78 ° C, slowly increase the temperature to 20 ~ 30 ° C after 3 ~ 4h, and stir well, add water to quench; then concentrate the reaction solution, adjust the pH to 2 ~ 3, then extract , the organic layer is combined and purified by column chromatography to obtain intermediate 6; the n-butyl lithium is a catalytic amount;
e)将中间体6溶于DCM中，于常温下缓慢加入三氟乙酸(TFA)，常温反应2～4h后，加入水与稀氢氧化钠溶液调节pH为11～12，然后萃取，弃去有机层，水层中再加入醋酸调节pH为弱酸性，然后萃取，合并有机层，经洗涤、干燥、过滤后，滤液除去有机相，得到中间体7；所述TFA为催化量；e) Dissolve Intermediate 6 in DCM, slowly add trifluoroacetic acid (TFA) at room temperature, react at room temperature for 2 to 4 hours, add water and dilute sodium hydroxide solution to adjust the pH to 11-12, then extract and discard. The organic layer is further added with acetic acid to adjust the pH to be weakly acidic, and then extracted, and the organic layer is combined, washed, dried, filtered, and the organic phase is removed by the filtrate to obtain intermediate 7; the TFA is a catalytic amount;
f)将中间体7和醋酸甲脒在乙二醇甲醚中，于110～140℃，反应8～12h；反应完全后，浓缩反应液，加入饱和碳酸钠溶液，然后用乙酸乙酯进行萃取，合并有机层，用饱和食盐水洗涤，有机层经干燥、过滤后，滤液除去有机相，得到中间体8；所述中间体7和醋酸甲脒的摩尔比为1:3；f) Intermediate 7 and formamidine acetate are reacted in ethylene glycol methyl ether at 110-140 ° C for 8-12 hours; after the reaction is completed, the reaction solution is concentrated, saturated sodium carbonate solution is added, and then extracted with ethyl acetate. , the organic layer was combined, washed with saturated brine, the organic layer was dried, filtered, the organic phase was removed to give intermediate 8; the molar ratio of intermediate 7 and methyl acetate was 1:3;
g)在甲醇溶液中加入金属钠碎片，常温搅拌，然后将中间体8加入其中，升高温度至40～60℃，反应6～10h；反应完全后，将反应液调节pH至中性，经柱层析提纯，得到中间体9；所述中间体8、金属钠的摩尔比为1:6；g) adding sodium metal fragments to the methanol solution, stirring at room temperature, then adding intermediate 8 thereto, raising the temperature to 40-60 ° C, and reacting for 6-10 hours; after the reaction is complete, adjusting the pH to neutrality of the reaction solution, Purification by column chromatography to obtain intermediate 9; the molar ratio of the intermediate 8, metal sodium is 1:6;
h)将中间体9、三氯氧磷和氯仿在60～80℃，反应0.5～2h后，向其中缓慢加入三乙胺，反应6～10h后，浓缩反应液，向其中加入DCM，然后将混合液缓慢的滴加到饱和碳酸氢钠的冰水液中；滴加完毕后，萃取，取DCM层合并，然后洗涤、干燥、过滤，滤液除去有机相，得到中间体10；所述三氯氧磷、三乙胺为催化量；h) Intermediate 9, phosphorus oxychloride and chloroform are reacted at 60-80 ° C for 0.5-2 h, then triethylamine is slowly added thereto, and after reacting for 6-10 h, the reaction solution is concentrated, DCM is added thereto, and then The mixture is slowly added dropwise to a saturated aqueous solution of sodium hydrogencarbonate; after the addition is completed, the mixture is extracted, and the DCM layer is combined, washed, dried, filtered, and the organic phase is removed from the filtrate to obtain intermediate 10; Phosphorus and triethylamine are catalytic amounts;
i)将中间体10和中间体3溶于丁酮，然后加入碳酸钾，于60～90℃反应4～6h后，浓缩反应液，经柱层析提纯，得到式Ⅲ化合物；所述中间体10和化合物3的摩尔比为5:6。i) Dissolving intermediate 10 and intermediate 3 in methyl ethyl ketone, then adding potassium carbonate, reacting at 60-90 ° C for 4-6 hours, concentrating the reaction solution, and purifying by column chromatography to obtain a compound of formula III; The molar ratio of 10 to compound 3 was 5:6.
路线二，式Ⅱ所示化合物的合成路线：Route 2, the synthetic route of the compound of formula II:
Figure PCTCN2018073793-appb-000045
Figure PCTCN2018073793-appb-000045
上述式Ⅱ所示化合物的合成步骤为：The synthetic steps of the compound of formula II above are:
j)将原料2和中间体3溶于丁酮，然后加入碳酸钾，于60～90℃反应4～6h后，浓缩反应液，经柱层析提纯，得到化合物式Ⅱ；所述原料2和中间体3的摩尔比为5:6；j) The raw material 2 and the intermediate 3 are dissolved in methyl ethyl ketone, then potassium carbonate is added, and after reacting at 60 to 90 ° C for 4 to 6 hours, the reaction liquid is concentrated and purified by column chromatography to obtain a compound of the formula II; The molar ratio of the intermediate 3 is 5:6;
或，k)将原料4和喹唑啉衍生物溶于N，N-二甲基甲酰胺，然后加入碳酸铯，于120～140℃反应4～6h后，浓缩反应液，经柱层析提纯，得到中间体12；所述原料4和喹唑啉衍生物的摩尔比为5：6；Or, k) dissolving the starting material 4 and the quinazoline derivative in N,N-dimethylformamide, then adding cesium carbonate, reacting at 120-140 ° C for 4-6 hours, concentrating the reaction solution, and purifying by column chromatography. , obtaining intermediate 12; the molar ratio of the starting material 4 and the quinazoline derivative is 5:6;
l)将中间体12和原料5溶于四氢呋喃，然后滴加三乙胺，升温至110～130℃反应2～4h后，浓缩反应液，经柱层析提纯，得到化合物式Ⅱ；所述中间体12和原料5的摩尔比为1：1.2。l) The intermediate 12 and the starting material 5 are dissolved in tetrahydrofuran, and then triethylamine is added dropwise, and the mixture is heated to 110-130 ° C for 2 to 4 hours, and then the reaction liquid is concentrated and purified by column chromatography to obtain a compound of the formula II; The molar ratio of the bulk 12 to the raw material 5 was 1:1.2.
路线三，式Ⅰ所示化合物的合成路线：Route 3, the synthetic route of the compound of formula I:
Figure PCTCN2018073793-appb-000046
Figure PCTCN2018073793-appb-000046
上述式Ⅰ所示化合物的合成步骤为：The synthetic steps of the compound of formula I above are:
m)将原料3和中间体3溶于丁酮，然后加入碳酸钾，于60～90℃反应4～6h后，浓缩反应液，经柱层析提纯，得到化合物式Ⅰ；所述原料3和中间体3的摩尔比为5：6；m) The raw material 3 and the intermediate 3 are dissolved in methyl ethyl ketone, then potassium carbonate is added, and after reacting at 60 to 90 ° C for 4 to 6 hours, the reaction liquid is concentrated and purified by column chromatography to obtain a compound of the formula I; The molar ratio of the intermediate 3 is 5:6;
或，n)将原料4和嘧啶衍生物溶于N，N-二甲基甲酰胺，然后加入碳酸铯，于120～140℃反应4～6h后，浓缩反应液，经柱层析提纯，得到中间体11；所述原料4和嘧啶衍生物的摩尔比为5：6；Or, n) the raw material 4 and the pyrimidine derivative are dissolved in N,N-dimethylformamide, and then the cesium carbonate is added, and the reaction is carried out at 120 to 140 ° C for 4 to 6 hours, and then the reaction liquid is concentrated and purified by column chromatography to obtain Intermediate 11; the molar ratio of the starting material 4 and the pyrimidine derivative is 5:6;
o)将中间体11和原料5溶于四氢呋喃，然后滴加三乙胺，升温至110～130℃反应2～4h后，浓缩反应液，经柱层析提纯，得到式Ⅰ化合物；所述中间体11和原料5的摩尔比为5：6。o) Dissolving intermediate 11 and starting material 5 in tetrahydrofuran, then adding triethylamine dropwise, raising the temperature to 110-130 ° C for 2 to 4 hours, then concentrating the reaction solution, and purifying by column chromatography to obtain a compound of formula I; The molar ratio of the bulk 11 to the raw material 5 was 5:6.
本发明还提供了上述取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物药学上可接受的盐和水合物。The present invention also provides pharmaceutically acceptable salts and hydrates of the above substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivatives.
本发明还提供了上述取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物药学上可接受的盐和水合物。The present invention also provides pharmaceutically acceptable salts and hydrates of the above substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivatives.
本发明还提供了式Ⅰ、式Ⅱ和式Ⅲ所示化合物的前药。依据本发明，前药是上述式Ⅰ、式Ⅱ和式Ⅲ所示化合物的衍生物，它们自身可能具有较弱的活性或甚至没有活性，但是在给药后，在生理条件下(例如通过代谢、溶剂分解或另外的方式)被转化成相应的生物活性形式。The invention also provides prodrugs of the compounds of Formula I, Formula II and Formula III. According to the invention, the prodrugs are derivatives of the compounds of the above formula I, formula II and formula III which may themselves have weak or even no activity, but after administration, under physiological conditions (for example by metabolism) , solvolysis or otherwise) is converted to the corresponding biologically active form.
本发明还提供一种组合物，是由本发明提供的上述取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物添加药学上可以接受的辅助性成分制备而成的。The present invention also provides a composition which is pharmaceutically acceptable by the above substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative provided by the present invention. Prepared from auxiliary ingredients.
本发明还提供了上述取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物、它的盐或水合物在制备FMS-样酪氨酸激酶3抑制剂中的用途。优选的，上述取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物、它的盐或水合物在针对FLT3酪氨酸激酶内部串联重复突变(FLT3-ITD)或在FLT3酪氨酸激酶原有内部串联重复突变(FLT3-ITD)基础上产生的单个氨基酸突变的耐药的FLT3-ITD/F691L或FLT3-ITD/D835Y抑制剂中的用途。The present invention also provides the above substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative, a salt or hydrate thereof for preparing FMS-like tyrosine Use in kinase 3 inhibitors. Preferably, the above substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative, its salt or hydrate is tandemly repeated in tandem against the FLT3 tyrosine kinase Mutation of FLT3-ITD/F691L or FLT3-ITD/D835Y inhibitors in a single amino acid mutation based on the original internal tandem repeat mutation (FLT3-ITD) of FLT3 tyrosine kinase use.
本发明还提供了上述取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物、它的盐或水合物在制备治疗急性髓性白血病药物中的用途。The present invention also provides the above-mentioned substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative, a salt or a hydrate thereof for preparing a medicament for treating acute myeloid leukemia Use in.
本发明还提供了上述取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物、它的盐或水合物在制备抗自身免疫性疾病药物中的用途。The present invention also provides the above-mentioned substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative, a salt or a hydrate thereof for preparing an anti-autoimmune disease drug Use in.
本发明还提供了上述取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物、它的盐或水合物在制备新生血管生成抑制剂中的用途。The present invention also provides the above substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative, its salt or hydrate in the preparation of a neovascularization inhibitor the use of.
本发明还提供了上述取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物、它的盐或水合物在制备抗肿瘤药物中的用途。The present invention also provides the use of the above substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative, a salt or hydrate thereof for preparing an antitumor drug .
本发明所述化合物有效地克服临床药物AC220产生的各种耐药，特别针对二次突变耐药FLT3-ITD/F691L和FLT3-ITD/D835Y具有较好的治疗作用。本发明提供了一类新型的吡唑并嘧啶的衍生物，其主要是4位有取代，并提供了本发明吡唑并嘧啶衍生物的简便、高效、成本低廉的制备方法。本发明1-(5-(叔丁基)异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物对多种激酶具有良好的抑制活性，对多种实体瘤、白血病以及自身免疫性疾病均具有抑制作用，为本领域 中的激酶抑制剂的制备、抗自身免疫性疾病药物的制备、新生血管生成抑制剂和抗肿瘤药物的制备提供了新的有效选择，具有很好的应用前景。The compound of the invention effectively overcomes various drug resistances produced by the clinical drug AC220, and has a better therapeutic effect especially for the secondary mutation resistance FLT3-ITD/F691L and FLT3-ITD/D835Y. The present invention provides a novel class of pyrazolopyrimidine derivatives which are predominantly substituted at the 4-position and which provide a simple, efficient and cost-effective process for the preparation of the pyrazolopyrimidine derivatives of the present invention. The 1-(5-(tert-butyl)isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative of the invention has good inhibitory activity against various kinases, and is applicable to various entities. Tumor, leukemia and autoimmune diseases have inhibitory effects, providing new effective options for the preparation of kinase inhibitors, preparation of anti-autoimmune diseases, preparation of neovascularization inhibitors and anti-tumor drugs. , has a good application prospects.
附图说明DRAWINGS
图1化合物SKLB707体外细胞凋亡检测实验结果。Figure 1 shows the results of in vitro apoptosis assay of compound SKLB707.
图2化合物SKLB707对FLT3信号通路体外检测实验结果。图2B为化合物SKLB707和AC220对FLT3信号通路各关键蛋白的抑制作用。Figure 2 shows the results of in vitro detection of FLT3 signaling pathway by compound SKLB707. Figure 2B shows the inhibitory effects of the compounds SKLB707 and AC220 on key proteins in the FLT3 signaling pathway.
图3化合物SKLB707体内药效检测实验结果。不同剂量的SKLB707都可以完全消除小鼠皮下瘤(图3A)，并且老鼠的体重没有下降(图3B)，老鼠的状态同样也没有明显变化。Figure 3 shows the results of in vivo pharmacodynamic test of compound SKLB707. Different doses of SKLB707 completely abolished mouse subcutaneous tumors (Fig. 3A), and the body weight of the mice did not decrease (Fig. 3B), and the state of the mice did not change significantly.
具体实施方式detailed description
实施例1 3-氨基-5-叔丁基异恶唑(中间体2)的制备Example 1 Preparation of 3-Amino-5-tert-butylisoxazole (Intermediate 2)
Figure PCTCN2018073793-appb-000047
Figure PCTCN2018073793-appb-000047
称取原料1(4,4-二甲基-3-氧代戊腈)(1.25g，10mM)于装有100mL水的250mL圆底烧瓶中，然后加入盐酸羟胺(759mg，11mM)和氢氧化钠(440mg，11mM)，将反应液在常温下搅拌半小时，后加入适量氢氧化钠调节pH至8～9，然后升温至50℃反应10h。TCL检测反应完全后，共用100mL甲基叔丁基醚分三次加入溶液中进行萃取，弃去有机层，水层再次分三次共加入150mL的DCM(二氯甲烷)进行萃取，收集DCM层，用饱和食盐水洗涤三次，随后加入无水硫酸钠干燥，过滤之后取滤液旋干，既得白色固体N'-羟基-4,4-二甲基-3-羰基戊脒(721mg，45.6％)，HPLC纯度96％。The starting material 1 (4,4-dimethyl-3-oxopentanonitrile) (1.25 g, 10 mM) was weighed into a 250 mL round bottom flask containing 100 mL of water, then hydroxylamine hydrochloride (759 mg, 11 mM) and hydroxide were added. Sodium (440 mg, 11 mM), the reaction solution was stirred at room temperature for half an hour, and then an appropriate amount of sodium hydroxide was added to adjust the pH to 8-9, and then the temperature was raised to 50 ° C for 10 h. After the TCL detection reaction was completed, 100 mL of methyl t-butyl ether was added to the solution in three portions for extraction, and the organic layer was discarded. The aqueous layer was again added to 150 mL of DCM (dichloromethane) for extraction, and the DCM layer was collected. The mixture was washed three times with saturated brine, then dried over anhydrous sodium sulfate and filtered, and the filtrate was evaporated to dryness to give white solid N--hydroxy-4,4-dimethyl-3-carbonylpentanthene (721 mg, 45.6%), HPLC The purity is 96%.
1H NMR(400MHz,CDCl 3)δ7.30(s,2H),5.52(s,1H),4.04(s,2H),1.30(s,9H)ppm.ESI-MS m/z:159.1[M+H] +。 1 H NMR (400 MHz, CDCl 3 ) δ 7.30 (s, 2H), 5.52 (s, 1H), 4.04 (s, 2H), 1.30 (s, 9H) ppm. ESI-MS m/z: 159.1 [M +H] + .
称取N'-羟基-4,4-二甲基-3-羰基戊脒(1.58g,10mM)于250mL的圆底烧瓶中，后加入75mL的水，然后用浓盐酸调节溶液的pH至4～5，然后升高温度到50℃，反应三小时。TCL检测反应完全后，将反应液冷却至室温，在冰水浴中缓慢加入适量氢氧化钠溶液调节pH＝12，随后会有白色沉淀析出，用减压泵抽滤后，分别用水和乙醚洗涤白色沉淀，低温烘干，得到灰色固体，即为中间体2(800mg，收率57.1％)，HPLC纯度95％。Weigh N'-hydroxy-4,4-dimethyl-3-carbonylpentanose (1.58 g, 10 mM) in a 250 mL round bottom flask, then add 75 mL of water, then adjust the pH of the solution to 4 with concentrated hydrochloric acid. ～5, then raise the temperature to 50 ° C and react for three hours. After the TCL detection reaction was completed, the reaction solution was cooled to room temperature, and an appropriate amount of sodium hydroxide solution was slowly added in an ice water bath to adjust the pH to 12, followed by a white precipitate, which was filtered with a reduced pressure pump and washed with water and diethyl ether respectively. Precipitation, drying at low temperature gave a gray solid, which was Intermediate 2 (800 mg, yield 57.1%), HPLC purity 95%.
1H NMR(400MHz,DMSO)δ7.66(s,1H),7.35(s,2H),1.24(s,9H)ppm。ESI-MS m/z:141.1[M+H] +。 1 H NMR (400 MHz, DMSO) δ 7.66 (s, 1 H), 7.35 (s, 2H), 1.24 (s, 9H) ppm. ESI-MS m/z: 141.1 [M+H] + .
实施例2 1-(5-(叔丁基)异恶唑-3-基)-3-(3-氟-4-羟苯基)脲(中间体3)的制备Example 2 Preparation of 1-(5-(tert-butyl)isoxazol-3-yl)-3-(3-fluoro-4-hydroxyphenyl)urea (Intermediate 3)
Figure PCTCN2018073793-appb-000048
Figure PCTCN2018073793-appb-000048
称取三光气(5.282g，17.8mM)于500mL圆底烧瓶中，然后加入乙酸乙酯200mL，然后在冰水浴环境中向烧瓶中缓慢滴加100mL中间体2(5g，35.7mM)的乙酸乙酯溶液，滴加完毕后再在冰水浴环境中缓慢滴加三乙胺(7.2g，71.4mM)，然后升温至70℃反应三小时。反应完毕后，直接用旋转蒸发仪将有机相除去，然后加入4-氨基-2-氟苯酚(4.4g，35mM)的乙酸乙酯溶液200mL，然后反应液于70℃下加热反应5h，TCL检测反应完毕后，将反应液中有机相用 旋转蒸发仪除去后加入200mL碳酸钾的饱和水溶液，萃取并取上层的有机层加入100～200mm的粗硅胶拌样。采用柱层析法，用300～400mm的硅胶拌样，体积比DCM︰MeOH(甲醇)＝50︰1作为洗脱剂，最终获得白色固体中间体3(7.34g，收率70.1％)，HPLC纯度96％。Weigh three phosgene (5.282 g, 17.8 mM) in a 500 mL round bottom flask, then add 200 mL of ethyl acetate, and then slowly add 100 mL of Intermediate 2 (5 g, 35.7 mM) of acetic acid B to the flask in an ice water bath environment. After the addition of the ester solution, triethylamine (7.2 g, 71.4 mM) was slowly added dropwise in an ice water bath environment, and then the temperature was raised to 70 ° C for three hours. After the reaction was completed, the organic phase was directly removed by a rotary evaporator, and then 200 mL of 4-amino-2-fluorophenol (4.4 g, 35 mM) in ethyl acetate was added, and then the reaction mixture was heated at 70 ° C for 5 h, TCL detection. After completion of the reaction, the organic phase in the reaction mixture was removed by a rotary evaporator, and then a saturated aqueous solution of 200 mL of potassium carbonate was added thereto, and the organic layer of the upper layer was taken and added to a crude silica gel of 100 to 200 mm. Column chromatography, using 300-400 mm silica gel, the volume ratio of DCM.. MeOH (methanol) = 50..1 as eluent, finally obtained white solid intermediate 3 (7.34g, yield 70.1%), HPLC The purity is 96%.
1H NMR(400MHz,DMSO)δ7.72(d,J＝12.5Hz,1H),7.27(s,1H),6.51(s,1H),6.53(s,1H),6.01(s,2H),5.70(s,1H),1.31(s,9H)ppm。ESI-MS m/z:294.2[M+H] +。 1 H NMR (400MHz, DMSO) δ7.72 (d, J = 12.5Hz, 1H), 7.27 (s, 1H), 6.51 (s, 1H), 6.53 (s, 1H), 6.01 (s, 2H), 5.70 (s, 1H), 1.31 (s, 9H) ppm. ESI-MS m/z: 294.2 [M+H] + .
实施例3 叔丁基(6-氟吡啶-3-基)氨基甲酸酯(中间体5)的制备Example 3 Preparation of tert-butyl(6-fluoropyridin-3-yl)carbamate (Intermediate 5)
Figure PCTCN2018073793-appb-000049
Figure PCTCN2018073793-appb-000049
在250mL的圆底烧瓶中加入中间体4(6-氟吡啶-3-胺)(2g，15.6mM)和BOC酸酐(二碳酸二叔丁酯，4g，18.7mM)，然后加入150mL二氧六环作溶剂，随后升温至100℃，反应约8h，直至TLC检测反应。然后减压浓缩反应液，加入水和稀盐酸溶液调节pH为3～4，随后加入150mL乙酸乙酯萃取，分三次加入到水层，收集乙酸乙酯层。然后用饱和食盐水将乙酸乙酯层洗涤三次，再用无水硫酸钠干燥乙酸乙酯层，随后过滤后，滤液直接用旋转蒸发仪将有机相除去，得白色固体中间体5(2.56g,收率71％)，HPLC纯度95％。Intermediate 250 (6-fluoropyridin-3-amine) (2 g, 15.6 mM) and BOC anhydride (di-tert-butyl dicarbonate, 4 g, 18.7 mM) were added to a 250 mL round bottom flask, followed by the addition of 150 mL of dioxane. The ring was used as a solvent, and then the temperature was raised to 100 ° C, and the reaction was carried out for about 8 hours until the reaction was detected by TLC. Then, the reaction liquid was concentrated under reduced pressure, and water and a dilute hydrochloric acid solution were added to adjust the pH to 3-4, and then extracted with ethyl acetate (150 mL), and added to the aqueous layer three times, and the ethyl acetate layer was collected. Then, the ethyl acetate layer was washed three times with brine, and the ethyl acetate layer was dried over anhydrous sodium sulfate. Yield 71%), HPLC purity 95%.
1H NMR(400MHz,DMSO)δ9.77(s,1H),8.47(s,1H),7.92(d,J＝8.1Hz,1H),7.42(d,J＝8.7Hz,1H),1.48(s,9H)ppm。ESI-MS m/z:213.2[M+H] +。 1 H NMR (400 MHz, DMSO) δ 9.77 (s, 1H), 8.47 (s, 1H), 7.92 (d, J = 8.1 Hz, 1H), 7.42 (d, J = 8.7 Hz, 1H), 1.48 ( s, 9H) ppm. ESI-MS m / z: 213.2 [M + H] +.
实施例4 5-叔丁氧基羰基氨基-2-氟异烟酸(中间体6)的制备Example 4 Preparation of 5-tert-Butoxycarbonylamino-2-fluoroisonicotinic acid (Intermediate 6)
Figure PCTCN2018073793-appb-000050
Figure PCTCN2018073793-appb-000050
在250mL三口烧瓶中加入中间体5(1g，4.375mM)，然后加入100mL重蒸无水的四氢呋喃溶液，随后用量程为-80℃到50℃的温度计以及橡胶塞、三通活塞将瓶口塞住密封。然后通过三通活塞将瓶内抽真空，同时用氮气充分置换。然后注入0.2mL的TMEDA(四甲基乙二胺)，并置于-80℃的冷阱中降温，直至瓶中温度低于-78℃，然后缓慢注入8mL正丁基锂，在-78℃下保持2h。随后将三口瓶取出在冰水浴条件下保持10min，然后再次放入-80℃的冷阱中，当温度低于-78℃时开始缓慢通入CO 2气体，3h后将三口瓶取出置于常温搅拌装置中，此时有大量气体放出。当反应瓶内温度上升至10℃左右时将三口瓶置于30℃油浴锅加热，并充分搅拌后加入50mL水淬灭，然后减压浓缩反应液，向其中加入稀盐酸溶液，调节pH为2～3，然后分三次共加入150mL乙酸乙酯萃取三次，合并有机层，用饱和食盐水洗涤三次后加入100～200mm的粗硅胶拌样，用DCM︰MeOH＝10︰1作为洗脱液最终得到淡黄色固体中间体6(300mg，产率为26％)，HPLC纯度97％。 Intermediate 5 (1 g, 4.375 mM) was added to a 250 mL three-necked flask, then 100 mL of a re-distilled anhydrous tetrahydrofuran solution was added, followed by a thermometer with a range of -80 ° C to 50 ° C and a rubber stopper and a three-way piston to stopper the bottle. Stay sealed. The inside of the bottle was then evacuated through a three-way piston while being sufficiently replaced with nitrogen. Then inject 0.2 mL of TMEDA (tetramethylethylenediamine) and cool it in a cold trap at -80 °C until the temperature in the bottle is below -78 °C, then slowly inject 8 mL of n-butyllithium at -78 °C. Keep it for 2 hours. The three bottles were then removed under ice-cooling to maintain 10min, then placed in the cold trap -80 ℃ again, when the temperature is lower than -78 deg.] C slowly into a CO 2 gas, 3h bottle was taken out at a normal temperature after three In the stirring device, a large amount of gas is released at this time. When the temperature inside the reaction flask rises to about 10 °C, the three-necked bottle is placed in a 30 ° C oil bath pot, and stirred thoroughly, and then quenched by adding 50 mL of water. Then, the reaction liquid is concentrated under reduced pressure, and a dilute hydrochloric acid solution is added thereto to adjust the pH to 2~3, and then added three times to add 150mL of ethyl acetate to extract three times, and the organic layer was combined, washed three times with saturated brine, and then added with 100-200mm of crude silica gel, using DCM..MeOH=10..1 as eluent. Intermediate 6 (300 mg, yield 26%) was obtained as a pale yellow solid.
1H NMR(400MHz,DMSO)δ10.04(s,1H),9.13(s,1H),7.77(s,1H),1.48(s,9H)ppm。ESI-MS m/z:257.2[M+H] +。 1 H NMR (400 MHz, DMSO) δ 10.04 (s, 1H), 9.13 (s, 1H), 7.77 (s, 1H), 1.48 (s, 9H) ppm. ESI-MS m/z: 257.2 [M+H] + .
实施例5 5-氨基-2-氟异烟酸(中间体7)的制备Example 5 Preparation of 5-amino-2-fluoroisonicotinic acid (Intermediate 7)
Figure PCTCN2018073793-appb-000051
Figure PCTCN2018073793-appb-000051
在100mL圆底烧瓶中加入中间体6(500mg,1.83mM)，然后加入30mL的DCM溶解， 于常温下缓慢加入10mL的三氟乙酸(TFA)，常温搅拌反应三小时后加入水与稀氢氧化钠溶液中和剩余的三氟乙酸并调节pH为11～12，然后萃取，弃去有几层，取水层。水层中再加入醋酸调节pH为弱酸性，然后分三次加入共150mL乙酸乙酯进行萃取，合并有机层，用饱和食盐水洗涤三次，合并乙酸乙酯层，然后用无水硫酸钠干燥，过滤后滤液直接用旋转蒸发仪将有机相除去，最终得淡黄色固体中间体7(200mg，产率63％)，HPLC纯度96％。Intermediate 6 (500 mg, 1.83 mM) was added to a 100 mL round bottom flask, and then dissolved in 30 mL of DCM. 10 mL of trifluoroacetic acid (TFA) was slowly added at normal temperature, and the reaction was stirred at room temperature for three hours, then water and dilute hydroxide were added. The sodium solution was neutralized with the remaining trifluoroacetic acid and the pH was adjusted to 11 to 12, and then extracted, and several layers were discarded, and the aqueous layer was taken. The aqueous layer was further added with acetic acid to adjust the pH to be weakly acidic. Then, a total of 150 mL of ethyl acetate was added in three portions for extraction. The organic layer was combined and washed three times with brine. The organic phase was removed directly on a rotary evaporator to give a pale yellow solid intermediate 7 (200 mg, yield 63%).
1H NMR(400MHz,DMSO)δ9.11(s,1H),8.03(s,1H),7.48(s,1H),6.27(s,2H)ppm。ESI-MS m/z:157.1[M+H] +。 1 H NMR (400 MHz, DMSO) δ 9.11 (s, 1 H), 8. s (s, 1H), 7.48 (s, 1H), 6.27 (s, 2H) ppm. ESI-MS m/z: 157.1 [M+H] + .
实施例6 6-氟吡啶并[3.4-D]嘧啶-4-酚的(中间体8)的制备Example 6 Preparation of 6-fluoropyrido[3.4-D]pyrimidin-4-ol (Intermediate 8)
Figure PCTCN2018073793-appb-000052
Figure PCTCN2018073793-appb-000052
称取中间体7(200mg，1.2mM)和醋酸甲脒(374mg，3.6mM)放入100mL的圆底烧瓶中，加入50mL的乙二醇甲醚，升高温度至120℃，反应10h。TCL检测反应完全后，减压浓缩，加入50mL的饱和碳酸钠溶液，然后分三次共100mL的乙酸乙酯进行萃取，合并有机层，用饱和食盐水洗涤三次，取有机层加入无水硫酸钠干燥，过滤后滤液直接用旋转蒸发仪将有机相除去，即得灰色固体中间体8(150mg，产率为69％)，HPLC纯度96％。Intermediate 7 (200 mg, 1.2 mM) and formazan acetate (374 mg, 3.6 mM) were weighed into a 100 mL round bottom flask, 50 mL of ethylene glycol methyl ether was added, and the temperature was raised to 120 ° C for 10 h. After the TCL reaction was completed, the mixture was concentrated under reduced pressure. 50 mL of saturated sodium carbonate solution was added, and then extracted with three portions of 100 mL of ethyl acetate. The organic layer was combined and washed three times with saturated brine. After filtration, the filtrate was directly subjected to a rotary evaporator to remove the organic phase to obtain a gray solid intermediate 8 (150 mg, yield 69%).
1H NMR(400MHz,DMSO)δ7.97(s,1H),7.86(s,1H),7.43(s,1H),3.96(s,1H)ppm。ESI-MS m/z:166.1[M+H] +。 1 H NMR (400 MHz, DMSO) δ 7.97 (s, 1H), 7.86 (s, 1H), 7.43 (s, 1H), 3.96 (s, 1H) ppm. ESI-MS m/z: 166.1 [M+H] + .
实施例7 6-甲氧基吡啶并[3.4-d]嘧啶-4-酚(中间体9)的制备Example 7 Preparation of 6-methoxypyrido[3.4-d]pyrimidin-4-phenol (Intermediate 9)
Figure PCTCN2018073793-appb-000053
Figure PCTCN2018073793-appb-000053
向100mL的圆底烧瓶中加入20mL的甲醇溶液，随后称取160mg金属钠，将其切成碎片加入其中，常温搅拌反应20min，然后称取中间体8(200mg，1.13mM)加入其中，升高温度至50℃，反应8h。反应完全后，加入稀盐酸溶液调节pH至中性，然后加入100～200mm的粗硅胶拌样，用DCM︰MeOH＝50︰1作为洗脱剂，使用柱层析法得到灰色固体中间体9(150mg，产率为70.1％)，HPLC纯度95％。20 mL of a methanol solution was added to a 100 mL round bottom flask, and then 160 mg of sodium metal was weighed, cut into pieces, and stirred at room temperature for 20 minutes, and then Intermediate 8 (200 mg, 1.13 mM) was weighed and added. The temperature was raised to 50 ° C and the reaction was carried out for 8 h. After the reaction is completed, the pH is adjusted to neutrality by adding a dilute hydrochloric acid solution, and then the mixture is mixed with 100-200 mm of crude silica gel, and DCM..MeOH = 50..1 is used as an eluent, and a gray solid intermediate 9 is obtained by column chromatography. 150 mg, yield 70.1%), HPLC purity 95%.
1H NMR(400MHz,DMSO)δ7.46(s,1H),7.22(s,1H),7.19(s,1H),3.87(s,1H),3.80(s,3H)ppm。ESI-MS m/z:178.1[M+H] +。 1 H NMR (400 MHz, DMSO) δ 7.46 (s, 1H), 7.22 (s, 1H), 7.19 (s, 1H), 3.87 (s, 1H), 3.80 (s, 3H) ppm. ESI-MS m/z: 178.1 [M+H] + .
实施例8 4-氯-6-甲氧基吡啶并[3,4-d]嘧啶(中间体10)的制备Example 8 Preparation of 4-chloro-6-methoxypyrido[3,4-d]pyrimidine (Intermediate 10)
Figure PCTCN2018073793-appb-000054
Figure PCTCN2018073793-appb-000054
称取中间体9(300mg，1.7mM)于100mL的圆底烧瓶中，然后加入10mL的氯仿和0.5mL的三氯氧磷，升温至70℃，反应1h后，向其中缓慢加入0.8mL三乙胺，反应8h后减压浓缩，向浓缩液中加入20mL的DCM将产品溶解，然后将混合液缓慢的滴加到100mL饱和碳酸氢钠的冰水液中。滴加完毕后，萃取，取下层的DCM层合并，然后用饱和食盐水洗涤三次，随后用无水硫酸钠干燥，过滤后滤液直接用旋转蒸发仪将有机相除去，得灰色固体中间体10(170mg，产率为51.3％)，HPLC纯度96％。Intermediate 9 (300 mg, 1.7 mM) was weighed into a 100 mL round bottom flask, then 10 mL of chloroform and 0.5 mL of phosphorus oxychloride were added, and the temperature was raised to 70 ° C. After 1 h of reaction, 0.8 mL of three B was slowly added thereto. The amine was reacted for 8 hours, and then concentrated under reduced pressure. To the concentrate was added 20 mL of DCM to dissolve the product, and then the mixture was slowly added dropwise to 100 mL of saturated aqueous sodium hydrogen carbonate solution. After the completion of the dropwise addition, the mixture was extracted, and the lower layer of the DCM layer was combined, and then washed three times with saturated brine, and then dried over anhydrous sodium sulfate, filtered, and the filtrate was directly removed from the organic phase by rotary evaporator to obtain a gray solid intermediate 10 ( 170 mg, yield 51.3%), HPLC purity 96%.
1H NMR(400MHz,DMSO)δ8.76(s,1H),7.63(s,1H),5.91(s,1H),3.77(s,3H)ppm。ESI-MS m/z:196.1[M+H] +。 1 H NMR (400 MHz, DMSO) δ 8.76 (s, 1 H), 7.63 (s, 1H), 5.91 (s, 1H), 3.77 (s, 3H) ppm. ESI-MS m/z: 196.1 [M+H] + .
实施例9 1-(5-(叔丁基)异恶唑-3-基)-3-(3-氟-4-((6-甲氧基吡啶并[3,4-d]嘧啶-4-基)氧)苯基)脲(化合物11)的制备Example 9 1-(5-(tert-butyl)isoxazol-3-yl)-3-(3-fluoro-4-((6-methoxypyrido[3,4-d]pyrimidine-4) Preparation of -yl)oxy)phenyl)urea (Compound 11)
Figure PCTCN2018073793-appb-000055
Figure PCTCN2018073793-appb-000055
称取中间体10(195mg,1mM)和化合物3(351mg，1.2mM)于100mL圆底烧瓶中，加入40mL的丁酮溶解混合物，然后加入碳酸钾(165mg，1.2mM)的作为缚酸剂，升高温度至80℃反应5h后，减压浓缩加入100-200mm的粗硅胶拌样，用DCM︰MeOH＝150︰1作为洗脱剂，使用柱层析方法最终得白色的化合物11(80mg,产率为17.7％)，HPLC纯度95％。Intermediate 10 (195 mg, 1 mM) and Compound 3 (351 mg, 1.2 mM) were weighed into a 100 mL round bottom flask, 40 mL of a butanone dissolution mixture was added, and then potassium carbonate (165 mg, 1.2 mM) was added as an acid binding agent. After raising the temperature to 80 ° C for 5 h, the mixture was concentrated under reduced pressure to a mixture of 100-200 mm of crude silica gel, using DCM.. MeOH = 150..1 as eluent, using column chromatography to give white compound 11 (80 mg, The yield was 17.7%) and the HPLC purity was 95%.
1H NMR(400MHz,DMSO)δ9.67(s,1H),9.22(s,1H),9.13(s,1H),8.71(s,1H),7.72(d,J＝12.5Hz,1H),7.52(s,1H),7.45(s,1H),7.27(s,1H),6.52(s,1H),4.05(s,3H),1.30(s,9H)ppm。 13C NMR(100MHz,CDCl 3)δ181.66,165.28,162.35,158.18,152.37,152.19,150.74,141.78,137.35,135.04,123.69,123.08,115.82,109.19,108.96,98.06,91.76,67.72,64.67,56.14,54.76,32.97,28.61.ESI-MS m/z:453.2[M+H] +。 1 H NMR (400MHz, DMSO) δ9.67 (s, 1H), 9.22 (s, 1H), 9.13 (s, 1H), 8.71 (s, 1H), 7.72 (d, J = 12.5Hz, 1H), 7.52 (s, 1H), 7.45 (s, 1H), 7.27 (s, 1H), 6.52 (s, 1H), 4.05 (s, 3H), 1.30 (s, 9H) ppm. 13 C NMR (100 MHz, CDCl 3 ) δ 181.66, 165.28, 162.35,158.18,152.37,152.19,150.74,141.78,137.35,135.04,123.69,123.08,115.82,109.19,108.96,98.06,91.76,67.72,64.67,56.14,54.76 , 32.97, 28.61. ESI-MS m/z: 453.2 [M+H] + .
实施例10 1-(5-(叔丁基)异恶唑-3-基)-3-(4-((6-氯吡啶并[3,4-d]嘧啶-4-基)氧)-3-氟苯基)脲(化合物11a)的制备Example 10 1-(5-(tert-butyl)isoxazol-3-yl)-3-(4-((6-chloropyrido[3,4-d]pyrimidin-4-yl)oxy)- Preparation of 3-fluorophenyl)urea (Compound 11a)
Figure PCTCN2018073793-appb-000056
Figure PCTCN2018073793-appb-000056
合成方法与实施例9相同(4,6-二氯吡啶[3,4-d]嘧啶作为反应原料)，反应处理得到灰白色粉末化合物11a，反应产率为20.11％，HPLC纯度96％。The synthesis was carried out in the same manner as in Example 9 (4,6-dichloropyridine [3,4-d]pyrimidine as a starting material for the reaction), and the reaction mixture gave an off-white powder compound 11a. The reaction yield was 20.11%, and the HPLC purity was 96%.
1H NMR(400MHz,DMSO)δ9.67(s,1H),9.36(s,1H),9.14(s,1H),8.95(s,1H),8.41(s,1H),7.73(d,J＝12.7Hz,1H),7.45(t,J＝8.6Hz,1H),7.27(d,J＝8.6Hz,1H),6.52(s,1H),1.30(s,9H)ppm. 13C NMR(100MHz,DMSO)δ180.79,166.04,164.80,158.71,155.62,151.87,147.34,139.66,138.81,133.89,127.24,125.38,124.81,118.72,115.30,112.48,107.44,93.01,28.83ppm。ESI-MS m/z:457.1[M+H]+。 1 H NMR (400MHz, DMSO) δ9.67 (s, 1H), 9.36 (s, 1H), 9.14 (s, 1H), 8.95 (s, 1H), 8.41 (s, 1H), 7.73 (d, J =12.7 Hz, 1H), 7.45 (t, J = 8.6 Hz, 1H), 7.27 (d, J = 8.6 Hz, 1H), 6.52 (s, 1H), 1.30 (s, 9H) ppm. 13 C NMR ( 100 MHz, DMSO) δ 180.79, 166.04, 164.80, 158.71, 155.62, 151.87, 147.34, 139.66, 138.81, 133.89, 127.24, 125.38, 124.81, 118.72, 115.30, 112.48, 107.44, 93.01, 28.83 ppm. ESI-MS m/z: 457.1 [M+H]+.
实施例11 1-(5-(叔丁基)异恶唑-3-基)-3-(3-氟-4-((6-(2-甲氧基乙氧基)吡啶并[3,4-d]嘧啶-4-基)氧)苯基)脲(化合物11b)的制备Example 11 1-(5-(tert-butyl)isoxazol-3-yl)-3-(3-fluoro-4-((6-(2-methoxyethoxy))pyridin[3, Preparation of 4-d]pyrimidin-4-yl)oxy)phenyl)urea (Compound 11b)
Figure PCTCN2018073793-appb-000057
Figure PCTCN2018073793-appb-000057
合成方法与实施例9相同，将实施例9中的4-氯-6-甲氧基吡啶并[3,4-d]嘧啶替换为4-氯-6-乙基吡啶并[3,4-d]嘧啶，反应处理得到灰白色粉末化合物11b，反应产率为18.6％，HPLC纯度为96％。The synthesis method was the same as in Example 9, except that 4-chloro-6-methoxypyrido[3,4-d]pyrimidine in Example 9 was replaced with 4-chloro-6-ethylpyrido[3,4- d] Pyrimidine, the reaction treatment gave an off-white powder compound 11b, the reaction yield was 18.6%, and the HPLC purity was 96%.
1H NMR(400MHz,DMSO)δ9.66(s,1H),9.20(s,1H),9.13(s,1H),8.71(s,1H),7.72(d,J＝12.8Hz,1H),7.51(s,1H),7.44(t,J＝8.8Hz,1H),7.26(d,J＝8.7Hz,1H),6.52(s,1H),4.59–4.52(m,2H),3.78–3.69(m,2H),1.99(s,3H),1.30(s,9H)ppm。 13C NMR(100MHz,CDCl 3)δ181.64,165.32,161.66,158.26,155.25,152.42,150.41,141.80,137.43,135.05,123.67,123.15,115.79,109.18,108.94,99.16,91.81,63.23,32.97,28.61,14.73ppm.ESI-MS m/z:497.2[M+H] +。 1 H NMR (400MHz, DMSO) δ9.66 (s, 1H), 9.20 (s, 1H), 9.13 (s, 1H), 8.71 (s, 1H), 7.72 (d, J = 12.8Hz, 1H), 7.51 (s, 1H), 7.44 (t, J = 8.8 Hz, 1H), 7.26 (d, J = 8.7 Hz, 1H), 6.52 (s, 1H), 4.59 - 4.52 (m, 2H), 3.78 - 3.69 (m, 2H), 1.99 (s, 3H), 1.30 (s, 9H) ppm. 13 C NMR (100 MHz, CDCl 3 ) δ 181.64, 165.32, 161.66, 158.26, 155.25, 152.42, 150.41, 141.80, 137.43, 135.05, 123.67, 123.15, 115.79, 109.18, 108.94, 99.16, 91.81, 63.23, 32.97, 28.61, 14.73 ppm.ESI-MS m/z: 497.2 [M+H] + .
实施例12 1-(5-(叔丁基)异恶唑-3-基)-3-(4-((6-乙氧基吡啶并[3,4-d]嘧啶-4-基)氧)-3-氟苯基)脲(化合物11c)的制备Example 12 1-(5-(tert-butyl)isoxazol-3-yl)-3-(4-((6-ethoxypyrido[3,4-d]pyrimidin-4-yl)oxyl Preparation of 3-fluorophenyl)urea (Compound 11c)
Figure PCTCN2018073793-appb-000058
Figure PCTCN2018073793-appb-000058
合成方法与实施例9相同，将实施例9中的4-氯-6-甲氧基吡啶并[3,4-d]嘧啶替换为(2-甲氧基乙氧基)吡啶并[3,4-d]嘧啶作为反应物，反应处理得到白色粉末化合物11c，反应产率为16.6％，HPLC纯度97％。The synthesis method was the same as in Example 9, except that 4-chloro-6-methoxypyrido[3,4-d]pyrimidine in Example 9 was replaced with (2-methoxyethoxy)pyridine [3, 4-d]pyrimidine as a reactant, which was subjected to a reaction to give white powdery compound 11c, yield: 16.6%, HPLC purity 97%.
1H NMR(400MHz,DMSO)；δ9.66(s,1H),9.20(s,1H),9.13(s,1H),8.71(s,1H),7.72(d,J＝12.8Hz,1H),7.51(s,1H),7.44(t,J＝8.8Hz,1H),7.26(d,J＝8.7Hz,1H),6.52(s,1H),4.59–4.52(m,2H),3.78–3.69(m,2H),1.99(s,3H),1.30(s,9H)ppm。 13C NMR(101MHz,CDCl 3)δ181.63,165.32,161.65,158.24,155.28,152.41,150.42,141.80,137.43,135.05,123.67,123.15,115.79,109.18,108.94,99.16,91.82,71.02,66.27,59.19,28.61ppm。ESI-MS m/z:497.2[M+H]+. 1 H NMR (400MHz, DMSO) ; δ9.66 (s, 1H), 9.20 (s, 1H), 9.13 (s, 1H), 8.71 (s, 1H), 7.72 (d, J = 12.8Hz, 1H) , 7.51 (s, 1H), 7.44 (t, J = 8.8 Hz, 1H), 7.26 (d, J = 8.7 Hz, 1H), 6.52 (s, 1H), 4.59 - 4.52 (m, 2H), 3.78 - 3.69 (m, 2H), 1.99 (s, 3H), 1.30 (s, 9H) ppm. 13 C NMR (101 MHz, CDCl 3 ) δ 181.63, 165.32, 161.65, 158.24, 155.28, 152.41, 150.42, 141.80, 137.43, 135.05, 123.67, 123.15, 115.79, 109.18, 108.94, 99.16, 91.82, 71.02, 66.27, 59.19, 28.61 Ppm. ESI-MS m/z: 497.2 [M+H]+.
实施例13 1-(5-(叔丁基)异恶唑-3-基)-3-(3-氟-4-((6-(2-吗啉乙氧基)吡啶并[3,4-d]嘧啶-4-基)氧)苯基)脲(化合物11d)的制备Example 13 1-(5-(tert-butyl)isoxazol-3-yl)-3-(3-fluoro-4-((6-(2-morpholineethoxy))pyridin[3,4 -d]pyrimidin-4-yl)oxy)phenyl)urea (compound 11d)
Figure PCTCN2018073793-appb-000059
Figure PCTCN2018073793-appb-000059
合成方法与实施例9相同，将实施例9中的4-氯-6-甲氧基吡啶并[3,4-d]嘧啶替换为4-(2-((4-氯吡啶并[3,4-d]嘧啶-6-基)氧基)乙基)吗啉为反应物，反应处理得到灰白色固体化合物11d，反应产率为14.8％，HPLC纯度为96％。The synthesis method was the same as in Example 9, except that 4-chloro-6-methoxypyrido[3,4-d]pyrimidine in Example 9 was replaced with 4-(2-((4-chloropyridin[3, 4-d]pyrimidin-6-yl)oxy)ethyl)morpholine was used as the reaction product, and the reaction was afforded to give a white solid solid compound 11d, yield: 14.8%, HPLC purity: 96%.
1H NMR(400MHz,DMSO)；δ9.66(s,1H),9.20(s,1H),9.13(s,1H),8.71(s,1H),7.72(d,J＝12.8Hz,1H),7.51(s,1H),7.44(t,J＝8.8Hz,1H),7.26(d,J＝8.6Hz,1H),6.52(s,1H),4.54(s,2H),3.74(d,J＝7.0Hz,4H),3.64–3.52(m,4H),2.77(s,2H),1.30(s,9H)ppm。 13C NMR(100MHz,CDCl 3)δ181.67,165.30,161.67,158.18,155.27,152.42,152.10,150.56,141.80,123.64,123.12,115.79,109.16,108.93,98.84,91.75,66.93,64.65,57.60,54.06,28.61ppm。ESI-MS m/z:552.1。 1 H NMR (400MHz, DMSO) ; δ9.66 (s, 1H), 9.20 (s, 1H), 9.13 (s, 1H), 8.71 (s, 1H), 7.72 (d, J = 12.8Hz, 1H) , 7.51 (s, 1H), 7.44 (t, J = 8.8 Hz, 1H), 7.26 (d, J = 8.6 Hz, 1H), 6.52 (s, 1H), 4.54 (s, 2H), 3.74 (d, J = 7.0 Hz, 4H), 3.64 - 3.52 (m, 4H), 2.77 (s, 2H), 1.30 (s, 9H) ppm. 13 C NMR (100 MHz, CDCl 3 ) δ 181.67, 165.30, 161.67, 158.18, 155.27, 152.42, 152.10, 150.56, 141.80, 123.64, 123.12, 115.79, 109.16, 108.93, 98.84, 91.75, 66.93, 64.65, 57.60, 54.06, 28.61 Ppm. ESI-MS m/z: 552.1.
实施例14 1-(5-(叔丁基)异恶唑-3-基)-3-(4-((7-氯喹唑啉-4-基)氧基)-3-氟苯基)脲(化合物13a)的制备Example 14 1-(5-(tert-butyl)isoxazol-3-yl)-3-(4-((7-chloroquinazolin-4-yl)oxy)-3-fluorophenyl)urea Preparation of (Compound 13a)
Figure PCTCN2018073793-appb-000060
Figure PCTCN2018073793-appb-000060
称取中间体3(100mg,0.34mM)和氢氧化钠(11.05mg，0.28mM)溶于四氢呋喃和水(体积比＝5︰1)的混合溶液中，常温搅拌30分钟。然后加入原料2a(4，7-二氯喹唑啉，157.98mg，0.23mM)，升温至60～66℃，反应6h。将反应混合液真空浓缩后加入水，用乙酸乙酯萃取三次后，真空浓缩，用100～200mm粗硅胶拌样，用石油醚︰乙酸乙酯＝3︰1作为洗脱剂，得到最终化合物13a(64.1mg，产率为63.12％)。Intermediate 3 (100 mg, 0.34 mM) and sodium hydroxide (11.05 mg, 0.28 mM) were weighed and dissolved in a mixed solution of tetrahydrofuran and water (volume ratio = 5..1), and stirred at room temperature for 30 minutes. Then, the starting material 2a (4,7-dichloroquinazoline, 157.98 mg, 0.23 mM) was added, and the mixture was heated to 60 to 66 ° C for 6 h. After the reaction mixture was concentrated in vacuo, EtOAc (EtOAc) (EtOAc) (HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH (64.1 mg, yield 63.12%).
1H NMR(400MHz,DMSO)δ9.66(s,1H),9.12(s,1H),8.78(s,1H),8.51(dd,J＝9.0,6.1Hz,1H),7.84(d,J＝10.1Hz,1H),7.73(m,2H),7.44(t,J＝8.9Hz,1H),7.26(d,J＝8.6Hz,1H),6.52(s,1H),1.30(s,9H)。 13C NMR(100MHz,DMSO)δ180.79,166.04,164.80,158.71,155.62,151.87,147.34,139.66,138.81,133.89,127.24,125.38,124.81,118.72,115.30,112.48,107.44,93.01,28.83。ESI-MS m/z:456.1[M+H]+。 1 H NMR (400MHz, DMSO) δ9.66 (s, 1H), 9.12 (s, 1H), 8.78 (s, 1H), 8.51 (dd, J = 9.0,6.1Hz, 1H), 7.84 (d, J = 10.1 Hz, 1H), 7.73 (m, 2H), 7.44 (t, J = 8.9 Hz, 1H), 7.26 (d, J = 8.6 Hz, 1H), 6.52 (s, 1H), 1.30 (s, 9H) ). 13 C NMR (100 MHz, DMSO) δ 180.79, 166.04, 164.80, 158.71, 155.62, 151.87, 147.34, 139.66, 138.81, 133.89, 127.24, 125.38, 124.81, 118.72, 115.30, 112.48, 107.44, 93.01, 28.83. ESI-MS m/z: 456.1 [M+H]+.
实施例15 1-(5-(叔丁基)异恶唑-3-基)-3-(4-((6,7-二甲氧基吡啶-4-基)氧基)-3-氟苯基)脲(化合物SKLB707)的制备Example 15 1-(5-(tert-butyl)isoxazol-3-yl)-3-(4-((6,7-dimethoxypyridin-4-yl)oxy)-3-fluoro Preparation of phenyl)urea (compound SKLB707)
Figure PCTCN2018073793-appb-000061
Figure PCTCN2018073793-appb-000061
合成方法与实施例14相同，将实施例14中的4，7-二氯喹唑啉替换为4-氯-6,7-二甲氧基喹唑啉为反应物，反应处理得到灰白色固体化合物SKLB707，产率为78.34％。The synthesis method was the same as that in Example 14, and the 4,7-dichloroquinazoline in Example 14 was replaced with 4-chloro-6,7-dimethoxyquinazoline as a reaction product, and the reaction was carried out to obtain an off-white solid compound SKLB707. The yield was 78.34%.
1H NMR(400MHz,DMSO)δ9.71(s,1H),9.23(s,1H),8.58(s,1H),7.70(dd,J1＝12.8Hz,J2＝2.0Hz,1H),7.58(s,1H),7.42(s,1H),7.25(d,J＝8.7Hz,1H),6.89(s,1H),6.53(s,1H),4.01(s,3H),4.00(s,3H),1.31(s,9H)。 13C NMR(100MHz,DMSO)δ180.79,164.55,158.72,156.42,155.33,152.90,152.62,151.87,150.75,149.43,138.48,134.44,124.92,115.27,109.59,107.27,101.02,93.00,56.68,56.53,28.83。ESI-MS m/z:482.3[M+H]+。 1 H NMR (400MHz, DMSO) δ9.71 (s, 1H), 9.23 (s, 1H), 8.58 (s, 1H), 7.70 (dd, J1 = 12.8Hz, J2 = 2.0Hz, 1H), 7.58 ( s, 1H), 7.42 (s, 1H), 7.25 (d, J = 8.7 Hz, 1H), 6.89 (s, 1H), 6.53 (s, 1H), 4.01 (s, 3H), 4.00 (s, 3H) ), 1.31 (s, 9H). 13 C NMR (100 MHz, DMSO) δ 180.79, 164.55, 158.72, 156.42, 155.33, 152.90, 152.62, 151.87, 150.75, 149.43, 138.48, 134.44, 124.92, 115.27, 109.59, 107.27, 101.02, 93.00, 56.68, 56.53, 28.83. ESI-MS m/z: 482.3 [M+H]+.
实施例16 1-(4-((6,7-双(2-甲氧基乙氧基)喹唑啉-4-基)氧基)-3-氟苯基)-3-(5-(叔丁基)异恶唑-3-基)脲(化合物13c)的制备Example 16 1-(4-((6,7-bis(2-methoxyethoxy)quinazolin-4-yl)oxy)-3-fluorophenyl)-3-(5-( Preparation of tert-butyl)isoxazol-3-yl)urea (compound 13c)
Figure PCTCN2018073793-appb-000062
Figure PCTCN2018073793-appb-000062
合成方法与实施例14相同，将实施例14中的4，7-二氯喹唑啉替换为4-氯-6,7-双(2-甲氧基乙氧基)喹唑啉为反应物，反应处理得到灰白色固体化合物13c，产率为48.66％。The synthesis method was the same as in Example 14, and the 4,7-dichloroquinazoline in Example 14 was replaced with 4-chloro-6,7-bis(2-methoxyethoxy)quinazoline as a reactant. The reaction treatment gave Compound 13c as an off-white solid (yield: 48.66%).
1H NMR(400MHz,DMSO)δ9.64(s,1H),9.09(s,1H),8.56(s,1H),7.68(dd,J1＝12.8,J2＝2.4Hz,1H),7.61(s,1H),7.44(s,1H),7.39(d,J＝6.0Hz,1H),7.26–7.21(m,1H),6.52(s,1H),4.38–4.31(m,4H),3.78–3.74(m,4H),3.36(d,J＝6.4Hz,6H),1.30(s,9H)。 13C NMR(100MHz,DMSO)δ180.79,172.48,164.55,158.72,155.71,153.06,152.64,151.86,149.99,149.33, 124.92,115.25,109.61,108.15,107.20,102.58,102.28,93.00,70.57,68.90,58.81,28.83。ESI-MS m/z:507.2[M+H]+。 1 H NMR (400 MHz, DMSO) δ 9.64 (s, 1H), 9.09 (s, 1H), 8.56 (s, 1H), 7.68 (dd, J1 = 12.8, J2 = 2.4 Hz, 1H), 7.61 (s) , 1H), 7.44 (s, 1H), 7.39 (d, J = 6.0 Hz, 1H), 7.26 - 7.21 (m, 1H), 6.52 (s, 1H), 4.38 - 4.31 (m, 4H), 3.78 - 3.74 (m, 4H), 3.36 (d, J = 6.4 Hz, 6H), 1.30 (s, 9H). 13 C NMR (100 MHz, DMSO) δ 180.79, 172.48, 164.55, 158.72, 155.71, 153.06, 152.64, 151.86, 149.99, 149.33, 124.92, 115.25, 109.61, 108.15, 107.20, 102.58, 102.28, 93.00, 70.57, 68.90, 58.81, 28.83. ESI-MS m/z: 507.2 [M+H]+.
实施例17 1-(5-(叔丁基)异恶唑-3-基)-3-(4-((6,7-甲氧基喹啉-4-基)氧基)-3-氟苯基)脲(化合物13d)的制备Example 17 1-(5-(tert-butyl)isoxazol-3-yl)-3-(4-((6,7-methoxyquinolin-4-yl)oxy)-3-fluoro Preparation of phenyl)urea (compound 13d)
Figure PCTCN2018073793-appb-000063
Figure PCTCN2018073793-appb-000063
称取原料2d(4-氯-6,7-二甲氧基喹啉，500mg,1.0mM)和原料4(4-硝基-2-氟苯酚，704mg，2.0mM)于100mL圆底烧瓶中，加入10mL的2,6-二甲基吡啶溶解混合物，然后加入有机碱1，8-二氮杂二环十一碳-7-烯，升高温度至147℃反应8h后，减压浓缩加入300-400mm的粗硅胶拌样，用DCM︰MeOH＝50︰1作为洗脱剂，使用柱层析方法最终得黄色的化合物(2-氟-4-硝基苯氧基)-6,7-二甲氧基喹唑啉(产率为46.5％)，HPLC纯度95％。The raw material 2d (4-chloro-6,7-dimethoxyquinoline, 500 mg, 1.0 mM) and the starting material 4 (4-nitro-2-fluorophenol, 704 mg, 2.0 mM) were weighed into a 100 mL round bottom flask. Add 10 mL of 2,6-lutidine to dissolve the mixture, then add the organic base 1,8-diazabicycloundec-7-ene, raise the temperature to 147 ° C for 8 h, then concentrate under reduced pressure. 300-400 mm of crude silica gel was mixed with DCM..MeOH=50..1 as eluent, and the title compound (2-fluoro-4-nitrophenoxy)-6,7- Dimethoxyquinazoline (46.5% yield), HPLC purity 95%.
称取(2-氟-4-硝基苯氧基)-6,7-二甲氧基喹唑啉(100mg,1.0mM)和铁粉(81mg，5.0mM)于25mL圆底烧瓶中，加入5mL乙醇和5mL水溶解混合物，然后加入氯化铵(8mg，0.5mM)，升高温度至60℃反应3h后，反应液抽滤后减压浓缩，加入原料5(58mg，1.2mM)于25mL圆底烧瓶中，加入10mL的甲苯溶解混合物，然后加入滴加2-3滴三乙胺，升高温度至120℃反应5h后，加入300～400mm的粗硅胶拌样，用DCM︰MeOH＝50︰1作为洗脱剂，使用柱层析方法最终得淡黄色的化合物13d(产率为34.3％)，HPLC纯度95％。(2-Fluoro-4-nitrophenoxy)-6,7-dimethoxyquinazoline (100 mg, 1.0 mM) and iron powder (81 mg, 5.0 mM) were weighed into a 25 mL round bottom flask and added 5 mL of ethanol and 5 mL of water were used to dissolve the mixture, then ammonium chloride (8 mg, 0.5 mM) was added, and the temperature was raised to 60 ° C for 3 hours. After the reaction solution was suction filtered, concentrated under reduced pressure, and the starting material 5 (58 mg, 1.2 mM) was added to 25 mL. In a round bottom flask, add 10 mL of toluene to dissolve the mixture, then add 2-3 drops of triethylamine, add the temperature to 120 ° C for 5 h, add 300-400 mm of crude silica gel, and mix with DCM..MeOH=50 ..1 as an eluent, a pale yellow compound 13d (yield: 34.3%) was obtained by column chromatography, and the HPLC purity was 95%.
1H NMR(400MHz,DMSO)δ9.88(s,1H),8.90(s,1H),8.50(d,J＝5.2Hz,1H),8.20(s,1H),7.49(s,1H),7.43–7.33(m,2H),7.11(d,J＝9.6Hz,1H),6.55(d,J＝5.3Hz,1H),6.49(s,1H),3.94(d,J＝7.4Hz,6H),1.33–1.19(m,9H)。 1 H NMR (400MHz, DMSO) δ9.88 (s, 1H), 8.90 (s, 1H), 8.50 (d, J = 5.2Hz, 1H), 8.20 (s, 1H), 7.49 (s, 1H), 7.43–7.33 (m, 2H), 7.11 (d, J = 9.6 Hz, 1H), 6.55 (d, J = 5.3 Hz, 1H), 6.49 (s, 1H), 3.94 (d, J = 7.4 Hz, 6H) ), 1.33–1.19 (m, 9H).
实施例18 1-(5-(叔丁基)异恶唑-3-基)-3-(3-氟-4-(喹啉-4-基氧基)苯基)脲(化合物13e)的制备Example 18 1-(5-(tert-butyl)isoxazol-3-yl)-3-(3-fluoro-4-(quinolin-4-yloxy)phenyl)urea (Compound 13e) preparation
Figure PCTCN2018073793-appb-000064
Figure PCTCN2018073793-appb-000064
称取原料2e(4-氯喹啉，100mg,1.0mM)和中间体3(200mg，1.0mM)于100mL圆底烧瓶中，加入10mL的2,6-二甲基吡啶溶解混合物，然后加入有机碱1，8-二氮杂二环十一碳-7-烯，升高温度至147℃反应8h后，减压浓缩加入300-400mm的粗硅胶拌样，用DCM︰MeOH＝50︰1作为洗脱剂，使用柱层析方法最终得黄色的化合物13e(产率为62.5％)，HPLC纯度95％。Raw material 2e (4-chloroquinoline, 100 mg, 1.0 mM) and Intermediate 3 (200 mg, 1.0 mM) were weighed into a 100 mL round bottom flask, 10 mL of 2,6-lutidine was added to dissolve the mixture, and then the organic base was added. 1,8-diazabicycloundec-7-ene, after raising the temperature to 147 ° C for 8 h, concentrated under reduced pressure, adding 300-400 mm of crude silica gel, and using DCM..MeOH = 50..1 for washing Deprotection, using a column chromatography method, finally yielded yellow compound 13e (yield: 62.5%).
1H NMR(400MHz,DMSO)δ8.68(d,J＝5.1Hz,1H),8.33(d,J＝8.3Hz,1H),8.03(d,J＝8.4Hz,1H),7.82(t,J＝7.6Hz,1H),7.66(t,J＝7.6Hz,1H),7.10(t,J＝9.0Hz,1H),6.57(dd,J＝12.5,3.4Hz,2H),6.49(dd,J＝8.8,2.0Hz,1H),6.26(d,J＝32.2Hz,1H),5.51(s,2H),1.34–0.98(m,9H)。 1 H NMR (400MHz, DMSO) δ8.68 (d, J = 5.1Hz, 1H), 8.33 (d, J = 8.3Hz, 1H), 8.03 (d, J = 8.4Hz, 1H), 7.82 (t, J=7.6 Hz, 1H), 7.66 (t, J=7.6 Hz, 1H), 7.10 (t, J=9.0 Hz, 1H), 6.57 (dd, J=12.5, 3.4 Hz, 2H), 6.49 (dd, J = 8.8, 2.0 Hz, 1H), 6.26 (d, J = 32.2 Hz, 1H), 5.51 (s, 2H), 1.34 - 0.98 (m, 9H).
实施例19 1-(4-((7-(苄氧基)-6-甲基喹唑啉-4-基)氧基)-3-氟苯基)-3-(5-(叔丁基)异恶唑-3-基)(化合物13f)的制备Example 19 1-(4-((7-(Benzyloxy)-6-methylquinazolin-4-yl)oxy)-3-fluorophenyl)-3-(5-(tert-butyl) Preparation of isoxazolyl-3-yl) (Compound 13f)
Figure PCTCN2018073793-appb-000065
Figure PCTCN2018073793-appb-000065
称取原料2f(7-苄氧基-4-氯-6-甲氧基喹唑啉，100mg,1.0mM)和中间体3(98mg，1.0mM)于100mL圆底烧瓶中，加入10mL的乙腈溶解混合物，然后加入碳酸钾(132mg，2.0mM)，升高温度至80℃反应8h后，减压浓缩加入300-400mm的粗硅胶拌样，用DCM︰MeOH＝50︰1作为洗脱剂，使用柱层析方法最终得黄色的化合物13f(产率为62.5％)，HPLC纯度95％。Raw material 2f (7-benzyloxy-4-chloro-6-methoxyquinazoline, 100 mg, 1.0 mM) and intermediate 3 (98 mg, 1.0 mM) were weighed into a 100 mL round bottom flask, and 10 mL of acetonitrile was added. The mixture was dissolved, then potassium carbonate (132 mg, 2.0 mM) was added, and the mixture was heated to 80 ° C for 8 h, and concentrated under reduced pressure to a mixture of 300-400 mm of crude silica gel, using DCM..MeOH = 50. The yellow compound 13f (yield 62.5%) was obtained by column chromatography, and the HPLC purity was 95%.
1H NMR(400MHz,DMSO)δ8.56(d,J＝1.8Hz,1H),7.94(dd,J＝11.8,2.4Hz,1H),7.81–7.76(m,1H),7.62(s,1H),7.54(s,2H),7.52(s,2H),7.46(d,J＝2.9Hz,1H),7.44(s,2H),7.42(s,1H),7.40(s,1H),7.38(s,1H),5.37(s,2H),4.00(s,3H),1.28(s,9H)。 1 H NMR (400 MHz, DMSO) δ 8.56 (d, J = 1.8 Hz, 1H), 7.94 (dd, J = 11.8, 2.4 Hz, 1H), 7.81 - 7.76 (m, 1H), 7.62 (s, 1H) ), 7.54 (s, 2H), 7.52 (s, 2H), 7.46 (d, J = 2.9 Hz, 1H), 7.44 (s, 2H), 7.42 (s, 1H), 7.40 (s, 1H), 7.38 (s, 1H), 5.37 (s, 2H), 4.00 (s, 3H), 1.28 (s, 9H).
实施例20 4-(4-(3-(5-(叔丁基)异恶唑-3-基)脲基)-2-氟苯氧基)-7-二甲氧基喹唑啉-6-基乙酸酯(化合物13g)的制备Example 20 4-(4-(3-(5-(tert-butyl)isoxazol-3-yl)ureido)-2-fluorophenoxy)-7-dimethoxyquinazoline-6 -Base acetate (compound 13g) preparation
Figure PCTCN2018073793-appb-000066
Figure PCTCN2018073793-appb-000066
称取原料2g(3,4-二氢-7-甲氧基-4-氧代喹唑啉-6-醇乙酸酯，1g,1.0mM)于100mL圆底烧瓶中，加入10mL的三氯氧磷溶解混合物，然后加入2mL三乙胺升高温度至70℃反应4h后，减压浓缩得到黄色固体6-乙酰氧基-4-氯-7-甲氧基喹唑啉。称取6-乙酰氧基-4-氯-7-甲氧基喹唑啉(100mg,1.0mM)和中间体3(116mg，1.0mM)于100mL圆底烧瓶中，加入10mL的乙腈溶解混合物，然后加入碳酸钾(157mg，2.0mM)，升高温度至80℃反应8h后，减压浓缩加入300-400mm的粗硅胶拌样，用DCM︰MeOH＝50︰1作为洗脱剂，使用柱层析方法最终得黄色的化合物13g(产率为62.5％)，HPLC纯度95％。2 g of raw material (3,4-dihydro-7-methoxy-4-oxoquinazolin-6-ol acetate, 1 g, 1.0 mM) was weighed into a 100 mL round bottom flask, and 10 mL of trichlorobenzene was added. The mixture was dissolved in oxyphosphorus, and then 2 mL of triethylamine was added thereto to raise the temperature to 70 ° C for 4 hours, followed by concentration under reduced pressure to give 6-acetoxy-4-chloro-7-methoxy quinazoline as a yellow solid. 6-Acetoxy-4-chloro-7-methoxyquinazoline (100 mg, 1.0 mM) and Intermediate 3 (116 mg, 1.0 mM) were weighed into a 100 mL round bottom flask, and 10 mL of acetonitrile was added to dissolve the mixture. Then, potassium carbonate (157 mg, 2.0 mM) was added, and the temperature was raised to 80 ° C for 8 hours. After concentration and concentration under reduced pressure, 300-400 mm of crude silica gel was added, and DCM..MeOH = 50..1 was used as an eluent, and a column layer was used. The analysis method finally gave 13 g of a yellow compound (yield: 62.5%).
1H NMR(400MHz,DMSO)δ10.37(s,1H),9.62(s,1H),9.08(s,1H),8.49(s,1H),7.67(dd,J＝12.9,2.5Hz,1H),7.50(s,1H),7.38(d,J＝3.7Hz,1H),7.22(dd,J＝8.8,1.4Hz,1H),6.52(s,1H),4.00(s,3H),2.03(m,3H),1.30(d,J＝4.0Hz,9H)。 1 H NMR (400MHz, DMSO) δ10.37 (s, 1H), 9.62 (s, 1H), 9.08 (s, 1H), 8.49 (s, 1H), 7.67 (dd, J = 12.9,2.5Hz, 1H ), 7.50 (s, 1H), 7.38 (d, J = 3.7 Hz, 1H), 7.22 (dd, J = 8.8, 1.4 Hz, 1H), 6.52 (s, 1H), 4.00 (s, 3H), 2.03 (m, 3H), 1.30 (d, J = 4.0 Hz, 9H).
实施例21 1-(5-(叔丁基)异恶唑-3-基)-3-(4-((7,8-二氢-[1,4]二恶烷[2,3-g]喹唑啉-4-基)氧基)-3-氟苯基)脲(化合物13i)的制备Example 21 1-(5-(tert-butyl)isoxazol-3-yl)-3-(4-((7,8-dihydro-[1,4]dioxane [2,3-g] Of quinazolin-4-yl)oxy)-3-fluorophenyl)urea (compound 13i)
Figure PCTCN2018073793-appb-000067
Figure PCTCN2018073793-appb-000067
称取原料2i(4-氯-6,7-二亚甲基二羟喹唑啉，100mg,1.0mM)和中间体3(228mg，1.0mM)于100mL圆底烧瓶中，加入10mL的乙腈溶解混合物，然后加入碳酸钾(310mg，2.0mM)，升高温度至80℃反应8h后，减压浓缩加入300-400mm的粗硅胶拌样，用DCM︰MeOH＝50︰1 作为洗脱剂，使用柱层析方法最终得黄色的化合物13i(产率为62.5％)，HPLC纯度95％。Raw material 2i (4-chloro-6,7-dimethylene quinazoline, 100 mg, 1.0 mM) and Intermediate 3 (228 mg, 1.0 mM) were weighed into a 100 mL round bottom flask and dissolved in 10 mL of acetonitrile. The mixture was then added with potassium carbonate (310 mg, 2.0 mM), and the temperature was raised to 80 ° C for 8 h. After concentration, the mixture was concentrated under reduced pressure to a mixture of 300-400 mm of crude silica gel, using DCM..MeOH = 50.. The column chromatography method finally gave the yellow compound 13i (yield: 62.5%).
1H NMR(400MHz,DMSO)δ9.63(s,1H),9.11(s,1H),8.53(s,1H),7.67(q,J＝2.6Hz,2H),7.41(s,1H),7.37(d,J＝8.8Hz,1H),7.23(dd,J＝8.8,1.4Hz,1H),6.52(s,1H),4.50–4.40(m,4H),1.30(s,9H). 1 H NMR (400MHz, DMSO) δ9.63 (s, 1H), 9.11 (s, 1H), 8.53 (s, 1H), 7.67 (q, J = 2.6Hz, 2H), 7.41 (s, 1H), 7.37 (d, J = 8.8 Hz, 1H), 7.23 (dd, J = 8.8, 1.4 Hz, 1H), 6.52 (s, 1H), 4.50 - 4.40 (m, 4H), 1.30 (s, 9H).
实施例22 1-(5-(叔丁基)异恶唑-3-基)-3-(3-氟-4-((7-甲氧基-6-(3-吗啉代丙氧基)喹唑啉-4-基)氧基)脲(化合物13j)的制备Example 22 1-(5-(tert-butyl)isoxazol-3-yl)-3-(3-fluoro-4-((7-methoxy-6-(3-morpholinopropoxy)oxyl) Preparation of quinazolin-4-yl)oxy)urea (compound 13j)
Figure PCTCN2018073793-appb-000068
Figure PCTCN2018073793-appb-000068
称取原料2j(3,4-二氢-7-甲氧基-4-氧代喹唑啉-6-醇乙酸酯，1g(1.0mM)于100mL圆底烧瓶中，加入10mL的三氯氧磷溶解混合物，然后加入2mL三乙胺升高温度至70℃反应4h后，减压浓缩得到黄色固体4-氯-7-甲氧基喹唑啉-6-醇。称取4-氯-7-甲氧基喹唑啉-6-醇(150mg,1.0mM)和N-(3-氯丙基)吗啉(140mg，1.2mM)于100mL圆底烧瓶中，加入10mL的DMF溶解混合物，然后加入碳酸钾(284mg，2.0mM)，升高温度至130℃反应8h后，减压浓缩加入300-400mm的粗硅胶拌样，用DCM︰MeOH＝50︰1作为洗脱剂，使用柱层析方法得黄色的化合物4--氯-7-(2-甲氧基乙氧基)-6-(3-吗啉-4-基-丙氧基)-喹唑啉。称取4--氯-7-(2-甲氧基乙氧基)-6-(3-吗啉-4-基-丙氧基)-喹唑啉(100mg,1.0mM)和中间体3(92mg，1.0mM)于100mL圆底烧瓶中，加入10mL的DMF溶解混合物，然后加入碳酸钾(104mg，2.0mM)，升高温度至130℃反应8h后，减压浓缩加入300-400mm的粗硅胶拌样，用DCM︰MeOH＝50︰1作为洗脱剂，使用柱层析方法最终得黄色的化合物13j(产率为62.5％)，HPLC纯度95％。Weigh 2j (3,4-dihydro-7-methoxy-4-oxoquinazolin-6-ol acetate, 1 g (1.0 mM) in a 100 mL round bottom flask, and add 10 mL of trichlorochloride. The mixture was dissolved in oxyphosphorus, then 2 mL of triethylamine was added to raise the temperature to 70 ° C for 4 h, and then concentrated under reduced pressure to give 4-chloro-7-methoxyquinazoline-6-ol as a yellow solid. 7-Methoxyquinazolin-6-ol (150 mg, 1.0 mM) and N-(3-chloropropyl)morpholine (140 mg, 1.2 mM) were placed in a 100 mL round bottom flask, and 10 mL of DMF was added to dissolve the mixture. Then, potassium carbonate (284 mg, 2.0 mM) was added, and the temperature was raised to 130 ° C for 8 hours. After concentration and concentration under reduced pressure, 300-400 mm of crude silica gel was added, and DCM..MeOH = 50..1 was used as an eluent. The yellow compound 4-chloro-7-(2-methoxyethoxy)-6-(3-morpholin-4-yl-propoxy)-quinazoline was obtained. Chloro-7-(2-methoxyethoxy)-6-(3-morpholin-4-yl-propoxy)-quinazoline (100 mg, 1.0 mM) and Intermediate 3 (92 mg, 1.0 mM) In a 100 mL round bottom flask, 10 mL of DMF was added to dissolve the mixture, then potassium carbonate (104 mg, 2.0 mM) was added, and the temperature was raised to 130 ° C for 8 hours, and then concentrated under reduced pressure to a mixture of 300-400 mm of crude silica gel. DCM︰MeOH = 50︰1 as the eluent, using column chromatography methods to give the final compound as a yellow 13j (yield 62.5%), HPLC purity 95%.
1H NMR(400MHz,DMSO)δ9.63(s,1H),9.08(s,1H),8.56(s,1H),7.66(d,J＝2.4Hz,1H),7.57(s,1H),7.40(d,J＝3.7Hz,2H),7.24(dd,J＝8.9,1.4Hz,1H),6.52(s,1H),4.24(t,J＝6.6Hz,2H),4.00(s,3H),3.59–3.55(m,4H),2.46(d,J＝6.9Hz,2H),2.39(s,4H),2.00–1.94(m,2H),1.30(s,9H)。 1 H NMR (400MHz, DMSO) δ9.63 (s, 1H), 9.08 (s, 1H), 8.56 (s, 1H), 7.66 (d, J = 2.4Hz, 1H), 7.57 (s, 1H), 7.40 (d, J = 3.7 Hz, 2H), 7.24 (dd, J = 8.9, 1.4 Hz, 1H), 6.52 (s, 1H), 4.24 (t, J = 6.6 Hz, 2H), 4.00 (s, 3H) ), 3.59 - 3.55 (m, 4H), 2.46 (d, J = 6.9 Hz, 2H), 2.39 (s, 4H), 2.00 - 1.94 (m, 2H), 1.30 (s, 9H).
实施例23 1-(5-(叔丁基)异恶唑-3-基)-3-(3-氟-4–((6-甲氧基-7-(3-吗啉代丙氧基)喹唑啉-4-基)氧基)脲(化合物13k)的制备Example 23 1-(5-(tert-butyl)isoxazol-3-yl)-3-(3-fluoro-4-((6-methoxy-7-(3-morpholinopropoxy) Preparation of quinazolin-4-yl)oxy)urea (compound 13k)
Figure PCTCN2018073793-appb-000069
Figure PCTCN2018073793-appb-000069
称取原料2k(7-苄氧基-4-氯-6-甲氧基喹唑啉，1g,1.0mM)于100mL圆底烧瓶中，加入20mL的甲醇溶解，然后加入钯碳(706g,2.0mM)，氮气保护下升温至70℃反应4h后，反应液抽滤后减压浓缩得到黄色固体6-乙酰氧基-4-氯-7-甲氧基喹唑啉。称取6-乙酰氧基-4-氯-7-甲氧基喹唑啉(200mg,1.0mM)于100mL圆底烧瓶中，加入10mL NaOH水溶液和二氧六环(1:1)的混合溶液溶解该化合物，常温搅拌反应8h后，调PH至3-4左右，用乙酸乙酯萃取收集有机相减压浓缩的黄色固体4-氯-7-甲氧基喹唑啉-6-醇。称取4-氯-7-甲氧基喹唑啉-6-醇(150mg,1.0mM)和N-(3-氯丙基)吗啉(140mg，1.2mM)于100mL圆底烧瓶中，加入10mL的 DMF溶解混合物，然后加入碳酸钾(284mg，2.0mM)，升高温度至130℃反应8h后，减压浓缩加入300-400mm的粗硅胶拌样，用DCM︰MeOH＝50︰1作为洗脱剂，使用柱层析方法得黄色的化合物4--氯-7-(2-甲氧基乙氧基)-6-(3-吗啉-4-基-丙氧基)-喹唑啉。称取4--氯-7-(2-甲氧基乙氧基)-6-(3-吗啉-4-基-丙氧基)-喹唑啉(100mg,1.0mM)和中间体3(92mg，1.0mM)于100mL圆底烧瓶中，加入10mL的DMF溶解混合物，然后加入碳酸钾(104mg，2.0mM)，升高温度至130℃反应8h后，减压浓缩加入300-400mm的粗硅胶拌样，用DCM︰MeOH＝50︰1作为洗脱剂，使用柱层析方法最终得黄色的化合物13k(产率为62.5％)，HPLC纯度95％。The raw material 2k (7-benzyloxy-4-chloro-6-methoxyquinazoline, 1 g, 1.0 mM) was weighed into a 100 mL round bottom flask, dissolved in 20 mL of methanol, and then palladium on carbon (706 g, 2.0). mM), the temperature was raised to 70 ° C under nitrogen for 4 h, and the reaction mixture was suction filtered and concentrated under reduced pressure to give 6-acetoxy-4-chloro-7-methoxy quinazoline. 6-Acetoxy-4-chloro-7-methoxyquinazoline (200 mg, 1.0 mM) was weighed into a 100 mL round bottom flask, and a mixed solution of 10 mL of NaOH aqueous solution and dioxane (1:1) was added. The compound was dissolved, and the mixture was stirred at room temperature for 8 hours, and then the mixture was adjusted to pH 3-4, and the organic phase was concentrated to give a yellow solid 4-chloro-7-methoxyquinazoline-6-ol. 4-Chloro-7-methoxyquinazolin-6-ol (150 mg, 1.0 mM) and N-(3-chloropropyl)morpholine (140 mg, 1.2 mM) were weighed into a 100 mL round bottom flask and added 10 mL of DMF was dissolved in the mixture, then potassium carbonate (284 mg, 2.0 mM) was added, and the temperature was raised to 130 ° C for 8 hours. After concentration, the mixture was concentrated under reduced pressure to a mixture of 300-400 mm of crude silica gel, and washed with DCM..MeOH = 50. Deprotection, using column chromatography to give the yellow compound 4-chloro-7-(2-methoxyethoxy)-6-(3-morpholin-4-yl-propoxy)-quinazoline . 4-Chloro-7-(2-methoxyethoxy)-6-(3-morpholin-4-yl-propoxy)-quinazoline (100 mg, 1.0 mM) and intermediate 3 were weighed out (92mg, 1.0mM) in a 100mL round bottom flask, add 10mL of DMF to dissolve the mixture, then add potassium carbonate (104mg, 2.0mM), raise the temperature to 130 ° C for 8h, then concentrate under reduced pressure to add 300-400mm thick The mixture was chromatographed on silica gel eluting with DCM. MeOH = 50.
1H NMR(400MHz,DMSO)δ9.71(s,1H),9.10(s,1H),8.63(s,1H),7.66(d,J＝2.4Hz,1H),7.54(s,1H),7.45(d,J＝3.7Hz,2H),7.24(dd,J＝8.9,1.4Hz,1H),6.42(s,1H),4.26(t,J＝6.6Hz,2H),4.07(s,3H),3.62–3.59(m,4H),2.42(d,J＝6.9Hz,2H),2.37(s,4H),1.98(m,2H),1.33(s,9H)。 1 H NMR (400 MHz, DMSO) δ 9.71 (s, 1H), 9.10 (s, 1H), 8.63 (s, 1H), 7.66 (d, J = 2.4 Hz, 1H), 7.54 (s, 1H), 7.45 (d, J = 3.7 Hz, 2H), 7.24 (dd, J = 8.9, 1.4 Hz, 1H), 6.42 (s, 1H), 4.26 (t, J = 6.6 Hz, 2H), 4.07 (s, 3H) ), 3.62 - 3.59 (m, 4H), 2.42 (d, J = 6.9 Hz, 2H), 2.37 (s, 4H), 1.98 (m, 2H), 1.33 (s, 9H).
实施例24 1-(5-(叔丁基)异恶唑-3-基)-3-(3-氟-4-((7-甲氧基-6-(2-吗啉代乙基)喹唑啉-4-基)氧基)苯基)脲(化合物13l)的制备Example 24 1-(5-(tert-butyl)isoxazol-3-yl)-3-(3-fluoro-4-((7-methoxy-6-(2-morpholinoethyl)) Preparation of quinazolin-4-yl)oxy)phenyl)urea (compound 13l)
Figure PCTCN2018073793-appb-000070
Figure PCTCN2018073793-appb-000070
合成方法与实施例23相同，将实施例23中的3,4-二氢-7-甲氧基-4-氧代喹唑啉-6-醇乙酸酯替换为4-(2-((4-氯-7-甲氧基喹啉-6-基)氧基)乙基)吗啉为反应物，反应处理得到灰白色固体化合物13l。The synthesis method was the same as in Example 23, and the 3,4-dihydro-7-methoxy-4-oxoquinazolin-6-ol acetate in Example 23 was replaced with 4-(2-(( 4-Chloro-7-methoxyquinolin-6-yl)oxy)ethyl)morpholine was used as the reaction product, and the reaction was afforded to give a pale white solid compound 13l.
1H NMR(400MHz,DMSO)δ9.75(s,1H),9.21(s,1H),8.70(s,1H),7.63(d,J＝2.4Hz,1H),7.60(s,1H),7.54(d,J＝3.7Hz,2H),7.30(dd,J＝8.9,1.4Hz,1H),6.42(s,1H),δ4.19(s,2H),3.90(s,3H),3.59(s,4H),2.75(s,2H),1.21(d,J＝23.1Hz,4H),1.33(s,9H)。 1 H NMR (400MHz, DMSO) δ9.75 (s, 1H), 9.21 (s, 1H), 8.70 (s, 1H), 7.63 (d, J = 2.4Hz, 1H), 7.60 (s, 1H), 7.54 (d, J = 3.7 Hz, 2H), 7.30 (dd, J = 8.9, 1.4 Hz, 1H), 6.42 (s, 1H), δ 4.19 (s, 2H), 3.90 (s, 3H), 3.59 (s, 4H), 2.75 (s, 2H), 1.21 (d, J = 23.1 Hz, 4H), 1.33 (s, 9H).
实施例25 1-(3-氟-4-((6-甲氧基-7-(2-吗啉代乙基)喹唑啉-4-基)氧基)苯基)-3-(5-异丙基异恶唑-3-基)脲(化合物13m)的制备Example 25 1-(3-Fluoro-4-((6-methoxy-7-(2-morpholinoethyl)quinazolin-4-yl)oxy)phenyl)-3-(5) -Isopropyloxazolyl-3-yl)urea (Compound 13m) Preparation
Figure PCTCN2018073793-appb-000071
Figure PCTCN2018073793-appb-000071
合成方法与实施例24相同，将实施例23中的7-苄氧基-4-氯-6-甲氧基喹唑啉替换为4-(2-((4-氯-6-甲基喹唑啉-7-基)氧基)乙基)吗啉为反应物，反应处理得到灰白色固体化合物13m。The synthesis method was the same as in Example 24, and the 7-benzyloxy-4-chloro-6-methoxyquinazoline in Example 23 was replaced with 4-(2-((4-chloro-6-methylquin). Oxazolin-7-yl)oxy)ethyl)morpholine was used as the reactant, which was subjected to a reaction to give a pale white solid compound 13 m.
1H NMR(400MHz,DMSO)δ9.77(s,1H),9.25(s,1H),8.72(s,1H),7.64(d,J＝2.4Hz,1H),7.65(s,1H),7.58(d,J＝3.7Hz,2H),7.35(dd,J＝8.9,1.4Hz,1H),6.45(s,1H),δ4.23(s,2H),3.92(s,3H),3.57(s,4H),2.78(s,2H),1.25(d,J＝23.1Hz,4H),1.35(s,9H)。 1 H NMR (400 MHz, DMSO) δ 9.77 (s, 1H), 9.25 (s, 1H), 8.72 (s, 1H), 7.64 (d, J = 2.4 Hz, 1H), 7.65 (s, 1H), 7.58 (d, J = 3.7 Hz, 2H), 7.35 (dd, J = 8.9, 1.4 Hz, 1H), 6.45 (s, 1H), δ 4.23 (s, 2H), 3.92 (s, 3H), 3.57 (s, 4H), 2.78 (s, 2H), 1.25 (d, J = 23.1 Hz, 4H), 1.35 (s, 9H).
实施例26 1-(5-(叔丁基)异恶唑-3-基)-3-(3-氟-4-((7-甲氧基-6-(3-(哌啶-1-基)丙氧基)氧基)苯基)脲(化合物13n)的制备Example 26 1-(5-(tert-butyl)isoxazol-3-yl)-3-(3-fluoro-4-((7-methoxy-6-(3-(piperidin-1-) Preparation of propyloxy)oxy)phenyl)urea (Compound 13n)
Figure PCTCN2018073793-appb-000072
Figure PCTCN2018073793-appb-000072
合成方法与实施例23相同，将实施例23中的3,4-二氢-7-甲氧基-4-氧代喹唑啉-6-醇乙酸酯替换为4-(3-((4-氯-7-甲氧基喹啉-6-基)氧基)丙基)吗啉为反应物，反应处理得到灰白色固体化合物13n。The synthesis method was the same as in Example 23, and the 3,4-dihydro-7-methoxy-4-oxoquinazolin-6-ol acetate in Example 23 was replaced with 4-(3-(( 4-Chloro-7-methoxyquinolin-6-yl)oxy)propyl)morpholine was used as the reaction product, which was subjected to a reaction to give a pale white solid compound 13n.
1H NMR(400MHz,DMSO)δ9.71(s,1H),9.10(s,1H),8.63(s,1H),7.66(d,J＝2.4Hz,1H),7.54(s,1H),7.45(d,J＝3.7Hz,2H),7.24(dd,J＝8.9,1.4Hz,1H),6.42(s,1H),4.26(t,J＝6.6Hz,2H),3.62–3.59(m,4H),2.42(d,J＝6.9Hz,2H),2.37(s,6H),1.98(m,2H),1.33(s,9H)。 1 H NMR (400 MHz, DMSO) δ 9.71 (s, 1H), 9.10 (s, 1H), 8.63 (s, 1H), 7.66 (d, J = 2.4 Hz, 1H), 7.54 (s, 1H), 7.45 (d, J = 3.7 Hz, 2H), 7.24 (dd, J = 8.9, 1.4 Hz, 1H), 6.42 (s, 1H), 4.26 (t, J = 6.6 Hz, 2H), 3.62 - 3.59 (m) , 4H), 2.42 (d, J = 6.9 Hz, 2H), 2.37 (s, 6H), 1.98 (m, 2H), 1.33 (s, 9H).
实施例27 1-(3-氟-4-((6-甲氧基-7-(3-(哌啶-1-基)丙氧基)喹唑啉-4-基)氧基)苯基)-3-(5-异丙基异恶唑-3-基)基)脲(化合物13o)的制备Example 27 1-(3-Fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinazolin-4-yl)oxy)phenyl) Preparation of 3-(5-isopropylisoxazol-3-yl))urea (Compound 13o)
Figure PCTCN2018073793-appb-000073
Figure PCTCN2018073793-appb-000073
合成方法与实施例24相同，将实施例23中的7-苄氧基-4-氯-6-甲氧基喹唑啉替换为4-(4-((4-氯-6-甲基喹唑啉-7-基)氧基)丁基)吗啉为反应物，反应处理得到灰白色固体化合物13o。The synthesis method was the same as in Example 24, and the 7-benzyloxy-4-chloro-6-methoxyquinazoline in Example 23 was replaced with 4-(4-((4-chloro-6-methylquin). Oxazolin-7-yl)oxy)butyl)morpholine is the reactant, which is reacted to give the compound 13o as an off-white solid.
1H NMR(400MHz,DMSO)δ9.70(s,1H),9.10(s,1H),8.63(s,1H),7.66(d,J＝2.4Hz,1H),7.54(s,1H),7.45(d,J＝3.7Hz,2H),7.24(dd,J＝8.9,1.4Hz,1H),6.42(s,1H),4.27(t,J＝6.6Hz,2H),3.62–3.59(m,4H),2.44(d,J＝6.9Hz,2H),2.38(s,6H),1.98(m,2H),1.33(s,9H)。 1 H NMR (400MHz, DMSO) δ9.70 (s, 1H), 9.10 (s, 1H), 8.63 (s, 1H), 7.66 (d, J = 2.4Hz, 1H), 7.54 (s, 1H), 7.45 (d, J = 3.7 Hz, 2H), 7.24 (dd, J = 8.9, 1.4 Hz, 1H), 6.42 (s, 1H), 4.27 (t, J = 6.6 Hz, 2H), 3.62 - 3.59 (m) , 4H), 2.44 (d, J = 6.9 Hz, 2H), 2.38 (s, 6H), 1.98 (m, 2H), 1.33 (s, 9H).
实施例28 1-(5-(叔丁基)异恶唑-3-基)-3-(4-((6-(3-(二甲基氨基)丙氧基)-7-甲基喹唑啉-4-基)氧基)-3-氟苯基)脲(化合物13p)的制备Example 28 1-(5-(tert-Butyl)isoxazol-3-yl)-3-(4-((6-(3-(dimethylamino)propoxy)-7-methylquina) Preparation of oxazolin-4-yl)oxy)-3-fluorophenyl)urea (Compound 13p)
Figure PCTCN2018073793-appb-000074
Figure PCTCN2018073793-appb-000074
合成方法与实施例23相同，将实施例23中的3,4-二氢-7-甲氧基-4-氧代喹唑啉-6-醇乙酸酯替换为3-((4-氯-7-甲氧基喹啉-6-基)氧基)-N，N-二甲基丙-1-胺为反应物，反应处理得到灰白色固体化合物13p。The synthesis method was the same as in Example 23, and the 3,4-dihydro-7-methoxy-4-oxoquinazolin-6-ol acetate in Example 23 was replaced with 3-((4-chloro). -7-Methoxyquinolin-6-yl)oxy)-N,N-dimethylpropan-1-amine was obtained as a reaction mixture.
1H NMR(400MHz,DMSO)δ9.78(s,1H),9.20(s,1H),8.74(s,1H),7.67(d,J＝2.4Hz,1H),7.65(s,1H),7.59(d,J＝3.7Hz,2H),7.36(dd,J＝8.9,1.4Hz,1H),6.47(s,1H),δ4.09(s,2H),3.94(d,J＝24.0Hz,3H),2.41(s,2H),2.16(d,J＝19.4Hz,6H),1.82(s,2H),1.24(s,9H)。 1 H NMR (400MHz, DMSO) δ9.78 (s, 1H), 9.20 (s, 1H), 8.74 (s, 1H), 7.67 (d, J = 2.4Hz, 1H), 7.65 (s, 1H), 7.59 (d, J = 3.7 Hz, 2H), 7.36 (dd, J = 8.9, 1.4 Hz, 1H), 6.47 (s, 1H), δ 4.09 (s, 2H), 3.94 (d, J = 24.0 Hz) , 3H), 2.41 (s, 2H), 2.16 (d, J = 19.4 Hz, 6H), 1.82 (s, 2H), 1.24 (s, 9H).
实施例29 1-(5-(叔丁基)异恶唑-3-基)-3-(4-((7-(3-(二甲基氨基)丙氧基)-6-甲基喹唑啉-4-基)氧基)-3-氟苯基)脲(化合物13r)的制备Example 29 1-(5-(tert-butyl)isoxazol-3-yl)-3-(4-((7-(3-(dimethylamino)propoxy)-6-methylquina) Preparation of oxazolin-4-yl)oxy)-3-fluorophenyl)urea (Compound 13r)
Figure PCTCN2018073793-appb-000075
Figure PCTCN2018073793-appb-000075
合成方法与实施例24相同，将实施例23中的7-苄氧基-4-氯-6-甲氧基喹唑啉替换为4-((4-氯-6-甲基喹唑啉-7-基)氧基)-N，N-二甲基丁-1-胺为反应物，反应处理得到灰白色固体化合物13r。The synthesis method was the same as in Example 24, and the 7-benzyloxy-4-chloro-6-methoxyquinazoline in Example 23 was replaced with 4-((4-chloro-6-methylquinazoline- 7-yl)oxy)-N,N-dimethylbutan-1-amine is the reactant, which is reacted to give the compound 13r as an off-white solid.
1H NMR(400MHz,DMSO)δ9.78(s,1H),9.20(s,1H),8.74(s,1H),7.67(d,J＝2.4Hz,1H),7.65(s,1H),7.59(d,J＝3.7Hz,2H),7.36(dd,J＝8.9,1.4Hz,1H),6.47(s,1H),δ4.09(s,2H),3.94(d,J＝24.0Hz,3H),2.41(s,2H),2.16(d,J＝19.4Hz,6H),1.82(s,2H),1.24(s,9H)。 1 H NMR (400MHz, DMSO) δ9.78 (s, 1H), 9.20 (s, 1H), 8.74 (s, 1H), 7.67 (d, J = 2.4Hz, 1H), 7.65 (s, 1H), 7.59 (d, J = 3.7 Hz, 2H), 7.36 (dd, J = 8.9, 1.4 Hz, 1H), 6.47 (s, 1H), δ 4.09 (s, 2H), 3.94 (d, J = 24.0 Hz) , 3H), 2.41 (s, 2H), 2.16 (d, J = 19.4 Hz, 6H), 1.82 (s, 2H), 1.24 (s, 9H).
实施例30 4-(4-(3-(5-(叔丁基)异恶唑-3-基)脲基)-2-氟苯氧基)-N-甲基吡啶酰胺(化合物14a)的制备Example 30 4-(4-(3-(5-(tert-Butyl)isoxazol-3-yl)ureido)-2-fluorophenoxy)-N-methylpyridineamide (Compound 14a) preparation
Figure PCTCN2018073793-appb-000076
Figure PCTCN2018073793-appb-000076
称取原料3a(4,6-二氯-5-甲氧基嘧啶，168mg,1.2mM)和原料4(4-氨基-2-氟苯酚，100mg，1.0mM)于100mL圆底烧瓶中，加入10mL的四氢呋喃和10mL水溶解混合物，然后加入氢氧化钠(63mg，2.0mM)，升高温度至60℃反应5h后，减压浓缩加入300-400mm的粗硅胶拌样，用DCM︰MeOH＝150︰1作为洗脱剂，使用柱层析方法最终得白色的化合物中间体11a(4-((6-氯-5-甲氧基嘧啶-4-基)氧代)-3-氟苯胺，80mg，产率为40.3％)，HPLC纯度95％。Raw material 3a (4,6-dichloro-5-methoxypyrimidine, 168 mg, 1.2 mM) and starting material 4 (4-amino-2-fluorophenol, 100 mg, 1.0 mM) were weighed into a 100 mL round bottom flask and added. Dissolve the mixture in 10 mL of tetrahydrofuran and 10 mL of water, then add sodium hydroxide (63 mg, 2.0 mM), raise the temperature to 60 ° C for 5 h, concentrate under reduced pressure, add 300-400 mm of crude silica gel, and mix with DCM..MeOH = 150 ..1 as an eluent, using a column chromatography method to finally obtain a white compound intermediate 11a (4-((6-chloro-5-methoxypyrimidin-4-yl)oxy)-3-fluoroaniline, 80 mg The yield was 40.3%) and the HPLC purity was 95%.
称取中间体11a(80mg,1.0mM)和原料5(60mg，1.2mM)于25mL圆底烧瓶中，加入10mL的四氢呋喃溶解混合物，然后加入滴加2-3滴三乙胺，升高温度至65℃反应5h后，减压浓缩加入300-400mm的粗硅胶拌样，用DCM︰MeOH＝150︰1作为洗脱剂，使用柱层析方法最终得淡黄色的化合物14a(80mg,产率为40.3％)，HPLC纯度95％。Intermediate 11a (80 mg, 1.0 mM) and starting material 5 (60 mg, 1.2 mM) were weighed into a 25 mL round bottom flask, 10 mL of tetrahydrofuran was added to dissolve the mixture, then 2-3 drops of triethylamine were added dropwise, and the temperature was raised to After reacting at 65 ° C for 5 h, it was concentrated under reduced pressure and then added to a mixture of 300-400 mm of crude silica gel, using DCM..MeOH = 150..1 as eluent, using column chromatography to give pale yellow compound 14a (80 mg, yield 40.3%), HPLC purity 95%.
1H NMR(400MHz,DMSO)δ9.80(s,1H),9.50(s,1H),8.79(d,J＝4.5Hz,1H),8.53(d,J＝5.6Hz,1H),7.74(dd,J＝13.1,2.1Hz,1H),7.38(dd,J＝10.3,5.7Hz,2H),7.27(d,J＝9.0Hz,1H),7.19(dd,J＝5.5,2.6Hz,1H),6.52(s,1H),2.79(d,J＝4.8Hz,3H),1.30(s,9H)。 1 H NMR (400MHz, DMSO) δ9.80 (s, 1H), 9.50 (s, 1H), 8.79 (d, J = 4.5Hz, 1H), 8.53 (d, J = 5.6Hz, 1H), 7.74 ( Dd, J = 13.1, 2.1 Hz, 1H), 7.38 (dd, J = 10.3, 5.7 Hz, 2H), 7.27 (d, J = 9.0 Hz, 1H), 7.19 (dd, J = 5.5, 2.6 Hz, 1H) ), 6.52 (s, 1H), 2.79 (d, J = 4.8 Hz, 3H), 1.30 (s, 9H).
实施例31 1-(5-(叔丁基)异恶唑-3-基)-3-(4-((6-氯-5-甲氧基嘧啶-4-基)氧基)-3-氟苯基)脲(化合物14b)的制备Example 31 1-(5-(tert-butyl)isoxazol-3-yl)-3-(4-((6-chloro-5-methoxypyrimidin-4-yl)oxy)-3- Preparation of fluorophenyl)urea (compound 14b)
Figure PCTCN2018073793-appb-000077
Figure PCTCN2018073793-appb-000077
称取原料3b(4-氯-N-甲基甲基吡啶酰胺，500mg,1.0mM)和原料4(4-氨基-2-氟苯酚，745mg，2.0mM)于100mL圆底烧瓶中，加入10mL的N，N-二甲基甲酰胺溶解混合物，然后加入碳酸铯(3820mg，4.0mM)，升高温度至130℃反应5h后，减压浓缩加入300-400mm的 粗硅胶拌样，用DCM︰MeOH＝50︰1作为洗脱剂，使用柱层析方法最终得白色的化合物中间体11b(4-(4-氨基-2-氟苯氧基)-N-甲基甲基吡啶酰胺，产率为32.3％)，HPLC纯度95％。Raw material 3b (4-chloro-N-methylmethylpyridine amide, 500 mg, 1.0 mM) and starting material 4 (4-amino-2-fluorophenol, 745 mg, 2.0 mM) were weighed into a 100 mL round bottom flask, and 10 mL was added. The N,N-dimethylformamide was dissolved in the mixture, then cesium carbonate (3820 mg, 4.0 mM) was added, and the temperature was raised to 130 ° C for 5 h, and then concentrated under reduced pressure to a mixture of 300-400 mm of crude silica gel, using DCM. MeOH = 50..1 as eluent, using column chromatography to give white compound intermediate 11b (4-(4-amino-2-fluorophenoxy)-N-methylmethyl pyridine amide, yield It was 32.3%) and the HPLC purity was 95%.
称取化合物中间体11b(50mg,1.0mM)和原料5(20mg，1.2mM)于25mL圆底烧瓶中，加入10mL的四氢呋喃溶解混合物，然后滴加2-3滴三乙胺，升高温度至65℃反应5h后，减压浓缩加入300-400mm的粗硅胶拌样，用DCM︰MeOH＝50︰1作为洗脱剂，使用柱层析方法最终得淡黄色的化合物14b(产率为45.3％)，HPLC纯度95％。Compound intermediate 11b (50 mg, 1.0 mM) and starting material 5 (20 mg, 1.2 mM) were weighed into a 25 mL round bottom flask, 10 mL of tetrahydrofuran was added to dissolve the mixture, then 2-3 drops of triethylamine were added dropwise, and the temperature was raised to After reacting at 65 ° C for 5 h, it was concentrated under reduced pressure, and then added to a mixture of 300-400 mm of crude silica gel, using DCM.. MeOH=50..1 as eluent, using column chromatography to give pale yellow compound 14b (yield 45.3%) ), HPLC purity 95%.
1H NMR(400MHz,DMSO)δ9.66(s,1H),9.16(s,1H),8.35(s,1H),7.68(dd,J＝12.9,2.3Hz,1H),7.39(t,J＝8.8Hz,1H),7.22(d,J＝8.8Hz,1H),6.51(s,1H),4.01(s,3H),1.30(s,9H)。 1 H NMR (400MHz, DMSO) δ9.66 (s, 1H), 9.16 (s, 1H), 8.35 (s, 1H), 7.68 (dd, J = 12.9,2.3Hz, 1H), 7.39 (t, J = 8.8 Hz, 1H), 7.22 (d, J = 8.8 Hz, 1H), 6.51 (s, 1H), 4.01 (s, 3H), 1.30 (s, 9H).
实施例32 1-(4-((1H-吡咯并[2,3-b]吡啶-4-基)氧基)-3-氟苯基)-3-(5-(叔丁基)异恶唑-3-基)(化合物14c)的制备Example 32 1-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-3-(5-(tert-butyl)iso- Preparation of oxazol-3-yl) (Compound 14c)
Figure PCTCN2018073793-appb-000078
Figure PCTCN2018073793-appb-000078
称取原料3c(4-氯-1H-吡咯并[2,3-b]吡啶，500mg,1.0mM)和原料4(4-硝基-2-氟苯酚，516mg，1.2mM)于100mL圆底烧瓶中，加入10mL的2,6-二甲基吡啶溶解混合物，然后加入4-二甲氨基吡啶(401mg，1.0mM)，升高温度至147℃反应8h后，减压浓缩加入300-400mm的粗硅胶拌样，用DCM︰MeOH＝50︰1作为洗脱剂，使用柱层析方法最终得黄色的化合物中间体11c(4-((1H-吡咯并[2,3-b]吡啶-4-基)氧基)-3-氟苯胺，产率为62.5％)，HPLC纯度95％。Raw material 3c (4-chloro-1H-pyrrolo[2,3-b]pyridine, 500 mg, 1.0 mM) and starting material 4 (4-nitro-2-fluorophenol, 516 mg, 1.2 mM) were weighed into a 100 mL round bottom. In the flask, 10 mL of 2,6-lutidine was added to dissolve the mixture, then 4-dimethylaminopyridine (401 mg, 1.0 mM) was added, and the temperature was raised to 147 ° C for 8 hours, and then concentrated under reduced pressure to add 300-400 mm. The crude silica gel was mixed and used to obtain the yellow compound intermediate 11c (4-((1H-pyrrolo[2,3-b]pyridine-4) by column chromatography using DCM..MeOH = 50..1 as eluent. - oxy)-3-fluoroaniline, yield 62.5%), HPLC purity 95%.
称取中间体11c(140mg,1.0mM)和铁粉(145mg，5.0mM)于25mL圆底烧瓶中，加入5mL乙醇和5mL水溶解混合物，然后加入氯化铵(20mg，0.5mM)，升高温度至60℃反应3h，反应液抽滤，减压浓缩后加入原料4(4-氨基-2-氟苯酚，102mg，1.2mM)于25mL圆底烧瓶中，加入10mL的甲苯溶解混合物，然后加入滴加2-3滴三乙胺，升高温度至120℃反应5h后，加入300-400mm的粗硅胶拌样，用DCM︰MeOH＝50︰1作为洗脱剂，使用柱层析方法最终得淡黄色的化合物14c(产率为30.3％)，HPLC纯度95％。Intermediate 11c (140 mg, 1.0 mM) and iron powder (145 mg, 5.0 mM) were weighed into a 25 mL round bottom flask, and 5 mL of ethanol and 5 mL of water were added to dissolve the mixture, followed by ammonium chloride (20 mg, 0.5 mM). The reaction was carried out at a temperature of 60 ° C for 3 h, and the reaction mixture was filtered with suction, and concentrated under reduced pressure, and then the mixture of 4 (4-amino-2-fluorophenol, 102 mg, 1.2 mM) was added to a 25 mL round bottom flask, and 10 mL of toluene was added to dissolve the mixture, and then added. Add 2-3 drops of triethylamine dropwise, raise the temperature to 120 ° C for 5 h, add 300-400 mm of crude silica gel, and use DCM..MeOH=50..1 as eluent to obtain the column chromatography method. Light yellow compound 14c (30.3% yield), HPLC purity 95%.
1H NMR(400MHz,DMSO)δ10.04(s,1H),9.90(s,1H),9.77(s,1H),8.85(s,1H),8.16(s,1H),7.23(t,J＝8.7Hz,1H),7.05(t,J＝8.8Hz,1H),6.85～6.78(m,1H),6.70(s,1H),6.59(s,1H),5.22(d,J＝27.7Hz,1H),1.34～1.01(m,9H)。 1 H NMR (400MHz, DMSO) δ10.04 (s, 1H), 9.90 (s, 1H), 9.77 (s, 1H), 8.85 (s, 1H), 8.16 (s, 1H), 7.23 (t, J =8.7 Hz, 1H), 7.05 (t, J = 8.8 Hz, 1H), 6.85 to 6.78 (m, 1H), 6.70 (s, 1H), 6.59 (s, 1H), 5.22 (d, J = 27.7 Hz) , 1H), 1.34 to 1.01 (m, 9H).
实施例33 1-(5-(叔丁基)异恶唑-3-基)-3-(3-氟-4-(吡啶-4-基氧基)苯基)脲(化合物14d)的制备Example 33 Preparation of 1-(5-(tert-butyl)isoxazol-3-yl)-3-(3-fluoro-4-(pyridin-4-yloxy)phenyl)urea (Compound 14d)
Figure PCTCN2018073793-appb-000079
Figure PCTCN2018073793-appb-000079
称取原料3d(4-氯吡啶，100mg,1.0mM)和中间体3(259mg，1.0mM)于100mL圆底烧瓶中，加入10mL的乙腈溶解混合物，然后加入碳酸钾(352mg，2.0mM)，升高温度至80℃反应8h后，减压浓缩加入300-400mm的粗硅胶拌样，用DCM︰MeOH＝50︰1作为洗脱剂，使用 柱层析方法最终得黄色的化合物14d(产率为62.5％)，HPLC纯度95％。Raw material 3d (4-chloropyridine, 100 mg, 1.0 mM) and Intermediate 3 (259 mg, 1.0 mM) were weighed into a 100 mL round bottom flask, 10 mL of acetonitrile was added to dissolve the mixture, and then potassium carbonate (352 mg, 2.0 mM) was added. After raising the temperature to 80 ° C for 8 h, the mixture was concentrated under reduced pressure to a mixture of 300-400 mm of crude silica gel, using DCM..MeOH = 50..1 as eluent, using column chromatography to finally obtain yellow compound 14d (yield At 62.5%), the HPLC purity was 95%.
1H NMR(400MHz,DMSO)δ7.95(s,3H),6.77(d,J＝16.4Hz,2H),6.22(s,1H),5.51(s,1H),5.38(d,J＝14.1Hz,3H),1.22(s,9H)。 1 H NMR (400MHz, DMSO) δ7.95 (s, 3H), 6.77 (d, J = 16.4Hz, 2H), 6.22 (s, 1H), 5.51 (s, 1H), 5.38 (d, J = 14.1 Hz, 3H), 1.22 (s, 9H).
实施例34 1-(5-(叔丁基)异恶唑-3-基)-3-(3-氟-4-((6-吗啉代吡啶-4-基)氧基)苯基)脲(化合物14e)的制备Example 34 1-(5-(tert-butyl)isoxazol-3-yl)-3-(3-fluoro-4-((6-morpholinopyridin-4-yl)oxy)phenyl) Preparation of urea (compound 14e)
Figure PCTCN2018073793-appb-000080
Figure PCTCN2018073793-appb-000080
称取原料3e(4,6-二氯嘧啶，300mg,1.0mM)和吗啉(176mg，1.0mM)于100mL圆底烧瓶中，加入10mL的二氯甲烷溶解混合物，然后加入碳酸钾(560mg，2.0mM)，常温搅拌反应4h后，减压浓缩得到黄色固体4-(6-氯嘧啶-4-基)吗啉。称取该固体4-(6-氯嘧啶-4-基)吗啉(150mg,1.0mM)和中间体3(176mg,1.2mM)于100mL圆底烧瓶中，加入10mL的乙腈溶解混合物，然后加入碳酸钾加入(138mg，2.0mM)，升高温度至80℃反应8h后，减压浓缩后加入300-400mm的粗硅胶拌样，用DCM︰MeOH＝50︰1作为洗脱剂，使用柱层析方法最终得黄色的化合物14e(产率为62.5％)，HPLC纯度95％。Raw material 3e (4,6-dichloropyrimidine, 300 mg, 1.0 mM) and morpholine (176 mg, 1.0 mM) were weighed into a 100 mL round bottom flask, 10 mL of dichloromethane was added to dissolve the mixture, and then potassium carbonate (560 mg, 2.0 mM), the reaction was stirred at room temperature for 4 h, then concentrated under reduced pressure to give 4-(6-chloropyrimidin-4-yl)morpholine as a yellow solid. Weigh the solid 4-(6-chloropyrimidin-4-yl)morpholine (150 mg, 1.0 mM) and Intermediate 3 (176 mg, 1.2 mM) in a 100 mL round bottom flask, add 10 mL of acetonitrile to dissolve the mixture, then add Add potassium carbonate (138 mg, 2.0 mM), raise the temperature to 80 ° C for 8 h, concentrate under reduced pressure, add 300-400 mm of crude silica gel, and use DCM..MeOH=50..1 as eluent, use column. The analytical procedure resulted in a yellow compound 14e (yield 62.5%) with a HPLC purity of 95%.
1H NMR(400MHz,DMSO)δ8.35(s,1H),7.95(s,5H),6.96(s,1H),5.75(s,1H),3.64(d,J＝4.5Hz,8H),1.24(t,J＝13.3Hz,9H)。 1 H NMR (400MHz, DMSO) δ8.35 (s, 1H), 7.95 (s, 5H), 6.96 (s, 1H), 5.75 (s, 1H), 3.64 (d, J = 4.5Hz, 8H), 1.24 (t, J = 13.3 Hz, 9H).
实施例35 1-(5-(叔丁基)异恶唑-3-基)-3-(3-氟-4-((5-硝基吡啶-4-基)氧基)苯基)脲(化合物14f)的制备Example 35 1-(5-(tert-butyl)isoxazol-3-yl)-3-(3-fluoro-4-((5-nitropyridin-4-yl)oxy)phenyl)urea Preparation of (Compound 14f)
Figure PCTCN2018073793-appb-000081
Figure PCTCN2018073793-appb-000081
称取原料3f(4-氯-5-硝基嘧啶，100mg，1.0mM)和中间体3(184mg，1.0mM)于100mL圆底烧瓶中，加入10mL的乙腈溶解混合物，然后加入碳酸钾(250mg，2.0mM)，升高温度至80℃反应8h后，减压浓缩加入300-400mm的粗硅胶拌样，用DCM︰MeOH＝50︰1作为洗脱剂，使用柱层析方法最终得黄色的化合物14f(产率为62.5％)，HPLC纯度95％。Raw material 3f (4-chloro-5-nitropyrimidine, 100 mg, 1.0 mM) and intermediate 3 (184 mg, 1.0 mM) were weighed into a 100 mL round bottom flask, 10 mL of acetonitrile was added to dissolve the mixture, and then potassium carbonate (250 mg) was added. , 2.0 mM), the temperature was raised to 80 ° C for 8 h, concentrated under reduced pressure, added to 300-400 mm of crude silica gel, using DCM..MeOH = 50..1 as eluent, using column chromatography to finally obtain yellow Compound 14f (yield 62.5%), HPLC purity 95%.
1H NMR(400MHz,DMSO)δ10.09(s,1H),8.71(s,1H),8.54(t,J＝5.6Hz,1H),8.10(d,J＝5.4Hz,1H),7.65(dd,J＝12.6,2.5Hz,1H),7.51(dd,J＝8.8,1.3Hz,1H),7.08(t,J＝9.2Hz,1H),6.05(s,1H),1.22(s,9H)。 1 H NMR (400MHz, DMSO) δ10.09 (s, 1H), 8.71 (s, 1H), 8.54 (t, J = 5.6Hz, 1H), 8.10 (d, J = 5.4Hz, 1H), 7.65 ( Dd, J = 12.6, 2.5 Hz, 1H), 7.51 (dd, J = 8.8, 1.3 Hz, 1H), 7.08 (t, J = 9.2 Hz, 1H), 6.05 (s, 1H), 1.22 (s, 9H) ).
实施例36 本发明化合物体外酶活性实验Example 36 In vitro Enzyme Activity Experiment of the Compound of the Invention
本实验的目的是测定部分发明化合物11-11d和SKLB707在体外对突变的FLT3激酶(FLT3-ITD、FLT3(D835Y)、FLT3-ITD/F691L和FLT3-ITD/D835V)的亲和力(Kd值)。1)实验材料The purpose of this experiment was to determine the affinity (Kd value) of some of the inventive compounds 11-11d and SKLB707 against mutant FLT3 kinases (FLT3-ITD, FLT3 (D835Y), FLT3-ITD/F691L and FLT3-ITD/D835V) in vitro. 1) Experimental materials
T7噬菌体株、大肠杆菌、HEK-293细胞、链霉亲和素包被的磁性小珠、SeaBlock(Pierce公司，用于终止反应)、1％BSA(牛血清白蛋白)、0.05％Tween(吐温)20、1mM DTT(二硫苏糖醇)、0.17x PBS(磷酸盐缓冲液)、6mM DTT、聚苯乙烯96孔板、1x PBS、0.5 μM非生物素化亲和配体、100％DMSO(二甲基亚砜)，以上原料由DiscoverRX公司(美国)提供。AC220和Ponatinib(普纳替尼)购买于Selleck Chemicals(中国，上海)。T7 phage strain, Escherichia coli, HEK-293 cells, streptavidin-coated magnetic beads, SeaBlock (Pierce, for termination of reaction), 1% BSA (bovine serum albumin), 0.05% Tween (spit Temperature) 20, 1 mM DTT (dithiothreitol), 0.17x PBS (phosphate buffer), 6 mM DTT, polystyrene 96-well plate, 1 x PBS, 0.5 μM non-biotinylated affinity ligand, 100% DMSO (dimethyl sulfoxide), the above materials were supplied by DiscoverRX (USA). AC220 and Ponatinib (Punatinib) were purchased from Selleck Chemicals (Shanghai, China).
Figure PCTCN2018073793-appb-000082
Figure PCTCN2018073793-appb-000082
2)实验方法2) Experimental method
待大肠杆菌生长到对数生长期时，于低温下用携带有待测激酶DNA片段的T7噬菌体进行感染，随后在32℃下震荡孵育，直至大肠杆菌裂解(约孵育90-150min)。裂解物经过离心(6000g/min)和过滤(0.2μm)后去除细胞碎片，从而得到大量带有待测激酶DNA片段的T7噬菌体。将得到的噬菌体感染HEK-293细胞，利用宿主细胞中的蛋白表达体系生产待检测的激酶蛋白，用此方法生产出的激酶蛋白带有DNA片段，在后续实验中可以通过qPCR技术检测DNA含量从而间接测定激酶的含量。选取与待测激酶具有强亲和力，并且用生物素标记的小分子配体处理链霉亲和素包被的磁珠30min，从而产生亲和树脂用于激酶测定。随后用过量的生物素孵育处理后的磁珠，并用封闭缓冲液(SeaBlock，1％BSA，0.05％Tween20,1mM DTT)进行冲洗，以除去未结合的配体，并减少非特异性结合。接下来，将带有DNA片段的待测激酶，带有小分子配体的磁珠和特定浓度的化合物溶液共同加到结合缓冲液(20％SeaBlock，0.17x PBS，0.05％Tween 20,6mM DTT)中进行结合反应。所有反应均在聚苯乙烯96孔板中进行中，反应液的最终体积是0.135ml。将反应体系在室温下震荡1h，用冲洗缓冲液(1x PBS，0.05％Tween 20)冲洗亲和磁珠。然后将磁珠再悬浮在洗脱缓冲液(1x PBS,0.05％Tween 20,0.5μM非生物素化亲和配体)，并在室温下摇动30分钟。在洗脱液中的激酶浓度通过qPCR测量。各试验化合物3倍梯度稀释的11个测试点是在100％DMSO中制备，随后在测定时稀释至1x(最终DMSO浓度为1％)。When E. coli was grown to logarithmic growth phase, infection was carried out with T7 phage carrying the DNA fragment of the kinase to be tested at low temperature, followed by shaking incubation at 32 ° C until E. coli lysis (about 90-150 min incubation). The lysate was centrifuged (6000 g/min) and filtered (0.2 μm) to remove cell debris, thereby obtaining a large number of T7 phage carrying the DNA fragment of the kinase to be tested. The obtained phage is infected with HEK-293 cells, and the protein protein to be detected is produced by using the protein expression system in the host cell, and the kinase protein produced by the method has a DNA fragment, and in the subsequent experiments, the DNA content can be detected by qPCR technology. The amount of kinase was determined indirectly. The affinity to the kinase to be tested was selected and the streptavidin-coated magnetic beads were treated with biotin-labeled small molecule ligand for 30 min to produce an affinity resin for the kinase assay. The treated magnetic beads were then incubated with excess biotin and rinsed with blocking buffer (SeaBlock, 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and reduce non-specific binding. Next, the test enzyme with the DNA fragment, the magnetic beads with the small molecule ligand and the specific concentration of the compound solution are added to the binding buffer (20% SeaBlock, 0.17x PBS, 0.05% Tween 20, 6mM DTT). The binding reaction is carried out. All reactions were carried out in polystyrene 96-well plates and the final volume of the reaction solution was 0.135 ml. The reaction system was shaken at room temperature for 1 h, and the affinity magnetic beads were washed with a washing buffer (1 x PBS, 0.05% Tween 20). The beads were then resuspended in elution buffer (1 x PBS, 0.05% Tween 20, 0.5 μM non-biotinylated affinity ligand) and shaken for 30 minutes at room temperature. The kinase concentration in the eluate was measured by qPCR. Eleven test points for a 3-fold gradient dilution of each test compound were prepared in 100% DMSO and subsequently diluted to 1 x at the time of assay (final DMSO concentration was 1%).
3)化合物的亲和力测试结果3) Affinity test results of the compound
通过以上测试方法，测试了本发明中的化合物与突变的FLT3激酶(FLT3-ITD、FLT3(D835Y)、FLT3-ITD/F691L和FLT3-ITD/D835V)的结合能力(kd值)。具体部分化合物的Kd值见表1。其中，“-”表示未测试。The binding ability (kd value) of the compound of the present invention to the mutated FLT3 kinases (FLT3-ITD, FLT3 (D835Y), FLT3-ITD/F691L, and FLT3-ITD/D835V) was tested by the above test methods. The Kd values of specific compounds are shown in Table 1. Among them, "-" means not tested.
表1 化合物与突变的FLT3激酶[FLT3-ITD、FLT3(D835Y)、FLT3-ITD/F691L和FLT3-ITD/D835V]的亲和力(Kd值)Table 1 Affinity (Kd value) of compound with mutant FLT3 kinase [FLT3-ITD, FLT3 (D835Y), FLT3-ITD/F691L and FLT3-ITD/D835V]
Figure PCTCN2018073793-appb-000083
Figure PCTCN2018073793-appb-000083
Figure PCTCN2018073793-appb-000084
Figure PCTCN2018073793-appb-000084
表1结果表明，受试化合物在体外酶水平对FLT3-ITD、FLT3(D835Y)、FLT3-ITD/F691L和FLT3-ITD/D835V(其中，FLT3-ITD/F691L和FLT3-ITD/D835V为临床上发现的AC220耐药突变体)均具有较好的结合能力，其中，针对FLT3-ITD/F691L、FLT3-ITD/D835V突变体，受试化合物SKLB707的活性明显优于阳性化合物AC220；针对FLT3-ITD/D835V突变体，受试化合物SKLB707的活性也明显优于阳性化合物Ponatinib(普纳替尼)。The results in Table 1 indicate that the test compound is in vitro at the enzyme level for FLT3-ITD, FLT3 (D835Y), FLT3-ITD/F691L, and FLT3-ITD/D835V (where FLT3-ITD/F691L and FLT3-ITD/D835V are clinically The found AC220 resistant mutants have good binding ability. Among them, the activity of test compound SKLB707 is significantly better than that of positive compound AC220 for FLT3-ITD/F691L and FLT3-ITD/D835V mutants; for FLT3-ITD The /D835V mutant, the activity of the test compound SKLB707 was also significantly better than the positive compound Ponatinib (Punatinib).
实施例37 本发明化合物针对AC220耐药细胞株的体外增值抑制实验Example 37 In vitro Molecular Weight Inhibition Experiment of Compounds of the Invention Against AC220 Drug-Resistant Cell Lines
本实验的目的是检测本发明化合物对具有AC220耐药二次突变细胞株的增值抑制活性，采用的方法为MTT(四甲基偶氮唑盐)比色法。The purpose of this experiment was to test the potentiation inhibitory activity of the compound of the present invention against an AC220-resistant secondary mutant cell line using the MTT (tetramethylazozolium salt) colorimetric method.
1)实验材料：1) Experimental materials:
主要试剂：RPMI-1640培养基购自Gibco BRL公司(Invitrogen Corporation，USA)，胎牛血清购自Pan-Biotech公司(Germany)，IL-3(白介素-3)购自PeproTech公司，G418(遗传霉素)购自Life Science公司。四甲基偶氮唑盐(MTT)、十二烷基磺酸钠(SDS)和DMSO(二甲亚砜)为Sigma公司(USA)产品。化合物用100％DMSO配制成10mM储存液，置-20℃冰箱避光保存备用，临用时用完全培养液稀释至所需浓度。Primary reagents: RPMI-1640 medium was purchased from Gibco BRL (Invitrogen Corporation, USA), fetal bovine serum was purchased from Pan-Biotech (Germany), IL-3 (interleukin-3) was purchased from PeproTech, G418 (genetic mold) Originally purchased from Life Science. Tetramethylazozolium salt (MTT), sodium dodecyl sulfate (SDS) and DMSO (dimethyl sulfoxide) are products of Sigma (USA). The compound was formulated into a 10 mM stock solution in 100% DMSO, stored in a refrigerator at -20 ° C in the dark, and diluted to the desired concentration with a complete medium at the time of use.
细胞系及培养：本实验所用细胞系小鼠原B淋巴细胞Ba/F3和急性髓性白血病细胞MV4-11均购于美国ATCC(American type culture collection)，由本实验室保存。AC220敏感细胞株Ba/F3FLT3-ITD，AC220耐药细胞株Ba/F3FLT3-ITD/D835V和Ba/F3FLT3-ITD/F691L由Ba/F3细胞采用Amaxa Kit V(Lonza公司)试剂盒进行电转构建。Ba/F3细胞用含10％胎牛血清、100U/mL青霉素、100μg/mL链霉素、10ng/ml IL-3的RPMI-1640完全培养基在5％CO 2、37℃条件下培养。Ba/F3FLT3-ITD、Ba/F3FLT3-ITD/D835V和Ba/F3FLT3-ITD/F691L使用含10％胎牛血清、100U/mL青霉素、100μg/mL链霉素和500μg/mL G418的RPMI-1640完全培养基在5％CO 2、37℃条件下培养。 Cell line and culture: The cell line used in this experiment, mouse B lymphocyte Ba/F3 and acute myeloid leukemia cell MV4-11 were purchased from American ATCC (American type culture collection) and kept by our laboratory. AC220 sensitive cell line Ba/F3FLT3-ITD, AC220 resistant cell line Ba/F3FLT3-ITD/D835V and Ba/F3FLT3-ITD/F691L were electroporated from Ba/F3 cells using Amaxa Kit V (Lonza) kit. Ba/F3 cells were cultured in RPMI-1640 complete medium containing 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 10 ng/ml IL-3 at 5% CO 2 at 37 °C. Ba/F3FLT3-ITD, Ba/F3FLT3-ITD/D835V and Ba/F3FLT3-ITD/F691L using RPMI-1640 with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin and 500 μg/mL G418 culture medium at 5% CO 2, 37 ℃.
2)实验方法：2) Experimental method:
在96孔板中加入100μL梯度浓度的受试化合物，DMSO浓度为0.1％，每个剂量组设3个复孔。用完全细胞培养液调整细胞浓度为2×10 5个/mL的细胞悬液，接种于96孔板，每孔100μl细胞悬液。同时设不含药物的阴性对照组和等体积的完全培养基对照组，在37℃，5％CO 2条件下培养。48小时(MV4-11为72h)后，每孔加入浓度为5mg/mL的MTT试剂20μL，再培养2-4h后，每孔加入50μL 20％SDS(十二烷基硫酸钠)(m：v)，在37℃条件下孵育过夜，用酶标仪(λ＝570nm)测定吸光度(A)值(A值与活细胞数成正比)，取其平均值。相对细胞增殖抑制率＝(对照组A570-实验组A570)/(对照组A570-完全培养基A570)×100％。实验至少重复3次。实验数据用平均值表示，数据统计资料采用t检验，P<0.05为差异有统计学意义。以下各化合物对细胞增殖抑制作用均用IC 50表示。 A 100 [mu]L gradient of test compound was added to a 96-well plate at a DMSO concentration of 0.1% with 3 replicate wells per dose. A cell suspension having a cell concentration of 2 × 10 5 /mL was adjusted with a complete cell culture medium, and seeded in a 96-well plate, 100 μl of a cell suspension per well. At the same time, a drug-free negative control group and an equal volume of a complete medium control group were cultured at 37 ° C under 5% CO 2 . After 48 hours (72h for MV4-11), 20 μL of MTT reagent at a concentration of 5 mg/mL was added to each well, and after further 2-4 hours, 50 μL of 20% SDS (sodium dodecyl sulfate) was added to each well (m:v Incubation was carried out overnight at 37 ° C, and the absorbance (A) value (A value is proportional to the number of viable cells) was measured with a microplate reader (λ = 570 nm), and the average value was taken. Relative cell proliferation inhibition rate = (control group A570 - experimental group A570) / (control group A570 - complete medium A570) × 100%. The experiment was repeated at least 3 times. The experimental data were expressed as mean values, and the data were analyzed by t test. P<0.05 was considered statistically significant. The inhibition of cell proliferation by each of the following compounds was expressed by IC 50 .
3)实验结果：3) Experimental results:
采用MTT比色法，对Ba/F3FLT3-ITD、Ba/F3FLT3-ITD/F691L和Ba/F3FLT3-ITD/D835V进行了细胞增殖抑制活性测试，结果如表2和图2A所示。Cell proliferation inhibition activity tests were performed on Ba/F3FLT3-ITD, Ba/F3FLT3-ITD/F691L and Ba/F3FLT3-ITD/D835V by MTT colorimetry, and the results are shown in Table 2 and Figure 2A.
表2 化合物对各细胞株的增殖抑制活性(IC 50：nM) Table 2 Proliferation inhibitory activity of the compound on each cell line (IC 50 : nM)
Figure PCTCN2018073793-appb-000085
Figure PCTCN2018073793-appb-000085
表2和图2A结果表明，受试化合物不仅对AC220敏感的细胞株Ba/F3FLT3-ITD具有很强的抑制活性，对于具有AC220耐药突变的细胞株Ba/F3Ba/F3FLT3-ITD/D835V及FLT3-ITD/F691L同样具有很好的抑制能力，可以有效克服AC220在临床上产生的耐药突变。The results of Table 2 and Figure 2A show that the test compound not only has strong inhibitory activity against the AC220-sensitive cell line Ba/F3FLT3-ITD, but also the cell line with the AC220 resistance mutation Ba/F3Ba/F3FLT3-ITD/D835V and FLT3. -ITD/F691L also has good inhibition ability, which can effectively overcome the clinical resistance mutation of AC220.
实施例38 化合物SKLB707体外细胞凋亡检测实验Example 38 In vitro apoptosis assay of compound SKLB707
本实验的目的是检测本发明化合物SKLB707在体外引起MV4-11以及实验室自主构建细胞株Ba/F3FLT3-ITD、Ba/F3FLT3-ITD/D835V和Ba/F3FLT3-ITD/F691L凋亡的情况。加药处理细胞一段时间后，收集细胞进行Annexin V/PI双染，随后用流式细胞仪进行检测。1)实验材料：The purpose of this experiment was to detect the in vitro MV4-11 and the autonomously constructed cell lines Ba/F3FLT3-ITD, Ba/F3FLT3-ITD/D835V and Ba/F3FLT3-ITD/F691L in the laboratory. After the cells were treated with the drug for a while, the cells were collected for Annexin V/PI double staining, followed by flow cytometry. 1) Experimental materials:
主要试剂：RPMI-1640和IMDM培养基购自Gibco BRL公司(Invitrogen Corporation，USA)，胎牛血清购自Pan-Biotech公司(Germany)，G418购自Life Science公司。PBS购自北京中杉金桥生物技术有限公司。细胞凋亡检测试剂盒购自上海碧云天生物技术有限公司。化合物用100％DMSO配制成10mM储存液，置-20℃冰箱避光保存备用，临用时用完全培养液稀释至所需浓度。Primary reagents: RPMI-1640 and IMDM media were purchased from Gibco BRL (Invitrogen Corporation, USA), fetal bovine serum was purchased from Pan-Biotech (Germany), and G418 was purchased from Life Science. PBS was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. The apoptosis detection kit was purchased from Shanghai Biyuntian Biotechnology Co., Ltd. The compound was formulated into a 10 mM stock solution in 100% DMSO, stored in a refrigerator at -20 ° C in the dark, and diluted to the desired concentration with a complete medium at the time of use.
细胞系及培养：细胞株Ba/F3FLT3-ITD、Ba/F3FLT3-ITD/D835V和Ba/F3FLT3-ITD/F691L使用含10％胎牛血清、100U/mL青霉素、100μg/mL链霉素、500μg/ml G418的RPMI-1640完全培养基在5％CO 2、37℃条件下培养。MV4-11细胞使用含20％胎牛血清的IMDM完全培养基在5％CO 2、37℃条件下培养。 Cell lines and culture: cell lines Ba/F3FLT3-ITD, Ba/F3FLT3-ITD/D835V and Ba/F3FLT3-ITD/F691L containing 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 500 μg/ ml G418 in complete RPMI-1640 medium at 5% CO 2, 37 ℃ conditions. MV4-11 cells were cultured in IMDM complete medium containing 20% fetal bovine serum at 5% CO 2 at 37 °C.
2)实验方法：2) Experimental method:
将待测细胞以每孔2×10 5个/孔的数目接种于6孔板中，同时加入不同浓度的受试化合物SKLB707溶液，将6孔板置于细胞培养箱中培养24h。之后，将细胞悬液收集至相应流式管中，并用PBS洗涤两次，随后加入500μL Binding Buffer(结合缓冲液)重悬细胞。细胞重悬后向流式管中依次加入5μL Annexin V-FITC(磷脂结合蛋白-异硫氰酸荧光素)溶液和5μL PI(碘化丙啶)溶液，混匀。在室温下避光孵育5-10min，立即用流式细胞仪进行检测。 The cells to be tested were seeded in a 6-well plate at a number of 2 × 10 5 /well per well, while different concentrations of the test compound SKLB707 solution were added, and the 6-well plate was placed in a cell culture incubator for 24 hours. Thereafter, the cell suspension was collected into a corresponding flow tube and washed twice with PBS, followed by resuspension of the cells by adding 500 μL of Binding Buffer (binding buffer). After the cells were resuspended, 5 μL of Annexin V-FITC (phospholipid binding protein-fluorescein isothiocyanate) solution and 5 μL of PI (propidium iodide) solution were sequentially added to the flow tube, and the mixture was mixed. Incubate for 5-10 min in the dark at room temperature and immediately test by flow cytometry.
3)实验结果：3) Experimental results:
如图1所示，在细胞MV4-11和Ba/F3FLT3-ITD中，SKLB707可以诱导细胞产生明显的凋亡。在AC220耐药细胞株Ba/F3FLT3-ITD/D835V和Ba/F3FLT3-ITD/F691L中，SKLB707同样可以诱导细胞产生明显的凋亡，并且增加给药浓度，凋亡细胞的数目也随之增加。SKLB707在1、5、10、50和100nM时诱导MV4-11细胞凋亡的比例分别为38.2％、65.2％、70.3％、73.2％和81.25％；SKLB707在1、5、10、50和100nM时诱导Ba/F3FLT3-ITD细胞凋亡的比例分别为6.4％、39.1％、44.2％、73.3％和81.25％；SKLB707在1、5、10、50和100nM时诱导Ba/F3FLT3-ITD/F691L细胞凋亡的比例分别为5.18％、6.6％、11.67％、37.1％和45.35％；SKLB707在1、5、10、50和100nM时诱导Ba/F3FLT3-ITD/D835V细胞凋亡的比例分别为5.66％、7.72％、13.08％、39.11％和60.01％。As shown in Figure 1, in cells MV4-11 and Ba/F3FLT3-ITD, SKLB707 can induce significant apoptosis in cells. In the AC220-resistant cell lines Ba/F3FLT3-ITD/D835V and Ba/F3FLT3-ITD/F691L, SKLB707 can also induce significant apoptosis in cells, and increase the concentration of apoptotic cells. SKLB707 induced apoptosis in MV4-11 cells at 1, 5, 10, 50 and 100 nM, respectively, at 38.2%, 65.2%, 70.3%, 73.2% and 81.25%; SKLB707 at 1, 5, 10, 50 and 100 nM The proportions of Ba/F3FLT3-ITD cells induced apoptosis were 6.4%, 39.1%, 44.2%, 73.3% and 81.25%, respectively; SKLB707 induced Ba/F3FLT3-ITD/F691L cells at 1, 5, 10, 50 and 100 nM The proportion of death was 5.18%, 6.6%, 11.67%, 37.1% and 45.35%, respectively. The proportion of SKLB707 induced apoptosis in Ba/F3FLT3-ITD/D835V cells at 1, 5, 10, 50 and 100 nM was 5.66%. 7.72%, 13.08%, 39.11% and 60.01%.
实施例39 化合物SKLB707对FLT3信号通路体外检测实验Example 39 In vitro assay of FLT3 signaling pathway by compound SKLB707
本实验的目的是检测发明化合物SKLB707在体外细胞水平上对FLT3信号通路抑制情况。本实验使用MV4-11以及实验室自主构建细胞株Ba/F3FLT3-ITD、Ba/F3FLT3-ITD/D835V和Ba/F3FLT3-ITD/F691L，加药处理细胞一定时间后提取总蛋白，用Western blot的方法检测信号FLT3信号通路相关蛋白的表达水平。The purpose of this experiment was to examine the inhibition of the FLT3 signaling pathway by the inventive compound SKLB707 at the cellular level in vitro. In this experiment, MV4-11 and the laboratory self-constructed cell lines Ba/F3FLT3-ITD, Ba/F3FLT3-ITD/D835V and Ba/F3FLT3-ITD/F691L were used to treat the cells for a certain period of time, and the total protein was extracted by Western blot. The method detects the expression level of a signal related to the signal pathway of FLT3.
1)实验材料：1) Experimental materials:
主要试剂：RPMI-1640和IMDM培养基购自Gibco BRL公司(Invitrogen Corporation，USA)，胎牛血清购自Pan-Biotech公司(Germany)，G418购自Life Science公司，生理盐 水购自四川科伦药业股份有限公司，RIPA裂解液购自上海碧云天生物技术有限公司，Cocktail(蛋白酶抑制剂)和PMSF(苯甲基磺酰氟)购自Sigma公司，BCA蛋白定量试剂盒购自西安赫特生物科技有限公司。FLT3抗体购自Santa Cruz公司，β-actin抗体购自北京中杉金桥公司，其余抗体均购自Cell Signaling Technology公司。HRP(辣根过氧化酶)标记的二抗购自北京中山金桥公司，PVDF膜(聚偏二氟乙烯膜)和显影底物购自millipore公司。化合物用100％DMSO配制成10mM储存液，置-20℃冰箱避光保存备用，临用时用完全培养液稀释至所需浓度。Main reagents: RPMI-1640 and IMDM medium were purchased from Gibco BRL (Invitrogen Corporation, USA), fetal bovine serum was purchased from Pan-Biotech (Germany), G418 was purchased from Life Science, and physiological saline was purchased from Sichuan Kelun. RIPA lysate was purchased from Shanghai Biyuntian Biotechnology Co., Ltd., Cocktail (protease inhibitor) and PMSF (phenylmethylsulfonyl fluoride) were purchased from Sigma, and BCA protein quantification kit was purchased from Xi'an Hut Bio Technology Co., Ltd. The FLT3 antibody was purchased from Santa Cruz, the β-actin antibody was purchased from Beijing Zhongshan Jinqiao Company, and the remaining antibodies were purchased from Cell Signaling Technology. HRP (horseradish peroxidase)-labeled secondary antibody was purchased from Beijing Zhongshan Jinqiao Co., Ltd., PVDF membrane (polyvinylidene fluoride membrane) and developing substrate were purchased from Millipore. The compound was formulated into a 10 mM stock solution in 100% DMSO, stored in a refrigerator at -20 ° C in the dark, and diluted to the desired concentration with a complete medium at the time of use.
细胞系及培养：细胞株Ba/F3FLT3-ITD、Ba/F3FLT3-ITD/D835V和Ba/F3FLT3-ITD/F691L使用含10％胎牛血清、100U/mL青霉素、100μg/mL链霉素和500μg/ml G418的RPMI-1640完全培养基在5％CO 2、37℃条件下培养。MV4-11使用含20％胎牛血清的IMDM完全培养基在5％CO 2、37℃条件下培养。 Cell lines and culture: cell lines Ba/F3FLT3-ITD, Ba/F3FLT3-ITD/D835V and Ba/F3FLT3-ITD/F691L containing 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin and 500 μg/ The RPMI-1640 complete medium of ml G418 was cultured at 5% CO 2 at 37 °C. MV4-11 was cultured in IMDM complete medium containing 20% fetal bovine serum at 5% CO 2 at 37 °C.
2)实验方法：2) Experimental method:
将待测细胞接种到6孔板中，扩增到合适的细胞密度后，加入不同浓度的受试化合物，在37℃，5％CO 2条件下培养1个小时。将细胞离心收集起来，用预冷的生理盐水洗涤细胞两次，并同时将细胞转移到1.5mL离心管中，吸取并弃除上清，加入适当体积的RIPA裂解液(含1％cocktail和1％PMSF)。涡旋以使细胞完全重悬在裂解液中，并立即置于冰上裂解。约15min后，用超声破碎仪器进行细胞破碎。随后将处理后的细胞置于低温高速离心机中离心(13000r/min，15min)以除去细胞碎片。用BCA(二喹啉甲酸)法进行蛋白定量，同时用BSA(牛血清蛋白)标准蛋白制作标准曲线，根据标准曲线计算各组蛋白浓度，统一各组蛋白样品的浓度后，向其中加入5×蛋白上样缓冲液，随后放入100℃干式恒温器中保持10min，使蛋白变性。处理好的蛋白样品按所需量分装后保存于-20℃备用。蛋白样品避免反复冻融。 The cells to be tested were seeded into a 6-well plate, and after appropriate cell density was amplified, test compounds of different concentrations were added, and cultured at 37 ° C, 5% CO 2 for 1 hour. The cells were collected by centrifugation, the cells were washed twice with pre-cooled physiological saline, and the cells were simultaneously transferred to a 1.5 mL centrifuge tube, and the supernatant was aspirated and discarded, and an appropriate volume of RIPA lysate (containing 1% cocktail and 1) was added. %PMSF). Vortex to allow the cells to completely resuspend in the lysate and immediately lyse on ice. After about 15 min, cell disruption was performed using an ultrasonic disruption apparatus. The treated cells were then centrifuged (13000 r/min, 15 min) in a low temperature high speed centrifuge to remove cell debris. The protein was quantified by BCA (bisquinolinecarboxylic acid) method, and a standard curve was prepared by using BSA (bovine serum albumin) standard protein. The concentration of each group of proteins was calculated according to the standard curve, and the concentration of each group of protein samples was unified, and 5× was added thereto. The protein loading buffer was then placed in a 100 ° C dry thermostat for 10 min to denature the protein. The processed protein samples were dispensed in the required amount and stored at -20 ° C until use. Protein samples avoid repeated freeze-thaw cycles.
待检测样品时，采用聚丙烯酰胺凝胶电泳(SDS-PAGE)分离蛋白。电泳分离完毕，采用槽式湿转法将蛋白转移至PVDF膜上，再将PVDF膜置于含5％(m:v)脱脂奶粉的TBS/T(三羟甲基氨基甲烷-盐酸/吐温缓冲盐溶液)中封闭2h，剪开不同分子量的蛋白条带，按相应抗体说明书推荐的稀释比例稀释一抗并在4℃冰箱孵育条带过夜。次日，取出各条带，用TBS/T缓冲液漂洗后加入1:5000稀释的HRP标记的二抗，在37℃孵育1h，随后，用TBS/T洗脱除去过量抗体，在PVDF膜上均匀滴加HRP底物后，置于快速凝胶成像系统中显影。When the sample to be tested is subjected to polyacrylamide gel electrophoresis (SDS-PAGE), the protein is separated. After the electrophoresis was separated, the protein was transferred to the PVDF membrane by the trough wet transfer method, and the PVDF membrane was placed in TBS/T (trishydroxymethylaminomethane-hydrochloric acid/Tween containing 5% (m:v) skim milk powder. Block in the buffered saline solution for 2 h, cut the protein bands of different molecular weights, dilute the primary antibody in the dilution ratio recommended by the corresponding antibody instructions and incubate the bands overnight at 4 ° C in the refrigerator. On the next day, each band was taken out, rinsed with TBS/T buffer, and then added with a 1:5000 dilution of HRP-labeled secondary antibody, incubated at 37 ° C for 1 h, followed by elution with TBS/T to remove excess antibody on the PVDF membrane. The HRP substrate was evenly added dropwise and developed in a rapid gel imaging system.
3)实验结果：3) Experimental results:
图2B为化合物SKLB707和AC220对FLT3信号通路各关键蛋白的抑制作用。其中，pFLT3表示磷酸化的FLT3，Total FLT3表示总的FLT3水平，pSTAT5表示磷酸化的STAT5，Total STAT5表示总的STAT5含量，pErk1/2表示磷酸化的Erk1/2，Total Erk1/2表示总的Erk1/2含量，β-actin表示β-肌动蛋白的含量。Figure 2B shows the inhibitory effects of the compounds SKLB707 and AC220 on key proteins in the FLT3 signaling pathway. Among them, pFLT3 represents phosphorylated FLT3, Total FLT3 represents total FLT3 level, pSTAT5 represents phosphorylated STAT5, Total STAT5 represents total STAT5 content, pErk1/2 represents phosphorylated Erk1/2, and Total Erk1/2 represents total Erk1/2 content, β-actin indicates the content of β-actin.
根据图2B中结果所示，在AC220敏感细胞株Ba/F3FLT3-ITD和MV4-11上，SKLB707可以剂量依赖性地抑制FLT3的自磷酸化水平，而不影响FLT3总蛋白的表达水平。同样，FLT3激酶下游蛋白STAT5和Erk1/2的磷酸化也被明显抑制。AC220对这些蛋白的磷酸化也有抑制作用，但抑制效果与同等剂量的SKLB707相比较弱。According to the results in Figure 2B, on the AC220-sensitive cell lines Ba/F3FLT3-ITD and MV4-11, SKLB707 inhibited the autophosphorylation level of FLT3 in a dose-dependent manner without affecting the expression level of FLT3 total protein. Similarly, phosphorylation of STAT5 and Erk1/2, downstream proteins of FLT3 kinase, was also significantly inhibited. AC220 also inhibited the phosphorylation of these proteins, but the inhibitory effect was weaker than the equivalent dose of SKLB707.
根据图2B中结果所示，在AC220耐药细胞株Ba/F3FLT3-ITD/D835V和Ba/F3FLT3-ITD/F691L 中，SKLB707可以剂量依赖性地抑制FLT3受体的自磷酸化水平，而不影响FLT3总蛋白的表达水平。同样，FLT3激酶下游蛋白STAT5和Erk1/2的磷酸化也被明显抑制。而相同剂量的AC220则对这些蛋白的磷酸化水平没有明显的抑制效果，说明FLT3激酶在发生两次突变后，SKLB707依然能够结合于其活性区域，克服AC220耐药的现象。According to the results in Figure 2B, in the AC220-resistant cell lines Ba/F3FLT3-ITD/D835V and Ba/F3FLT3-ITD/F691L, SKLB707 can inhibit the autophosphorylation level of FLT3 receptor in a dose-dependent manner without affecting The expression level of total protein of FLT3. Similarly, phosphorylation of STAT5 and Erk1/2, downstream proteins of FLT3 kinase, was also significantly inhibited. The same dose of AC220 did not significantly inhibit the phosphorylation level of these proteins, indicating that after two mutations in FLT3 kinase, SKLB707 can still bind to its active region, overcoming AC220 resistance.
实施例40 化合物SKLB707体内药效检测实验Example 40 In vivo drug efficacy test of compound SKLB707
本实验的目的是检测发明化合物SKLB707在体内药效。将MV4-11皮下接种于NOD SCID小鼠，构建肿瘤异种移植模型，以AC220为阳性对照，检测SKLB707抑制皮下瘤生长情况。The purpose of this experiment was to test the in vivo efficacy of the inventive compound SKLB707. MV4-11 was subcutaneously inoculated into NOD SCID mice to construct a tumor xenograft model, and AC220 was used as a positive control to detect the inhibition of subcutaneous tumor growth by SKLB707.
1)实验材料：1) Experimental materials:
主要试剂：IMDM培养基购自Gibco BRL公司(Invitrogen Corporation，USA)，胎牛血清购自Pan-Biotech公司(Germany)。PEG400(聚乙二醇400)和DMSO购于西格玛奥德里奇(上海)贸易有限公司。p-FLT3，p-STAT5，p-ERK抗体均购于Cell Signaling Technology公司。Primary reagents: IMDM medium was purchased from Gibco BRL (Invitrogen Corporation, USA) and fetal calf serum was purchased from Pan-Biotech (Germany). PEG400 (polyethylene glycol 400) and DMSO were purchased from Sigma Aldrich (Shanghai) Trading Co., Ltd. p-FLT3, p-STAT5, and p-ERK antibodies were purchased from Cell Signaling Technology.
细胞及实验动物：MV4-11使用含20％胎牛血清的IMDM完全培养基在5％CO 2、37℃条件下培养。NOD SCID小鼠(雌性5-6周龄的免疫缺陷小鼠)购于北京维通利华实验动物技术有限公司。 Cells and laboratory animals: MV4-11 was cultured in IMDM complete medium containing 20% fetal bovine serum at 5% CO 2 at 37 °C. NOD SCID mice (female 5-6 week old immunodeficient mice) were purchased from Beijing Vital Lihua Experimental Animal Technology Co., Ltd.
2)实验方法：2) Experimental method:
扩大培养MV4-11细胞，收集处于对数生长周期的细胞，用不含血清及抗生素的IMDM培养基重悬细胞，离心、清洗三次，最后一次计数使细胞浓度为1×10  8个/mL。接种100μL的细胞悬液至NOD SCID小鼠右侧面的的背部皮下，构建MV4-11小鼠皮下瘤模型。待肿瘤长至约400mm 3的体积时，将小鼠随机分为5个组，每组6只，开始灌胃给药。这5个组分别为溶剂对照组、SKLB707 1mg/kg组、SKLB707 3mg/kg组、SKLB707 10mg/kg组以及AC220 3mg/kg组。以5％DMSO、25％的PEG400(用灭菌水配成50％的PEG400)和75％的灭菌水为溶剂，逐次加入称好的化合物中，配制相应不同浓度的药物储备液。配好后分装保存于4℃冰箱中备用。口服灌胃给药，每天给药一次，每三天测量一次肿瘤体积和小鼠体重，给药21天后结束实验。给药过程中观察动物的一般状况，如进食情况、精神状态、皮肤颜色及光泽以及有无腹泻状况等等。肿瘤体积计算公式：肿瘤体积＝L×W  2/2；其中，L和W分别为肿瘤长径和短径。 The MV4-11 cells were expanded and the cells in the logarithmic growth cycle were collected. The cells were resuspended in IMDM medium containing no serum and antibiotics, centrifuged and washed three times, and the final cell count was 1×10 8 cells/mL. A subcutaneous tumor model of MV4-11 mice was constructed by inoculating 100 μL of the cell suspension to the back of the right side of NOD SCID mice. When the tumor grew to a volume of about 400 mm 3 , the mice were randomly divided into 5 groups of 6 animals each, and the gavage administration was started. The five groups were the solvent control group, the SKLB707 1 mg/kg group, the SKLB707 3 mg/kg group, the SKLB707 10 mg/kg group, and the AC220 3 mg/kg group. The 5% DMSO, 25% PEG400 (50% PEG400 with sterilized water) and 75% sterilized water were used as solvents to sequentially add the weighed compounds to prepare different concentrations of the drug stock solution. After being prepared, store in a refrigerator at 4 ° C for use. Oral gavage was administered once a day, tumor volume and mouse body weight were measured every three days, and the experiment was terminated after 21 days of administration. The general condition of the animal, such as eating condition, mental state, skin color and luster, and presence or absence of diarrhea, was observed during the administration. Tumor volume calculation formula: tumor volume = L × W 2 /2; wherein L and W are the tumor long diameter and short diameter, respectively.
3)实验结果：3) Experimental results:
如图3所示，不同剂量的SKLB707都可以完全消除小鼠皮下瘤(图3A)，并且老鼠的体重没有下降(图3B)，老鼠的状态同样也没有明显变化。SKLB707 10mg/kg组在给药11天可以完全消掉肿瘤；SKLB707 1mg/kg组，SKLB707 3mg/kg组和AC220 3mg/kg组在给药12天都可以完全消掉肿瘤，表明SKLB707抑制肿瘤生长的效果同AC220相当。小鼠肿瘤异种移植模型实验表明，SKLB707在体内具有很好的抑瘤效果。As shown in Figure 3, different doses of SKLB707 completely abolished mouse subcutaneous tumors (Fig. 3A), and the body weight of the mice did not decrease (Fig. 3B), and the state of the mice did not change significantly. SKLB707 10mg/kg group can completely eliminate tumors after 11 days of administration; SKLB707 1mg/kg group, SKLB707 3mg/kg group and AC220 3mg/kg group can completely eliminate tumors after 12 days of administration, indicating that SKLB707 inhibits tumor growth The effect is equivalent to AC220. Mouse tumor xenograft model experiments show that SKLB707 has a good anti-tumor effect in vivo.
参考文献：references:
1.Levis,M.J.；Perl,A.E.；Dombret,H.；
Figure PCTCN2018073793-appb-000086
H.；Steffen,B.；Rousselot,P.；Martinelli,G.；Estey,E.H.；Burnett,A.K.；Gammon,G.,Final results of a phase 2open-label,monotherapy  efficacy and safety study of quizartinib(AC220)in patients with FLT3-ITD positive or negative relapsed/refractory acute myeloid leukemia after second-line chemotherapy or hematopoietic stem cell transplantation.Blood 2012,120,673-673. 1. Levis, MJ; Perl, AE; Dombret, H.;
Figure PCTCN2018073793-appb-000086
H.; Steffen, B.; Rousselot, P.; Martinelli, G.; Estey, EH; Burnett, AK; Gammon, G., Final results of a phase 2open-label, monotherapy efficacy and safety study of quizartinib (AC220) In patients with FLT3-ITD positive or negative relapsed/refractory acute myeloid leukemia after second-line chemotherapy or hematopoietic stem cell transplantation.Blood 2012,120,673-673.
2.Cortes,J.E.；Perl,A.E.；Dombret,H.；Kayser,S.；Steffen,B.；Rousselot,P.；Martinelli,G.；Estey,E.H.；Burnett,A.K.；Gammon,G.,Final results of a phase 2 open-label,monotherapy efficacy and safety study of quizartinib (AC220) in patients≥60 years of age with FLT3 ITD positive or negative relapsed/refractory acute myeloid leukemia.Blood 2012,120,48-48.2.Cortes, JE; Perl, AE; Dombret, H.; Kayser, S.; Steffen, B.; Rousselot, P.; Martinelli, G.; Estey, EH; Burnett, AK; Gammon, G., Final results Of a phase 2 open-label, monotherapy efficacy and safety study of quizartinib (AC220) in patients≥60 years of age with FLT3 ITD positive or negative relapsed/refractory acute myeloid leukemia.Blood 2012,120,48-48.
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Claims (16)

  1. 取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物，其结构式如式Ⅰ所示：Substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative having the structural formula shown in Formula I:
    Figure PCTCN2018073793-appb-100001
    Figure PCTCN2018073793-appb-100001
    其中，X为碳或氮；Y为氮、氧或硫；Wherein X is carbon or nitrogen; Y is nitrogen, oxygen or sulfur;
    R 1、R 2独立地为-H、-NO 2、卤素、
    Figure PCTCN2018073793-appb-100002
    苯基、5～12元饱和或不饱和的杂环基；或者R 1和R 2组合成环，所述的环为取代的苯基、取代的5～12元饱和或不饱和的杂环基；所述5～12饱和或不饱和的杂环基含有1～2个杂原子；所述的杂原子为氮、氧或硫；     R 1 and R 2 are independently -H, -NO 2 , halogen,
    Figure PCTCN2018073793-appb-100002
    a phenyl group, a 5- to 12-membered saturated or unsaturated heterocyclic group; or a combination of R 1 and R 2 to form a ring, the ring being a substituted phenyl group, a substituted 5 to 12 membered saturated or unsaturated heterocyclic group The 5-12 saturated or unsaturated heterocyclic group contains 1 to 2 hetero atoms; the hetero atom is nitrogen, oxygen or sulfur;
    所述取代的苯基、取代的5～12元饱和或不饱和杂环基的取代基为-H、卤素、C 1～C 6烷基、C 1～C 6烷氧基、
    Figure PCTCN2018073793-appb-100003
    n＝1～4；     The substituent of the substituted phenyl group or the substituted 5- to 12-membered saturated or unsaturated heterocyclic group is -H, halogen, C 1 -C 6 alkyl group, C 1 -C 6 alkoxy group,
    Figure PCTCN2018073793-appb-100003
    n=1～4;
    R 6为C 1～C 6烷基、卤素、C 1～C 6烷氧基、
    Figure PCTCN2018073793-appb-100004
    -N(CH 3) 2、
    Figure PCTCN2018073793-appb-100005
    或苯基。     R 6 is a C 1 -C 6 alkyl group, a halogen, a C 1 -C 6 alkoxy group,
    Figure PCTCN2018073793-appb-100004
    -N(CH 3 ) 2 ,
    Figure PCTCN2018073793-appb-100005
    Or phenyl.
  2. 根据权利要求1所述的取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物，其特征在于：The substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative according to Claim 1 which is characterized in that:
    X为碳或氮；Y为氧；X is carbon or nitrogen; Y is oxygen;
    R 1、R 2独立地为-H、-NO 2、卤素、
    Figure PCTCN2018073793-appb-100006
    苯基、5～12元饱和或不饱和的杂环基；或者R 1和R 2组合成环，所述的环为取代的苯基、取代的5～12元饱和或不饱和的杂环基；所述5～12饱和或不饱和的杂环基含有1～2个杂原子；所述的杂原子为氮、氧或硫；     R 1 and R 2 are independently -H, -NO 2 , halogen,
    Figure PCTCN2018073793-appb-100006
    a phenyl group, a 5- to 12-membered saturated or unsaturated heterocyclic group; or a combination of R 1 and R 2 to form a ring, the ring being a substituted phenyl group, a substituted 5 to 12 membered saturated or unsaturated heterocyclic group The 5-12 saturated or unsaturated heterocyclic group contains 1 to 2 hetero atoms; the hetero atom is nitrogen, oxygen or sulfur;
    所述取代的苯基、取代的5～12元饱和或不饱和杂环基的取代基为-H、卤素、C 1～C 6烷基、C 1～C 6烷氧基、
    Figure PCTCN2018073793-appb-100007
    n＝1～4；     The substituent of the substituted phenyl group or the substituted 5- to 12-membered saturated or unsaturated heterocyclic group is -H, halogen, C 1 -C 6 alkyl group, C 1 -C 6 alkoxy group,
    Figure PCTCN2018073793-appb-100007
    n=1～4;
    R 6为C 1～C 6烷基、卤素、C 1～C 6烷氧基、
    Figure PCTCN2018073793-appb-100008
    -N(CH 3) 2、
    Figure PCTCN2018073793-appb-100009
    或苯基；     R 6 is a C 1 -C 6 alkyl group, a halogen, a C 1 -C 6 alkoxy group,
    Figure PCTCN2018073793-appb-100008
    -N(CH 3 ) 2 ,
    Figure PCTCN2018073793-appb-100009
    Or phenyl;
    优选的，X为碳或氮；Y为氧；Preferably, X is carbon or nitrogen; Y is oxygen;
    R 1、R 2独立地为-H、-NO 2、卤素、
    Figure PCTCN2018073793-appb-100010
    或5～10元饱和或不饱和的杂环基；或者R 1和R 2组合成环，所述的环为取代的苯基、取代的5～10元饱和或不饱和的杂环基；所述的5～10元饱和或不饱和的杂环基含有1～2个杂原子；所述的杂原子为氮、氧或硫；     R 1 and R 2 are independently -H, -NO 2 , halogen,
    Figure PCTCN2018073793-appb-100010
    Or a 5- to 10-membered saturated or unsaturated heterocyclic group; or R 1 and R 2 are combined to form a ring, said ring being a substituted phenyl group, a substituted 5 to 10 membered saturated or unsaturated heterocyclic group; The 5- to 10-membered saturated or unsaturated heterocyclic group has 1 to 2 hetero atoms; the hetero atom is nitrogen, oxygen or sulfur;
    所述取代的苯环、取代的5～10元饱和或不饱和的杂环基上的取代基为-H、卤素、C 1～C 4烷氧基、
    Figure PCTCN2018073793-appb-100011
    n＝1～4；     The substituent on the substituted benzene ring, the substituted 5- to 10-membered saturated or unsaturated heterocyclic group is -H, halogen, C 1 -C 4 alkoxy,
    Figure PCTCN2018073793-appb-100011
    n=1～4;
    R 6为C 1～C 6烷基、卤素、C 1～C 6烷氧基、
    Figure PCTCN2018073793-appb-100012
    -N(CH 3) 2、
    Figure PCTCN2018073793-appb-100013
    或苯基；     R 6 is a C 1 -C 6 alkyl group, a halogen, a C 1 -C 6 alkoxy group,
    Figure PCTCN2018073793-appb-100012
    -N(CH 3 ) 2 ,
    Figure PCTCN2018073793-appb-100013
    Or phenyl;
    进一步优选的，X为碳或氮；Y为氧；Further preferably, X is carbon or nitrogen; Y is oxygen;
    R 1、R 2独立地为-H、-NO 2、卤素、
    Figure PCTCN2018073793-appb-100014
    或5～6元饱和的杂环基；或者R 1和R 2组合成环，所述的环为取代的苯基、取代的5～10元不饱和的杂环基；所述的5～10元饱和或不饱和杂环基含有1～2个杂原子；所述的杂原子为氮或氧；     R 1 and R 2 are independently -H, -NO 2 , halogen,
    Figure PCTCN2018073793-appb-100014
    Or a 5- to 6-membered saturated heterocyclic group; or R 1 and R 2 are combined to form a ring, said ring being a substituted phenyl group, a substituted 5 to 10 membered unsaturated heterocyclic group; said 5 to 10 a mono-saturated or unsaturated heterocyclic group containing 1 to 2 hetero atoms; the hetero atom is nitrogen or oxygen;
    所述取代的苯基、取代的5～6元不饱和的杂环基上的取代基为-H、卤素、C 1～C 4烷氧基、
    Figure PCTCN2018073793-appb-100015
    n＝1～4；     The substituent on the substituted phenyl group or the substituted 5- to 6-membered unsaturated heterocyclic group is -H, halogen, C 1 -C 4 alkoxy group,
    Figure PCTCN2018073793-appb-100015
    n=1～4;
    R 6为C 1～C 6烷基、卤素、C 1～C 6烷氧基、
    Figure PCTCN2018073793-appb-100016
    -N(CH 3) 2、
    Figure PCTCN2018073793-appb-100017
    或苯基；     R 6 is a C 1 -C 6 alkyl group, a halogen, a C 1 -C 6 alkoxy group,
    Figure PCTCN2018073793-appb-100016
    -N(CH 3 ) 2 ,
    Figure PCTCN2018073793-appb-100017
    Or phenyl;
    更进一步优选的，X为碳或氮；Y为氧；Still more preferably, X is carbon or nitrogen; Y is oxygen;
    R 1、R 2独立地为-H、-NO 2、卤素、
    Figure PCTCN2018073793-appb-100018
    或5～6元饱和的杂环基；或者R 1和R 2组合成环，所述的环为取代的苯基、取代的5～6元不饱和的杂环基；所述的5～6元饱和杂环基或5～10元不饱和杂环基含有1～2个杂原子；所述的杂原子为氮或氧；     R 1 and R 2 are independently -H, -NO 2 , halogen,
    Figure PCTCN2018073793-appb-100018
    Or a 5- to 6-membered saturated heterocyclic group; or R 1 and R 2 are combined to form a ring, said ring being a substituted phenyl group, a substituted 5- to 6-membered unsaturated heterocyclic group; said 5 to 6 a monosaturated heterocyclic group or a 5 to 10 membered unsaturated heterocyclic group having 1 to 2 hetero atoms; the hetero atom is nitrogen or oxygen;
    所述取代的苯基、取代的5～10元不饱和的杂环基上的取代基为卤素、C 1～C 4烷氧基、
    Figure PCTCN2018073793-appb-100019
    n＝1～4；     The substituent on the substituted phenyl group or the substituted 5- to 10-membered unsaturated heterocyclic group is a halogen, a C 1 -C 4 alkoxy group,
    Figure PCTCN2018073793-appb-100019
    n=1～4;
    R 6为C 1～C 6烷基、卤素、C 1～C 6烷氧基、
    Figure PCTCN2018073793-appb-100020
    -N(CH 3) 2、
    Figure PCTCN2018073793-appb-100021
    或苯基；     R 6 is a C 1 -C 6 alkyl group, a halogen, a C 1 -C 6 alkoxy group,
    Figure PCTCN2018073793-appb-100020
    -N(CH 3 ) 2 ,
    Figure PCTCN2018073793-appb-100021
    Or phenyl;
    再进一步优选的，X为碳或氮；Y为氧；Still further preferably, X is carbon or nitrogen; Y is oxygen;
    R 1、R 2独立地为-H、-NO 2、卤素、
    Figure PCTCN2018073793-appb-100022
    或5～6元饱和的杂环基；或者R 1和R 2组合成环，所述的环为取代的苯基、取代的5～6元不饱和的杂环基；所述的5～6元饱和杂环基或5～10元不饱和杂环基含有1～2个杂原子；所述的杂原子为氮或氧；     R 1 and R 2 are independently -H, -NO 2 , halogen,
    Figure PCTCN2018073793-appb-100022
    Or a 5- to 6-membered saturated heterocyclic group; or R 1 and R 2 are combined to form a ring, said ring being a substituted phenyl group, a substituted 5- to 6-membered unsaturated heterocyclic group; said 5 to 6 a monosaturated heterocyclic group or a 5 to 10 membered unsaturated heterocyclic group having 1 to 2 hetero atoms; the hetero atom is nitrogen or oxygen;
    所述苯基、取代的5～10元不饱和的杂环基上的取代基为-H、卤素、C 1～C 4烷氧基、
    Figure PCTCN2018073793-appb-100023
    Figure PCTCN2018073793-appb-100024
    n＝1～4；     The substituent on the phenyl group, the substituted 5- to 10-membered unsaturated heterocyclic group is -H, a halogen, a C 1 -C 4 alkoxy group,
    Figure PCTCN2018073793-appb-100023
    Figure PCTCN2018073793-appb-100024
    n=1～4;
    R 6为卤素、C 1～C 6烷氧基、
    Figure PCTCN2018073793-appb-100025
    -N(CH 3) 2、
    Figure PCTCN2018073793-appb-100026
    或苯基；     R 6 is halogen, C 1 -C 6 alkoxy,
    Figure PCTCN2018073793-appb-100025
    -N(CH 3 ) 2 ,
    Figure PCTCN2018073793-appb-100026
    Or phenyl;
    最优的，X为碳或氮；Y为氧；Optimally, X is carbon or nitrogen; Y is oxygen;
    R 1、R 2独立地为-H、-NO 2、卤素、
    Figure PCTCN2018073793-appb-100027
    或5～6元饱和的杂环基；或者R 1和R 2组合成环， 所述的环为取代的苯基、取代的5～6元不饱和的杂环基；；所述的5～6元饱和杂环基或5～10元不饱和杂环基含有1个氮原子；     R 1 and R 2 are independently -H, -NO 2 , halogen,
    Figure PCTCN2018073793-appb-100027
    Or a 5- to 6-membered saturated heterocyclic group; or R 1 and R 2 are combined to form a ring, the ring being a substituted phenyl group, a substituted 5- to 6-membered unsaturated heterocyclic group; a 6-membered saturated heterocyclic group or a 5 to 10 membered unsaturated heterocyclic group containing 1 nitrogen atom;
    所述苯基、取代的5～10元不饱和的杂环基上的取代基为-H、-Cl、C 1～C 4烷氧基、
    Figure PCTCN2018073793-appb-100028
    Figure PCTCN2018073793-appb-100029
    n＝1～4；     The substituent on the phenyl group, the substituted 5- to 10-membered unsaturated heterocyclic group is -H, -Cl, C 1 -C 4 alkoxy,
    Figure PCTCN2018073793-appb-100028
    Figure PCTCN2018073793-appb-100029
    n=1～4;
    R 6为C 1～C 6烷氧基、
    Figure PCTCN2018073793-appb-100030
    -N(CH 3) 2、
    Figure PCTCN2018073793-appb-100031
    或苯基。     R 6 is a C 1 -C 6 alkoxy group,
    Figure PCTCN2018073793-appb-100030
    -N(CH 3 ) 2 ,
    Figure PCTCN2018073793-appb-100031
    Or phenyl.
  3. 根据权利要求1或2所述的取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物，其特征在于：当X为氮，Y为氧，R 1和R 2环合为取代的苯基时，所述取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物的结构式如式Ⅱ所示： The substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative according to claim 1 or 2, wherein when X is nitrogen, Y is When oxygen, R 1 and R 2 are cyclized to a substituted phenyl group, the structural formula of the substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative is as Formula II shows:
    Figure PCTCN2018073793-appb-100032
    Figure PCTCN2018073793-appb-100032
    其中，R 3、R 4独立地为-H、卤素、C 1～C 4烷氧基、
    Figure PCTCN2018073793-appb-100033
    n＝1～4；     Wherein R 3 and R 4 are independently -H, halogen, C 1 -C 4 alkoxy,
    Figure PCTCN2018073793-appb-100033
    n=1～4;
    R 6为C 1～C 6烷基、卤素、C 1～C 6烷氧基、
    Figure PCTCN2018073793-appb-100034
    -N(CH 3) 2、
    Figure PCTCN2018073793-appb-100035
    或苯基。     R 6 is a C 1 -C 6 alkyl group, a halogen, a C 1 -C 6 alkoxy group,
    Figure PCTCN2018073793-appb-100034
    -N(CH 3 ) 2 ,
    Figure PCTCN2018073793-appb-100035
    Or phenyl.
  4. 根据权利要求3所述的取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物，其特征在于：R 3、R 4独立地为-H、卤素、C 1～C 4烷氧基、
    Figure PCTCN2018073793-appb-100036
    n＝1～4；R 6为卤素、C 1～C 6烷氧基、
    Figure PCTCN2018073793-appb-100037
    -N(CH 3) 2、
    Figure PCTCN2018073793-appb-100038
    或苯基；     The substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative according to claim 3, wherein R 3 and R 4 are independently - H, halogen, C 1 -C 4 alkoxy,
    Figure PCTCN2018073793-appb-100036
    n=1～4; R 6 is halogen, C 1 -C 6 alkoxy,
    Figure PCTCN2018073793-appb-100037
    -N(CH 3 ) 2 ,
    Figure PCTCN2018073793-appb-100038
    Or phenyl;
    优选的，R 3、R 4独立地为-H、卤素、C 1～C 4烷氧基、
    Figure PCTCN2018073793-appb-100039
    n＝1～4；R 6为C 1～C 6烷氧基、
    Figure PCTCN2018073793-appb-100040
    -N(CH 3) 2、
    Figure PCTCN2018073793-appb-100041
    或苯基；     Preferably, R 3 and R 4 are independently -H, halogen, C 1 -C 4 alkoxy,
    Figure PCTCN2018073793-appb-100039
    n=1 to 4; R 6 is a C 1 -C 6 alkoxy group,
    Figure PCTCN2018073793-appb-100040
    -N(CH 3 ) 2 ,
    Figure PCTCN2018073793-appb-100041
    Or phenyl;
    最优的，R 3、R 4独立地为-H、C 1～C 4烷氧基、
    Figure PCTCN2018073793-appb-100042
    n＝1～4；R 6为C 1～C 6烷氧基、
    Figure PCTCN2018073793-appb-100043
    -N(CH 3) 2、
    Figure PCTCN2018073793-appb-100044
    或苯基。     Most preferably, R 3 and R 4 are independently -H, C 1 -C 4 alkoxy,
    Figure PCTCN2018073793-appb-100042
    n=1 to 4; R 6 is a C 1 -C 6 alkoxy group,
    Figure PCTCN2018073793-appb-100043
    -N(CH 3 ) 2 ,
    Figure PCTCN2018073793-appb-100044
    Or phenyl.
  5. 根据权利要求1或2所述的取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物，其特征在于：当X为氮，Y为氧，R 1和R 2环合为含有1个氮原子的取代不饱和6元杂环基时，所述取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物的结构式如式Ⅲ所示： The substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative according to claim 1 or 2, wherein when X is nitrogen, Y is When oxygen, R 1 and R 2 are cyclized to a substituted unsaturated 6-membered heterocyclic group containing 1 nitrogen atom, the substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4) The structural formula of the -phenyl)urea derivative is as shown in formula III:
    Figure PCTCN2018073793-appb-100045
    Figure PCTCN2018073793-appb-100045
    其中，R 5为-H、卤素、C 1～C 4烷氧基、
    Figure PCTCN2018073793-appb-100046
    n＝1～4；     Wherein R 5 is -H, halogen, C 1 -C 4 alkoxy,
    Figure PCTCN2018073793-appb-100046
    n=1～4;
    R 6为C 1～C 6烷基、卤素、C 1～C 6烷氧基、
    Figure PCTCN2018073793-appb-100047
    -N(CH 3) 2、
    Figure PCTCN2018073793-appb-100048
    或苯基。     R 6 is a C 1 -C 6 alkyl group, a halogen, a C 1 -C 6 alkoxy group,
    Figure PCTCN2018073793-appb-100047
    -N(CH 3 ) 2 ,
    Figure PCTCN2018073793-appb-100048
    Or phenyl.
  6. 根据权利要求5所述的取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物，其特征在于：R 5为-H、卤素、C 1～C 4烷氧基或
    Figure PCTCN2018073793-appb-100049
    n＝1～4；R 6为C 1～C 6烷基、卤素、C 1～C 6烷氧基、
    Figure PCTCN2018073793-appb-100050
    -N(CH 3) 2、
    Figure PCTCN2018073793-appb-100051
    或苯基；     The substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative according to claim 5, wherein R 5 is -H, halogen, C 1 to C 4 alkoxy or
    Figure PCTCN2018073793-appb-100049
    n=1 to 4; R 6 is a C 1 -C 6 alkyl group, a halogen, a C 1 -C 6 alkoxy group,
    Figure PCTCN2018073793-appb-100050
    -N(CH 3 ) 2 ,
    Figure PCTCN2018073793-appb-100051
    Or phenyl;
    优选的，R 5为-H、卤素、C 1～C 4烷氧基或
    Figure PCTCN2018073793-appb-100052
    n＝1～4；R 6为卤素、C 1～C 6烷氧基、
    Figure PCTCN2018073793-appb-100053
    或苯基；     Preferably, R 5 is -H, halogen, C 1 -C 4 alkoxy or
    Figure PCTCN2018073793-appb-100052
    n=1～4; R 6 is halogen, C 1 -C 6 alkoxy,
    Figure PCTCN2018073793-appb-100053
    Or phenyl;
    最优的，R 5为-H、卤素、C 1～C 4烷氧基或
    Figure PCTCN2018073793-appb-100054
    n＝1～4；R 6为卤素、C 1～C 6烷氧基或
    Figure PCTCN2018073793-appb-100055
    Most preferably, R 5 is -H, halogen, C 1 -C 4 alkoxy or
    Figure PCTCN2018073793-appb-100054
    n=1～4; R 6 is halogen, C 1 -C 6 alkoxy or
    Figure PCTCN2018073793-appb-100055
  7. 取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物，其结构式为：Substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative having the structural formula:
    Figure PCTCN2018073793-appb-100056
    Figure PCTCN2018073793-appb-100057
    Figure PCTCN2018073793-appb-100056
    Figure PCTCN2018073793-appb-100057
  8. 权利要求1～7任一项所述的取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物药学上可接受的盐或水合物。A pharmaceutically acceptable salt or hydrate of a substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative according to any one of claims 1 to 7.
  9. 权利要求1～7任一项所述的取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物的前药。A prodrug of a substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative according to any one of claims 1 to 7.
  10. 一种组合物，是由权利要求1～7任一项所述的取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物添加药学上可以接受的辅助性成分制备而成的。A composition obtained by adding a substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative according to any one of claims 1 to 7 to a pharmaceutically acceptable form Prepared from acceptable auxiliary ingredients.
  11. 权利要求1～7任一项所述的取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物、权利要求8所述的盐或水合物在制备FMS-样酪氨酸激酶3抑制剂中的用途。The substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative according to any one of claims 1 to 7, the salt or hydrated according to claim 8. Use of the substance in the preparation of an FMS-like tyrosine kinase 3 inhibitor.
  12. 根据权利要求11所述的用途，其特征在于：所述的FMS-样酪氨酸激酶3抑制剂，为针对FLT3酪氨酸激酶内部串联重复突变(FLT3-ITD)或在FLT3酪氨酸激酶原有内部串联重复突变(FLT3-ITD)基础上产生的单个氨基酸突变的耐药的FLT3-ITD-F691L或FLT3-ITD-D835Y抑制剂。The use according to claim 11, characterized in that the FMS-like tyrosine kinase 3 inhibitor is directed against FLT3 tyrosine kinase internal tandem repeat mutation (FLT3-ITD) or in FLT3 tyrosine kinase The original internal tandem repeat mutation (FLT3-ITD) produces a single amino acid mutant resistant FLT3-ITD-F691L or FLT3-ITD-D835Y inhibitor.
  13. 权利要求1～7任一项所述的取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物、权利要求8所述的盐或水合物在制备治疗急性髓性白血病药物中的用途。The substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative according to any one of claims 1 to 7, the salt or hydrated according to claim 8. The use of the substance in the preparation of a medicament for treating acute myeloid leukemia.
  14. 权利要求1～7任一项所述的取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物、权利要求8所述的盐或水合物在制备抗自身免疫性疾病药物中的用途。The substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative according to any one of claims 1 to 7, the salt or hydrated according to claim 8. The use of the substance in the preparation of a medicament against autoimmune diseases.
  15. 权利要求1～7任一项所述的取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物、权利要求8所述的盐或水合物在制备新生血管生成抑制剂中的用途。The substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative according to any one of claims 1 to 7, the salt or hydrated according to claim 8. Use of the substance in the preparation of a neonatal angiogenesis inhibitor.
  16. 权利要求1～7任一项所述的取代的1-(异恶唑-3-基)-3-(3-氟-4-苯基)脲衍生物、权利要求8所述的盐或水合物在制备抗肿瘤药物中的用途。The substituted 1-(isoxazol-3-yl)-3-(3-fluoro-4-phenyl)urea derivative according to any one of claims 1 to 7, the salt or hydrated according to claim 8. The use of the substance in the preparation of an antitumor drug.
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