WO2020063856A1 - 作为flt3和axl抑制剂的3,9-二氮杂螺[5,5]十一烷类化合物 - Google Patents

作为flt3和axl抑制剂的3,9-二氮杂螺[5,5]十一烷类化合物 Download PDF

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WO2020063856A1
WO2020063856A1 PCT/CN2019/108522 CN2019108522W WO2020063856A1 WO 2020063856 A1 WO2020063856 A1 WO 2020063856A1 CN 2019108522 W CN2019108522 W CN 2019108522W WO 2020063856 A1 WO2020063856 A1 WO 2020063856A1
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compound
acid
flt3
mmol
degrees celsius
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French (fr)
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付翔宇
丁照中
胡利红
陆剑宇
江文
黎健
陈曙辉
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Medshine Discovery Inc
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Medshine Discovery Inc
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Priority to JP2021517760A priority Critical patent/JP7201800B2/ja
Priority to CN201980064621.7A priority patent/CN112839930B/zh
Priority to EP19865038.4A priority patent/EP3858819A4/en
Priority to US17/281,479 priority patent/US11691974B2/en
Publication of WO2020063856A1 publication Critical patent/WO2020063856A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/08—Bridged systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/10—Spiro-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A61K31/497—Non-condensed pyrazines containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia

Definitions

  • the present invention relates to a class of compounds that are inhibitors of FLT3 and AXL, and specifically discloses the compound represented by formula (I) and its pharmaceutically acceptable salt, and its application in the preparation of AML drugs.
  • AML Acute myeloid leukemia
  • AML Acute myeloid leukemia
  • the incidence of AML is 3.4 / 100,000, and the median age of the patients is 67 years.
  • the treatment of AML still depends on chemotherapy, and about 70% of patients who have remitted eventually relapse and become refractory leukemia.
  • the prognosis of AML is poor, especially for elderly patients and patients with poor physical fitness.
  • Drug resistance is the leading cause of failure to treat AML, but the mechanism of leukemia resistance is unknown. Therefore, finding new targets and their inhibitors is of great significance for improving the efficacy of AML and changing the prognosis.
  • the FLT3 receptor is a member of the type III receptor tyrosine kinase family.
  • FLT3 mutations are the most common genetic mutations in AML, mainly including tandem repeat mutations (ITDs) and point mutations (TKDs) at the loops of the membrane membrane region of FLT3. These mutations cause the downstream signaling pathways to be continuously activated and the mutant cells to proliferate.
  • FLT3 has been considered as an important target for the treatment of AML, and FLT3 inhibitors are also considered as the most promising molecular targeted drugs for the treatment of AML.
  • AXL is also called Ufo, Ark or Tyro7. Its abnormal expression can activate antagonizing tumor cell apoptosis, promote tumor cell invasion and metastasis, and promote tumor blood vessel formation. All of the above effects have promoted the occurrence and development of tumor. For AML patients, high AXL expression results in reduced survival and worsened prognosis. In addition, AXL overexpression is closely related to drug resistance of targeted drugs and chemotherapy drugs. AXL has also recently been found to have potential in immunotherapy. Therefore, the development of dual inhibitors of FLT3 and AXL is expected to achieve better efficacy in the treatment of AML.
  • WO2012053606A1 reports compound A (Example 176 in WO2012053606A1), and it is mentioned that such molecules have FLT3 inhibitory activity and can be used for the treatment of AML, but no specific test data is given.
  • WO2010128659A1 reports compound B having FLT3 inhibitory activity (Example 547 in WO2010128659A1). A Phase III clinical trial of this compound in the treatment of relapsed or refractory AML is ongoing.
  • the present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,
  • the invention also provides the use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cancer.
  • the cancer is acute myeloid leukemia.
  • the invention provides a novel FLT3 / AXL double inhibitor. Compared with the prior art, it has unexpectedly higher in vitro enzyme activity and cell activity, especially in the FLT3 mutant enzyme activity test. The pharmacokinetic properties are superior to the prior art. In MV4-11 in vivo experiments, low doses have shown good tumor suppressive activity. The drug withdrawal-rebound test proves that the compound of the present invention has a strong ability to sustain tumor inhibition. In Molm-13 in vivo experiments, it has unexpectedly excellent tumor suppressive effect, which is obviously superior to the existing technology.
  • pharmaceutically acceptable refers to those compounds, materials, compositions, and / or dosage forms that are within the scope of sound medical judgment and are suitable for use in contact with human and animal tissues Without excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit / risk ratio.
  • pharmaceutically acceptable salt refers to a salt of a compound of the present invention, prepared from a compound having a specific substituent and a relatively non-toxic acid or base found in the present invention.
  • base addition salts can be obtained by contacting such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent.
  • Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts.
  • acid addition salts can be obtained by contacting such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent.
  • Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydroiodic acid, phosphorous acid, etc .; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Similar acids such as fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid; also include salts of amino acids (such as arginine, etc.) , And salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain basic and acidic functional groups
  • the pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound containing an acid group or a base by a conventional chemical method. Generally, such salts are prepared by reacting these compounds in the form of a free acid or base with a stoichiometric appropriate base or acid in water or an organic solvent or a mixture of the two.
  • aq stands for water
  • HATU O- (7-azabenzotriazol-1-yl) -N, N, N ', N'-tetramethylurea hexafluorophosphate
  • EDC stands for N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride
  • m-CPBA stands for 3-chloroperoxybenzoic acid
  • eq stands for equivalent, equivalent
  • CDI stands for Carbonyl diimidazole
  • DCM stands for dichloromethane
  • PE stands for petroleum ether
  • DIAD diisopropyl azodicarboxylate
  • DMF stands for N, N-dimethylformamide
  • DMSO stands for dimethyl sulfoxide
  • EtOAc stands for ethyl acetate EtOH for ethanol; MeOH for methanol
  • CBz benzyloxycarbonyl, an amine protecting group
  • Step A Add compound 1-1 (30 g, 230.52 mmol, 28.57 ml, 1 equivalent) to water (600 ml), and then add sodium hydroxide (11.99 g, 299.67 mmol, 1.3 equivalent), 20 degrees Celsius Stir for 16 hours. Reduce the temperature of the system to between 0 ° C and 5 ° C, and then slowly add a solution of sodium nitrite (17.50 g, 253.57 mmol, 1.1 equivalents) in water (60 ml), adjust the pH of the system to 4 with sulfuric acid, and then Stir for 12 hours at 20 degrees Celsius.
  • sodium hydroxide 11.99 g, 299.67 mmol, 1.3 equivalent
  • Step B Dissolve Compound 1-2 (20 g, 197.882 mmol, 1 eq) in isopropanol (400 mL), and add Compound 1-3 (50 g, 197.41 mmol, 0.998 eq, p-toluenesulfonate Acid salt), the mixed system was stirred at 20 degrees Celsius for 16 hours.
  • the reaction solution was poured into water (300 ml) and extracted with ethyl acetate (500 ml x 3). The organic phases were combined, washed with saturated brine (800 ml), dried over sodium sulfate, and concentrated to obtain compound 1-4.
  • Step C Compound 1-4 (31 g, 188.84 mmol, 1 equivalent) was dissolved in N, N-dimethylformamide (300 ml), the temperature was lowered to 0 degrees Celsius, and phosphorous trichloride (78.52) was slowly added dropwise. G, 512.09 mmol, 47.59 ml, 2.71 equivalents), keeping the temperature below 5 degrees Celsius. After the addition was complete, the system was heated to 80 degrees Celsius and stirred for 2 hours. The reaction solution was added dropwise to ice (900 g), and the mixture was stirred at 20 ° C for 16 hours. A solid precipitated, filtered, and the filter cake was collected and dried under vacuum to give compound 1-5.
  • Step D Dissolve tert-butyl nitrite (20.61 g, 199.88 mmol, 23.77 ml, 2.5 eq) and copper bromide (21.43 g, 95.94 mmol, 4.49 ml, 1.2 eq) in N, N-dimethyl In formamide (200 ml), the system was heated to 65 ° C, and a solution of compound 1-5 (14.6 g, 79.95 mmol, 1 equivalent) in N, N-dimethylformamide (150 ml) was added dropwise.
  • Step E Dissolve compound 1-6 (4 g, 16.23 mmol, 1 equivalent) and compound 1-7 (1.97 g, 14.31 mmol, 0.882 equivalent) in 1,4 dioxane (50 ml), Further N, N-diisopropylethylamine (5.03 g, 38.95 mmol, 6.78 ml, 2.4 equivalents) was added. The mixture was heated to 65 degrees Celsius and stirred for 12 hours. Water (100 ml) was poured into the reaction solution, and stirred at 20 ° C for 0.5 hours. The mixture was filtered, and the filter cake was washed with water and dried under vacuum to obtain compound 1-8. MS (ESI) m / z: 310.9, 312.9 [M + H + ].
  • Step F Add ammonium acetate (2.04 g, 26.42 mmol, 0.1 equivalent) to compound 1-10 (89.65 g, 792.59 mmol, 84.58 ml, 3 equivalent) of methanol (100 To the solution, compound 1-9 (50 g, 264.20 mmol, 49.02 ml, 1 equivalent) was added. Then, below 10 degrees Celsius, aqueous ammonia (51.85 g, 369.87 mmol, 56.98 ml, 25%, 1.4 equivalents) was added to the mixture. The mixture was stirred for 1 hour between 0 ° C and 5 ° C, and then the reaction mixture was warmed to 20 ° C and stirred for 20 hours.
  • Step G To a mixture of sulfuric acid (161.92 g, 1.65 mol, 88 ml, 10.65 eq) and water (12.00 g, 666.10 mmol, 12 ml, 4.30 eq) was added compound 1-11 (49.95 g, 154.95 mmol) , 1 equivalent), at this time the temperature rose to 40 degrees Celsius. The mixture was heated to 80 degrees Celsius and stirred for 2 hours. Then, water (20.00 g, 1.11 moles, 20 ml, 7.16 equivalents) was added, and the mixture was heated to 100 degrees Celsius and stirred for 1.5 hours.
  • Step H Compound 1-12 (39.14 g, 114.66 mmol, 1 equivalent) was added to an aqueous sodium hydroxide solution (5 mol / liter, 183.45 ml, 8 equivalents), and the mixture was heated to 80 degrees Celsius and stirred for 2 hours. .
  • the temperature of the system was cooled to 60 degrees Celsius, hydrochloric acid (12 moles / liter, 75 ml, 7.85 equivalents) was slowly added, the temperature was heated to 75 degrees Celsius and hydrochloric acid (12 moles / liter, 15 ml, 1.57 equivalents) was added dropwise.
  • the mixture was heated to 85 ° C and stirred for 1 hour, then cooled to 25 ° C and stirred for 16 hours.
  • Step I Compound 1-13 (28 g, 102.81 mmol, 1 equivalent) was dissolved in tetrahydrofuran (300 ml), the mixture was heated to 70 degrees Celsius, and then lithium tetrahydroaluminum (15.61 g, 411.25 mmol, 4 (Equivalent) was added to the solution in portions. The mixture was stirred at 70 degrees Celsius for 12 hours. After cooling to room temperature, a saturated sodium sulfate solution (30 ml) was slowly added dropwise to the reaction solution, followed by filtration, and the filter cake was washed with ethyl acetate (100 ml). The filtrates were combined and concentrated to give compound 1-14. MS (ESI) m / z: 245.1 [M + H + ].
  • Step J Compound 1-14 (0.5 g, 2.05 mmol, 1 equivalent) and compound 1-15 (317.39 mg, 2.05 mmol, 1 equivalent) were dissolved in N, N-dimethylformamide (10 ml ), Potassium carbonate (565.55 mg, 4.09 mmol, 2 equivalents) was added. The mixture was heated to 80 degrees Celsius and stirred for 12 hours. The reaction solution was poured into water (60 mL), and extracted with ethyl acetate (60 mL ⁇ 2). The organic phases were combined, washed with saturated brine (60 mL), dried, and concentrated to obtain compound 1-16. MS (ESI) m / z: 380.0 [M + H + ].
  • Step K To a solution of compound 1-16 (550 mg, 1.45 mmol, 1 equivalent) in dichloromethane (15 mL) was added methyl iodide (246.85 mg, 1.74 mmol, 108.27 microliters, 1.2 equivalents), and the mixture Stir for 12 hours at 25 degrees Celsius. The reaction solution was concentrated to obtain compound 1-17. MS (ESI) m / z: 394.1 [M + H + ].
  • Step L To a solution of compound 1-17 (620 mg, 1.19 mmol, 1 equivalent) in ethanol (20 ml) was added wet palladium on carbon (100 mg, 10%), and after replacing with hydrogen, the mixture was heated to 60 In Celsius, it reacts for 12 hours under a hydrogen pressure of 50 pounds per square inch. It was then filtered and the filtrate was concentrated to give compound 1-18.
  • Step M To a solution of compound 1-18 (300 mg, 1.10 mmol, 1 equivalent) and compound 1-8 (341.43 mg, 1.10 mmol, 1 equivalent) in 1,4-dioxane (10 mL) Add palladium acetate (24.63 mg, 109.72 micromoles, 0.1 equivalent), 4,5-bis (diphenylphosphonium) -9,9-dimethyloxanthene (63.49 mg, 109.72 micromoles, 0.1 equivalent) and Potassium carbonate (303.29 mg, 2.19 mmol, 2 eq). The system was replaced with nitrogen, then heated to 80 degrees Celsius, and stirred under a nitrogen atmosphere for 12 hours.
  • Step N Dissolve compound 1-19 (200 mg, 397.08 micromolar, 1 equivalent) in dimethyl sulfoxide (2 ml) and ethanol (6 ml), cool the system to 0 degrees Celsius, and then add sodium hydroxide (4 moles per liter, 297.81 microliters, 3 equivalents) and hydrogen peroxide (135.06 mg, 1.19 mmol, 114.46 microliters, purity 30%, 3 equivalents). The reaction solution was naturally heated to 25 degrees Celsius and stirred for 12 hours.
  • Method 1 Pour the reaction solution into water (30 ml) and extract with ethyl acetate (40 ml x 3). Combine the organic phases, wash with saturated brine (40 ml), dry over sodium sulfate, and concentrate the resulting crude product. , Separation and purification (preparative high performance liquid chromatography, chromatography column: PhenomenexSynergi C18 150 * 25 * 10 microns; mobile phase: [water (0.1% trifluoroacetic acid) -acetonitrile]; acetonitrile%: 10% -37%, 10 minutes) Trifluoroacetate of the compound of formula (I) is obtained.
  • Method 2 Add water (20 ml) to the reaction solution, stir for 30: minutes, filter, wash the filter cake with water (10 ml), beat the cake with ethanol (5 ml), filter, and dry under reduced pressure to obtain the formula (I) Compound.
  • FLT3 Kinase Enzyme System was purchased from Promega. Envision Multi-Label Analyzer (PerkinElmer).
  • buffer solution in the kit to dilute enzymes, substrates, ATP (adenosine triphosphate) and inhibitors.
  • the test compound was diluted 5 times with an exhaust gun to the eighth concentration, that is, from 5 micromoles per liter to 0.065 nanomoles per liter, and the final concentration of dimethyl sulfoxide was 5%.
  • the final compound concentration gradient was diluted from 1 micromole per liter to 0.013 nanomole per liter.
  • the reaction system was left at 30 degrees Celsius for 120 minutes.
  • reaction After the reaction, add 5 microliters of ADP-Glo reagent to each well, and continue the reaction at 30 degrees Celsius for 40 minutes. After the reaction, add 10 microliters of kinase detection reagent to each well. After 30 minutes of reaction, use PerkinElmer Envision multi-label analyzer reading Chemiluminescence, integration time 0.5 seconds.
  • Table 1 provides the FLT3 enzymatic inhibitory activity of the compounds of the invention.
  • the compound of the present invention has excellent in vitro inhibitory activity on FLT3.
  • AXL Kinase Enzyme System was purchased from Promega. Envision Multi-Label Analyzer (PerkinElmer).
  • the test compound was diluted 5 times with an exhaust gun to the eighth concentration, that is, from 5 micromoles per liter to 0.065 nanomoles per liter, and the final concentration of dimethyl sulfoxide was 5%.
  • the final compound concentration gradient was diluted from 1 micromole per liter to 0.013 nanomole per liter.
  • the reaction system was left at 30 degrees Celsius for 60 minutes.
  • reaction After the reaction, add 5 microliters of ADP-Glo reagent to each well, and continue the reaction at 30 degrees Celsius for 40 minutes. After the reaction, add 10 microliters of kinase detection reagent to each well. After 30 minutes of reaction, use PerkinElmer Envision multi-label analyzer reading Chemiluminescence, integration time 0.5 seconds.
  • Table 2 provides the compounds of the invention for AXL enzymatic inhibitory activity.
  • the compounds of the present invention have excellent in vitro inhibitory activity on AXL.
  • the compounds of the present invention have excellent in vitro inhibitory activity against mutated FLT3 targets. In all 10 mutations, it showed higher activity than the known compound B, among which FLT3 (ITD, F691L) activity was 3.6 times higher and FLT3 (K663Q) activity was 5.9 times higher. Considering that point mutation is an important reason for the resistance of FLT3 inhibitors, the higher activity on mutant FLT3 has extremely high clinical significance.
  • IMDM medium fetal calf serum, penicillin / streptomycin antibiotic were purchased from Promega (Madison, WI).
  • the MV-4-11 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences. Envision Multi-Label Analyzer (PerkinElmer).
  • MV-4-11 cells were seeded in white 96-well plates with 80 microliters of cell suspension per well, which contained 10,000 MV-4-11 cells. Cell plates were cultured overnight in a carbon dioxide incubator.
  • test compound 5 Dilute the test compound 5 times with an exhaust gun to the 8th concentration, that is, dilute from 2 mmol to 26 nanomoles per liter, and set up a double-complex experiment.
  • Cell plates were cultured in a carbon dioxide incubator for 3 days.
  • Table 3 provides the inhibitory activity of the compounds of the present invention on the proliferation of MV-4-11 cells.
  • the compound of the present invention has excellent inhibitory activity on the proliferation of MV-4-11 cells.
  • the purpose of this experiment was to evaluate the pharmacokinetic behavior of the compound after a single intravenous injection and intragastric administration, and to investigate the bioavailability after intragastric administration.
  • CD-1 male mice aged 7 to 10 weeks were selected for intravenous and oral administration at 1 mg / kg and 2.5 mg / kg, respectively. Mice were fasted for at least 12 hours before dosing, and resumed feeding 4 hours after dosing, with free access to water throughout the experiment.
  • mice The results of evaluating PK properties in mice are shown in Table 5.
  • the compound of the present invention has a proper clearance rate in mice, oral AUC, good bioavailability, and good pharmacokinetic properties. There is an unexpected improvement in PK properties compared to compound A.
  • This test uses a human biphenotypic B bone marrow mononuclear leukemia cell MV4-11 subcutaneous xenograft tumor nude mouse model to evaluate the antitumor effect of the compound.
  • Human phenotype B bone marrow mononuclear leukemia cells MV4-11 were cultured in suspension in vitro.
  • the in vitro culture conditions were RPMI1640 medium plus 10% fetal calf serum, 100 U / mL penicillin and 100 ⁇ g / mL streptomycin.
  • Tumor diameter was measured twice a week with vernier calipers.
  • the compound of the present invention has a significant inhibitory effect on the growth of human phenotype B bone marrow mononuclear leukemia cells MV4-11 xenograft tumors.
  • a tumor suppressive effect equivalent to that of Compound A at a higher dose (20 mg / kg) was exhibited.
  • the same dose (5 mg / kg) has a more obvious antitumor effect than Compound B.
  • the purpose of this experiment was to evaluate the pharmacokinetic behavior of the compound after a single intravenous injection and intragastric administration, and to investigate the bioavailability after intragastric administration.
  • Rats 7 to 10 week old SD male rats were selected, and the doses were intravenously and orally administered at 1 mg / kg and 2.5 mg / kg, respectively. Rats were fasted for at least 12 hours before dosing, and resumed feeding 4 hours after dosing, with free access to water throughout the test.
  • the compound of the present invention is orally administered AUC in rats, has excellent bioavailability, and has good pharmacokinetic properties. There is an unexpected improvement in PK properties compared to compound B.
  • MOLM-13 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum. Collect exponential growth MOLM-13 cells and resuspend PBS to a suitable concentration for inoculation of subcutaneous tumors in nude mice.
  • mice were subcutaneously inoculated with 5 ⁇ 10 6 MOLM-13 cells on the right back, and the cells were resuspended in 0.1ml PBS (0.1ml / head). The tumor growth was regularly observed. When the tumor grew to an average volume of 98mm 3 , according to the tumor size And mouse weights were randomly divided into groups.
  • tumor volume (mm 3 ) 1/2 ⁇ (a ⁇ b 2 ) (where a represents the long diameter and b represents the short diameter).
  • the compound of the present invention has a significant inhibitory effect on the growth of human-derived Molm-13 xenograft tumors. At the same dose (15 mg / kg), it showed better tumor suppressive effect than Compound B. Tumor volume shrank to zero at a dose of 50 mg / kg.

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Abstract

本发明公开了新的一类作为FLT3和AXL抑制剂的化合物，具体公开了式(I)所示化合物及其药学上可接受的盐。

Description

作为FLT3和AXL抑制剂的3,9-二氮杂螺[5,5]十一烷类化合物
本申请主张如下优先权：
CN201811157842.2，申请日2018年9月30日；
CN201910193150.1，申请日2019年3月14日。
技术领域
本发明涉及一类作为FLT3和AXL抑制剂的化合物，具体公开了式(I)所示化合物及其药学上可接受的盐及其在制备治疗AML药物中的应用。
背景技术
急性髓性白血病(AML)是成年人中最常见的急性白血病，是由骨髓造血细胞恶性增殖引起的疾病。AML的发病率为3.4/10万，患者年龄中位数是67岁。目前，AML的治疗依然需要依赖化疗，且70％左右得到缓解的患者最终复发并变成难治性白血病。另外，AML的预后较差，尤其是对于老年患者以及身体素质较差的患者。耐药性是治疗AML失败的最主要原因，但是白血病耐药的机制还不明了。所以，寻找新的靶标及其抑制剂，对于改善AML的疗效以及改变预后具有重要意义。
FLT3受体是III型受体酪氨酸激酶家族中的成员。FLT3突变是AML中最常见的基因突变，主要包括FLT3近膜区内部串联重复突变(ITD)以及环处的点突变(TKD)。这些突变引起下游信号通路被持续激活，变异细胞也过度增殖。目前，FLT3已经被认为是治疗AML的重要靶标，FLT3抑制剂也被认为是当前最有研究前景的治疗AML的分子靶向药物。
AXL也叫Ufo，Ark或Tyro7，它的异常表达能够激活拮抗肿瘤细胞凋亡，促进肿瘤细胞的侵袭和转移，并促进肿瘤血管的生成，以上作用都推动了肿瘤的发生和发展。对AML患者而言，AXL高表达会导致生存期减小，预后变差。此外，AXL的过表达与靶向药物以及化疗药物的耐药密切相关。近期AXL还被发现在免疫治疗中具有潜力。因此，发展FLT3和AXL的双抑制剂有望在AML治疗上获得更好疗效。
WO2012053606A1报道了化合物A(WO2012053606A1中实施例176)，提到这类分子具有FLT3抑制活性，可用于AML的治疗，但没有给出具体测试数据。
Figure PCTCN2019108522-appb-000001
WO2010128659A1报道了具有FLT3抑制活性的化合物B(WO2010128659A1中实施例547)。该化合物治疗复发或难治行AML的临床III期试验正在进行中。
发明内容
本发明提供式(I)所示化合物或其药学上可接受的盐，
Figure PCTCN2019108522-appb-000002
本发明还提供式(I)所示化合物或其药学上可接受的盐在制备治疗癌症药物中的应用。
在本发明的一些方案中，上述癌症是指急性髓性白血病。
技术效果
本发明提供了一种新型的FLT3/AXL双抑制剂。与现有技术相比，具有意想不到的更高的体外酶活性、细胞活性，尤其在FLT3突变的酶活性测试中优势明显。药代动力学性质优于现有技术。在MV4-11体内实验中，低剂量即表现出与良好的肿瘤抑制活性。停药-反弹实验证明本发明化合物具有较强的持续抑瘤能力。在Molm-13体内实验中，具有意想不到的优秀的肿瘤抑制效果，明显优于现有技术。
定义和说明
除非另有说明，本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。
这里所采用的术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
术语“药学上可接受的盐”是指本发明化合物的盐，由本发明发现的具有特定取代基的化合物与相对无毒的酸或碱制备。当本发明的化合物中含有相对酸性的功能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的碱与这类化合物接触的方式获得碱加成盐。药学上可接受的碱加成盐包括钠、钾、钙、铵、有机胺或镁盐或类似的盐。当本发明的化合物中含有相对碱性的官能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的酸与这类化合物接触的方式获得酸加成盐。药学上可接受的酸加成盐的实例包括无机酸盐，所述无机酸包括例如盐酸、氢溴酸、硝酸、碳酸，碳酸氢根，磷酸、磷酸一氢根、磷酸二氢根、硫酸、硫酸氢根、氢碘酸、亚磷酸等；以及有机酸盐，所述有机酸包括如乙酸、丙酸、异丁酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、酒石酸和甲磺酸等类似的酸；还包括氨基酸(如精氨酸等)的盐，以及如葡糖醛酸等有机酸的盐。本发明的某些特定的化合物含有碱性和酸性的官能团，从而可以被转换成任一碱或酸加成盐。
本发明的药学上可接受的盐可由含有酸根或碱基的母体化合物通过常规化学方法合成。一般情况下，这样的盐的制备方法是：在水或有机溶剂或两者的混合物中，经由游离酸或碱形式的这些化合物与化学计量的适当的碱或酸反应来制备。
本发明采用下述缩略词：aq代表水；HATU代表O-(7-氮杂苯并三唑-1-基)-N，N，N'，N'-四甲基 脲六氟磷酸盐；EDC代表N-(3-二甲基氨基丙基)-N'-乙基碳二亚胺盐酸盐；m-CPBA代表3-氯过氧苯甲酸；eq代表当量、等量；CDI代表羰基二咪唑；DCM代表二氯甲烷；PE代表石油醚；DIAD代表偶氮二羧酸二异丙酯；DMF代表N，N-二甲基甲酰胺；DMSO代表二甲亚砜；EtOAc代表乙酸乙酯；EtOH代表乙醇；MeOH代表甲醇；CBz代表苄氧羰基，是一种胺保护基团；BOC代表叔丁氧羰基是一种胺保护基团；HOAc代表乙酸；NaCNBH 3代表氰基硼氢化钠；r.t.代表室温；O/N代表过夜；THF代表四氢呋喃；Boc 2O代表二-叔丁基二碳酸酯；TFA代表三氟乙酸；DIPEA代表二异丙基乙基胺；SOCl 2代表氯化亚砜；CS 2代表二硫化碳；TsOH代表对甲苯磺酸；NFSI代表N-氟-N-(苯磺酰基)苯磺酰胺；NCS代表1-氯吡咯烷-2,5-二酮；n-Bu 4NF代表氟化四丁基铵；iPrOH代表2-丙醇；mp代表熔点；LDA代表二异丙基胺基锂。
化合物依据本领域常规命名原则或者使用
Figure PCTCN2019108522-appb-000003
软件命名，市售化合物采用供应商目录名称。
具体实施方式
下面通过实施例对本发明进行详细描述，但并不意味着对本发明任何不利限制。本发明的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。对本领域的技术人员而言，在不脱离本发明精神和范围的情况下针对本发明具体实施方式进行各种变化和改进将是显而易见的。
实施例1
Figure PCTCN2019108522-appb-000004
步骤A：将化合物1-1(30克，230.52毫摩尔，28.57毫升，1当量)加入到水(600毫升)中，再加入氢氧化钠(11.99克，299.67毫摩尔，1.3当量)，20摄氏度搅拌16小时。将体系温度降到0摄氏 度到5摄氏度之间，再缓慢加入亚硝酸钠(17.50克，253.57毫摩尔，1.1当量)的水(60毫升)溶液，将体系的pH用硫酸调节到4，再在20摄氏度搅拌12小时。该水相用乙酸乙酯(400毫升×2)萃取，合并有机相，用饱和食盐水(100毫升×2)洗涤，硫酸钠干燥，浓缩，得到化合物1-2。 1H NMR(400MHz,CDCl 3)δ＝8.83-8.54(m,1H),7.56(s,1H),2.80(q,J＝7.2Hz,2H),1.13(t,J＝7.2Hz,3H).
步骤B：将化合物1-2(20克，197.82毫摩尔，1当量)溶于异丙醇(400毫升)中，再加入化合物1-3(50克，197.41毫摩尔，0.998当量，对甲苯磺酸盐),该混合体系在20摄氏度搅拌16小时。将反应液倒入水(300毫升)中，用乙酸乙酯(500毫升×3)萃取，合并有机相，用饱和食盐水(800毫升)洗涤，硫酸钠干燥，浓缩，得到化合物1-4。MS(ESI)m/z:165.3[M+H +].
步骤C：将化合物1-4(31克，188.84毫摩尔，1当量)溶于N,N-二甲基甲酰胺(300毫升)，降温到0摄氏度，再缓慢滴加三氯氧磷(78.52克，512.09毫摩尔，47.59毫升，2.71当量),保持温度低于5摄氏度。滴加完后将体系加热到80摄氏度搅拌2小时。将反应液滴加到冰(900克)里，在自然升温到20摄氏度搅拌16小时。有固体析出，过滤，收集滤饼并真空干燥，得到化合物1-5。
步骤D：将亚硝酸叔丁酯(20.61克，199.88毫摩尔，23.77毫升，2.5当量)和溴化铜(21.43克，95.94毫摩尔，4.49毫升，1.2当量)溶于N,N-二甲基甲酰胺(200毫升)中，将体系加热到65摄氏度，再滴加化合物1-5(14.6克，79.95毫摩尔，1当量)的N,N-二甲基甲酰胺(150毫升)溶液。在65摄氏度反应0.5小时后，将反应液倒入冰水(1000克)中，将析出的固体过滤，滤饼溶于乙酸乙酯(300毫升)中，再过滤，将滤液浓缩得到化合物1-6。 1H NMR(400MHz,DMSO-d 6)δ＝2.92(q,J＝7.2Hz,2H),1.23(t,J＝7.2Hz,3H).
步骤E：将化合物1-6(4克，16.23毫摩尔，1当量)和化合物1-7(1.97克，14.31毫摩尔，0.882当量)溶于1,4二氧六环(50毫升)中，再加入N,N-二异丙基乙基胺(5.03克，38.95毫摩尔，6.78毫升，2.4当量)。将该混合物加热到65摄氏度搅拌12小时。将水(100毫升)倒入反应液中，并在20摄氏度搅拌0.5小时。将该混合物过滤，滤饼用水洗涤，真空干燥，得到化合物1-8。MS(ESI)m/z:310.9,312.9[M+H +].
Figure PCTCN2019108522-appb-000005
Figure PCTCN2019108522-appb-000006
步骤F：在5摄氏度到8摄氏度之间，将醋酸铵(2.04克，26.42毫摩尔，0.1当量)加入到化合物1-10(89.65克，792.59毫摩尔，84.58毫升，3当量)的甲醇(100毫升)溶液中，再加入化合物1-9(50克，264.20毫摩尔，49.02毫升，1当量)。然后，在10摄氏度以下，将氨水(51.85克，369.87毫摩尔，56.98毫升，25％，1.4当量)加入到该混合液中。该混合液在0摄氏度到5摄氏度之间搅拌1小时，然后反应混合物升温至20摄氏度，并搅拌20小时。将水(100毫升)加入到该体系中，加热到55摄氏度。用盐酸(12摩尔/升)调节pH到4，并保持温度不超过70摄氏度。之后将混合物冷却到10摄氏度，搅拌30分钟后过滤，滤饼用水洗涤并减压干燥，得到化合物1-11。MS(ESI)m/z:323.1[M+H +].
步骤G：向硫酸(161.92克，1.65摩尔，88毫升，10.65当量)和水(12.00克，666.10毫摩尔，12毫升，4.30当量)的混合液中加入化合物1-11(49.95克，154.95毫摩尔，1当量)，此时温度升至40摄氏度。再将该混合物加热到80摄氏度并搅拌2小时。然后加入水(20.00克，1.11摩尔，20毫升，7.16当量)，再加热到100摄氏度搅拌1.5小时。向该反应液中加入水(250毫升)并在30摄氏度搅拌12小时，然后过滤，滤饼用水洗涤，减压干燥得到化合物1-12。MS(ESI)m/z:342.0[M+H +].
步骤H：向氢氧化钠水溶液(5摩尔/升，183.45毫升，8当量)中加入化合物1-12(39.14克，114.66毫摩尔，1当量)，将该混合液加热到80摄氏度并搅拌2小时。冷却该体系温度到60摄氏度，缓慢加入盐酸(12摩尔/升，75毫升，7.85当量)，将温度加热到75摄氏度并滴加盐酸(12摩尔/升，15毫升，1.57当量)。再将该混合物加热到85摄氏度搅拌1小时，然后冷却至25摄氏度，搅拌16小时。向反应液中加入水(200毫升)，冷却到10摄氏度，过滤，滤饼用水(300毫升)洗涤，减压干燥得到化合物1-13。MS(ESI)m/z:273.1[M+H +].
步骤I：将化合物1-13(28克，102.81毫摩尔，1当量)溶于四氢呋喃(300毫升),将该混合物加热到70摄氏度，然后将四氢铝锂(15.61克，411.25毫摩尔，4当量)分批加入到该溶液中。该混合物在70摄氏度搅拌12小时。冷却到室温后，将饱和硫酸钠溶液(30毫升)缓慢滴加到反应液中，然后过滤，用乙酸乙酯(100毫升)洗涤滤饼。将滤液合并并浓缩得到化合物1-14。MS(ESI)m/z:245.1[M+H +].
步骤J：将化合物1-14(0.5克，2.05毫摩尔，1当量)和化合物1-15(317.39毫克，2.05毫摩尔， 1当量)溶于N,N-二甲基甲酰胺中(10毫升)，加入碳酸钾(565.55毫克，4.09毫摩尔，2当量)。将该混合物加热到80摄氏度并搅拌12小时。将反应液倒入水(60毫升)中，用乙酸乙酯(60毫升×2)萃取，合并有机相，用饱和食盐水(60毫升)洗涤，干燥后浓缩，得到化合物1-16。MS(ESI)m/z:380.0[M+H +].
步骤K：向化合物1-16(550毫克，1.45毫摩尔，1当量)的二氯甲烷(15毫升)溶液中加入碘甲烷(246.85毫克，1.74毫摩尔，108.27微升，1.2当量)，该混合物在25摄氏度搅拌12小时。将反应液浓缩得到化合物1-17。MS(ESI)m/z:394.1[M+H +].
步骤L：向化合物1-17(620毫克，1.19毫摩尔，1当量)的乙醇(20毫升)溶液中加入湿钯碳(100毫克，10％)，用氢气置换后，将该混合物加热到60摄氏度，在氢气压力为50磅每平方英寸的条件下反应12小时。然后过滤，将滤液浓缩得到化合物1-18。MS(ESI)m/z:274.1[M+H +].
步骤M：向化合物1-18(300毫克，1.10毫摩尔，1当量)和化合物1-8(341.43毫克，1.10毫摩尔，1当量)的1,4-二氧六环(10毫升)的溶液中加入醋酸钯(24.63毫克，109.72微摩尔，0.1当量)，4,5-双(二苯基磷)-9,9-二甲基氧杂蒽(63.49毫克，109.72微摩尔，0.1当量)和碳酸钾(303.29毫克，2.19毫摩尔，2当量)。该体系用氮气置换，然后加热到80摄氏度，在氮气氛围下搅拌12小时。将反应液过滤，滤饼用乙酸乙酯(60毫升)洗涤，滤液浓缩得到的粗品分离纯化，得到化合物1-19。MS(ESI)m/z:504.2[M+H +].
步骤N：将化合物1-19(200毫克，397.08微摩尔，1当量)溶于二甲基亚砜(2毫升)和乙醇(6毫升)中，冷却该体系到0摄氏度，再加入氢氧化钠(4摩尔每升，297.81微升，3当量)和双氧水(135.06毫克，1.19毫摩尔，114.46微升，纯度30％，3当量)。该反应液自然升温到25摄氏度搅拌12小时。
方法1：将反应液倒入水(30毫升)中，再用乙酸乙酯(40毫升×3)萃取，合并有机相，用饱和食盐水(40毫升)洗涤，硫酸钠干燥，浓缩得到的粗品，分离纯化(制备高效液相色谱，色谱柱:PhenomenexSynergi C18 150*25*10微米；流动相:[水(0.1％三氟乙酸)-乙腈]；乙腈％:10％-37％,10分钟)得到式(I)化合物的三氟乙酸盐。 1H NMR(400MHz,DMSO-d 6)δ＝11.13(s,1H),9.23(br s,1H),7.61-7.40(m,3H),7.28-7.11(m,2H),6.85(d,J＝7.2Hz,1H),4.18-4.06(m,1H),3.99-3.91(m,2H),3.41-3.37(m,2H),3.30-3.27(m,2H),3.13-2.91(m,6H),2.79(d,J＝4.4Hz,3H),2.62-2.55(m,2H),2.33-2.28(m,3H),2.03-1.78(m,6H),1.73-1.44(m,6H),1.19(t,J＝7.2Hz,3H).MS(ESI)m/z:522.0[M+H +].
方法2：将水(20毫升)加入到反应液中，搅拌30:分钟后，过滤，滤饼用水(10毫升)洗涤，将滤饼用乙醇(5毫升)打浆，过滤，减压干燥得到式(I)化合物。 1H NMR(400MHz,DMSO-d 6)δ＝11.01(s,1H),7.52(d,J＝2.8Hz,1H),7.43(d,J＝2.4Hz,1H),7.36(dd,J＝8.8Hz,2.4Hz,1H),7.20(d,J＝2.4Hz,1H),6.99(d,J＝8.8Hz,1H),6.79(d,J＝7.6Hz,1H),4.15-4.06(m,1H),3.95-3.92(m,2H),3.42-3.39(m,2H),2.74-2.71(m,4H),2.56(q,J＝7.6Hz,2H),2.28-2.25(m,4H),2.22(s,3H),2.14(s,3H),1.88-1.84(m,2H),1.69-1.45(m,10H),1.18(t,J＝7.2Hz,3H).MS(ESI)m/z:522.3[M+H +].
实验例1：FLT3体外抑制活性实验
实验材料：
FLT3 Kinase Enzyme System(激酶系统)购自Promega。Envision多标记分析仪(PerkinElmer)。
实验方法：
使用试剂盒里的缓冲溶液稀释酶，底物，ATP(腺苷酸三磷酸)和抑制剂。
将待测化合物用排枪进行5倍稀释至第8个浓度，即从5微摩尔每升稀释至0.065纳摩尔每升，二甲亚砜终浓度为5％，设置双复孔实验。向微孔板中加入1微升抑制剂各浓度梯度，2微升FLT3酶(15纳克)，2微升底物和ATP的混合物(50微摩尔每升ATP，0.1微克每微升MBP)，此时化合物终浓度梯度为1微摩尔每升稀释至0.013纳摩尔每升。反应体系置于30摄氏度度反应120分钟。反应结束后，每孔加入5微升ADP-Glo试剂，30摄氏度继续反应40分钟，结束反应后每孔加入10微升的激酶检测试剂，30摄氏度反应30分钟后采用PerkinElmer Envision多标记分析仪读数化学发光，积分时间0.5秒。
数据分析：
原始数据换算成抑制率，IC 50的值可通过四参数进行曲线拟合得出。表1提供了本发明的化合物对FLT3酶学抑制活性。
实验结果：见表1。
结论：本发明化合物对FLT3具有优异的体外抑制活性。
表1
样品 FLT3 IC 50(纳摩尔每升)
化合物A的三氟乙酸盐 4.02
化合物B 0.81
式(I)化合物的三氟乙酸盐 0.42
实验例2：AXL体外抑制活性实验
实验材料：
AXL Kinase Enzyme System(激酶系统)购自Promega。Envision多标记分析仪(PerkinElmer)。
实验方法：
使用试剂盒里的缓冲溶液稀释酶，底物，ATP和抑制剂。
将待测化合物用排枪进行5倍稀释至第8个浓度，即从5微摩尔每升稀释至0.065纳摩尔每升，二甲亚砜终浓度为5％，设置双复孔实验。向微孔板中加入1微升抑制剂各浓度梯度，2微升AXL酶(6纳克)，2微升底物和ATP的混合物(50微摩尔每升ATP，0.2微克每微升Axltide)，此时化合物终浓度梯度为1微摩尔每升稀释至0.013纳摩尔每升。反应体系置于30摄氏度度反应60分钟。反应结束后，每孔加入5微升ADP-Glo试剂，30摄氏度继续反应40分钟，结束反应后每孔加入10微升的激酶检测试剂，30摄氏度反应30分钟后采用PerkinElmer Envision多标记分析仪读数化学发光，积分时间0.5秒。
数据分析：
原始数据换算成抑制率，IC 50的值可通过四参数进行曲线拟合得出。表2提供了本发明的化合物对AXL酶学抑制活性。
实验结果：见表2。
结论：本发明化合物对AXL具有优异的体外抑制活性。
表2
样品 AXL IC 50(纳摩尔每升)
化合物A的三氟乙酸盐 5.76
化合物B 1.37
式(I)化合物的三氟乙酸盐 1.22
实验例3：FLT3突变体外抑制增殖实验
实验方法：
使用KINOMEscan TM技术进行测试。实验化合物保存在100％DMSO中。通过3倍稀释，取11个点拟合的方式进行测试。所有用于Kd测量的化合物都通过超声分散，然后这些化合物被直接稀释并进行实验。所有反应都在聚丙烯384孔板中进行。每一份最终体积为0.02毫升，在室温下摇匀孵育1小时，处理，最后用qPCR法测定洗脱液中激酶浓度，拟合得到Kd。
实验结果：见表3。
表3
Figure PCTCN2019108522-appb-000007
结论：本发明化合物对突变的FLT3靶点具有优异的体外抑制活性。在所有10个突变中均表现出比已知化合物B更高的活性，其中FLT3(ITD,F691L)活性高出3.6倍，FLT3(K663Q)活性高出5.9倍。考虑到点突变是FLT3抑制剂耐药的重要原因，对突变型FLT3更高的活性在临床上具有极高的意义。
实验例4：MV-4-11体外抑制增殖实验
实验材料：
IMDM培养基，胎牛血清，盘尼西林/链霉素抗生素购自Promega(Madison,WI)。MV-4-11细胞系购自中国科学院细胞库。Envision多标记分析仪(PerkinElmer)。
实验方法：
将MV-4-11细胞种于白色96孔板中，80微升细胞悬液每孔，其中包含10000个MV-4-11细胞。 细胞板置于二氧化碳培养箱中过夜培养。
将待测化合物用排枪进行5倍稀释至第8个浓度，即从2毫摩尔每升稀释至26纳摩尔每升，设置双复孔实验。向中间板中加入78微升培养基，再按照对应位置，转移2微升每孔的梯度稀释化合物至中间板，混匀后转移20微升每孔到细胞板中。细胞板置于二氧化碳培养箱中培养3天。
向细胞板中加入每孔25微升的Promega CellTiter-Glo试剂，室温孵育10分钟使发光信号稳定。采用PerkinElmer Envision多标记分析仪读数。
数据分析：
原始数据换算成抑制率，IC 50的值可通过四参数进行曲线拟合得出。表3提供了本发明的化合物对MV-4-11细胞增殖的抑制活性。
实验结果：见表4。
结论：本发明化合物对MV-4-11细胞增殖有优异的抑制活性。
表4
样品 MV-4-11 IC 50(纳摩尔每升)
化合物A 5.4
化合物B 4.65
式(I)化合物的三氟乙酸盐 3.02
实验例5：小鼠体内药代动力学研究
实验目的：
本实验目的是评价化合物单次静脉注射和灌胃给药后的药代动力学行为，考察灌胃给药后的生物利用度。
实验操作：
选取7至10周龄的CD-1雄性小鼠，静脉和口服给药的剂量分别为1毫克每公斤和2.5毫克每公斤。小鼠在给药前禁食至少12小时，给药4小时后恢复供食，整个试验期间自由饮水。
实验当天静脉组动物通过尾静脉单次注射给予相应化合物，给药体积为5mL/kg；口服组和通过单次灌胃给予相应化合物，给药体积为10mL/kg。在给药前称量动物体重，根据体重计算给药体积。样品采集时间为：0.083(注射组)，0.25,0.5,1,2,4,8,24h。每个时间点通过隐静脉采集大约30μL全血用于制备血浆供高效液相色谱-串联质谱(LC-MS/MS)进行浓度测定。所有动物在采集完最后一个时间点的PK样品后进行CO 2麻醉安乐死。采用WinNonlin TM Version 6.3(Pharsight,Mountain View,CA)药动学软件的非房室模型处理血浆浓度，使用线性对数梯形法方法计算药动学参数。
实验结果：小鼠体内PK性质评价结果见表5。
实验结论：
本发明化合物在小鼠体内清除率适当，口服AUC、生物利用度较好，具有良好的药代动力学性质。与化合物A相比有意想不到的PK性质改善。
表5体内药代动力学性质评价结果
Figure PCTCN2019108522-appb-000008
Figure PCTCN2019108522-appb-000009
实验例6：MV4-11皮下异种移植肿瘤抑制体内实验
实验目的：
本试验使用人双表型B骨髓单核白血病细胞MV4-11皮下异种移植肿瘤裸小鼠模型评价化合物的抗肿瘤作用。
实验操作：
人双表型B骨髓单核白血病细胞MV4-11体外悬浮培养，其体外培养条件为RPMI1640培养基中加10％胎牛血清，100U/mL青霉素和100μg/mL链霉素，温度为37摄氏度，在5％CO2细胞培养箱中培养。一周两次进行常规传代，收取对数生长期内的细胞，计数后用于接种。
在每只小鼠的右侧颈背部皮下接种10×106个MV4-11细胞，接种体积为0.2mL，细胞悬液为PBS加基质胶(体积比为1:1)。体内药效实验在细胞接种后第13天，肿瘤平均体积达到181mm3，采用随机分组法分组并开始给药，每组6只小鼠。
每周两次用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5a×b2，a和b分别表示肿瘤的长径和短径。
给药14天后，开始停止给药，观察肿瘤的反弹情况。
实验结果：化合物肿瘤抑制效果见表6。
表6 MV4-11异种异位移植实验结果
Figure PCTCN2019108522-appb-000010
实验结论：
本发明化合物对人双表型B骨髓单核白血病细胞MV4-11异种移植瘤的生长有显著抑制作用。在低剂量(5毫克每公斤)下即表现出比化合物A高剂量(20毫克每公斤)等同的肿瘤抑制效果。停药-反弹实验中，同剂量(5毫克每公斤)下比化合物B具有更明显的持续抑瘤效果。
实验例7：大鼠体内药代动力学研究
实验目的：
本实验目的是评价化合物单次静脉注射和灌胃给药后的药代动力学行为，考察灌胃给药后的生物利用度。
实验操作：
选取7至10周龄的SD雄性大鼠，静脉和口服给药的剂量分别为1毫克每公斤和2.5毫克每公斤。大鼠在给药前禁食至少12小时，给药4小时后恢复供食，整个试验期间自由饮水。
实验当天静脉组动物通过尾静脉单次注射给予相应化合物，给药体积为5mL/kg；口服组和通过单次灌胃给予相应化合物，给药体积为10mL/kg。在给药前称量动物体重，根据体重计算给药体积。样品采集时间为：0.083(注射组)，0.25,0.5,1,2,4,6,8,24h。每个时间点通过颈静脉采集大约200μL全血用于制备血浆供高效液相色谱-串联质谱(LC-MS/MS)进行浓度测定。所有动物在采集完最后一个时间点的PK样品后进行CO 2麻醉安乐死。采用WinNonlin TM Version 6.3(Pharsight,Mountain View,CA)药动学软件的非房室模型处理血浆浓度，使用线性对数梯形法方法计算药动学参数。
实验结果：大鼠体内PK性质评价结果见表7。
表7体内药代动力学性质评价结果
Figure PCTCN2019108522-appb-000011
实验结论：
本发明化合物在大鼠体内口服AUC、生物利用度优异，具有良好的药代动力学性质。与化合物B相比有意想不到的PK性质改善。
实验例8：Molm-13皮下异种移植肿瘤抑制体内实验
实验目的：
化合物在人急性骨髓瘤MOLM-13细胞株皮下异种移植NOD/SCID雌性鼠模型中的药效学评价。
实验操作：
MOLM-13细胞培养在含10％胎牛血清的RPMI-1640培养液中。收集指数生长期的MOLM-13细胞，PBS重悬至适合浓度用于裸鼠皮下肿瘤接种。
实验小鼠于右侧背部皮下接种5×10 6 MOLM-13细胞，细胞重悬在0.1ml PBS中(0.1ml/只)定期观察肿瘤生长情况，待肿瘤生长至平均体积98mm 3时根据肿瘤大小和小鼠体重随机分组给药。
开始给药后，每周测量三次小鼠的体重和肿瘤的大小。肿瘤体积计算公式：肿瘤体积(mm 3)＝1/2×(a×b 2)(其中a表示长径，b表示短径)。
实验结果：化合物肿瘤抑制效果见表8。
表8 Molm-13异种异位移植实验结果
Figure PCTCN2019108522-appb-000012
实验结论：
本发明化合物对人源Molm-13异种移植瘤的生长有显著抑制作用。在同等剂量(15毫克每公斤)下表现出比化合物B更优的肿瘤抑制效果。剂量为50毫克/公斤时肿瘤体积缩小至0。

Claims (3)

  1. 式(I)所示化合物或其药学上可接受的盐，
    Figure PCTCN2019108522-appb-100001
  2. 式(I)所示化合物或其药学上可接受的盐在制备治疗癌症药物中的应用。
  3. 根据权利要求2所述的应用，其中，癌症是指急性髓性白血病。
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