CN110407812B - Indazole piperidine pyrimidine derivative and preparation method and application thereof - Google Patents
Indazole piperidine pyrimidine derivative and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of medicines, and particularly relates to an indazole piperidine pyrimidine derivative, and a preparation method and application thereof. The indazole piperidine pyrimidine derivative provided by the invention replaces an acrylamide structure forming a covalent bond on the basis of keeping a third-generation EGFR inhibitor pyrimidine parent ring, so that a non-covalent bond combined inhibitor is obtained. The results of the drug activity tests show that the compounds show excellent in-vitro inhibition effect on human lung cancer H1975 cells. Meanwhile, most compounds have less toxicity to normal human cells than lung cancer cells, show better selectivity and can be used for further developing anti-cancer drugs.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to an indazole piperidine pyrimidine derivative, and a preparation method and application thereof.
Background
Human cancer cells are often associated with overexpression of growth factors and their receptors. The Epidermal Growth Factor Receptor (EGFR) family is also known as the type i tyrosine kinase receptor or ErbB tyrosine kinase receptor. This receptor family consists of four classes of homologous receptors: epidermal growth factor receptor (ErbB1/EGFr/HER1), ErbB2(HER2), ErbB3(HER3), and ErbB4(HER 4).
Over-expression of the epidermal growth factor receptor exists in more than 80% of patients with non-small cell lung cancer, so that the receptor protein becomes an important research target for resisting small cell lung cancer. EGFR inhibitors can be broadly divided into two broad categories: 1) non-covalently bound epidermal growth factor receptor inhibitors such as gefitinib and erlotinib; 2) covalently bound epidermal growth factor receptor inhibitors, such as norcetin and oxitinib. The EGFR inhibitor combined by non-covalent bonds can produce better treatment effect in the process of early taking, but the binding force of the medicines with receptors in vivo is not very strong, and the T790M drug-resistant strain is produced after long-term taking, so that the treatment effect is greatly reduced. The EGFR inhibitor combined by covalent bonds can generate Michael addition reaction with the amino acid Cys797 to form covalent bonds with strong acting force, thereby having better activity on various variants including T790M. However, the irreversible inhibitor has strong binding with receptors in vivo, so that the medicine stays in vivo for too long, and most of the compounds have large toxic and side effects, such as rash, diarrhea and other serious side effects. Therefore, a new high-efficiency low-toxicity small-molecule compound is urgently needed to be found as a candidate drug for treating the non-small cell lung cancer.
Most of the first-generation EGFR inhibitors are quinazoline parent ring structures, which show good treatment effects in early clinical application, but the drug resistance caused by mutation greatly reduces the curative effect of the compounds. The parent ring of the second generation EGFR inhibitor still retains the quinazoline structure, and the strong covalent interaction is formed between the parent ring and the amino acid residue through the introduced unsaturated bond, so that the drug resistance caused by mutation is reduced, but most of the compounds have large toxic and side effects. On the basis of keeping unsaturated bonds, the third-generation EGFR inhibitor uses a pyrimidine ring to replace quinazoline to obtain a new inhibitor, and also shows good properties in clinical research, but most of the compounds still have large toxic and side effects.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides an indazole piperidine pyrimidine derivative, and a preparation method and application thereof.
The technical scheme provided by the invention is as follows:
an indazole piperidine pyrimidine derivative, which is a compound shown as a formula (I):
wherein R is positioned at ortho position, meta position or para position of the ether group, and R is mono-substituted or di-substituted; r is methyl, trifluoromethyl, halogen or cyano.
The derivative provided by the technical scheme replaces an acrylamide structure forming a covalent bond on the basis of keeping a third-generation EGFR inhibitor pyrimidine parent ring, so that a non-covalent bond combined inhibitor is obtained. The results of the drug activity tests show that the compounds show excellent in-vitro inhibition effect on human lung cancer H1975 cells. Meanwhile, most compounds have less toxicity to normal human cells than lung cancer cells, show better selectivity and can be used for further developing anti-cancer drugs.
The invention also provides a preparation method of the indazole piperidine pyrimidine derivative, which comprises the following steps:
1) dissolving raw material intermediates I-a and 1- (tert-butyloxycarbonyl) -3- (bromomethyl) -indazole I-b in a first solvent, stirring until all raw materials are fully dissolved, adding alkali, stirring at room temperature until all raw materials are completely consumed, and separating and purifying to obtain an intermediate I-c;
2) suspending or dissolving the obtained intermediate I-c in a second solvent, slowly dropwise adding acid while stirring in an ice bath to remove a tert-butoxycarbonyl protecting group, removing the ice bath after dropwise adding is finished, stirring at normal temperature until the raw materials react completely, adjusting the pH of a reaction solution to 6.0-7.5 by using an alkali solution, and separating and purifying to obtain a target product I;
the reaction formula is as follows:
wherein R is positioned at ortho position, meta position or para position of the ether group, and R is mono-substituted or di-substituted; r is methyl, trifluoromethyl, halogen or cyano.
The compound of formula (I) provided by the invention can be prepared by the technical scheme.
Specifically, the first solvent in step 1) is a mixed solvent composed of any one or more of dichloromethane, chloroform, 1, 4-dioxane, N-dimethylformamide, dimethyl sulfoxide or N-methylpyrrolidone.
Specifically, the second solvent in step 2) is a mixed solvent composed of any one or more of dichloromethane, chloroform, carbon tetrachloride, 1, 2-dichloroethane, 1, 4-dioxane, tetrahydrofuran, N-dimethylformamide, and dimethyl sulfoxide.
Specifically, the alkali is any one or a mixture of more of potassium carbonate, sodium carbonate, cesium carbonate, N-diisopropylethylamine, sodium hydroxide, potassium hydroxide or sodium hydrogen.
Specifically, the acid in the step 2) is one or a mixture of trifluoroacetic acid and concentrated hydrochloric acid.
The invention also provides application of the indazole piperidine pyrimidine derivative in preventing or treating cancer.
The test result of the drug activity shows that the compound has good in-vitro inhibition effect on various cancer cells, particularly has excellent in-vitro inhibition effect on human lung cancer H1975 cells, and the activity of the compounds is stronger than that of a reference drug gefitinib. Meanwhile, the toxicity of a plurality of compounds to human normal cells is less than that of cancer cells such as lung cancer, and the like, so that the compounds show better selectivity and can be used for preventing or treating cancers.
Detailed Description
The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.
Example 1: synthesis of intermediate I-c 1
I-a 1(1mmol) and I-b (1mmol) were weighed into 1, 4-dioxane (10mL) and K was added with stirring at room temperature2CO3Stirring at room temperature for 4h, detecting by thin-layer chromatography to show that two raw material points basically completely react, stopping stirring, pouring the reaction liquid into 50mL of water, extracting by using ethyl acetate (2 × 50mL), washing by using saturated saline solution (2 × 40mL), drying by using organic phase anhydrous sodium sulfate, performing suction filtration, performing spin-drying on the filtrate to obtain oily matter, and performing column chromatography purification to obtain I-c 1 with the yield of 35.2%.
Example 2: synthesis of intermediate I-c 1
I-a 1(1mmol) and I-b (1mmol) were weighed into N-methylpyrrolidone (10mL), DIEA was added with stirring at room temperature and stirred at room temperature for 4 h. Thin layer chromatography detection showed that the two material spots were essentially completely reacted and the stirring was stopped. The reaction mixture was poured into 50mL of water, extracted with ethyl acetate (2X 50mL), washed with saturated brine (2X 50mL), dried over anhydrous sodium sulfate as the organic phase, filtered under suction, and the filtrate was spin-dried to give an oil, which was purified by column chromatography to give I-c 1 in 42.3% yield.
Example 3: synthesis of intermediate I-c 2
I-a 2(1mmol) and I-b (1mmol) were weighed into DCM (10mL), and sodium bicarbonate was added with stirring at RT and stirred at RT for 3 h. Thin layer chromatography detection showed that the two material spots were essentially completely reacted and the stirring was stopped. The reaction mixture was poured into 50mL of water, extracted with DCM (2X 50mL), washed with saturated brine (2X 50mL), dried over anhydrous sodium sulfate as the organic phase, filtered under suction, and the filtrate was spin-dried to give an oil, which was purified by column chromatography to give I-c 2 in 34.4% yield.
Example 4: synthesis of intermediate I-c 2
I-a 2(1mmol) and I-b (1mmol) were weighed into chloroform (10mL), and sodium bicarbonate was added with stirring at room temperature and stirred at room temperature for 3 h. Thin layer chromatography detection showed that the two material spots were essentially completely reacted and the stirring was stopped. The reaction mixture was poured into 50mL of water, extracted with DCM (2X 50mL), washed with saturated brine (2X 50mL), dried over anhydrous sodium sulfate as the organic phase, filtered under suction, and the filtrate was spin-dried to give an oil, which was purified by column chromatography to give I-c 2 in 36.0% yield.
Example 5: synthesis of intermediate I-c 3
I-a 3(1mmol) and I-b (1mmol) were weighed into N, N-dimethylformamide (10mL), sodium hydrogen was added slowly with stirring in an ice bath and stirred for 3h in an ice bath. Thin layer chromatography detection showed that the two material spots were essentially completely reacted and the stirring was stopped. The reaction mixture was poured into 50mL of water, extracted with ethyl acetate (2X 50mL), washed with saturated brine (2X 50mL), dried over anhydrous sodium sulfate as the organic phase, filtered under suction, and the filtrate was spin-dried to give an oil, which was purified by column chromatography to give I-c 3 in 40.2% yield.
Example 6: synthesis of intermediate I-c 4
I-a 4(1mmol) and I-b (1mmol) were weighed into dimethyl sulfoxide (10mL), and cesium carbonate was added with stirring at room temperature for 4 h. Thin layer chromatography detection showed that the two material spots were essentially completely reacted and the stirring was stopped. Pouring the reaction solution into 50mL of water, extracting with ethyl acetate (2X 50mL), washing with saturated saline (2X 50mL), drying with anhydrous sodium sulfate, filtering, spin-drying the filtrate to obtain oily substance, and purifying by column chromatography to obtain I-c 4 with yield of 44.2%
Example 7: synthesis of intermediate I-c 5
I-a 5(1mmol) and I-b (1mmol) were weighed into N-methylpyrrolidone (10mL), sodium carbonate was added with stirring at room temperature, and stirring was carried out at room temperature for 4 h. Thin layer chromatography detection showed that the two material spots were essentially completely reacted and the stirring was stopped. Pouring the reaction solution into 50mL of water, extracting with ethyl acetate (2X 50mL), washing with saturated saline (2X 50mL), drying with anhydrous sodium sulfate, filtering, spin-drying the filtrate to obtain oily substance, and purifying by column chromatography to obtain I-c 5 with yield of 36.9%
Example 8: synthesis of target Compound I-1
I-c 1(0.8mmol) was weighed out and suspended in dichloromethane (10mL) and added slowly with stirring in an ice bath to the solutionRemoving the ice bath with fluoroacetic acid (16.1mmol) for 0.5h, stirring at normal temperature for 3h, detecting with thin layer chromatography to show that the reaction at the raw material point is complete, stopping stirring, slowly adding saturated sodium bicarbonate solution into the reaction solution under the ice bath until no bubbles are generated, adding 60mL of dichloromethane solution for extraction and liquid separation, washing the organic phase with saturated saline solution (2 × 50mL), drying with anhydrous sodium sulfate, performing suction filtration, spin-drying the filtrate to obtain a crude product, pulping the crude product with n-hexane/dichloromethane mixed solvent (6:1, volume ratio), performing suction filtration, and performing vacuum drying on the filter cake to obtain a white powdery solid I-1, wherein the yield is 53.3%, m p 182.9-187.3 ℃;1H NMR(400MHz,DMSO-d6):(ppm)12.80(s,1H,IndNH),8.16(s,1H,PyH),7.84-7.07(m,8H,ArH),6.19-6.18(s,1H,PyH)，3.79-3.70(m,3H,-CH2-and PipH),2.84-2.79(m,2H,PipH),2.07-1.34(m,6H,PipH)；13C NMR(200MHz,DMSO-d6):168.55,161.63,160.27,159.84,148.25,140.87,130.32,129.90,128.56,128.20,126.99,125.86,124.54,122.15,120.60,119.72,110.03,95.82,54.91,54.41,52.09,31.04；HRMS(ESI),C23H23ClN6O,[M+H]+the theoretical calculation is 434.1622, and the actual measurement is 435.1698.
Example 9: synthesis of target Compound I-1
I-c 1(0.8mmol) is weighed and suspended in chloroform (10mL), trifluoroacetic acid (16.1mmol) is slowly added into the reaction solution under the stirring of an ice bath, the ice bath is removed after 0.5h, the reaction solution is stirred for 3h at normal temperature, the thin-layer chromatography detection shows that the raw material point reaction is complete, the stirring is stopped, saturated sodium bicarbonate solution is slowly added into the reaction solution under the ice bath until no bubble is generated, 60mL dichloromethane solution is added for extraction and liquid separation, the organic phase is washed by saturated saline (2 × 50mL), dried by anhydrous sodium sulfate, filtered, the filtrate is dried by spinning to obtain a crude product, the crude product is pulped by n-hexane/dichloromethane mixed solvent (6:1, volume ratio), filtered, the filter cake is dried in vacuum to obtain white powdery solid I-1, the yield of the white solid is 55.2%, m p 183.4,1H NMR(400MHz,DMSO-d6):(ppm)12.80(s,1H,IndNH),8.16(s,1H,PyH),7.84-7.07(m,8H,ArH),6.19-6.18(s,1H,PyH)，3.79-3.70(m,3H,-CH2-and PipH),2.84-2.79(m,2H,PipH),2.07-1.34(m,6H,PipH)；13C NMR(200MHz,DMSO-d6):168.55,161.63,160.27,159.84,148.25,140.87,130.32,129.90,128.56,128.20,126.99,125.86,124.54,122.15,120.60,119.72,110.03,95.82,54.91,54.41,52.09,31.04.
example 10: synthesis of target Compound I-2
I-c 2(0.8mmol) is weighed and suspended in carbon tetrachloride (10mL), trifluoroacetic acid (16.1mmol) is slowly added into the reaction solution under the stirring of an ice bath, the ice bath is removed after 0.5h, the reaction solution is stirred for 3h at normal temperature, the thin layer chromatography detection shows that the raw material point reaction is complete, the stirring is stopped, saturated sodium bicarbonate solution is slowly added into the reaction solution under the ice bath until no bubble is generated, 60mL dichloromethane solution is added for extraction and liquid separation, the organic phase is washed by saturated saline (2 × 50mL), dried by anhydrous sodium sulfate, filtered, the filtrate is dried by spinning to obtain a crude product, the crude product is pulped by n-hexane/dichloromethane mixed solvent (6:1, volume ratio), filtered, the filter cake is dried in vacuum to obtain white powdery solid I-2, the yield is 51.9%, m p 194.3.3-196..1H NMR(400MHz,DMSO-d6):(ppm)12.80(s,1H,IndNH),8.14(s,1H,PyH).7.84-7.05(m,8H,ArH),6.10-6.15(d,1H,PyH),3.77(m,3H,-CH2-and PipH),2.81(s,2H,PipH),2.03-1.40(m,6H,PipH)；13C NMR(150MHz,DMSO-d6):169.57,161.65,159.56,150.12,140.88,134.37,130.03,126.10,122.39,121.52,120.45,120.20,110.28,96.39,53.03,46.88,51.51,30.06,20.40；HRMS(ESI),C23H23ClN6O,[M+H]+The theoretical calculation is 434.1622, and the actual measurement is 435.1715.
Example 11: synthesis of target Compound I-2
Weighing I-c 2(0.8mmol) and suspending in 1, 2-dichloroethane (10mL), slowly adding trifluoroacetic acid (16.1mmol) under stirring in an ice bath, removing the ice bath for 0.5h, stirring at normal temperature for 3h, detecting by thin layer chromatography to show that the raw material point reaction is complete, stopping stirring, slowly adding saturated sodium bicarbonate solution into the reaction solution under ice bath until no bubble is generated, adding 60mL dichloromethane solution for extraction and liquid separation, washing the organic phase with saturated saline (2 × 50mL), drying with anhydrous sodium sulfate, performing suction filtration, and spin-drying the filtrate to obtain a crude product, and using n-hexane to obtain the crude productPulping the mixed solvent of alkane and dichloromethane (6:1, volume ratio), filtering, and drying the filter cake in vacuum to obtain white powdery solid I-2, 47.4%. m p 195.1-196.9 ℃.1H NMR(400MHz,DMSO-d6):(ppm)12.80(s,1H,IndNH),8.14(s,1H,PyH).7.84-7.05(m,8H,ArH),6.10-6.15(d,1H,PyH),3.77(m,3H,-CH2-andPipH),2.81(s,2H,PipH),2.03-1.40(m,6H,PipH)；13C NMR(150MHz,DMSO-d6):169.57,161.65,159.56,150.12,140.88,134.37,130.03,126.10,122.39,121.52,120.45,120.20,110.28,96.39,53.03,46.88,51.51,30.06,20.40.
Example 12: synthesis of target Compound I-3
Weighing I-c 3(0.8mmol) and suspending in 1, 4-dioxane (10mL), slowly adding concentrated hydrochloric acid (16.1mmol) under stirring in an ice bath, removing the ice bath for 0.5h, stirring at normal temperature for 3h, detecting by thin layer chromatography to show that the raw material point reaction is complete, stopping stirring, slowly adding saturated sodium bicarbonate solution into the reaction solution under ice bath until no bubble is generated, adding 60mL ethyl acetate to dilute the reaction solution, washing with water (2 × 50mL), separating, drying the organic phase with anhydrous sodium sulfate, performing suction filtration, spin-drying the filtrate to obtain a crude product, pulping the crude product with a mixed solvent of n-hexane and dichloromethane (6:1, volume ratio), performing suction filtration, and performing vacuum drying on the filter cake to obtain a white powdery solid I-3, 65.6%, m p 179.2.2-173.6 ℃;1H NMR(400MHz,DMSO-d6):(ppm)13.65(s,1H,IndNH),10.58-10.45(d,1H,-NH-),8.24-8.23(d,1H,PyH),8.02-7.19(m,8H,ArH),6.34-6.33(d,1H,PyH),4.65(s,2H,-CH2-),4.06-3.90(m,1H,PipH),3.53(s,2H,PipH),3.24-2.94(m,2H,PipH),2.08-1.38(m,6H,PipH)；13C NMR(200MHz,DMSO-d6):(ppm),168.76,161.10,159.53,158.30,155.30,140.73,134.42,126.84,126.37,125.70,124.68,122.49,120.87,119.94,115.62,110.46,97.14,54.76,50.66,46.48,27.04；HRMS(ESI),C24H23F3N6O,[M+H]+the theoretical calculation is 468.1885, and the actual measurement is 469.1983.
Example 13: synthesis of target Compound I-4
Weighing I-c 4(0.8mmol) and suspending in tetrahydrofuran (10mL), slowly adding concentrated hydrochloric acid (16.1mmol) under the stirring of an ice bath, removing the ice bath for 0.5h, stirring at normal temperature for 3h, detecting by thin-layer chromatography to show that the raw material point reaction is complete, stopping stirring, slowly adding a saturated sodium bicarbonate solution into the reaction solution under the ice bath until no bubbles are generated, adding 60mL of ethyl acetate to dilute the reaction solution, washing with water (2 × 50mL), separating, drying the organic phase with anhydrous sodium sulfate, performing suction filtration, spin-drying the filtrate to obtain a crude product, pulping the crude product with a n-hexane/dichloromethane mixed solvent (6:1, volume ratio), performing suction filtration, and performing vacuum drying on the filter cake to obtain a white powdery solid I-4, wherein the yield is 53.9%, m p 202.2.2-205.1 ℃;1H NMR(400MHz,DMSO-d6):(ppm)12.78(s,1H,IndNH),8.19(s,1H,PyH),7.91-7.06(m,8H,ArH),6.21(s,1H,PyH),3.79-3.71(m,3H,-CH2-and PipH),2.83(s,2H,PipH),2.06-1.39(m,6H,PipH)；13C NMR(200MHz,DMSO-d6):168.49,161.35,160.23,155.28,142.22,140.88,134.16,125.85,122.98,122.12,120.58,119.70,118.52,110.03,107.74,95.92,54.58,52.31,48.02,29.90；HRMS(ESI),C24H23N7O,[M+H]+the theoretical calculation is 425.1964, and the actual measurement is 426.2068.
Example 14: synthesis of target Compound I-5
Weighing I-c 5(0.8mmol) and suspending in N, N-dimethylformamide (10mL), slowly adding concentrated hydrochloric acid (16.1mmol) under ice bath stirring, removing ice bath for 0.5h, stirring at normal temperature for 3h, detecting by thin layer chromatography to show that the raw material point reaction is complete, stopping stirring, slowly adding saturated sodium bicarbonate solution into the reaction solution under ice bath till no bubble is generated, adding 60mL ethyl acetate to dilute the reaction solution, washing with water (2 × 50mL), separating, drying the organic phase with anhydrous sodium sulfate, performing suction filtration, and spin-drying the filtrate to obtain a crude product, and using N-hexane/dichloromethane mixed solvent (6:1, volume 1) to obtain the crude productVolume ratio), pumping filtration and vacuum drying of filter cake to obtain white powdery solid I-5 with yield of 53.9%. m p 167.1.1-169.8 deg.C;1H NMR(400MHz,DMSO-d6):(ppm)12.78(s,1H,IndNH),8.19(s,1H,PyH),7.90-7.06(m,8H,ArH),6.24-6.23(d,1H,PyH),3.78-3.70(m,2H,-CH2-and PipH),2.82(m,2H,PipH),2.04-1.41(m,6H,PipH)；13C NMR(200MHz,DMSO-d6)168.26,160.66,160.14,153.05,142.08,140.86,134.71,134.26,128.92,125.80,125.05,122.14,120.59,119.67,116.90,113.26,110.02,95.11,54.51,52.40,48.47,28.43；HRMS(ESI),C24H22ClN7O,[M+H]+the theoretical calculation is 459.1974, and the actual measurement is 460.1657.
Example 15: synthesis of target Compound I-5
Weighing I-c 5(0.8mmol) and suspending in dimethyl sulfoxide (10mL), slowly adding concentrated hydrochloric acid (16.1mmol) under the stirring of an ice bath, removing the ice bath for 0.5h, stirring at normal temperature for 3h, detecting by thin-layer chromatography to show that the raw material point reaction is complete, stopping stirring, slowly adding a saturated sodium bicarbonate solution into the reaction solution under the ice bath until no bubbles are generated, adding 60mL of ethyl acetate to dilute the reaction solution, washing with water (2 × 50mL), separating, drying the organic phase with anhydrous sodium sulfate, performing suction filtration, spin-drying the filtrate to obtain a crude product, pulping the crude product with a n-hexane/dichloromethane mixed solvent (6:1, volume ratio), performing suction filtration, and performing vacuum drying on the filter cake to obtain a white powdery solid I-5, wherein the yield is 55.7%, m p 167.1.1-169.8 ℃;1H NMR(400MHz,DMSO-d6):(ppm)12.78(s,1H,IndNH),8.19(s,1H,PyH),7.90-7.06(m,8H,ArH),6.24-6.23(d,1H,PyH),3.78-3.70(m,2H,-CH2-andPipH),2.82(m,2H,PipH),2.04-1.41(m,6H,PipH)；13C NMR(200MHz,DMSO-d6)168.26,160.66,160.14,153.05,142.08,140.86,134.71,134.26,128.92,125.80,125.05,122.14,120.59,119.67,116.90,113.26,110.02,95.11,54.51,52.40,48.47,28.43
example 16: MTT method for measuring and calculating influence on cell proliferation rate
Cell lines: human lung cancer cells A549 and H1975 were purchased from Shanghai cell biology institute of Chinese academy of sciences, and human lung cancer cells H1299, human normal bronchial epithelial cells HBE, human liver cancer cells HEPG-2 and human colon cancer cells HCT-116 were purchased from Shanghai Fuji Biotech Limited, and were stored and subcultured in the laboratory according to the culture data. Each cell was inoculated into a 50mL cell culture flask, and cultured in DMEM/RPI-1640 medium containing 10% fetal bovine serum at 37 ℃ under 5% CO2 and 100% humidity, and the medium was changed every day. When the growth reaches 80-90%, digesting with pancreatin EDTA mixed liquor, and carrying out passage 1: 3.
The experimental method comprises the steps of adopting an MTT method to investigate the influence of a target compound on the viability of each cell in an in-vitro cell activity proliferation inhibition experiment, digesting each cell in a logarithmic growth phase by pancreatin, collecting, uniformly blowing by using a pipette, counting, and carrying out the concentration of cells with the density of 1 × 104cells/mL were seeded at 100. mu.L/well in 96-well plates at 37 ℃ with 5% CO2After overnight incubation at 100% humidity, the stock culture was discarded and the cultures were incubated for 48h (n-3) in serum-free blank medium containing the target compound (0-40 μ M) at various final concentrations. Then 5mg/mL MTT solution 10. mu.L/well was added, and after further incubation for 4h 100. mu.L of the triple was added per well and left overnight in the incubator. The absorbance at 570nm was measured with a microplate reader and the survival rate of five cells after administration was calculated.
The results of the experiment are shown in table 1.
TABLE 1 antitumor cell proliferation Activity
As can be seen from Table 1, 5 compounds tested showed different levels of inhibitory activity against six cancer cells, and excellent in vitro inhibitory effect against human lung cancer H1975 cells among them, and the activity of a plurality of compounds (I-1, I-3, I-4, I-5) was stronger than that of the reference drug gefitinib. Meanwhile, comparing the antiproliferative activity of a plurality of compounds on H1975 with the antiproliferative activity on HBE5, the compounds have lower toxicity to normal cells of human than lung cancer cells, show better selectivity, and are expected to be further developed into anticancer drugs based on the structure.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (7)
1. An indazole piperidine pyrimidine derivative, which is a compound shown as a formula (I):
wherein R is positioned at ortho position, meta position or para position of the ether group, and R is mono-substituted or di-substituted; r is methyl, trifluoromethyl, halogen or cyano.
2. A preparation method of indazole piperidine pyrimidine derivatives is characterized by comprising the following steps:
1) dissolving raw material intermediates I-a and 1- (tert-butyloxycarbonyl) -3- (bromomethyl) -indazole I-b in a first solvent, stirring until all raw materials are fully dissolved, adding alkali, stirring at room temperature until all raw materials are completely consumed, and separating and purifying to obtain an intermediate I-c;
2) suspending or dissolving the obtained intermediate I-c in a second solvent, slowly dropwise adding acid under the stirring of an ice bath, removing the ice bath after dropwise adding is finished, stirring at normal temperature until the raw materials completely react, then adjusting the pH of a reaction solution to 6.0-7.5 by using an alkali solution, and separating and purifying to obtain a target product I;
the reaction formula is as follows:
wherein R is positioned at ortho position, meta position or para position of the ether group, and R is mono-substituted or di-substituted; r is methyl, trifluoromethyl, halogen or cyano.
3. The method of preparing an indazole-piperidinopyrimidine derivative according to claim 2, characterized in that: the first solvent in the step 1) is a mixed solvent composed of any one or more of dichloromethane, chloroform, 1, 4-dioxane, N-dimethylformamide, dimethyl sulfoxide or N-methylpyrrolidone.
4. The method of preparing an indazole-piperidinopyrimidine derivative according to claim 2, characterized in that: the second solvent in the step 2) is a mixed solvent consisting of any one or more of dichloromethane, chloroform, carbon tetrachloride, 1, 2-dichloroethane, 1, 4-dioxane, tetrahydrofuran, N-dimethylformamide or dimethyl sulfoxide.
5. The method of preparing an indazole-piperidinopyrimidine derivative according to claim 2, characterized in that: the alkali is any one or a mixture of more of potassium carbonate, sodium carbonate, cesium carbonate, N-diisopropylethylamine, sodium hydroxide, potassium hydroxide or sodium hydrogen.
6. The method of preparing an indazole-piperidinopyrimidine derivative according to any one of claims 2 to 5, characterized in that: the acid in the step 2) is one or a mixture of trifluoroacetic acid and concentrated hydrochloric acid.
7. The use of an indazole piperidinopyrimidine derivative according to claim 1 for the preparation of a medicament for the prevention or treatment of cancer.
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