CN110407812B - Indazole piperidine pyrimidine derivative and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of medicine, and in particular relates to an indazole piperidine pyrimidine derivative and a preparation method and application thereof. The indazole piperidine pyrimidine derivatives provided by the invention replace the covalently bonded acrylamide structure on the basis of retaining the pyrimidine parent ring of the third-generation EGFR inhibitor to obtain a class of non-covalently bonded inhibitors . The drug activity test results showed that the compounds showed excellent in vitro inhibitory effect on human lung cancer H1975 cells. At the same time, most of the compounds are less toxic to human normal cells than lung cancer cells, showing good selectivity and can be used for further development of anticancer drugs.

Description

A kind of indazole piperidine pyrimidine derivative and its preparation method and use

technical field

The invention belongs to the technical field of medicine, and in particular relates to an indazole piperidine pyrimidine derivative and a preparation method and application thereof.

Background technique

Human cancer cells are often accompanied by overexpression of growth factors and their receptors. The epidermal growth factor receptor family (epidermal growth factor receptor, EGFR) is also known as type I tyrosine kinase receptors or ErbB tyrosine kinase receptors. This receptor family consists of four homologous receptors: epidermal growth factor receptor (ErbB1/EGFr/HER1), ErbB2 (HER2), ErbB3 (HER3), ErbB4 (HER4).

Epidermal growth factor receptor is overexpressed in more than 80% of non-small cell lung cancer patients, so this receptor protein has become an important research target for anti-small cell lung cancer. EGFR inhibitors can be roughly divided into two categories: 1) non-covalently bound EGFR inhibitors, such as gefitinib and erlotinib; 2) covalently bound EGFR inhibitors Inhibitors such as nositinib and osimertinib. Non-covalently bound EGFR inhibitors can produce better therapeutic effects in the process of early administration, but the binding force of such drugs to receptors in the body is not very strong, and long-term use will lead to the emergence of T790M drug-resistant strains , the treatment effect is greatly reduced. The covalently bound EGFR inhibitor can undergo Michael addition reaction with the amino acid Cys797 to form a covalent bond with strong force, which has good activity against a variety of mutants including T790M. However, this irreversible inhibitor binds too strongly to the receptor in the body, causing the drug to stay in the body for too long. Most of these compounds have large toxic side effects, such as rash, diarrhea and other serious side effects. Therefore, there is an urgent need to find a new small molecule compound with high efficiency and low toxicity as a candidate drug for the treatment of non-small cell lung cancer.

Most of the first-generation EGFR inhibitors have a quinazoline parent ring structure, which has shown good therapeutic effects in early clinical applications, but the drug resistance caused by mutation greatly reduces the efficacy of such compounds. The parent ring of the second-generation EGFR inhibitor still retains the quinazoline structure, and forms a strong covalent interaction with amino acid residues through the introduction of unsaturated bonds, so as to reduce the drug resistance caused by variation, but the large Most of these compounds have large toxic and side effects. On the basis of retaining the unsaturated bond, the third-generation EGFR inhibitor replaced quinazoline with a pyrimidine ring to obtain a new class of inhibitors, which also showed good properties in clinical research, but most of these compounds still exist greater toxic side effects.

SUMMARY OF THE INVENTION

In order to solve the deficiencies of the prior art, the present invention provides an indazole piperidine pyrimidine derivative and a preparation method and application thereof.

The technical scheme provided by the present invention is as follows:

An indazole piperidine pyrimidine derivative, characterized in that it is a compound of formula (I):

wherein R is at the ortho, meta or para position of the ether group, R is mono- or di-substituted; R is methyl, trifluoromethyl, halogen or cyano.

The derivative provided by the above technical solution replaces the acrylamide structure forming a covalent bond on the basis of retaining the pyrimidine parent ring of the third-generation EGFR inhibitor, thereby obtaining a class of non-covalently bonded inhibitors. The drug activity test results showed that the compounds showed excellent in vitro inhibitory effect on human lung cancer H1975 cells. At the same time, most of the compounds are less toxic to human normal cells than lung cancer cells, showing good selectivity and can be used for further development of anticancer drugs.

The present invention also provides a preparation method of indazole piperidine pyrimidine derivatives, comprising the following steps:

1) Dissolve raw material intermediate I-a and 1-(tert-butoxycarbonyl)-3-(bromomethyl)-indazole I-b in the first solvent, stir until each raw material is fully dissolved, then add alkali, and at room temperature Stir until the raw materials are completely consumed, and separate and purify to obtain Intermediate I-c;

2) Suspend or dissolve the obtained intermediate I-c in the second solvent, slowly add acid dropwise with stirring in an ice bath to remove the tert-butoxycarbonyl protecting group, remove the ice bath after the dropwise addition, and stir at room temperature until the raw materials react Complete, then adjust the pH of the reaction solution to 6.0-7.5 with an alkali solution, and separate and purify to obtain the target product I;

The general reaction formula is as follows:

Wherein, R is located at the ortho, meta or para position of the ether group, R is mono- or di-substituted; R is methyl, trifluoromethyl, halogen or cyano.

The compound of formula (I) provided by the present invention can be prepared through the above technical solutions.

Specifically, the first solvent in step 1) is dichloromethane, chloroform, 1,4-dioxane, N,N-dimethylformamide, dimethyl sulfoxide or N-methylpyrrolidone A mixed solvent composed of any one or several of them.

Specifically, the second solvent in step 2) is dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, 1,4-dioxane, tetrahydrofuran, N,N-dimethylene A mixed solvent composed of any one or several of dimethyl sulfoxide or dimethyl sulfoxide.

Specifically, the alkali is any one or a mixture of potassium carbonate, sodium carbonate, cesium carbonate, N,N-diisopropylethylamine, sodium hydroxide, potassium hydroxide or sodium hydrogen.

Specifically, the acid in step 2) is any one of trifluoroacetic acid or concentrated hydrochloric acid or a mixture of the two.

The present invention also provides the application of the indazole piperidine pyrimidine derivatives provided by the present invention in preventing or treating cancer.

The results of drug activity test showed that these compounds showed good in vitro inhibitory effect on a variety of cancer cells, especially on human lung cancer H1975 cells, and the activities of many compounds were stronger than the reference drug gefitinib. At the same time, many compounds are less toxic to human normal cells than cancer cells such as lung cancer, show good selectivity, and can be used to prevent or treat cancer.

Detailed ways

The principles and features of the present invention are described below, and the examples are only used to explain the present invention, but not to limit the scope of the present invention.

Example 1: Synthesis of Intermediate I-c1

I-a1 (1 mmol) and I-b (1 mmol) were weighed and dissolved in 1,4-dioxane (10 mL), K ₂ CO ₃ was added under stirring at room temperature, and the mixture was stirred at room temperature for 4 h. Thin-layer chromatography showed that the two starting materials had basically reacted completely, and the stirring was stopped. The reaction solution was poured into 50 mL of water, extracted with ethyl acetate (2×50 mL), washed with saturated brine (2×40 mL), the organic phase was dried over anhydrous sodium sulfate, filtered with suction, and the filtrate was spin-dried to obtain an oily substance, which was subjected to column chromatography After purification, I-c1 was obtained in a yield of 35.2%.

Example 2: Synthesis of Intermediate I-c1

Weigh I-a1 (1 mmol) and I-b (1 mmol) and dissolve them in N-methylpyrrolidone (10 mL), add DIEA under stirring at room temperature, and stir at room temperature for 4 h. Thin-layer chromatography showed that the two starting materials had basically reacted completely, and the stirring was stopped. The reaction solution was poured into 50 mL of water, extracted with ethyl acetate (2×50 mL), washed with saturated brine (2×50 mL), the organic phase was dried over anhydrous sodium sulfate, filtered with suction, and the filtrate was spin-dried to obtain an oily substance, which was subjected to column chromatography After purification, I-c1 was obtained with a yield of 42.3%.

Example 3: Synthesis of Intermediate I-c2

Weigh I-a2 (1 mmol) and I-b (1 mmol) and dissolve in DCM (10 mL), add sodium bicarbonate with stirring at room temperature, and stir at room temperature for 3 h. Thin-layer chromatography showed that the two starting materials had basically reacted completely, and the stirring was stopped. The reaction solution was poured into 50 mL of water, extracted with DCM (2×50 mL), washed with saturated brine (2×50 mL), the organic phase was dried over anhydrous sodium sulfate, filtered with suction, and the filtrate was spin-dried to obtain an oily substance, which was purified by column chromatography to obtain I-c2, yield 34.4%.

Example 4: Synthesis of Intermediate I-c2

Weigh I-a2 (1 mmol) and I-b (1 mmol) and dissolve them in chloroform (10 mL), add sodium bicarbonate with stirring at room temperature, and stir at room temperature for 3 h. Thin-layer chromatography showed that the two starting materials had basically reacted completely, and the stirring was stopped. The reaction solution was poured into 50 mL of water, extracted with DCM (2×50 mL), washed with saturated brine (2×50 mL), the organic phase was dried over anhydrous sodium sulfate, filtered with suction, and the filtrate was spin-dried to obtain an oily substance, which was purified by column chromatography I-c2 was obtained in a yield of 36.0%.

Example 5: Synthesis of Intermediate I-c3

Weigh I-a3 (1 mmol) and I-b (1 mmol) and dissolve them in N,N-dimethylformamide (10 mL), add sodium hydrogen slowly with stirring in an ice bath, and stir in an ice bath for 3 h. Thin-layer chromatography showed that the two starting materials had basically reacted completely, and the stirring was stopped. The reaction solution was poured into 50 mL of water, extracted with ethyl acetate (2×50 mL), washed with saturated brine (2×50 mL), the organic phase was dried over anhydrous sodium sulfate, filtered with suction, and the filtrate was spin-dried to obtain an oily substance, which was subjected to column chromatography After purification, I-c3 was obtained in a yield of 40.2%.

Example 6: Synthesis of Intermediate I-c4

Weigh I-a4 (1 mmol) and I-b (1 mmol) and dissolve them in dimethyl sulfoxide (10 mL), add cesium carbonate under stirring at room temperature, and stir at room temperature for 4 h. Thin-layer chromatography showed that the two starting materials had basically reacted completely, and the stirring was stopped. The reaction solution was poured into 50 mL of water, extracted with ethyl acetate (2×50 mL), washed with saturated brine (2×50 mL), the organic phase was dried over anhydrous sodium sulfate, filtered with suction, and the filtrate was spin-dried to obtain an oily substance, which was subjected to column chromatography After purification, I-c4 was obtained with a yield of 44.2%.

Example 7: Synthesis of Intermediate I-c5

Weigh I-a5 (1 mmol) and I-b (1 mmol) and dissolve in N-methylpyrrolidone (10 mL), add sodium carbonate under stirring at room temperature, and stir at room temperature for 4 h. Thin-layer chromatography showed that the two starting materials had basically reacted completely, and the stirring was stopped. The reaction solution was poured into 50 mL of water, extracted with ethyl acetate (2×50 mL), washed with saturated brine (2×50 mL), the organic phase was dried over anhydrous sodium sulfate, filtered with suction, and the filtrate was spin-dried to obtain an oily substance, which was subjected to column chromatography After purification, I-c5 was obtained with a yield of 36.9%.

Example 8: Synthesis of target compound I-1

Weigh I-c1 (0.8 mmol) and suspend it in dichloromethane (10 mL), slowly add trifluoroacetic acid (16.1 mmol) under stirring in an ice bath, remove the ice bath for 0.5 h, stir at room temperature for 3 h, TLC detection shows the raw material When the reaction is complete, stop stirring. Saturated sodium bicarbonate solution was slowly added to the reaction solution under ice bath until no bubbles were generated, 60 mL of dichloromethane solution was added to extract and separate the layers, the organic phase was washed with saturated brine (2×50 mL) and then dried with anhydrous sodium sulfate. Filtration, and the filtrate was spin-dried to obtain the crude product. The crude product was slurried with n-hexane/dichloromethane mixed solvent (6:1, volume ratio), suction filtered, and the filter cake was vacuum-dried to obtain white powdery solid I-1 with a yield of 53.3%. mp 182.9-187.3°C; ¹ H NMR (400 MHz, DMSO-d ₆ ): δ (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, -CH ₂ -and PipH), 2.84-2.79(m, 2H, PipH), 2.07-1.34(m, 6H, PipH); ¹³ C NMR(200MHz,DMSO-d ₆ ):δ168.55,161.63,160.27,159.84,148.25,140.87,130.32,129.90,128.56,128.20,126.99,125.86,124.54,122.15,120.60,119.72,110.03,95.82,54.91, 54.41, 52.09, 31.04; HRMS (ESI), C ₂₃ H ₂₃ ClN ₆ O, [M+H] ⁺ Theoretical calculation: 434.1622, found: 435.1698.

Example 9: Synthesis of target compound I-1

Weigh I-c1 (0.8 mmol) and suspend it in chloroform (10 mL), slowly add trifluoroacetic acid (16.1 mmol) under stirring in an ice bath, remove the ice bath for 0.5 h, stir at room temperature for 3 h, TLC detection shows that the starting material is reacted Complete, stop stirring. Saturated sodium bicarbonate solution was slowly added to the reaction solution under ice bath until no bubbles were generated, 60 mL of dichloromethane solution was added to extract and separate the layers, the organic phase was washed with saturated brine (2×50 mL) and then dried with anhydrous sodium sulfate. Filtration, and the filtrate was spin-dried to obtain the crude product. The crude product was slurried with n-hexane/dichloromethane mixed solvent (6:1, volume ratio), suction filtered, and the filter cake was vacuum-dried to obtain white powdery solid I-1, the yield of white solid was 55.2%. mp 183.4-185.1°C, ¹ H NMR (400MHz, DMSO-d ₆ ): δ (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, -CH ₂ -and PipH), 2.84-2.79(m, 2H, PipH), 2.07-1.34(m, 6H, PipH); ¹³ C NMR(200MHz,DMSO-d ₆ ):δ168.55,161.63,160.27,159.84,148.25,140.87,130.32,129.90,128.56,128.20,126.99,125.86,124.54,122.15,120.60,119.72,110.03,95.82,54.91, 54.41, 52.09, 31.04.

Example 10: Synthesis of target compound I-2

Weigh I-c2 (0.8 mmol) and suspend it in carbon tetrachloride (10 mL), slowly add trifluoroacetic acid (16.1 mmol) under stirring in an ice bath, remove the ice bath for 0.5 h, stir at room temperature for 3 h, TLC detection shows that The reaction at the starting point was complete, and the stirring was stopped. Saturated sodium bicarbonate solution was slowly added to the reaction solution under ice bath until no bubbles were generated, 60 mL of dichloromethane solution was added to extract and separate the layers, the organic phase was washed with saturated brine (2×50 mL) and then dried with anhydrous sodium sulfate. Filtration, and the filtrate was spin-dried to obtain the crude product. The crude product was slurried with n-hexane/dichloromethane mixed solvent (6:1, volume ratio), suction filtered, and the filter cake was vacuum-dried to obtain white powdery solid I-2 with a yield of 51.9%. mp 194.3-196.4°C. ¹ H NMR (400 MHz, DMSO-d ₆ ): δ (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, -CH ₂ -and PipH), 2.81 (s, 2H, PipH), 2.03-1.40 (m, 6H, PipH); ¹³ C NMR ( 150MHz,DMSO-d ₆ ):δ169.57,161.65,159.56,150.12,140.88,134.37,130.03,126.10,122.39,121.52,120.45,120.20,110.28,96.39,53.03,46.88,51.51,30.06,20.40；HRMS(ESI) , C ₂₃ H ₂₃ ClN ₆ O, [M+H] ⁺ Theoretical calculation: 434.1622, found: 435.1715.

Example 11: Synthesis of target compound I-2

Weigh I-c2 (0.8 mmol) and suspend it in 1,2-dichloroethane (10 mL), slowly add trifluoroacetic acid (16.1 mmol) with stirring in an ice bath, remove the ice bath for 0.5 h, stir at room temperature for 3 h, thin Layer chromatography showed that the reaction at the starting point was complete, and the stirring was stopped. Saturated sodium bicarbonate solution was slowly added to the reaction solution under ice bath until no bubbles were generated, 60 mL of dichloromethane solution was added to extract and separate the layers, the organic phase was washed with saturated brine (2×50 mL) and then dried with anhydrous sodium sulfate. Filtration, and the filtrate was spin-dried to obtain the crude product. The crude product was slurried with n-hexane/dichloromethane mixed solvent (6:1, volume ratio), suction filtered, and the filter cake was vacuum-dried to obtain white powdery solid I-2, 47.4%. mp 195.1-196.9°C. ¹ H NMR (400 MHz, DMSO-d ₆ ): δ (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, -CH ₂ -and PipH), 2.81 (s, 2H, PipH), 2.03-1.40 (m, 6H, PipH); ¹³ C NMR (150MHz) ,DMSO-d ₆ ):δ169.57,161.65,159.56,150.12,140.88,134.37,130.03,126.10,122.39,121.52,120.45,120.20,110.28,96.39,53.03,46.8,2,0.0.01.51

Example 12: Synthesis of target compound I-3

Weigh Ⅰ-c3 (0.8mmol) and suspend it in 1,4-dioxane (10mL), slowly add concentrated hydrochloric acid (16.1mmol) under ice bath stirring, remove ice bath for 0.5h, stir at room temperature for 3h, thin layer Chromatographic detection showed that the reaction at the starting point was complete, and the stirring was stopped. Saturated sodium bicarbonate solution was slowly added to the reaction solution under ice bath until no bubbles were formed, 60 mL of ethyl acetate was added to dilute the reaction solution, washed with water (2 × 50 mL), the layers were separated, the organic phase was dried over anhydrous sodium sulfate, filtered with suction, The filtrate was spin-dried to obtain the crude product. The crude product was slurried with n-hexane/dichloromethane mixed solvent (6:1, volume ratio), suction filtered, and the filter cake was vacuum-dried to obtain white powdery solid I-3, 65.6%. mp 179.2-173.6°C; ¹ H NMR (400 MHz, DMSO-d ₆ ): δ (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, -CH ₂ -), 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); ¹³ C NMR (200 MHz, DMSO-d ₆ ): δ (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),C ₂₄ H ₂₃ F ₃ N ₆ O, [M+H] ⁺ theoretical calculation: 468.1885, actual measurement: 469.1983.

Example 13: Synthesis of target compound I-4

Weigh I-c4 (0.8 mmol) and suspend it in tetrahydrofuran (10 mL), slowly add concentrated hydrochloric acid (16.1 mmol) under stirring in an ice bath, remove the ice bath for 0.5 h, stir at room temperature for 3 h, TLC detection shows that the reaction at the starting point is complete , stop stirring. Saturated sodium bicarbonate solution was slowly added to the reaction solution under ice bath until no bubbles were formed, 60 mL of ethyl acetate was added to dilute the reaction solution, washed with water (2 × 50 mL), the layers were separated, the organic phase was dried over anhydrous sodium sulfate, filtered with suction, The filtrate was spin-dried to obtain the crude product. The crude product was slurried with n-hexane/dichloromethane mixed solvent (6:1, volume ratio), suction filtered, and the filter cake was vacuum-dried to obtain white powdery solid I-4 with a yield of 53.9%. mp 202.2-205.1°C; ¹ H NMR (400 MHz, DMSO-d ₆ ): δ (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, -CH ₂ -and PipH), 2.83(s, 2H, PipH), 2.06-1.39(m, 6H, PipH); ¹³ C NMR( 200MHz,DMSO-d ₆ ):δ168.49,161.35,160.23,155.28,142.22,140.88,134.16,125.85,122.98,122.12,120.58,119.70,118.52,110.03,107.74,95.92,54.58,52.31,48.02,29.90；HRMS( ESI), C ₂₄ H ₂₃ N ₇ O, [M+H] ⁺ theoretical calculation: 425.1964, found: 426.2068.

Example 14: Synthesis of target compound I-5

Weigh I-c5 (0.8mmol) and suspend it in N,N-dimethylformamide (10mL), slowly add concentrated hydrochloric acid (16.1mmol) under ice bath stirring, remove the ice bath for 0.5h, stir at room temperature for 3h, thin Layer chromatography showed that the reaction at the starting point was complete, and the stirring was stopped. Saturated sodium bicarbonate solution was slowly added to the reaction solution under ice bath until no bubbles were formed, 60 mL of ethyl acetate was added to dilute the reaction solution, washed with water (2 × 50 mL), the layers were separated, the organic phase was dried over anhydrous sodium sulfate, filtered with suction, The filtrate was spin-dried to obtain the crude product. The crude product was slurried with n-hexane/dichloromethane mixed solvent (6:1, volume ratio), suction filtered, and the filter cake was vacuum-dried to obtain white powdery solid I-5 with a yield of 53.9%. mp 167.1-169.8°C; ¹ H NMR (400 MHz, DMSO-d ₆ ): δ (ppm) 12.78 (s, 1H, IndNH), 8.19 (s, 1H, PyH), 7.90-7.06 (m, 8H, ArH) ₁₃ ^C NMR(200MHz,DMSO-d ₆ )δ168.26,160.66,160.14,153.05,142.08,140.86,134.71,134.26,128.92,125.80,125.05,122.14,120.59,119.67,116.90,113.26,110.02,95.11,54.51,52.40,48.47 , 28.43; HRMS (ESI), C ₂₄ H ₂₂ ClN ₇ O, [M+H] ⁺ theoretical calculation: 459.1974, found: 460.1657.

Example 15: Synthesis of target compound I-5

Weigh I-c5 (0.8 mmol) and suspend it in dimethyl sulfoxide (10 mL), slowly add concentrated hydrochloric acid (16.1 mmol) under stirring in an ice bath, remove the ice bath for 0.5 h, stir at room temperature for 3 h, TLC detection shows that The reaction at the starting point was complete, and the stirring was stopped. Saturated sodium bicarbonate solution was slowly added to the reaction solution under ice bath until no bubbles were formed, 60 mL of ethyl acetate was added to dilute the reaction solution, washed with water (2 × 50 mL), the layers were separated, the organic phase was dried over anhydrous sodium sulfate, filtered with suction, The filtrate was spin-dried to obtain the crude product. The crude product was slurried with n-hexane/dichloromethane mixed solvent (6:1, volume ratio), suction filtered, and the filter cake was vacuum-dried to obtain white powdery solid I-5 with a yield of 55.7%. mp 167.1-169.8°C; ¹ H NMR (400 MHz, DMSO-d ₆ ): δ (ppm) 12.78 (s, 1H, IndNH), 8.19 (s, 1H, PyH), 7.90-7.06 (m, 8H, ArH) ₁₃ C ^NMR (200MHz,DMSO-d ₆ )δ168.26,160.66,160.14,153.05,142.08,140.86,134.71,134.26,128.92,125.80,125.05,122.14,120.59,119.67,116.90,113.26,110.02,95.11,54.51,52.40,48.47, 28.43

Example 16: Influence of MTT method on cell proliferation rate

Cell lines: human lung cancer cells A549 and H1975 were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences, 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 Fu Heng Biotechnology Co., Ltd. was preserved and subcultured in our laboratory according to its culture data. Each cell was routinely inoculated into a 50 mL cell culture flask, and cultured with DMEM/RPI-1640 medium containing 10% fetal bovine serum at 37°C, 5% CO2, and 100% humidity, and the medium was changed every day. When it grows to 80-90%, it is digested with trypsin-EDTA mixture and passaged 1:3.

Experimental methods: In vitro cell viability and proliferation inhibition assay MTT assay was used to investigate the effect of target compounds on the viability of each cell. Digest the cells in logarithmic growth phase with trypsin, blow evenly with a pipette and count after collection, inoculate cells with a density of 1×10 ⁴ cells/mL in a 96-well plate with 100 μL per well, and inoculate them in a 96-well plate at 37°C, 5 After overnight incubation under %CO ₂ and 100% humidity conditions, the original culture medium was discarded, and serum-free blank medium containing different final concentrations of target compounds (0-40 μM) was added to culture for 48 hours (n=3). Then, 10 μL/well of 5 mg/mL MTT solution was added, and after incubation for 4 h, 100 μL triple solution was added to each well, and it was incubated overnight in the incubator. The absorbance was measured at a wavelength of 570 nm with a microplate reader and the survival rate of the five cells after administration was calculated.

The experimental results are shown in Table 1.

Table 1 Anti-tumor cell proliferation activity

It can be seen from Table 1 that the tested 5 compounds showed different intensities of inhibitory activities on six cancer cells, and showed excellent in vitro inhibitory effect on human lung cancer H1975 cells, and multiple compounds (I-1, I-3 , I-4, I-5) activity was stronger than the reference drug gefitinib. At the same time, comparing the anti-proliferative activities of multiple compounds on H1975 and anti-proliferative activities on HBE5, it can be seen that the toxicity of multiple compounds to human normal cells is less than that of lung cancer cells, showing good selectivity, which is expected to be further developed based on this structure. Anti-cancer drugs.

The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

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.