CN113387935B - Compound for inhibiting triple-mutation epidermal growth factor receptor tyrosine kinase and application thereof - Google Patents
Compound for inhibiting triple-mutation epidermal growth factor receptor tyrosine kinase and application thereof Download PDFInfo
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- C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
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Abstract
The invention discloses a compound for inhibiting triple mutation epidermal growth factor receptor tyrosine kinase, which is characterized by having the following structural formula:
wherein: substituent R1Is composed of
One of (1); substituent R2Is composed of
One of (a) and (b); the substituent X is H or Cl; substituent R3Is H,
One kind of (1).
Description
Technical Field
The invention discloses a compound for inhibiting triple mutation epidermal growth factor receptor tyrosine kinase and application thereof.
Background
Lung cancer is one of the most common malignant tumors in the world, and about 80% -85% of lung cancers are non-small cell lung cancers (NSCLC), and the disease condition of patients is approximately diagnosed at a locally advanced or advanced stage.
The epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) discovered by research with the Epidermal Growth Factor Receptor (EGFR) as a target spot can obviously improve the survival period of non-small cell lung cancer patients. The epidermal growth factor receptor is a transmembrane protein and is divided into an extracellular ligand binding region, a transmembrane region and an intracellular region containing a TK structural domain. When the epidermal growth factor receptor is combined with a corresponding extracellular ligand, kinase activation is induced, so that a downstream signal transduction pathway is activated, and the proliferation, survival, differentiation, metastasis and the like of tumor cells are caused.
Currently, the approved EGFR-TKI drugs on the market are gefitinib, erlotinib, afatinib, axitinib and amitinib.
Gefitinib and erlotinib are the first reversible EGFR-TKIs, and are mainly used for treating lung cancer caused by sensitive mutations of EGFR.
Afatinib is the second generation irreversible EGFR-TKI and is mainly used for treating lung cancer caused by EGFR-T790M gene mutation.
Oxcetitinib and amitinib are third-generation irreversible EGFR-TKI and are mainly used for treating lung cancer caused by EGFR-T790M gene mutation after drug resistance.
Recently, most patients have been found to develop a new EGFR-C797S mutation after some time of drug administration, thereby developing drug resistance. Therefore, the discovery of new small molecule inhibitors for the treatment of mutant EGFR is a problem to be solved urgently.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims at solving the problems existing in the prior art, namely the invention discloses a compound for inhibiting triple mutant epidermal growth factor receptor tyrosine kinase and application thereof.
The technical scheme is as follows: a compound for inhibiting triple mutant epidermal growth factor receptor tyrosine kinase, which has the following structural formula:
substituent R1Is composed of
One of (a) and (b);
substituent R2Is composed of
One of (1);
the substituent X is H or Cl;
substituent R3Is H,
One kind of (1).
Further, the compound for inhibiting the triple mutant epidermal growth factor receptor tyrosine kinase is one of the compounds 1 to 33, and the substituents of the compounds 1 to 33 are shown in the following table:
further, the compound for inhibiting the triple mutant epidermal growth factor receptor tyrosine kinase is one of compound 12, compound 13, compound 14, compound 16, compound 28, compound 30, compound 31, compound 32 and compound 33.
Further, the three-mutation epidermal growth factor receptor tyrosine kinase is EGFRL858R/T790M/C797SA kinase.
The above compounds as EGFRL858R/T790M/C797SInhibitors of kinases.
Has the advantages that: the compound for inhibiting the triple-mutation epidermal growth factor receptor tyrosine kinase disclosed by the invention can well inhibit EGFRL858R/T790M/C797SA kinase.
Drawings
FIG. 1 shows EGFR of compound 12, compound 32, compound 33 for inhibiting triple mutant epidermal growth factor receptor tyrosine kinase disclosed in the present inventionL858R/T790M/C797SKinase Activity IC50A graph.
The specific implementation mode is as follows:
the following describes in detail specific embodiments of the present invention.
A compound for inhibiting triple mutant epidermal growth factor receptor tyrosine kinase, the structural formula of which is as follows:
substituent R1Is composed of
One of (1);
substituent R2Is composed of
One of (1);
the substituent X is H or Cl;
substituent R3Is H,
One kind of (1).
Further, the compound for inhibiting the triple mutant epidermal growth factor receptor tyrosine kinase is one of the compounds 1 to 33, and the substituents of the compounds 1 to 33 are shown in the following table:
further, the compound for inhibiting the triple mutant epidermal growth factor receptor tyrosine kinase is one of compound 12, compound 13, compound 14, compound 16, compound 28, compound 30, compound 31, compound 32 and compound 33.
Further, the three-mutation epidermal growth factor receptor tyrosine kinase is EGFRL858R/T790M/C797SA kinase.
The above compounds as EGFRL858R/T790M/C797SInhibitors of kinases.
Synthesis of Compound 1-Compound 33
1. Synthesis of Compounds 1 to 4
R3The synthetic route of the H-substituted compound is as follows, compounds 1, 2, 3 and 4, 4 of which the total number is 4, are synthesized through the synthetic route, and R of the compounds3All fragments are substituted with H.
Synthesis of Compound 1-a
Placing 5-fluoro-2-nitro-anisole (430mg, 2.00mmoL) in a 25mL two-neck reaction flask, vacuumizing, adding 12mL anhydrous DMA solvent under nitrogen protection, adding DIPEA (662 μ L, 4.00mmoL) dropwise, stirring at room temperature for 5min, and adding N dropwise1，N1，N2-trimethylethane-1, 2-diamine (327 μ L, 2.00mmoL), then gradually heating to 60 ℃, reacting for 6-8h, after TLC detection reaction is completed, cooling the reaction solution, adding a proper amount of 1M hydrochloric acid solution to adjust the pH value to be alkaline, and then adding a proper amount of saturated NaHCO3Adjusting pH of the solution to neutral, adding distilled water, extracting with dichloromethane for three times, mixing organic phases, washing with saturated NaCl solution, and mixing organic phases with anhydrous NaSO4After drying, concentration to dryness under reduced pressure gave a crude product as a yellow oily liquid, which was separated by silica gel chromatography (eluting with dichloromethane: methanol-30: 1 to 15: 1) to give compound 1-a (396mg, yield 78.2%) as a yellow solid.
Synthesis of Compound 1-b
Compound 1-a (420mg, 1.66mmoL) was placed in a 50mL single-neck reaction flask, 15mL of anhydrous methanol solvent was added, followed by a dilution of Pd/C (80.0mg, 20% by mass) anhydrous methanol (5mL) to give a suspension, and the suspension was purified in H2Stirring at room temperature for 4-5h under ambient conditions, after TLC detection reaction is complete, pouring the suspension into a funnel containing filter paper and diatomaceous earth, suction-filtering under reduced pressure, concentrating the obtained filtrate under reduced pressure to dryness to obtain a crude purple solid, and separating by silica gel chromatography (using dichloromethane: methanol 15:1 to 10: 1) to give compound 1-b (320mg, yield 86.3%) as a pale purple foamy solid.
Synthesis of Compound 1-c
Placing anhydrous aluminum chloride (1.80g, 13.4mmoL) in a 50mL two-mouth reaction bottle, vacuumizing, then carrying out nitrogen protection, adding 20mL of anhydrous DME solvent, dropwise adding 2, 4-dichloropyrimidine (2.00g, 13.4mmoL), stirring at room temperature for 15min, dropwise adding 1-methylindole (1.67mL, 13.4mmoL), then gradually heating to 80 ℃, reacting for 2-4h, cooling the reaction solution after TLC detection reaction is completed, and dropwise adding saturated NaHCO which is vigorously stirred within 20min3Stirring the solution (300mL) at room temperature for about 30min, vacuum filtering with Buchner funnel, washing the solid with water for 3 times, adding appropriate amount of water and dichloromethane into the solid until the solid is completely dissolved, extracting with dichloromethane for three times, combining organic phases, washing with saturated NaCl solution, and washing the organic phase with anhydrous NaSO4Drying and concentration to dryness under reduced pressure gave a crude product as a reddish brown solid which was separated by silica gel chromatography (eluting with petroleum ether: ethyl acetate: 6: 1 to 2: 1) to give compound 1-c (1.48g, yield 45.3%) as a beige solid powder.
Synthesis of Compound 1
Placing compound 1-c (120mg, 0.490mmoL) and compound 1-b (110mg, 0.490mmoL) in a 10mL two-mouth reaction flask, vacuumizing, adding 3mL anhydrous 2-pentanol solvent, stirring at room temperature for 5min, dropwise adding 4-methylbenzenesulfonic acid monohydrate (186mg, 0.980mmoL), gradually heating to 105 ℃, reacting for 4h, cooling the reaction solution after the reaction is completely detected by TLC, adding a proper amount of saturated NaHCO, and stirring3Adjusting pH of the solution to neutral, adding appropriate amount of distilled water, extracting with dichloromethane for three times, and mixingAnd the organic phase is washed with saturated NaCl solution and the organic phase is anhydrous NaSO4After drying, concentration to dryness under reduced pressure gave a crude product as a yellow solid, which was separated by silica gel chromatography (dichloromethane: methanol 25: 1 to 20: 1 elution) to give compound 1(117mg, yield 55.4%) as a yellow solid.
Synthesis of Compound 2-a
Placing 2, 4-dichloropyrimidine (559mg, 3.75mmoL) in a 25mL two-mouth reaction bottle, vacuumizing, then protecting with nitrogen, adding 11mL of anhydrous DMA solvent, dropwise adding DIPEA (1550 mu L, 9.38mmoL), stirring at room temperature for 5min, dropwise adding 1, 2, 3, 4-tetrahydroquinoline (471 mu L, 3.75mmoL), gradually heating to 100 ℃, reacting for 6-8h, detecting by TLC after the reaction is completed, cooling the reaction solution, adding an appropriate amount of 1M hydrochloric acid solution to adjust the pH value to be alkaline, then adding an appropriate amount of saturated NaHCO solution to adjust the pH value to be alkaline3Adjusting pH of the solution to neutral, adding distilled water, extracting with dichloromethane for three times, mixing organic phases, washing with saturated NaCl solution, and mixing organic phases with anhydrous NaSO4After drying, concentration to dryness under reduced pressure gave a crude product as a yellow oily liquid, which was separated by silica gel chromatography (eluting with petroleum ether: ethyl acetate ═ 6: 1 to 4: 1) to give compound 2-a (330mg, yield 36.0%) as a pale yellow solid.
Synthesis of Compound 2
The synthesis procedure was the same as for compound 1, and the crude pale yellow product obtained from the reaction was separated by silica gel chromatography (eluting with dichloromethane: methanol 25: 1 to 20: 1) to give compound 2
Synthesis of Compound 3-a
The synthesis procedure was the same as compound 2-a, and the resulting yellow crude product was isolated by silica gel chromatography (eluting with petroleum ether: ethyl acetate ═ 6: 1 to 4: 1) to give compound 3-a (452mg, yield 43.0%) as a pale yellow solid.
Synthesis of Compound 3
The synthesis procedure was the same as compound 1, and the crude pale yellow product obtained from the reaction was separated by silica gel chromatography (eluting with dichloromethane: methanol 25: 1 to 20: 1) to give compound 3(49.2mg, yield 21.5%) as a yellow solid.
Synthesis of Compound 4-a
The synthesis procedure was the same as for compound 2-a, and the resulting yellow crude product was isolated by silica gel chromatography (eluting with petroleum ether: ethyl acetate ═ 6: 1 to 4: 1) to give compound 4-a (498mg, 45.0% yield) as a pale yellow solid.
Synthesis of Compound 4
The synthesis procedure was the same as compound 1, and the crude pale yellow product obtained from the reaction was separated by silica gel chromatography (eluting with dichloromethane: methanol 25: 1 to 20: 1) to give compound 4(53.0mg, 22.5% yield) as a yellow solid.
2. Synthesis of Compounds 5 to 10
Synthesis of Compound 5-a
5-fluoro-2-nitrobenzyl ether (2.00g, 11.7mmoL) was placed in a 100mL single-neck reaction flask, 65mL of anhydrous methanol solvent was added, and then a diluted solution of Pd/C (300mg, 15% by mass) anhydrous methanol (5mL) was added to obtain a suspension, which was then subjected to high-pressure distillation in H2Stirring at room temperature for 4-5h under ambient conditions, after TLC detection reaction was complete, pouring the suspension into a funnel with filter paper and diatomaceous earth, suction filtering under reduced pressure, concentrating the filtrate under reduced pressure to dryness to give a crude purple solid, which was separated by silica gel chromatography (eluting with petroleum ether: ethyl acetate: 15: 1 to 10: 1) to give compound 5-a (1.42g, 86.0% yield) as a pale purple solid.
Synthesis of Compound 5-b
Placing compound 5-a (675mg, 4.78mmoL) in a single-mouth reaction bottle, vacuumizing, then protecting with nitrogen, adding 5mL of concentrated sulfuric acid solvent under the condition of ice-water bath, stirring for 40min, adding potassium nitrate (512mg, 4.78mmoL) into the system, continuously stirring for 2-3h at 0-5 ℃, after TLC detection reaction is completed, dropwise adding the reaction solution into vigorously stirred 250mL of ice water, then adding a proper amount of 25% ammonia water, adjusting pH to 8-9, extracting with dichloromethane for three times, combining organic phases, washing with saturated NaCl solution, and using anhydrous NaSO to wash the organic phase4After drying, concentration to dryness under reduced pressure gave a tan crude product which was isolated by silica gel chromatography (eluting with petroleum ether: ethyl acetate 4: 1 to 2: 1) to give compound 5-b (447mg, 50.2% yield) as a reddish brown solid.
Synthesis of Compound 5-c
Placing compound 5-b (360mg, 1.93mmoL) and compound 1-c (390mg, 1.93mmoL) in a 25mL two-mouth reaction bottle, vacuumizing, adding 12mL of anhydrous 2-pentanol solvent, stirring at room temperature for 5min, adding 4-methylbenzenesulfonic acid monohydrate (736mg, 3.87mmoL), gradually heating to 105 ℃, reacting for 4h, detecting the reaction by TLC, cooling the reaction solution, performing suction filtration under reduced pressure, washing the filter cake twice with 2-pentanol, dissolving with dichloromethane, concentrating under reduced pressure to dryness, adding an appropriate amount of acetonitrile solvent, performing centrifugation after ultrasound (3000r/min, 10min), discarding the supernatant, dissolving the obtained solid with dichloromethane, concentrating under reduced pressure to dryness to obtain a yellow solid crude product, separating by a silica gel chromatographic column (eluting with dichloromethane: methanol ═ 30: 1 to 20: 1), compound 5-c (720mg, 94.8% yield) was obtained as a yellow solid.
Synthesis of Compound 5-d
Placing compound 5-c (520mg, 1.32mmoL) in a 50mL two-mouth reaction bottle, vacuumizing, then protecting with nitrogen, adding 16mL anhydrous DMA solvent, then dropwise adding DIPEA (436 mu L, 2.64mmoL), stirring at room temperature for 5min, then dropwise adding N1, N1, N2-trimethylethane-1, 2-diamine (172 mu L, 1.32mmoL), then gradually heating to 100 ℃, reacting for 6-8h, after TLC detects the reaction is complete, cooling the reaction solution, adding an appropriate amount of 1M hydrochloric acid solution to adjust the pH value to be alkaline, then adding an appropriate amount of saturated NaHCO3Adjusting pH of the solution to neutral, adding distilled water, extracting with dichloromethane for three times, mixing organic phases, washing with saturated NaCl solution, and mixing organic phases with anhydrous NaSO4After drying, concentration to dryness under reduced pressure gave a crude product as a yellow oily liquid, which was separated by silica gel chromatography (eluting with dichloromethane: methanol 20: 1 to 15: 1) to give compound 5-d (488mg, yield 77.7%) as an orange solid.
Synthesis of Compound 5-e
Compound 5-d (700mg, 1.47mmoL) was placed in a 50mL single-neck reaction flask, 12mL of anhydrous ethanol solvent was added, and then a diluted solution of Pd/C (105mg, 15% by mass) anhydrous ethanol (5mL) was added to obtain a suspension, which was then subjected to suspension in H2After completion of the TLC detection, the suspension was poured into a funnel containing filter paper and celite, filtered under reduced pressure and the resulting filtrate was concentrated to dryness under reduced pressure to give a crude purple solid which was separated by silica gel chromatography (eluting with dichloromethane: methanol 15: 1 to 10: 1) to give compound 5-e (540mg, 84.7% yield) as a pale green foamy solid.
Synthesis of Compound 5
Placing compound 5-e (65.0mg, 0.146mmoL) in a 10mL single-mouth reaction bottle, vacuumizing, then protecting with nitrogen, adding 3mL anhydrous DCM solvent, dropwise adding DIPEA (48.0 muL, 0.274mmoL) in ice-water bath, stirring for 5min at 0 ℃, dropwise adding acryloyl chloride (11.5 muL, 0.146mmoL), reacting for 2-3h at 0 ℃, after TLC detection reaction is completed, adding proper amount of 1M hydrochloric acid solution to adjust pH value to alkalinity, then adding proper amount of NaHCO saturated NaHCO3Adjusting pH of the solution to neutral, adding distilled water, extracting with dichloromethane for three times, mixing organic phases, washing with saturated NaCl solution, and mixing organic phases with anhydrous NaSO4After drying, concentration to dryness under reduced pressure gave a yellow crude product which was isolated by silica gel chromatography (eluting with dichloromethane: methanol 20: 1 to 15: 1) to give compound 5(25.0mg, 34.2% yield) as a yellow solid.
Synthesis of Compound 6
The synthesis procedure was the same as compound 5, and the resulting yellow crude product was reacted and separated by silica gel chromatography (eluting with dichloromethane: methanol 20: 1 to 15: 1) to give compound 6(31.0mg, yield 42.5%) as a pale yellow solid.
Synthesis of Compound 7
Placing compound HATU (81.7mg, 0.215mmoL) in a 10mL single-mouth reaction flask, vacuumizing, then protecting with nitrogen, adding 2mL anhydrous DCM solvent, dropwise adding 2-methacrylic acid (16.7 muL, 0.197mmoL) in ice-water bath, dropwise adding DIPEA (59.2 muL, 0.358mmoL), stirring at 0 deg.C for 30min, dropwise adding compound 5-e (80.0mg, 0.197mmoL), slowly heating to room temperature, reacting overnight for 18-20h, after TLC detection reaction is completed, adding appropriate 1M hydrochloric acid solution to adjust pH to be alkaline, adding appropriate saturated NaHCO3Adjusting pH of the solution to neutral, adding distilled water, extracting with dichloromethane for three times, mixing organic phases, washing with saturated NaCl solution, and mixing organic phases with anhydrous NaSO4After drying, concentration to dryness under reduced pressure gave a yellow crude product which was isolated by silica gel chromatography (eluting with dichloromethane: methanol 20: 1 to 15: 1) to give compound 7(45.0mg, yield 44.2%) as a yellow solid.
Synthesis of Compound 8
The synthesis procedure was the same as compound 7, and the resulting dark brown crude product was isolated by silica gel chromatography (eluting with dichloromethane: methanol 20: 1 to 15: 1) to give compound 8(30.0mg, 29.5% yield) as a pale yellow solid.
Synthesis of Compound 9
The synthesis procedure was the same as for compound 7, and the crude pale yellow product obtained from the reaction was separated by silica gel chromatography (eluting with dichloromethane: methanol 20: 1 to 15: 1) to give compound 9
Synthesis of Compound 10-a
Ethyl fluoroacetate (916 μ L, 9.42mmoL) was placed in a 50mL single-neck reaction flask, evacuated and then blanketed with nitrogen, and 14mL of ethanol: adding water (9:1) solvent, dropwise adding sodium hydroxide (448mg, 11.2mmoL), reacting at room temperature for 20h, detecting by TLC, concentrating the reaction solution under reduced pressure to dryness, dissolving in 8mL of 3M diluted hydrochloric acid solution, adding appropriate amount of NaCl to the system until the solution is saturated, extracting with ethyl acetate for three times, combining organic phases, and adding anhydrous NaSO4After drying, concentration to dryness under reduced pressure was carried out to obtain compound 10-a (460mg, yield 52.2%) as white crystals.
Synthesis of Compound 10
The synthesis procedure was the same as compound 7, and the resulting pale yellow crude product was isolated by silica gel chromatography (eluting with dichloromethane: methanol 20: 1 to 15: 1) to give compound 10(67.0mg, 67.4% yield) as a yellow solid.
3. Synthesis of Compound 11-Compound 27
Synthesis of Compound 11-a
Placing 5-fluoro-2-nitrobenzyl ether (257mg, 1.50mmoL) in a 25mL two-mouth reaction bottle, vacuumizing, then protecting with nitrogen, adding 8mL anhydrous DMA solvent, then dropwise adding DIPEA (540 mu L, 3.00mmoL), stirring at room temperature for 5min, dropwise adding 1-methyl-4- (4-piperidyl) piperazine (300mg, 1.63mmoL), then gradually heating to 100 ℃, reacting for 6-8h, after TLC detection reaction is completed, cooling the reaction solution, adding an appropriate amount of 1M hydrochloric acid solution to adjust the pH value to be alkaline, then adding an appropriate amount of saturated NaHCO3Adjusting pH of the solution to neutral, adding distilled water, extracting with dichloromethane for three times, mixing organic phases, washing with saturated NaCl solution, and mixing organic phases with anhydrous NaSO4After drying, concentration to dryness under reduced pressure gave a crude product as a yellow oily liquid, which was separated by silica gel chromatography (eluting with dichloromethane: methanol 25: 1 to 20: 1) to give compound 11-a (145mg, yield 62.0%) as an orange solid.
Synthesis of Compound 11-b
Compound 11-a (360mg, 1.08mmoL) was placed in a 25mL single-neck reaction flask, 7mL of anhydrous methanol solvent was added, followed by a dilution of Pd/C (54.0mg, 15% by mass) anhydrous ethanol (4mL), and the resulting suspension was purified in H2After completion of the TLC reaction, the suspension was poured into a funnel containing filter paper and celite, filtered under reduced pressure and the resulting filtrate was concentrated to dryness under reduced pressure to give a crude purple solid which was separated by silica gel chromatography (eluting with dichloromethane: methanol 15: 1 to 10: 1) to give compound 11-b (276mg, 91.0% yield) as a pale yellow foamy solid.
Synthesis of Compound 11
Placing compound 1-c (82.0mg, 0.334mmoL) and compound 11-b (101mg, 0.334mmoL) in a 10mL two-mouth reaction flask, vacuumizing, adding 2mL anhydrous 2-pentanol solvent, stirring at room temperature for 5min, dropwise adding 4-methylbenzenesulfonic acid monohydrate (105mg, 0.668mmoL), gradually heating to 105 ℃, reacting for 4h, cooling the reaction solution after TLC detection reaction is complete, adding a proper amount of saturated NaHCO3Adjusting pH to neutral, adding appropriate amount of distilled water, extracting with dichloromethane for three times, mixing organic phases, washing with saturated NaCl solution, and mixing organic phases with anhydrous NaSO4After drying, it was concentrated to dryness under reduced pressure to give a pale yellow crude product, which was separated by silica gel chromatography (dichloromethane: methanol ═ 20: 1 to 15: 1 for elution) to give compound 11(55.7mg, yield 32.0%) as a yellow solid.
Synthesis of Compound 12
The synthesis procedure was the same as compound 11, and the crude product was reacted to give a yellow solid, which was separated by silica gel chromatography (eluting with dichloromethane: methanol 20: 1 to 15: 1) to give compound 12(50mg, yield 30.1%) as a yellow solid.1H NMR(400MHz,Chloroform-d)δ8.24(d,J＝8.5Hz,1H),7.93(d,J＝5.9Hz,1H),7.42–7.36(m,2H),7.20–7.16(m,2H),7.04(t,J＝7.5Hz,1H),6.53(d,J＝9.1Hz,2H),6.44(d,J＝6.0Hz,1H),3.98(t,J＝6.5Hz,2H),3.87(s,3H),3.64(d,J＝12.0Hz,2H),2.96–2.50(m,13H),2.48(s,3H),2.04–1.93(m,4H),1.75(apparent q,J＝11.3,10.5Hz,2H).13C NMR(100MHz,Chloroform-d)δ162.37,159.87,156.08,148.96,146.67,139.52,132.64,129.04,126.04,124.00,123.38,123.28,119.66,108.94,101.48,97.80,62.08,55.79,54.44,50.64,47.87,45.11,45.00,28.05,27.33,24.22.
Synthesis of Compound 13
The synthesis procedure was the same as compound 11, and the resulting pale yellow crude product was isolated by silica gel chromatography (eluting with dichloromethane: methanol 20: 1 to 15: 1) to give compound 13(47.6mg, 26.0% yield) as a yellow solid.1H NMR(400MHz,Chloroform-d)δ8.13(d,J＝8.8Hz,1H),8.08(s,1H),7.41(s,1H),7.13(d,J＝7.5Hz,1H),7.07(t,J＝7.8Hz,1H),6.96(t,J＝7.5Hz,1H),6.77(d,J＝8.1Hz,1H),6.52(s,1H),6.48(d,J＝8.7Hz,1H),3.89(t,J＝6.4Hz,2H),3.86(s,3H),3.63(d,J＝11.7Hz,2H),2.92–2.50(m,13H),2.47(s,3H),2.10–1.92(m,4H),1.72(apparent q,J＝10.9Hz,2H).13C NMR(100MHz,Methanol-d4)δ159.54,158.24,157.99,148.91,147.00,140.23,129.39,128.65,128.62,125.87,122.70,120.67,119.33,110.03,108.81,101.30,62.04,55.78,54.55,50.50,47.96,47.28,45.19,28.08,26.96,23.97.
Synthesis of Compound 14-a
The synthesis procedure was the same as for compound 2-a, and the resulting yellow crude product was isolated by silica gel chromatography (eluting with petroleum ether: ethyl acetate ═ 6: 1 to 4: 1) to give compound 14-a (321mg, yield 33.0%) as a pale yellow solid.
Synthesis of Compound 14
The synthesis procedure was the same as compound 11, and the resulting pale yellow crude product was isolated by silica gel chromatography (eluting with dichloromethane: methanol 20: 1 to 15: 1) to give compound 14(51.0mg, yield 29.0%) as a yellow solid.1H NMR(400MHz,Chloroform-d)δ8.27(d,J＝8.6Hz,1H),7.93(d,J＝6.0Hz,1H),7.42–7.32(m,2H),7.26(d,J＝9.5Hz,1H),7.18(t,J＝7.7Hz,1H),7.09(t,J＝7.5Hz,1H),6.55(d,J＝9.8Hz,2H),6.43(d,J＝6.0Hz,1H),3.99(apparent q,J＝6.2,5.6Hz,2H),3.88(s,3H),3.65(d,J＝12.0Hz,2H),2.92–2.43(m,12H),2.40(s,3H),2.18–2.08(m,1H),1.97(d,J＝12.4Hz,2H),1.81–1.60(m,3H),1.33(apparent d,J＝6.9Hz,3H).13C NMR(100MHz,Chloroform-d)δ162.35,159.97,156.17,148.90,146.77,138.86,137.39,126.89,126.04,124.02,123.31,123.19,119.62,108.87,101.41,97.69,63.76,61.96,55.76,54.92,50.67,48.40,45.54,43.64,32.31,31.19,28.17,20.01.
Synthesis of Compound 15
The synthesis procedure was the same as compound 11, and the resulting pale yellow crude product was isolated by silica gel chromatography (eluting with dichloromethane: methanol 20: 1 to 15: 1) to give compound 15(50.7mg, yield 27.0%) as a yellow solid.
Synthesis of Compound 16-a
The synthesis procedure was the same as for compound 2-a, and the resulting yellow crude product was isolated by silica gel chromatography (eluting with petroleum ether: ethyl acetate ═ 6: 1 to 4: 1) to give compound 16-a (534mg, 52.0% yield) as a pale yellow solid.
Synthesis of Compound 16
The synthesis procedure was the same as compound 11, and the resulting pale yellow crude product was isolated by silica gel chromatography (eluting with dichloromethane: methanol 20: 1 to 15: 1) to give compound 16(47.6mg, 26.3% yield) as a yellow solid.1H NMR(400MHz,Chloroform-d)δ8.26(d,J＝8.7Hz,1H),7.92(d,J＝5.9Hz,1H),7.34(d,J＝6.5Hz,3H),7.10(dt,J＝26.5,7.6Hz,2H),6.58–6.51(m,2H),6.43(d,J＝5.9Hz,1H),4.01(t,J＝6.6Hz,2H),3.86(s,3H),3.63(d,J＝11.8Hz,2H),2.75–2.32(m,11H),2.29(s,3H),1.93(d,J＝12.4Hz,2H),1.83–1.65(m,4H),1.28(s,6H).13C NMR(100MHz,Chloroform-d)δ162.36,160.06,156.33,148.83,146.87,140.01,138.08,125.86,125.85,123.86,123.40,123.04,119.56,108.75,101.29,97.80,61.81,55.67,55.40,50.69,48.93,45.97,42.60,38.53,33.31,29.09,28.29.
Synthesis of Compound 17-a
The synthesis procedure was the same as compound 2-a, and the resulting yellow crude product was isolated by silica gel chromatography (eluting with petroleum ether: ethyl acetate ═ 6: 1 to 4: 1) to give compound 17-a (631mg, yield 54.6%) as a pale yellow solid.
Synthesis of Compound 17
The synthesis procedure was the same as compound 11, and the resulting pale yellow crude product was isolated by silica gel chromatography (eluting with dichloromethane: methanol 20: 1 to 15: 1) to give compound 17(41.7mg, 21.7% yield) as a yellow solid.
Synthesis of Compound 18-a
The synthesis procedure was the same as compound 2-a, and the resulting yellow crude product was isolated by silica gel chromatography (eluting with petroleum ether: ethyl acetate ═ 6: 1 to 4: 1) to give compound 18-a (436mg, yield 47.0%) as a pale yellow solid.
Synthesis of Compound 18
The synthesis procedure was the same as compound 11, and the resulting pale yellow crude product was isolated by silica gel chromatography (eluting with dichloromethane: methanol 20: 1 to 15: 1) to give compound 18(53.0mg, 31.0% yield) as a yellow solid.
Synthesis of Compound 19-a
The synthesis procedure was the same as compound 2-a, and the resulting yellow crude product was isolated by silica gel chromatography (eluting with petroleum ether: ethyl acetate ═ 6: 1 to 4: 1) to give compound 19-a (466mg, yield 44.0%) as a pale yellow solid.
Synthesis of Compound 19
The synthesis procedure was the same as compound 11, and the resulting pale yellow crude product was isolated by silica gel chromatography (eluting with dichloromethane: methanol 20: 1 to 15: 1) to give compound 19(46.0mg, 25.0% yield) as a yellow solid.
Synthesis of Compound 20-a
The synthesis procedure was the same as compound 2-a, and the resulting yellow crude product was isolated by silica gel chromatography (eluting with petroleum ether: ethyl acetate ═ 6: 1 to 4: 1) to give compound 20-a (317mg, yield 32.5%) as a pale yellow solid.
Synthesis of Compound 20
The synthesis procedure was the same as compound 11, and the resulting pale yellow crude product was isolated by silica gel chromatography (eluting with dichloromethane: methanol 20: 1 to 15: 1) to give compound 20(38.2mg, 21.7% yield) as a yellow solid.
Synthesis of Compound 21-a
The synthesis procedure was the same as compound 2-a, and the resulting yellow crude product was isolated by silica gel chromatography (eluting with petroleum ether: ethyl acetate ═ 6: 1 to 4: 1) to give compound 21-a (479mg, yield 43.4%) as a pale yellow solid.
Synthesis of Compound 21
The synthesis procedure was the same as compound 11, and the resulting pale yellow crude product was isolated by silica gel chromatography (eluting with dichloromethane: methanol 20: 1 to 15: 1) to give compound 21(42.1mg, 22.4% yield) as a yellow solid.
Synthesis of Compound 22-a
The synthesis procedure was the same as compound 2-a, and the resulting yellow crude product was isolated by silica gel chromatography (eluting with petroleum ether: ethyl acetate ═ 6: 1 to 4: 1) to give compound 22-a (413mg, yield 41.8%) as a pale yellow solid.
Synthesis of Compound 22
The synthesis procedure was the same as compound 11, and the resulting pale yellow crude product was isolated by silica gel chromatography (eluting with dichloromethane: methanol 20: 1 to 15: 1) to give compound 22(40.7mg, 22.9% yield) as a yellow solid.
Synthesis of Compound 23-a
The synthesis procedure was the same as compound 2-a, and the resulting yellow crude product was isolated by silica gel chromatography (eluting with petroleum ether: ethyl acetate ═ 6: 1 to 4: 1) to give compound 23-a (454mg, 40.6% yield) as a pale yellow solid.
Synthesis of Compound 23
The synthesis procedure was the same as compound 11, and the resulting pale yellow crude product was isolated by silica gel chromatography (eluting with dichloromethane: methanol 20: 1 to 15: 1) to give compound 23(44.6mg, 23.6% yield) as a yellow solid.
Synthesis of Compound 24-a
The synthesis procedure was the same as compound 2-a, and the resulting yellow crude product was isolated by silica gel chromatography (eluting with petroleum ether: ethyl acetate ═ 6: 1 to 4: 1) to give compound 24-a (274mg, yield 31.7%) as a pale yellow solid.
Synthesis of Compound 24
The synthesis procedure was the same as compound 11, and the resulting pale yellow crude product was isolated by silica gel chromatography (eluting with dichloromethane: methanol 20: 1 to 15: 1) to give compound 24(30.9mg, yield 18.6%) as a yellow solid.
Synthesis of Compound 25-a
The synthesis procedure was the same as compound 2-a, and the resulting yellow crude product was isolated by silica gel chromatography (eluting with petroleum ether: ethyl acetate ═ 6: 1 to 4: 1) to give compound 25-a (301mg, yield 30.3%) as a pale yellow solid.
Synthesis of Compound 25
The synthesis procedure was the same as compound 11, and the resulting pale yellow crude product was isolated by silica gel chromatography (eluting with dichloromethane: methanol 20: 1 to 15: 1) to give compound 25(30.6mg, yield 17.2%) as a yellow solid.
Synthesis of Compound 26-a
The synthesis procedure was the same as for compound 2-a, and the resulting yellow crude product was isolated by silica gel chromatography (eluting with petroleum ether: ethyl acetate: 6: 1 to 4: 1) to give compound 26-a (411mg, yield 47.3%) as a pale yellow solid.
Synthesis of Compound 26
The synthesis procedure was the same as compound 11, and the resulting pale yellow crude product was isolated by silica gel chromatography (eluting with dichloromethane: methanol 20: 1 to 15: 1) to give compound 26(37.6mg, 22.5% yield) as a yellow solid.
Synthesis of Compound 27-a
The synthesis procedure was the same as compound 2-a, and the resulting yellow crude product was isolated by silica gel chromatography (eluting with petroleum ether: ethyl acetate ═ 6: 1 to 4: 1) to give compound 27-a (461mg, 46.4% yield) as a pale yellow solid.
Synthesis of Compound 27
The synthesis procedure was the same as compound 11, and the resulting pale yellow crude product was isolated by silica gel chromatography (eluting with dichloromethane: methanol 20: 1 to 15: 1) to give compound 27(36.2mg, yield 20.3%) as a yellow solid.
4. Synthesis of Compound 28-Compound 33
Synthesis of Compound 28-a
Placing compound 5-b (360mg, 1.93mmoL) and compound 2-a (474mg, 1.93mmoL) in a 25mL two-mouth reaction bottle, vacuumizing, adding 12mL of anhydrous 2-pentanol solvent, stirring at room temperature for 5min, adding 4-methylbenzenesulfonic acid monohydrate (736mg, 3.87mmoL), gradually heating to 105 ℃, reacting for 4h, detecting the reaction by TLC, cooling the reaction solution, performing suction filtration under reduced pressure, washing the filter cake twice with 2-pentanol, dissolving with dichloromethane, concentrating under reduced pressure to dryness, adding an appropriate amount of acetonitrile solvent, performing centrifugation after ultrasonic treatment (3000r/min, 10min), discarding the supernatant, dissolving the obtained solid with dichloromethane, concentrating under reduced pressure to dryness to obtain a yellow solid crude product, separating by a silica gel chromatographic column (eluting with dichloromethane: methanol ═ 30: 1 to 20: 1), compound 28-a (690mg, 90.4% yield) was obtained as a yellow solid.
Synthesis of Compound 28-b
Placing compound 28-a (522mg, 1.32mmoL) in a 50mL two-mouth reaction bottle, vacuumizing, then protecting with nitrogen, adding 16mL of anhydrous DMA solvent, then dropwise adding DIPEA (436 mu L, 2.64mmoL), stirring at room temperature for 5min, then dropwise adding 1-methyl-4- (4-piperidyl) piperazine (135mg, 1.32mmoL), then gradually heating to 100 ℃, reacting for 6-8h, after TLC detects that the reaction is complete, cooling the reaction solution, adding a proper amount of 1M hydrochloric acid solution to adjust the pH value to be alkaline, then adding a proper amount of saturated NaHCO solution to adjust the pH value to be alkaline3Adjusting pH of the solution to neutral, adding distilled water, extracting with dichloromethane for three times, mixing organic phases, washing with saturated NaCl solution, and mixing organic phases with anhydrous NaSO4After drying, concentration to dryness under reduced pressure gave a crude product as a yellow oily liquid, which was separated by silica gel chromatography (eluting with dichloromethane: methanol 20: 1 to 15: 1) to give compound 28-d (542mg, yield 73.5%) as an orange solid.
Synthesis of Compound 28-c
Compound 28-b (822mg, 1.47mmoL) was placed in a 50mL single-neck reaction flask, 12mL of anhydrous ethanol solvent was added, and then a diluted solution of Pd/C (105mg, 15% by mass) anhydrous ethanol (5mL) was added to obtain a suspension, which was then subjected to suspension in H2After completion of the TLC reaction, the suspension was poured into a funnel containing filter paper and celite, filtered under reduced pressure, and the resulting filtrate was concentrated to dryness under reduced pressure to give a crude purple solid which was separated by silica gel chromatography (eluting with dichloromethane: methanol 15: 1 to 10: 1) to give compound 28-c (677mg, 87.1% yield) as a pale yellow foamy solid.
Synthesis of Compound 28
Placing compound HATU (81.7mg, 0.215mmoL) in a 10mL single-mouth reaction flask, vacuumizing, then protecting with nitrogen, adding 2mL anhydrous DCM solvent, dropwise adding fluoroacetic acid (15.4mg, 0.197mmoL) in ice-water bath, dropwise adding DIPEA (59.2 muL, 0.358mmoL), stirring at 0 deg.C for 30min, dropwise adding compound 28-c (104mg, 0.197mmoL), slowly heating to room temperature, reacting overnight for 18-20h, after TLC detection reaction is completed, adding appropriate 1M hydrochloric acid solution to adjust pH value to alkalinity, adding appropriate saturated NaHCO3Adjusting pH of the solution to neutral, adding distilled water, extracting with dichloromethane for three times, mixing organic phases, washing with saturated NaCl solution, and mixing organic phases with anhydrous NaSO4After drying, concentration to dryness under reduced pressure gave a yellow crude product which was isolated by silica gel chromatography (eluting with dichloromethane: methanol 20: 1 to 15: 1) to give compound 28(37.7mg, yield 32.5%) as a yellow solid.
Synthesis of Compound 29
The synthesis procedure was the same as compound 28, and the resulting yellow crude product was reacted and separated by silica gel chromatography (eluting with dichloromethane: methanol 20: 1 to 15: 1) to give compound 29(38.3mg, yield 31.7%) as a yellow solid.
Synthesis of Compound 30
The synthesis procedure was the same as compound 28, and the resulting yellow crude product was reacted and separated by silica gel chromatography (eluting with dichloromethane: methanol 20: 1 to 15: 1) to give compound 30(40.7mg, yield 34.4%) as a yellow solid.1H NMR(400MHz,Chloroform-d)δ9.42(s,1H),9.37(s,1H),7.92(d,J＝6.0Hz,1H),7.44(s,1H),7.34(d,J＝8.0Hz,1H),7.18–7.07(m,2H),7.01(t,J＝7.4Hz,1H),6.70(s,1H),6.50(d,J＝6.0Hz,1H),4.13(t,J＝6.3Hz,2H),4.00(s,2H),3.84(s,3H),3.50(s,3H),3.03(d,J＝11.5Hz,2H),2.76–2.64(m,8H),2.57–2.38(m,5H),2.32(s,3H),2.02–1.92(m,4H),1.79–1.67(m,2H).
Synthesis of Compound 31
The synthesis procedure was the same as compound 28, and the resulting yellow crude product was reacted and separated by silica gel chromatography (eluting with dichloromethane: methanol 20: 1 to 15: 1) to give compound 31(37.4mg, yield 30.8%) as a yellow solid.1H NMR(400MHz,Chloroform-d)δ9.79(s,1H),9.45(s,1H),7.93(d,J＝5.9Hz,1H),7.46(s,1H),7.35(d,J＝8.0Hz,1H),7.18–7.10(m,2H),7.02(t,J＝7.5Hz,1H),6.73(s,1H),6.51(d,J＝6.0Hz,1H),4.13(t,J＝6.4Hz,2H),3.85(s,3H),3.35(s,2H),3.03(d,J＝11.3Hz,2H),2.78–2.64(m,8H),2.60–2.34(m,5H),2.33(s,3H),2.18(s,3H),2.05–1.95(m,4H),1.87–1.75(m,2H).
Synthesis of Compound 32
The synthesis procedure was the same as compound 28, and the resulting yellow crude product was reacted and separated by silica gel chromatography (eluting with dichloromethane: methanol 20: 1 to 15: 1) to give compound 32(40.2mg, yield 35.1%) as a yellow solid.
Synthesis of Compound 33
The synthesis procedure was the same as compound 28, and the resulting yellow crude product was reacted and separated by silica gel chromatography (eluting with dichloromethane: methanol 20: 1 to 15: 1) to give compound 33(43.5mg, 37.8% yield) as a yellow solid.
Second, Kinase-gloMax Kinase assay
1. Kinase-gloMax Kinase experimental principle
In the Kinase assay, a reagent for detecting the Kinase activity of Kinase-Glo Max is used, which is a homogeneous luciferase assay method, under the catalysis of luciferase, luciferin can react with oxygen, the process consumes ATP, and generates excited oxyluciferin, and when the excited oxyluciferin returns to the ground state from the excited state, a light signal is released, and the light signal is proportional to ATP in the system. The kinase phosphorylation reaction needs to consume ATP, and after a competitive inhibitor is added, the kinase phosphorylation reaction can be inhibited, the consumption of ATP can be reduced, and the optical signal is inversely proportional to the kinase activity in a system.
2. Kinase-gloMax Kinase experimental procedure
1. Firstly, ATP, 50x PTK substrate, Kinase-gloMax and the like are thawed, the room temperature is balanced, 5x Kinase assay buffer, DTT, high purity water and the like are slowly thawed under crushed ice, and the crushed ice is placed for standby.
2. Adding 15 mu L of DTT solution into 1.5mL of 5x kinase assay buffer, and fully mixing for later use; then 500. mu.L of 5x kinase assay buffer (containing DTT solution) was added to 2mL of high purity water to prepare 2.5mL of 1 × kinase assay buffer for use.
3. The compounds to be tested are prepared in advance to corresponding concentration gradients, and the buffer is diluted with high-purity water.
4. Table 1 lists the amounts of each reagent required to be added to each well in each of the test, control and blank groups, with small volumes of reagent added first, with EGFR added last after each reagent is added in sequenceL858R/T790M/C797SA triple mutant kinase.
TABLE 1 test, control, blank groups of test reagents
5. After adding the reagents to each well, the 96-well plate was placed in a 30 ℃ constant temperature shaker and allowed to react for 45 minutes.
6. After the reaction is finished, 50 mu L of Kinase-Glo Max detection reagent is added into each hole, the hole plate is covered by aluminum foil paper, the mixture is incubated for 15 minutes at room temperature, then a 96 micropore plate luminescence detector is used for detecting optical signals of each hole, and the data are collected and processed.
3. Results of Small molecule inhibitor kinase Activity
Table 2 shows that Compound 1-Compound 33 are directed against EGFRL858R/T790M/C797SResults of kinase activity. Wherein: the compound with the activity of A represents that the inhibition rate of the kinase is more than 50 percent, the compound with the activity of B represents that the inhibition rate of the kinase is between 30 percent and 50 percent, and the compound with the activity of C represents that the inhibition rate of the kinase is less than 30 percent.
TABLE 2 Compounds 1-33 for EGFRL858R/T790M/C797SResults of kinase Activity
In conclusion, in the kinase activity assayCompounds 12, 13, 14, 28, 30, 31, 32, 33 on EGFRL858R /T790M/C797SThe kinase activity inhibitory effect is excellent.
Third, Kinase-GloMax Kinase experiment
1. Kinase-GloMax Kinase experiment principle
As above, no further description is given.
2. Kinase assay procedure
1. First, ATP, 50 xPTTKsubstrate, Kinase-GloMax and the like are thawed, equilibrated at room temperature, and 5xkinase assaybuffer, DTT, high purity water and the like are slowly thawed under crushed ice, and placed in the crushed ice for later use.
2. Adding 15 mu L of DTT solution into 1.5mL of 5xkinaseassaybuffer, and fully mixing for later use; then 500. mu.L of 5xkinase assaybuffer (containing DTT solution) was added to 2mL of high-purity water to prepare 2.5mL of 1xkinase assaybuffer for use.
3. The compounds to be tested need to be prepared into corresponding concentration gradients in advance, and the buffer is diluted with high-purity water.
4. Table 3 lists the amounts of each reagent required to be added to each well in each of the test, control and blank groups, with small volumes of reagent added first, with EGFR added last after each reagent is added in sequenceL858R/T790M/C797SA kinase.
TABLE 3 test, control, blank groups of test reagents
5. After adding the reagents to each well, the 96-well plate was placed in a 30 ℃ constant temperature shaker and allowed to react for 45 minutes.
6. After the reaction is finished, 50 mu L of Kinase-GloMax detection reagent is added into each hole, the hole plate is covered by aluminum foil paper, the mixture is incubated for 15 minutes at room temperature, then a 96 micropore plate luminescence detector is used for detecting the optical signals of each hole, and the data are collected and processed.
EGFR based on each of the above compoundsL858R/T790M/C797SResults of kinase Activity experiments, we selected compound 12, compounds 32 and 33, on which EGFR was performedL858R/T790M/C797SKinase IC50And (4) measuring the value. The positive control compound for the kinase assay was AZD9291 (Oxitinib), the concentration gradient range was set to 1-10000nM, the concentration gradient range for compounds 12, 32 and 33 was set to 1-1000nM, and 4 wells were set for each concentration to perform the assay, the results of which are shown in FIG. 1 and Table 4.
As can be seen from FIG. 1, compounds 12, 32 and 33 are shown to be active against EGFR compared to the positive control AZD9291L858R/T790M/C797SThe kinase activity inhibition was significantly enhanced, with compound 32 having the strongest kinase inhibitory activity.
TABLE 4 EGFRL858R/T790M/C797SResults of kinase Activity
| Kinase activity inhibition IC50Value of | |
| AZD9291 | 305±73.5 |
| |
43.5±9.2 |
| |
16.6±6.1 |
| |
27.6±7.3 |
As shown in Table 4, the positive control AZD9291 compound is shown to act on EGFRL858R/T790M/C797SKinase activity inhibition IC50The value was 305nM, consistent with data known in the literature. In addition, compounds 12, 32 and 33 are active against EGFRL858R/T790M/C797SKinase activity inhibition IC50The values were 43.5nM, 16.6nM and 27.6nM, respectively, and the inhibition of kinase activity by Compound 12, Compound 32 and Compound 33 was enhanced by about 7, 19 and 11 fold, respectively, compared to the control AZD9291, indicating that Compound 12, Compound 32, Compound 33, respectively, are inhibitory to EGFRL858R/T790M/C797SThe kinase activity inhibitory effect is excellent.
The embodiments of the present invention have been described in detail. However, the present invention is not limited to the above-described embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present invention.
Claims (2)
1. A compound for inhibiting triple mutant epidermal growth factor receptor tyrosine kinase, which has the following structural formula:
2. the compound for inhibiting triple mutant epidermal growth factor receptor tyrosine kinase according to claim 1, wherein the triple mutant epidermal growth factor receptor tyrosine kinase is EGFRL858R/T790M/C797SA kinase.
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