WO2021196655A1 - Compound containing benzimidazole structure, preparation method therefor and application thereof - Google Patents

Abstract

The invention relates to the pharmaceutical chemistry field and discloses a compound containing a benzimidazole structure, a preparation method therefor, and an application thereof. A compound represented by general formula (I) or a pharmaceutically acceptable salt thereof is disclosed. R1 represents H, COOH, COOR4 or CONHR4, where R4 represents hydrogen or C1-C6 alkyl, R2 represents hydrogen or C1-C5 alkyl, and R3 represents optionally substituted benzene ring or aromatic heterocyclic ring. The aromatic heterocyclic ring is a six-membered or five-membered aromatic heterocyclic ring. The substituent is halogen, C1-C3 alkyl, alkoxy, haloalkyl, haloalkoxy, OH, NR5R6 or CN, where R5 and R6 represent hydrogen or C1-C6 alkyl. The compound of the present invention can be used to treat tumors.

Description

Compound containing benzimidazole structure and preparation method and application thereof Technical field

The invention relates to the field of medicinal chemistry, and discloses a compound containing a benzimidazole structure and a preparation method and application thereof.

technical background

c-ROS proto-oncogene 1 tyrosine kinase (c-ros oncogene 1 tyrosine kinase, ROS1) was discovered in avian sarcoma RNA tumor virus in 1987. It is a kind of gene encoding receptor tyrosine kinase (RTK). Oncogenes belong to the insulin receptor family. Studies have found that ROS1 is not expressed in normal human lung tissue, but it is expressed in the kidney, cerebellum, peripheral nerve tissue, stomach, small intestine, and colon in normal adults, especially the highest expression in the kidney. It was first found in tissues of patients with lung adenocarcinoma in 2007. Studies have found that the mutation ratio of ROS1 rearrangement in non-small cell lung cancer accounts for about 1%-2%, mainly in lung adenocarcinoma. As the kinase activity region of ROS1 and ALK are 70% similar, the US FDA approved the ALK inhibitor crizotinib for the treatment of metastatic non-small cell lung cancer with ROS1 mutation on March 11, 2016 based on clinical research data. The patient is also the first FDA-approved targeted drug for the treatment of patients with metastatic non-small cell lung cancer with ROS1 mutations. On August 15, 2019, the U.S. Food and Drug Administration accelerated the approval of Entratinib for the treatment of adult patients with ROS1-positive metastatic non-small cell lung cancer (NSCLC). Therefore, ROS1, as a new lung cancer driver gene, has become a clear lung cancer treatment target.

Although crizotinib has been successfully used in clinical trials of NSCLC for the treatment of ROS1 mutations, the current clinical results have found that, like most anti-tumor drugs, long-term application can easily lead to drug resistance. In addition, crizotinib and entritinib are both non-specific ROS1 inhibitors, so far there is no highly selective ROS1 inhibitor. Therefore, it is of great practical significance to find specific ROS1 inhibitors with strong activity and high safety.

Summary of the invention

Objective of the invention: The present invention discloses a class of compounds containing a benzimidazole ring structure, and provides a specific preparation method of this class of compounds and their pharmaceutical applications as selective ROS1 inhibitors.

Technical Solution: The present invention discloses a compound of general formula (I) or a pharmaceutically acceptable salt thereof:

Wherein: R ¹ represents H, COOH, COOR ⁴ or CONHR ⁴ , where R ⁴ represents hydrogen, C ₁ ～C ₆ alkyl;

R ² represents hydrogen, a C ₁ ～C ₅ alkyl group;

R ³ represents an optionally substituted benzene ring or aromatic heterocyclic ring, the aromatic heterocyclic ring is a six-membered or five-membered aromatic heterocyclic ring, the substituent is a halogen, a C ₁ ～C ₃ alkyl, alkoxy, Halogenated alkyl, halogenated alkoxy, OH, NR ⁵ R ⁶ or CN, where R ⁵ and R ⁶ represent hydrogen, C ₁ ～C ₆ alkyl;

As a preference of the present invention:

R ¹ represents H, COOH, COOR ⁴ or CONHR ⁴ , wherein R ⁴ represents hydrogen, C ₁ ～C ₆ alkyl;

R ² represents hydrogen, a C ₁ ～C ₃ alkyl group;

R ³ represents an optionally substituted benzene ring or aromatic heterocyclic ring, the aromatic heterocyclic ring is a six-membered or five-membered aromatic heterocyclic ring, and the substituents are F, Cl, Br, I, CH ₃ , C ₂ H ₅ , OH, NR ⁵ R ⁶ , OCH ₃ , OCF ₃ , CF ₃ or CN, where R ⁵ and R ⁶ represent hydrogen, C ₁ ～C ₆ alkyl;

Wherein: R ^{1 is} more preferably CONH ₂ or CONHCH ₃ ;

R ^{2 is} more preferably H or CH ₃ ;

R ^{3 is} further preferably an optionally substituted phenyl group, and the substituent is preferably F, Cl, Br, CH ₃ , OH, NH ₂ , OCH ₃ , OCF ₃ or CF ₃ .

Among them, R ^{1 is} more preferably CONH ₂ or CONHCH ₃ ;

R ^{2 is} more preferably CH ₃ ,

R ^{3 is} more preferably an optionally substituted phenyl group, and the substituents are F, CH ₃ , CH ₂ CH ₃ , and OCH ₃ .

More preferably, the benzimidazole compound of the present invention is selected from I-1 to I-8:

The pharmaceutically acceptable salt of the above compound is an acid addition salt of the compound of general formula (I), wherein the acid used for salt formation is: hydrogen chloride, hydrogen bromide, sulfuric acid, carbonic acid, oxalic acid, citric acid, succinic acid, tartaric acid , Phosphoric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or ferulic acid.

The compound of general formula (I) of the present invention can be prepared by the following method:

Among them: ^{the definitions of R 1} , R ² , and R ³ are the same as before;

Compound IV is prepared by reacting compound II with compound III, the base used is selected from sodium hydrogen, potassium carbonate, sodium carbonate, potassium tert-butoxide, sodium hydroxide, potassium hydroxide, sodium ethoxide, preferably sodium hydrogen; the solvent used is selected from N, N-dimethylformamide, N,N-dimethylacetamide, acetone, 1,4-dioxane, tetrahydrofuran, ethanol, acetonitrile, acetone, water or a mixed solvent composed of any two, preferably N, N-dimethylformamide;

Compound VI is prepared by reacting compound IV with compound V, the solvent used is selected from toluene, N,N-dimethylformamide, ethylene glycol dimethyl ether, 1,4-dioxane, tetrahydrofuran, ethanol, acetonitrile, acetone , Water or a mixed solvent composed of any two, preferably tetrahydrofuran/water; the base used is selected from sodium ethoxide, potassium acetate, sodium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate or triethylamine, preferably potassium carbonate; the catalyst used It is Pd(PPh ₃ ) ₄ , Pd(dppf)Cl ₂ , Pd(PPh ₃ ) ₂ Cl ₂ or Pd(OAc) ₂ , preferably Pd(PPh ₃ ) ₄ .

Compound VII is prepared from compound VI through hydrolysis reaction, the base used is selected from lithium hydroxide, sodium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, preferably sodium hydroxide; the solvent used is selected from water, methanol, ethanol, tetrahydrofuran or any A mixed solvent composed of the two is preferably methanol/water.

Compound IX is prepared by reacting compound VII with compound VIII. The condensing agent used is selected from carbonyldiimidazole (CDI), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-(3). -Dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea six Fluorophosphate (HATU) or hexafluorophosphate benzotriazol-1-yl-oxytripyrrolidinyl phosphorus (PyBOP), preferably HATU; the acid binding agent is selected from N,N-diisopropylethylamine or tris Ethylamine, preferably N,N-diisopropylethylamine. The dehydrating agent is selected from glacial acetic acid, polyphosphoric acid, hydrochloric acid, acetic anhydride, preferably glacial acetic acid;

Compound IA is prepared by reduction reaction of compound IX, the solvent used is selected from methanol, ethanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, 1,4-dioxide A mixed solvent composed of six rings, water or any two, preferably methanol/tetrahydrofuran; the reducing agent used is selected from iron powder/dilute hydrochloric acid, stannous chloride, sodium sulfide, palladium carbon, platinum carbon, Raney nickel, preferably palladium carbon ；

The present invention also discloses a pharmaceutical composition, which contains the compound of the above general formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. The compounds can be added with pharmaceutically acceptable carriers to prepare common medicinal preparations, such as tablets, capsules, syrups, suspensions, injections, and can be added with flavors, sweeteners, liquid or solid fillers or diluents, etc. medical supplements.

The application of the compound of general formula (I) and its hydrate, solvate or crystal in the preparation of selective ROS1 target inhibitor drugs is also within the protection scope of the present invention.

Use of the compound of the present invention, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition in the preparation of ROS1 inhibitor drugs.

Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

The compound of the present invention can selectively inhibit ROS1, and can be used to prepare drugs for treating cancers such as non-small cell lung cancer. The use of selective ROS1 inhibitors can reduce off-target effects, reduce toxic side effects, and improve treatment safety.

Detailed ways

The application will be described in detail below in conjunction with specific embodiments.

Example 1 Synthesis of Compound I-1

2-(1-((2-amino-5-(4-methylphenyl)pyridin-3-yl)oxy)ethyl)-N-methyl-1H-benzo[d]imidazole-4- Synthesis of formamide (I-1: R ¹ ＝CONHCH ₃ , R ² ＝CH ₃ , R ³ ＝4-methylphenyl)

Synthesis of 2-((5-bromo-2-nitropyridin-3-yl)oxy) ethyl propionate (IV-1)

Dissolve compound II (5g, 22.8mmol) in 50mL N,N-dimethylformamide, cool in an ice bath, add sodium hydride (1.10g, 0.0274mol), stir, remove the ice bath, slowly add 2-bromo Ethyl acetate (III-1) (1.50mL×3,34.20mmol), after the dripping, was heated to 80°C for constant temperature reaction for 12h, TLC (petroleum ether: ethyl acetate=4:1) detected that the raw material had reacted completely. Stop heating, after cooling to room temperature, add 120mL of water and stir, extract with ethyl acetate (30mL×3), combine the organic layers, wash with saturated sodium chloride solution (30mL×2), dry with anhydrous magnesium sulfate, filter with suction, and reduce The solvent was removed by pressure evaporation, and purified by column chromatography (petroleum ether: ethyl acetate = 50:1) to obtain 6.88 g of beige solid, yield: 94.43%, mp 84.2-85.0°C. ¹ H NMR (300MHz, DMSO-d ₆ ) δ (ppm): 8.36 (d, J = 1.6 Hz, 1H, ArH), 8.33 (d, J = 1.8 Hz, 1H, ArH), 5.48 (q, J = 6.8Hz,1H, CH CH ₃ ), 4.15(q,J=7.1Hz,2H, CH ₂ CH ₃ ), 1.54(d,J=6.8Hz,3H,CH CH ₃ ),1.17(t,J=7.1 Hz, 3H, CH ₂ CH ₃ ).

Synthesis of ethyl 2-((2-nitro-5-(4-methylphenyl)pyridin-3-yl)oxy)propionate (VI-1)

Dissolve IV-1 (1.00g, 3.13mmol) and 4-methylphenylboronic acid (V-1) (0.64g, 4.70mmol) in 20mL of tetrahydrofuran, and use 2mL of water to dissolve potassium carbonate (1.21g, 8.77mmol) Potassium carbonate solution was added dropwise to the reaction solution, then tetrakis(triphenylphosphine)palladium (0.36g, 0.31mmol) was added, and the reaction was refluxed under nitrogen protection for 24h. TLC (petroleum ether: ethyl acetate=4:1) was used to detect the raw material The reaction is complete. The heating was stopped and cooled to room temperature. The reaction system was evaporated to remove the solvent under reduced pressure, 20 mL ethyl acetate and 15 mL were added to separate the layers, the aqueous phase was extracted with ethyl acetate once (15 mL), and the organic phases were combined and washed with saturated sodium chloride (15 mL). ×3), dried over anhydrous magnesium sulfate, filtered with suction, evaporated under reduced pressure to remove the solvent, and purified by column chromatography (petroleum ether: ethyl acetate = 50:1 to 10:1 gradient elution) to obtain 1.03 g of light yellow solid. Yield: 99.62%, mp 99.6-102.2°C. ¹ H NMR(300MHz, CDCl ₃ )δ(ppm): 8.34(d,J=1.7Hz,1H,ArH), 7.56(d,J=1.6Hz,1H,ArH),7.46(d,J=8.2Hz ,2H,ArH),7.34(d,J=8.0Hz,2H,ArH),4.94(q,J=6.8Hz,1H,CH ₃ CH ), 4.26(q,J=7.1Hz,2H,CH ₃ CH ₂ ), 2.45 (s, 3H, Ar CH ₃ ), 1.75 (d, J = 6.8 Hz, 3H, CH CH ₃ ), 1.25 (t, J = 7.2 Hz, 3H, CH ₂ CH ₃ ).

Synthesis of 2-((2-nitro-5-(4-methylphenyl)pyridin-3-yl)oxy)propionic acid (VII-1)

Dissolve VI-1 (1.02g, 3.09mmol) in 15mL methanol and 55mL water, add 3mL 20% sodium hydroxide solution, heat to 70℃ for 1h, TLC (petroleum ether: ethyl acetate = 4:1) to detect the raw material The reaction is complete. The heating was stopped, the reaction system was cooled to room temperature, 30mL ethyl acetate was added for extraction, the aqueous phase was collected, adjusted to pH 5 with 6mol/L hydrochloric acid solution, extracted with ethyl acetate (30mL×2), dried with anhydrous magnesium sulfate, filtered , The solvent was evaporated under reduced pressure to obtain 0.81 g of light yellow-brown solid, yield: 86.74%, mp 182.8-185.6°C. ¹ H-NMR(300MHz, CDCl ₃ )δ(ppm): 8.38(d,J=1.7Hz,1H,ArH), 7.60(d,J=1.6Hz,1H,ArH),7.48(d,J=8.0 Hz, 2H, ArH), 7.35 (d, J = 8.3 Hz, 2H, ArH), 5.05 (q, J = 6.9 Hz, 1H, CH ₃ CH ), 2.46 (s, 3H, Ar CH ₃ ), 1.81 ( d, J = 6.8 Hz, 3H, CH CH ₃ ).

N-methyl-2-(1-((2-nitro-5-(4-methylphenyl)pyridin-3-yl)oxy)ethyl)-1H-benzo[d]imidazole-4 -Synthesis of formamide (IX-1)

Compound VII-1 (0.48g, 1.57mmol), VIII-1 (0.26g, 1.57mmol) and HATU (0.60g, 1.57mmol) were dissolved in 6mL N,N-dimethylformamide, and DIPEA (5.56 mL, 3.14 mmol), the reaction solution was placed at room temperature and stirred for 5 hours, TLC (petroleum ether: ethyl acetate = 1:1) detected the complete reaction of the raw materials. The reaction system was added dropwise to 35 mL of water, a white solid precipitated, filtered, the filter cake was washed with a small amount of water, dried to obtain a light yellow solid, dissolved in 10 mL of glacial acetic acid, refluxed at 120 °C for 1 h, TLC (petroleum ether: ethyl acetate = 1:1) Check that the reaction of the raw materials is complete, stop heating, cool the reaction system to room temperature, add 40mL of water to dilute, extract with ethyl acetate (20mL×3), combine the organic phases, and wash with saturated sodium chloride solution (20mL×3). Water magnesium sulfate drying. Suction filtration, evaporation of the solvent under reduced pressure, purification by column chromatography (petroleum ether: ethyl acetate = 10:1～3:1 gradient elution) to obtain 0.39 g of light yellow solid, yield: 86.09%, mp184-187°C . ¹ H NMR (400MHz, DMSO-d ₆ ) δ (ppm): 13.29 (s, 1H, NH), 9.52 (q, J = 3.9 Hz, 1H, CO NH CH ₃ ), 8.45 (s, 2H, ArH) ,7.86(d,J=7.6Hz,1H,ArH),7.70(dd,J=7.5,5.3Hz,3H,ArH),7.35(t,J=7.2Hz,3H,ArH),6.42(q,J ＝6.7Hz,1H,CH ₃ CH ), 2.90(d,J＝4.7Hz,3H,NH CH ₃ ), 2.37(s,3H,Ar CH ₃ ), 1.89(d,J＝6.5Hz,3H,CH CH ₃ ).

2-(1-((2-amino-5-(4-methylphenyl)pyridin-3-yl)oxy)ethyl)-N-methyl-1H-benzo[d]imidazole-4- Synthesis of formamide (I-1)

Dissolve IX-1 (0.39g, 0.90mmol) in 10mL of tetrahydrofuran, add 0.1g of 10% Pd-C, ventilate with hydrogen three times, and let it react at room temperature. TLC (dichloromethane: methanol = 25:1) detects After the raw materials are reacted, they are filtered and the solvent is evaporated under reduced pressure. The residue obtained is slurried with ethyl acetate to obtain 200 mg of white solid, yield: 55.35%, mp185-188°C. ¹ H NMR (400MHz, DMSO-d ₆ ) δ (ppm): 13.18 (s, 1H, NH), 9.63 (q, J = 4.6 Hz, 1H, CO NH CH ₃ ), 7.91-7.83 (m, 2H, ArH), 7.71 (d, J = 7.9 Hz, 1H, ArH), 7.59 (d, J = 1.5 Hz, 1H, ArH), 7.46 (d, J = 8.1 Hz, 2H, ArH), 7.35 (t, J =7.8Hz,1H,ArH),7.19(d,J=7.9Hz,2H,ArH),6.14–6.06(m,1H,CH ₃ CH ),6.03(s,2H,ArNH ₂ ),2.97(d, J=4.7Hz,3H,NH CH ₃ ),2.31(s,3H,ArCH ₃ ),1.78(d,J=6.4Hz,3H,CH CH ₃ ).HR-MS(ESI)m/z[M+ H] ⁺ Calcd for C ₂₃ H ₂₄ N ₅ O ₂ ,402.1852; Found: 402.1923

Example 2 Synthesis of Compound I-2

2-(1-((2-amino-5-(4-fluorophenyl)pyridin-3-yl)oxy)ethyl)-N-methyl-1H-benzo[d]imidazole-4-methyl Synthesis of amide (I-2: R ¹ ＝CONHCH ₃ , R ² ＝CH ₃ , R ³ ＝4-fluorophenyl)

Synthesis of ethyl 2-((5-(4-fluorophenyl)-2-nitropyridin-3-yl)oxy)propionate (VI-2)

Dissolve IV-1 (0.50g, 1.57mmol) and 4-fluorophenylboronic acid (V-2) (0.33g, 2.35mmol) in 10mL of tetrahydrofuran, use 1mL of water to dissolve potassium carbonate (0.61g, 4.39mmol) Potassium carbonate solution was added dropwise to the reaction solution, then tetrakis(triphenylphosphine)palladium (0.18g, 0.16mmol) was added, and the reaction was refluxed under nitrogen protection for 24h. TLC (petroleum ether: ethyl acetate=4:1) was used to detect the raw material The reaction is complete. The heating was stopped and cooled to room temperature. The reaction system was evaporated to remove the solvent under reduced pressure, and 10 mL ethyl acetate and 15 mL were added to separate the layers. The aqueous phase was extracted with ethyl acetate once (10 mL). The organic phases were combined and washed with saturated sodium chloride (10 mL). ×3), dried over anhydrous magnesium sulfate, filtered with suction, evaporated under reduced pressure to remove the solvent, and purified by column chromatography to obtain 0.44 g of light yellow solid, yield: 84.00%, mp 130-132°C. ¹ H NMR (300MHz, DMSO-d ₆ ), δ (ppm): 8.47 (d, J = 1.7 Hz, 1H, ArH), 8.18 (d, J = 1.7 Hz, 1H, ArH), 7.91 (dd, J ＝8.9,5.3Hz,2H,ArH),7.42(t,J＝8.9Hz,2H,ArH), 5.63(q,J＝6.8Hz,1H,CH ₃ CH ), 4.14(q,J＝7.1Hz, 2H, CH ₃ CH ₂ ), 1.57 (d, J = 6.8 Hz, 3H, CH CH ₃ ), 1.15 (t, J = 7.1 Hz, 3H, CH ₂ CH ₃ ).

Synthesis of 2-((5-(4-fluorophenyl)-2-nitropyridin-3-yl)oxy)propionic acid (VII-2)

Dissolve VI-2 (0.44g, 1.32mmol) in 5mL methanol and 20mL water, add 1.5mL 20% sodium hydroxide solution, heat to 70℃ and react for 1h, TLC (petroleum ether: ethyl acetate = 4:1) detection The raw material reaction is complete. The heating was stopped, the reaction system was cooled to room temperature, and 10 mL of ethyl acetate was added for extraction and liquid separation. The aqueous phase was collected, adjusted to pH 5 with 6 mol/L hydrochloric acid solution, extracted with ethyl acetate (10 mL×2), dried with anhydrous magnesium sulfate, and filtered , The solvent was evaporated under reduced pressure to obtain 0.37 g of yellow solid, yield: 91.79%, mp 167-169°C. ¹ H NMR (300MHz, DMSO-d ₆ ) δ (ppm): 13.29 (s, 1H, COOH), 8.45 (d, J = 1.6 Hz, 1H, ArH), 8.11 (d, J = 1.6 Hz, 1H, ArH), 7.90 (dd, J = 8.8, 5.4 Hz, 2H, ArH), 7.42 (t, J = 8.9 Hz, 2H, ArH), 5.53 (q, J = 6.7 Hz, 1H, CH ₃ CH ), 1.56 (d,J=6.8Hz,3H,CH CH ₃ ).

N-methyl-2-(1-((2-nitro-5-(4-fluorophenyl)pyridin-3-yl)oxy)ethyl)-1H-benzo[d]imidazole-4- Synthesis of formamide (IX-2)

Compound VII-2 (0.56g, 1.68mmol), VIII-1 (0.25g, 1.52mmol) and HATU (0.64g, 1.68mmol) were dissolved in 6mL N,N-dimethylformamide, and DIPEA (5.95 mL, 3.36 mmol), the reaction solution was placed at room temperature and stirred for 5 hours, TLC (petroleum ether: ethyl acetate = 1:1) detected the complete reaction of the raw materials. The reaction system was added dropwise to 35mL of water, a white solid precipitated, filtered, the filter cake was washed with a small amount of water, dried to obtain a light yellow solid, the solid was dissolved in 5mL of glacial acetic acid, 120℃ refluxed for 1h, TLC detected the raw material reaction is complete, stop After heating, the reaction system was cooled to room temperature, diluted with 20 mL of water, extracted with ethyl acetate (10 mL×3), combined the organic phases, washed with saturated sodium chloride solution (10 mL×3), and dried with anhydrous magnesium sulfate. Suction filtration, evaporation of the solvent under reduced pressure, purification by column chromatography to obtain 0.15 g of light yellow solid, yield: 24.09%, mp113-116°C. ¹ H NMR (300MHz, DMSO-d ₆ ) δ (ppm): 113.27 (s, 1H, NH), 9.49 (s, 1H, CO NH CH ₃ ), 8.48 (d, J = 8.4 Hz, 2H, ArH) ,7.90(dd,J=9.0,5.5Hz,3H,ArH),7.85(d,J=7.5Hz,1H,ArH),7.69(d,J=7.6Hz,1H,ArH),7.41(d,J =8.8Hz,2H,ArH),6.44(q,J=6.4Hz,1H,CH ₃ CH ), 2.88(d,J=4.6Hz,3H,NH CH ₃ ),1.88(d,J=6.3Hz, 3H,CH CH ₃ )

2-(1-((2-amino-5-(4-fluorophenyl)pyridin-3-yl)oxy)ethyl)-N-methyl-1H-benzo[d]imidazole-4-methyl Synthesis of amide (I-2)

Dissolve IX-2 (0.15g, 0.34mmol) in 10mL of tetrahydrofuran, add 0.05g of 10% Pd-C, ventilate with hydrogen three times, and let it react at room temperature. TLC (dichloromethane: methanol = 25:1) detects After the raw materials have reacted, they are filtered and the solvent is evaporated under reduced pressure. The residue obtained is slurried with ethyl acetate to obtain 120 mg of white solid, yield: 87.05%, mp198-201°C. ¹ H NMR(400MHz,DMSO-d ₆ )δ(ppm): 13.25(s,1H,NH),9.71-9.64(m,1H,CO NH CH ₃ ),7.91(d,J=5.6Hz,2H, ArH), 7.75 (d, J = 7.9 Hz, 1H, ArH), 7.69–7.58 (m, 3H, ArH), 7.38 (t, J = 7.7 Hz, 1H, ArH), 7.23 (t, J = 8.8 Hz ,2H,ArH),6.23–6.00(m,3H,ArNH _2, CH ₃ CH ), 2.99(d,J=4.1Hz,3H,NH CH ₃ ),1.81(d,J=6.4Hz,3H,CH CH ₃ ).HR-MS(ESI)m/z[M+Na] ⁺ Calcd for C ₂₂ H ₂₀ FN ₅ O ₂ Na,428.1601; Found: 428.1495.

Example 3 Synthesis of Compound I-3

2-(1-((2-amino-5-(3-methylphenyl)pyridin-3-yl)oxy)ethyl)-N-methyl-1H-benzo[d]imidazole-4- Synthesis of formamide (I-3: R ¹ ＝CONHCH ₃ , R ² ＝CH ₃ , R ³ ＝3-methylphenyl)

Synthesis of 2-((2-nitro-5-(3-methylphenyl)pyridin-3yl)-oxy) ethyl propionate (VI-3)

Dissolve IV-1 (1.00g, 3.13mmol) and 3-methylphenylboronic acid (V-3) (0.64g, 4.70mmol) in 20mL of tetrahydrofuran, and use 2mL of water to dissolve potassium carbonate (1.22g, 8.77mmol) Potassium carbonate solution was added dropwise to the reaction solution, then tetrakis(triphenylphosphine)palladium (0.36g, 0.33mmol) was added, and the reaction was refluxed under nitrogen protection for 24h. TLC (petroleum ether: ethyl acetate = 4:1) detected the raw material The reaction is complete. The heating was stopped and cooled to room temperature. The reaction system was evaporated to remove the solvent under reduced pressure, 15 mL ethyl acetate and 20 mL were added to separate the layers, the aqueous phase was extracted with ethyl acetate once (15 mL), and the organic phases were combined and washed with saturated sodium chloride (15 mL ×3), dried over anhydrous magnesium sulfate, filtered with suction, evaporated under reduced pressure to remove the solvent, and purified by column chromatography to obtain 0.89 g of light yellow solid, yield: 86.08%, mp 103-106°C. ¹ H NMR (300MHz, CDCl ₃ ) δ (ppm): 8.46 (d, J = 1.6 Hz, 1H, ArH), 8.14 (d, J = 1.7 Hz, 1H, ArH), 7.68-7.58 (m, 2H, ArH),7.45(t,J=7.6Hz,1H,ArH),7.33(d,J=7.4Hz,1H,ArH), 5.64(q,J=6.7Hz,1H,CH ₃ CH ), 4.15(q ,J=7.2Hz,2H,CH ₃ CH ₂ ),2.41(s,3H,ArCH ₃ ),1.58(d,J=6.8Hz,3H,CH CH ₃ ),1.16(t,J=7.1Hz,3H ,CH ₂ CH ₃ ).

2-((2-nitro-5-(3-methylphenyl)pyridin-3-yl)oxy)propionic acid (VII-3)

VI-3 (0.89 g, 2.69 mmol) was dissolved in 15 mL methanol and 55 mL water, 3 mL 20% sodium hydroxide solution was added, and the reaction was heated to 70° C. for 1 h. TLC detected the complete reaction of the raw materials. The heating was stopped, the reaction system was cooled to room temperature, 30mL ethyl acetate was added for extraction, the aqueous phase was collected, adjusted to pH 5 with 6mol/L hydrochloric acid solution, extracted with ethyl acetate (30mL×2), dried with anhydrous magnesium sulfate, filtered , The solvent was evaporated under reduced pressure to obtain 0.70 g of light yellow-brown solid, yield: 86.09%, mp187-190°C. ¹ H NMR (300MHz, CDCl ₃ ) δ (ppm): 8.36 (d, J = 1.6 Hz, 1H, ArH), 7.58 (d, J = 1.1 Hz, 1H, ArH), 7.40 (d, J = 7.2 Hz ,1H,ArH),7.37–7.28(m,3H,ArH),5.04(q,J=7.1Hz,1H,CH ₃ CH ), 2.45(s,3H,ArCH ₃ ),1.78(d,J=6.6 Hz, 3H, CH CH ₃ ).

N-methyl-2-(1-((2-nitro-5-(3-methylphenyl)pyridin-3-yl)oxy)ethyl)-1H-benzo[d]imidazole-4 -Synthesis of formamide (IX-3)

Dissolve compound VII-2 (0.70g, 2.32mmol), VIII-1 (0.37g, 2.32mmol) and HATU (0.88g, 2.32mmol) in 6mL N,N-dimethylformamide, slowly add DIPEA (8.22 mL, 4.64 mmol), the reaction solution was placed at room temperature and stirred for 5 hours, TLC (petroleum ether: ethyl acetate = 1:1) detected the complete reaction of the raw materials. The reaction system was added dropwise to 35 mL of water, a white solid precipitated, filtered, the filter cake was washed with a small amount of water, dried to obtain a light yellow solid, dissolved in 20 mL of glacial acetic acid, refluxed at 120 °C for 1 h, TLC (petroleum ether: ethyl acetate = 1:1) Check that the reaction of the raw materials is complete, stop heating, cool the reaction system to room temperature, add 40mL of water to dilute, extract with ethyl acetate (20mL×3), combine the organic phases, and wash with saturated sodium chloride solution (20mL×3). Water magnesium sulfate drying. Suction filtration, evaporation of the solvent under reduced pressure, column chromatography purification to obtain 0.50 g of light yellow solid, yield: 55.45%, m.p. 193-196°C.

¹ H-NMR (300MHz, DMSO-d ₆ ) δ (ppm): 13.26 (s, 1H, NH), 9.50 (d, J = 4.1 Hz, 1H, CO NH CH ₃ ), 8.42 (d, J = 9.5 Hz, 2H, ArH), 7.86 (d, J = 7.9 Hz, 1H, ArH), 7.70 (d, J = 7.7 Hz, 1H, ArH), 7.57 (d, J = 6.4 Hz, 2H, ArH), 7.41 (t,J=7.8Hz,1H,ArH),7.38–7.27(m,2H,ArH),6.41(q,J=6.3Hz,1H,CH ₃ CH ), 2.88(d,J=4.6Hz,3H ,NH CH ₃ ), 2.38 (s, 3H, ArCH ₃ ), 1.89 (d, J = 5.9 Hz, 3H, CH CH ₃ ).

2-(1-((2-amino-5-(3-methylphenyl)pyridin-3-yl)oxy)ethyl)-N-methyl-1H-benzo[d]imidazole-4- Synthesis of formamide (I-3)

Dissolve IX-3 (0.50g, 1.16mmol) in 20mL of tetrahydrofuran, add 0.10g of 10% Pd-C, ventilate with hydrogen three times, and let it react at room temperature. TLC (dichloromethane: methanol = 25:1) detects After the raw materials are reacted, they are filtered, and the solvent is evaporated under reduced pressure. The residue obtained is slurried with ethyl acetate to obtain 140 mg of off-white solid, yield: 30.06%, mp185-188°C. ¹ H NMR (400MHz, DMSO-d ₆ ) δ (ppm): 13.19 (s, 1H, NH), 9.63 (q, J = 4.6 Hz, 1H, CO NH CH ₃ ), 7.98 (d, J = 1.9, 1H,ArH), 7.87(dd,J=7.6,1.0Hz,1H,ArH),7.72(dd,J=8.0,1.0Hz,1H,ArH), 7.70(d,J=1.9Hz,1H,ArH) ,7.52–7.38(m,3H,ArH),7.36(t,J=7.8Hz,1H,ArH), 7.08(t,J=6.5Hz,1H,ArH), 6.21(s,2H,ArNH ₂ ), 6.15(q,J=6.3Hz,1H,CH ₃ CH ), 2.96(d,J=4.7Hz,3H,NH CH ₃ ),2.51(s,3H,Ar CH ₃ ),1.77(d,J=6.4 Hz,3H,CH CH ₃ ).HR-MS(ESI)m/z[M+H] ⁺ Calcd for C ₂₃ H ₂₄ N ₅ O ₂ ,402.1852; Found: 402.1926.

Example 4 Synthesis of Compound I-4

2-(1-((2-amino-5-(3-fluorophenyl)pyridin-3-yl)oxy)ethyl)-N-methyl-1H-benzo[d]imidazole-4-methyl Synthesis of amide (I-4: R ¹ ＝CONHCH ₃ , R ² ＝CH ₃ , R ³ ＝3-fluorophenyl)

Synthesis of ethyl 2-((5-(3-fluorophenyl)-2-nitropyridin-3-yl)-oxy)propionate (VI-4)

Dissolve IV-1 (1.00g, 3.13mmol) and 3-fluorophenylboronic acid (V-4) (0.66g, 4.70mmol) in 20mL of tetrahydrofuran, and use 2mL of water to dissolve potassium carbonate (1.22g, 8.77mmol) to prepare carbonic acid Potassium solution was added dropwise to the reaction solution, then tetrakis(triphenylphosphine)palladium (0.36g, 0.33mmol) was added, and the reaction was refluxed under nitrogen protection for 24h. TLC (petroleum ether: ethyl acetate = 4:1) was used to detect the reaction of raw materials completely. Stop heating, cool to room temperature, evaporate the solvent under reduced pressure, add 15 mL ethyl acetate and 20 mL to separate the layers, extract the aqueous phase with ethyl acetate once (15 mL), combine the organic phases, and wash with saturated sodium chloride (15 mL×3 ), dried over anhydrous magnesium sulfate, filtered with suction, evaporated under reduced pressure to remove the solvent, and purified by column chromatography to obtain 0.79 g of pale yellow solid, yield: 75.50%, mp 100-105°C. ¹ H NMR (300MHz, CDCl ₃ ) δ (ppm): 8.33 (d, J = 1.8 Hz, 1H, ArH), 7.56 (d, J = 1.8 Hz, 1H, ArH), 7.52 (dd, J = 8.0, 2.2Hz,1H,ArH),7.35(d,J=7.8Hz,1H,ArH),7.30–7.19(m,2H,ArH), 4.96(q,J=6.8Hz,1H,CH ₃ CH ), 4.27 (q,J=7.1Hz,2H,CH ₃ CH ₂ ), 1.75(d, J=6.8Hz, 3H, CH CH ₃ ), 1.28(t, J=7.1Hz, 3H, CH ₂ CH ₃ ).

Synthesis of 2-((5-(3-fluorophenyl)-2-nitropyridin-3-yl)oxy)propionic acid (VII-4)

Dissolve VI-4 (0.79 g, 2.36 mmol) in 15 mL methanol and 55 mL water, add 3 mL 20% sodium hydroxide solution, and heat to 70° C. to react for 1 hour. TLC detects that the raw material has reacted completely. The heating was stopped, the reaction system was cooled to room temperature, 30mL ethyl acetate was added for extraction, the aqueous phase was collected, adjusted to pH 5 with 6mol/L hydrochloric acid solution, extracted with ethyl acetate (30mL×2), dried with anhydrous magnesium sulfate, filtered The solvent was evaporated under reduced pressure to obtain 0.62 g of light yellow solid, yield: 85.78%, mp 188-190°C. ¹ H NMR (300MHz, DMSO-d ₆ ) δ (ppm): 13.35 (s, 1H, COOH), 8.50 (d, J = 1.8 Hz, 1H, ArH), 8.17 (d, J = 1.8 Hz, 1H, ArH), 7.76 (dd, J = 10.4, 2.3 Hz, 1H, ArH), 7.70 (d, J = 7.4 Hz, 1H, ArH), 7.61 (td, J = 8.0, 6.1 Hz, 1H, ArH), 7.36 (td,J=8.5,2.6Hz,1H,ArH), 5.54(q,J=6.9Hz,1H,CH ₃ CH ), 1.56(d,J=6.7Hz, 3H, CH CH ₃ ).

2-(1-((5-(3-Fluorophenyl)-2-nitro-pyridin-3-yl)oxy)ethyl)-N-methyl-1H-benzo[d]imidazole-4 -Synthesis of formamide (IX-4)

Compound VII-4 (0.62g, 2.02mmol), VIII-1 (0.31g, 2.02mmol) and HATU (0.77g, 2.02mmol) were dissolved in 6mL N,N-dimethylformamide, and DIPEA (7.16 mL, 4.04 mmol), the reaction solution was placed at room temperature and stirred for 5 hours, TLC (petroleum ether: ethyl acetate = 1:1) detected the complete reaction of the raw materials. The reaction system was added dropwise to 35mL of water, a white solid precipitated, filtered, the filter cake was washed with a small amount of water, dried to obtain a brown-yellow solid, dissolved in 20mL of glacial acetic acid, refluxed at 120°C for 1h, TLC (petroleum ether: ethyl acetate = 1:1) Check that the reaction of the raw materials is complete, stop heating, cool the reaction system to room temperature, add 40mL of water to dilute, extract with ethyl acetate (20mL×3), combine the organic phases, and wash with saturated sodium chloride solution (20mL×3). Water magnesium sulfate drying. Suction filtration, evaporation of the solvent under reduced pressure, purification by column chromatography to obtain 0.30 g of light yellow solid, yield: 48.87%, mp 210-213°C. ¹ H NMR (300MHz, DMSO-d ₆ ) δ (ppm): 13.27 (s, 1H, NH), 9.62 (q, J = 5.4 Hz, 1H, CO NH CH ₃ ) 8.53 (d, J = 8.6 Hz, 1H,ArH), 8.10 (s, 1H, ArH), 7.90 (s, 1H, ArH), 7.85 (d, J = 7.3 Hz, 1H, ArH), 7.75 (d, J = 9.8 Hz, 1H, ArH) ,7.67(d,J=7.9Hz,1H,ArH),7.47(d,J=4.7Hz,2H,ArH),7.34(d,J=7.1Hz,1H,ArH), 6.09(q,J=6.2 Hz, 1H, CH ₃ CH ), 2.92 (d, J = 4.0 Hz, 3H, NH CH ₃ ), 1.85 (d, J = 6.5 Hz, 3H, CH CH ₃ ).

2-(1-((2-amino-5-(3-fluorophenyl)pyridin-3-yl)oxy)ethyl)-N-methyl-1H-benzo[d]imidazole-4-methyl Synthesis of amide (I-4)

Dissolve IX-4 (0.30g, 0.34mmol) in 20mL of tetrahydrofuran, add 0.10g of 10% Pd-C, ventilate with hydrogen three times, and let it react at room temperature. TLC (dichloromethane: methanol = 25:1) detects After the raw materials were reacted, they were filtered and the solvent was evaporated under reduced pressure. The residue obtained was slurried with ethyl acetate to obtain 15 mg of off-white solid, yield: 5.36%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ (ppm): 13.19 (s, 1H, NH), 9.76-9.51 (m, 1H, CO NH CH ₃ ), 7.89 (t, J = 8.8Hz, 2H, ArH), 7.72 (d, J = 8.1 Hz, 1H, ArH), 7.60 (d, J = 8.8 Hz, 1H, ArH), 7.38-7.32 (m, 2H, ArH), 7.27 (d, J = 7.5 Hz ,1H,ArH),7.14–7.02(m,1H,ArH),6.95–6.84(m,1H,ArH),6.16–5.96(m,3H,ArNH ₂ , CH CH ₃ ), 2.96(d,J= 4.6Hz,3H,CONH CH ₃ ),1.79(d,J=6.0Hz,3H,CH CH ₃ ).HR-MS(ESI)m/z[M+H] ⁺ Calcd for C ₂₂ H ₂₁ FN ₅ O ₂ ,406.1601; Found:406.1680.

Example 5 Synthesis of Compound I-5

2-(1--((2-Amino-5-(2-fluorophenyl)pyridin-3-yl)oxy)ethyl)-N-methyl-1H-benzo[d]imidazole-4-methyl Synthesis of amide (I-5: R ¹ ＝CONHCH ₃ , R ² ＝CH ₃ , R ³ ＝2-fluorophenyl)

Synthesis of ethyl 2-((5-(2-fluorophenyl)-2-nitropyridin-3-yl)oxy)propionate (VI-5)

Dissolve IV-1 (1.00g, 3.13mmol) and 2-fluorophenylboronic acid (V-5) (0.66g, 4.70mmol) in 20mL of tetrahydrofuran, dissolve potassium carbonate (1.22g, 8.77mmol) with 2mL of water to prepare carbonic acid Potassium solution was added dropwise to the reaction solution, then tetrakis(triphenylphosphine)palladium (0.36g, 0.33mmol) was added, and the reaction was refluxed under nitrogen protection for 24h. TLC (petroleum ether: ethyl acetate = 4:1) was used to detect the reaction of raw materials completely. Stop heating, cool to room temperature, evaporate the solvent under reduced pressure, add 15 mL ethyl acetate and 20 mL to separate the layers, extract the aqueous phase with ethyl acetate once (15 mL), combine the organic phases, and wash with saturated sodium chloride (15 mL×3 ), dried over anhydrous magnesium sulfate, filtered with suction, evaporated under reduced pressure to remove the solvent, and purified by column chromatography to obtain 0.83 g of light yellow solid, yield: 79.32%, mp 106-109°C. ¹ H NMR (300MHz, CDCl ₃ ), δ (ppm): 8.30 (d, J = 1.2 Hz, 1H, ArH), 7.62 (d, J = 1.5 Hz, 1H, ArH), 7.50-7.43 (m, 2H ,ArH),7.33(dd,J=7.8,1.3Hz,1H,ArH),7.27–7.20(m,1H,ArH),4.92(q,J=6.8Hz,1H,CH ₃ CH ), 4.26(q , J = 7.1Hz, 2H, CH ₃ CH ₂ ), 1.74 (d, J = 6.8 Hz, 3H, CH CH ₃ ), 1.27 (t, J = 7.1 Hz, 3H, CH ₂ CH ₃ ).

Synthesis of 2-((5-(2-fluorophenyl)-2-nitropyridin-3-yl)oxy)propionic acid (VII-5)

VI-5 (0.83 g, 2.48 mmol) was dissolved in 15 mL methanol and 55 mL water, 3 mL 20% sodium hydroxide solution was added, and the reaction was heated to 70° C. for 1 h. TLC detected the complete reaction of the raw materials. The heating was stopped, the reaction system was cooled to room temperature, 30mL ethyl acetate was added for extraction, the aqueous phase was collected, adjusted to pH 5 with 6mol/L hydrochloric acid solution, extracted with ethyl acetate (30mL×2), dried with anhydrous magnesium sulfate, filtered The solvent was evaporated under reduced pressure to obtain 0.30 g of light yellow solid, yield: 39.50%, mp 165-168°C. ¹ H NMR (300MHz, DMSO-d ₆ ) δ (ppm): 13.40 (s, 1H, COOH), 8.33 (s, 1H, ArH), 8.04 (s, 1H, ArH), 7.68 (t, J = 7.8 Hz, 1H, ArH), 7.56 (t, J = 7.1 Hz, 1H, ArH), 7.47–7.35 (m, 2H, ArH), 5.40 (q, J = 6.8 Hz, 1H, CH ₃ CH ), 1.55 ( d, J = 6.5 Hz, 3H, CH CH ₃ ).

2-(1-((5-(2-Fluorophenyl)-2-nitropyridin-3-yl)oxy)ethyl)-N-methyl-1H-benzo[d]imidazole-4- Synthesis of formamide (IX-5)

Compound VII-5 (0.44g, 1.43mmol), VIII-1 (0.22g, 1.43mmol) and HATU (0.54g, 1.43mmol) were dissolved in 6mL N,N-dimethylformamide, and DIPEA (5.03 mL, 2.86 mmol), the reaction solution was placed at room temperature and stirred for 5 hours, TLC (petroleum ether: ethyl acetate = 1:1) detected the complete reaction of the raw materials. The reaction system was added dropwise to 35mL of water, a white solid precipitated out, filtered, the filter cake was washed with a small amount of water, dried to obtain a brownish-yellow solid, dissolved in 10mL of glacial acetic acid, refluxed at 120℃ for 1h, TLC (petroleum ether: ethyl acetate = 1:1) Check that the reaction of the raw materials is complete, stop heating, cool the reaction system to room temperature, add 20mL of water to dilute, extract with ethyl acetate (10mL×3), combine the organic phases, and wash with saturated sodium chloride solution (10mL×3). Water magnesium sulfate drying. Suction filtration, evaporation of the solvent under reduced pressure, purification by column chromatography to obtain 0.11 g of light yellow solid, yield: 32.81%, mp 196-199°C. ¹ H NMR (400MHz, DMSO-d ₆ ) δ (ppm): 13.26 (s, 1H, NH), 9.48 (d, J = 4.8 Hz, 1H, CO NH CH ₃ ), 8.41 (s, 1H, ArH) ,8.34(s,1H,ArH),7.86(d,J=7.7Hz,1H,ArH),7.73-7.62(m,2H,ArH),7.57(d,J=6.4Hz,1H,ArH),7.42 –7.35(m,3H,ArH),6.32(q,J=6.4Hz,1H,CH ₃ CH ), 2.88(d,J=4.7Hz,3H,NH CH ₃ ), 1.87(d,J=6.5Hz ,3H,CH CH ₃ ).

2-(1--((2-Amino-5-(2-fluorophenyl)pyridin-3-yl)oxy)ethyl)-N-methyl-1H-benzo[d]imidazole-4-methyl Synthesis of amide (I-5)

Dissolve IX-5 (0.11g, 0.25mmol) in 10mL of tetrahydrofuran, add 0.02g of 10% Pd-C, ventilate with hydrogen for three times, and let it react at room temperature. TLC (dichloromethane: methanol = 25:1) detects After the reaction of the raw materials is completed, filter, evaporate the solvent under reduced pressure, and purify by column chromatography to obtain 15 mg of white solid, yield: 14.80%, mp201-204°C. ¹ H NMR (400MHz, DMSO-d ₆ ) δ (ppm): 13.18 (s, 1H, NH), 9.72-9.51 (m, 1H, CO NH CH ₃ ), 7.87 (d, J = 7.3Hz, 1H, ArH), 7.79 (s, 1H, ArH), 7.72 (d, J = 8.0 Hz, 1H, ArH), 7.55-7.43 (m, 2H, ArH), 7.39-7.29 (m, 2H, ArH), 7.28- 7.20 (m, 2H, ArH), 6.18 (s, 2H, ArNH ₂ ), 5.99 (q, J = 6.0 Hz, 1H, CH ₃ CH ), 2.95 (d, J = 3.8 Hz, 3H, NH CH ₃ ) ,1.78(d,J=6.2Hz,3H,CH CH ₃ ).HR-MS(ESI)m/z[M+H] ⁺ Calcd for C ₂₂ H ₂₁ FN ₅ O ₂ ,406.1601; Found: 406.1680.

Example 6 Synthesis of Compound I-6

2-(1--((2-Amino-5-(4-ethylphenyl)pyridin-3-yl)oxy)ethyl)-N-methyl-1H-benzo[d]imidazole-4- Synthesis of formamide (I-6: R ¹ ＝CONHCH ₃ , R ² ＝CH ₃ , R ³ ＝4-ethylphenyl)

Synthesis of 2-((5-(4-ethylphenyl)-2-nitropyridin-3-yl)oxy) ethyl propionate (VI-6)

Dissolve IV-1 (1.00g, 3.13mmol) and 4-ethylphenylboronic acid (V-6) (0.70g, 4.70mmol) in 20mL of tetrahydrofuran, use 2mL of water to dissolve potassium carbonate (1.22g, 8.77mmol) Potassium carbonate solution was added dropwise to the reaction solution, then tetrakis(triphenylphosphine)palladium (0.36g, 0.33mmol) was added, and the reaction was refluxed under nitrogen protection for 24h. TLC (petroleum ether: ethyl acetate = 4:1) detected the raw material The reaction is complete. Stop heating, cool to room temperature, evaporate the solvent under reduced pressure, add 15 mL ethyl acetate and 20 mL to separate the layers, extract the aqueous phase with ethyl acetate once (15 mL), combine the organic phases, and wash with saturated sodium chloride (15 mL×3 ), dried over anhydrous magnesium sulfate, filtered with suction, evaporated under reduced pressure to remove the solvent, and purified by column chromatography to obtain 0.87 g of light yellow solid, yield: 80.71%, mp 75-78°C. ¹ H NMR(300MHz, CDCl ₃ )δ(ppm): 8.35(d,J=1.7Hz,1H,ArH), 7.56(d,J=1.7Hz,1H,ArH),7.49(d,J=8.2Hz ,2H,ArH),7.37(d,J=8.1Hz,2H,ArH),4.94(q,J=6.8Hz,1H,CH ₃ CH ), 4.26(q,J=7.1Hz,2H,O CH ₂ ), 2.75 (q, J = 7.6 Hz, 2H, Ar CH ₂ CH ₃ ), 1.75 (d, J = 6.8 Hz, 3H, CH CH ₃ ), 1.31 (t, J = 7.6 Hz, 3H, ArCH ₂ CH ₃ ), 1.27 (t, J = 7.1 Hz, 3H, OCH ₂ CH ₃ ).

Synthesis of 2-((5-(4-ethylphenyl)-2-nitropyridin-3-yl)oxy)propionic acid (VII-6)

VI-6 (0.87 g, 2.53 mmol) was dissolved in 15 mL methanol and 55 mL water, 3 mL 20% sodium hydroxide solution was added, and the reaction was heated to 70° C. for 1 h. TLC detected that the raw material had reacted completely. The heating was stopped, the reaction system was cooled to room temperature, 30mL ethyl acetate was added for extraction, the aqueous phase was collected, adjusted to pH 5 with 6mol/L hydrochloric acid solution, extracted with ethyl acetate (30mL×2), dried with anhydrous magnesium sulfate, filtered The solvent was evaporated under reduced pressure to obtain 0.65 g of light yellow solid, yield: 81.22%, mp 172-175°C. ¹ H NMR (300MHz, DMSO-d ₆ ) δ (ppm): 13.42 (s, 1H, COOH), 8.43 (d, J = 1.7 Hz, 1H, ArH), 8.05 (d, J = 1.4 Hz, 1H, ArH), 7.74 (d, J = 8.2 Hz, 2H, ArH), 7.39 (d, J = 8.2 Hz, 2H, ArH), 5.50 (q, J = 6.8 Hz, 1H, CH ₃ CH ), 2.68 (q , J=7.4Hz, 2H, CH ₃ CH ₂ ), 1.56(d, J=6.8Hz, 3H, CH CH ₃ ), 1.22(t, J=7.6Hz, 3H, CH ₂ CH ₃ ).

2-(1-(5-(4-ethylphenyl)-2-nitro-pyridin-3-yl)oxy)ethyl)-N-methyl-1H-benzo[d]imidazole-4 -Synthesis of formamide (IX-6)

Compound VII-6 (0.65g, 2.05mmol), VIII-1 (0.31g, 2.05mmol) and HATU (0.78g, 2.05mmol) were dissolved in 6mL N,N-dimethylformamide, and DIPEA (7.27 mL, 4.10 mmol), the reaction solution was placed at room temperature and stirred for 5 hours, TLC (petroleum ether: ethyl acetate = 1:1) detected the complete reaction of the raw materials. The reaction system was added dropwise to 35mL of water, a white solid precipitated out, filtered, the filter cake was washed with a small amount of water, dried to obtain a brownish-yellow solid, dissolved in 10mL of glacial acetic acid, refluxed at 120℃ for 1h, TLC (petroleum ether: ethyl acetate = 1:1) Check that the reaction of the raw materials is complete, stop heating, cool the reaction system to room temperature, add 20mL of water to dilute, extract with ethyl acetate (10mL×3), combine the organic phases, and wash with saturated sodium chloride solution (10mL×3). Water magnesium sulfate drying. Suction filtration, evaporation of the solvent under reduced pressure, purification by column chromatography to obtain 0.53 g of light yellow solid, yield: 66.47%, mp 200-203°C. ¹ H NMR (400MHz, DMSO-d ₆ ) δ (ppm): 13.27 (s, 1H, NH), 9.49 (q, J = 4.3 Hz, 1H, CO NH CH ₃ ), 8.44 (d, J = 2.2 Hz ,2H,ArH),7.85(dd,J=7.5,0.9Hz,1H,ArH),7.74-7.66(m,3H,ArH),7.40-7.35(m,2H,ArH),7.34(d,J= 7.8Hz, 1H, ArH), 6.41 (q, J = 6.4 Hz, 1H, CH ₃ CH ), 2.88 (d, J = 4.8 Hz, 3H, NH CH ₃ ), 2.67 (q, J = 7.6 Hz, 2H ,CH ₃ CH ₂ ), 1.88 (d, J = 6.5 Hz, 3H, CH CH ₃ ), 1.21 (t, J = 7.6 Hz, 3H, CH ₂ CH ₃ ).

2-(1--((2-Amino-5-(4-ethylphenyl)pyridin-3-yl)oxy)ethyl)-N-methyl-1H-benzo[d]imidazole-4- Synthesis of formamide (I-6)

Dissolve IX-6 (0.53g, 1.19mmol) in 20mL of tetrahydrofuran, add 0.05g of 10% Pd-C, ventilate with hydrogen for three times, then stand at room temperature for reaction, TLC (dichloromethane: methanol = 25:1) detection After the reaction of the raw materials, filter, evaporate the solvent under reduced pressure, and purify by column chromatography to obtain 100 mg of off-white solid, yield: 20.22%, mp 200-203°C. ¹ H NMR (400MHz, DMSO-d ₆ ) δ (ppm): 13.17 (s, 1H, NH), 9.62 (q, J = 4.5 Hz, 1H, CO NH CH ₃ ), 7.86 (dd, J = 6.0, 1.5Hz, 2H, ArH), 7.71 (dd, J = 8.0 Hz, 1.0 Hz, 1H, ArH), 7.59 (d, J = 1.8 Hz, 1H, ArH), 7.47 (d, J = 8.2 Hz, 2H, ArH),7.35(t,J=7.8Hz,1H,ArH),7.21(d,J=8.2Hz,2H,ArH),6.07(q,J=6.4Hz,1H,CH ₃ CH ),6.03(s ,2H,ArNH ₂ ),2.96(d,J=4.7Hz,3H,NH CH ₃ ), 2.60(q,J=7.6Hz,2H,CH ₃ CH ₂ ),1.78(d,J=6.5Hz,3H ,CH CH ₃ ),1.18(t,J=7.6Hz,3H,CH ₂ CH ₃ ).HR-MS(ESI)m/z[M+H] ⁺ Calcd for C ₂₄ H ₂₆ N ₅ O ₂ ,416.2008 ;Found:416.2081.

Example 7 Synthesis of Compound I-7

2-(1--((2-Amino-5-(4-methoxyphenyl)pyridin-3-yl)oxy)ethyl)-N-methyl-1H-benzo[d]imidazole-4 -Synthesis of formamide (I-7: R ¹ ＝CONHCH ₃ , R ² ＝CH ₃ , R ³ ＝4-methoxyphenyl)

Synthesis of ethyl 2-((5-(4-methoxyphenyl)-2-nitropyridin-3-yl)oxy)propionate (VI-7)

Dissolve IV-1 (1.00g, 3.13mmol) and 4-methoxyphenylboronic acid (V-7) (0.72g, 4.70mmol) in 20mL of tetrahydrofuran, use 2mL of water to dissolve potassium carbonate (1.22g, 8.77mmol) Prepare potassium carbonate solution and add it dropwise to the reaction solution, then add tetrakis(triphenylphosphine) palladium (0.36g, 0.33mmol), reflux under nitrogen protection for 24h, TLC (petroleum ether: ethyl acetate = 4:1) detection The raw material reaction is complete. Stop heating, cool to room temperature, evaporate the solvent under reduced pressure, add 15 mL ethyl acetate and 20 mL to separate the layers, extract the aqueous phase with ethyl acetate once (15 mL), combine the organic phases, and wash with saturated sodium chloride (15 mL×3 ), dried over anhydrous magnesium sulfate, filtered with suction, evaporated under reduced pressure to remove the solvent, and purified by column chromatography to obtain 0.85 g of light yellow solid, yield: 78.41%, mp 72-75°C. ¹ H NMR (300MHz, CDCl ₃ ) δ (ppm): 8.30 (s, 1H, ArH), 7.51 (s, 2H, ArH), 7.48 (s, 1H, ArH), 7.03 (d, J = 8.6 Hz, 2H, ArH), 4.92 (q, J = 6.4 Hz, 1H, CH ₃ CH ), 4.24 (q, J = 7.1 Hz, 2H, CH ₃ CH ₂ ), 3.88 (s, 3H, ArO CH ₃ ), 1.72 (d, J = 6.9 Hz, 3H, CH CH ₃ ), 1.25 (t, J = 7.1 Hz, 3H, CH ₂ CH ₃ ).

Synthesis of 2-((5-(4-methoxyphenyl)-2-nitropyridin-3-yl)oxy)propionic acid (VII-7)

Dissolve VI-7 (0.85 g, 2.45 mmol) in 15 mL methanol and 55 mL water, add 3 mL 20% sodium hydroxide solution, and heat to 70° C. to react for 1 hour. TLC detects that the raw material has reacted completely. The heating was stopped, the reaction system was cooled to room temperature, 30mL ethyl acetate was added for extraction, the aqueous phase was collected, adjusted to pH 5 with 6mol/L hydrochloric acid solution, extracted with ethyl acetate (30mL×2), dried with anhydrous magnesium sulfate, filtered The solvent was evaporated under reduced pressure to obtain 0.71 g of yellow solid, yield: 91.10%, mp168-171°C. ¹ H NMR (300MHz, DMSO-d ₆ ) δ (ppm): 13.37 (s, 1H, COOH), 8.42 (d, J = 1.7 Hz, 1H, ArH), 8.03 (d, J = 1.8 Hz, 1H, ArH),7.80(d,J=8.7Hz,2H,ArH),7.12(d,J=8.8Hz,2H,ArH),5.52(q,J=6.8Hz,1H,CH ₃ CH ), 3.83(s ,3H,OCH ₃ ),1.56(d,J=6.8Hz,3H,CH CH ₃ ).

2-(1-((5-(4-methoxyphenyl)-2-nitropyridin-3-yl)oxy)ethyl)-N-methyl-1H-benzo[d]imidazole- Synthesis of 4-formamide (IX-7)

Compound VII-7 (0.71g, 2.23mmol), VIII-1 (0.34g, 2.23mmol) and HATU (0.85g, 2.23mmol) were dissolved in 6mL N,N-dimethylformamide, and DIPEA (7.90 mL, 4.46 mmol), the reaction solution was placed at room temperature and stirred for 5 hours, TLC (petroleum ether: ethyl acetate = 1:1) detected the complete reaction of the raw materials. The reaction system was added dropwise to 35mL of water, a white solid precipitated out, filtered, the filter cake was washed with a small amount of water, dried to obtain a brownish-yellow solid, dissolved in 10mL of glacial acetic acid, refluxed at 120℃ for 1h, TLC (petroleum ether: ethyl acetate = 1:1) Check that the reaction of the raw materials is complete, stop heating, cool the reaction system to room temperature, add 20mL of water to dilute, extract with ethyl acetate (10mL×3), combine the organic phases, and wash with saturated sodium chloride solution (10mL×3). Water magnesium sulfate drying. Suction filtration, evaporation of the solvent under reduced pressure, purification by column chromatography to obtain 0.40 g of light yellow solid, yield: 47.43%, mp 202-204°C. ¹ H NMR (400MHz, DMSO-d ₆ ) δ (ppm): 13.23 (s, 1H, NH), 9.61 (s, 1H, CO NH CH ₃ ), 8.73 (d, J = 6.1Hz, 1H, ArH) ,8.60(d,J=6.1Hz,2H,ArH), 8.20(d,J=1.9Hz,1H,ArH), 8.04(d,J=1.1Hz,1H,ArH),7.78(d,J=7.4 Hz, 1H, ArH), 7.68 (dd, J = 4.5, 1.5 Hz, 2H, ArH), 7.25 (t, J = 7.7 Hz, 1H, ArH), 6.06 (q, J = 6.4 Hz, 1H, CH ₃ CH ), 3.94 (s, 3H, O CH ₃ ), 2.92 (d, J = 4.7 Hz, 3H, NH CH ₃ ), 1.85 (d, J = 6.4 Hz, 3H, CH CH ₃ ).

2-(1--((2-Amino-5-(4-methoxyphenyl)pyridin-3-yl)oxy)ethyl)-N-methyl-1H-benzo[d]imidazole-4 -Synthesis of formamide (I-7)

Dissolve IX-7 (0.40g, 0.96mmol) in 20mL of tetrahydrofuran, add 0.05g of 10% Pd-C, ventilate with hydrogen three times, and let it react at room temperature. TLC (dichloromethane: methanol = 25:1) detects After the reaction of the raw materials is completed, filter, evaporate the solvent under reduced pressure, and purify by column chromatography to obtain 200 mg of white solid, yield: 49.90%, mp 184-187°C. ¹ H NMR (300MHz, DMSO-d ₆ ) δ (ppm): 13.19 (s, 1H, NH), 9.63 (q, J = 4.6 Hz, 1H, CO NH CH ₃ ), 7.92–7.78 (m, 2H, ArH), 7.72(d,J=7.8Hz,1H,ArH),7.58(s,1H,ArH),7.50(d,J=8.7Hz,2H,ArH),7.35(t,J=7.8Hz,1H ,ArH),6.95(d,J=8.8Hz,2H,ArH),6.16-5.89(m,3H,Ar NH ₂ ,CH ₃ CH ),3.77(s,3H,OCH ₃ ),2.96(d,J ＝4.5Hz,3H,NH CH ₃ ),1.77(d,J＝6.4Hz,3H,CH CH ₃ ).HR-MS(ESI)m/z[M+H] ⁺ Calcd for C ₂₃ H ₂₄ N ₅ O ₃ ,418.1801; Found: 418.1880.

Example 8 Synthesis of Compound I-8

2-(1-((2-amino-5-(1-methyl-1H-pyrazol-3-yl)pyridin-3-yl)oxy)ethyl)-N-methyl-1H-benzo [d] Synthesis of imidazole-4-carboxamide (I-8: R ¹ ＝CONHCH ₃ , R ² ＝CH ₃ , R ³ ＝1-methylpyrazol-3-yl)

Synthesis of 2-((5-(1-methyl-1H-pyrazol-3-yl)-2-nitropyridin-3-yl)oxy) ethyl propionate (VI-8)

Dissolve IV-1 (1.00g, 3.13mmol) and 1-methylpyrazole-4 boronic acid (V-8) (0.59g, 4.70mmol) in 20mL of tetrahydrofuran, use 2mL of water to dissolve potassium carbonate (1.22g, 8.77 mmol) Prepare potassium carbonate solution, add it dropwise to the reaction solution, then add tetrakis(triphenylphosphine) palladium (0.36g, 0.33mmol), nitrogen protection reflux reaction for 24h, TLC (petroleum ether: ethyl acetate = 4:1 ) It is detected that the raw material reaction is complete. Stop heating, cool to room temperature, evaporate the solvent under reduced pressure, add 15 mL ethyl acetate and 20 mL to separate the layers, extract the aqueous phase with ethyl acetate once (15 mL), combine the organic phases, and wash with saturated sodium chloride (15 mL×3 ), dried over anhydrous magnesium sulfate, filtered with suction, evaporated under reduced pressure to remove the solvent, and purified by column chromatography to obtain 0.50 g of light yellow solid, yield: 49.87%, mp 105-108°C. ¹ H NMR (300MHz, CDCl ₃ ), δ (ppm): 8.25 (s, 1H, ArH), 7.80 (d, J = 1.4 Hz, 1H, ArH), 7.75 (d, J = 1.4 Hz, 1H, ArH ),7.45(s,1H,ArH),4.91(q,J=6.6Hz,1H,CH ₃ CH ), 4.26(q,J=7.1Hz,2H,CH ₃ CH ₂ ),4.01(s,3H, NCH ₃ ), 1.74 (d, J = 6.8 Hz, 3H, CH CH ₃ ), 1.27 (t, J = 7.1 Hz, 3H, CH ₂ CH ₃ ).

Synthesis of 2-((5-(1-methyl-1H-pyrazol-3-yl)-2-nitropyridin-3-yl)oxy)propionic acid (VII-8)

VI-8 (1.13 g, 3.35 mmol) was dissolved in 15 mL methanol and 55 mL water, 3 mL 20% sodium hydroxide solution was added, and the reaction was heated to 70° C. for 1 h. TLC detected the complete reaction of the raw materials. The heating was stopped, the reaction system was cooled to room temperature, 30mL ethyl acetate was added for extraction, the aqueous phase was collected, adjusted to pH 5 with 6mol/L hydrochloric acid solution, extracted with ethyl acetate (30mL×2), dried with anhydrous magnesium sulfate, filtered , The solvent was distilled off under reduced pressure to obtain 0.64 g of brown-yellow solid, yield: 62.04%, mp 242-244°C. ¹ H NMR(300MHz,DMSO-d ₆ )δ(ppm): 8.20-8.08(m,2H,ArH), 7.53(d,J=7.4Hz,1H,ArH), 7.24(d,J=7.1Hz, 1H, ArH), 6.63 (q, J = 7.8 Hz, 1H, CH ₃ CH ), 3.94 (s, 3H, NCH ₃ ), 2.24 (d, J = 3.7 Hz, 3H, CH CH ₃ ).

N-methyl-2-(1-(5-((1-methyl-1H-pyrazol-3-yl)-2-nitro-pyridin-3-yl)oxy)ethyl)-1H- Synthesis of Benzo[d]imidazole-4-carboxamide (IX-8)

Compound VII-8 (0.64g, 2.19mmol), VIII-1 (0.36g, 2.19mmol) and HATU (0.83g, 2.19mmol) were dissolved in 6mL N,N-dimethylformamide, and DIPEA (7.76 mL, 4.38 mmol), the reaction solution was placed at room temperature and stirred for 5 hours, TLC (petroleum ether: ethyl acetate = 1:1) detected the complete reaction of the raw materials. The reaction system was added dropwise to 35mL of water, a white solid precipitated out, filtered, the filter cake was washed with a small amount of water, dried to obtain a brownish-yellow solid, dissolved in 10mL of glacial acetic acid, refluxed at 120℃ for 1h, TLC (petroleum ether: ethyl acetate = 1:1) Check that the reaction of the raw materials is complete, stop heating, cool the reaction system to room temperature, add 20mL of water to dilute, extract with ethyl acetate (10mL×3), combine the organic phases, and wash with saturated sodium chloride solution (10mL×3). Water magnesium sulfate drying. Suction filtration, evaporation of the solvent under reduced pressure, purification by column chromatography to obtain 0.21 g of light yellow solid, yield: 31.74%, mp 180-183°C. ¹ H NMR (300MHz, DMSO-d ₆ ) δ (ppm): 13.22 (s, 1H, NH), 9.52 (s, 1H, CO NH CH ₃ ), 8.43-8.33 (m, 3H, ArH), 8.10 ( s, 1H, ArH), 7.85 (d, J = 8.4 Hz, 1H, ArH), 7.68 (d, J = 7.1 Hz, 1H, ArH), 7.40-7.27 (m, 1H, ArH), 6.32 (q, J = 6.4Hz, 1H, CH ₃ CH ), 3.89 (s, 3H, N CH ₃ ), 2.93 (d, J = 2.2 Hz, 3H, NH CH ₃ ), 1.87 (d, J = 4.8 Hz, 3H, CH CH ₃ ).

2-(1-((2-amino-5-(1-methyl-1H-pyrazol-3-yl)pyridin-3-yl)oxy)ethyl)-N-methyl-1H-benzo [d] Synthesis of imidazole-4-carboxamide (I-8)

Dissolve IX-8 (0.21g, 0.50mmol) in 10mL of tetrahydrofuran, add 0.02g of 10% Pd-C, ventilate with hydrogen three times, and let it react at room temperature. TLC (dichloromethane: methanol = 25:1) detects After the reaction of the raw materials is completed, filter, evaporate the solvent under reduced pressure, and purify by column chromatography to obtain 10 mg of brown solid, yield: 5.11%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ (ppm): 13.17 (s, 1H, NH), 9.62 (q, J = 4.1 Hz, 1H, CO NH CH ₃ ), 7.99 (s, 1H, ArH) ,7.87(d,J=7.6Hz,1H,ArH),7.82(d,J=1.6Hz,1H,ArH),7.75-7.69(m,2H,ArH),7.55(d,J=1.4Hz,1H ,ArH),7.36(t,J=7.8Hz,1H,ArH),6.03(q,J=6.4Hz,1H,CH ₃ CH ),5.91(s,2H,ArNH ₂ ),3.83(s,3H, NCH ₃ ), 2.98(d,J=4.7Hz,3H,NH CH ₃ ),1.75(d,J=6.4Hz,3H,CH CH ₃ ).HR-MS(ESI)m/z[M+Na] ⁺ Calcd for C ₂₀ H ₂₁ N ₇ O ₂ Na,414.1757; Found:414.1652.

Example 9

2-(1-((2-amino-5-(4-methylphenyl)pyridin-3-yl)oxy)ethyl)-1H-benzo(d)imidazole-4-carboxamide (I- 9: ^{Synthesis of R 1} ＝CONH ₂ , R ² ＝CH ₃ , R ³ ＝4-methylphenyl)

2-(1-((2-nitro-5-(4-methylphenyl)pyridin-3-yl)oxy)ethyl)-1H-benzo[d]imidazole-4-carboxamide (IX -9) Synthesis

Compound VII-1 (0.81g, 2.68mmol), VIII-2 (0.40g, 2.68mmol) and HATU (1.02g, 2.68mmol) were dissolved in 6mL N,N-dimethylformamide, and DIPEA (9.6 mL, 5.36 mmol), the reaction solution was placed at room temperature and stirred for 5 h, TLC (petroleum ether: ethyl acetate = 1:1) detected the complete reaction of the raw materials. The reaction system was added dropwise to 35mL of water, a white solid precipitated out, filtered, the filter cake was washed with a small amount of water, dried to obtain a brownish-yellow solid, dissolved in 10mL of glacial acetic acid, refluxed at 120℃ for 1h, TLC (petroleum ether: ethyl acetate = 1:1) Check that the reaction of the raw materials is complete, stop heating, cool the reaction system to room temperature, add 20mL of water to dilute, extract with ethyl acetate (10mL×3), combine the organic phases, and wash with saturated sodium chloride solution (10mL×3). Water magnesium sulfate drying. Suction filtration, evaporation of the solvent under reduced pressure, column chromatography purification to obtain 0.95 g of yellow solid, yield: 98.95%, mp 180.4-182.1°C. ¹ H NMR (300MHz, DMSO-d ₆ ) δ (ppm): 13.26 (s, 1H, NH), 9.11 (s, 1H, CO NH ₂ ), 8.48-8.34 (m, 2H, ArH), 7.91-7.81 (m, 2H, ArH, CO NH ₂ ), 7.69 (dt, J = 8.0, 4.1 Hz, 3H, ArH), 7.37 (d, J = 8.0 Hz, 3H, ArH), 6.39 (q, J = 6.6 Hz ,1H,CH ₃ CH ), 2.38(s,3H,ArCH ₃ ), 1.87(d,J=6.4Hz,3H,CH CH ₃ ).

2-(1-((2-amino-5-(4-methylphenyl)pyridin-3-yl)oxy)ethyl)-1H-benzo(d)imidazole-4-carboxamide (I- 9) Synthesis

Dissolve IX-9 (0.95 g, 2.28 mmol) in 10 mL of tetrahydrofuran, add 0.10 g of 10% Pd-C, ventilate with hydrogen three times, and let it react at room temperature. TLC (dichloromethane: methanol = 25:1) detects After the reaction of the raw materials is completed, filter, evaporate the solvent under reduced pressure, and purify by column chromatography to obtain 780 mg of white solid, yield: 88.30%, purity: 99.85%, mp 227.5-228.1°C. ¹ H NMR (300MHz, DMSO-d ₆ ) δ (ppm): 13.14 (s, 1H, NH), 9.18 (s, 1H, CONH ₂ ), 7.89-7.72 (m, 3H, ArH), 7.69 (d, J = 7.2Hz, 1H, CONH ₂ ), 7.57-7.47 (m, 1H, ArH), 7.41 (d, J = 8.1 Hz, 2H, ArH), 7.32 (t, J = 7.7 Hz, 1H, ArH), 7.16(d,J=8.1Hz,2H,ArH),6.08–5.89(m,3H,ArNH ₂ , CH CH ₃ ), 2.27(s,3H,ArCH ₃ ), 1.73(d,J=6.4Hz,3H ,CH CH ₃ ).HR-MS(ESI)m/z[M+H] ⁺ Calcd for C ₂₂ H ₂₂ N ₅ O ₂ ,388.1695; Found: 388.1773.

Example 10

The pharmacological experiments and results of some of the compounds prepared above are as follows:

1. ROS1, ALK and c-Met enzyme inhibitory activity experiment

①Experimental method

The Caliper Mobility Shift Assay method was used to detect the inhibitory effects of the compounds on the kinases ROS1, ALK and c-Met. The compound test started with a concentration of 10 μM or 5 μM, and a 3-fold dilution of 7 or 8 concentrations. Use a dispenser Echo 550 to transfer 250 nL of 100-fold final concentration compound to a 384-well reaction plate, add 10 μL of kinase solution with a final concentration of 1.25 nM ALK or 1.25 nM c-Met or 0.3 nM ROS1, and pre-incubate at room temperature for 10 minutes (negative The control well contains 10 μL kinase buffer and 250 nL 100% DMSO; the positive control well contains 10 μL kinase solution and 250 nL 100% DMSO). Add 15μL of ATP with a final concentration of 30μM and 3μM substrate No. 22 peptide mixed solution to ALK to start the reaction, and react at room temperature for 25 minutes; add 15μL of ATP with a final concentration of 40.84μM and 3μM substrate No. 2 peptide on c-Met Start the reaction with the mixed solution and react at room temperature for 60 minutes; add 15μL of ATP with a final concentration of 26.7μM and 3μM substrate peptide No. 22 mixed solution to ROS1 to start the reaction, react at room temperature for 15 minutes, add 30μL of EDTA-containing stop detection solution to stop Kinase reaction. Use Caliper EZ Reader to read the conversion rate. Conversion inhibition rate%=(average positive control conversion rate%-sample conversion rate%/(average positive control conversion rate%-negative control conversion rate%). Among them: negative control wells represent the conversion rate readings without enzyme active wells; Positive control wells represent the conversion rate readings of wells without compound inhibition. Take the log value of the concentration as the X-axis, and the percentage inhibition rate on the Y-axis, using the analysis software GraphPad Prism 5 log(inhibitor) vs.response-Variable slope fitting amount Efficacy curve, thereby obtaining the IC ₅₀ value of each compound on the enzyme activity.

Calculation formula: Y=Bottom+(Top-Bottom)/(1+10^((LogIC ₅₀ -X)*HillSlope)).

②Test results:

In vitro ROS1, c-Met, and ALK inhibitory activities were screened for some compounds of the present invention, and the results are shown in Table 1.

IC ₅₀ : 0.01～0.5 μM (denoted as: A); IC ₅₀ : 0.5～5.0 _{μM (denoted as: B); IC 50} : >5.0 μM (denoted as: C).

Table 1. Inhibitory activity of some compounds on ROS1, c-Met and ALK

The results in Table 1 show that the compounds of the present invention have good inhibitory activity on ROS1, and weaker activity on ALK and c-Met. Among them, compounds I-1, I-2, I-3, I-6, I- 7 and I-9 have higher selectivity to ROS1.

2. Ba/F3-CD74-ROS1 cell anti-proliferation experiment

①Experimental method

Cell culture: Resuspend the cells and count them with an automatic cell counter. According to the Ba/F3 seeding density, 2000 cells per well, the cell suspension is diluted to the required density. Pour 95μL of cells in each well and incubate at 37°C for a stable equilibrium.

Compound preparation: Dissolve the compound in DMSO, prepare a 20mM stock solution, and store at -20°C in the dark until use. After the cells are cultured, the compound is prepared into a diluted solution with a final concentration of 200 times. The compound was diluted with culture medium to prepare a compound of 20 times the final concentration. Add 5 μL of compound to each well, use the same volume of DMSO as the control, and incubate at 37° C., 5% CO _{2 for} 72 hours.

Cell detection: equilibrate the cell plate to room temperature. Add 40μL per well

Reagent, shake for 2 minutes and let stand for 10 minutes. Use SpectraMax Paradigm to detect.

data analysis:

Calculated using GraphPad Prism 5 IC _50. Among them, Max signal is the positive control well, with only the same volume of DMSO as the compound. Min signal is a negative control well with only medium.

②Test results:

Some compounds of the present invention were screened for the inhibitory activity of Ba/F3-CD74-ROS1 cells, and the results are shown in Table 2. IC ₅₀ :10.0～100.0nM (denoted as: A); IC ₅₀ : 100.0～1000.0nM (denoted as: B); IC ₅₀ :>1000nM (denoted as: C).

Table 2 Inhibitory activity of some compounds on Ba/F3-CD74-ROS1 cells

The results in Table 2 show that the compounds of the present invention have good inhibitory activity on cells with high ROS1 expression.

Claims (10)

1. A compound represented by general formula (I) or a pharmaceutically acceptable salt thereof:

   

   in:

   R ¹ represents H, COOH, COOR ⁴ or CONHR ⁴ , wherein R ⁴ represents hydrogen, C ₁ ～C ₆ alkyl;

   R ² represents hydrogen, a C ₁ ～C ₅ alkyl group;

   R ³ represents an optionally substituted benzene ring or aromatic heterocyclic ring, the aromatic heterocyclic ring is a six-membered or five-membered aromatic heterocyclic ring, the substituent is a halogen, a C ₁ ～C ₃ alkyl, alkoxy, Halogenated alkyl, halogenated alkoxy, OH, NR ⁵ R ⁶ or CN, where R ⁵ and R ⁶ represent hydrogen and C ₁ ～C ₆ alkyl.
2. The compound of general formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein R ¹ represents H, COOH, COOR ⁴ or CONHR ⁴ , wherein R ⁴ represents hydrogen, C ₁ ～C ₆ alkyl;

   R ² represents hydrogen, a C ₁ ～C ₃ alkyl group;

   R ³ represents an optionally substituted benzene ring or aromatic heterocyclic ring, the aromatic heterocyclic ring is a six-membered or five-membered aromatic heterocyclic ring, and the substituents are F, Cl, Br, I, CH ₃ , C ₂ H ₅ , OH, NR ⁵ R ⁶ , OCH ₃ , OCF ₃ , CF ₃ or CN, where R ⁵ and R ⁶ represent hydrogen and C ₁ ～C ₆ alkyl.
3. The compound according to claim 2 or a pharmaceutically acceptable salt thereof, characterized in that R ¹ represents CONH ₂ or CONHCH ₃ ; R ² represents H or CH ₃ , R ³ represents an optionally substituted phenyl group, and the substituent is F, Cl, Br, CH ₃ , CH ₂ CH ₃ , OH, NH ₂ , OCH ₃ , OCF ₃ or CF ₃ .
4. The compound according to claim 2 or a pharmaceutically acceptable salt thereof, characterized in that R ¹ is CONH ₂ or CONHCH ₃ , R ² is CH ₃ , and R ³ is F, methyl, ethyl or methoxy substituted benzene base.
5. The compound according to claim 2 or a pharmaceutically acceptable salt thereof, characterized in that the compound of general formula (I) is selected from the following compounds:

   

   
6. The compound of any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt is an acid addition salt formed by the compound of general formula (I) of claim 1 with the following acids: hydrogen chloride, Hydrogen bromide, sulfuric acid, carbonic acid, oxalic acid, citric acid, succinic acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, or ferulic acid.
7. The preparation method of the compound of general formula (I) according to any one of claims 1 to 5, characterized in that the synthetic route is as follows:

   
8. A pharmaceutical composition characterized by containing the compound of any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
9. Use of the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to claim 8 in the preparation of ROS1 inhibitor drugs.
10. Use of the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to claim 8 in the preparation of a medicament for the treatment of tumors.