Preparation and purification method and application of quinoline derivative with biological activity

Abstract

The invention discloses a preparation and purification method and application of a quinoline derivative with biological activity, and belongs to the technical field of medicine synthesis. According to the key points of the technical scheme, the quinoline derivative molecule has a structure # imgabs0 #, wherein X is NH or empty (benzene rings are directly connected); r is an aromatic ring derivative or alkyl or other groups. The invention designs and synthesizes a quinoline derivative with a novel structure, and the compound has a relieving effect on LPS-induced microglial cell inflammation and has a certain inhibition effect on cervical cancer cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical synthesis, and particularly relates to a preparation, purification method and application of a quinoline derivative with biological activity. Background Art

[0002] Depression is a neuropsychiatric disorder characterized by low mood, slowed thinking, and reduced movement. Its main features are low mood, decreased pleasure or interest, psychomotor agitation or retardation, and sleep disturbances. In 2019, 264 million people worldwide suffered from depression, which can affect people of all ages and accounts for almost 17% of the world's population. According to statistics, approximately 800,000 people with depression die by suicide worldwide, and depression is a major cause of disability. The pathogenesis of depression is complex and the course of the disease is difficult to predict. Although many antidepressant drugs have been approved for marketing, most drug interventions take weeks to months to produce effects, with poor treatment prognosis and a high failure rate of newly developed drugs in clinical trials. Therefore, there is an urgent need to find new therapies with fewer side effects and higher efficacy, which can not only solve the problems of traditional antidepressant drugs, relieve the pain of patients, but also generate huge economic benefits, and have important theoretical significance and application value.

[0003] Drug treatment is the main treatment method for depression. Most of the antidepressants currently used in clinical practice are related to the monoamine neurotransmitter hypothesis. This hypothesis suggests that the occurrence of depression is closely related to the deficiency of monoamine neurotransmitters such as serotonin (5-HT), dopamine (DA), and norepinephrine (NE). Tryptophan is the precursor of 5-HT and can be metabolized through two main pathways: one pathway is the conversion of tryptophan to 5-hydroxytryptophan by tryptophan hydroxylase, and then to 5-HT by aromatic amino acid decarboxylase. The other pathway is through the kynurenine pathway (KP), in which indoleamine-2,3-dioxygenase 1 (IDO1) is the rate-limiting enzyme of this pathway. IDO-1 indirectly affects the availability of 5-HT by altering the metabolic pathway of tryptophan, and may thus affect mood and behavior.

[0004] In recent years, studies have found that IDO1 may play an important role in the pathogenesis of depression. In patients with depression, neuroinflammation occurs in the brain. Inflammatory stimuli increase the expression level and activity of IDO1, leading to more tryptophan being shunted into the kynurenine pathway and converted into kynurenine, thereby reducing the amount of tryptophan available for 5-HT synthesis. This competitive metabolism may lead to a decrease in 5-HT levels in the central nervous system and the production of neuroimmune regulators such as kynurenine, which is closely related to the occurrence and development of depression. Therefore, the overactivation of IDO1 reduces the synthesis of serotonin and may promote the emergence of depressive symptoms. On the other hand, the activation of IDO1 further induces the production of inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), exacerbating the neuroinflammatory response. By affecting neural plasticity and neurogenesis, it exacerbates depressive symptoms and further promotes the pathological process of depression. Therefore, IDO1 not only affects mood regulation by influencing the synthesis of neurotransmitters but may also participate in the development of depression by promoting inflammatory responses. This provides a potential target for developing new antidepressant treatment strategies, and inhibiting the activity of IDO1 may help improve the clinical symptoms of patients with depression. The team, in collaboration with Henan Gulf Stream, designed and synthesized two derivatives based on the quinoline structure. Henan Gulf Stream was responsible for compound design and synthesis. The team used LPS-induced microglial inflammation experiments to judge their antidepressant effects. We found that one of the derivatives could produce antidepressant effects by targeting IDO1, and the other derivative could promote the growth of microglia by activating glutamate receptors to produce antidepressant effects. Summary of the Invention

[0005] The preparation method and application of a quinoline derivative with antidepressant activity according to the present invention are characterized in that the molecular structure of the quinoline derivative is:

[0006] wherein X or Y is NH or empty (directly connected to the benzene ring), Z is methyl or empty; R is an aromatic ring derivative, an alkyl group or other groups.

[0007] The preparation method and application of a quinoline derivative with antidepressant activity according to the present invention are characterized in that:

[0008] (1) Add a certain amount of 4-chloro-6,7-bis(2-methoxyethoxy)quinazoline and triethylamine to N,N-dimethylformamide, then add 3-aminophenylacetylene, heat to 80 °C, react for a period of time, add water and stir, then extract with dichloromethane multiple times, combine the organic phases, vacuum concentrate to evaporate the organic solvent, and then separate by silica gel column chromatography to obtain N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine.

[0009] (2) Add a certain amount of 4-chloro-6,7-bis(2-methoxyethoxy)quinazoline and triethylamine to N,N-dimethylformamide, then add propargylamine, heat to 80 °C, stir for a period of time after the reaction, add water and stir, then extract with dichloromethane for multiple times, combine the organic phases, concentrate under vacuum to remove the organic solvent, and then separate by silica gel column chromatography to obtain N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine.

[0010] (3) Take a certain amount of N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine, benzyl azide compound, L-ascorbic acid sodium salt and anhydrous CuSO4 and add them to a round-bottom flask. Subsequently, add a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) to the flask, stir at room temperature for 10 min to completely dissolve them, keep at room temperature, stir and react for a period of time, extract with dichloromethane for multiple times, and collect the lower organic phase. Then back-extract with saturated brine for multiple times, collect the lower organic phase, add anhydrous sodium sulfate to it, stir well and let stand for 30 min, and then filter to remove anhydrous sodium sulfate. Distill off dichloromethane under reduced pressure at 35 °C to obtain the crude product, and then separate and purify the obtained crude product by column chromatography, or carry out a reaction to remove cuprous ions, and finally obtain the product; the method for removing cuprous ions is as follows: add sulfuric acid solution dropwise to the reaction system dried with anhydrous sodium sulfate, adjust the pH of the reaction system to 1.5, first stir at room temperature for 5 h, then slowly add saturated sodium sulfide solution dropwise to the reaction system, pay attention to the formation of solids in the reaction system during the dropping process, stop dropping after no more solids appear after continuous dropping, stir at room temperature and then filter the reaction system, separate the organic phase, and concentrate the organic phase to obtain the pure product; or add saturated sodium hypochlorite solution dropwise to the reaction system dried with anhydrous sodium sulfate, stir at room temperature and then add saturated potassium iodide solution, stir and then filter the reaction system, separate the organic phase, and concentrate the organic phase to obtain the target compound.

[0011] (4) 4-chloro-6,7-di(2-methoxyethoxy)quinazoline, 3-alkynylphenylboronic acid, palladium acetate, dbpf, and potassium phosphate were added to a mixed solution of ethanol, water, and dimethyl sulfoxide, heated to 60°C and stirred for a period of time, then extracted with dichloromethane, concentrated, added with a mixed solvent of water: tert-butyl alcohol: THF = 1:1:1 (v / v / v), then added with benzyl azide compounds, sodium ascorbate, and CuSO4, stirred at room temperature, heated under reflux and stirred for a period of time, detected that after the reaction was completed, extracted with dichloromethane for multiple times, collected the organic phase, and then back-extracted with saturated brine for multiple times, and collected the lower layer. The organic phase is added with anhydrous sodium sulfate, stirred thoroughly and allowed to stand, and then filtered to remove the anhydrous sodium sulfate, and distilled under reduced pressure to remove dichloromethane to obtain a crude product, and then the crude product is separated and purified by column chromatography to finally obtain the target compound; the molar ratio of the feeding amount of 4-chloro-6,7-bis(2-methoxyethoxy)quinazoline to 3-alkynylphenylboronic acid to palladium acetate to dbpf to potassium phosphate is 1:1.2:0.2:0.2:1; the molar ratio of the feeding amount of 4-chloro-6,7-bis(2-methoxyethoxy)quinazoline to benzyl azide compounds to sodium ascorbate to CuSO4 is 1:1.2:2:1.

[0012] (5) Preparation method of N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine: 4-chloro-6,7-bis(2-methoxyethoxy)quinazoline and triethylamine are added to N,N-dimethylformamide, propargylamine is added, heated to 80°C, reacted for 5 hours, water is added and stirred, and then extracted with dichloromethane for multiple times, the organic phases are combined, vacuum concentrated to evaporate the organic solvent, and then separated by silica gel column chromatography to obtain N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine; 6,7-dihydroxyquinazolin-4(3H)-one and DBU are added to N,N-dimethylformamide, PyBOP is added, stirred, and then dropped into the reaction system. propargylamine, after the addition is complete, stir at room temperature for 24 hours, remove the solvent under vacuum, add 100 mL of N,N-dimethylformamide, potassium carbonate and 1-chloro-2-methoxyethane, heat to 90°C, react, and concentrate in vacuum to obtain N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine; add a magnetic stirrer, a graphite anode (6.0 mm graphite rod), a platinum electrode and an Ag / Ag+ reference electrode to a reaction tube; add 2-amino-4,5-dihydroxybenzamide, formaldehyde, p-toluenesulfonic acid·hydrate and a solution of tetrabutylammonium perchlorate in acetonitrile (0.1 M, 8.0 mL); at room temperature, at 1.0 V (relative to Ag / Ag +) Carry out an electrolysis reaction for 1.5 h at the controlled potential, vacuum concentrate the reaction mixture, add N,N-dimethylformamide, then add benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate and propargylamine), slowly add DBU dropwise under stirring, and stir the reaction mixture at 110 °C for 16 h. After cooling the reaction, add N,N-dimethylformamide, potassium carbonate and 1-chloro-2-methoxyethane, heat to 90 °C, react for 2 h, wash with brine, extract and vacuum concentrate to obtain N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine; Add 2-nitro-4,5-dihydroxybenzonitrile to water, add sodium sulfite, and heat the reaction mixture to 50 °C, stir at this temperature for 3 - 4 hours, then raise the temperature to 65 °C, slowly add concentrated hydrochloric acid dropwise, after complete addition, cool to 20 °C, and adjust the pH to about 10 with 50% aqueous sodium hydroxide solution. The formed precipitate is dissolved in toluene after filtration, washing and drying, add acetic acid and DMF-DMA, and heat the reaction under reflux conditions for 4 - 6 hours. After the reaction is completed, evaporate the excess DMF-DMA under vacuum, cool to room temperature, add propargylamine and acetic acid; stir the reaction mixture at 60 °C for 2 - 3 hours, raise the temperature to reflux, and continue to stir the reaction for 2 - 3 hours. Cool the reaction mixture to room temperature, extract the organic layer with ethyl acetate. Combine the organic extracts, dry, filter and concentrate under vacuum, add N,N-dimethylformamide, potassium carbonate and 1-chloro-2-methoxyethane, heat to 90 °C, react for 5 h, wash with brine, extract and vacuum concentrate to obtain N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine.

[0013] (6) Take a certain amount of N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine, benzyl azide compound, L-ascorbic acid sodium salt and anhydrous CuSO4 and add them to a round-bottom flask. Subsequently, add a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) to the flask. Stir at room temperature until all are dissolved, keep at room temperature, stir the reaction for a period of time, extract with dichloromethane multiple times, and collect the lower organic phase. Then back-extract with saturated brine multiple times, collect the lower organic phase, add anhydrous sodium sulfate to it, stir well and let stand, then filter to remove anhydrous sodium sulfate, distill off dichloromethane under reduced pressure to obtain the crude product, and then separate and purify the obtained crude product by column chromatography to finally obtain the product.

[0014] The quinoline derivatives of the present invention have the following technical advantages: (1) The present invention modifies the molecular structure of the reported marketed drug erlotinib to obtain compounds with novel structures; (2) The triazole in these compounds can bind to the heme in the IDO1 target, thereby inhibiting its activity and having a certain anti-inflammatory effect; (3) These compounds can act as N-methyl-D-aspartic acid receptor agonists, thereby promoting the growth of microglia; (4) The present invention can obtain products not contaminated by cuprous ions, which can improve the activity; (5) This compound can reduce the inflammation of microglia and promote the growth of microglia, and can be used to relieve depression. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the hydrogen spectrum of compound 3d obtained in Example 7.

[0016] Figure 2 It is the hydrogen spectrum of compound 5j obtained in Example 39.

[0017] Figure 3 Molecular docking diagram of compound 3d and IDO1.

[0018] Figure 4 Molecular docking diagram of compound 5f and IDO1.

[0019] Figure 5 Molecular docking diagram of compound 5j and IDO1.

[0020] Figure 6 Molecular docking diagram of compound 5f and NMDA.

[0021] Figure 7 Molecular docking diagram of compound 5j and NMDA. DETAILED DESCRIPTION OF THE INVENTION

[0022] The following further elaborates on the above content of the present invention through examples, but this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention.

[0023] Example 1

[0024]

[0025] In a reaction flask equipped with stirring, 31.2 g of 4-chloro-6,7-bis(2-methoxyethoxy)quinazoline and 10 g of triethylamine were added to 300 mL of N,N-dimethylformamide. Then, 11.7 g of 3-aminophenylacetylene was added. The mixture was heated to 80 °C and reacted for 5 h. Then, 300 mL of water was added and stirred. Subsequently, it was extracted multiple times with 200 mL of dichloromethane. The organic phases were combined, and after concentrating and evaporating the organic solvents under vacuum, N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine was obtained through silica gel column chromatography.

[0026] Example 2

[0027]

[0028] Take N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 4-fluorobenzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4 (1 mmol) and add them to a 100 mL round-bottom flask equipped with a magnetic stirrer. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. Stir at room temperature for 10 min to completely dissolve them. Heat under reflux and stir. After detecting the completion of the reaction by TLC, extract 3 times with dichloromethane and collect the lower organic phase. Then, back-extract 3 times with saturated brine and collect the lower organic phase. Add anhydrous sodium sulfate to it, stir well and let stand for 30 min. Then, filter to remove anhydrous sodium sulfate. Distill off dichloromethane under reduced pressure at 35 °C to obtain the crude product. Then, separate and purify the obtained crude product by column chromatography to finally obtain the solid product 3a with a yield of about 39.75%.

[0029] Example 3

[0030] Take N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 3-fluorobenzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4 (1 mmol) and add them to a 100 mL round-bottom flask equipped with a magnetic stirrer. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. Stir at room temperature for 10 min to completely dissolve them. Heat under reflux and stir. After detecting the completion of the reaction by TLC, extract 3 times with dichloromethane and collect the lower organic phase. Then, back-extract 3 times with saturated brine and collect the lower organic phase. Add anhydrous sodium sulfate to it, stir well and let stand for 30 min. Then, filter to remove anhydrous sodium sulfate. Distill off dichloromethane under reduced pressure at 35 °C to obtain the crude product. Then, separate and purify the obtained crude product by column chromatography to finally obtain the solid product 3b with a yield of about 55.89%.

[0031] Example 4

[0032] Take N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), benzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4 (1 mmol) and add them into a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, add 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) to the flask. Stir at room temperature for 10 min to dissolve all of them completely. Then stir under reflux with heating. After detecting the completion of the reaction by TLC, extract with dichloromethane three times and collect the lower organic phase. Back-extract with saturated brine three times and collect the lower organic phase. Add anhydrous sodium sulfate to it, stir well and let it stand for 30 min, and then filter off the anhydrous sodium sulfate. Distill off dichloromethane under reduced pressure at 35 °C to obtain the crude product, and then separate and purify the obtained crude product by column chromatography to finally obtain the solid product 3c with a yield of about 62.34%.

[0033] Example 5

[0034]

[0035] Take N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 2-bromobenzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4 (1 mmol) and add them into a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, add 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) to the flask. Stir at room temperature for 10 min to dissolve all of them completely. Then stir under reflux with heating. After detecting the completion of the reaction by TLC, extract with dichloromethane three times and collect the lower organic phase. Back-extract with saturated brine three times and collect the lower organic phase. Add anhydrous sodium sulfate to it, stir well and let it stand for 30 min, and then filter off the anhydrous sodium sulfate. Distill off dichloromethane under reduced pressure at 35 °C to obtain the crude product, and then separate and purify the obtained crude product by column chromatography to finally obtain the solid product 3d with a yield of about 51.49%.

[0036] Example 6

[0037] Take N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 2-bromobenzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4(1 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min to completely dissolve it, and then stirred under reflux heating. After detecting the completion of the reaction by TLC, it was extracted with dichloromethane three times, and the lower organic phase was collected. It was then back-extracted with saturated brine three times, and the lower organic phase was collected. Sulfuric acid solution was added dropwise to the reaction system to adjust the pH of the reaction system to 1.5. Then it was stirred at room temperature for 5 h, and then a saturated sodium sulfide solution was slowly added dropwise to the reaction system. During the addition process, pay attention to the formation of solids in the reaction system. After continuing to add and no more solids appeared, stop adding. Stir at room temperature for 1 h, filter the reaction system, separate the organic phase, and after concentrating the organic phase, pure product 3d was obtained with a yield of about 71.05%.

[0038] Example 7

[0039] Take N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 2-bromobenzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4 (1 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min to completely dissolve it, and then stirred under reflux heating. After detecting the completion of the reaction by TLC, it was extracted with dichloromethane three times, and the lower organic phase was collected. It was then back-extracted with saturated brine three times, and the lower organic phase was collected. A saturated sodium hypochlorite solution was added dropwise to the reaction system, stirred at room temperature for 2 h, then a saturated potassium iodide solution was added, stirred for 10 min, filtered the reaction system, separated the organic phase, and after concentrating the organic phase, pure product 3d was obtained with a yield of about 62.33%.

[0040] Example 8

[0041]

[0042] Take N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 4-bromobenzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4(1 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min to completely dissolve it, and then stirred under reflux for 2 h. After detecting the completion of the reaction by TLC, it was extracted with dichloromethane three times, and the lower organic phase was collected. It was then back-extracted with saturated brine three times, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and after stirring thoroughly, it was allowed to stand for 30 min, and then the anhydrous sodium sulfate was removed by filtration. Dichloromethane was removed by distillation under reduced pressure at 35 °C to obtain the crude product, and then the obtained crude product was separated and purified by column chromatography to finally obtain the solid product 3e with a yield of about 57.54%.

[0043] Example 9

[0044] Take N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 2-(trifluoromethyl)benzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4 (1 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min to completely dissolve it, and then stirred under reflux until the reaction was completed. It was then extracted with dichloromethane three times, and the lower organic phase was collected. It was then back-extracted with saturated brine three times, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and after stirring thoroughly, it was allowed to stand for 30 min, and then the anhydrous sodium sulfate was removed by filtration. Dichloromethane was removed by distillation under reduced pressure at 35 °C to obtain the crude product, and then the obtained crude product was separated and purified by column chromatography to finally obtain the solid product 3f with a yield of 44.92%.

[0045] Example 10

[0046] Take N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 3-(trifluoromethyl)benzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4(1 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min to completely dissolve it. After the reaction was completed with heating under reflux and stirring, it was extracted with dichloromethane three times, and the lower organic phase was collected. It was then back-extracted with saturated brine three times, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and after sufficient stirring, it was allowed to stand for 30 min, and then the anhydrous sodium sulfate was removed by filtration. Dichloromethane was removed by distillation under reduced pressure at 35 °C to obtain the crude product, and then the obtained crude product was separated and purified by column chromatography. Finally, 3 g of a solid product was obtained, and the yield was 35.72%.

[0047] Example 11

[0048] Take N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 4-trifluoromethylbenzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4 (1 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min to completely dissolve it. After the reaction was completed with heating under reflux and stirring, it was extracted with dichloromethane three times, and the lower organic phase was collected. It was then back-extracted with saturated brine three times, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and after sufficient stirring, it was allowed to stand for 30 min, and then the anhydrous sodium sulfate was removed by filtration. Dichloromethane was removed by distillation under reduced pressure at 35 °C to obtain the crude product, and then the obtained crude product was separated and purified by column chromatography. Finally, 3 h of a solid product was obtained, and the yield was 56.86%.

[0049] Example 12

[0050]

[0051] Take N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 4-chlorobenzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4(1 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min to completely dissolve it. After the reaction was completed with heating under reflux and stirring, it was extracted with dichloromethane three times, and the lower organic phase was collected. It was then back-extracted with saturated brine three times, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and after sufficient stirring, it was allowed to stand for 30 min, and then the anhydrous sodium sulfate was removed by filtration. Dichloromethane was removed by distillation under reduced pressure at 35 °C to obtain the crude product, and then the obtained crude product was separated and purified by column chromatography to finally obtain the solid product 3i with a yield of 80.69%.

[0052] Example 13

[0053]

[0054] Take N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 2-chloro-6-fluorobenzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4 (1 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min to completely dissolve it. After the reaction was completed with heating under reflux and stirring, it was extracted with dichloromethane three times, and the lower organic phase was collected. It was then back-extracted with saturated brine three times, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and after sufficient stirring, it was allowed to stand for 30 min, and then the anhydrous sodium sulfate was removed by filtration. Dichloromethane was removed by distillation under reduced pressure at 35 °C to obtain the crude product, and then the obtained crude product was separated and purified by column chromatography to finally obtain the solid product 3j with a yield of 56.38%.

[0055] Example 14

[0056]

[0057] Take N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 3-nitrobenzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4(1 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min to completely dissolve it. After the reaction was completed with heating under reflux and stirring, it was extracted 3 times with dichloromethane, and the lower organic phase was collected. It was then back-extracted 3 times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and after sufficient stirring, it was allowed to stand for 30 min, and then the anhydrous sodium sulfate was removed by filtration. Dichloromethane was removed by distillation under reduced pressure at 35 °C to obtain a crude product, and then the obtained crude product was separated and purified by column chromatography to finally obtain a solid product 3k with a yield of 61.07%.

[0058] Example 15

[0059]

[0060] Take N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 3,5-dimethylbenzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4 (1 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min to completely dissolve it. After the reaction was completed with heating under reflux and stirring, it was extracted 3 times with dichloromethane, and the lower organic phase was collected. It was then back-extracted 3 times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and after sufficient stirring, it was allowed to stand for 30 min, and then the anhydrous sodium sulfate was removed by filtration. Dichloromethane was removed by distillation under reduced pressure at 35 °C to obtain a crude product, and then the obtained crude product was separated and purified by column chromatography to finally obtain a solid product 3l with a yield of 49.82%.

[0061] Example 16

[0062] Take N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 2-acetonitrile benzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4(1 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min to completely dissolve it. After the reaction was completed with heating under reflux and stirring, it was extracted 3 times with dichloromethane, and the lower organic phase was collected. It was then back-extracted 3 times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and after sufficient stirring, it was allowed to stand for 30 min, and then the anhydrous sodium sulfate was removed by filtration. Dichloromethane was removed by distillation under reduced pressure at 35 °C to obtain a crude product, and then the obtained crude product was separated and purified by column chromatography to finally obtain a solid product 3m with a yield of 47.17%.

[0063] Example 17

[0064]

[0065] Take N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 3-chlorobenzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4 (1 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min to completely dissolve it. After the reaction was completed with heating under reflux and stirring for 2 h and detected by TLC, it was extracted 3 times with dichloromethane, and the lower organic phase was collected. It was then back-extracted 3 times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and after sufficient stirring, it was allowed to stand for 30 min, and then the anhydrous sodium sulfate was removed by filtration. Dichloromethane was removed by distillation under reduced pressure at 35 °C to obtain a crude product, and then the obtained crude product was separated and purified by column chromatography to finally obtain a solid product 3n with a yield of 70.08%.

[0066] Example 18

[0067]

[0068] Take N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 4-methylbenzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4(1 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min to completely dissolve it, and then stirred under reflux for 2 h. After the reaction was completed as detected by TLC, it was extracted with dichloromethane three times, and the lower organic phase was collected. It was then back-extracted with saturated brine three times, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and after sufficient stirring, it was allowed to stand for 30 min, and then the anhydrous sodium sulfate was removed by filtration. Dichloromethane was removed by distillation under reduced pressure at 35 °C to obtain a crude product, and then the obtained crude product was separated and purified by column chromatography to finally obtain a solid product 3o with a yield of 27.53%.

[0069] Example 19

[0070]

[0071] 4-Chloro-6,7-bis(2-methoxyethoxy)quinazoline (1 mmol), 3-ethynylphenylboronic acid (1.2 mmol), palladium acetate (0.02 mmol), dbpf (0.02 mmol), and potassium phosphate (1 mmol) were added to a mixed solution of ethanol, water, and dimethyl sulfoxide. The mixture was heated to 60 °C and stirred for 4 h, then extracted with dichloromethane. After concentration, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added, and then 2-bromobenzyl azide (1.2 mmol), sodium ascorbate (2 mmol), and CuSO 4 (1 mmol) were added. The mixture was stirred at room temperature for 10 min to completely dissolve it, and then stirred under reflux for 2 h. After the reaction was completed as detected by TLC, it was extracted with dichloromethane three times, and the lower organic phase was collected. It was then back-extracted with saturated brine three times, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and after sufficient stirring, it was allowed to stand for 30 min, and then the anhydrous sodium sulfate was removed by filtration. Dichloromethane was removed by distillation under reduced pressure at 35 °C to obtain a crude product, and then the obtained crude product was separated and purified by column chromatography to finally obtain a solid product 3p.

[0072] Example 20

[0073] Detection of NO level in cell supernatant by Griess method: BV2 cells were seeded at 2×10 4 cells / well in a 96-well culture plate and placed in an incubator containing 5% CO 2Cultured in a 37°C constant temperature incubator. After 24 hours of culture, the corresponding concentration of the test compound was added to the dosing group and incubated for 2 hours. Subsequently, LPS with a final concentration of 100 ng / ml was added to both the dosing group and the LPS model group. Incubated in the incubator for 24 hours, 50 μL of supernatant and 50 μl of Greiss buffer were taken from each well and reacted at room temperature for 15 min. The OD values of each group at a wavelength of 540 nm were detected using an ELISA reader (Biotek).

[0074] Improvement effect of the compound on LPS-induced inflammation in BV2 cells

[0075]

[0076] Example 21

[0077] BV2 cells were seeded at 2×10 4 cells / well in a 96-well culture plate and placed in a 37°C constant temperature incubator containing 5% CO 2 . After 24 hours of culture, the corresponding concentration of the test compound was added to the dosing group and incubated for 2 hours. Subsequently, LPS with a final concentration of 100 ng / mL was added to both the dosing group and the LPS model group. Incubated for another 24 hours, MTT with a final concentration of 0.5 mg / mL was added to each well for live cell staining. After incubating in the incubator for 1 hour, the culture medium was discarded, 100 μL of DMSO was added to each well, and it was shaken on a shaker to dissolve it completely. The OD values of each group at a wavelength of 490 nm were detected using an ELISA reader (Biotek).

[0078] Toxic effect of the compound on BV2 cells

[0079]

[0080] Example 22

[0081] From CO 2Remove the culture dish of viable human cervical cancer Hela cells from the incubator and perform the following operations respectively: Conduct aseptic operation beside the alcohol lamp, open the dish cover, aspirate the culture medium into the waste liquid tank, wash the culture medium in the culture flask twice with 2 mL of PBS, digest with 0.25% trypsin, use a pipette to pipette the bottom of the culture flask to make the cells detached, transfer the obtained cell suspension to a sterile centrifuge tube, set the centrifuge to 1000 r / min for 3 min for centrifugation, then slowly pour out the supernatant in the centrifuge tube, add 2 - 5 mL of culture medium, and perform cell counting under an inverted microscope. According to the counting results, seed the viable human cervical cancer Hela cells in the logarithmic growth phase at a number of 50,000 cells per well in a 96-well cell culture plate, culture with a medium containing 10% fetal bovine serum for 5 - 6 hours, add 100 μL of compound 3d diluted with the medium (concentration of 1.0 μM) and recombinant human interferon γ (final concentration of 100 ng / mg) to activate the expression of IDO1 in Hela cells. After the operation, place the 96-well cell culture plate in a 37 °C cell incubator rich in 5% carbon dioxide and culture for 18 hours, then terminate the reaction with a certain amount of 3.05 N trichloroacetic acid, and then incubate at 50 °C for 30 minutes. After the cell culture medium is precipitated, take the supernatant, color it with p-(N,N-dimethyl)benzaldehyde, and then detect the absorbance at 480 nm with a multifunctional microplate reader. Use the group treated with the medium containing only IFNγ without drugs as 100% (At), and the group treated with the medium containing only 0.1% DMSO as the blank control 0% (Ab); calculate the absorbance under different treatment conditions according to the following formula: Absorbance% = (A - Ab) / (At - Ab), A: drug treatment + 100 ng / mL IFNγ, Ab: blank control, At: no drug and only 100 ng / mL IFNγ. Compound 3d has the best inhibitory activity against IDO1, and the IDO1 enzyme activity is approximately 36.22% (Example 5), 20.73% (Example 6), 17.87% (Example 7). We performed molecular docking of compound 3d with IDO1 and found that compound 3d can effectively enter the active pocket of IDO1. The triazole structure in compound 3d is located directly above the heme of the IDO1 target, and can coordinate with the ferrous ion in the heme, competitively inhibiting the activity of IDO1. The IDO1 enzyme activity of compound 3p is approximately 15.21%; due to the 3,5-dimethylbenzyl structure of compound 3l, the steric hindrance of the two methyl groups is relatively large, and it is difficult to enter the active pocket of IDO1, so the inhibitory activity against IDO1 is very small (82.9%) under the condition of 1.0 μM.

[0082] Example 23

[0083]

[0084] In a reaction flask equipped with stirring, 4-chloro-6,7-bis(2-methoxyethoxy)quinazoline (0.01 mol) and triethylamine (0.02 mol) were added to 100 mL of N,N-dimethylformamide, and then propargylamine (0.013 mol) was added. The mixture was heated to 80 °C and reacted for 5 h. Then 100 mL of water was added and stirred, and then extracted multiple times with 200 mL of dichloromethane. The organic phases were combined, and after concentrating and evaporating the organic solvent under vacuum, N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine was obtained by silica gel column chromatography with a yield of 40.8%.

[0085] Example 24

[0086]

[0087] In a reaction flask equipped with stirring, 6,7-dihydroxyquinazolin-4(3H)-one (3 mmol) and DBU (3 mmol) were added to 50 mL of N,N-dimethylformamide, and then PyBOP (6 mmol) was added. The mixture was stirred and reacted for 10 min, and then propargylamine (7 mmol) was added dropwise to the reaction system. After the addition, the mixture was stirred at room temperature for 24 h. The solvent was removed under vacuum, and then 100 mL of N,N-dimethylformamide, potassium carbonate (6 mmol) and 1-chloro-2-methoxyethane (7 mmol) were added. The mixture was heated to 90 °C and reacted for 25 h. After vacuum concentration, N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine was obtained with a yield of about 77.2%.

[0088] Example 25

[0089]

[0090] A magnetic stir bar, a graphite anode (6.0 mm graphite rod), a platinum electrode (1 cm x 1 cm x 0.1 cm) and an Ag / Ag+ reference electrode were added to a reaction test tube; 2-amino-4,5-dihydroxybenzamide (0.2 mmol), formaldehyde (0.3 mmol), p-toluenesulfonic acid hydrate (0.02 mmol) and a solution of tetrabutylammonium perchlorate in acetonitrile (0.1 M, 8.0 mL) were added; at room temperature, an electrolytic reaction was carried out at a controlled potential of 1.0 V (relative to Ag / Ag + ) for 1.5 h. The reaction mixture was concentrated under vacuum, 7 mL of N,N-dimethylformamide was added, and then benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (0.25 mmol) and propargylamine (0.3 mmol) were added. DBU (0.3 mmol) was slowly added dropwise under stirring, and at 110 oStir the reaction mixture at C for a period of time, cool the reaction, add 5 mL of N,N-dimethylformamide, potassium carbonate (0.5 mmol) and 1-chloro-2-methoxyethane (0.5 mmol), heat to 90 °C, react for a period of time, wash with brine, extract and concentrate in vacuo, and the yield is about 96.2%.

[0091] Example 26

[0092]

[0093] Add 2-nitro-4,5-dihydroxybenzonitrile (5 mmol) to water (10 mL), add sodium sulfite (10 mmol), and heat the reaction mixture to 50 °C. Stir at this temperature for 3 - 4 hours, then raise the temperature to 65 °C, slowly add concentrated hydrochloric acid (3 mL). After complete addition, cool to 20 °C, and adjust the pH to about 10 using 50% aqueous sodium hydroxide solution. The formed precipitate is dissolved in toluene (5 mL) after filtration, washing and drying. Add acetic acid (2.5 mL) and DMF-DMA (10 mmol), and heat the reaction under reflux conditions. After the reaction is completed, evaporate the excess DMF-DMA in vacuo, cool to room temperature, add propargylamine (5 mmol) and acetic acid (10 mmol), stir the reaction mixture at 60 °C for 2 - 3 hours, raise the temperature to reflux, and continue to stir the reaction for a period of time. Cool the reaction mixture to room temperature, extract the organic layer with ethyl acetate (3×15 mL). Combine the organic extracts, dry, filter and concentrate in vacuo. Add 50 mL of N,N-dimethylformamide, potassium carbonate (10 mmol) and 1-chloro-2-methoxyethane (12 mmol), heat to 90 °C, wash with brine after complete reaction, extract and concentrate in vacuo, and the yield is about 72.03%.

[0094] Example 27

[0095]

[0096] Take N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 4-fluorobenzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4(1 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min, then stirred under reflux. After the reaction was completed as detected by TLC, it was extracted with dichloromethane three times, and the lower organic phase was collected. Then it was back-extracted with saturated brine three times, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and after stirring thoroughly, it was allowed to stand for 30 min. Then the anhydrous sodium sulfate was removed by filtration. Dichloromethane was removed by distillation under reduced pressure at 35 °C to obtain the crude product, and then the obtained crude product was separated and purified by column chromatography to finally obtain the solid product 5a with a yield of 83.94%.

[0097] Example 28

[0098]

[0099] Take N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 3-fluorobenzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4 (1 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min, then stirred under reflux. After the reaction was completed, it was extracted with dichloromethane three times, and the lower organic phase was collected. Then it was back-extracted with saturated brine three times, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and after stirring thoroughly, it was allowed to stand for 30 min. Then the anhydrous sodium sulfate was removed by filtration. Dichloromethane was removed by distillation under reduced pressure at 35 °C to obtain the crude product, and then the obtained crude product was separated and purified by column chromatography to finally obtain the solid product 5b with a yield of 67.29%.

[0100] Example 29

[0101] Take N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), benzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4(1 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min, then heated under reflux with stirring. After the reaction was completed as detected by TLC, it was extracted with dichloromethane three times, and the lower organic phase was collected. It was then back-extracted with saturated brine three times, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and after sufficient stirring, it was allowed to stand for 30 min. Then, the anhydrous sodium sulfate was removed by filtration, and dichloromethane was removed by distillation under reduced pressure to obtain a crude product. Then, the obtained crude product was separated and purified by column chromatography to finally obtain the solid product 5c with a yield of 39.56%.

[0102] Example 30

[0103]

[0104] Take N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 2-bromobenzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4 (1 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min, then heated under reflux with stirring. After the reaction was completed as detected by TLC, it was extracted with dichloromethane three times, and the lower organic phase was collected. It was then back-extracted with saturated brine three times, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and after sufficient stirring, it was allowed to stand for 30 min. Then, the anhydrous sodium sulfate was removed by filtration, and dichloromethane was removed by distillation under reduced pressure to obtain a crude product. Then, the obtained crude product was separated and purified by column chromatography to finally obtain the solid product 5d with a yield of 66.28%.

[0105] Example 31

[0106] Take N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 4-bromobenzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4(1 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min, then heated under reflux with stirring. After the reaction was completed as detected by TLC, it was extracted with dichloromethane three times, and the lower organic phase was collected. It was then back-extracted with saturated brine three times, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and after stirring thoroughly, it was allowed to stand for 30 min. Then, the anhydrous sodium sulfate was removed by filtration, and dichloromethane was removed by distillation under reduced pressure to obtain a crude product. Then, the obtained crude product was separated and purified by column chromatography, and finally, solid product 5e was obtained with a yield of 37.97%.

[0107] Example 32

[0108]

[0109] Take N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 2-(trifluoromethyl)benzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4 (1 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min, then heated under reflux with stirring. After the reaction was completed as detected by TLC, it was extracted with dichloromethane three times, and the lower organic phase was collected. It was then back-extracted with saturated brine three times, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and after stirring thoroughly, it was allowed to stand for 30 min. Then, the anhydrous sodium sulfate was removed by filtration, and dichloromethane was removed by distillation under reduced pressure to obtain a crude product. Then, the obtained crude product was separated and purified by column chromatography, and finally, solid product 5f was obtained with a yield of 44.29%.

[0110] Example 33

[0111] Take N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 2-(trifluoromethyl)benzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4(1 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min to completely dissolve it. After the reaction was completed with heating under reflux and stirring, it was extracted with dichloromethane three times, and the lower organic phase was collected. Then it was back-extracted with saturated brine three times, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and after sufficient stirring, it was allowed to stand for 30 min. Then the anhydrous sodium sulfate was removed by filtration. A sulfuric acid solution was added dropwise to the reaction system to adjust the pH of the reaction system to 1.5, and it was stirred at room temperature for 5 h. Then a saturated sodium sulfide solution was slowly added dropwise to the reaction system. During the addition process, pay attention to the formation of solids in the reaction system. When no more solids appeared after continuous addition, the addition was stopped. It was stirred at room temperature for 1 h, the reaction system was filtered, and the organic phase was separated. After concentration of the organic phase, the pure product 5f was obtained, and the yield was about 47.96%.

[0112] Example 34

[0113] Take N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 2-trifluoromethylbenzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4 (1 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min to completely dissolve it. After the reaction was completed with heating under reflux and stirring, it was extracted with dichloromethane three times, and the lower organic phase was collected. Then it was back-extracted with saturated brine three times, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and after sufficient stirring, it was allowed to stand for 30 min. Then the anhydrous sodium sulfate was removed by filtration. A saturated hydrogen peroxide solution was added dropwise to the reaction system, and then it was stirred at room temperature for 2 h. The reaction system was filtered, and the organic phase was separated. After concentration of the organic phase, the pure product 5f was obtained, and the yield was about 69.03%.

[0114] Example 35

[0115] Take N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 2-fluorobenzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4(1 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min to completely dissolve it. After the reaction was completed with heating under reflux and stirring, it was extracted 3 times with dichloromethane, and the lower organic phase was collected. It was then back-extracted 3 times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and after sufficient stirring, it was allowed to stand for 30 min. Then, the anhydrous sodium sulfate was removed by filtration. A saturated solution of sodium hypochlorite was added dropwise to the reaction system, and the mixture was stirred at room temperature for 2 h. Then, a saturated solution of potassium iodide was added, and the mixture was stirred for 10 min. The reaction system was filtered, and the organic phase was separated. After concentration of the organic phase, the pure product 5f was obtained with a yield of approximately 46.61%.

[0116] Example 36

[0117] Take N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 2-trifluoromethylbenzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4 (1 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min to completely dissolve it. After the reaction was completed with heating under reflux and stirring, it was extracted 3 times with dichloromethane, and the lower organic phase was collected. It was then back-extracted 3 times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and after sufficient stirring, it was allowed to stand for 30 min. Then, the anhydrous sodium sulfate was removed by filtration. A solution of potassium dichromate (1 mmol) was added dropwise to the reaction system, and the mixture was stirred at room temperature for 1 h. Then, a saturated solution of sodium sulfide was slowly added dropwise to the reaction system, and it was noted that a solid was formed in the reaction system during the dropping process. After continued dropping, when the solid no longer appeared, the dropping was stopped, and the mixture was stirred at room temperature for 1 h. The reaction system was filtered, and the organic phase was separated. After concentration of the organic phase, the pure product 5f was obtained.

[0118] Example 37

[0119]

[0120] Take N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 3-trifluoromethylbenzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4(1 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min to completely dissolve it. After the reaction was completed with heating under reflux and stirring, it was extracted 3 times with dichloromethane, and the lower organic phase was collected. It was then back-extracted 3 times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and after sufficient stirring, it was allowed to stand for 30 min. Then, the anhydrous sodium sulfate was removed by filtration, and dichloromethane was removed by distillation under reduced pressure to obtain a crude product. Then, the obtained crude product was separated and purified by column chromatography, and finally 5 g of a solid product was obtained, with a yield of about 55.62%.

[0121] Example 38

[0122] Take N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 4-trifluoromethylbenzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4 (1 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min to completely dissolve it. After the reaction was completed with heating under reflux and stirring, it was extracted 3 times with dichloromethane, and the lower organic phase was collected. It was then back-extracted 3 times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and after sufficient stirring, it was allowed to stand for 30 min. Then, the anhydrous sodium sulfate was removed by filtration, and dichloromethane was removed by distillation under reduced pressure to obtain a crude product. Then, the obtained crude product was separated and purified by column chromatography, and finally 5 h of a solid product was obtained, with a yield of about 30.39%.

[0123] Example 39

[0124] Take N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 4-chlorobenzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4 (1 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min to completely dissolve it. After the reaction was completed with heating under reflux and stirring, it was extracted 3 times with dichloromethane, and the lower organic phase was collected. It was then back-extracted 3 times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and after sufficient stirring, it was allowed to stand for 30 min. Then, the anhydrous sodium sulfate was removed by filtration, and dichloromethane was removed by distillation under reduced pressure to obtain a crude product. Then, the obtained crude product was separated and purified by column chromatography, and finally 5 i of a solid product was obtained.

[0125] Example 40

[0126]

[0127] Take N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 2-chloro-6-fluorobenzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4 (1 mmol) and add them to a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, add 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) to the flask. Stir at room temperature for 10 min to completely dissolve them. Heat under reflux and stir for 2 h. After detecting the completion of the reaction by TLC, extract with dichloromethane three times and collect the lower organic phase. Then back-extract with saturated brine three times and collect the lower organic phase. Add anhydrous sodium sulfate to it, stir well and let stand for 30 min. Then filter to remove anhydrous sodium sulfate and remove dichloromethane by rotary evaporation to obtain the crude product. Then separate and purify the obtained crude product by column chromatography to finally obtain the solid product 5j.

[0128] Example 41

[0129]

[0130] Take N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 3-nitrobenzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4 (1 mmol) and add them to a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, add 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) to the flask. Stir at room temperature for 10 min to completely dissolve them. Heat under reflux and stir until the reaction is complete. Then extract with dichloromethane three times and collect the lower organic phase. Back-extract with saturated brine three times and collect the lower organic phase. Add anhydrous sodium sulfate to it, stir well and let stand for 30 min. Then filter to remove anhydrous sodium sulfate and remove dichloromethane by rotary evaporation to obtain the crude product. Then separate and purify the obtained crude product by column chromatography to finally obtain the solid product 5k.

[0131] Example 42

[0132] Take N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 3,5-dimethylbenzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4(1 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min to completely dissolve it. After the reaction was completed with heating under reflux and stirring, it was extracted 3 times with dichloromethane, and the lower organic phase was collected. It was then back-extracted 3 times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and after sufficient stirring, it was allowed to stand for 30 min. Then, the anhydrous sodium sulfate was removed by filtration, and dichloromethane was removed by distillation under reduced pressure to obtain a crude product. Then, the obtained crude product was separated and purified by column chromatography, and finally, a solid product 5l was obtained with a yield of about 33.85%.

[0133] Example 43

[0134]

[0135] Take N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 2-acetonitrile benzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4 (1 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min to completely dissolve it. After the reaction was completed with heating under reflux and stirring for 2 h and detected by TLC, it was extracted 3 times with dichloromethane, and the lower organic phase was collected. It was then back-extracted 3 times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and after sufficient stirring, it was allowed to stand for 30 min. Then, the anhydrous sodium sulfate was removed by filtration, and dichloromethane was removed by distillation under reduced pressure to obtain a crude product. Then, the obtained crude product was separated and purified by column chromatography, and finally, a solid product 5m was obtained with a yield of about 72.04%.

[0136] Example 44

[0137]

[0138] Take N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 3-chlorobenzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4(1 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stirrer. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min to completely dissolve it, and then stirred under reflux for 2 h. After the reaction was completed as detected by TLC, it was extracted with dichloromethane three times, and the lower organic phase was collected. It was then back-extracted with saturated brine three times, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and after sufficient stirring, it was allowed to stand for 30 min. Then, the anhydrous sodium sulfate was removed by filtration, and dichloromethane was removed by distillation under reduced pressure to obtain a crude product. Then, the obtained crude product was separated and purified by column chromatography, and finally, a solid product 5n was obtained with a yield of about 77.95%.

[0139] Example 45

[0140]

[0141] Take N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine (1 mmol), 4-methylbenzyl azide (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4 (1 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stirrer. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min to completely dissolve it, and then stirred under reflux for 2 h. After the reaction was completed as detected by TLC, it was extracted with dichloromethane three times, and the lower organic phase was collected. It was then back-extracted with saturated brine three times, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and after sufficient stirring, it was allowed to stand for 30 min. Then, the anhydrous sodium sulfate was removed by filtration, and dichloromethane was removed by distillation under reduced pressure to obtain a crude product. Then, the obtained crude product was separated and purified by column chromatography, and finally, a solid product 5o was obtained with a yield of about 56.28%.

[0142] Example 46

[0143]

[0144] Take 4-chloro-6,7-bis(2-methoxyethoxy)quinazoline (1 mmol), sodium azide (2 mmol), add them to 50 mL of N,N-dimethylformamide, stir at room temperature for 3 h, pour it into water, filter by suction, and the filter cake and phenylacetylene (1.2 mmol), sodium ascorbate (2 mmol) and CuSO 4(1 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min to completely dissolve it, and then stirred under reflux for 2 h. After the reaction was completed as detected by TLC, it was extracted with dichloromethane three times, and the lower organic phase was collected. It was then back-extracted with saturated brine three times, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and after sufficient stirring, it was allowed to stand for 30 min. Then, the anhydrous sodium sulfate was removed by filtration, and dichloromethane was removed by distillation under reduced pressure to obtain a crude product. Then, the obtained crude product was separated and purified by column chromatography, and finally, the solid product 5p was obtained.

[0145] Example 47

[0146]

[0147] 4-Chloro-6,7-bis(2-methoxyethoxy)quinazoline (1 mmol) and sodium azide (2 mmol) were taken and added to 50 mL of N,N-dimethylformamide. The mixture was stirred at room temperature for 3 h, poured into water, and filtered by suction. The filter cake, 4-nitrophenylacetylene (1.2 mmol), sodium ascorbate (2 mmol), and CuSO 4 (1 mmol) was added to a 100 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, 50 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min to completely dissolve it, and then stirred under reflux for 2 h. After the reaction was completed as detected by TLC, it was extracted with dichloromethane three times, and the lower organic phase was collected. It was then back-extracted with saturated brine three times, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and after sufficient stirring, it was allowed to stand for 30 min. Then, the anhydrous sodium sulfate was removed by filtration, and dichloromethane was removed by distillation under reduced pressure to obtain a crude product. Then, the obtained crude product was separated and purified by column chromatography, and finally, the solid product 5q was obtained.

[0148] Example 48

[0149]

[0150] 4-Chloro-6,7-bis(2-methoxyethoxy)quinazoline (1 mmol), bis(triphenylphosphine)palladium(II) dichloride (0.1 mmol), and copper(I) iodide (20 mmol) were added to 20 mL of diisopropylamine. The mixture was stirred at room temperature for 10 min, then TMS-acetylene (1.2 mmol) was added, and the mixture was heated at 70 °C for 10 h. Then 10 mL of water and 10 mL of tert-butanol were added. After stirring, 4-nitrophenyl azide (1 mmol) was added, and the mixture was continuously heated under reflux with stirring. After the reaction was completed, the mixture was extracted with dichloromethane three times, and the lower organic phase was collected. Then it was back-extracted with saturated brine three times, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and the mixture was stirred thoroughly and allowed to stand for 30 min. Then anhydrous sodium sulfate was removed by filtration, and dichloromethane was removed by distillation under reduced pressure to obtain a crude product. Then the obtained crude product was separated and purified by column chromatography, and finally the solid product 5r was obtained.

[0151] Example 49

[0152] 4-Chloro-6,7-bis(2-methoxyethoxy)quinazoline (1 mmol), bis(triphenylphosphine)palladium(II) dichloride (0.1 mmol), and copper(I) iodide (20 mmol) were added to 20 mL of diisopropylamine. The mixture was stirred at room temperature for 10 min, then TMS-acetylene (1.2 mmol) was added, and the mixture was heated at 70 °C for 10 h. Then 10 mL of water and 10 mL of tert-butanol were added. After stirring, phenyl azide (1 mmol) was added, and the mixture was continuously heated under reflux with stirring. After the reaction was completed, the mixture was extracted with dichloromethane three times, and the lower organic phase was collected. Then it was back-extracted with saturated brine three times, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and the mixture was stirred thoroughly and allowed to stand for 30 min. Then anhydrous sodium sulfate was removed by filtration, and dichloromethane was removed by distillation under reduced pressure to obtain a crude product. Then the obtained crude product was separated and purified by column chromatography, and finally the solid product 5s was obtained.

[0153] Example 50

[0154] 4-Chloro-6,7-bis(2-methoxyethoxy)quinazoline (1 mmol), bis(triphenylphosphine)palladium(II) dichloride (0.1 mmol), and copper(I) iodide (20 mmol) were added to 20 mL of diisopropylamine. The mixture was stirred at room temperature for 10 min, then TMS-acetylene (1.2 mmol) was added, and the mixture was heated at 70 °C for 10 h. Then 10 mL of water and 10 mL of tert-butanol were added. After stirring, 4-azidopyridine (1 mmol) was added, and the mixture was continuously heated under reflux with stirring. After the reaction was completed, the mixture was extracted with dichloromethane three times, and the lower organic phase was collected. Then it was back-extracted with saturated brine three times, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and the mixture was stirred thoroughly and allowed to stand for 30 min. Then anhydrous sodium sulfate was removed by filtration, and dichloromethane was removed by distillation under reduced pressure to obtain a crude product. Then the obtained crude product was separated and purified by column chromatography, and finally the solid product 5t was obtained.

[0155] Example 51

[0156]

[0157] 4-Chloro-6,7-bis(2-methoxyethoxy)quinazoline (1 mmol), bis(triphenylphosphine)palladium(II) dichloride (0.1 mmol), and copper(I) iodide (20 mmol) were added to 20 mL of diisopropylamine. The mixture was stirred at room temperature for 10 min, then TMS-acetylene (1.2 mmol) was added, and the mixture was heated at 70 °C for 10 h. Then 10 mL of water and 10 mL of tert-butanol were added. After stirring, TMS-azide (1.5 mmol) was added, and the mixture was heated under reflux with stirring. After the reaction was completed, the mixture was extracted with dichloromethane three times, and the lower organic phase was collected. Then it was back-extracted with saturated brine three times, and the lower organic phase was collected. Anhydrous sodium sulfate was added thereto, and after sufficient stirring, the mixture was allowed to stand for 30 min. Then the anhydrous sodium sulfate was removed by filtration, and dichloromethane was removed by distillation under reduced pressure to obtain a crude product. Then the obtained crude product was separated and purified by column chromatography to finally obtain a solid product 5u.

[0158] Example 52

[0159] Detection of NO level in cell supernatant by Griess method: BV2 cells were seeded at 2×10 4 cells / well in a 96-well culture plate and placed in a 37 °C constant temperature incubator containing 5% CO 2 . After culturing for 24 h, the corresponding concentration of the test compound was added to the dosing group and incubated for 2 h. Then, LPS with a final concentration of 100 ng / ml was added to both the dosing group and the LPS model group. After culturing in the incubator for 24 h, 50 μL of the supernatant from each well was taken and mixed with 50 μl of Greiss buffer, and the reaction was carried out at room temperature for 15 min. The OD values of each group at a wavelength of 540 nm were detected by an enzyme-linked immunosorbent assay (ELISA) reader (Biotek).

[0160] Improvement effect of the compound on LPS-induced inflammation in BV2 cells

[0161]

[0162]

[0163] BV2 cells were seeded at 2×10 4 cells / well in a 96-well culture plate and placed in a 37 °C constant temperature incubator containing 5% CO 2Cultured in a 37°C constant temperature incubator. After 24 hours of culture, the corresponding concentration of the test compound was added to the dosing group and incubated for 2 hours. Subsequently, LPS with a final concentration of 100 ng / mL was added to both the dosing group and the LPS model group. Incubation continued for 24 hours, and MTT with a final concentration of 0.5 mg / mL was added to each well for live cell staining. After incubating in the incubator for 1 hour, the culture medium was discarded, 100 μL of DMSO was added to each well, and the plate was shaken on a shaker to fully dissolve the crystals. The OD values of each group at a wavelength of 490 nm were measured using an ELISA reader (Biotek).

[0164] Toxic effects of compounds on BV2 cells

[0165]

[0166] Example 53

[0167] From CO 2Take out the viable human cervical cancer Hela cell culture dish from the incubator and perform the following operations respectively: Perform aseptic operation beside the alcohol lamp, open the dish cover, aspirate the culture medium into the waste liquid tank, wash the culture medium in the culture bottle twice with 2 mL of PBS, digest with 0.25% trypsin, use a pipette to pipette the bottom of the culture bottle to make the cells detached, transfer the obtained cell suspension to a sterile centrifuge tube, set the centrifuge to 1000 r / min for 3 min, centrifuge, then slowly pour out the supernatant in the centrifuge tube, add 2 - 5 mL of culture medium, and perform cell counting under an inverted microscope. According to the counting results, seed the viable human cervical cancer Hela cells in the logarithmic growth phase at a density of 50,000 cells per well in a 96-well cell culture plate, culture with a medium containing 10% fetal bovine serum for 5 - 6 hours, add 100 μL of compound 5f and 5j (concentration 1.0 μM) diluted with the medium and recombinant human interferon γ (final concentration 100 ng / mg) to activate the IDO1 expression in Hela cells. After the operation, place the 96-well cell culture plate in a 37 °C cell incubator containing 5% carbon dioxide and culture for 18 hours, then terminate the reaction with a certain amount of 3.05 N trichloroacetic acid, and then incubate at 50 °C for 30 minutes. After the cell culture medium is precipitated, take the supernatant, color it with p-(N,N-dimethyl)benzaldehyde, and detect the absorbance at 480 nm with a multi-functional microplate reader. Use the group treated with the medium containing only IFNγ without drugs as 100% (At), and the group treated with the medium containing only 0.1% DMSO as the blank control 0% (Ab); calculate the absorbance under different treatment conditions according to the following formula: Absorbance%=(A - Ab) / (At - Ab), where A: drug treatment + 100 ng / mL IFNγ, Ab: blank control, At: no drug only containing 100 ng / mL IFNγ. Compounds 5p, 5q, 5r, 5s, 5t and 5u inhibited the IDO1 enzyme activity by approximately 52.17%, 56.29%, 37.85%, 29.63%, 20.67%, 11.38%; compounds 5f and 5j had no inhibitory activity against IDO1, and the IDO1 enzyme activities were 77.35% and 91.07% respectively. We performed molecular docking of compounds 5f and 5j with IDO1 and found that 5f and 5g could not effectively enter the active pocket of IDO1, and the triazole structure in compounds 5f and 5j was far from the heme of the IDO1 target, outside the active pocket.

[0168] We found that the N-methyl-D-aspartic acid (NMDA) receptor belongs to the ligand-gated ionotropic glutamate receptor family and is an important excitatory receptor in the central nervous system, which is closely related to neuropathic pain, learning, memory, etc. We used the whole-cell patch-clamp technique to test the agonist effects of compounds 5f and 5j on the human NMDA receptor. The cell line used was the HEK-293 cell line stably expressing the human NR1 / NR2B NMDA receptor. The method for recording the effects of 1.0 μM solutions of compounds 5f and 5j on NMDA receptor current was as follows: after forming a whole-cell seal, the cell membrane voltage was clamped at -70 mV, and NMDA receptor current was recorded in the Gap-free mode. Since the NMDA receptor requires the simultaneous presence of glycine and L-glutamate to be activated, in order to verify whether the compounds designed and synthesized in this study can enhance the current generated by the NMDA receptor in the presence of glycine and L-glutamate in the low concentration range, the concentrations of glycine and L-glutamate used in this study were both the concentrations corresponding to their EC10, that is, glycine 0.05 μmol·L －1 and L-glutamate 0.3 μmol·L －1 , glycine 0.05 μmol·L － 1 + L-glutamate 0.3 μmol·L －1 When present simultaneously, the corresponding membrane current was 100%. The effects of compounds 5f and 5j on the current of the human NR1 / NR2B NMDA receptor were measured at three concentrations of 1.0 μmol·L －1 in the presence of glycine 0.05 μmol·L －1 + L-glutamate 0.3 μmol·L －1 simultaneously. It was found that at this concentration, the membrane current could be effectively enhanced, and the increments were 182.36% (Example 32), 315.23 (Example 33), 290.48 (Example 34), 307.31 (Example 35), 322.26 (Example 36) and 207.35% (Example 40), indicating that they have an agonist effect on the human NR1 / NR2B NMDA receptor. We further performed molecular docking of compounds 5f and 5j with related targets (PDB: 5I56) and found that compounds 5f and 5j could effectively act on the active pocket, and the triazole structure was located at the active position of the target. Due to the large steric hindrance of the benzene ring in compound 3d, it could not enter the effective position of the target, and the increment of the membrane current at a concentration of 1.0 μmol·L －1 was 4.21%.

[0169] Example 54

[0170] We conducted an inhibitory activity experiment on the obtained compounds against cervical cancer cells and found that some compounds exhibited superior activity compared to erlotinib itself. Among them, compound 3h showed good performance, and we further evaluated the effect of compound 3h on the MAPK signaling pathway. The phosphorylation and total protein levels of ERK1 / 2, JNK, and p38 in Hela cells were detected by Western blot. Compound 3h dose-dependently inhibited the levels of p-ERK1 / 2 and p-JNK. The results indicated that compound 3h could downregulate the activation of the ERK and JNK signaling pathways.

[0171]

[0172] The above embodiments describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the scope of the principles of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for preparing and purifying a biologically active quinoline derivative and its application, characterized in that The structure of the quinoline derivative is: Wherein X or Y is NH or empty (benzene rings are directly connected), Z is methyl or empty; R is an aromatic ring derivative or an alkyl group or other groups.

2. The method for preparing a quinoline derivative according to claim 1, characterized in that The specific process is as follows: a certain amount of 4-chloro-6,7-bis(2-methoxyethoxy)quinazoline and triethylamine are added to N,N-dimethylformamide, 3-aminophenylacetylene is added, the mixture is heated to 80°C, water is added and stirred after a period of reaction, and then the mixture is extracted with dichloromethane for multiple times, the organic phases are combined, the organic solvent is evaporated off in a vacuum concentration, and then the mixture is separated by silica gel column chromatography to obtain N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine; a certain amount of 4-chloro-6,7-bis(2-methoxyethoxy)quinazoline and triethylamine are added to N,N-dimethylformamide, propargylamine is added, the mixture is heated to 80°C, water is added and stirred after a period of reaction, and then the mixture is extracted with dichloromethane for multiple times, the organic phases are combined, the organic solvent is evaporated off in a vacuum concentration, and then the mixture is separated by silica gel column chromatography to obtain N-(3-propargyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine.

3. The method for preparing a quinoline derivative according to claim 1, characterized in that The specific process is: take a certain amount of N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine, benzyl azide compounds, sodium L-ascorbate and anhydrous CuSO4 and add them into a round-bottom flask, then add a mixed solvent of water: tert-butanol: THF = 1:1:1 (v / v / v) into the flask, stir at room temperature, heat to react for a period of time, extract with dichloromethane several times, and collect the lower organic phase. The mixture is then back-extracted with saturated saline for multiple times, the lower organic phase is collected, anhydrous sodium sulfate is added thereto, the mixture is fully stirred and allowed to stand, and then the anhydrous sodium sulfate is removed by filtering, and distillation under reduced pressure is performed to remove dichloromethane to obtain a crude product, and then the obtained crude product is separated and purified by column chromatography, or a reaction to remove cuprous ions is performed to finally obtain the product; wherein the method for removing cuprous ions is as follows: a sulfuric acid solution is added dropwise to the reaction system after drying with anhydrous sodium sulfate, the pH value of the reaction system is adjusted to 1.5, the reaction system is stirred at room temperature, and then a saturated sodium sulfide solution is slowly added dropwise to the reaction system, and during the addition, attention is paid to the generation of solids in the reaction system, and when the solids no longer appear after continued addition, the addition is stopped, the reaction system is filtered after stirring at room temperature, the organic phase is separated, and the pure product is obtained after the organic phase is concentrated; or a saturated sodium hypochlorite solution is added dropwise to the reaction system after drying with anhydrous sodium sulfate, a saturated potassium iodide solution is added after stirring at room temperature, the reaction system is filtered after stirring, the organic phase is separated, and the target compound is obtained after the organic phase is concentrated.

4. The method for preparing a quinoline derivative according to claim 1, characterized in that The specific process is as follows: 4-chloro-6,7-di(2-methoxyethoxy)quinazoline, 3-alkynylphenylboronic acid, palladium acetate, dbpf, potassium phosphate are added to a mixed solution of ethanol, water and dimethyl sulfoxide, heated to 60°C and stirred for a period of time, then dichloromethane is added for extraction, concentrated and then a mixed solvent of water: tert-butyl alcohol: THF = 1:1:1 (v / v / v) is added, then benzyl azide compounds, sodium ascorbate and CuSO4 are added, stirred at room temperature and then heated under reflux for a period of time, after the reaction is completed, extracted with dichloromethane for multiple times, the organic phase is collected, and then back-extracted with saturated brine for multiple times, and the lower phase is collected. The organic phase is prepared by mixing anhydrous sodium sulfate, stirring thoroughly, standing and filtering to remove the anhydrous sodium sulfate, distilling under reduced pressure to remove dichloromethane to obtain a crude product, and then separating and purifying the obtained crude product by column chromatography to finally obtain the target compound; the molar ratio of the feeding amounts of 4-chloro-6,7-bis(2-methoxyethoxy)quinazoline to 3-alkynylphenylboronic acid to palladium acetate to dbpf to potassium phosphate is 1:1.2:0.2:0.2:1; the molar ratio of the feeding amounts of 4-chloro-6,7-bis(2-methoxyethoxy)quinazoline to benzyl azide compounds to sodium ascorbate to CuSO4 is 1:1.2:2:

1.

5. The method for preparing a quinoline derivative according to claim 1, characterized in that The specific process is as follows: Preparation method of N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine: 4-chloro-6,7-bis(2-methoxyethoxy)quinazoline and triethylamine are added to N,N-dimethylformamide, propargylamine is added, heated to 80°C, reacted for 5 hours, water is added and stirred, and then extracted with dichloromethane for multiple times, the organic phases are combined, vacuum concentrated and evaporated to remove the organic solvent, and then separated by silica gel column chromatography to obtain N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine; 6,7-dihydroxyquinazolin-4(3H)-one and DBU are added to N,N-dimethylformamide, PyBOP is added, stirred, and the mixture is stirred for 5 hours. After stirring, propargylamine is added dropwise to the reaction system, and after the addition is completed, the mixture is stirred at room temperature, the solvent is removed under vacuum, and N,N-dimethylformamide, potassium carbonate and 1-chloro-2-methoxyethane are added, and the mixture is heated to 90°C for reaction for a period of time, and N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine is obtained after vacuum concentration; a magnetic stirrer, a graphite anode (6.0 mm graphite rod), a platinum electrode and an Ag / Ag+ reference electrode are added to a reaction tube; a solution of 2-amino-4,5-dihydroxybenzamide, formaldehyde, p-toluenesulfonic acid·hydrate and tetrabutylammonium perchlorate in acetonitrile is added; at room temperature, the mixture is heated to 1.0 V (relative to Ag / Ag + ) for a period of time, the reaction mixture was concentrated in vacuo, N,N-dimethylformamide was added, and then benzotriazol-1-yloxy tris(dimethylamino)phosphonium hexafluorophosphate and propargylamine were added, DBU was slowly added dropwise under stirring, and the reaction mixture was stirred at 110°C for a period of time, N,N-dimethylformamide, potassium carbonate and 1-chloro-2-methoxyethane were added after cooling the reaction, and the mixture was heated to 90°C for a period of time, washed with brine, extracted and concentrated in vacuo to obtain N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine; 2-nitro-4,5-dihydroxybenzonitrile was added to water In the reaction mixture, sodium sulfite is added, and the reaction mixture is heated to 50°C, kept stirring at this temperature for a period of time, then heated to 65°C, concentrated hydrochloric acid is slowly added dropwise, cooled to room temperature after complete addition, and the pH is adjusted to about 10 using a 50% sodium hydroxide aqueous solution. The formed precipitate is filtered, washed, dried, dissolved in toluene, acetic acid and DMF-DMA are added, and heated under reflux conditions. After the reaction is completed, the excess DMF-DMA is removed in vacuo, cooled to room temperature, propargylamine and acetic acid are added; the reaction mixture is stirred at 60°C, the temperature is raised to reflux, and the reaction is continued to be stirred, the reaction mixture is cooled to room temperature, and the organic layer is extracted with ethyl acetate. The organic extracts are combined, dried, filtered, concentrated under vacuum, N,N-dimethylformamide, potassium carbonate and 1-chloro-2-methoxyethane are added, heated to 90°C, reacted for a period of time, washed with brine, extracted and concentrated in vacuo to obtain N-(3-propynyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine.

6. The method for preparing a quinoline derivative according to claim 1, characterized in that The specific process is as follows: a certain amount of N-(3-propargyl)-6,7-bis(2-methoxyethoxy)-4-quinolinamine, benzyl azide compounds, sodium L-ascorbate and anhydrous CuSO4 are added to a round-bottom flask, and then a mixed solvent of water: tert-butanol: THF = 1:1:1 (v / v / v) is added to the flask, and the mixture is heated to react for a period of time after stirring at room temperature, extracted with dichloromethane for multiple times, and the lower organic phase is collected, and then back-extracted with saturated brine for multiple times, and the lower organic phase is collected, anhydrous sodium sulfate is added thereto, and the mixture is fully stirred and allowed to stand, and then the anhydrous sodium sulfate is filtered out, and the dichloromethane is removed by reduced pressure distillation to obtain a crude product, and then the crude product is separated and purified by column chromatography to finally obtain the product.

7. The method for preparing a quinoline derivative according to claim 1, characterized in that The specific process is: take 4-chloro-6,7-di(2-methoxyethoxy)quinazoline and sodium azide, add them to N,N-dimethylformamide, stir at room temperature for a period of time, pour into water, filter, add the filter cake and phenylacetylene, sodium ascorbate and CuSO4 into a round-bottom flask, then add a mixed solvent of water: tert-butanol: THF = 1:1:1 (v / v / v) into the flask, stir at room temperature and then heat under reflux to react, after the reaction is completed, extract with dichloromethane, collect the lower organic phase, then back-extract with saturated brine, collect the lower organic phase, add anhydrous sodium sulfate thereto, stir well and let stand, then filter to remove the anhydrous sodium sulfate, distill under reduced pressure to remove dichloromethane to obtain a crude product, and then separate and purify the obtained crude product by column chromatography to finally obtain a solid product.

8. The method for preparing a quinoline derivative according to claim 1, characterized in that The specific process is as follows: a certain amount of 4-chloro-6,7-bis(2-methoxyethoxy)quinazoline, bistriphenylphosphine palladium dichloride and cuprous iodide are added to diisopropylamine, TMS-acetylene is added after stirring at room temperature, water and tert-butyl alcohol are added after heating at 70°C for reaction, azide compounds are added after stirring, heating and reflux stirring are continued, extraction is performed with dichloromethane after the reaction is completed, the lower organic phase is collected, and then back-extraction is performed with saturated saline, the lower organic phase is collected, anhydrous sodium sulfate is added thereto, the mixture is fully stirred and allowed to stand, anhydrous sodium sulfate is removed by filtration, dichloromethane is removed by reduced pressure distillation to obtain a crude product, and then the obtained crude product is separated and purified by column chromatography to finally obtain a solid product; the molar ratio of the feed amount of 4-chloro-6,7-bis(2-methoxyethoxy)quinazoline to bistriphenylphosphine palladium dichloride to cuprous iodide to TMS-acetylene to azide compounds is 1:0.1:20-50:1.

2.

9. The quinoline derivatives as claimed in claim 1 can be used in targeted anti-inflammatory applications.

10. The quinoline derivatives as claimed in claim 1 can be used in targeted anti-tumor applications.

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