1,4-Benzoxazinone derivatives with biological activity and preparation method and application thereof

Abstract

The present invention discloses a biologically active 1,4-benzoxazinone derivative and a preparation method and application thereof, wherein the biologically active 1,4-benzoxazinone derivative has the structural formula: utilizing the ability of 1,2,3-triazole ring to coordinate with metal ions, wherein the N atom of triazole and heme ferrous iron can form a coordination interaction, and a batch of novel compounds containing triazole are designed. The prepared compounds are tested for inhibiting IDO1 enzyme activity, and it is found that some compounds have an inhibitory effect on IDO1, and also have a good inhibitory effect on some tumor cells, and can reduce some inflammatory indicators at the same time, and can be further used to prepare IDO1 inhibitory drugs, anti-tumor drugs and anti-inflammatory drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of preparation and application of 1,4-benzoxazinone derivatives, and specifically relates to a 1,4-benzoxazinone derivative with biological activity, a preparation method and application thereof. Background Art

[0002] Heterocyclic compounds are the largest branch of organic compounds. They are widely distributed in nature, with a wide variety of chemical structures and unique properties and uses. 1,4-Benzoxazinone (2H-1,4-Benzoxazin-3(4H)-one, 1) is an important N- and O-containing heterocyclic compound, and its related derivatives are mainly distributed in plants such as Aegilops, Arundo donax, and Triticeae in nature. Different types of plants contain different types of compounds, such as oats mainly contain DIBOA2, and wheat and corn both contain DIMBOA3. In nature, 2-hydroxy-2H-1,4-benzoxazin-3(4H)-one (HBOA) and 2-hydroxy-7-methyl-2H-1,4-benzoxazin-3(4H)-one (HMBOA) are stable forms of 2H-1,4-benzoxazinone compounds. The 2-hydroxyl group and 7-methoxy group in the structure are both conducive to the breakage and rearrangement of the CO bond of benzoxazinone and the formation of cations. An important mechanism of the physiological effects of benzoxazinone compounds is the ability to react with nucleophiles. For example, its electrophilic center can react with the C-8 carbon atom of guanine to cause DNA mutations. It can also combine with amino acid derivatives to modify proteins, which is one of the main reasons why this type of compound has insecticidal and antibacterial activities.

[0003]

[0004] Since 1,4-benzoxazinone has a wide range of biological activities and low biological toxicity, a large number of derivatives based on it have been synthesized, especially in the field of pesticides, with a large number of reports on its insecticide, fungicide, and weed control. For example, Sumitomo has developed a very classic herbicide, fluazifop-butyl, for soybeans, peanuts, and cotton. This herbicide can effectively control many difficult-to-control grass weeds, and has the characteristics of flexible fertilization time, excellent rotation conditions, and environmental friendliness. It also performs well in medicine. For example, Anderluh et al. synthesized a 1,4-benzoxazinone derivative 5 that has anticoagulant effect and inhibits fibrinogen receptor function.

[0005]

[0006] Feng et al. synthesized 1,4-benzoxazinone derivative 6, which not only has antibacterial and antifungal effects, but also has potential antidepressant function and can be used to treat schizophrenia. Compound 7 synthesized by Niemeyer et al. by introducing glycoside groups into the 1,4-benzoxazinone skeleton framework and compound 8 synthesized by Frechette et al. both have good inhibitory effects on bacteria. The 1,4-benzoxazinone derivative 9c designed by Manojit's team reduced the viability of A549 cells, DLD-1 cells and MV4-11 cells to 21.7%, 12.1% and 0.6% at a concentration of 20 μM, which is better than erlotinib's 68.5%, 55.4% and 16.8%. The 1,4-benzoxazinone derivative 14n designed by Shoji's team can be used as an excellent mineralocorticoid receptor (MR) with an inhibition rate of IC 50 The value reached 41nM, and the inhibition rate IC of highly homologous androgen receptor (AR), progesterone receptor (PR) and glucocorticoid receptor (GR) was 50 The values ​​were all above 1800nM, showing good selectivity. The Yellu research group obtained a series of 1,4-benzoxazinone derivatives containing 1,2,3-triazole structure, among which compound 5b had an inhibition rate of IC on MCF-7 cells and Hela cells. 50 The values ​​were 17.08μg / mL and 15.38μg / mL, respectively, and showed a certain inhibitory effect in mouse in vivo experiments. The compound 8d-1 designed by Li's research group with 1,4-benzoxazinone as the skeleton is a pan-class I PI3K / mTOR inhibitor with an IC50 of 0.63nM for PI3Ka. The mouse in vivo experiments showed good oral bioavailability and showed a certain inhibitory effect on Hela cells and A549 cells, with an IC 50 The values ​​were 1.35μM and 1.22μM, respectively. The inhibitory effect of 1,4-benzoxazinone derivative 3c designed by Miao's research group on A549 cells IC 50 The value is 3.29μM, and it can upregulate the expression of LC3 protein in A549 cells, causing autophagy, and at the same time, it can block A549 cells in the G1 phase and cause the A549 cell nucleus to shrink. Summary of the invention

[0007] The technical problem solved by the present invention is to provide a biologically active 1,4-benzoxazinone derivative and a preparation method thereof. The method utilizes the ability of 1,2,3-triazole ring to coordinate with metal ions, wherein the N atom of triazole and heme ferrous iron can form a coordination interaction, and a batch of novel compounds containing triazole are designed. The prepared compounds were tested for inhibiting IDO1 enzyme activity, and it was found that some compounds have an inhibitory effect on IDO1 and also have a good inhibitory effect on some tumor cells. At the same time, some inflammatory indicators can be reduced, and they can be further used to prepare IDO1 inhibitory drugs, anti-tumor drugs and anti-inflammatory drugs.

[0008] The present invention adopts the following technical solution to solve the above technical problems. The biologically active 1,4-benzoxazinone derivative is characterized by its structural formula:

[0009]

[0010] Where n is 0 or 1; R 1 , R 2 , R 3 , R 4 and R 5 It is one or more of H, halogen, trifluoromethyl, ethyl, phenyl, hydroxyl, cyano, nitro or aromatic ring.

[0011] The method for preparing the biologically active 1,4-benzoxazinone derivatives of the present invention is characterized by the following specific steps:

[0012] Step S1, adding 2-amino-4-Bocamino-phenol and potassium carbonate to a dichloromethane solution, the reaction system is heated to 0°C, and then a dichloromethane solution containing chloroacetyl chloride is added dropwise to the system to maintain a stable constant state. After the addition is complete, the reaction is continued to be stirred and the reaction is completed by detecting by TLC, and the reaction solution is cooled to room temperature. The reaction solution is transferred to a separatory funnel in batches, extracted with saturated brine, and the upper organic phase is collected. Anhydrous sodium sulfate is added thereto, and the mixture is stirred thoroughly and allowed to stand. The anhydrous sodium sulfate is removed by filtration, and the organic solvent is removed by reduced pressure distillation using a rotary evaporator at 35°C. The obtained crude product is then separated and purified by column chromatography to finally obtain 6-Bocamino-2H-1,4-benzoxazine-3(4H)-one;

[0013] Step S2, adding the 6-Bocamino-2H-1,4-benzoxazine-3(4H)-one obtained in step S1 to dichloromethane, stirring to completely dissolve, then adding trifluoroacetic acid, stirring to react at room temperature, and adjusting the pH of the reaction system to 6 with a saturated sodium carbonate solution, separating the organic phase, and concentrating to obtain 6-Bocamino-2H-1,4-benzoxazine-3(4H)-one;

[0014] Step S3, adding 6-Bocamino-2H-1,4-benzoxazine-3(4H)-one, 3-ethynylaniline, HATU and DIPEA obtained in step S2 to a round-bottom flask in sequence, and then adding N,N-dimethylformamide to the system, stirring the reaction at room temperature, and adding water to the reaction system. During the stirring process, solids are precipitated, which are filtered and dried to obtain a terminal acetylene compound;

[0015] Step S4, adding the terminal acetylene compound, azide compound, sodium L-ascorbate and anhydrous CuSO4 obtained in step S3 to a reaction container, then adding a mixed solvent of water, tert-butyl alcohol and THF to the reaction container, stirring at room temperature to dissolve all of them, maintaining room temperature, and detecting the completion of the reaction by TLC, laying diatomaceous earth in the funnel, filtering under reduced pressure to remove solid impurities, collecting the filtrate, transferring it to a separatory funnel, extracting with dichloromethane for multiple times, collecting the lower organic phase, and then back-extracting with saturated brine for multiple times, collecting the lower organic phase, adding anhydrous sodium sulfate thereto, stirring thoroughly and letting it stand, filtering to remove anhydrous sodium sulfate, and removing organic solvents such as DCM by reduced pressure distillation at 35°C to obtain a crude product, and then separating and purifying the obtained crude product by column chromatography to finally obtain the target compound.

[0016] It is further defined that the molar ratio of 2-amino-4-Bocamino-phenol to chloroacetyl chloride in step S1 is 1:1.

[0017] It is further defined that the molar ratio of 6-Bocamino-2H-1,4-benzoxazine-3(4H)-one, 3-ethynylaniline, HATU and DIPEA in step S3 is 1:1.5:1.5:3.

[0018] The invention discloses an application of the biologically active 1,4-benzoxazinone derivative in the preparation of IDO1 inhibitory drugs, anti-tumor drugs or anti-inflammatory drugs.

[0019] It is further defined that the tumor is human cancer cell A549, Huh7, MCF-7, HCT-116 or SKOV3.

[0020] It is further defined that by using ionic liquids in combination with DMSO to dissolve biologically active 1,4-benzoxazinone derivatives, the inhibitory activity of 1,4-benzoxazinone derivatives on lymphoma cells and IDO1 enzyme activity is effectively increased through the synergistic effect of ionic liquids. The specific process is: dissolving the biologically active 1,4-benzoxazinone derivatives in a mixed solution of ionic liquid / DMSO with a volume ratio of 1:1, ultrasonically reacting at 25°C for 30 minutes, and fully mixing the 1,4-benzoxazinone derivatives and the ionic liquid, wherein the ionic liquid is one or more of [BMIM]PF6, [BMIM]Ac or [BMIM]NO3.

[0021] It is further defined that a supercritical device is used for drug mixing, and a biologically active 1,4-benzoxazinone derivative is added to a mixed solution of an ionic liquid / DMSO with a volume ratio of 1:1 to 1.5 to prepare a concentration of 10 mmol / L, and then all of it is transferred into a supercritical reactor, the outlet valve is closed, and CO2 fluid is introduced into the reactor at a flow rate of 15 L / min until the pressure reaches 7 to 8 MPa. The temperature is set to 30 to 50°C, and the mixture is stirred at a speed of 300 r / min. After 0.5 h, CO2 is slowly released to obtain a mixed solution, and the ionic liquid is one or more of [BMIM]PF6, [BMIM]Ac or [BMIM]NO3.

[0022] The invention discloses an application of the biologically active 1,4-benzoxazinone derivative in the preparation of a protective preparation for HT22 nerve cell damage caused by glutamate.

[0023] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0024] 1. The present invention has obtained a series of novel 1,4-benzoxazinone compounds, some of which have an inhibitory effect on IDO1 and also have a good inhibitory effect on some tumor cells. At the same time, some of the compounds can reduce some inflammatory indicators, and can be further used to prepare IDO1 inhibitory drugs, anti-tumor drugs and anti-inflammatory drugs;

[0025] 2. This type of compound has a good inhibitory effect on IDO1. The 1,2,3-triazole structure can form a relatively strong effect with the Fe ions in heme, competitively inhibiting the activity of IDO1. The phenyl structure with a methoxy substituent can enter the hydrophilic pocket, and the benzyl structure can enter the hydrophobic pocket. The compound molecules can combine well with the IDO1 target pocket, further proving that this type of compound is a rationally designed IDO1 inhibitor.

[0026] 3. This type of compound can inhibit the growth of tumor cells by increasing the content of reactive oxygen in tumor cells.

[0027] 4. The present invention finds that the activity of the target compound can be improved through the synergistic effect of ionic liquids. DETAILED DESCRIPTION

[0028] The above contents of the present invention are further described in detail below through examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples, and all technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

[0029] Example 1

[0030]

[0031] In a 500 mL clean round-bottom flask equipped with a stirrer, 2-amino-4-Bocamino-phenol (0.10 mol) and potassium carbonate (0.10 mol) were added to a dichloromethane solution, the reaction system was heated to 0°C, and then a dichloromethane solution containing chloroacetyl chloride (0.10 mol) was slowly added dropwise to the system, and the mixture was kept stable and constant. After the addition was complete, the reaction was continued to stir for 30 min. After the reaction was completed by TLC, the reaction was cooled to room temperature and the reaction solution was transferred to a separate The mixture was added into a liquid funnel and extracted with saturated brine (200 mL×3), the upper organic phase was collected, anhydrous sodium sulfate was added thereto, the mixture was stirred thoroughly and allowed to stand for 30 min, anhydrous sodium sulfate was removed by filtration, and the organic solvent DCM was removed by reduced pressure distillation at 35°C using a rotary evaporator, and then the crude product was separated and purified by column chromatography (PE:EA=4:1, v / v, the chromatography column was filled with silica gel) to finally obtain 6-Bocamino-2H-1,4-benzoxazine-3(4H)-one.

[0032] Example 2

[0033]

[0034] 6-Bocamino-2H-1,4-benzoxazine-3(4H)-one (0.1 mol) was added to 200 mL of dichloromethane and stirred to completely dissolve. Then 20 mL of trifluoroacetic acid was added and the reaction was stirred at room temperature for 2 h. The pH of the reaction system was adjusted to 6 with a saturated sodium carbonate solution. The organic phase was separated and concentrated to obtain 6-Bocamino-2H-1,4-benzoxazine-3(4H)-one.

[0035] Example 3

[0036]

[0037] Into a 500mL clean round-bottom flask equipped with a stirrer, 6-Bocamino-2H-1,4-benzoxazine-3(4H)-one (3.3g, 0.02mol), 3-ethynylaniline (3.69g, 0.03mol), HATU (12.96g, 0.03mol) and DIPEA (8.13g, 0.06mol) were added in sequence. Then, 300mL of N,N-dimethylformamide was added to the reaction system and stirred at room temperature for 1h. 200mL of water was added to the reaction system. A large amount of solid precipitated during the stirring process. The terminal acetylene compound was obtained by filtration and drying. 1H NMR (400MHz, DMSO-d6): 10.76 (s, 1H), 10.26 (s, 1H), 8.04 (s, 1H), 7.96 (d, J=8.0Hz, 1H), 7.68 (d, J= 8.0Hz,1H),7.54-7.52(m,2H),7.25-7.22(m,1H),6.93(d,J=8.0Hz,1H),4.54(s,2H),4.30(s,1H).

[0038] Example 4

[0039]

[0040] Take the terminal acetylene compound (2mmol), 4-bromobenzyl azide (4mmol), sodium L-ascorbate (0.72g, 3.63mmol) and anhydrous CuSO4 (0.3g, 1.88mmol) and add them to a 250mL round-bottom flask equipped with a stirrer, then add 100mL of a mixed solvent of water: tert-butyl alcohol: THF = 1:1:1 (v / v / v) to the flask, stir at room temperature for 10min, and dissolve it completely. Keep room temperature and stir overnight for 12h. After the reaction is completed by TLC detection, diatomaceous earth is laid in the funnel, and the solid impurities are removed by vacuum filtration. Collect the filtrate, transfer it to a separatory funnel, extract it 3 times with DCM, and collect the lower organic phase. Then extract it 3 times with saturated brine, collect the lower organic phase, add anhydrous sodium sulfate to it, stir it thoroughly and let it stand for 30min, then filter to remove the anhydrous sodium sulfate. DCM and other organic solvents were removed by reduced pressure distillation at 35°C to obtain a crude product, which was then separated and purified by column chromatography (DCM:MeOH=500:1, v / v, the chromatography column was filled with neutral-Al2O3) to finally obtain a white solid compound m1 (yield 93.6%). 1 H NMR (400MHz, DMSO-d6): 10.78(s,1H),10.29(s,1H),8.72(s,1H),8.38(s,1H),8.04(d,J=8.0Hz,1H),7.88(d,J=8.0Hz, 1H),7.62-7.53(m,4H),7.34(d,J=8.0Hz,2H),7.26(d,J=12.0Hz,1H),6.93(d,J=12.0Hz,1H),5.67(s,2H),4.55(s,2H).

[0041] Example 5

[0042]

[0043] The synthesis method of compound m2 is similar to that of compound m1. The crude product is separated and purified by column chromatography to obtain white solid D2 (yield 87.1%).

[0044] Example 6

[0045]

[0046] The synthesis method of compound m3 is similar to that of compound m1. The crude product is separated and purified by column chromatography to obtain white solid D3 (yield 86.5%).

[0047] Example 7

[0048]

[0049] The synthesis method of compound m4 is similar to that of compound m1. The crude product is separated and purified by column chromatography to obtain white solid D4 (yield 90.2%).

[0050] Example 8

[0051]

[0052] The synthesis method of compound m5 is similar to that of compound m1. The crude product is separated and purified by column chromatography to obtain white solid D5 (yield 88.1%).

[0053] Example 9

[0054]

[0055] The synthesis method of compound m6 is similar to that of compound m1. The crude product is separated and purified by column chromatography to obtain white solid D6 (yield 79.9%).

[0056] Example 10

[0057]

[0058] The synthesis method of compound m7 is similar to that of compound m1. The crude product is separated and purified by column chromatography to obtain white solid D7 (yield 76.8%).

[0059] Embodiment 11

[0060]

[0061] The synthesis method of compound m8 is similar to that of compound m1. The crude product is separated and purified by column chromatography to obtain white solid D8 (yield 82.4%).

[0062] Example 12

[0063]

[0064] The synthesis method of compound m9 is similar to that of compound m1. The crude product is separated and purified by column chromatography to obtain white solid D9 (yield 93.7%).

[0065] Embodiment 13

[0066]

[0067] The synthesis method of compound m10 is similar to that of compound m1. The crude product is separated and purified by column chromatography to obtain a white solid D10 (yield 77.4%).

[0068] Embodiment 14

[0069]

[0070] The synthesis method of compound m11 is similar to that of compound m1. The crude product is separated and purified by column chromatography to obtain a white solid D11 (yield 80.9%).

[0071] Embodiment 15

[0072]

[0073] The synthesis method of compound m12 is similar to that of compound m1. The crude product is separated and purified by column chromatography to obtain white solid m12 (yield 72.5%).

[0074] Example 16

[0075]

[0076] The synthesis method of compound m13 is similar to that of compound m1. The crude product is separated and purified by column chromatography to obtain white solid m13 (yield 84.2%).

[0077] Embodiment 17

[0078]

[0079] The synthesis method of compound m14 is similar to that of compound m1. The crude product is separated and purified by column chromatography to obtain white solid m14 (yield 87.1%).

[0080] Embodiment 18

[0081]

[0082] The synthesis method of compound m15 is similar to that of compound m1. The crude product is separated and purified by column chromatography to obtain white solid m15 (yield 77.8%).

[0083] Embodiment 19

[0084]

[0085] The synthesis method of compound m16 is similar to that of compound m1. The crude product is separated and purified by column chromatography to obtain white solid m16 (yield 73.1%).

[0086] Embodiment 20

[0087]

[0088] The synthesis method of compound m17 is similar to that of compound m1. The crude product is separated and purified by column chromatography to obtain white solid m17 (yield 79.0%).

[0089] Embodiment 21

[0090]

[0091] The synthesis method of compound m18 is similar to that of compound m1. The crude product is separated and purified by column chromatography to obtain white solid m18 (yield 91.5%).

[0092] Embodiment 22

[0093]

[0094] The synthesis method of compound m19 is similar to that of compound m1. The crude product is separated and purified by column chromatography to obtain white solid m19 (yield 89.4%).

[0095] Embodiment 23

[0096]

[0097] The synthesis method of compound m20 is similar to that of compound m1. The crude product is separated and purified by column chromatography to obtain white solid m20 (yield 87.6%).

[0098] Embodiment 24

[0099]

[0100] The synthesis method of compound m21 is similar to that of compound m1. The crude product is separated and purified by column chromatography to obtain white solid m21 (yield 78.2%).

[0101] Embodiment 25

[0102]

[0103] The synthesis method of compound m22 is similar to that of compound m1. The crude product is separated and purified by column chromatography to obtain white solid m22 (yield 81.5%).

[0104] Embodiment 26

[0105]

[0106] The synthesis method of compound m23 is similar to that of compound m1. The crude product is separated and purified by column chromatography to obtain white solid m23 (yield 85.6%).

[0107] Embodiment 27

[0108]

[0109] The synthesis method of compound m24 is similar to that of compound m1. The crude product is separated and purified by column chromatography to obtain white solid m24 (yield 76.3%).

[0110] Embodiment 28

[0111]

[0112] The synthesis method of compound m25 is similar to that of compound m1. The crude product is separated and purified by column chromatography to obtain white solid m25 (yield 80.2%).

[0113] Embodiment 29

[0114] Antitumor activity test: Cells were cultured in DMEM or RPMI 1640 medium containing 10wt% fetal bovine serum (FBS) and 1wt% penicillin / streptomycin at 37°C and 5% CO2 (volume fraction). According to the CCK-8 manual, CCK-8 was tested in several human cancer cell lines including A549, Huh7, MCF-7, HCT-116, and SKOV3. Cells were plated at 5×10 cells per well. 3 The density of cells was plated on a 96-well plate. After incubation for 24 hours, the cells were treated with different compounds at a concentration of 40 μM for 48 hours. 10 μL of CCK-8 reagent was added to each well, and the CCK-8 reagent was dissolved in the cell culture medium for 1 hour to measure the cell viability (Table 1). The absorbance was measured at 450 nm using an enzyme reader. The IC 50 The dose-response curves were drawn using five concentration sequences of 0, 10, 20, 40, 80, and 160 μM, and the IC was calculated using GraphPad Prism software. 50 Values ​​(Table 2).

[0115] Table 1

[0116]

[0117]

[0118] Table 2

[0119] CompdNo. <![CDATA[IC 50 (μM)]]> CompdNo. <![CDATA[IC 50 (μM)]]> m3 >100 m14 88.58±18.03 m5 >100 m17 29.38±3.49 m6 55.25±1.56 m22 25.06±4.62 m8 51.12±2.54 m23 24.05±3.83 m10 43.45±5.67 m24 38.68±14.89 m12 >100 m25 29.06±1.15

[0120] Embodiment 30

[0121] The cells were 5×10 3 The cells were seeded on a 96-well plate at a density of 100 cells and allowed to adhere overnight. The compounds were added to the cells at concentrations of 0, 5, 10, and 20 μM for 24 hours. The culture medium was then removed and new culture medium without FBS but containing 10 μM DCFH-DA was added and incubated in the dark for 30 minutes. The cells were washed three times with culture medium without FBS to completely remove excess probes, and then ROS levels were measured and photographed using a confocal microscope. It was found that compounds 22 and 23 could effectively increase the level of reactive oxygen species in A549 cells.

[0122] Embodiment 31

[0123] For the inhibitory activity of lymphoma cells: (1) Collect the cells in the logarithmic growth phase with a 15mL centrifuge tube and centrifuge them at 1000rpm for 5min in a desktop high-speed small centrifuge. After discarding the supernatant, add culture medium to resuspend the cells, count them using a cell counting plate and plate them on a 96-well plate, with 2000 cells plated in each well and 100μL solution in one well. (2) Dissolve the compound with dimethyl sulfoxide, first prepare a drug concentration of 10mM, then dilute it 100 times with the corresponding cell culture medium to obtain a 100uM solution, which is then diluted 2 times to obtain a 50uM solution. The 50uM solution is diluted 5 times in sequence, and 7 concentrations are prepared for each medicinal culture medium. Add the prepared solution to a 96-well plate, add 100μL to each well, so that the final drug concentration ranges from 3.2nM to 50μM (5-fold increase), and repeat three wells for each sample. (3) Set up a cell-free, drug-free, culture medium group and a cell-free, drug-free, culture medium group as control groups. (4) After wiping the 96-well plate with an alcohol cotton ball, place it in a constant temperature cell culture incubator and culture it for 72 hours at 37°C, 5% CO2 and saturated humidity. (5) Perform numerical measurements on the cells in the 96-well plate. For suspended cells (specifically REC-1 cells and Ramos cells in this article), add 20 μL of CellTiter-Glo luminescence detection reagent directly to each well, incubate in a 37°C incubator for 10 minutes, and then perform fluorescence detection. (6) Subtract the value of the blank control group from the value measured in each well, calculate the percentage between the drug concentration and the absorbance and luminescence intensity, and use Prism software to draw a curve graph to obtain the half-inhibitory concentration (IC50) of the drug. 50 , Table 3).

[0124] Table 3

[0125]

[0126]

[0127] Embodiment 32

[0128] During the test, it was found that some compounds had poor solubility in DMSO, which had a certain impact on the test results. Therefore, three ionic liquids were selected in this embodiment, and the compounds were dissolved in a mixed solution of ionic liquid: DMSO = 1:1 (v / v). Ultrasonic reaction was carried out at 25°C for 30 minutes to fully mix the compounds and ionic liquids. On the one hand, the problem of poor drug solubility was solved, and on the other hand, it was hoped that the ionic liquids and drugs could interact with each other to form new active ingredients (API-ILs).

[0129] The three ionic liquids selected were: IL1 = [BMIM] PF6; IL2 = [BMIM] Ac; IL3 = [BMIM] NO3. The Ramos antiproliferative activity of the three ionic liquids was studied in the same manner as in Example 31. It was found that the combination of IL3 and the compound had the best antiproliferative effect. The antiproliferative activity of the ionic liquid alone was very weak, but the drug IC after adding the ionic liquid IL3 was 50 The IC of compound m5 against Ramos after combining with IL1 and IL2 was significantly decreased, indicating that the anti-proliferative activity was enhanced, indicating that the ionic liquid had a synergistic effect. 50 The specific comparison data are shown in Table 4 below.

[0130] Table 4

[0131]

[0132]

[0133] Embodiment 33

[0134] Materials used: HeLa cells, DMEM culture medium, fetal bovine serum, double antibody (penicillin-streptomycin), high-purity water, DMSO, human interferon IFN-γ, 3.05N trichloroacetic acid, p-dimethylaminobenzaldehyde, acetic acid.

[0135] Operation method: 1. Drug dilution: The medium used for drug dilution is 0.1wt% DMSO, so the final concentration of DMSO is 0.1wt% in all treatments. 2. Cell culture medium formula: DMEM medium, 10wt% fetal bovine serum, 1wt% double antibody. 3. Detection method: Add 100μL of medium containing HeLa cells to a 96-well plate, 50,000 cells / well. The cells are cultured at 37°C and 5% CO2 for 24h. Add 100μL of the test compound diluted with culture medium (concentrations are 3.2nM, 16nM, 80nM, 0.4μM, 2μM, 10μM, 50μM) and recombinant human interferon γ to activate IDO1 expression in Hela cells. After the operation, place the 96-well cell culture plate in a 37°C cell culture incubator rich in 5% carbon dioxide and culture for 18 hours. Take out 140 μL of culture medium to a new 96-well plate, and add 20 μL of 3.05N trichloroacetic acid to each well. Incubate the 96-well plate at 50°C for 30 min. Centrifuge the 96-well plate at 2500 rpm for 10 min, take 100 μL of supernatant from each well and transfer it to a new 96-well plate, and add 100 μL of 2% (w / v) p-dimethylaminobenzaldehyde (acetic acid as solvent) to each well. Incubate the 96-well plate at room temperature for 10 min, and detect OD480 with an enzyme-labeled instrument. 4. Data processing: Three parallel experiments were performed for each concentration of the experiment. Graphpad Prism was used to analyze the absorbance data. The one without drug treatment but containing 100 ng / mL IFN-γ was taken as 100% (At). The blank experiment with only culture medium was taken as 0% (Ab). The absorbance at each drug treatment was calculated according to the following equation: Absorbance% = (A-Ab) / (At-Ab), A: drug treatment + 100ng / mL IFN-γ, Ab: blank control, At: no drug treatment but containing 100ng / mL IFN-γ. The values ​​of absorbance% and the values ​​of different drug concentrations were used to make a nonlinear regression of the sigmoidal dose-response curve. The regression equation is: Y = B + (TB) / 1 + 10 ((LogEC50-X)×HillSlope) , Y = absorbance%, B = minimum absorbance%, T = maximum absorbance%, X = Log drug concentration, Hill Slope = slope (Hill coefficient). Compounds 5 and 13 were selected to test the anti-proliferative activity of IDO1 in combination with ionic liquid IL3. If ultrasound-assisted dissolution was used, it was found that compound 5 and compound 13 had a synergistic effect of ionic liquid IL3 on IC 50The values ​​were 1.582μM and 0.921μM, respectively. Compound 5, ionic liquid IL3 and DMSO were mixed in a volume ratio of 1:1 to a concentration of 10mmol / L, and then all were transferred into a supercritical reactor. The outlet valve was closed, and CO2 fluid was introduced into the reactor at a flow rate of 15L / min until the pressure reached 8MPa. The temperature was set to 50°C and stirred at a speed of 300r / min. CO2 was slowly released after 0.5h. The treated compound 5 solution was reconfigured into a concentration gradient for the inhibitory activity test against IDO1. IC 50 The IC value was 0.357 μM after dissolution with ionic liquid IL3 alone. 50 It is 0.095 μM; thus, it can be found that the ionic liquid IL3 specifically improves the function of drug activity.

[0136] Table 5

[0137] Comp. <![CDATA[IC 50 (μM)]]> m1 11.235 m2 45.987 m3 37.717 m4 ＞50 m5 3.635 m6 8.021 m7 10.082 m8 9.987 m9 30.588 m10 ＞50 m11 14.388 m12 8.250 m13 8.457 m14 29.009 m15 14.426

[0138] Embodiment 34

[0139] Detection of the protective effect of compounds on HT22 neuronal cell damage induced by glutamate

[0140] HT22 cells were cultured at 5×10 3 Each well was inoculated on a 96-well culture plate and cultured in a 37°C constant temperature incubator containing 5% CO2 by volume. After 24 hours of culture, the corresponding concentration of the test compound was added to the drug-treated group and incubated for 2 hours, and then 10 mM glutamate was added to the drug-treated group and the glutamate model group. The culture was continued for 24 hours, and a final concentration of 0.5 mg / mL MTT was added to each well for live cell staining. After incubation in the incubator for 3 hours, the culture medium was discarded, 100 μL DMSO was added to each well, and it was shaken on a shaker to fully dissolve. The OD value of each group at a wavelength of 490 nm was detected by an enzyme marker (Biotek).

[0141] Protective effects of compounds on glutamate-induced HT22 cell injury

[0142]

[0143]

[0144] In the cell survival rate test, the cell survival rate of the blank control group (Control group) was set as 100% (n=3). The data in the table are expressed as mean±SEM. *P<0.05, **P<0.01, ***P<0.001 relative to the glutamate model group; ###P<0.001 relative to the blank control group.

[0145] 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 to the above embodiments. The above embodiments and descriptions are only for illustrating the principles of the present invention. Without departing from the scope of the principles of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of protection of the present invention.

Claims

1. A biologically active 1,4-benzoxazinone derivative, characterized in that: Its structural formula is: ； Where n is 0 or 1; R 1 , R 2 , R 3 , R 4 and R 5 is one or more of H, halogen, trifluoromethyl, ethyl, phenyl, hydroxyl, cyano or nitro.

2. A method for preparing the biologically active 1,4-benzoxazinone derivative according to claim 1, characterized in that: The specific steps are: The terminal acetylene compound, the azide compound, the sodium L-ascorbate and the anhydrous CuSO4 are added to the reaction container, and then a mixed solvent of water, tert-butyl alcohol and THF is added to the reaction container, and stirred at room temperature to dissolve all of them. The room temperature is maintained, and after the reaction is completed by TLC detection, diatomaceous earth is laid in the funnel, and the solid impurities are removed by vacuum filtration. The filtrate is collected and transferred to a separatory funnel, and 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, and anhydrous sodium sulfate is added thereto, and after sufficient stirring and standing, the anhydrous sodium sulfate is filtered to remove the anhydrous sodium sulfate, and the organic solvent is removed by vacuum distillation at 35°C to obtain a crude product, and then the obtained crude product is separated and purified by column chromatography to finally obtain the target compound; wherein the structure of the terminal acetylene compound is: ; The structure of azide compounds is: .

3. Use of the biologically active 1,4-benzoxazinone derivative according to claim 1 in the preparation of IDO1 inhibitory drugs, anti-tumor drugs or anti-inflammatory drugs.

4. The use according to claim 3, characterized in that: The tumor is human cancer cell A549, Huh7, MCF-7, HCT-116 or SKOV3.

5. Use of the biologically active 1,4-benzoxazinone derivative according to claim 1 in the preparation of a protective agent against glutamate-induced HT22 neuronal cell damage.

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