CN113980022B - Theophylline acetic acid derivative with antitumor activity and preparation method thereof - Google Patents

Abstract

Theophylline acetic acid derivative with anti-tumor activity and application thereofA preparation method. The theophylline acetic acid derivative has a structural formula shown in a formula (J). The preparation method comprises the following steps: (1) taking theophylline acetic acid as a raw material, and carrying out condensation reaction with 3-aminophenylacetylene under the action of a condensation reagent to obtain N-3-alkynyl phenyl-theophylline acetamide; (2) the N-3-alkynyl phenyl-theophylline acetamide reacts with azide compounds with different substituent groups to obtain a target compound. The theophylline acetic acid derivatives have good inhibition effect on various tumor cells. The derivatives have simple preparation method, easily obtained raw materials and important significance in finding anti-tumor active compounds.

Description

Theophylline acetic acid derivative with antitumor activity and preparation method thereof

Technical Field

The invention belongs to the technical field of preparation of antitumor drugs. In particular to a theophylline acetic acid derivative with anti-tumor activity and a preparation method thereof.

Background

Malignant tumor seriously harms human health, and the latest cancer data issued by the international agency for research on cancer (IARC) of the world health organization on the whole world show that 1929 ten thousands of new cancers are discovered in the world in 2020, and 996 thousands of deaths are realized. Almost 1 out of 5 people will develop cancer in their lifetime, while 1 out of every 8 men and every 11 women will die due to cancer. Tumor treatment is a worldwide problem, and the current treatment means mainly comprise surgical operation, chemotherapy and radiotherapy. Chemotherapy is the most widely used method for treating tumors, but has the defects of large adverse reaction, easy generation of drug resistance and the like, so that the tumor drug which is designed to be safe, effective and small in side effect has a good application prospect.

Theophylline derivatives have been widely studied for their biological effects. They represent an important class of therapeutic agents with a variety of biological activities, such as antitumor activity, antimicrobial, anti-inflammatory, central nervous system stimulants, adenosine receptor antagonism, Phosphodiesterase (PDE) inhibition, antiasthmatic and antitussive, immunomodulating and hypoglycemic activities. In addition, the medicine also has a remarkable treatment effect on improving renal function of asphyxia neonates, chronic bronchia and chronic obstructive pulmonary diseases.

The theophylline and the carmustine or the cyclophosphamide are used together, and can generate a synergistic anti-tumor effect by increasing the adenosine cyclophosphate. Theophylline and caffeine can enhance the toxicity of doxorubicin to tumor cells. When theophylline was used in combination with gemcitabine or cisplatin, it was found that theophylline induced apoptosis in a variety of human cancer cell lines and malignantly transformed granular cell lines. The mechanism of theophylline pro-apoptotic action has been found to include either reduction of the level of the intracellular anti-apoptotic mediator Bcl-2, or as a potent inhibitor of the aldehyde dehydrogenase 1 family member a1(ALDH1a1) protein to inhibit proliferation of a variety of malignant tumors and cancer stem cells. The theophylline derivative diprophylline is combined with morphine to carry out combined treatment on patients with lung cancer dyspnea, can effectively improve the dyspnea degree of the patients and improve the treatment effect, and is a common clinical dyspnea relieving medicine. The mechanism is that theophylline antiasthmatic drugs can inhibit Phosphodiesterase (PDEs), so that decomposition of CAMP is reduced, and tracheal smooth muscle is relaxed to appropriately relieve tracheal mucosal hemorrhage. In addition, the doxofylline is an effective and safe method for treating the lung cancer dyspnea, can obviously improve the lung function of a patient, relieve clinical symptoms and reduce the incidence rate of adverse reactions, and has good clinical popularization value.

1,2, 3-triazole plays an important role in drug design and synthesis as an important nitrogen heterocyclic structure unit. Compounds with 1,2, 3-triazole units generally show good inhibitory activity against cancer, inflammation and microorganisms. In addition, the triazole ring can be easily constructed by carrying out cycloaddition reaction on azide and alkyne under the catalysis of copper, so that the synthesis difficulty is reduced, and the application potential is further improved. Some compounds containing 1,2, 3-triazole are widely designed, of which ceftriazine and carboxyamidotriazole have been used clinically or in clinical evaluation for cancer treatment, and tazobactam is also used for antimicrobial. In a word, the 1,2, 3-triazole can be hybridized with other anticancer pharmacophores through different linkers or serves as a linker for connecting two anticancer pharmacophores, so that the designed novel compound has better antitumor activity.

Therefore, based on the multiple functions of theophylline and the strong attractive structural scaffold thereof, by combining the advantages of 1,2, 3-triazole, a series of theophylline derivatives are designed by cooperation of a laboratory and a Jinan Aisi medical science and technology limited company, and the theophylline derivatives have good research prospects in the aspect of antitumor activity.

Disclosure of Invention

The invention aims to find a theophylline acetic acid derivative and apply the theophylline acetic acid derivative to the research of antitumor activity. The invention provides a theophylline acetic acid derivative with anti-tumor activity and a preparation method thereof, wherein the theophylline acetic acid derivative has excellent inhibition effect on various tumors by changing substituent groups on 1,2, 3-triazole.

The technical scheme of the invention is as follows:

a theophylline acetic acid derivative J has a structural formula:

in the formula: r is aryl, nitrogen-containing heterocycle or sulfur-containing heterocycle compounds such as phenyl compounds, tert-butyl, pyridine compounds, thiophene compounds and the like.

The preparation method of the theophylline acetic acid derivative is characterized in that the specific preparation process of the compound J comprises the following steps:

(1) reacting theophylline acetic acid with 3-aminophenylacetylene to obtain a condensation intermediate;

(2) and reacting the condensation intermediate of theophylline acetic acid and 3-aminophenylacetylene with a compound with azide to obtain a target compound.

Further limiting, the specific process of step (1) is as follows: at room temperature, a certain amount of theophylline acetic acid, 3-aminophenylacetylene, HATU, DIPEA and a solvent DMF were added together into a reaction flask, and stirred for 24 hours under the protection of nitrogen. The progress of the reaction was monitored by Thin Layer Chromatography (TLC). After completion of the reaction, DMF was removed by an oil pump and the product was extracted with dichloromethane. The organic layer was concentrated to dryness, washed with dichloromethane and saturated aqueous sodium chloride solution, and the organic phases were combined, dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and recrystallized in methanol. Obtaining a condensation intermediate of theophylline acetic acid and 3-aminophenylacetylene, wherein the molar ratio of the theophylline acetic acid to the 3-aminophenylacetylene to the HATU to the DIPEA is 1: 1.5: 1.5: 3.

further limiting, the specific process of step (2) is as follows: sequentially adding a condensation intermediate of theophylline acetic acid and 3-aminophenylacetylene, an active group with an azide structure, tert-butyl alcohol, water, tetrahydrofuran, anhydrous copper sulfate and sodium ascorbate into a reaction bottle, reacting for a period of time at 70 ℃, completely reacting raw materials, adding dichloromethane, filtering reaction liquid to obtain yellow liquid, separating out an organic phase, extracting a water phase twice by using dichloromethane, combining the organic phases, drying by using anhydrous magnesium sulfate, concentrating under reduced pressure to obtain a solid, and separating by using a chromatographic column to obtain a target compound; the molar ratio of the condensation intermediate of the theophylline acetic acid and the 3-aminophenylacetylene to the active group of the azide structure is 1: 1 to 2.

According to the invention, a series of compounds with novel structures are obtained by connecting theophylline acetic acid and other active groups, the compounds have excellent anti-tumor performance and can be better applied to medicines, and the compounds have the advantages of simple process, easiness in control and high yield of target products in the preparation process.

The invention has the advantages and beneficial effects that:

(1) the theophylline acetic acid derivative obtained by the invention has a molecular structure containing a theophylline structure and a1, 2, 3-triazole structure, wherein different substituents are linked on the 1,2, 3-triazole structure, so that the molecular structure is novel;

(2) the theophylline acetic acid derivative obtained by the invention has good inhibition effect on various tumor cells and has small toxicity on normal cells;

(3) the theophylline acetic acid derivative obtained by the invention can promote tumor cell apoptosis by inhibiting tumor cell proliferation.

(4) The theophylline acetic acid derivative obtained by the invention can play an anti-tumor role by inhibiting IDO1 target.

Drawings

FIG. 1 is a drawing of Compound J25¹H NMR chart;

FIG. 2 is a drawing of Compound J25¹³C NMR chart;

FIG. 3 shows Compound J6 before refinement¹H NMR chart;

FIG. 4 shows the results of the fine treatment of Compound J6¹H NMR chart;

FIG. 5 is a 2D plot of Compound J6 docking with IDO1 target;

FIG. 6 is a 3D map of compound J6 docking with IDO1 target.

Detailed Description

Example 1

Theophylline acetic acid (compound 1(5g, 0.02mol)), 3-aminophenylacetylene (3.69g, 0.03mol), HATU (12.96g, 0.03mol) and DIPEA (8.13g, 0.06mol) were added together with the solvent DMF250mL to a 500mL reaction flask at room temperature, and stirred under nitrogen for 24 hours. After 24 hours, the reaction was complete, as monitored by Thin Layer Chromatography (TLC), and the reaction solution was seen to be light brown. DMF was removed by concentration under reduced pressure, the reaction solution (150mLx3) was extracted by adding dichloromethane, all organic phases were combined and washed with saturated sodium chloride (150ml x2) to pH 7Left and right, concentration gave a very viscous, tan-colored product. Under ultrasonic oscillation, methanol is added dropwise and slowly, and solid is separated out. Then standing, filtering, and drying to obtain a condensation intermediate (compound 2, 3.64g) of theophylline acetic acid and 3-aminophenylacetylene. LC-MS M/z (%). 337[ M + H]⁺

Example 2

Adding compound 2(1g), 3-chloro-4-fluorobenzyl azide (1g), tert-butyl alcohol (70 mL), water (70 mL), tetrahydrofuran (70 mL), anhydrous copper sulfate (0.5 g) and sodium ascorbate (1g) in sequence into a reaction flask, reacting at 70 ℃ for 6 hours until the raw materials react completely, adding dichloromethane (100 mL), filtering the reaction solution to obtain a yellow liquid, separating an organic phase, extracting the aqueous phase twice with dichloromethane (50 mL), combining the organic phases, drying with anhydrous magnesium sulfate, concentrating under reduced pressure to obtain a solid, and separating with a chromatographic column to obtain compound J1(1.03 g).¹H NMR(400MHz,DMSO-d₆)δ10.55(s,1H),8.64(s,1H),8.18(s,1H),8.09(s,1H),7.67(d,J＝9.0,1H),7.52(d,J＝7.5,2H),7.47–7.37(m,3H),5.65(s,2H),5.23(s,2H),3.46(s,3H),3.20(s,3H).

Example 3

Adding compound 2(1g), 2-trifluoromethyl benzyl azide 1g, tert-butyl alcohol 70mL, water 70mL, tetrahydrofuran 70mL, anhydrous copper sulfate 0.5g and sodium ascorbate 1g in sequence into a reaction bottle, reacting for 6h at 70 ℃, adding dichloromethane 100mL, filtering the reaction solution to obtain yellow liquid, separating out an organic phase, extracting the aqueous phase twice with dichloromethane 50mL, combining the organic phases, drying over anhydrous magnesium sulfate, concentrating under reduced pressure to obtain a solid, and separating by using a chromatographic column to obtain compound J2(0.96 g).¹H NMR(400MHz,DMSO-d6)δ10.55(s,1H),8.61(s,1H),8.18(s,1H),8.09(s,1H),7.83(d,J＝7.7,1H),7.70(d,J＝15.1,1H),7.57(d,J＝32.7,3H),7.40(d,J＝15.8,1H),7.24(d,J＝7.7,1H),5.84(s,2H),5.23(s,2H),3.46(s,3H),3.20(s,3H)。

Example 4

Adding compound 2(1g), 3-trifluoromethyl benzyl azide 1g, tert-butyl alcohol 70mL, water 70mL, tetrahydrofuran 70mL, anhydrous copper sulfate 0.5g and sodium ascorbate 1g in sequence into a reaction bottle, reacting at 70 ℃ for 6h until the raw materials react completely, adding dichloromethane 100mL, filtering the reaction liquid to obtain yellow liquid, separating out an organic phase, extracting the aqueous phase twice with dichloromethane 50mL, combining the organic phases, drying with anhydrous magnesium sulfate, concentrating under reduced pressure to obtain a solid, and separating with a chromatographic column to obtain compound J3(1.26 g).¹H NMR(400MHz,DMSO-d₆)δ10.57(s,1H),8.70(s,1H),8.21(s,1H),8.10(s,1H),7.80(s,1H),7.73(d,J＝6.4,1H),7.66(s,2H),7.54(d,J＝11.3,2H),7.40(d,J＝15.8,1H),5.78(s,2H),5.25(s,2H),3.47(s,3H),3.21(s,3H)。

Example 5

Adding compound 2(1g), 4-trifluoromethyl benzyl azide 1g, tert-butyl alcohol 70mL, water 70mL, tetrahydrofuran 70mL, anhydrous copper sulfate 0.5g and sodium ascorbate 1g in sequence into a reaction flask, reacting at 70 ℃ for 6h until the raw materials react completely, adding dichloromethane 100mL, filtering the reaction solution to obtain a yellow liquid, separating an organic phase, extracting the aqueous phase twice with dichloromethane 50mL, combining the organic phases, drying over anhydrous magnesium sulfate, concentrating under reduced pressure to obtain a solid, and separating with a chromatographic column to obtain compound J4(1.29 g).¹H NMR(400MHz,DMSO-d₆)δ10.56(s,1H),8.67(s,1H),8.20(s,1H),8.09(s,1H),7.77(d,J＝8.1,2H),7.59–7.49(m,4H),7.40(d,J＝15.8,1H),5.77(s,2H),5.23(s,2H),3.46(s,3H),3.20(s,3H)。

Example 6

Adding compound 2(1g), 2-chlorobenzyl azide 1g, tert-butyl alcohol 70mL, water 70mL, tetrahydrofuran 70mL, anhydrous copper sulfate 0.5g and sodium ascorbate 1g in sequence into a reaction flask, reacting for 6h at 70 ℃, adding dichloromethane 100mL, filtering the reaction solution to obtain yellow liquid, separating out an organic phase, extracting the aqueous phase twice with dichloromethane 50mL, combining the organic phases, drying over anhydrous magnesium sulfate, concentrating under reduced pressure to obtain a solid, and separating with a chromatographic column to obtain compound J5(0.74 g).¹H NMR(400MHz,DMSO-d₆)δ10.54(s,1H),8.58(s,1H),8.17(s,1H),8.09(s,1H),7.53(d,J＝7.9,3H),7.40(d,J＝28.8,3H),7.29(d,J＝9.2,1H),5.75(d,J＝3.9,2H),5.23(s,2H),3.46(s,3H),3.20(s,3H)。

Example 7

The reaction solvent is tert-butyl alcohol in volume ratio: tetrahydrofuran: water 1: 1: 1, the preparation is finished. The method comprises the following steps of sequentially adding a compound 2(1g), 4-bromobenzyl azide (1g), anhydrous copper sulfate (0.5 g) and a reaction solvent (200 mL) into a 500mL reaction bottle, finally rapidly adding sodium ascorbate (1g), pumping out air by a vacuum pump, carrying out nitrogen protection, stirring and refluxing at 70 ℃ for 6 hours, wherein a reaction solution is yellow, and monitoring by Thin Layer Chromatography (TLC) to ensure that raw materials are completely reacted. Adding dichloromethane 100mL of extraction reaction liquid for 3 times, combining all organic phases, washing with saturated sodium chloride (100mLx2) until the pH value is about 7, combining the organic phases, drying with anhydrous magnesium sulfate, adding 0.1g of coconut shell activated carbon, stirring for 30min, performing suction filtration, and concentrating under reduced pressure to obtain 1.51g of a solid compound, wherein the organic phases are slightly light yellow and contain cuprous ions; using nuclear magnetic spectrograms¹H NMR is characterized in that as shown in figure 3, DMSO is used as a deuteration reagent, a dichloromethane peak exists at a chemical shift of 5.76, and a plurality of high-boiling-point substances peaks exist at chemical shifts of 0.8-1.5.

And then recrystallizing with ethyl acetate, namely putting the concentrated sample in a centrifuge tube, dropwise adding ethyl acetate under the condition of ultrasonic oscillation until the sample is just completely dissolved, then dropwise and slowly adding petroleum ether (the amount of the petroleum ether is 2-3 times that of the ethyl acetate), separating out white solid, taking out, standing, centrifuging, pouring off the solvent, adding a small amount of isopropanol, ultrasonically oscillating, filtering, and drying to obtain 1.34 g. Using nuclear magnetic spectrograms¹And H NMR characterization. Peaks at chemical shifts 5.76 and 0.8-1.5 disappeared. As shown in fig. 4.¹H NMR(400MHz,DMSO-d₆)δ10.56(s,1H),8.62(s,1H),8.13(d,J＝35.2,2H),7.55(dd,J＝31.8,7.5,4H),7.35(dd,J＝27.7,11.4,3H),5.63(s,2H),5.23(s,2H),3.46(s,3H),3.20(s,3H)。

Example 8

Adding compound 2(1g), 2-bromobenzyl azide 1g, tert-butyl alcohol 70mL, water 70mL, tetrahydrofuran 70mL, anhydrous copper sulfate 0.5g and sodium ascorbate 1g in sequence into a reaction bottle, reacting for 6h at 70 ℃, adding dichloromethane 100mL, filtering the reaction solution to obtain yellow liquid, separating out an organic phase, extracting the aqueous phase twice with dichloromethane 50mL, combining the organic phases, drying over anhydrous magnesium sulfate, concentrating under reduced pressure to obtain a solid, and separating by using a chromatographic column to obtain compound J7(1.13 g).¹H NMR(400MHz,DMSO-d6)δ10.43(s,1H),8.45(s,1H),8.04(s,1H),7.96(s,1H),7.58(d,J＝7.9,1H),7.41(d,J＝7.8,2H),7.28(dt,J＝15.1,7.7,2H),7.20(t,J＝7.3,1H),7.10(d,J＝7.4,1H),5.60(s,2H),5.10(s,2H),3.34(s,3H),3.07(s,3H).

Example 9

The reaction solvent is tert-butyl alcohol in volume ratio: tetrahydrofuran: water 1: 1: 1, the preparation is finished. In a 500mL reaction flask, Compound 2(1g), 2, 6-dichloro were added in that order1g of benzyl azide, 0.5g of anhydrous copper sulfate and 200mL of reaction solvent, finally, rapidly adding 1g of sodium ascorbate, pumping out air by a vacuum pump, carrying out nitrogen protection, stirring and refluxing for 6h at 70 ℃, wherein the reaction solution is yellow, and monitoring by Thin Layer Chromatography (TLC) to ensure that the raw materials are completely reacted. Adding dichloromethane 100mL of extraction reaction liquid for 3 times, combining all organic phases, washing with saturated sodium chloride (100mLx2) until the pH value is about 7, combining the organic phases, drying with anhydrous magnesium sulfate, wherein the organic phases are light yellow and contain cuprous ions, adding coal activated carbon 0.8g, stirring for 30min, performing suction filtration, and concentrating under reduced pressure to obtain a solid compound 1.49 g; and then recrystallizing with ethyl acetate, namely putting the concentrated sample in a centrifuge tube, dropwise adding ethyl acetate under the condition of ultrasonic oscillation until the sample is just completely dissolved, then dropwise and slowly adding petroleum ether (the amount of the petroleum ether is 2-3 times that of the ethyl acetate), separating out white solid, taking out, standing, centrifuging, pouring off the solvent, adding a small amount of isopropanol, ultrasonically oscillating, filtering, and drying to obtain 1.28 g.¹H NMR(400MHz,DMSO-d₆)δ10.55(s,1H),8.55(s,1H),8.10(d,J＝13.5,2H),7.56(d,J＝30.5,4H),7.49(d,J＝16.1,1H),7.38(d,J＝15.8,1H),5.84(s,2H),5.22(s,2H),3.46(s,3H),3.20(s,3H)。

Example 10

Adding compound 2(1g), 4-methylbenzyl azide (1g), tert-butyl alcohol (70 mL), water (70 mL), tetrahydrofuran (70 mL), anhydrous copper sulfate (0.5 g) and sodium ascorbate (1g) in sequence into a reaction flask, reacting at 70 ℃ for 6 hours until the raw materials react completely, adding dichloromethane (100 mL), filtering the reaction solution to obtain a yellow liquid, separating an organic phase, extracting the aqueous phase twice with dichloromethane (50 mL), combining the organic phases, drying over anhydrous magnesium sulfate, concentrating under reduced pressure to obtain a solid, and separating by using a chromatographic column to obtain compound J9(1.28 g).¹H NMR(400MHz,DMSO-d₆)δ10.55(s,1H),8.58(s,1H),8.13(d,J＝31.2,2H),7.51(d,J＝7.5,2H),7.38(d,J＝15.7,1H),7.31–7.14(m,4H),5.57(s,2H),5.23(s,2H),3.46(s,3H),3.20(s,3H),2.28(s,3H)。

Example 11

Adding compound 2(1g), 2-fluorobenzyl azide 1g, tert-butyl alcohol 70mL, water 70mL, tetrahydrofuran 70mL, anhydrous copper sulfate 0.5g and sodium ascorbate 1g in sequence into a reaction flask, reacting for 6h at 70 ℃, adding dichloromethane 100mL, filtering the reaction solution to obtain a yellow liquid, separating an organic phase, extracting an aqueous phase twice with dichloromethane 50mL, combining the organic phases, drying over anhydrous magnesium sulfate, concentrating under reduced pressure to obtain a solid, and separating with a chromatographic column to obtain compound J10(0.85 g).¹H NMR(400MHz,DMSO-d₆)δ10.53(s,1H),8.58(s,1H),8.12(d,J＝30.9,2H),7.55–7.21(m,7H),5.73(d,J＝22.6,2H),5.23(s,2H),3.46(s,3H),3.20(s,3H)。

Example 12

Adding compound 2(1g), 2-fluoro-4-bromobenzyl azide (1g), tert-butyl alcohol (70 mL), water (70 mL), tetrahydrofuran (70 mL), anhydrous copper sulfate (0.5 g) and sodium ascorbate (1g) in sequence into a reaction flask, reacting at 70 ℃ for 6 hours until the raw materials react completely, adding dichloromethane (100 mL), filtering the reaction solution to obtain a yellow liquid, separating an organic phase, extracting the aqueous phase twice with dichloromethane (50 mL), combining the organic phases, drying with anhydrous magnesium sulfate, concentrating under reduced pressure to obtain a solid, and separating with a chromatographic column to obtain compound J11(0.91 g).¹H NMR(400MHz,DMSO-d₆)δ10.55(s,1H),8.60(s,1H),8.13(d,J＝30.9,2H),7.64(d,J＝11.0,1H),7.50(d,J＝29.8,3H),7.37(d,J＝25.7,2H),5.68(s,2H),5.23(s,2H),3.46(s,3H),3.20(s,3H)。

Example 13

Adding compound 2(1g), 3-fluorobenzyl azide 1g, tert-butyl alcohol 70mL, water 70mL, tetrahydrofuran 70mL, anhydrous copper sulfate 0.5g and sodium ascorbate 1g in sequence into a reaction flask, reacting for 6h at 70 ℃, adding dichloromethane 100mL, filtering the reaction solution to obtain a yellow liquid, separating an organic phase, extracting an aqueous phase twice with dichloromethane 50mL, combining the organic phases, drying over anhydrous magnesium sulfate, concentrating under reduced pressure to obtain a solid, and separating with a chromatographic column to obtain compound J12(1.06 g).¹H NMR(400MHz,DMSO-d₆)δ10.55(s,1H),8.64(s,1H),8.19(s,1H),8.09(s,1H),7.53(d,J＝7.7,2H),7.47(s,2H),7.20(d,J＝32.3,3H),5.67(s,2H),5.24(s,2H),3.46(s,3H),3.20(s,3H)。

Example 14

In a reaction bottle, compound 2(1g), 3,4, 5-trifluorobenzyl azide 1g, tert-butyl alcohol 70mL, water 70mL, tetrahydrofuran 70mL, anhydrous copper sulfate 0.5g and sodium ascorbate 1g are sequentially added, the reaction is carried out for 6h at 70 ℃, raw materials are completely reacted, dichloromethane 100mL is added, the reaction solution is filtered to obtain yellow liquid, an organic phase is separated, the aqueous phase is extracted twice by dichloromethane 50mL, the combined organic phase is dried by anhydrous magnesium sulfate and then concentrated under reduced pressure to obtain a solid, and the solid is separated by a chromatographic column to obtain compound J13(1.07 g).¹H NMR(400MHz,DMSO-d₆)δ10.55(s,1H),8.63(s,1H),8.19(s,1H),8.09(s,1H),7.52(d,J＝7.6,2H),7.40(d,J＝14.8,3H),5.65(s,2H),5.24(s,2H),3.47(s,3H),3.20(s,3H)。

Example 15

In a reaction bottle, compound 2(1g), 3, 5-difluorobenzyl azide 1g, tert-butyl alcohol 70mL, water 70mL, tetrahydrofuran 70mL, anhydrous copper sulfate 0.5g and sodium ascorbate 1g are added in sequence,reacting at 70 ℃ for 6h until the raw materials react completely, adding 100mL of dichloromethane, filtering the reaction solution to obtain a yellow liquid, separating an organic phase, extracting an aqueous phase twice with 50mL of dichloromethane, combining the organic phases, drying the organic phases with anhydrous magnesium sulfate, concentrating under reduced pressure to obtain a solid, and separating by using a chromatographic column to obtain a compound J14(1.33 g).¹H NMR(400MHz,DMSO-d₆)δ10.55(s,1H),8.66(s,1H),8.19(s,1H),8.09(s,1H),7.53(d,J＝8.0,2H),7.40(d,J＝15.8,1H),7.24(d,J＝18.8,1H),7.11(d,J＝6.3,2H),5.68(s,2H),5.24(s,2H),3.47(s,3H),3.20(s,3H)。

Example 16

Adding compound 2(1g), 2, 6-difluorobenzyl azide 1g, tert-butyl alcohol 70mL, water 70mL, tetrahydrofuran 70mL, anhydrous copper sulfate 0.5g and sodium ascorbate 1g in sequence into a reaction flask, reacting at 70 ℃ for 6h until the raw materials react completely, adding dichloromethane 100mL, filtering the reaction solution to obtain a yellow liquid, separating an organic phase, extracting the aqueous phase twice with dichloromethane 50mL, combining the organic phases, drying over anhydrous magnesium sulfate, concentrating under reduced pressure to obtain a solid, and separating with a chromatographic column to obtain compound J15(0.94 g).¹H NMR(400MHz,DMSO-d₆)δ10.54(s,1H),8.58(s,1H),8.15(s,1H),8.09(s,1H),7.53(d,J＝30.5,3H),7.38(d,J＝15.8,1H),7.20(d,J＝16.0,2H),5.71(s,2H),5.23(s,2H),3.46(s,3H),3.20(s,3H)。

Example 17

Adding compound 2(1g), 3-methoxybenzyl azide (1g), tert-butyl alcohol (70 mL), water (70 mL), tetrahydrofuran (70 mL), anhydrous copper sulfate (0.5 g) and sodium ascorbate (1g) in sequence into a reaction flask, reacting at 70 ℃ for 6h until the raw materials react completely, adding dichloromethane (100 mL), filtering the reaction solution to obtain yellow liquid, separating out an organic phase, extracting the aqueous phase twice with dichloromethane (50 mL),the combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to give a solid, which was separated by a column chromatography to give compound J16(1.15 g).¹H NMR(400MHz,DMSO-d₆)δ10.53(s,1H),8.60(s,1H),8.17(s,1H),8.09(s,1H),7.52(d,J＝7.8,2H),7.38(d,J＝15.8,1H),7.30(d,J＝15.8,1H),6.92(d,J＝24.2,3H),5.60(s,2H),5.23(s,2H),3.74(s,3H),3.46(s,3H),3.20(s,3H)。

Example 18

Adding compound 2(1g), 3-chlorobenzyl azide 1g, tert-butyl alcohol 70mL, water 70mL, tetrahydrofuran 70mL, anhydrous copper sulfate 0.5g and sodium ascorbate 1g in sequence into a reaction flask, reacting for 6h at 70 ℃, adding dichloromethane 100mL, filtering the reaction solution to obtain yellow liquid, separating out an organic phase, extracting the aqueous phase twice with dichloromethane 50mL, combining the organic phases, drying over anhydrous magnesium sulfate, concentrating under reduced pressure to obtain a solid, and separating with a chromatographic column to obtain compound J17(0.93 g).¹H NMR(400MHz,DMSO-d₆)δ10.55(s,1H),8.65(s,1H),8.19(s,1H),8.09(s,1H),7.53(d,J＝7.7,2H),7.39(d,J＝62.2,5H),5.66(s,2H),5.24(s,2H),3.46(s,3H),3.20(s,3H)。

Example 19

Adding compound 2(1g), 2-cyanobenzylazide 1g, tert-butanol 70mL, water 70mL, tetrahydrofuran 70mL, anhydrous copper sulfate 0.5g and sodium ascorbate 1g in sequence into a reaction flask, reacting at 70 ℃ for 6h until the raw materials react completely, adding dichloromethane 100mL, filtering the reaction solution to obtain a yellow liquid, separating an organic phase, extracting an aqueous phase twice with dichloromethane 50mL, combining the organic phases, drying over anhydrous magnesium sulfate, concentrating under reduced pressure to obtain a solid, and separating with a chromatographic column to obtain compound J18(0.77g).¹H NMR(400MHz,DMSO-d₆)δ10.55(s,1H),8.64(s,1H),8.17(s,1H),8.09(s,1H),7.93(d,J＝7.4,1H),7.74(d,J＝16.2,1H),7.56(d,J＝28.5,3H),7.41(d,J＝29.1,2H),5.86(s,2H),5.23(s,2H),3.46(s,3H),3.20(s,3H)。

Example 20

In a reaction flask, compound 2(1g), 1g of 2-cyano-5-fluorobenzyl azide, 70mL of tert-butanol, 70mL of water, 70mL of tetrahydrofuran, 0.5g of anhydrous copper sulfate and 1g of sodium ascorbate are sequentially added, the mixture is reacted for 6 hours at 70 ℃, dichloromethane is added for complete reaction of raw materials, the reaction solution is filtered to obtain yellow liquid, an organic phase is separated, the aqueous phase is extracted twice by using dichloromethane (50 mL), the combined organic phases are dried by anhydrous magnesium sulfate and then concentrated under reduced pressure to obtain a solid, and the solid is separated by using a chromatographic column to obtain compound J19(1.28 g).¹H NMR(400MHz,DMSO-d₆)δ10.56(s,1H),8.66(s,1H),8.18(t,J＝1.9,1H),8.04(s,2H),7.55(t,J＝7.7,2H),7.48(d,J＝19.5,1H),7.41(t,J＝7.6,2H),5.87(s,2H),5.24(s,2H),3.46(s,3H),3.20(s,3H)。

Example 21

The reaction solvent is tert-butyl alcohol in volume ratio: tetrahydrofuran: water 1: 1: 1, the preparation is finished. In a 500mL reaction flask, sequentially adding compound 2(1g), 2-fluoro-5-chlorobenzyl azide (1g), anhydrous copper sulfate (0.5 g) and a reaction solvent (200 mL), finally, rapidly adding sodium ascorbate (1g), pumping out air by a vacuum pump, protecting with nitrogen, stirring and refluxing at 70 ℃ for 6h to ensure that the reaction solution is yellow, and monitoring by thin-layer chromatography (TLC) to ensure that the raw materials are completely reacted. Adding dichloromethane 100mL extraction reaction liquid for 3 times, combining all organic phases, washing with saturated sodium chloride (100mLx2) until pH is about 7, combining the organic phases, drying with anhydrous magnesium sulfate, adding 0.1g of coconut shell activated carbon containing cuprous ions into the organic phases which are light yellow, stirring for 30min, filtering, and concentrating under reduced pressure to obtain solidBody compound 1.40 g; and then recrystallizing with ethyl acetate, namely putting the concentrated sample into a centrifuge tube, dropwise adding ethyl acetate under the condition of ultrasonic oscillation until the concentrated sample is completely dissolved, slowly adding petroleum ether drop by drop to precipitate white solid, taking out, standing, centrifuging, pouring out the solvent, adding a small amount of isopropanol, oscillating with ultrasonic waves, carrying out suction filtration, and drying to obtain 1.29g of the product.¹H NMR(400MHz,DMSO-d₆)δ10.55(s,1H),8.63(s,1H),8.17(s,1H),8.09(s,1H),7.53(s,4H),7.37(d,J＝36.2,2H),5.70(s,2H),5.23(s,2H),3.46(s,3H),3.20(s,3H)。

Example 22

Adding compound 2(1g), 2-ethyl phenyl azide (1g), tert-butyl alcohol (70 mL), water (70 mL), tetrahydrofuran (70 mL), anhydrous copper sulfate (0.5 g) and sodium ascorbate (1g) in sequence into a reaction flask, reacting at 70 ℃ for 6h until the raw materials react completely, adding dichloromethane (100 mL), filtering the reaction solution to obtain a yellow liquid, separating an organic phase, extracting the aqueous phase twice with dichloromethane (50 mL), combining the organic phases, drying over anhydrous magnesium sulfate, concentrating under reduced pressure to obtain a solid, and separating by using a chromatographic column to obtain compound J21(1.19 g).¹H NMR(400MHz,DMSO-d₆)δ＝10.59(s,1H),8.92(s,1H),8.26(s,1H),8.10(s,1H),7.57(d,J＝35.9,4H),7.46(d,J＝30.7,3H),5.25(s,2H),3.47(s,3H),3.21(s,3H),2.47(s,1H),1.24(s,1H),1.05(d,J＝15.1,3H).

Example 23

Adding compound 2(1g), 3-chlorphenyl azide 1g, tertiary butanol 70mL, water 70mL, tetrahydrofuran 70mL, anhydrous copper sulfate 0.5g and sodium ascorbate 1g in sequence into a reaction bottle, reacting for 6h at 70 ℃, adding dichloromethane 100mL, filtering the reaction solution to obtain yellow liquid, separating out an organic phase, and using methylene dichloride for an aqueous phaseThe mixture was dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to give a solid, which was then separated by a column chromatography to give compound J22(1.06 g).¹H NMR(400MHz,DMSO-d₆)δ10.61(s,1H),9.36(s,1H),8.31(s,1H),8.11(d,J＝5.9,2H),7.99(d,J＝9.0,1H),7.61(d,J＝51.1,4H),7.46(d,J＝15.7,1H),5.25(s,2H),3.47(s,3H),3.20(s,3H)。

Example 24

Adding compound 2(1g), 3-methylphenyl azide 1g, tert-butyl alcohol 70mL, water 70mL, tetrahydrofuran 70mL, anhydrous copper sulfate 0.5g and sodium ascorbate 1g in sequence into a reaction flask, reacting at 70 ℃ for 6h until the raw materials react completely, adding dichloromethane 100mL, filtering the reaction solution to obtain a yellow liquid, separating an organic phase, extracting an aqueous phase twice with dichloromethane 50mL, combining the organic phases, drying over anhydrous magnesium sulfate, concentrating under reduced pressure to obtain a solid, and separating with a chromatographic column to obtain compound J23(1.22 g).¹H NMR(400MHz,DMSO-d₆)δ10.60(s,1H),9.25(s,1H),8.30(s,1H),8.10(s,1H),7.84–7.72(m,2H),7.62(d,J＝7.6,1H),7.58–7.43(m,3H),7.33(d,J＝7.6,1H),5.25(s,2H),3.47(s,3H),3.21(s,3H),2.44(s,3H)。

Example 25

Adding compound 2(1g), 3-bromophenyl azide 1g, tert-butyl alcohol 70mL, water 70mL, tetrahydrofuran 70mL, anhydrous copper sulfate 0.5g and sodium ascorbate 1g in sequence into a reaction flask, reacting at 70 ℃ for 6h until the raw materials react completely, adding dichloromethane 100mL, filtering the reaction solution to obtain a yellow liquid, separating out an organic phase, extracting the aqueous phase twice with dichloromethane 50mL, combining the organic phases, drying over anhydrous magnesium sulfate, concentrating under reduced pressure to obtain a solid, and separating with a chromatographic column to obtain compound J24(1.27 g).¹H NMR(400MHz,DMSO-d₆)δ10.60(s,1H),9.36(s,1H),8.31(s,1H),8.24(s,1H),8.10(s,1H),8.04(d,J＝8.1,1H),7.71(d,J＝8.1,1H),7.64–7.54(m,3H),7.46(d,J＝15.7,1H),5.26(s,2H),3.47(s,3H),3.21(s,3H)。

Example 26

Adding compound 2(1g), 3-fluorophenyl azide 1g, tert-butyl alcohol 70mL, water 70mL, tetrahydrofuran 70mL, anhydrous copper sulfate 0.5g and sodium ascorbate 1g in sequence into a reaction bottle, reacting for 6h at 70 ℃, adding dichloromethane 100mL, filtering the reaction solution to obtain a yellow liquid, separating an organic phase, extracting an aqueous phase twice with dichloromethane 50mL, combining the organic phases, drying over anhydrous magnesium sulfate, concentrating under reduced pressure to obtain a solid, and separating by using a chromatographic column to obtain compound J25(1.03 g).¹H NMR(400MHz,DMSO-d₆)δ10.61(s,1H),9.33(s,1H),8.31(s,1H),8.10(s,1H),7.90(d,J＝23.5,2H),7.71–7.61(m,2H),7.57(d,J＝8.5,1H),7.47(d,J＝15.7,1H),7.37(d,J＝19.0,1H),5.26(s,2H),3.47(s,3H),3.21(s,3H)。

Example 27

Adding compound 2(1g), 4-chlorophenylazide 1g, tert-butanol 70mL, water 70mL, tetrahydrofuran 70mL, anhydrous copper sulfate 0.5g and sodium ascorbate 1g in sequence into a reaction flask, reacting at 70 ℃ for 6h until the raw materials react completely, adding dichloromethane 100mL, filtering the reaction solution to obtain a yellow liquid, separating an organic phase, extracting an aqueous phase twice with dichloromethane 50mL, combining the organic phases, drying with anhydrous magnesium sulfate, concentrating under reduced pressure to obtain a solid, and separating with a chromatographic column to obtain compound J26(1.27 g).¹H NMR(400MHz,DMSO-d₆)δ10.61(s,1H),9.30(s,1H),8.30(s,1H),8.10(s,1H),8.01(d,J＝8.9,2H),7.70(d,J＝8.9,2H),7.59(dd,J＝19.3,8.3,2H),7.46(d,J＝15.8,1H),5.25(s,2H),3.46(s,3H),3.20(s,3H)。

Example 28

In a reaction flask, compound 2(1g), 1g of 2, 5-trifluoromethylphenyl azide, 70mL of tert-butanol, 70mL of water, 70mL of tetrahydrofuran, 0.5g of anhydrous copper sulfate and 1g of sodium ascorbate are sequentially added, the reaction is carried out for 6h at 70 ℃, the raw materials are completely reacted, 100mL of dichloromethane is added, the reaction solution is filtered to obtain yellow liquid, an organic phase is separated, the aqueous phase is extracted twice by 50mL of dichloromethane, the combined organic phase is dried by anhydrous magnesium sulfate and then concentrated under reduced pressure to obtain a solid, and the solid is separated by a chromatographic column to obtain compound J27(1.09 g).¹H NMR(400MHz,DMSO-d₆)δ10.60(s,1H),9.08(s,1H),8.42(s,1H),8.31(d,J＝21.5,3H),8.10(s,1H),7.60(d,J＝17.9,2H),7.46(d,J＝15.7,1H),5.25(s,2H),3.47(s,3H),3.21(s,3H)。

Example 29

Adding compound 2(1g), 2-trifluoromethoxyphenyl azide (1g), tert-butyl alcohol (70 mL), water (70 mL), tetrahydrofuran (70 mL), anhydrous copper sulfate (0.5 g) and sodium ascorbate (1g) in sequence into a reaction bottle, reacting at 70 ℃ for 6h until the raw materials react completely, adding dichloromethane (100 mL), filtering the reaction liquid to obtain a yellow liquid, separating an organic phase, extracting an aqueous phase twice by using dichloromethane (50 mL), combining the organic phases, drying by using anhydrous magnesium sulfate, concentrating under reduced pressure to obtain a solid, and separating by using a chromatographic column to obtain compound J28(1.20 g).¹H NMR(400MHz,DMSO-d₆)δ10.60(s,1H),9.04(s,1H),8.26(s,1H),8.10(s,1H),7.92(d,J＝8.0,1H),7.76(d,J＝4.4,2H),7.69(d,J＝16.7,1H),7.61(d,J＝17.1,2H),7.46(d,J＝15.8,1H),5.26(s,2H),3.47(s,3H),3.21(s,3H)。

Example 30

Adding compound 2(1g), 2-chlorphenyl azide 1g, tert-butyl alcohol 70mL, water 70mL, tetrahydrofuran 70mL, anhydrous copper sulfate 0.5g and sodium ascorbate 1g in sequence into a reaction bottle, reacting for 6h at 70 ℃, adding dichloromethane 100mL, filtering the reaction solution to obtain yellow liquid, separating out an organic phase, extracting the aqueous phase twice with dichloromethane 50mL, combining the organic phases, drying over anhydrous magnesium sulfate, concentrating under reduced pressure to obtain a solid, and separating by using a chromatographic column to obtain compound J29(0.77 g).¹H NMR(400MHz,DMSO-d₆)δ10.61(s,1H),9.03(s,1H),8.27(s,1H),8.11(s,1H),7.80(d,J＝14.4,2H),7.65(d,J＝27.5,4H),7.45(d,J＝15.7,1H),5.26(s,2H),3.46(s,3H),3.21(s,3H)。

Example 31

Adding compound 2(1g), 3-trifluoromethylphenyl azide (1g), tert-butyl alcohol (70 mL), water (70 mL), tetrahydrofuran (70 mL), anhydrous copper sulfate (0.5 g) and sodium ascorbate (1g) in sequence into a reaction flask, reacting at 70 ℃ for 6h until the raw materials react completely, adding dichloromethane (100 mL), filtering the reaction solution to obtain a yellow liquid, separating an organic phase, extracting the aqueous phase twice with dichloromethane (50 mL), combining the organic phases, drying over anhydrous magnesium sulfate, concentrating under reduced pressure to obtain a solid, and separating by using a chromatographic column to obtain compound J30(1.18g).¹H NMR(400MHz,DMSO-d₆)δ10.61(s,1H),9.46(s,1H),8.35(d,J＝10.7,3H),8.10(s,1H),7.88(d,J＝6.2,2H),7.64(d,J＝7.6,1H),7.56(d,J＝8.7,1H),7.47(d,J＝15.7,1H),5.26(s,2H),3.47(s,3H),3.21(s,3H)。

Example 32

In a reaction flask, compound 2(1g) and 1g of 2-fluorophenyl azide were added in this order70mL of tert-butyl alcohol, 70mL of water, 70mL of tetrahydrofuran, 0.5g of anhydrous copper sulfate and 1g of sodium ascorbate, reacting for 6 hours at 70 ℃, completely reacting raw materials, adding 100mL of dichloromethane, filtering the reaction solution to obtain a yellow liquid, separating an organic phase, extracting an aqueous phase twice with 50mL of dichloromethane, combining the organic phases, drying the organic phases with anhydrous magnesium sulfate, concentrating under reduced pressure to obtain a solid, and separating by using a chromatographic column to obtain a compound J31(0.94 g).¹H NMR(400MHz,DMSO-d₆)δ10.61(s,1H),9.06(d,J＝1.5,1H),8.27(s,1H),8.10(s,1H),7.91(d,J＝15.3,1H),7.62(d,J＝18.4,4H),7.46(d,J＝25.0,2H),5.25(s,2H),3.47(s,3H),3.21(s,3H)。

Example 33

The reaction solvent is tert-butyl alcohol in volume ratio: tetrahydrofuran: water 1: 1: 1, the preparation is finished. In a 500mL reaction flask, compound 2(1g), 3-methoxyphenyl azide (1g), anhydrous copper sulfate (0.5 g) and reaction solvent (200 mL) are sequentially added, and finally sodium ascorbate (1g) is rapidly added, air is pumped out by a vacuum pump, nitrogen is used for protection, stirring and refluxing are carried out at 70 ℃ for 6 hours, the reaction solution is yellow, and Thin Layer Chromatography (TLC) is adopted for monitoring, so that the raw materials are completely reacted. Adding dichloromethane 100mL of extraction reaction liquid for 3 times, combining all organic phases, washing with saturated sodium chloride (100mLx2) until the pH value is about 7, combining the organic phases, drying with anhydrous magnesium sulfate, adding 0.1g of coconut shell activated carbon, stirring for 30min, performing suction filtration, and concentrating under reduced pressure to obtain 1.35g of a solid compound, wherein the organic phases are slightly light yellow and contain cuprous ions; and then recrystallizing with ethyl acetate, namely putting the concentrated sample in a centrifuge tube, dropwise adding ethyl acetate under the condition of ultrasonic oscillation until the sample is just completely dissolved, then dropwise and slowly adding petroleum ether, precipitating white solid, taking out, standing, centrifuging, pouring out the solvent, adding a small amount of isopropanol, ultrasonically oscillating, carrying out suction filtration, and drying to obtain 1.24 g.¹H NMR(400MHz,DMSO-d₆)δ10.56(s,1H),8.61(s,1H),8.18(s,1H),8.09(s,1H),7.52(s,2H),7.35(d,J＝37.2,2H),6.93(d,J＝20.2,3H),5.24(s,2H),3.75(s,3H),3.46(s,3H),3.20(s,3H)。

Example 34

Adding compound 2(1g), 4-fluorophenyl azide (1g), tert-butyl alcohol (70 mL), water (70 mL), tetrahydrofuran (70 mL), anhydrous copper sulfate (0.5 g) and sodium ascorbate (1g) in sequence into a reaction flask, reacting at 70 ℃ for 6h until the raw materials react completely, adding dichloromethane (100 mL), filtering the reaction solution to obtain a yellow liquid, separating an organic phase, extracting an aqueous phase twice with dichloromethane (50 mL), combining the organic phases, drying over anhydrous magnesium sulfate, concentrating under reduced pressure to obtain a solid, and separating by using a chromatographic column to obtain compound J33(1.26 g).¹H NMR(400MHz,DMSO-d6)δ10.61(s,1H),9.26(s,1H),8.29(s,1H),8.06(d,J＝34.6,3H),7.64–7.42(m,5H),5.25(s,2H),3.47(s,3H),3.21(s,3H)。

Example 35

Adding compound 2(1g), 2-iodophenyl azide (1g), tert-butyl alcohol (70 mL), water (70 mL), tetrahydrofuran (70 mL), anhydrous copper sulfate (0.5 g) and sodium ascorbate (1g) in sequence into a reaction flask, reacting at 70 ℃ for 6h until the raw materials react completely, adding dichloromethane (100 mL), filtering the reaction solution to obtain a yellow liquid, separating an organic phase, extracting the aqueous phase twice with dichloromethane (50 mL), combining the organic phases, drying over anhydrous magnesium sulfate, concentrating under reduced pressure to obtain a solid, and separating by using a chromatographic column to obtain compound J34(0.76 g).¹H NMR(400MHz,DMSO-d₆)δ10.62(s,1H),9.28(s,1H),8.29(d,J＝12.6,1H),8.11(s,1H),7.98(d,J＝7.8,1H),7.61(d,J＝27.4,4H),7.54–7.44(m,2H),5.26(s,2H),3.47(s,3H),3.21(s,3H)。

Example 36

In a reaction flask, compound 2(1g), 3, 5-trifluoromethylphenyl azide (1g), tert-butanol (70 mL), water (70 mL), tetrahydrofuran (70 mL), anhydrous copper sulfate (0.5 g) and sodium ascorbate (1g) are sequentially added, the reaction is carried out for 6h at 70 ℃, raw materials are completely reacted, dichloromethane (100 mL) is added, the reaction solution is filtered to obtain yellow liquid, an organic phase is separated, an aqueous phase is extracted twice by dichloromethane (50 mL), the combined organic phase is dried by anhydrous magnesium sulfate and then concentrated under reduced pressure to obtain a solid, and the solid is separated by a chromatographic column to obtain compound J35(1.02 g).¹H NMR(400MHz,DMSO-d₆)δ10.63(s,1H),9.60(s,1H),8.71(s,2H),8.37(s,1H),8.30(s,1H),8.10(s,1H),7.64(d,J＝7.4,1H),7.56–7.46(m,2H),5.26(s,2H),3.47(s,3H),3.21(s,3H)。

Example 37

Adding compound 2(1g), 2-methyl-3-nitrophenylazide (1g), tert-butyl alcohol (70 mL), water (70 mL), tetrahydrofuran (70 mL), anhydrous copper sulfate (0.5 g) and sodium ascorbate (1g) in sequence into a reaction flask, reacting at 70 ℃ for 6 hours until the raw materials react completely, adding dichloromethane (100 mL), filtering the reaction solution to obtain a yellow liquid, separating an organic phase, extracting the aqueous phase twice with dichloromethane (50 mL), combining the organic phases, drying with anhydrous magnesium sulfate, concentrating under reduced pressure to obtain a solid, and separating with a chromatographic column to obtain compound J36(0.87 g).¹H NMR(400MHz,DMSO-d₆)δ10.61(s,1H),9.04(s,1H),8.28(s,1H),8.19(d,J＝8.2,1H),8.10(s,1H),7.93(d,J＝7.9,1H),7.72(d,J＝16.1,1H),7.60(d,J＝14.3,2H),7.45(d,J＝15.7,1H),5.25(s,2H),3.46(s,3H),3.21(s,3H),2.24(s,3H)。

Example 38

The reaction solvent is tert-butyl alcohol in volume ratio: tetrahydrofuran: water 1: 1: 1, the preparation is finished.In a 500mL reaction flask, compound 2(1g), 2-methoxyphenyl azide (1g), anhydrous copper sulfate (0.5 g) and reaction solvent (200 mL) are sequentially added, and finally sodium ascorbate (1g) is rapidly added, air is pumped out by a vacuum pump, nitrogen is used for protection, stirring and refluxing are carried out at 70 ℃ for 6 hours, the reaction solution is yellow, and Thin Layer Chromatography (TLC) is adopted for monitoring, so that the raw materials are completely reacted. Adding dichloromethane 100mL of extraction reaction liquid for 3 times, combining all organic phases, washing with saturated sodium chloride (100mLx2) until the pH value is about 7, combining the organic phases, drying with anhydrous magnesium sulfate, wherein some organic phases show light yellow and contain cuprous ions, adding coal activated carbon 0.8g, stirring for 30min, performing suction filtration, and concentrating under reduced pressure to obtain a solid compound 1.28 g; and then recrystallizing with ethyl acetate, namely putting the concentrated sample into a centrifuge tube, dropwise adding ethyl acetate under the condition of ultrasonic oscillation until the concentrated sample is completely dissolved, slowly adding petroleum ether drop by drop to precipitate white solid, taking out, standing, centrifuging, pouring out the solvent, adding a small amount of isopropanol, oscillating with ultrasonic waves, carrying out suction filtration, and drying to obtain 1.15g of the product.¹H NMR(400MHz,DMSO-d6)δ10.59(s,1H),8.87(s,1H),8.23(s,1H),8.10(s,1H),7.68(d,J＝7.8,1H),7.61(d,J＝12.1,2H),7.56(d,J＝15.8,1H),7.43(d,J＝15.7,1H),7.34(d,J＝8.4,1H),7.17(d,J＝15.3,1H),5.25(s,2H),3.88(s,3H),3.46(s,3H),3.21(s,3H)。

Example 39

The reaction solvent is tert-butyl alcohol in volume ratio: tetrahydrofuran (tetrahydrofuran): water 1: 1: 1, the preparation is finished. In a 500mL reaction flask, sequentially adding a compound 2(1g), 2-methoxyphenyl azide, anhydrous copper sulfate 0.5g and a reaction solvent 200mL, finally rapidly adding sodium ascorbate 1g, pumping out air by a vacuum pump, protecting nitrogen, stirring and refluxing at 70 ℃ for 6h, wherein the reaction solution is yellow, and monitoring by Thin Layer Chromatography (TLC) is adopted, and the raw materials are completely reacted. Adding dichloromethane 100mL for extraction, 3 times, combining all organic phases, washing with saturated sodium chloride (100mLx2) to pH 7, combining the organic phases, drying with anhydrous magnesium sulfate, and obtaining organic phase with a little amount of colorShowing light yellow, adding 0.1g of coal activated carbon into the light yellow containing cuprous ions, stirring for 30min, performing suction filtration, and performing reduced pressure concentration to obtain 1.33g of a light yellow solid compound; and then recrystallizing with ethyl acetate, namely putting the concentrated sample in a centrifuge tube, dropwise adding ethyl acetate under the condition of ultrasonic oscillation until the sample is just completely dissolved, then dropwise and slowly adding petroleum ether, separating out a slightly light yellow solid, taking out, standing, centrifuging, pouring out the solvent, adding a small amount of isopropanol, ultrasonically oscillating, carrying out suction filtration, and drying to obtain 1.19 g.¹H NMR(400MHz,DMSO-d6)δ10.59(s,1H),8.87(s,1H),8.23(s,1H),8.10(s,1H),7.68(d,J＝7.8,1H),7.61(d,J＝12.1,2H),7.56(d,J＝15.8,1H),7.43(d,J＝15.7,1H),7.34(d,J＝8.4,1H),7.17(d,J＝15.3,1H),5.25(s,2H),3.88(s,3H),3.46(s,3H),3.21(s,3H)。

Example 40

Adding compound 2(1g), 4-trifluoromethylphenyl azide (1g), tert-butyl alcohol (70 mL), water (70 mL), tetrahydrofuran (70 mL), anhydrous copper sulfate (0.5 g) and sodium ascorbate (1g) in sequence into a reaction flask, reacting at 70 ℃ for 6h until the raw materials react completely, adding dichloromethane (100 mL), filtering the reaction solution to obtain a yellow liquid, separating an organic phase, extracting the aqueous phase twice with dichloromethane (50 mL), combining the organic phases, drying over anhydrous magnesium sulfate, concentrating under reduced pressure to obtain a solid, and separating by using a chromatographic column to obtain compound J38(1.26 g).¹H NMR(400MHz,DMSO-d₆)δ10.63(s,1H),9.43(s,1H),8.33(s,1H),8.24(d,J＝8.4,2H),8.11(s,1H),8.02(d,J＝8.5,2H),7.64(d,J＝7.6,1H),7.58(d,J＝8.1,1H),7.48(d,J＝15.7,1H),5.27(s,2H),3.47(s,3H),3.21(s,3H)。

EXAMPLE 41

In a reaction flask, compound 2(1g), 2-bromophenyl azide 1g, tert-butanol 7 were added in this order0mL, 70mL of water, 70mL of tetrahydrofuran, 0.5g of anhydrous copper sulfate and 1g of sodium ascorbate, reacting for 6h at 70 ℃, completely reacting the raw materials, adding 100mL of dichloromethane, filtering the reaction solution to obtain a yellow liquid, separating an organic phase, extracting an aqueous phase twice with 50mL of dichloromethane, combining the organic phases, drying the organic phases with anhydrous magnesium sulfate, concentrating under reduced pressure to obtain a solid, and separating by using a chromatographic column to obtain a compound J39(0.84 g).¹H NMR(400MHz,DMSO-d₆)δ10.73–10.48(m,1H),9.00(d,J＝5.8,1H),8.32–8.21(m,1H),8.09(d,J＝5.8,1H),7.94(s,1H),7.77–7.40(m,6H),5.32–5.19(m,2H),3.46(d,J＝5.8,3H),3.20(d,J＝5.8,3H)。

Example 42

Theophylline acetic acid (compound 1(4.8g, 0.02mol), 3-aminophenylacetylene (2.4g, 0.02mol), HATU (12.96g, 0.03mol) and DIPEA (8.13g, 0.06mol), 4-bromobenzylazide (4.8 g), anhydrous copper sulfate (1g) and sodium ascorbate (2 g) were sequentially added to 500mL of tetrahydrofuran at room temperature, air was evacuated by a vacuum pump under nitrogen protection, stirring was carried out at room temperature for 8 hours, then 200mL of water and 200mL of t-butanol were added, the reaction was continued to be stirred for 0.5 hour, the reaction solution was filtered, 300mL of dichloromethane was added and extracted several times, all organic phases were combined, saturated sodium chloride was washed to pH 7 or so, the organic phases were combined, anhydrous magnesium sulfate was dried, concentrated under reduced pressure, the concentrated sample was taken, placed in a centrifuge tube, ethyl acetate was added dropwise with ultrasonic oscillation until just completely dissolved, petroleum ether was slowly added, white solid is separated out, taken out, kept stand, centrifuged, the solvent is poured off, a small amount of isopropanol is added, ultrasonic oscillation is carried out, suction filtration and drying are carried out, and 8.7 g of product is obtained.

Example 43

In a reaction flask, adding into the reaction flask in sequenceCompound 2(1g), 1g of 2, 4, 6-trimethylphenylazide, 70mL of tert-butanol, 70mL of water, 70mL of tetrahydrofuran, 0.5g of anhydrous copper sulfate and 1g of sodium ascorbate, reacting at 70 ℃ for 6h until the raw materials react completely, adding 100mL of dichloromethane, filtering the reaction solution to obtain a yellow liquid, separating out an organic phase, extracting an aqueous phase twice with 50mL of dichloromethane, combining the organic phases, drying over anhydrous magnesium sulfate, concentrating under reduced pressure to obtain a solid, and separating by a chromatographic column to obtain compound J40(1.17 g).¹H NMR(400MHz,DMSO-d₆)δ10.59(s,1H),8.77(q,J＝8.4,7.7,1H),8.31–8.03(m,2H),7.65–7.39(m,3H),7.12(d,J＝6.5,2H),5.26(d,J＝6.5,2H),3.46(d,J＝12.9,3H),3.21(d,J＝12.9,3H),2.34(t,J＝6.1,3H),1.94(t,J＝6.2,6H)。

Example 44

In order to screen out compounds with good antitumor activity from 40 theophylline acetic acid derivatives, we selected three tumor cell lines a549, MCF-7 and Caco-2 as subjects for treatment. CCK-8 was used to evaluate the effect of the series of theophylline acetic acid derivatives on the proliferative activity of A549, MCF-7 and Caco-2. The specific operation is as follows:

collecting lung cancer cells A549, MCF-7 and Caco-2 in a growth phase, taking 0.25% trypsin digestion solution to enable adherent cells to fall off, preparing cell suspension, and inoculating the cells in a logarithmic growth phase to a 96-well plate according to 2000-4000 cells/hole. After 24 hours of plating, cells were treated with different concentrations of drug (1 μ M, 2 μ M, 8 μ M, 16 μ M, 32 μ M) for 72 hours, with 0.1% DMSO as a negative control. Finally, CCK-8 reagent is added for incubation for 1-4 hours, and the absorbance (OD value) of each well is measured at the wavelength of 450 nm. The cell viability of the negative control group was taken as 100%, and compound IC was calculated using non-linear regression with the aid of Graph Pad prism8.0 software₅₀The value is obtained. The following table shows the anti-proliferative activity of 40 synthesized compounds and theophylline acetate on tumor cells A549, MCF-7 and Caco-2 by using the CCK-8 method.

Example 45

To confirm the antitumor activity of compound J25, the cell line most sensitive to compound J25 was selected, and 7 cell lines H460, a2780, LOVO, MB-231, OVCAR3, SW480, and PC9 were added as subjects for treatment. The results indicated that compound J25 was active against the 10 cancer cell lines A549 (IC)₅₀＝5.924±4.45μM)、H460(IC₅₀＝9.980±2.57μM)、Caco-2(IC₅₀＝9.37±9.63μM),A2780(IC₅₀＝46.1±1.47μM)、LOVO(IC₅₀＝24.5±2.58μM)、MB-231(IC₅₀＝7.588±1.25μM)、MCF-7(IC₅₀＝24.94±5.23μM)、OVCAR3(IC₅₀＝18.20±2.02μM)、SW480(IC₅₀17.16 ± 2.48 μ M) and PC9 (IC)₅₀17.59 ± 4.38 μ M) showed antiproliferative and cytotoxic properties. In addition, we also measured the cytotoxicity of compound J25 on normal hepatocytes LO2, which indicates that J25 has little cytotoxicity on normal hepatocytes at therapeutically effective concentrations, with IC' s₅₀＝85.31±32.7μM。

Example 46

Compounds J3 and J6 were each molecularly interfaced with IDO1 target (PDB: 4PK5) using molecular docking software, because the m-aminophenylacetylene and the 1, 3-disubstituted 1,2, 3-triazole are used, the space bending structure is good, further, it was found that compounds J3 and J6 could enter IDO1 target pocket (if m-aminophenylacetylene was replaced by p-aminophenylacetylene, the rigid elongation of the molecular structure could not effectively enter IDO1 pocket), wherein the triazole structure can have an action relationship with heme in the target protein, the 3-trifluoromethyl benzyl structure and the 4-bromobenzyl structure can be used for carrying out a hydrophobic pocket, wherein trifluoromethyl has stronger electron-withdrawing ability than bromine, theophylline structure can react with hydrophilic amino acid, the compound can generate an action relation with IDO1 target spot to generate the action of inhibiting tumor cells.

From CO₂Taking out the human cervical carcinoma Hela cell culture dish with vitality from the incubator, and respectively carrying out the steps asThe following operations are carried out: aseptic operation is carried out beside an alcohol lamp, a dish cover is opened, culture solution is sucked out of a waste liquid cylinder, the culture solution in a culture bottle is washed twice by 2mL PBS, 0.25% trypsin is used for digestion, digestion is stopped when observation shows that cell gaps are increased and cells are changed into small circle shapes, a liquid transfer gun is used for blowing the bottom of the culture bottle to enable the cells to fall off, the obtained cell suspension is transferred into an aseptic centrifuge tube, a centrifuge is set to be 1000r/min and 3min for centrifugation, then supernatant in the centrifuge tube is poured slowly, 2-5 mL culture solution is added, and cell counting is carried out under an inverted microscope. According to the counting result, viable human cervical carcinoma Hela cells growing in logarithmic phase are paved in a 96-well cell culture plate according to the number of 50000 cells per well, are cultured for 5-6 hours by RPMI1640 containing 10% fetal bovine serum, and 100 mu L of a test compound (the concentration is 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6) diluted by a culture medium and a recombinant human interferon gamma (the final concentration is 100ng/mg) are added to activate the expression of IDO1 in the Hela cells. After the operation, the 96-well cell culture plate was placed in a cell culture chamber at 37 ℃ and enriched with 5% carbon dioxide for 18 hours, and then the reaction was terminated with a certain amount of 3.05N trichloroacetic acid, followed by incubation at 50 ℃ for 30 minutes. After the cell culture solution is precipitated, the supernatant is taken and is colored by p- (N, N-dimethyl) benzaldehyde, and then the absorbance at 480nm is detected by a multifunctional microplate reader. The group treated with IFN γ -only medium without drug was taken as 100% (At), and the group treated with DMSO-only medium 0.1% was taken as a blank 0% (Ab); the absorbance at different conditions was calculated according to the following formula: absorbance% (a-Ab)/(At-Ab), a: drug treatment +100ng/mL IFN γ, Ab: blank control, At: no drug contained 100ng/mL IFN γ alone; generation of the IC Using Graph Pad Prism8.0 software₅₀Inhibition curve of values. Through research on inhibition of IDO1 activity of HeLa cells over-expressed by IDO1, we found that compounds J3 and J6 have inhibition activity IC of IDO1₅₀17.1 μ M and 664.7 μ M respectively, whereas with M-aminophenylacetylene replaced by p-aminophenylacetylene, the compounds corresponding to J3 and J6 both had an activity against IDO1 enzyme of more than 1000 μ M.

The foregoing embodiments illustrate the principles, principal features and advantages of the invention, and it will be understood by those skilled in the art that the invention is not limited to the foregoing embodiments, which are merely illustrative of the principles of the invention, and that various changes and modifications may be made therein without departing from the scope of the principles of the invention.

Claims (6)

1. A theophylline acetic acid derivative J, characterized by the structural formula:

in the formula: r is

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Or

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2. A process for the preparation of the theophylline acetic acid derivative J as claimed in claim 1, which comprises:

(1) the theophylline acetic acid reacts with the 3-aminophenylacetylene to obtain

；

（2）、

Reacting with a compound with azide to obtain a target compound.

3. The process for the preparation of theophylline acetic acid derivative J as claimed in claim 2, wherein the embodiment of step (1) comprises: adding a certain amount of theophylline acetic acid, 3-aminophenylacetylene, HATU, DIPEA and DMF into a reaction bottle at room temperature, stirring for a period of time under the protection of nitrogen, monitoring the reaction process by adopting thin-layer chromatography, removing DMF by using an oil pump after the reaction is completed, extracting a product by using dichloromethane, concentrating an organic layer to be dry, washing by using dichloromethane and saturated sodium chloride aqueous solution, combining organic phases, drying by using anhydrous sodium sulfate, concentrating under reduced pressure, and recrystallizing in methanol to obtain the theophylline acetic acid, the 3-aminophenylacetylene, the HATU, the DIPEA and the DMF

SaidThe mol ratio of the theophylline acetic acid, the 3-aminophenylacetylene, the HATU and the DIPEA is 1: 1.5: 1.5: 3.

4. the process for producing theophylline acetic acid derivative J according to claim 2, wherein the specific embodiment of step (2) comprises: in a reaction flask, adding into the reaction flask in sequence

Reacting a compound with azide, tert-butyl alcohol, water, tetrahydrofuran, anhydrous copper sulfate and sodium ascorbate at a certain temperature for a period of time, completely reacting the raw materials, adding dichloromethane, filtering the reaction solution to obtain a yellow liquid, separating an organic phase, extracting a water phase twice by using dichloromethane, combining the organic phases, drying the organic phases by using anhydrous magnesium sulfate, concentrating under reduced pressure to obtain a solid, and separating by using a chromatographic column to obtain a target compound; said

The molar ratio of the compound to the compound with the azide is 1: 1 to 2.

5. The process for preparing theophylline acetic acid derivative J according to claim 2, wherein the embodiment of step (2) comprises: the reaction solvent is tert-butyl alcohol in advance according to the volume ratio: tetrahydrofuran: water = 1: 1: 1, preparing, and sequentially adding into a reaction bottle

Extracting air by a vacuum pump, protecting nitrogen, stirring and refluxing at 70 ℃ for a period of time, wherein the reaction solution is yellow, monitoring by adopting thin-layer chromatography, completely reacting raw materials, adding dichloromethane to extract the reaction solution for multiple times, combining all organic phases, washing by saturated sodium chloride until the pH is =7, combining the organic phases, drying the anhydrous magnesium sulfate, adding activated carbon into the organic phases, stirring, performing suction filtration, performing reduced pressure concentration to obtain a solid compound, and then using ethyl acetate to perform weight reductionCrystallizing, placing the concentrated sample in a centrifuge tube, dropwise adding ethyl acetate under the condition of ultrasonic oscillation until the sample is completely dissolved, dropwise and slowly adding petroleum ether, separating out white solid, taking out, standing, centrifuging, pouring off the solvent, adding a small amount of isopropanol, ultrasonically oscillating, filtering, and drying to obtain a pure product; said

The mass ratio of the compound with azide to the anhydrous copper sulfate to the sodium ascorbate is 1: 1: 0.5: 1; the mass ratio of the theophylline acetic acid to the active carbon is 1: 0.1 to 0.8, the activated carbon is coconut shell activated carbon or coal activated carbon, and the amount of the petroleum ether is 2 to 3 times of that of the ethyl acetate.

6. Use of the theophylline acetic acid derivative J of claim 1 in the manufacture of a medicament for the treatment of cancer.

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