阅读说明：本技术 一种含葡萄糖三氮唑结构的异噁唑衍生物及其制备方法和应用 (Isoxazole derivative containing glucose triazole structure and preparation method and application thereof ) 是由 王玮 李少婷 邱木清 杨鹏 孙传庆 李明梓 刘江 于 2021-05-13 设计创作，主要内容包括：本发明公开了一种含葡萄糖三氮唑结构的异噁唑衍生物及其制备方法和应用,首先制备化合物4乙酰葡萄糖三氮唑水杨醛肟,将3-硝基-1-甲磺酰基-7-氮杂吲哚和化合物4溶于无水乙醇中,加入氯胺T,导入葡萄糖三氮唑和异噁唑结构,然后氮气保护下缓慢滴加浓度为1.0mol/L甲醇钠的甲醇溶液,过滤,滤液减压除去甲醇后得淡黄色固体,柱层析分离得到含葡萄糖三氮唑结构的异噁唑衍生物。本发明制备的含葡萄糖三氮唑结构的异噁唑衍生物具有较强的肿瘤细胞抑制效果和体外抗乙肝病毒活性,为其进一步的医药领域应用提供了基础。(The invention discloses an isoxazole derivative containing a glucose triazole structure and a preparation method and application thereof, which comprises the steps of firstly preparing a compound 4 acetyl glucose triazole salicylaldoxime, dissolving 3-nitro-1-methylsulfonyl-7-azaindole and the compound 4 in absolute ethyl alcohol, adding chloramine T, introducing glucose triazole and the isoxazole structure, then slowly dropwise adding a methanol solution with the concentration of 1.0mol/L sodium methoxide under the protection of nitrogen, filtering, decompressing the filtrate to remove methanol to obtain a light yellow solid, and performing column chromatography separation to obtain the isoxazole derivative containing the glucose triazole structure. The isoxazole derivative containing the glucose triazole structure has strong tumor cell inhibition effect and in-vitro anti-hepatitis B virus activity, and provides a basis for further application in the medical field.)

1. The isoxazole derivative containing the glucose triazole structure is characterized by having the following chemical structural formula:

2. the preparation method of the isoxazole derivative containing the glucose triazole structure according to claim 1, wherein the preparation method of the isoxazole derivative containing the glucose triazole structure comprises the following steps:

adding hydroxylamine hydrochloride and water into a reaction bottle, stirring until the hydroxylamine hydrochloride is dissolved, adding acetyl glucose triazole salicylaldehyde and absolute ethyl alcohol into the reaction bottle, stirring vigorously for 2-3 hours, and after the reaction is finished, adding 20% Na₂CO₃Regulating the pH value of the reaction solutionAnd (3) when the solution is neutral, standing and cooling to room temperature to generate white precipitate, putting the white precipitate into a refrigerator for overnight, filtering under reduced pressure, and drying at room temperature to obtain granular white crystalline compound 4: acetyl glucose triazole salicylaldoxime;

dissolving 3-nitro-1-methylsulfonyl-7-azaindole and the compound 4 in absolute ethyl alcohol, adding chloramine T, refluxing for 4-6 hours, carrying out 1, 3-dipolar cycloaddition reaction, introducing structures of glucose triazole and isoxazole, and recrystallizing with methanol to obtain a compound 5: (2R, 3R, 4S, 5R, 6R) -2- (acetoxymethyl) -6- (4- ((2- (4- (methylsulfonyl) -8 b-nitro-3 a, 8 b-dihydro-4H-isoxazolo [5 ', 4': 4,5] pyrrolo [2,3-b ] pyridin-3-yl) phenoxy) methyl) -1H-1,2, 3-triazol-1-yl) tetrahydro-2H-pyran-3, 4, 5-triacetate;

suspending the compound 5 in methanol, cooling the mixture to 0 ℃ with ice water, slowly dropwise adding a methanol solution with the concentration of 1.0mol/L sodium methoxide under the protection of nitrogen, continuously reacting for 4-5 hours at room temperature, monitoring by TLC (thin layer chromatography) until the raw material point disappears, adjusting the system to be neutral with 732 strong acid styrene cation exchange resin, filtering, removing the methanol from the filtrate under reduced pressure to obtain a light yellow solid, and performing column chromatography separation to obtain the isoxazole derivative containing the glucose triazole structure: (2R, 3S, 4S, 5R, 6R) -2- (hydroxymethyl) -6- (4- ((2- (4- (methylsulfonyl) -8 b-nitro-3 a, 8 b-dihydro-4H-isoxazolo [5 ', 4': 4,5] pyrrolo [2,3-b ] pyridin-3-yl) phenoxy) methyl) -1H-1,2, 3-triazol-1-yl) tetrahydro-2H-pyran-3, 4, 5-triol.

3. The preparation method of the isoxazole derivative containing the glucose triazole structure as claimed in claim 2, wherein the ratio of the acetyl glucose triazole salicylaldehyde to the hydroxylamine hydrochloride is 1: 1.20-1: 1.30.

4. the preparation method of the isoxazole derivative containing the triazole structure of glucose according to claim 2, wherein the ratio of the amounts of the 3-nitro-1-methanesulfonyl-7-azaindole, the acetyl triazole salicylaldoxime, and the chloramine T is 1: 0.9-1: 1.2 to 1.3.

5. The preparation method of isoxazole derivatives containing a glucose triazole structure according to claim 2, wherein the compound 5 is suspended in methanol, ice water is cooled to 0 ℃, and a methanol solution of sodium methoxide with a concentration of 1.0mol/L is slowly dropped under the protection of nitrogen, and the method comprises the following steps:

adding 5mmol of compound 5 into 20-30 mL of methanol solvent;

0.6mL of a methanol solution of sodium methoxide with the concentration of 1.0mol/L is added into every 20-30 mL of the methanol solvent.

6. The preparation method of the isoxazole derivative containing the glucose triazole structure as claimed in claim 2, wherein the eluent used for the column chromatography separation is chloroform and methanol, and the volume ratio of chloroform to methanol is 20: 1.

7. An application of the isoxazole derivative containing a glucose triazole structure according to claim 1 in preparation of antitumor drugs.

8. An application of the isoxazole derivative containing the glucose triazole structure of claim 1 in preparing anti-hepatitis B virus drugs.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an isoxazole derivative containing a glucose triazole structure, and a preparation method and application thereof.

Background

Chemical name: 3-nitro-1-methylsulfonyl-7-azaindole, the chemical structural formula is shown in figure 1. In the process of drug discovery, 7-azaindole is an important structural unit, and many natural compounds with biological activity all contain 7-azaindole structures, and people find that the structural compounds have wide application and can be used for anticancer, antibacterial, antiviral, antidepressant, hypertension treatment and the like.

The triazole has aromaticity and abundant electrons in a molecular structure, can interact with enzymes and receptors in organisms by forming hydrogen bonds, and has various biological activities. Glycoside compounds widely exist in organisms and play important physiological functions, and introduction of glycoside into compound molecules can increase water solubility and guidance of the compounds and improve pharmacological properties of the compounds. Recent studies have shown that some compounds containing glycosylated triazole show better inhibition effects on carbonic anhydrase, glycosyltransferase and protein tyrosine phosphatase.

The isoxazoline skeleton is an important pharmacophore in the application of medicines and has obvious physiological and pharmacological activity. In addition, spiroisoxazolines synthesized by 1, 3-dipolar cycloaddition of nitrile oxides to exocyclic double bonds have attracted attention by pharmacologists because of exhibiting some important physiological properties.

Disclosure of Invention

The application aims to provide an isoxazole derivative containing a glucose triazole structure, a preparation method and application thereof, has a strong tumor cell inhibition effect and in-vitro anti-hepatitis B virus activity, and provides a foundation for further application in the field of medicines.

In order to achieve the purpose, the technical scheme of the invention is as follows:

an isoxazole derivative containing a glucose triazole structure has a chemical structural formula as follows:

the application also provides a preparation method of the isoxazole derivative containing the glucose triazole structure, which comprises the following steps:

adding hydroxylamine hydrochloride and water into a reaction bottle, stirring until the hydroxylamine hydrochloride is dissolved, adding acetyl glucose triazole salicylaldehyde and absolute ethyl alcohol into the reaction bottle, stirring vigorously for 2-3 hours, and after the reaction is finished, adding 20% Na₂CO₃Adjusting the pH value of the reaction solution to be neutral, standing and cooling to room temperature to generate white precipitate, putting the white precipitate into a refrigerator for overnight, filtering under reduced pressure, and drying at room temperature to obtain granular white crystal compound 4: acetyl glucose triazole salicylaldoxime;

dissolving 3-nitro-1-methylsulfonyl-7-azaindole and the compound 4 in absolute ethyl alcohol, adding chloramine T, refluxing for 4-6 hours, carrying out 1, 3-dipolar cycloaddition reaction, introducing structures of glucose triazole and isoxazole, and recrystallizing with methanol to obtain a compound 5: (2R, 3R, 4S, 5R, 6R) -2- (acetoxymethyl) -6- (4- ((2- (4- (methylsulfonyl) -8 b-nitro-3 a, 8 b-dihydro-4H-isoxazolo [5 ', 4': 4,5] pyrrolo [2,3-b ] pyridin-3-yl) phenoxy) methyl) -1H-1,2, 3-triazol-1-yl) tetrahydro-2H-pyran-3, 4, 5-triacetate;

suspending the compound 5 in methanol, cooling the mixture to 0 ℃ with ice water, slowly dropwise adding a methanol solution with the concentration of 1.0mol/L sodium methoxide under the protection of nitrogen, continuously reacting for 4-5 hours at room temperature, monitoring by TLC (thin layer chromatography) until the raw material point disappears, adjusting the system to be neutral with 732 strong acid styrene cation exchange resin, filtering, removing the methanol from the filtrate under reduced pressure to obtain a light yellow solid, and performing column chromatography separation to obtain the isoxazole derivative containing the glucose triazole structure: (2R, 3S, 4S, 5R, 6R) -2- (hydroxymethyl) -6- (4- ((2- (4- (methylsulfonyl) -8 b-nitro-3 a, 8 b-dihydro-4H-isoxazolo [5 ', 4': 4,5] pyrrolo [2,3-b ] pyridin-3-yl) phenoxy) methyl) -1H-1,2, 3-triazol-1-yl) tetrahydro-2H-pyran-3, 4, 5-triol.

Further, the weight ratio of the acetyl glucose triazole salicylaldehyde to the hydroxylamine hydrochloride substance is 1: 1.20-1: 1.30.

further, the ratio of the 3-nitro-1-methylsulfonyl-7-azaindole to the acetyl glucose triazole salicylaldoxime to the chloramine T substance is 1: 0.9-1: 1.2 to 1.3.

Further, the compound 5 is suspended in methanol, ice water is cooled to 0 ℃, and a methanol solution with the concentration of 1.0mol/L sodium methoxide is slowly dropped under the protection of nitrogen, and the method comprises the following steps:

adding 5mmol of compound 5 into 20-30 mL of methanol solvent;

0.6mL of a methanol solution of sodium methoxide with the concentration of 1.0mol/L is added into every 20-30 mL of the methanol solvent.

Furthermore, the eluent adopted by the column chromatography separation is chloroform and methanol, and the volume ratio of the chloroform to the methanol is 20: 1.

The application also provides an application of the isoxazole derivative containing the glucose triazole structure in preparation of antitumor drugs.

The application also provides an application of the isoxazole derivative containing the glucose triazole structure in preparing anti-hepatitis B virus medicines.

The invention provides an isoxazole derivative containing a glucose triazole structure, and a preparation method and application thereof, and the prepared (2R, 3S, 4S, 5R, 6R) -2- (hydroxymethyl) -6- (4- ((2- (4- (methylsulfonyl) -8 b-nitro-3 a, 8 b-dihydro-4H-isoxazolo [5 ', 4': 4,5] pyrrolo [2,3-b ] pyridin-3-yl) phenoxy) methyl) -1H-1,2, 3-triazol-1-yl) tetrahydro-2H-pyran-3, 4, 5-triol has strong tumor cell inhibition effect and in-vitro anti-hepatitis B virus activity, and provides a foundation for further application in the field of medicine.

Drawings

FIG. 1 is a chemical structural formula of 3-nitro-1-methanesulfonyl-7-azaindole;

FIG. 2 is a chemical structural formula of an isoxazole derivative containing a glucose triazole structure according to the application;

FIG. 3 is a chemical formula schematic diagram of the preparation method of the application.

Detailed Description

The technical solutions of the present invention are further described in detail below with reference to the drawings and examples, which should not be construed as limiting the present invention.

The 1,2, 3-triazole compound has various biological activities of resisting bacteria, tumors, tuberculosis, viruses, convulsion and the like. Because the structure of the aromatic biodegradable polyester is aromatic, the biodegradable polyester is not easy to be biodegraded; is rich in electrons, can be tightly combined with biomacromolecules through hydrogen bonds and dipole interaction, and is often used as an effective functional group to be introduced into the structure of the existing medicament so as to improve the physicochemical property and pharmacokinetic parameters of the medicament and improve the biological activity of the medicament. The glucoside compound has good antibacterial and anticancer activities, and the introduction of a glucoside structure into the compound can enhance the water solubility and targeting property of the compound and improve the pharmacological properties of the compound.

The general idea of the application is to skillfully introduce a glucoside with biological activity and a 1,2, 3-triazole pharmacodynamic structure into a molecular structure of 3-nitro-1-methylsulfonyl-7-azaindole, and highly specifically prepare (2R, 3S, 4S, 5R, 6R) -2- (hydroxymethyl) -6- (4- ((2- (4- (methylsulfonyl) -8 b-nitro-3 a, 8 b-dihydro-4H-isoxazolo [5 ', 4': 4,5] pyrrolo [2,3-b ] pyridin-3-yl) phenoxy) methyl) -1H-1,2, 3-triazol-1-yl) tetrahydro-2H-pyran-3, 4, 5-triol, improving pharmacological activity.

The chemical structural formula of the isoxazole derivative containing a glucose triazole structure is shown in figure 2, and the isoxazole derivative is named as (2R, 3S, 4S, 5R, 6R) -2- (hydroxymethyl) -6- (4- ((2- (4- (methylsulfonyl) -8 b-nitro-3 a, 8 b-dihydro-4H-isoxazolo [5 ', 4': 4,5] pyrrolo [2,3-b ] pyridin-3-yl) phenoxy) methyl) -1H-1,2, 3-triazol-1-yl) tetrahydro-2H-pyran-3, 4, 5-triol. Hereinafter, the isoxazole derivatives containing the glucose triazole structure are all referred to as isoxazole derivatives.

In one embodiment, as shown in fig. 3, a method for preparing an isoxazole derivative containing a glucose triazole structure includes:

adding hydroxylamine hydrochloride and water into a reaction bottle, stirring until the hydroxylamine hydrochloride is dissolved, adding acetyl glucose triazole salicylaldehyde and absolute ethyl alcohol into the reaction bottle, stirring vigorously for 2-3 hours, and after the reaction is finished, adding 20% Na₂CO₃Adjusting the pH value of the reaction solution to be neutral, standing and cooling to room temperature to generate white precipitate, putting the white precipitate into a refrigerator for overnight, filtering under reduced pressure, and drying at room temperature to obtain granular white crystal compound 4: acetyl glucose triazole salicylaldoxime;

dissolving 3-nitro-1-methylsulfonyl-7-azaindole and the compound 4 in absolute ethyl alcohol, adding chloramine T, refluxing for 4-6 hours, carrying out 1, 3-dipolar cycloaddition reaction, introducing structures of glucose triazole and isoxazole, and recrystallizing with methanol to obtain a compound 5: (2R, 3R, 4S, 5R, 6R) -2- (acetoxymethyl) -6- (4- ((2- (4- (methylsulfonyl) -8 b-nitro-3 a, 8 b-dihydro-4H-isoxazolo [5 ', 4': 4,5] pyrrolo [2,3-b ] pyridin-3-yl) phenoxy) methyl) -1H-1,2, 3-triazol-1-yl) tetrahydro-2H-pyran-3, 4, 5-triacetate;

suspending the compound 5 in methanol, cooling the mixture to 0 ℃ with ice water, slowly dropwise adding a methanol solution with the concentration of 1.0mol/L sodium methoxide under the protection of nitrogen, continuously reacting for 4-5 hours at room temperature, monitoring by TLC (thin layer chromatography) until the raw material point disappears, adjusting the system to be neutral with 732 strong acid styrene cation exchange resin, filtering, removing the methanol from the filtrate under reduced pressure to obtain a light yellow solid, and performing column chromatography separation to obtain the isoxazole derivative containing the glucose triazole structure: (2R, 3S, 4S, 5R, 6R) -2- (hydroxymethyl) -6- (4- ((2- (4- (methylsulfonyl) -8 b-nitro-3 a, 8 b-dihydro-4H-isoxazolo [5 ', 4': 4,5] pyrrolo [2,3-b ] pyridin-3-yl) phenoxy) methyl) -1H-1,2, 3-triazol-1-yl) tetrahydro-2H-pyran-3, 4, 5-triol.

Of these, 3-nitro-1-methanesulfonyl-7-azaindole is also referred to in the examples as compound 3, compound 3 has been prepared by a prior art method, as briefly described below:

adding 1.18g of 7-azaindole (compound 1) and 10mL of DMF (dimethyl formamide) into a 50mL round-bottom flask, slowly adding 0.6g of NaH at 0-5 ℃, stirring for 10 minutes, adding 5mL of DMF solution dissolved with 1.15g of methanesulfonyl chloride, stirring for 1 hour at room temperature, monitoring the reaction end point by TLC (thin layer chromatography), adding 20mL of water into the reaction solution after the reaction is finished, extracting for 3 times by using 20mL of ethyl acetate each time, combining organic layers, and evaporating the solvent to obtain N-methanesulfonyl-7-azaindole (compound 2);

adding 20mL of acetic anhydride into a 50mL round-bottom flask, slowly dropwise adding 0.2mL of concentrated nitric acid at 0-5 ℃, stirring for 10 minutes, dropwise adding the reaction liquid into 30mL of acetic anhydride solution dissolved with 0.59 g of N-methylsulfonyl-7-azaindole, stirring overnight at room temperature after the dropwise adding is finished, monitoring the reaction end point by TLC, pouring the reaction liquid into 50g of ice after the reaction is finished, stirring for 1 hour, extracting for 3 times by using 20mL of ethyl acetate each time, combining organic layers, drying by using anhydrous sodium sulfate, evaporating the solvent to dryness, and performing column chromatography by using an eluent (petroleum ether: ethyl acetate ═ 5: 1) to obtain 3-nitro-1-methylsulfonyl-7-azaindole (compound 3).

As shown in fig. 3, chemical formula 1 is 7-azaindole (compound 1), chemical formula 2 is N-methanesulfonyl-7-azaindole (compound 2), chemical formula 3 is 3-nitro-1-methanesulfonyl-7-azaindole (compound 3), chemical formula 4 is acetylglucose triazole salicylaldoxime (compound 4), chemical formula 5 is a product (compound 5) obtained by dipolar cycloaddition reaction under mild conditions, compound 5 is deacetylated to generate (2R, 3S, 4S, 5R, 6R) -2- (hydroxymethyl) -6- (4- ((2- (4- (methylsulfonyl) -8 b-nitro-3 a, 8 b-dihydro-4H-isoxazolo [ 5', 4': 4,5] pyrrolo [2,3-b ] pyridin-3-yl) phenoxy) methyl) -1H-1,2, 3-triazol-1-yl) tetrahydro-2H-pyran-3, 4, 5-triol (Compound 6).

In one example, the ratio of the amounts of the acetyl glucose triazole salicylaldehyde to the hydroxylamine hydrochloride substance is 1: 1.20-1: 1.30.

for example: into a reaction flask, 3.56g (0.05mol) of hydroxylamine hydrochloride and 90mL to 100mL of H were charged₂And O, magnetically stirring until the hydroxylamine hydrochloride is dissolved (usually stirring by a magnetic stirrer). Under stirring, 18.4g (0.04mol) of acetyl glucose triazole salicylaldehyde and 50-60 mL of absolute ethyl alcohol are weighed and poured into a reaction bottle, and the mixture is vigorously stirred for 2-3 hours. After the reaction is finished, 20% Na is used₂CO₃The solution adjusts the pH value of the reaction solution to be neutral, and the reaction solution is placed and cooled to room temperature to generateWhite precipitate, putting into refrigerator overnight, filtering under reduced pressure, and drying at room temperature to obtain granular white crystal acetyl glucose triazole salicylaldoxime (compound 4).

In another example, the ratio of the amounts of the 3-nitro-1-methanesulfonyl-7-azaindole, the acetyl-glucose triazole salicylaldoxime, and the chloramine T substance is 1: 0.9-1: 1.2 to 1.3.

For example, 10mmol of 3-nitro-1-methanesulfonyl-7-azaindole (compound 3) and 10mmol of acetylglucose triazole salicylaldoxime (compound 4) are dissolved in 30mL to 40mL of anhydrous ethanol, 12mmol of chloramine T is added, the mixture is refluxed for 4 to 6 hours, 1, 3-dipolar cycloaddition reaction is carried out, a structure of glucose triazole and isoxazole is introduced, and recrystallization is carried out with methanol to obtain (2R, 3R, 4S, 5R, 6R) -2- (acetoxymethyl) -6- (4- ((2- (4- (methylsulfonyl) -8 b-nitro-3 a, 8 b-dihydro-4H-isoxazolo [5 ', 4': 4,5] pyrrolo [2,3-b ] pyridin-3-yl) phenoxy) methyl) -1H-1,2, 3-triazol-1-yl) tetrahydro-2H-pyran-3, 4, 5-triacetate (Compound 5).

In another example, compound 5 is suspended in methanol, ice water is cooled to 0 ℃, and a methanol solution with a concentration of 1.0mol/L sodium methoxide is slowly added dropwise under the protection of nitrogen, wherein the methanol solution comprises:

adding 5mmol of compound 5 into 20-30 mL of methanol solvent;

0.6mL of a methanol solution of sodium methoxide with the concentration of 1.0mol/L is added into every 20-30 mL of the methanol solvent.

For example, 5mmol of compound 5 is suspended in 20mL to 30mL of methanol, ice water is cooled to 0 ℃, 0.6mL of methanol solution of sodium methoxide with the concentration of 1.0mol/L is slowly dropped under the protection of nitrogen, the reaction is continued for 4 hours at room temperature, TLC monitors that the raw material point disappears, 732 strong acid styrene cation exchange resin is used for adjusting the system to be neutral, filtration is carried out, the filtrate is decompressed to remove the methanol, light yellow solid is obtained, and column chromatography separation [ eluent: volume ratio (chloroform: methanol ═ 20: 1) ], to give (2R, 3S, 4S, 5R, 6R) -2- (hydroxymethyl) -6- (4- ((2- (4- (methylsulfonyl) -8 b-nitro-3 a, 8 b-dihydro-4H-isoxazolo [5 ', 4': 4,5] pyrrolo [2,3-b ] pyridin-3-yl) phenoxy) methyl) -1H-1,2, 3-triazol-1-yl) tetrahydro-2H-pyran-3, 4, 5-triol (compound 6).

The experimental data are as follows: (2R, 3S, 4S, 5R, 6R) -2- (hydroxymethyl) -6- (4- ((2- (4- (methylsulfonyl) -8 b-nitro-3 a, 8 b-dihydro-4H-isoxazolo [5 ', 4': 4,5] pyrrolo [2,3-b ] pyridin-3-yl) phenoxy) methyl) -1H-1,2, 3-triazol-1-yl) tetrahydro-2H-pyran-3, 4, 5-triol (Compound 6) as a pale yellow powder in 31.4% yield, m.p.142-143 ℃ melting point, with the following nuclear magnetic, infrared and elemental analysis data:

δ:¹H NMR(DMSO-d₆)δ:8.39～8.37(m，1H)，8.03(d，J＝7.6Hz，2H)，

7.72(dd，J＝7.6，1.2Hz，1H)，7.57(t，J＝7.2Hz，1H)，7.47-7.38(m,3H,Ar-H),7.33(m,1H),7.26(t，J＝8.0Hz，2H),6.97(dd，J＝7.6,5.2Hz，1H)，5.56(dd，J＝6.4,3.6Hz，1H)，5.30(2H,s),4.85-3.58(m,7H,4×Galactosyl H,OH)，3.28(3H,m)；

IR(KBr)v/cm^-1 3446,3430,2983,1708,1633,1576,1462,1209,1160,1093,751

m/e:619(100.0％)。

Anal.calcd.for C₂₄H₂₅N₇O₁₁S C,46.53；H,4.07；N,15.83；found C,46.54；H,4.07；N,15.81。

in this example, the MTT method was used to determine the in vitro inhibitory effect of compound 6 on different tumor strains, and the antitumor activity of (2R, 3S, 4S, 5R, 6R) -2- (hydroxymethyl) -6- (4- ((2- (4- (methylsulfonyl) -8 b-nitro-3 a, 8 b-dihydro-4H-isoxazolo [5 ', 4': 4,5] pyrrolo [2,3-b ] pyridin-3-yl) phenoxy) methyl) -1H-1,2, 3-triazol-1-yl) tetrahydro-2H-pyran-3, 4, 5-triol (compound 6) was determined as follows:

compound 6 was diluted with DMSO, and tumor cells HepG2 (liver cancer cells), A375 (melanoma cells), SW620 (human colorectal adenocarcinoma cells), A549 (lung adenocarcinoma cells), SGC7901 (stomach cancer cells), SKOV3 (ovarian cancer cells) were seeded in 4000/200. mu.L/well in 96-well plates, 2. mu.L of compound was added to each well to a final concentration of 12.0. mu.M, 6.0. mu.M, 3.0. mu.M, 1.5. mu.M, together at 37 ℃ with 5% CO₂The cells were incubated in an incubator for 72 hours, with DMSO (1%) as a blank control. After 72 hours, a final concentration of 0.25mg/mL was addedMTT, 5% CO at 37 ℃₂After 4 hours in the cell incubator, the solvent was blotted, 100. mu.L of DMSO was added to each well, absorbance (OD value) was measured at 570nm with an enzyme-linked immunosorbent assay, and the data obtained was used to calculate IC₅₀The value is obtained. Selecting compounds with high inhibitory activity, and determining the influence of different action times of the compounds at different concentrations on the human tumor cell cycle and apoptosis.

The test compounds of different concentrations were coarse-screened in 96-well plates and IC was calculated from the resulting inhibition₅₀Values, results are given in the following table:

TABLE 1 IC of Compound 6 against six tumor cell lines₅₀Value of

In Table 1, (2R, 3S, 4S, 5R, 6R) -2- (hydroxymethyl) -6- (4- ((2- (4- (methylsulfonyl) -8 b-nitro-3 a, 8 b-dihydro-4H-isoxazolo [5 ', 4': 4, 5)]Pyrrolo [2,3-b]IC of pyridin-3-yl) phenoxy) methyl) -1H-1,2, 3-triazol-1-yl) tetrahydro-2H-pyran-3, 4, 5-triol (Compound 6) on six tumor cell lines₅₀The value shows that the compound 6 has stronger tumor cell inhibition effect on A375 (melanoma cells), and provides a foundation for further application in the medical field.

The target compounds were tested for anti-HBV activity. Taking HepG22.2.15 cells in logarithmic phase, washing with 0.02% EDTA for 2 times, digesting with 0.25% trypsin, blowing uniformly, and counting to 2.5 × 10 cells/mL^-1And inoculated into 24-well plates, 0.5mL per well, and administration is started after the cells adhere to the wall. Samples were prepared at 12.5, 25, 50. mu.g/mL in DMSO-containing medium^-13 concentrations of DMS0 were added to 24-well culture plates, 0.6mL per well, and 2 wells per concentration, and cells containing the same amount of DMS0 were used as control groups instead of the drug solution. The 3 rd day was administered with the same concentration of the drug solution, and the 6 th day was administered to collect the cells. After washing 2 times with Phosphate Buffered Saline (PBS), extraction was performed with a reagent for extracting virus core particles. The Taqman probe is used for fluorescent quantitative PCR to determine the HBV DNA content in the cell. According to the formula, the HBV DNA inhibition ratio (%) is equal toShellfish number-number of copies in the administered group)/number of copies in the control group x 100%, and the inhibition rate of HBV DNA replication in the cells by the sample was calculated. The compound 6 has inhibition effect on replication of HBV DNA in HepG22.2.15 cells and presents a certain dose-effect relationship. The results are shown in table 2:

TABLE 2 replication inhibition of HBV DNA cells by Compound 6

As can be seen, Compound 6 was at 50. mu.g/mL^-1The inhibition rate to HBV DNA is 77.34%, and the in vitro anti-HBV activity is better.

The above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and those skilled in the art can make various corresponding changes and modifications according to the present invention without departing from the spirit and the essence of the present invention, but these corresponding changes and modifications should fall within the protection scope of the appended claims.