Triazole neuraminidase inhibitor and preparation method and application thereof

文档序号：1793154 发布日期：2021-11-05 浏览：32次中文

阅读说明：本技术 一种三唑类神经氨酸酶抑制剂及其制备方法与应用 (Triazole neuraminidase inhibitor and preparation method and application thereof ) 是由程利平石林庞婉于 2021-07-02 设计创作，主要内容包括：本发明涉及一种三唑类神经氨酸酶抑制剂及其制备方法与应用,该抑制剂具有式(I)所示的结构：制备为：(1)将取代苯胺和氯乙酰氯形成反应体系,得到式(Ⅱ)中间体；(2)将呋喃酰氯和氨基硫脲溶于有机溶剂中形成反应体系,得到式(Ⅲ)中间体；(3)取步骤(2)得到的式(Ⅲ)中间体溶于氢氧化钠溶液中形成反应体系,得到式(Ⅳ)中间体；(4)取步骤(3)得到的式(Ⅳ)中间体和步骤(1)得到的式(Ⅱ)中间体与氢氧化钠溶液溶于有机溶剂中,反应经后处理得到式(Ⅰ)所示的抑制剂。本发明的化合物结构新颖,实验表明具有较好的神经氨酸酶抑制活性,可用于制备抑制神经氨酸酶活性的药物。(The invention relates to a triazole neuraminidase inhibitor and a preparation method and application thereof, wherein the inhibitor has a structure shown in a formula (I):)

1. A triazole neuraminidase inhibitor having the structure of formula (I):

wherein the content of the first and second substances,

ar is selected from Any one of them.

2. The triazole neuraminidase inhibitor according to claim 1,

ar is selected from Any one of them.

3. The triazole neuraminidase inhibitor according to claim 2, wherein the inhibitor has the formula:

4. the method for preparing a triazole neuraminidase inhibitor according to claim 1, wherein the formula of the preparation method is as follows:

the preparation method specifically comprises the following steps:

(1) forming a reaction system by substituted aniline and chloracetyl chloride, and carrying out post-treatment after reaction to obtain an intermediate of a formula (II);

(2) dissolving furan acyl chloride and thiosemicarbazide in an organic solvent to form a reaction system, and carrying out post-treatment after reaction to obtain an intermediate of a formula (III);

(3) dissolving the intermediate of the formula (III) obtained in the step (2) in a sodium hydroxide solution to form a reaction system, and carrying out post-treatment on the reaction system to obtain an intermediate of the formula (IV);

(4) and (3) dissolving the intermediate of the formula (IV) obtained in the step (3), the intermediate of the formula (II) obtained in the step (1) and sodium hydroxide solution in an organic solvent, reacting, and carrying out aftertreatment to obtain the inhibitor shown in the formula (I).

5. The preparation method of the triazole neuraminidase inhibitor according to claim 4, wherein triethylamine is used as an acid-binding agent in the step (1), dichloromethane is used as an organic solvent, the reaction system is placed in an ice-water bath for reaction, nitrogen is used for protection, the reaction temperature is 0 ℃, the reaction time is 3-4.5 hours, and the post-treatment process specifically comprises the following steps: the reaction was diluted with 20ml of dichloromethane, washed successively with 1M hydrochloric acid, saturated sodium bicarbonate solution and brine and the dichloromethane removed by rotary evaporation to give the intermediate of formula (II).

6. The preparation method of the triazole neuraminidase inhibitor according to claim 4, wherein in the step (2), the organic solvent is dichloromethane, the reaction system is placed in an ice-water bath for reaction, the reaction temperature is 0 ℃, the reaction time is 12-18h, and the post-treatment process is as follows: removal of the dichloromethane by rotary evaporation gives the intermediate of formula (III).

7. The preparation method of the triazole neuraminidase inhibitor according to claim 4, wherein in the step (3), the sodium hydroxide solution is a 5% sodium hydroxide solution, the reaction system is placed in an oil bath for reaction, the reaction temperature is 90-120 ℃, the reaction time is 2.5-4.5h, and the post-treatment comprises the following specific processes: and (3) taking out the reaction system, cooling, adjusting the pH to 4 by using acid (hydrochloric acid), separating out a precipitate, and sequentially filtering, washing and drying the precipitate to obtain the intermediate shown in the formula (IV).

8. The preparation method of the triazole neuraminidase inhibitor according to claim 4, wherein in the step (4), the organic solvent is methanol, the reaction system is placed at room temperature for 3-5 hours at 20-30 ℃, and the post-treatment comprises the following specific steps: adjusting the pH value to 7 by adopting acid, separating out a precipitate, and sequentially filtering, washing, drying and recrystallizing by using ethanol to obtain the inhibitor shown in the formula (I).

9. The preparation method of the triazole neuraminidase inhibitor according to claim 4, wherein the adding amount ratio of the substituted aniline, the chloroacetyl chloride, the triethylamine and the organic solvent in the step (1) is (4-8) mmol, (8-15) mL;

the adding amount ratio of the furoyl chloride, the thiosemicarbazide and the organic solvent in the step (2) is (4-8) mmol, (15-25) mL;

the adding amount ratio of the intermediate of the formula (IV), the intermediate of the formula (II), sodium hydroxide and the organic solvent in the step (4) is 1mmol (1.0-1.1) to 1mmol (5-10) mL.

10. Use of a triazole neuraminidase inhibitor according to any one of claims 1 to 3 for the preparation of a medicament capable of inhibiting neuraminidase activity.

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a triazole neuraminidase inhibitor as well as a preparation method and application thereof.

Background

Neuraminidase is an important functional glycoprotein embedded in the surface of an influenza virus envelope, and has a main function of cracking glycosidic bonds in the replication process of influenza viruses, so that the viruses are separated from host cells to achieve the purpose of transmission. Therefore, neuraminidase is one of important targets for developing anti-influenza virus drugs. Anti-influenza drugs developed with neuraminidase as a target can be classified into the following classes according to structure: neuraminic acid analogs, cyclohexenes, cyclopentanes, benzoates, pyrrolyclines to indole alkaloids, multimers, fusion proteins, polycyclic compounds, and the like. The most widely used anti-influenza drug is tamiflu at present, but with the wide use of the drug, the drug resistance of influenza virus to tamiflu also appears, the drug resistance of tamiflu also appears, the production raw material of tamiflu is extremely expensive, and the synthesis process is complex. Therefore, it is urgent to develop a neuraminidase inhibitor having a novel structure and a good inhibitory effect.

Disclosure of Invention

The invention aims to solve the problems and provide a triazole neuraminidase inhibitor and a preparation method and application thereof.

The purpose of the invention is realized by the following technical scheme:

a triazole neuraminidase inhibitor having the structure shown in formula (I):

wherein Ar is selected from Any one of them.

Preferably, Ar is selected from Any one of them.

Further preferably, the inhibitor has the formula:

a preparation method of the triazole neuraminidase inhibitor, wherein the formula of the preparation method is as follows:

the preparation method specifically comprises the following steps:

(1) forming a reaction system by substituted aniline and chloracetyl chloride, and carrying out post-treatment after reaction to obtain an intermediate of a formula (II);

(2) dissolving furan acyl chloride and thiosemicarbazide in an organic solvent to form a reaction system, and carrying out post-treatment after reaction to obtain an intermediate of a formula (III);

In the step (1), triethylamine is used as an acid-binding agent, and the organic solvent is dichloromethane.

In the step (1), the reaction system is placed in an ice-water bath for reaction, nitrogen is used for protection, the reaction temperature is 0 ℃, and the reaction time is 3-4.5h, preferably 4 h.

In the step (1), the post-treatment process specifically comprises the following steps: the reaction was diluted with 20ml of dichloromethane, washed successively with 1M hydrochloric acid, saturated sodium bicarbonate solution and brine and the dichloromethane removed by rotary evaporation to give the intermediate of formula (II).

In the step (1), the adding amount ratio of the substituted aniline, the chloracetyl chloride, the triethylamine and the organic solvent is (4-8) mmol, (8-15) mL, preferably 5mmol:5mmol:5mmol:10 mL.

In the step (2), dichloromethane is used as the organic solvent.

In the step (2), the reaction system is placed in an ice-water bath for reaction, the reaction temperature is 0 ℃, and the reaction time is 12-18h, preferably 16 h.

In the step (2), the addition ratio of the furoyl chloride to the thiosemicarbazide to the organic solvent is (4-8) mmol, (15-25) mL, preferably 5mmol, and 20 mL.

In the step (2), the post-treatment process is as follows: removal of the dichloromethane by rotary evaporation gives the intermediate of formula (III).

In the step (3), the sodium hydroxide solution is a 5% sodium hydroxide solution.

In the step (3), the reaction system is placed in an oil bath for reaction, the reaction temperature is 90-120 ℃, the reaction time is preferably 110 ℃, and the reaction time is 2.5-4.5h, preferably 4 h.

In the step (3), the post-treatment comprises the following specific processes: and (3) taking out the reaction system, cooling, adjusting the pH to 4 by using acid (hydrochloric acid), separating out a precipitate, and sequentially filtering, washing and drying the precipitate to obtain the intermediate shown in the formula (IV).

In the step (4), the organic solvent is methanol.

In the step (4), the reaction system is placed at room temperature for reaction, the reaction temperature is 20-30 ℃, the reaction time is preferably 25 ℃, and the reaction time is 3-5 h, preferably 5 h.

In the step (4), the adding amount ratio of the intermediate of the formula (IV), the intermediate of the formula (II), sodium hydroxide and the organic solvent is 1mmol (1.0-1.1) to 1mmol (5-10) mL, preferably 1mmol to 1.1mmol to 1mmol to 8 mL.

In the step (4), the post-treatment comprises the following specific processes: adjusting the pH value to 7 by adopting acid (hydrochloric acid), separating out a precipitate, and sequentially filtering, washing, drying and recrystallizing by using ethanol to obtain the inhibitor shown in the formula (I).

The application of the triazole neuraminidase inhibitor in preparing the medicine capable of inhibiting the neuraminidase activity.

The invention utilizes a receptor-based molecular docking virtual screening method to screen 250000 compounds from a ZINC database to obtain one compound theoretically having neuraminidase inhibition activity, then modifies the structure of the compound, designs more reasonable compounds, carries out neuraminidase test on nine compounds, takes oseltamivir acid (OSC) as a positive control, wherein IC of the OSC is IC₅₀IC of compound with best inhibition effect with value of 0.10 μ M₅₀The value is 0.11 mu M, which shows that the compound has excellent neuraminidase inhibitory activity, is equivalent to the activity of oseltamivir acid, has a novel skeleton structure, is a neuraminidase inhibitor, and has a simple synthesis method, a good neuraminidase inhibitory effect and can be applied to preparation of an anti-avian influenza medicament for inhibiting the neuraminidase activity.

Detailed Description

The present invention is described in detail below with reference to specific examples, but the present invention is not limited thereto in any way.

A triazole neuraminidase inhibitor having the structure shown in formula (I):

wherein Ar is selected from Any one of them.

Ar is

A preparation method of the triazole neuraminidase inhibitor is as follows:

the preparation method specifically comprises the following steps:

(1) forming a reaction system by substituted aniline and chloracetyl chloride, and carrying out post-treatment after reaction to obtain an intermediate of a formula (II);

(2) dissolving furan acyl chloride and thiosemicarbazide in an organic solvent to form a reaction system, and carrying out post-treatment after reaction to obtain an intermediate of a formula (III);

In the step (1), triethylamine is used as an acid-binding agent, and the organic solvent is dichloromethane. The reaction system is placed in an ice-water bath for reaction, nitrogen is used for protection, the reaction temperature is 0 ℃, and the reaction time is 3-4.5 h. The post-treatment process specifically comprises the following steps: the reaction was diluted with 20ml of dichloromethane, washed successively with 1M hydrochloric acid, saturated sodium bicarbonate solution and brine and the dichloromethane removed by rotary evaporation to give the intermediate of formula (II). The adding amount ratio of the substituted aniline to the chloracetyl chloride to the triethylamine to the organic solvent is (4-8) mmol, (4-8) mmol and (8-15) mL.

In the step (2), the organic solvent is dichloromethane, the reaction system is placed in an ice-water bath for reaction at the temperature of 0 ℃ for 12-18h, and the post-treatment process comprises the following steps: removal of the dichloromethane by rotary evaporation gives the intermediate of formula (III). The adding amount ratio of the 2-furoyl chloride to the thiosemicarbazide to the organic solvent is (4-8) mmol, (15-25) mL.

In the step (3), the sodium hydroxide solution is 5% sodium hydroxide solution, the reaction system is placed in an oil bath for reaction, the reaction temperature is 90-120 ℃, preferably 110 ℃, the reaction time is 2.5-4.5h, and the post-treatment specific process comprises the following steps: and (3) taking out the reaction system, cooling, adjusting the pH to 4 by using acid (hydrochloric acid), separating out a precipitate, and sequentially filtering, washing and drying the precipitate to obtain the intermediate shown in the formula (IV).

In the step (4), the organic solvent is methanol, the reaction system is carried out at room temperature, the reaction temperature is 20-30 ℃, preferably 25 ℃, the reaction time is 3-5 h, and the post-treatment comprises the following specific processes: adjusting the pH value to 7 by adopting acid (hydrochloric acid), separating out a precipitate, and sequentially filtering, washing, drying and recrystallizing by using ethanol to obtain the inhibitor shown in the formula (I). The adding amount ratio of the intermediate of the formula (IV), the intermediate of the formula (II), sodium hydroxide and an organic solvent is 1mmol (1.0-1.1) to 1mmol (5-10) mL. The steps of filtering, washing, drying and absolute ethyl alcohol recrystallization, which are involved in each step, are all conventional operations, and the operation parameters are selected according to parameters commonly adopted in laboratories to reach the required target values.

The application of the triazole neuraminidase inhibitor in preparing the medicine capable of inhibiting the neuraminidase activity.

The following are specific examples:

example 1

A preparation method of a triazole neuraminidase inhibitor is disclosed, and the structural formula of the inhibitor is shown as a formula I:

the specific synthesis steps are as follows:

(1) accurately weighing 0.61g (5mmol) of 3-ethylaniline and 0.51g (5mmol) of triethylamine in a 50mL round bottom flask, adding 10mL of dichloromethane, adding 0.56g (5mmol) of chloroacetyl chloride under nitrogen protection, placing in an ice water bath, stirring for reaction at 0 ℃ for 4 hours, adding 20mL of dichloromethane to dilute the reaction, washing with 1M hydrochloric acid, saturated sodium bicarbonate solution and brine in sequence, and removing dichloromethane by rotary evaporation to obtain the intermediate of formula (II).

(2) Accurately weighing 0.65g (5mmol) of 2-furoyl chloride and 0.46g (5mmol) of thiosemicarbazide into a 50mL round-bottomed flask, adding 20mL of dichloromethane, placing in an ice-water bath, stirring for reaction at 0 ℃ for 16h, and after the reaction is finished, removing the dichloromethane in vacuum to obtain the intermediate of the formula (III).

(3) 0.93g (5mmol) of the intermediate of the formula (III) is taken out of a round-bottom flask, 20mL of 5% sodium hydroxide solution is added into the round-bottom flask, the round-bottom flask is placed in a constant temperature oil bath kettle, and the round-bottom flask is reacted for 4 hours at 110 ℃. And (3) after the reaction is finished, taking out the reaction liquid to cool, regulating the pH value to 4 by using hydrochloric acid, standing to separate out a large amount of precipitate, filtering the precipitate to obtain a filter cake, washing the filter cake for multiple times by using a large amount of ice water, drying in a vacuum drying oven, and recrystallizing by using ethanol to obtain the intermediate shown in the formula (IV).

(4) 0.17g (1mmol) of the intermediate of formula (IV), 0.40g (1mmol) of sodium hydroxide and 2mL of water are taken in a 50mL round-bottom flask, 0.20g (1mmol) of the intermediate of formula (II) and 8mL of methanol are added, and the reaction is stirred at room temperature for 4h at 25 ℃. After the reaction is finished, hydrochloric acid is used for adjusting the PH value to 7, standing is carried out to separate out a precipitate, the precipitate is filtered to obtain a filter cake, the filter cake is washed by distilled water for many times and then dried, and the filter cake is recrystallized by ethanol to obtain the inhibitor shown in the formula (I), wherein the specific structural formula is shown in the formula A, and the inhibitor is N- (3-ethylphenyl) -2- ((5- (furan-2-yl) -4H-1,2, 4-triazole-3-yl) sulfenyl) acetamide, is white solid and has the yield of 85%.

The prepared inhibitor is tested for inhibiting the activity of neuraminidase:

1. laboratory instruments and materials

A multifunctional fluorescent microplate reader, model SP-Max 3500FL, Shanghai flash spectrum Biotech limited;

an ultra-clean bench;

bond A3Pipette manual single-channel adjustable pipettor, 0.5-10ul, 10-100ul, 100 and 1000ul of tylosin science and technology;

96-well plate (black), sterilized, kangning;

H5N1 neuraminidase available from Beijing Yi Qiao Shen science and technology, Inc.; the fluorogenic substrate 2' - (4-methylumbelliferone) - α -D-acetylneuraminic acid sodium hydrate (4-MUNANA) (Sigma, M8639) used in the enzyme inhibition experiments was purchased from Sigma; 2- (N-morpholine) ethanesulfonic acid (MES), calcium chloride, sodium hydroxide, absolute ethanol, purchased from Tatan technology.

A positive control drug, Oseltamivir acid (abbreviated as OSC), shanghai haokang biotechnology limited.

2. Experimental methods

Dissolving a positive control drug and a target compound (namely prepared N- (3-ethylphenyl) -2- ((5- (furan-2-yl) -4H-1,2, 4-triazole-3-yl) sulfenyl) acetamide) in DMSO, preparing the initial concentration into 1000 mu m/l, diluting the initial concentration into 6 concentration gradients according to a multiple ratio, and preparing three groups of concentration gradients sequentially from 200 mu m/l, 40 mu m/l, 8 mu m/l, 1.6 mu m/l, 0.32 mu m/l and 0.064 mu m/l;

2.1 sample preparation for detection

a. Buffer (33mM MES, 4mM CaCl)₂) Adding 70 mu L of the enzyme-linked immunosorbent assay (ELISA) plate into each hole;

b. adding 10 mu L of neuraminidase into each hole;

c. adding 10 mu L of a prepared neuraminidase inhibitor sample to be detected or a positive control drug sample with the concentration into each hole, and simultaneously setting three groups of blank test controls;

d. neuraminidase substrate (100. mu.M.L)^-14-MUNANA) 10. mu.L per well.

2.2 detection

a. Placing the 96-well plate in a multifunctional fluorescent microplate reader, and shaking and uniformly mixing for 1 minute;

b. setting the temperature to be 37 ℃, and incubating for 15 minutes to ensure that the neuraminidase and the sample to be detected are fully mixed and interacted;

c. taking out the 96-well plate, and adding 10 mu L of neuraminidase fluorescent substrate into each well;

d. placing the mixture in a multifunctional fluorescent microplate reader again, and shaking and uniformly mixing for 1 minute;

e. after incubation at 37 ℃ for 60 minutes, the cells were removed and 150. mu.L of stop solution (14 mM. multidot.L) was added to each well^-183% ethanol water solution of NaOH), placing the mixture in a multifunctional fluorescent microplate reader again, shaking and uniformly mixing for 1 minute, setting the excitation wavelength to be 355nm and the emission wavelength to be 460nm, and starting fluorescence intensity (RFU) measurement after the incubation is finished;

f. the above procedure was repeated to perform 3 parallel experiments.

Note: the first well in the 96-well plate was used as a blank, no sample to be tested was added, and 10 μ l of DMSO solution was added.

Calculating the average value of the inhibition rate of the sample under each gradient concentration in each parallel experiment, and then fitting the corresponding IC through Origin₅₀The value is obtained.

2.3 results of the experiment

The positive control drug and the target compound are prepared into mixed solutions with initial concentrations of 1000 mu m/L by DMSO solutions, the two mixed solutions are diluted into 6 concentration gradients according to a multiple ratio, the concentration gradients are 200 mu m/L, 40 mu m/L, 8 mu m/L, 1.6 mu m/L, 0.32 mu m/L and 0.064 mu m/L in sequence, and three groups are prepared in sequence for each concentration gradient. 70 mu L of neuraminidase buffer solution, 10 mu L of neuraminidase and positive control drug samples of each gradient concentration to be detected are added into a 96-hole black fluorescent enzyme label plate, and three groups of blank test controls are arranged at the same time. Shaking in a multifunctional fluorescent microplate reader for 1 min, mixing, and incubating at 37 deg.C for 15 min; taking out 96-well enzyme-linked immunosorbent assay plate, adding 10 μ L neuraminidase substrate into each well, shaking for 1 min, mixing, incubating at 37 deg.C for 60min, taking out, adding 150 μ L stop solution (14 mM. L) into each well^-1And (3) putting the NaOH aqueous solution in 83% ethanol solution) into the multifunctional luciferase reader again, uniformly mixing the NaOH aqueous solution and the NaOH aqueous solution by shaking for 1 minute, setting the excitation wavelength to be 355nm and the emission wavelength to be 460nm, and starting to measure the fluorescence intensity (RFU) after the incubation is finished. Three experiments were performed in parallel. Calculating the average value of the inhibition rate of the sample under each gradient concentration in each parallel experiment, and then fitting the corresponding IC through Origin₅₀The inhibition rate of each sample was calculated and thenCorresponding IC was fitted by Origin₅₀The value is obtained. IC thereof₅₀Value of 0.11. mu.M, IC of positive control₅₀The value was 0.10. mu.M.

N- (3-ethylphenyl) -2- ((5- (furan-2-yl) -4H-1,2, 4-triazol-3-yl) thio) acetamide as a white solid in 85% yield.

¹H NMR(500MHz,DMSO)δ14.43(s,1H),10.22(s,1H),7.86(s,1H),7.48(d,J＝10.00Hz,2H),7.13(d,J＝10.00Hz,2H),6.99(s,1H),6.66(s,1H),4.11(s,2H),2.56–2.52(m,2H),1.14(t,J＝15.00,10.00Hz,3H)。¹³C NMR(125MHz,DMSO)δ165.80,144.41,138.94,136.56,127.95,119.26,111.96,110.19,36.57,27.60,15.63.。HRMS(ESI):329.1070[M-H]+。

Example 2