CN115232079B - Pyrimidine neuraminidase inhibitor and preparation method and application thereof - Google Patents
Pyrimidine neuraminidase inhibitor and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a pyrimidine neuraminidase inhibitor, a preparation method and application thereof, wherein the structural formula of the inhibitor is as follows:
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a pyrimidine neuraminidase inhibitor and a preparation method and application thereof.
Background
Neuraminidase is a glycoprotein distributed on the envelope of influenza virus, which hydrolyzes the glycosidic bond between sialic acid and glycoprotein on the surface of host cells upon viral infection, causing mature virus to be released from the infected cell surface and thereby continue to infect new cells. Meanwhile, the method can prevent the filial generation of virus particles from agglomerating after being released from host cells, and can hydrolyze sialic acid in respiratory tract mucous membrane, so that the filial generation of virus particles is prevented from inactivating, the transmission of viruses in respiratory tracts is promoted, and important roles are played in the replication and infection period of the viruses, so that neuraminidase is one of important targets for the development and design of anti-influenza virus medicines.
Currently, there are only 6, 2M 2 proton channel inhibitors (amantadine and rimantadine), 3 neuraminidase inhibitors (zanamivir, oseltamivir, and peramivir) and 1 RNA-dependent RNA polymerase inhibitor (sof Lu Za) in the U.S. FDA approved anti-influenza virus drugs. Anti-influenza virus drugs developed with neuraminidase as a target point can be classified into the following classes according to structures: cyclohexene, cyclopentane, pyran, pyrrolidine, benzoic acid derivatives, natural extracts, and the like.
In recent years, because neuraminidase inhibition drugs are widely applied to clinic, drug-resistant virus strains appear, duffy is an oral preparation, but the application is most extensive, the drug resistance of viruses is also most serious, the raw materials for producing duffy are extremely expensive, the synthesis process is complex, and zanamivir, peramivir and ranimivir also have drug-resistant virus strains to begin to appear. Therefore, the development of novel, efficient and safe neuraminidase inhibitors is still a research hotspot in the field of anti-influenza viruses.
Disclosure of Invention
The invention aims to provide a pyrimidine neuraminidase inhibitor, and a preparation method and application thereof.
The aim of the invention can be achieved by the following technical scheme: a pyrimidine neuraminidase inhibitor having a chemical formula represented by formula (I):
wherein R is 1 Selected from any one of the following structural formulas:
(X=F, cl, br or I), ->
R 2 Selected from any one of the following structural formulas:
(X=F, cl, br or I),
Preferably, R 1 Selected from any one of the following structural formulas:
R 2 selected from any one of the following structural formulas:
further preferably, the inhibitor has one of the following chemical formulas:
the preparation method of the pyrimidine neuraminidase inhibitor specifically comprises the following steps:
(1) Forming a reaction system by using substituted benzaldehyde, acetaldehyde and an organic solvent, and performing post-treatment after the reaction to obtain an intermediate shown in a formula (II);
(2) Dissolving the intermediate of the formula (II) obtained in the step (1) in an organic solvent and zinc powder to form a reaction system, and reacting to obtain the intermediate of the formula (III);
(3) Dissolving the intermediate shown in the formula (III) obtained in the step (2) in an organic solvent, adding phosphorus tribromide to form a reaction system, and performing post-treatment after the reaction to obtain the intermediate shown in the formula (IV);
(4) Forming a reaction system by substituted phenethylamine, chloroacetyl chloride and triethylamine, and performing post-treatment after the reaction to obtain an intermediate shown in a formula (V);
(5) Taking the intermediate of the formula (V) obtained in the step (4), reacting with 4, 6-dihydroxyl-2-mercaptopyrimidine under alkaline conditions, and performing aftertreatment to obtain an intermediate shown in the formula (VI);
(6) And (3) taking the intermediate of the formula (VI) obtained in the step (5), forming a reaction system with the intermediate of the formula (IV) obtained in the step (3) under the weak alkaline condition, and performing post-treatment after the reaction to obtain the pyrimidine neuraminidase inhibitor shown in the formula (I).
The equation of the preparation method is as follows:
preferably, in step (1), the temperature of the reaction is 25-60 ℃, preferably 25 ℃, and the time of the reaction is 8-16 hours, preferably 10 hours.
Preferably, in step (1), anhydrous K is used 2 CO 3 As the catalyst, pyridine is used as the organic solvent.
Preferably, in step (1), the substituted benzaldehyde, acetaldehyde, anhydrous K 2 CO 3 And the organic solvent is added in an amount ratio of (20-30) mmoL (40-100) mmoL (30-45) mmoL (20-60) mL, preferably 20mmoL:40mmoL:30mmoL:20mL.
Preferably, in the step (1), the post-treatment process specifically includes: adding 100mL of distilled water into a reaction system, regulating the pH to 3 by using concentrated hydrochloric acid, adding 150mL of ethyl acetate into the reaction solution for extraction and liquid separation, taking an organic phase, and performing reduced pressure distillation on the organic phase to obtain a crude product of the intermediate of the formula (II), and performing column chromatography to obtain a pure intermediate of the formula (II).
Preferably, in step (2), the temperature of the reaction is from 0 to 40 ℃, preferably 25 ℃, and the time of the reaction is from 9.5 to 24 hours, preferably 12 hours.
Preferably, in step (2), the reduction is carried out using a zinc powder and ammonium chloride system, and the organic solvent is methanol.
Preferably, in the step (2), the addition amount ratio of the intermediate of the formula (II), zinc powder, ammonium chloride and organic solvent is (10-30) mmoL: (10-100) mmoL: (20-40) mL, preferably 15mmoL:90mmoL:20mL.
Preferably, in step (3), the reaction temperature is from 0 to 30 ℃, preferably 25 ℃, and the reaction time is from 9 to 17.5 hours, preferably 12 hours.
Preferably, in step (3), the organic solvent is diethyl ether, acetonitrile, toluene, tetrahydrofuran or dichloromethane, preferably dichloromethane.
Preferably, the addition ratio of the intermediate of formula (III), phosphorus tribromide and organic solvent is 5mmoL: (1-5) mmoL: (10-20) mL, preferably 5 mmol/2.5 mmol/15 mL.
Preferably, in step (4), the temperature of the reaction is from 0 to 35 ℃, preferably 0 ℃, and the time of the reaction is from 0.5 to 2 hours, preferably 1.5 hours.
Preferably, in step (4), the organic solvent used for extraction is acetone, ethyl acetate, tetrahydrofuran or dichloromethane, preferably dichloromethane.
Preferably, in the step (4), the addition amount ratio of the substituted phenethylamine, chloroacetyl chloride, triethylamine and the organic solvent used for extraction is 5mmoL: (5-5.5) mmoL: (5-5.5) mmoL: (20-50) mL, preferably 5mM L:5.1mM L:30mL.
Preferably, in step (5), the temperature of the reaction is 25-65 ℃, preferably 25 ℃, and the time of the reaction is 8-12 hours, preferably 10 hours.
Preferably, in step (5), the base is sodium hydroxide, potassium hydroxide, sodium ethoxide, sodium methoxide or potassium tert-butoxide, preferably sodium hydroxide; the solvent is ethyl acetate, methanol, ethanol or distilled water, preferably distilled water.
Preferably, in the step (5), the addition amount ratio of the intermediate of formula (V), 4, 6-dihydroxy-2-mercaptopyrimidine, sodium hydroxide and solvent is (5-5.5) mmoL: (5-5.5) mmoL: (5-10) mmoL: (10-50) mL, preferably 5.5mmol:5mmol:10mmoL:15mL.
Preferably, in the step (5), the post-treatment process specifically includes: the pH of the reaction solution was adjusted to 7 with concentrated hydrochloric acid, and the reaction solution was allowed to stand to precipitate a solid, which was collected by filtration, washed with a large amount of distilled water, and dried at room temperature to obtain an intermediate represented by the formula (VI).
Preferably, in step (6), the temperature of the reaction is 25-65 ℃, preferably 25 ℃, and the time of the reaction is 10-20 hours, preferably 18 hours.
Preferably, in step (6), the base is triethylamine, diethylamine, sodium bicarbonate, cesium carbonate, potassium carbonate or sodium carbonate, preferably potassium carbonate; the organic solvent is ethyl acetate, methanol, ethanol or DMF, preferably DMF.
Preferably, in step (6), the addition ratio of the intermediate of formula (VI), the intermediate of formula (IV), the base and the organic solvent is (5-5.5) mmoL: (5-5.5) mmoL: (7.5-15) mmoL: (10-30) mL, preferably 5.5mmol:11mmoL:15mL.
Preferably, in the step (6), the post-treatment process specifically includes: the reaction solution was poured into saturated saline, ethyl acetate was added to extract for a plurality of times, the organic phase was washed with saturated saline for a plurality of times and dried over anhydrous sodium sulfate, and the inhibitor represented by the formula (I) was obtained by column chromatography.
The pyrimidine neuraminidase inhibitor is applied to the preparation of medicines capable of inhibiting neuraminidase activity.
The invention utilizes a receptor-based molecular docking virtual screening method to screen 600000 compounds from a ZINC database to obtain a compound theoretically having neuraminidase inhibitory activity, then modifies the structure of the compound to design more reasonable compounds, and performs neuraminidase test on 7 compounds, takes Oseltamivir carboxlate (OSC) as a positive control, wherein the IC of the OSC 50 The value was 1.12. Mu.M.
IC of the four compounds synthesized according to the invention 50 Values are all close to 1.12 μm:
among them, the compounds with the best inhibition effectIC thereof 50 The value is 1.36+/-0.56 mu M, and the neuraminidase has excellent neuraminidase inhibition activity.
Compared with the prior art, the neuraminidase inhibitor with the novel framework structure, the preparation method and the application thereof are provided, the synthesis method is simple, and the prepared inhibitor has good neuraminidase inhibition activity and excellent neuraminidase inhibition effect, and can be applied to preparation of medicines for inhibiting neuraminidase activity.
Detailed Description
The following examples of the present invention are described in detail, and are given by way of illustration of the present invention, but the scope of the present invention is not limited to the following examples.
A pyrimidine neuraminidase inhibitor having a structure according to formula (I):
wherein R is 1 Selected from the group consisting of(X=F, cl, br or I), Any one of them;
R 2 selected from the group consisting of (X=F, cl, br or I), -> Any one of the following.
The preparation method of the pyrimidine neuraminidase inhibitor is as follows:
the preparation method specifically comprises the following steps:
(1) Forming a reaction system by substituted benzaldehyde, acetaldehyde and pyridine, and performing post-treatment after the reaction to obtain an intermediate shown in a formula (II);
(2) Dissolving the intermediate of the formula (II) obtained in the step (1) in an organic solvent and zinc powder to form a reaction system, and reacting to obtain the intermediate of the formula (III);
(3) Dissolving the intermediate shown in the formula (III) obtained in the step (2) in an organic solvent, adding phosphorus tribromide to form a reaction system, and performing post-treatment after the reaction to obtain the intermediate shown in the formula (IV);
(4) Forming a reaction system by substituted phenethylamine and chloroacetyl chloride, and performing post-treatment after the reaction to obtain an intermediate of the formula (V);
(5) Taking the intermediate of the formula (V) obtained in the step (4), reacting with 4, 6-dihydroxyl-2-mercaptopyrimidine under alkaline conditions, and performing aftertreatment to obtain an intermediate shown in the formula (VI);
(6) Taking the intermediate of the formula (VI) obtained in the step (5) and the intermediate of the formula (IV) obtained in the step (3) to form a reaction system under the weak alkaline condition, and performing post-treatment after the reaction to obtain the pyrimidine inhibitor shown in the formula (I);
in the step (1), anhydrous K is adopted 2 CO 3 As a catalyst, pyridine is adopted as the organic solvent, the reaction system is placed at the temperature of 25-60 ℃, preferably 25 ℃, the reaction time is 8-16 hours, preferably 10 hours, and the post-treatment process specifically comprises: adding 100mL of distilled water into a reaction system, regulating the pH to 3 by using concentrated hydrochloric acid, adding 150mL of ethyl acetate into the reaction solution for extraction and liquid separation, taking an organic phase, and performing reduced pressure distillation on the organic phase to obtain a crude product of the intermediate in the formula (II), wherein the pure intermediate in the formula (II) is obtained by column chromatography, and the addition amount ratio of the substituted benzaldehyde to the acetaldehyde to the anhydrous potassium carbonate to the organic solvent is (20-30) mmoL (40-100 mmoL) (30-45 mmoL): (20-60 mL), preferably 20mmoL:40mmoL:30mmoL:20mL.
In the step (2), zinc powder and an ammonium chloride system are adopted for reduction, the organic solvent adopts methanol, the reaction system is placed at the temperature of 0-40 ℃, preferably 25 ℃ for 9.5-24 hours, preferably 12 hours, and the addition amount ratio of the intermediate of the formula (II), the zinc powder, the ammonium chloride and the organic solvent is (10-30) mmoL (10-30 mmoL): (10-100) mmoL: (20-40) mL, preferably 15mmoL:90mmoL:20mL.
In the step (3), diethyl ether, acetonitrile, toluene, tetrahydrofuran or dichloromethane, preferably dichloromethane are adopted as the organic solvent, the reaction system is placed at the temperature of 0-30 ℃, preferably 25 ℃ for 9-17.5 hours, preferably 12 hours, and the addition ratio of the intermediate of the formula (III), phosphorus tribromide and the organic solvent is 5mmoL: (1-5) mmoL: (10-20) mL, preferably 5mmoL:2.5mmoL:15mL.
In the step (4), the organic solvent adopts acetone, ethyl acetate, tetrahydrofuran or dichloromethane, preferably dichloromethane, the reaction system is placed at the temperature of 0-35 ℃, preferably 0 ℃, the reaction time is 0.5-2h, preferably 1.5h, and the addition ratio of the substituted phenethylamine, chloroacetyl chloride, triethylamine and the organic solvent is 5mmoL: (5-5.5) mmoL: (5-5.5) mmoL: (20-50) mL, preferably 5mM L:5.1mM L:30mL.
In the step (5), sodium hydroxide, potassium hydroxide, sodium ethoxide, sodium methoxide or potassium tert-butoxide is used as a base, ethyl acetate, methanol, ethanol or distilled water, preferably distilled water is used as the solvent, the reaction system is placed at the temperature of 25-65 ℃, preferably 25 ℃, the reaction time is 8-12 hours, preferably 10 hours, and the post-treatment process specifically comprises: the pH of the reaction solution was adjusted to 7 with concentrated hydrochloric acid, and the reaction solution was allowed to stand to precipitate a solid, which was collected by filtration, washed with a large amount of distilled water, and dried at room temperature to obtain an intermediate represented by the formula (VI). The addition amount ratio of the intermediate of the formula (V), 4, 6-dihydroxy-2-mercaptopyrimidine, sodium hydroxide and solvent is (5-5.5) mmoL: (5-5.5) mmoL: (5-10) mmoL: (20-50) mL, preferably 5.5mmol:5mmol:10mmoL:15mL.
In the step (6), triethylamine, diethylamine, sodium bicarbonate, potassium bicarbonate, sodium carbonate or potassium carbonate is used as a base, preferably potassium carbonate; the organic solvent is methanol, ethanol or DMF, preferably DMF. The reaction temperature is 25-65 ℃, preferably 25 ℃, and the reaction time is 10-20h, preferably 18h. The post-treatment process specifically comprises the following steps: the reaction solution was poured into saturated saline, ethyl acetate was added to extract for a plurality of times, the organic phase was washed with saturated saline for a plurality of times and dried over anhydrous sodium sulfate, and the inhibitor represented by the formula (I) was obtained by column chromatography. The addition amount ratio of the intermediate of the formula (VI), the intermediate of the formula (IV), the anhydrous potassium carbonate and the organic solvent is (5-5.5) mmoL: (5-5.5) mmoL: (7.5-15) mmoL: (10-30) mL, preferably 5.5mmol:11mmoL:15mL.
The prepared inhibitor is subjected to a neuraminidase activity inhibition test, and the specific test method is as follows:
1. laboratory instrument and materials
Multifunctional fluorescent enzyme-labeled instrument, SP-Max 3500FL type Shanghai flash Spectrum biotechnology Co., ltd;
an ultra-clean workbench;
bond A3Pipette manual single-channel adjustable pipettor, 0.5-10ul,10-100ul,100-1000ul of Thai technology;
96-well plates (black), sterilized, corning;
H5N1 neuraminidase from beijing, samsungzhushun technologies inc; fluorogenic substrate 2' - (4-methylumbelliferone) -alpha-D-acetylneuraminic acid sodium hydrate (4-MUNANA) (Sigma, M8639) used in enzyme inhibition experiments was purchased from Sigma company; 2- (N-morpholinoethanesulfonic acid (MES), calcium chloride, sodium hydroxide, absolute ethanol, purchased from Tatans technology.
A positive control drug, oseltamivir acid (Oseltamivir carboxlate, abbreviated OSC), shanghai He Kang biotechnology limited.
2. Experimental method
Dissolving a positive control drug and a target compound prepared in the example with DMSO, preparing an initial concentration of 1000 mu m/l, diluting the initial concentration into 6 concentration gradients according to a doubling ratio, and sequentially preparing three groups of the concentration gradients, namely 50 mu m/l, 25 mu m/l, 12.5 mu m/l, 6.25 mu m/l, 3.13 mu m/l and 1.56 mu m/l;
2.1 sample detection preparation
a. Buffer solution (33mM MES,4mM CaCl) 2 ) 70 mu L of each well is added into a 96-well ELISA plate;
b. 10. Mu.L of neuraminidase was added per well;
c. adding 10 mu L of neuraminidase inhibitor sample or positive control medicine sample to be tested with prepared concentration into each hole, and setting three groups of blank test controls at the same time;
d. neuraminidase substrate (100. Mu.M.L) -1 4-Munana) was added at 10. Mu.L per well.
2.2 detection
a. Placing the 96-well plate in a multifunctional fluorescence enzyme-labeled instrument, and vibrating and uniformly mixing for 1 minute;
b. setting the temperature to 37 ℃ and incubating for 5 minutes to ensure that neuraminidase and a sample to be tested are fully mixed and interacted;
c. taking out the 96-well plate, and adding 10 mu L of neuraminidase fluorogenic substrate into each well;
d. placing the mixture in a multifunctional fluorescence enzyme labeling instrument again, and vibrating and uniformly mixing the mixture for 1 minute;
e. incubation was carried out at 37℃for 30min, and then removed, and 150. Mu.L of stop solution (14 mM. Multidot.L) was added to each well -1 83% ethanol aqueous solution of NaOH), placing in a multifunctional fluorescence enzyme-labeling instrument again,shaking and mixing for 1 minute, setting the excitation wavelength to 355nm, setting the emission wavelength to 460nm, and starting fluorescence intensity (RFU) measurement after incubation;
f. repeating the above operation steps, and performing 3 groups of parallel experiments.
Note that: the first well in a 96-well plate served as a blank, and no sample to be tested was added, and 10. Mu.l of DMSO solution was added.
Calculating average value of inhibition rate of sample under each gradient concentration in each parallel experiment, and fitting corresponding IC by Origin 50 Values.
The positive control medicine and the target compound are prepared into mixed solution with initial concentration of 1000 mu m/L by using DMSO solution, and then the mixed solution is diluted into 6 concentration gradients according to the double ratio, wherein the concentration gradients are sequentially 50 mu m/L, 25 mu m/L, 12.5 mu m/L, 6.25 mu m/L, 3.13 mu m/L and/1.56 mu m/L, and three groups are sequentially prepared for each concentration gradient. 70 mu L of neuraminidase buffer solution and 10 mu L of neuraminidase and positive control medicine samples with each gradient concentration to be tested are added into a 96-hole black fluorescent ELISA plate, and three groups of blank test controls are simultaneously arranged. Shaking in a multifunctional fluorescence enzyme labeling instrument for 1 minute, uniformly mixing, and incubating for 5 minutes at 37 ℃; taking out 96-well ELISA plate, adding 10 μl of neuraminidase substrate into each well, shaking for 1 min, mixing, incubating at 37deg.C for 30min, taking out, adding 150 μl of stop solution (14 mM. Multidot.L) -1 83% ethanol aqueous solution of NaOH), again placed in a multifunctional fluorescence microplate reader, mixed with shaking for 1 min, excitation wavelength set at 355nm, emission wavelength set at 460nm, and fluorescence intensity (RFU) measurement started after incubation was completed. Performing three experiments in parallel, calculating average value of inhibition rate of samples at each gradient concentration in each parallel experiment, and fitting corresponding IC by Origin 50 The inhibition rate of each sample is calculated, and the corresponding IC is fitted through Origin 50 Values.
The following are specific examples:
example 1
2- ((5-cinnamyl-6-hydroxy-4-oxo-1, 4-dihydropyrimidin-2-yl) thio) -N- (4-methoxyphenethyl) acetamide having a structural formula as shown in formula I:
the specific synthesis steps are as follows:
(1) 2.04mL (20 mmoL) of benzaldehyde and 2.25mL (40 mmoL) of acetaldehyde are accurately measured by using a measuring cylinder, poured into a 50mL round bottom flask, 2.76g (30 mmoL) of anhydrous potassium carbonate is accurately weighed, added into the round bottom flask, 20mL of pyridine is poured into the round bottom flask, and the mixture is placed at room temperature and stirred for reaction for 10 hours at 25 ℃, and after the reaction is finished. Adding 100mL of distilled water into a reaction system, regulating the pH to 3 by using concentrated hydrochloric acid, adding 150mL of ethyl acetate into the reaction solution for extraction and liquid separation, taking an organic phase, and performing reduced pressure distillation on the organic phase to obtain a crude product of the intermediate of the formula (II), and performing column chromatography to obtain a pure intermediate of the formula (II).
(2) 1.98g (15 mmoL) of the intermediate of formula (II), 0.98g (15 mmoL) of Zn powder and 4.81g (90 mmoL) of ammonium chloride were accurately weighed into a 50mL round bottom flask, and reacted at 25℃for 12 hours with 20mL of methanol. After the reaction, the reaction mixture was filtered, and the cake was washed with 10mL of methanol, and the solvent was removed in vacuo to give the intermediate of formula (III).
(3) 0.98g (5 mmoL) of intermediate of formula (III) was taken in a 25mL round bottom flask, 15mL of methylene chloride was added, 0.24mL (2.5 mmoL) of phosphorus tribromide was slowly dropped in a constant pressure liquid funnel, after the dropping was completed, stirring was carried out at 25℃for 12 hours, after the completion of the reaction, the reaction solution was carefully poured into 50mL of ice water, 3X30mL of methylene chloride was added for extraction, the organic phase was combined, and the organic phase was washed with 3X50mL of saturated sodium bicarbonate solution, and the organic phase was collected. Drying over anhydrous sodium sulfate, and removing the solvent under reduced pressure to obtain an intermediate represented by formula (IV).
(4) 0.76g (5 mmoL) of 4-methoxyphenylethylamine and 0.52g (5.1 mmoL) of triethylamine were accurately weighed into a 50mL round-bottomed flask, stirred in an ice bath for 15 minutes, and then 0.41mL (5.1 mmoL) of chloroacetyl chloride was slowly added dropwise via a constant pressure ground funnel, and after the addition was completed, the mixture was allowed to react at 0℃for 1.5 hours with stirring. After the completion of the reaction, the reaction mixture was poured into 30mL of water, 30mL of methylene chloride was added to extract, and the mixture was separated by a separating funnel to obtain an organic phase. The organic phase is washed with a saturated sodium bicarbonate solution, 1moL/L hydrochloric acid solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, the solvent is removed in vacuo, and column chromatography gives the pure intermediate of formula (V).
(5) 1.25g (5.5 mmoL) of the intermediate of the formula (V) and 0.72g (5 mmoL) of 4, 6-dihydroxy-2-mercaptopyrimidine were weighed accurately in a 25mL round-bottomed flask, 15mL of distilled water was added, 0.4g (10 mmoL) of sodium hydroxide was added in portions, and the mixture was stirred at 25℃for 10 hours to react, after the reaction was completed. The reaction solution was adjusted to pH 7 with concentrated hydrochloric acid, allowed to stand, and the solid was precipitated, collected by filtration, washed with a large amount of distilled water, and dried at room temperature to obtain an intermediate represented by formula (VI).
(6) 1.95g (5.5 mmoL) of the intermediate of formula (VI), 1.08g (5.5 mmoL) of the intermediate of formula (IV) and 1.52g (11 mmoL) of anhydrous potassium carbonate are accurately weighed into a 25mL round bottom flask, 15mM of LDMF is added, and the mixture is stirred at 25℃for 18 hours, after which the reaction is completed. The reaction solution was poured into saturated saline, ethyl acetate was added to extract for a plurality of times, the organic phase was washed with saturated saline for a plurality of times and dried over anhydrous sodium sulfate, and the inhibitor represented by the formula (I) was obtained by column chromatography.
Experimental results:
2- ((5-cinnamyl-6-hydroxy-4-oxo-1, 4-dihydropyrimidin-2-yl) thio) -N- (4-methoxyphenylethyl) acetamide as a pale yellow solid, 55% yield, IC 50 IC of positive control drug with value of 1.36+ -0.56 μm and melting point of 256.2-257.3 deg.C 50 The value was 1.12. Mu.M.
1 H NMR(500MHz,DMSO-d 6 )δ7.32–7.28(m,2H),7.23–7.17(m,5H),6.80(d,J＝7.5Hz,2H),6.47(dt,J＝15.1,1.1Hz,1H),6.32(s,1H),5.93(s,1H),5.82(s,1H),3.80(s,3H),3.76(s,2H),3.33(t,J＝5.1Hz,2H),2.75–2.71(m,2H),2.63(dd,J＝6.0,0.9Hz,2H). 13 C NMR(125MHz,DMSO-d 6 )δ171.17,165.45,162.83,159.49,157.83,137.10,131.63,130.34,129.77,128.90,128.56,126.97,126.51,113.78,88.20,55.35,40.53,35.37,34.26,30.03.
Example 2
2- ((5-cinnamyl-6-hydroxy-4-oxo-1, 4-dihydropyrimidin-2-yl) thio) -N- (4-hydroxyphenylethyl) acetamide, having the following structural formula, was prepared by a method similar to that of example 1.
Pale yellow solid, yield 41%, IC 50 The value is 1.65+/-0.23 mu M, and the melting point is 310.2-312.1 ℃.
1 H NMR(500MHz,DMSO-d 6 )δ7.30(dd,J＝6.8,3.1Hz,2H),7.20(qd,J＝4.0,1.5Hz,2H),7.04–6.99(m,2H),6.74(s,1H),6.71–6.65(m,1H),6.55(dt,J＝15.1,5.9Hz,2H),6.46(d,J＝15.2Hz,1H),5.80(s,1H),4.51(s,1H),3.76(s,2H),3.33(t,J＝5.4Hz,2H),2.73(ddd,J＝6.4,4.9,1.1Hz,2H),2.63(d,J＝5.9Hz,2H). 13 C NMR(125MHz,DMSO-d 6 )δ171.17,165.45,162.83,159.49,155.93,137.10,130.58,130.44,130.34,129.77,128.56,126.97,126.51,116.03,88.20,40.53,35.37,34.26,30.03.
Example 3
2- ((5-cinnamyl-6-hydroxy-4-oxo-1, 4-dihydropyrimidin-2-yl) thio) -N- (4-hydroxy-3, 5-dimethoxyphenethyl) acetamide has the following structural formula and is prepared by a method similar to that of example 1.
Pale yellow solid, yield 49%, IC 50 The value is 2.89+/-0.97 mu M, and the melting point is 235.2-236.5 ℃.
1 H NMR(500MHz,DMSO-d 6 )δ7.35–7.27(m,2H),7.20(dd,J＝5.0,1.8Hz,3H),6.57(dt,J＝15.0,1.1Hz,1H),6.44(d,J＝1.2Hz,2H),6.33(s,1H),6.24(d,J＝15.2Hz,1H),5.93(s,1H),5.26(s,1H),3.87(s,6H),3.76(s,2H),3.33(t,J＝7.6Hz,2H),2.78(tt,J＝7.7,1.1Hz,2H),2.63(dd,J＝6.2,1.1Hz,2H). 13 C NMR(125MHz,DMSO-d 6 )δ171.17,165.45,162.83,159.49,147.61,137.10,133.29,132.76,130.34,129.77,128.56,126.97,126.51,107.50,88.20,56.35,40.53,34.66,34.26,30.03.
Example 4
2- ((5-cinnamyl-6-hydroxy-4-oxo-1, 4-dihydropyrimidin-2-yl) thio) -N- (4-hydroxy-3-methoxyphenylethyl) acetamide, having the following structural formula, was prepared by a method similar to that of example 1.
Pale yellow solid, 39% yield, IC 50 The value is 1.87+ -0.49 μm, and the melting point is 240.8-241.2 ℃.
1 H NMR(500MHz,DMSO-d 6 )δ7.30(qd,J＝3.0,1.7Hz,2H),7.20(qd,J＝4.0,1.4Hz,3H),6.80–6.73(m,1H),6.67(ddt,J＝7.5,2.2,1.1Hz,1H),6.59(dt,J＝2.0,1.1Hz,1H),6.53–6.42(m,2H),5.71(s,1H),5.62(s,1H),3.95(s,3H),3.76(s,2H),3.33(t,J＝5.2Hz,2H),2.68–2.60(m,4H). 13 C NMR(125MHz,DMSO-d 6 )δ171.17,165.45,162.83,159.49,148.78,144.71,137.10,131.60,130.34,129.77,128.56,126.97,126.51,121.82,115.88,112.94,88.20,56.05,40.53,39.04,34.26,30.03.
Example 5
2- ((5-cinnamyl-6-hydroxy-4-oxo-1, 4-dihydropyrimidin-2-yl) thio) -N- (4-fluorophenethyl) acetamide has the following structural formula and is prepared by a method similar to that of example 1.
Pale yellow solid, 36% yield, IC 50 The value is 4.79 + -0.97 mu M, and the melting point is 236.4-237.2 ℃.
1 H NMR(500MHz,DMSO-d 6 )δ7.33–7.23(m,4H),7.21(td,J＝4.3,1.5Hz,3H),6.65(s,2H),6.44(d,J＝14.9Hz,1H),6.33(s,1H),5.68(s,1H),3.76(s,2H),3.33(t,J＝7.7Hz,2H),2.73(t,J＝7.6Hz,2H),2.63(dd,J＝6.0,0.9Hz,2H). 13 C NMR(125MHz,DMSO-d 6 )δ171.17,165.45,162.83,162.29,160.27,159.49,137.10,133.63,133.61,130.34,130.31,130.25,129.77,128.56,126.97,126.51,115.18,115.02,88.20,40.53,35.37,34.26,30.03.
Example 6
(E) -2- ((5- (3- (3-bromophenyl) allyl) -6-hydroxy-4-oxo-1, 4-dihydropyrimidin-2-yl) thio) -N- (3, 4-dimethoxyphenethyl) acetamide having the formula as follows and prepared by a method analogous to that of example 1.
Pale yellow solid, yield 48%, IC 50 The value is 2.24+/-0.24 mu M, and the melting point is 265.8-267.2 ℃.
1 H NMR(500MHz,DMSO-d 6 )δ7.53–7.48(m,1H),7.42(dt,J＝7.3,2.0Hz,1H),6.94(t,J＝7.5Hz,1H),6.87(dq,J＝7.9,1.9Hz,1H),6.83–6.75(m,2H),6.65(ddt,J＝7.6,2.1,1.1Hz,1H),6.47(s,1H),6.42(dt,J＝15.1,1.0Hz,1H),6.23(dt,J＝15.0,6.2Hz,1H),5.06(s,1H),3.89(d,J＝10.1Hz,6H),3.76(s,2H),3.33(t,J＝7.7Hz,2H),2.68–2.59(m,4H). 13 C NMR(125MHz,DMSO-d 6 )δ171.17,165.45,162.83,159.49,148.98,147.79,139.49,131.92,130.89,129.73,129.24,129.04,127.31,126.03,122.45,120.71,112.04,111.83,88.20,56.03,55.95,40.53,39.04,34.26,30.03.
Example 7
(E) -2- ((5- (3- (4-hydroxyphenyl) allyl) -6-hydroxy-4-oxo-1, 4-dihydropyrimidin-2-yl) thio) -N- (3, 4-dimethoxyphenethyl) acetamide having the formula as follows and prepared by a method analogous to that of example 1.
Pale yellow solid, 43% yield, IC 50 The value is 1.74+/-0.54 mu M, and the melting point is 320.4-322.4 ℃.
1 H NMR(500MHz,DMSO-d 6 )δ7.24–7.17(m,2H),6.80(d,J＝7.5Hz,1H),6.77(dt,J＝2.1,1.0Hz,1H),6.75–6.68(m,2H),6.65(ddt,J＝7.5,2.0,1.1Hz,1H),6.47–6.36(m,1H),6.15(dt,J＝15.2,6.2Hz,1H),5.85(s,1H),5.12(s,1H),3.89(d,J＝10.1Hz,6H),3.76(s,2H),3.33(t,J＝5.4Hz,2H),2.67–2.61(m,4H). 13 C NMR(125MHz,DMSO-d 6 )δ171.17,165.45,162.83,159.87,159.49,148.98,147.79,131.92,129.78,128.80,127.52,126.97,120.71,116.05,112.04,111.83,88.20,56.03,55.95,40.53,39.04,34.26,30.03.
The inhibitor has novel structure, and experiments show that the inhibitor has good neuraminidase inhibition activity and can be used for preparing medicines for inhibiting neuraminidase activity.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.
Claims (10)
1. A pyrimidine neuraminidase inhibitor, which is characterized by having a chemical structural formula shown in formula (I):
wherein R is 1 Selected from any one of the following structural formulas:
x=f, cl, br or I, +.>
R 2 Selected from any one of the following structural formulas:
x=f, cl, br or I.
2. The pyrimidine neuraminidase inhibitor according to claim 1 wherein R 1 Selected from any one of the following structural formulas:
R 2 selected from any one of the following structural formulas:
3. the pyrimidine neuraminidase inhibitor according to claim 1 wherein the inhibitor has one of the following chemical formulas:
4. a process for the preparation of a pyrimidine neuraminidase inhibitor according to any one of claims 1 to 3, which comprises the following steps:
(1) Forming a reaction system by using substituted benzaldehyde, acetaldehyde and an organic solvent, and performing post-treatment after the reaction to obtain an intermediate of a formula (II);
(2) Dissolving the intermediate of the formula (II) obtained in the step (1) in an organic solvent and zinc powder to form a reaction system, and reacting to obtain the intermediate of the formula (III);
(3) Dissolving the intermediate shown in the formula (III) obtained in the step (2) in an organic solvent, adding phosphorus tribromide to form a reaction system, and performing post-treatment after the reaction to obtain the intermediate shown in the formula (IV);
(4) Forming a reaction system from substituted phenethylamine, chloroacetyl chloride and triethylamine in an organic solvent, and performing post-treatment after the reaction to obtain an intermediate shown in a formula (V);
(5) Taking the intermediate of the formula (V) obtained in the step (4), reacting with 4, 6-dihydroxyl-2-mercaptopyrimidine under alkaline conditions, and performing aftertreatment to obtain an intermediate shown in the formula (VI);
(6) Taking the intermediate of the formula (VI) obtained in the step (5) and the intermediate of the formula (IV) obtained in the step (3) to form a reaction system under the weak alkaline condition, and performing post-treatment after the reaction to obtain the pyrimidine neuraminidase inhibitor shown in the formula (I);
the equation of the preparation method is as follows:
5. the process for producing a pyrimidine neuraminidase inhibitor according to claim 4, wherein in the step (1), the reaction is carried out at 25 to 60℃for 8 to 16 hours at an anhydrous K 2 CO 3 As the catalyst, pyridine is adopted as the organic solvent;
the substituted benzaldehyde, acetaldehyde and anhydrous K 2 CO 3 And the addition amount ratio of the organic solvent is (20-30) mmoL (40-100) mmoL (30-45) mmoL (20-60) mL.
6. The method for preparing a pyrimidine neuraminidase inhibitor according to claim 5, wherein in step (1), the post-treatment is carried out by: adding distilled water into a reaction system, regulating the pH value by using concentrated hydrochloric acid, adding ethyl acetate into the reaction solution for extraction and liquid separation, taking an organic phase, and performing reduced pressure distillation on the organic phase to obtain a crude product of the intermediate of the formula (II), and performing column chromatography to obtain a pure intermediate of the formula (II).
7. The method for preparing a pyrimidine neuraminidase inhibitor according to claim 4, wherein in the step (2), the reaction temperature is 0-40 ℃, the reaction time is 9.5-24 hours, zinc powder and an ammonium chloride system are adopted for reduction, and methanol is adopted as the organic solvent;
the addition amount ratio of the intermediate of the formula (II), zinc powder, ammonium chloride and organic solvent is (10-30) mmoL (10-30): (10-100) mmoL: (20-40) mL;
in the step (3), the reaction temperature is 0-30 ℃, the reaction time is 9-17.5h, and the organic solvent adopts diethyl ether, acetonitrile, toluene, tetrahydrofuran or dichloromethane;
the addition ratio of the intermediate of the formula (III), the phosphorus tribromide and the organic solvent is 5mmoL: (1-5) mmoL: (10-20) mL.
8. The method for preparing a pyrimidine neuraminidase inhibitor according to claim 4, wherein in the step (4), the reaction temperature is 0-35 ℃, the reaction time is 0.5-2 hours, and the organic solvent is acetone, ethyl acetate, tetrahydrofuran or dichloromethane;
the addition ratio of the substituted phenethylamine, chloroacetyl chloride, triethylamine and the organic solvent is 5mmoL: (5-5.5) mmoL: (5-5.5) mmoL: (20-50) mL.
9. The method for preparing a pyrimidine neuraminidase inhibitor according to claim 4, wherein in the step (5), the reaction temperature is 25-65 ℃, the reaction time is 8-12 hours, sodium hydroxide, potassium hydroxide, sodium ethoxide, sodium methoxide or potassium tert-butoxide is used as the base, and ethyl acetate, methanol, ethanol or distilled water is used as the solvent;
the addition amount ratio of the intermediate of the formula (V), 4, 6-dihydroxy-2-mercaptopyrimidine, alkali and solvent is (5-5.5) mmoL: (5-5.5) mmoL: (5-10) mmoL: (10-50) mL;
in the step (6), the reaction temperature is 25-65 ℃, the reaction time is 10-20h, triethylamine, diethylamine, sodium bicarbonate, cesium carbonate, potassium carbonate or sodium carbonate are adopted as the base, and ethyl acetate, methanol, ethanol or DMF is adopted as the solvent;
the addition ratio of the intermediate of the formula (VI), the intermediate of the formula (IV), the alkali and the organic solvent is (5-5.5) mmoL: (5-5.5) mmoL: (7.5-15) mmoL: (10-30) mL.
10. Use of a pyrimidine neuraminidase inhibitor according to any one of claims 1 to 3 for the preparation of a medicament for inhibiting neuraminidase activity.
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