CN110872263B

CN110872263B - Compound, preparation method and application

Info

Publication number: CN110872263B
Application number: CN201811022661.9A
Authority: CN
Inventors: 王振亚; 徐海伟; 牟晓东; 胡晓宁; 李媛媛; 刘宏民
Original assignee: Zhengzhou University
Current assignee: Zhengzhou University
Priority date: 2018-09-03
Filing date: 2018-09-03
Publication date: 2021-09-28
Anticipated expiration: 2038-09-03
Also published as: CN110872263A

Abstract

The application discloses a compound, the structural formula of which is shown as the following formula:

wherein R is¹Selected from F, Cl, Br, C₁～C₅Any one of an alkyl group of (1) and a group having a structural formula shown in formula (1); n is 0, 1, 2, 3,4 or 5; r²Is selected from any one of a group having a structural formula shown in a formula (1), a group having a structural formula shown in a formula (2) and a group having a structural formula shown in a formula (3). The compound has simple preparation method, can be used as a neuraminidase inhibitor and has good antiviral activity.

Description

Compound, preparation method and application

Technical Field

The application relates to a compound, a preparation method and application thereof, belonging to the field of chemistry.

Background

Influenza virus has become one of the most serious diseases threatening human life and health, causes hundreds of thousands of deaths each year, and causes enormous loss to social property each year. At present, although many drugs are on the market, the development of new anti-influenza virus drugs with high efficacy and low toxicity is urgent because influenza viruses are very likely to produce resistant virus strains.

Neuraminidase has attracted a great deal of medical attention as one of the targets for treating influenza virus. Neuraminidase is closely related to virus replication, and inhibition of neuraminidase can inhibit influenza virus replication to a certain extent. Therefore, designing and synthesizing a novel neuraminidase inhibitor is very important for preventing and treating influenza virus.

Disclosure of Invention

According to one aspect of the present application, there is provided a compound useful as a neuraminidase inhibitor.

The compound is characterized in that the structural formula is shown as a formula I:

wherein R is¹Selected from F, Cl, Br, C₁～C₅Any one of an alkyl group of (1) and a group having a structural formula shown in formula (1); n is 0, 1, 2, 3,4 or 5;

R²any one selected from the group having a structural formula shown in formula (1), the group having a structural formula shown in formula (2), and the group having a structural formula shown in formula (3);

*-O-M¹¹formula (1)

Wherein M is¹¹Is selected from H or C₁～C₅Alkyl groups of (a);

wherein M is²¹、M²²、M²³、M²⁴、M²⁵、M²⁶、M²⁷、M²⁸Independently selected from H or C₁～C₅Alkyl groups of (a);

wherein M is³¹Selected from H, F, Cl, Br, C₁～C₅Any one of an alkyl group of (1) and a group having a structural formula shown in formula (1); m³²、M³³、M³⁴Independently selected from H or C₁～C₅Alkyl group of (1).

Optionally, wherein R¹Any one selected from F, Cl, Br, methyl, ethyl and a group having a structural formula shown in formula (1); n is 0, 1, 2, 3,4 or 5.

Optionally, n ═ 0 in formula I.

Alternatively, R in formula I¹One selected from F, Cl, Br, methyl, ethyl, methoxy and ethoxy; n is 1 or 2.

Alternatively, R in formula I¹Selected from 4-F, 4-Cl, 4-Br, 4-CH₃、4-OCH₃3-F, 2-F, 3, 4-2F.

Optionally, M in formula (1)¹¹Is selected from C₁～C₄Alkyl group of (1).

Optionally, M in formula (1)¹¹Selected from methyl or ethyl.

Optionally, M in formula (2)²¹、M²²、M²³、M²⁴、M²⁵、M²⁶、M²⁷、M²⁸Are all hydrogen.

Optionally, M in formula (2)²¹、M²²、M²³、M²⁴、M²⁵、M²⁶、M²⁷、M²⁸Independently selected from C₁～C₄Alkyl group of (1).

Optionally, M in formula (2)²¹、M²²、M²³、M²⁴、M²⁵、M²⁶、M²⁷、M²⁸Independently selected from hydrogen, methyl or ethyl.

Optionally, M in formula (3)³¹Selected from H, F, Cl, Br, C₁～C₄Any one of the alkyl group of (1) and a group having a structural formula shown in formula (1).

Optionally, M in formula (3)³¹Selected from any one of H, F, Cl, Br, methyl, ethyl and a group with a structural formula shown in a formula (1).

Optionally, M in formula (3)³¹Selected from any one of H, F, Cl, Br, methyl, ethyl, methoxy and ethoxy.

Optionally, M in formula (3)³²、M³³、M³⁴Independently selected from H,Methyl or ethyl.

Alternatively, R¹Selected from F, Cl, Br, — CH₃、*-OCH₃At least one of (1).

Alternatively, R¹Selected from F, Cl, Br, — CH₃、*-OCH₃N is 2.

Alternatively, R¹Selected from F, Cl, Br, — CH₃、*-OCH₃Wherein n is 1.

Alternatively, R in formula I²Is selected from: -OCH₃、

Or a group having a structural formula represented by formula (3); wherein, M in formula (3)³²、M³³、M³⁴Are all H, M³¹Is selected from any one of H, F, Cl, Br, methoxyl and methyl.

Alternatively, R in formula I²is-OCH₃。

As a specific embodiment, the structural formula of the compound is shown in formula I-5:

alternatively, R¹Selected from 4-F, 4-Cl, 4-Br, 4-CH₃、4-OCH₃3-F, 2-F or 3,4-2F, and n-1 or n-0.

Alternatively, the structural formula is shown in formula I-1:

alternatively, the R is¹Selected from F, Cl, — CH₃、*-OCH₃And n ═ 1; or n ═ 0.

Alternatively, the R is¹Selected from 4-F, 4-Cl, 4-OCH₃、4-CH₃Or 3-F.

Alternatively, the structural formula is shown in formula I-2:

alternatively, the structural formula is shown in formula I-3:

optionally, the M³¹Selected from H, 4-F, 4-Cl, 4-Br, 4-OCH₃、4-CH₃3-F or 2-F.

Alternatively, the structural formula is shown in formula I-4:

alternatively, R in formula I-4¹Selected from Cl,. about. -OCH₃At least one of (1).

Alternatively, R in formula I-4¹Is selected from 4-Cl or 4-OCH₃。

In another aspect of the present application, there is provided a method for preparing a compound according to any one of the above, comprising:

(I) carrying out esterification reaction on a mixture I containing substituted phenylacetic acid and halogenated ethyl acetate in the presence of a catalyst to obtain an intermediate I, wherein the structural formula of the intermediate I is shown as a formula II-1:

(II) carrying out Dieckmann reaction on a raw material I containing an intermediate I to obtain an intermediate II, wherein the structural formula of the intermediate II is shown as a formula II-2:

optionally, the mixture I in the step (I) further comprises a solvent I and an acid-binding agent;

the molar ratio of the substituted phenylacetic acid to the halogenated ethyl acetate to the acid-binding agent is 1-2: 1-2: 1-2;

the esterification reaction conditions are as follows: and carrying out reflux reaction at the temperature of 60-80 ℃.

Optionally, the solvent I comprises at least one of tetrahydrofuran, dichloromethane, DMF, ethyl acetate, acetone, methanol, ethanol;

the acid-binding agent comprises at least one of triethylamine, pyridine, anhydrous potassium carbonate, sodium acetate and anhydrous sodium carbonate;

the molar ratio of the substituted phenylacetic acid to the halogenated ethyl acetate to the acid-binding agent is 1:1.2: 1.2;

the esterification reaction conditions are as follows: heated to reflux at 75 ℃ with stirring.

As a specific embodiment, the synthesis route of the intermediate I is as follows:

wherein, the reaction conditions and yield are as follows:

a：(Et)₃N，THF，75℃，98％。

wherein, the reaction conditions and yield are as follows:

a：(Et)₃N，THF，75℃，98％。

wherein, the reaction conditions and yield are as follows:

a：(Et)₃N，THF，75℃，98％。

optionally, the synthesis method of the intermediate I comprises: heating and refluxing a mixture containing substituted phenylacetic acid, halogenated ethyl acetate, a solvent I and an acid-binding agent under the condition of stirring; TCL monitors the extent of reaction; and after the reaction is finished, cooling, removing triethylamine salt, extracting, washing, drying and filtering to obtain a target product.

As a specific implementation mode, commercially available substituted phenylacetic acid and ethyl chloroacetate are used as raw materials, tetrahydrofuran is used as a solvent, triethylamine is used as an acid-binding agent, and the raw materials are fed in a ratio of n (substituted phenylacetic acid): n (ethyl chloroacetate): n (triethylamine) ═ 1:1.2:1.2, stirring, and heating at 75 ℃ under reflux. The reaction was monitored by TLC as a single point quantitative reaction, which was complete in about 6 hours. After the reaction is finished, cooling to room temperature, and filtering to remove triethylamine salt generated in the reaction. Concentrated under reduced pressure, extracted with ethyl acetate, washed three times with saturated brine, and dried over anhydrous sodium sulfate overnight. And (4) carrying out suction filtration and reduced pressure concentration to obtain dark red liquid, which is directly used for the next reaction without purification.

Optionally, the raw material I in the step (II) further comprises a solvent II and a base I;

the molar ratio of the intermediate I to the base I is 1: 1.5 to 3;

the Dieckmann reaction conditions are as follows: stirring in an ice bath for 20-50 min, and then continuing to react at room temperature.

Alternatively, the solvent II comprises DMF, THF, t-BuOH, methanol, acetone, CH₂Cl₂At least one of DMSO, EtOAc;

the base I comprises potassium tert-butoxide and anhydrous K₂CO₃Anhydrous Na₂CO₃、CH₃ONa、C₂H₅ONa、NaOH、NaHCO₃At least one of NaH;

the molar ratio of the intermediate I to the base I is 1: 2;

the Dieckmann reaction conditions are as follows: stirring in ice bath for 30min, and then continuing to react for 4-6 hours at room temperature.

Alternatively, the intermediate I has an active methylene structure and is synthesized by a Dieckmann reaction.

As a specific embodiment, the synthetic route of the intermediate II is as follows:

wherein, the reaction conditions and the yield are as follows:

b: t-BuOK, DMF, 0-room temperature, 80-90%.

wherein, the reaction conditions and the yield are as follows:

b: t-BuOK, DMF, 0-room temperature, 80-90%.

wherein, the reaction conditions and the yield are as follows:

b: t-BuOK, DMF, 0-room temperature, 80-90%.

Optionally, the preparation method of the intermediate II comprises: adding a base I into a mixture containing the intermediate I and the base I under ice bath conditions, stirring, reacting at room temperature, and stopping the reaction after the reaction is completely detected by thin layer chromatography.

Mixing a mixture containing an intermediate I, a base I and a solvent II

As a specific embodiment, the synthesis method of the intermediate II comprises: DMF (or t-BuOH and THF) is taken as a solvent, and the feeding ratio n (an intermediate I): n (potassium tert-butoxide) ═ 1:2, under the ice-bath condition, the potassium tert-butoxide in the reaction system is added in 6 times and a half hours. And (3) stirring and reacting for 30min under an ice bath condition, and then placing the system at room temperature for further reaction for 4-6 hours. The reaction is a single-point quantitative reaction, and the reaction is stopped after the thin-layer chromatography detection reaction is completed.

Alternatively, in the post-treatment, 1-fold amount of water was added to the reaction system to dilute the reaction system. And (3) dropwise adding 5% dilute hydrochloric acid into the system under the ice-bath stirring condition to adjust the pH to about 2-3, continuously separating out solids in the pH adjusting process, and adding a large amount of water into the system until no solids are separated out when the pH is adjusted. And (5) placing the mixture in a refrigerator for cooling for about 2 hours, carrying out suction filtration, and recrystallizing dichloromethane to obtain an intermediate 3. However, this method of work-up is not suitable for the substrates used, and some products do not precipitate as solids during the pH adjustment. If no solid is formed, saturated NaCl extraction and anhydrous Na₂SO₄Drying overnight and purifying by column chromatography.

Optionally, the method of preparing the compound further comprises:

(III-1) carrying out methylation reaction on a mixture II containing an intermediate II and dimethyl sulfate to obtain an intermediate III-1, wherein the structural formula of the intermediate III-1 is shown as a formula II-3-1:

(IV-1) reacting a mixture III containing the intermediate III-1, sodium methoxide and paraformaldehyde to obtain a compound shown in the formula I; wherein R in the formula I²is-OCH₃. Namely the compound shown as the formula I-5.

Optionally, the mixture II in the step (III-1) further comprises a solvent III and a base II;

the molar ratio of the intermediate II to the alkali II to the dimethyl sulfate is 1: 1-2: 1-2;

the methylation reaction conditions are as follows: the reaction was carried out at room temperature under protection from light.

Optionally, the solvent III comprises acetone, methanol, CH₂Cl₂At least one of THF, DMF, EtOAc;

the base II comprises anhydrous K₂CO₃Anhydrous Na₂CO₃、CH₃ONa、NaOH、NaHCO₃At least one of NaH;

the molar ratio of the intermediate II to the alkali II to the dimethyl sulfate is 1:1.2: 1.2;

the methylation reaction conditions are as follows: and reacting for 8-10 hours at room temperature under the condition of keeping out of the light.

As a specific embodiment, the synthetic route of the intermediate III-1 is as follows:

wherein, the reaction conditions and the yield are as follows:

c1：Me₂SO₄，K₂CO₃and 70-80% of acetone.

wherein, the reaction conditions and the yield are as follows:

c1：Me₂SO₄，K₂CO₃and 70-80% of acetone.

wherein, the reaction conditions and the yield are as follows:

c1：Me₂SO₄，K₂CO₃and 70-80% of acetone.

Alternatively, the preparation method of the intermediate III-1 comprises the following steps: under the condition of keeping out of the sun, dripping dimethyl sulfate into a mixed solution containing an intermediate II, alkali II and a solvent III, and reacting at room temperature; completion of the reaction was checked by thin layer chromatography. And concentrating, extracting, washing, drying, performing suction filtration and rotary evaporation, and recrystallizing the product obtained after the reaction to obtain an intermediate III-1.

As a specific embodiment, the preparation method of the intermediate III-1 comprises the following steps: using acetone as solvent, andwater K₂CO₃The reaction needs to slowly dropwise add an acetone solution of dimethyl sulfate by using a constant-pressure dropping funnel under the condition of keeping out of the sun because dimethyl sulfate is easy to decompose under the action of light, and the material ratio n (an intermediate II): n (anhydrous K)₂CO₃): n (dimethyl sulfate) ═ 1:1.2: 1.2. Reacting at room temperature in dark for about 9h, detecting by thin layer chromatography, concentrating under reduced pressure to remove acetone, extracting with ethyl acetate, washing with saturated NaCl, and adding anhydrous Na₂SO₄Dry overnight. And (3) carrying out suction filtration and rotary evaporation to obtain a solid, wherein the solid is ethyl acetate: recrystallizing with petroleum ether at a ratio of 1:1 to obtain the intermediate compound III-1.

Optionally, the mixture III in the step (IV-1) further comprises a solvent IV;

the molar ratio of the intermediate III-1 to the sodium methoxide to the paraformaldehyde is 1: 0.1-0.5;

the reaction conditions are as follows: reflux reaction at 60-100 ℃.

Optionally, the reaction conditions are 60-80 ℃.

Optionally, the solvent IV comprises methanol, acetone, CH₂Cl₂At least one of THF, DMF, EtOAc;

the reaction conditions are as follows: and (3) feeding the intermediate III-1, sodium methoxide and paraformaldehyde according to the molar ratio of 1:0.6:0.3, refluxing and stirring at 75 ℃, adding paraformaldehyde and sodium methoxide at intervals, and reacting for 70-75 hours.

Optionally, during the reaction: 0.3 mol of paraformaldehyde and sodium methoxide are added at intervals of 8 hours, and the reaction is carried out for 72 hours.

Alternatively, the synthetic route of step (IV-1) is as follows:

wherein, the reaction conditions and yield are as follows:

d：(CH₂O)n，CH₃ONa，75℃，45～50％。

alternatively, the synthetic route of step (IV-1) is as follows:

wherein, the reaction conditions and yield are as follows:

d：(CH₂O)n，CH₃ONa，75℃，45～50％。

alternatively, the synthetic route of step (IV-1) is as follows:

wherein, the reaction conditions and yield are as follows:

d：(CH₂O)n，CH₃ONa，75℃，45～50％。

optionally, the step (IV-1) comprises: stirring a mixture containing a solvent IV, sodium methoxide and an intermediate III-1 for reflux reaction, and adding paraformaldehyde and sodium methoxide at intervals; and (3) after TLC monitoring that the reactant completely disappears, removing methanol, extracting, washing, drying, carrying out suction filtration, concentrating and purifying to obtain the target product.

As a specific embodiment, the step (IV-1) includes: taking methanol as a solvent and sodium methoxide as a base, and feeding the mixture according to a feeding ratio n (an intermediate III-1): n (sodium methoxide): n (paraformaldehyde) ═ 1:0.6:0.3, and the mixture was stirred under reflux at 75 ℃. 0.3N paraformaldehyde and sodium methoxide are added every 8 hours for reaction for about 48 hours, after TLC monitors that the reactants completely disappear, the methanol is removed by decompression and concentration, ethyl acetate extraction is carried out, the mixture is washed by saturated saline solution, and dried by anhydrous sodium sulfate overnight. Filtering, concentrating, and performing column chromatography to obtain the target compound.

Optionally, the gamma position of butenolide in the intermediate III-1 can attack carbonyl positive ions in paraformaldehyde to perform a condensation reaction, so as to synthesize a target compound.

As a specific embodiment, the synthetic route of the compound represented by formula I-5 is as follows:

wherein, the reaction and yield of each step are as follows: a: (Et)₃N, THF, 75 ℃, 98%; b: t-BuOK, DMF, 0-room temperature, 80-90%; c 1: me₂SO₄，K₂CO₃70-80% of acetone; d: (CH)₂O)n，CH₃ONa，75℃，45～50％。

Alternatively, R in the synthetic route of the compound represented by the above formula I-5¹And H is substituted.

Optionally, the method of preparing the compound further comprises:

(III-2) carrying out chloro nucleophilic reaction on the raw material II containing the intermediate II to obtain an intermediate III-2; the structural formula of the intermediate III-2 is shown as a formula II-3-2:

(IV-2) carrying out nucleophilic substitution on the mixture IV containing the intermediate III-2 and morpholine under alkaline conditions to obtain an intermediate IV-1; the structural formula of the intermediate IV-1 is shown as a formula II-4-1:

(V-2) reacting the mixture V containing the intermediate IV-1, sodium methoxide and paraformaldehyde to obtain the compound shown in the formula I-1.

Optionally, the raw material II in step (III-2) further includes: solvent V and nucleophile I;

the molar ratio of the intermediate III-2 to the nucleophilic reagent I is 1: 1-4;

the conditions of the chloro nucleophilic reaction are as follows: stirring in ice bath for 20-50 min, and then reacting at room temperature.

Alternatively, the solvent V is selected from 1:1 of DMF and CH₂Cl₂At least one of methanol, acetone, THF, DMF, EtOAc;

the nucleophilic reagent I is selected from oxalyl chloride and POCl₃At least one of thionyl chloride;

the molar ratio of the intermediate III-2 to the nucleophile I is 1: 2;

the conditions of the chloro nucleophilic reaction are as follows: the mixture was stirred in an ice bath for 30min, and then reacted at room temperature for 6 hours.

Alternatively, the chloro nucleophilic reaction may be obtained by other means, such as: in POCl₃With NaOH and H under the conditions of₂Taking O mixed solvent as reaction solvent to perform chlorination and Me₃SiCl as chloro reagent, DMSO as catalyst, POCl₃As chlorinating reagent in the reaction, POCl is used in the reaction₃Also as a reaction solvent, diisopropylamine is used as a base, and acid is generated in the neutralization reaction.

Alternatively, the synthetic route of the intermediate III-2 is as follows:

wherein, the reaction conditions and yield are as follows:

c 2: oxalyl chloride, DMF CH₂Cl₂(volume ratio 1:1), 95%.

Alternatively, the synthetic route of the intermediate III-2 is as follows:

wherein, the reaction conditions and yield are as follows:

c 2: oxalyl chloride, DMF CH₂Cl₂(volume ratio 1:1), 95%.

Alternatively, the preparation method of the intermediate III-2 comprises the following steps: and under the ice bath condition, adding the nucleophilic reagent I into a mixture containing the intermediate II and the solvent V, stirring, reacting at room temperature, completely detecting by TLC, extracting, washing, drying, performing suction filtration, and purifying to obtain an intermediate III-2.

As a specific embodiment, DMF/CH is used₂Cl₂(1:1) is used as a solvent, oxalyl chloride is slowly dropped under the ice bath condition, and the feeding ratio n (intermediate II): n (oxalyl chloride) ═ 1: 2. After stirring in an ice bath for about half an hour, the reaction was continued for about 6h with the system brought to room temperature. After TLC detection of reaction completion, saturated NaHCO was used₃Adjusting the pH of the system to be neutral, and then using CH₂Cl₂Extracting for three times, combining organic phases, washing with 15ml of saturated saline solution, drying over night through anhydrous sodium sulfate, filtering, and carrying out column chromatography to obtain a pure intermediate compound III-2.

Optionally, the mixture IV in the step (IV-2) further comprises a solvent VI and a base III;

the molar ratio of the intermediate III-2 to the base III to the morpholine is 1: 1-4: 1-4;

the reaction time of the nucleophilic substitution is 3-6 hours.

Alternatively, the solvent VI comprises THF, methanol, acetone, CH₂Cl₂At least one of DMF and EtOAc;

the base III comprises: anhydrous K₂CO₃Anhydrous Na₂CO₃、CH₃ONa、NaOH、NaHCO₃At least one of NaH;

the molar ratio of the intermediate III-2, the base III and the morpholine is 1:2: 2;

the reaction time for the nucleophilic substitution was 4 h.

Alternatively, the synthetic route of the intermediate IV-1 is as follows:

wherein, the reaction conditions are as follows:

d 1: morpholine, K₂CO₃，THF。

Alternatively, the synthetic route of the intermediate IV-1 is as follows:

wherein, the reaction conditions are as follows:

d 1: morpholine, K₂CO₃，THF。

Alternatively, the preparation method of the intermediate IV-1 comprises the following steps: adding alkali III into a solution containing an intermediate III-2 and a solvent VI, then dropwise adding morpholine, stirring and reacting; and (3) after the TLC spot plate detection reaction is completed, carrying out suction filtration/concentration, extraction, washing, drying and purification to obtain the product.

As a specific embodiment, the preparation method of the intermediate IV-1 comprises the following steps: THF as solvent, anhydrous K₂CO₃Adding intermediate compound III-2 into 2ml THF solution as alkali to obtain a completely soluble solution, and adding anhydrous K into the system₂CO₃Slowly dropwise adding morpholine under the stirring condition, wherein the feeding ratio n (an intermediate III-2): n (anhydrous K)₂CO₃): (morpholine) ═ 1:2: 2. During the dropping process, the system gradually turns into yellow, and a yellow solid is continuously separated out during the stirring process, and the reaction lasts for about 4 hours. And after TLC plate counting detection reaction is completed, carrying out suction filtration to obtain an intermediate compound 8, and if a solid cannot be separated out, concentrating, extracting with ethyl acetate, washing with saturated saline solution, drying with anhydrous sodium sulfate, and carrying out column chromatography purification to obtain the compound.

Optionally, the mixture V in the step (V-2) further comprises a solvent VII;

the molar ratio of the intermediate IV-1 to the sodium methoxide to the paraformaldehyde is 1: 0.1-1: 0.1 to 0.5;

the reaction conditions are as follows: reflux reaction at 60-100 ℃.

Alternatively, the solvent VII comprises methanol, THF, acetone, CH₂Cl₂At least one of DMF and EtOAc;

the reaction conditions are as follows: feeding the intermediate IV-1, sodium methoxide and paraformaldehyde according to the molar ratio of 1:0.6:0.3, refluxing and stirring at 75 ℃, adding paraformaldehyde and sodium methoxide at intervals, and reacting for 70-75 hours.

Alternatively, the synthetic route of the step (V-2) is as follows:

wherein, the reaction conditions and yield are as follows:

e1：(CH₂O)n，CH₃ONa，75℃，30～40％。

alternatively, the synthetic route of the step (V-2) is as follows:

wherein, the reaction conditions and yield are as follows:

e1：(CH₂O)n，CH₃ONa，75℃，30～40％。

alternatively, the preparation method of step (V-2) comprises: stirring a mixture containing a solvent VII, sodium methoxide and an intermediate IV-1 for reflux reaction, and adding paraformaldehyde and sodium methoxide at intervals; and (3) after TLC monitoring that the reactant completely disappears, removing methanol, extracting, washing, drying, carrying out suction filtration, concentrating and purifying to obtain the target product.

As a specific embodiment, the step (V-2) specifically includes: taking methanol as a solvent and sodium methoxide as a base, and feeding the mixture according to a feeding ratio n (an intermediate IV-1): n (sodium methoxide): n (paraformaldehyde) ═ 1:0.6:0.3, and the mixture was stirred under reflux at 75 ℃. 0.3N paraformaldehyde and sodium methoxide are added every 8 hours for reaction for about 72 hours, after the reaction is monitored by a thin layer, the mixture is concentrated under reduced pressure to remove methanol, extracted by ethyl acetate (3X 15ml), washed by saturated saline solution and dried over night by anhydrous sodium sulfate. Filtering, concentrating, and performing column chromatography to obtain the target compound.

As a specific embodiment, the synthetic route of the compound represented by formula I-1 is as follows:

wherein, the reaction conditions and the yield are as follows:

a：(Et)₃n, THF, 75 ℃, 98%; b: 80-90% of t-BuOK, DMF, 0-room temperature; c 2: oxalyl chloride, DMF CH₂Cl₂(volume ratio 1:1), 95%; d 1: morpholine, K₂CO₃，THF；e1：(CH₂O)n，CH₃ONa，75℃，30～40％。

Alternatively, R in the synthetic route of the compound represented by the above formula I-1¹And H is substituted.

Optionally, the method of preparing the compound further comprises:

(IV-3) carrying out nucleophilic substitution on the mixture VI containing the intermediate III-2 and benzylamine or substituted benzylamine under alkaline conditions to obtain an intermediate IV-2; the structural formula of the intermediate IV-2 is shown as a formula II-4-2:

(V-3) reacting a mixture VII containing the intermediate IV-2, sodium methoxide and paraformaldehyde to obtain the compound shown in the formula I-2.

Optionally, the mixture VI in step (IV-3) further comprises a solvent VIII and a base IV;

the molar ratio of the intermediate III-2 to the base IV to the benzylamine or the substituted benzylamine is 1: 1-4: 1-4;

the reaction time of the nucleophilic substitution is 3-6 hours.

Alternatively, the solvent VIII comprises THF, methanol, acetone, CH₂Cl₂At least one of DMF and EtOAc;

the base IV comprises: anhydrous K₂CO₃Anhydrous Na₂CO₃、CH₃ONa、NaOH、NaHCO₃At least one of NaH, NaH;

the molar ratio of the intermediate III-2 to the base IV to the benzylamine or the substituted benzylamine is 1:2: 2;

the reaction time for the nucleophilic substitution was 4 h.

Alternatively, the synthetic route of the intermediate IV-2 is as follows:

wherein, the reaction conditions are as follows:

d 3: benzylamine or substituted benzylamine (e.g. 4-methylbenzylamine or benzylamine), K₂CO₃，THF。

Alternatively, the preparation method of the intermediate IV-2 comprises the following steps: and (2) dropwise adding benzylamine or substituted benzylamine into a mixture containing the intermediate III-2, the solvent VIII and the alkali IV, stirring at room temperature for reaction, performing suction filtration and washing after TLC detection reaction is complete, thus obtaining the compound.

As a specific embodiment, the preparation method of the intermediate IV-2 comprises the following steps: THF is added into an intermediate III-2 container, and anhydrous K is added under stirring₂CO₃Slowly adding benzylamine or substituted benzylamine (such as 4-methylbenzylamine) dropwise, continuously generating yellow solid in the dropwise adding process, stirring at room temperature for 3h after dropwise adding is finished, performing suction filtration after detection reaction is completed, and washing with saturated salt solution to obtain the product.

Optionally, the mixture VII in step (V-3) further comprises a solvent IX;

the molar ratio of the intermediate IV-2 to the sodium methoxide to the paraformaldehyde is 1: 0.1-1: 0.1 to 0.5;

the reaction conditions are as follows: reflux reaction at 60-100 ℃.

Optionally, the solvent IX comprises methanol, acetone, CH₂Cl₂At least one of THF, DMF, EtOAc;

the reaction conditions are as follows: and (3) feeding the intermediate IV-2, sodium methoxide and paraformaldehyde according to the molar ratio of 1:0.6:0.3, refluxing and stirring at 75 ℃, adding paraformaldehyde and sodium methoxide at intervals, and reacting for 70-75 hours.

Optionally, the reaction conditions further comprise: 0.3 mol of paraformaldehyde and sodium methoxide are added at intervals of 8 hours, and the reaction is carried out for 72 hours.

Alternatively, the synthetic route of the step (V-3) is as follows:

wherein, the reaction conditions and yield are as follows:

e2：(CH₂O)n，CH₃ONa，75℃，30～40％。

alternatively, the preparation method of step (V-2) comprises: stirring a mixture containing a solvent IX, sodium methoxide and an intermediate IV-2 for reflux reaction, and adding paraformaldehyde and sodium methoxide at intervals; and (3) after TLC monitoring that the reactant completely disappears, removing methanol, extracting, washing, drying, carrying out suction filtration, concentrating and purifying to obtain the target product.

As a specific embodiment, the step (V-3) specifically includes: taking methanol as a solvent and sodium methoxide as a base, and feeding the mixture according to a feeding ratio n (an intermediate IV-2): n (sodium methoxide): n (paraformaldehyde) ═ 1:0.6:0.3, and the mixture was stirred under reflux at 75 ℃. 0.3N paraformaldehyde and sodium methoxide are added every 8 hours for reaction for about 72 hours, after the reaction is monitored by a thin layer, the mixture is concentrated under reduced pressure to remove methanol, extracted by ethyl acetate (3X 15ml), washed by saturated saline solution and dried over night by anhydrous sodium sulfate. Filtering, concentrating, and performing column chromatography to obtain the target compound.

Alternatively, the synthetic route for the compound of formula I-2 is as follows:

wherein, the reaction conditions and yield are as follows:

a：(Et)₃n, THF, 75 ℃, 98%; b: 80-90% of t-BuOK, DMF, 0-room temperature; c 2: oxalyl chloride, DMF CH₂Cl₂(volume ratio is 1:1), 95%; d 2: benzylamine or substituted benzylamine, K₂CO₃，THF；e2：(CH₂O)n，CH₃ONa，75℃，30-40％。

Alternatively, R in the synthetic route of the compound represented by the formula I-2 described above¹And H is substituted.

wherein, the reaction conditions and yield are as follows:

d 2': benzylamine, K₂CO₃，THF；e：(CH₂O)n，CH₃ONa，75℃，30-40％。

d 2': 4-methylbenzylamine, K₂CO₃，THF；e：(CH₂O)n，CH₃ONa，75℃，42.6％。

d2' ″: 4-methylbenzylamine, K₂CO₃，THF；e：(CH₂O)n，CH₃ONa，75℃，44.3％。

In yet another aspect of the present application, there is provided a neuraminidase inhibitor characterized by comprising at least one compound of any one of the compounds described above, or a compound prepared according to any one of the methods described above.

Optionally, the neuraminidase inhibitor is at least one of the compound described in any of the above, or a compound prepared according to the method described in any of the above.

In-vivo animal experiments of the neuraminidase inhibitor also prove that the compound has a better treatment effect and has significance for further research.

In the present application, the alkyl group is a group formed by an alkane compound having any one hydrogen atom removed. The alkane compound comprises straight-chain alkane, branched-chain alkane and cycloalkane.

In this application, C₁～C₅Represents the number of carbon atoms comprising the group.

In the present application, "4-F", "3-F", etc. are substitution positions of the respective groups in the compounds described in the present application; wherein, the number of the substitution position is the relative position of the substituent on the benzene ring, and the specific structure is as follows:

for example, "3-F" means that a hydrogen atom above carbon 3 of the benzene ring of the compound is replaced by FAnd (4) generation. The 3,4-2F is that the hydrogen atoms on the carbon 3 and the carbon 4 of the benzene ring of the compound are replaced by F.

In the present application, "CC₅₀"refers to the ability of a drug to inhibit viral replication, and is the concentration of drug that inhibits 50% of viral replication.

In the present application, "EC₅₀By "is meant the half-effect concentration of a dose of drug that causes 50% of the subjects to produce a particular effect.

The beneficial effects that this application can produce include:

1) the compound provided by the application takes butenolide as a mother nucleus and can be used as a neuraminidase inhibitor; the compound has no cytotoxicity, has a higher safety index, and has the same inhibition rate on neuraminidase as oseltamivir.

2) EC of Compounds provided herein₅₀Can reach 6.68 +/-1.32 mu M and is superior to a positive medicament ribavirin (EC)₅₀105 ± 51 μ M) and also has a higher safety index SI of 85.6.

3) The preparation method of the compound provided by the application is simple, and raw materials are easy to obtain.

Drawings

FIG. 1 is a drawing of compound Ia in one embodiment of the present application¹HNMR spectrogram;

FIG. 2 is a drawing of compound Ia in one embodiment of the present application¹³CNMR spectrogram;

FIG. 3 shows the preparation of compound Ib according to an embodiment of the present application¹H NMR spectrum;

FIG. 4 shows an embodiment of the present application of compound Ib¹³C NMR spectrum;

FIG. 5 is a scheme showing the preparation of compound Ic in one embodiment of the present application¹H NMR spectrum;

FIG. 6 shows the preparation of compound Ic in one embodiment of the present application¹³C NMR spectrum;

FIG. 7 shows one embodiment of the present application for compound Id¹H NMR spectrum;

FIG. 8 shows one embodiment of the present application for compound Id¹³C NMR spectrum;

FIG. 9 is a drawing of compound Ie in one embodiment of the present application¹H NMR spectrum;

FIG. 10 is a drawing of compound Ie in one embodiment of the present application¹³C NMR spectrum;

FIG. 11 is a 1H NMR spectrum of compound If according to one embodiment of the disclosure;

FIG. 12 is a drawing of compound If in one embodiment of the present application¹³C NMR spectrum;

FIG. 13 shows the preparation of compound Ig in one embodiment of the present application¹H NMR spectrum;

FIG. 14 shows the preparation of compound Ig in one embodiment of the present application¹³C NMR spectrum;

FIG. 15 is a drawing of compound Ih in one embodiment of the present application¹H NMR spectrum;

FIG. 16 is a drawing of compound Ih in one embodiment of the present application¹³C NMR spectrum;

FIG. 17 is a representation of compound Ii in one embodiment of the present application¹H NMR spectrum;

FIG. 18 shows one embodiment of the present application of Compound Ii¹³C NMR spectrum;

FIG. 19 is a drawing of compound II-1 in one embodiment of the present application¹H NMR spectrum;

FIG. 20 is a drawing of compound II-1 in one embodiment of the present application¹³C NMR spectrum;

FIG. 21 is a drawing of compound II-2 in one embodiment of the present application¹H NMR spectrum;

FIG. 22 is a drawing of compound II-2 in one embodiment of the present application¹³C NMR spectrum;

FIG. 23 is a drawing of compound II-3 in one embodiment of the present application¹H NMR spectrum;

FIG. 24 is a drawing of compound II-3 in one embodiment of the present application¹³C NMR spectrum;

FIG. 25 is a drawing of compound II-4 in one embodiment of the present application¹H NMR spectrum;

FIG. 26 is a drawing of compound II-4 in one embodiment of the present application¹³C NMR spectrum;

FIG. 27 is a drawing of compound II-5 in one embodiment of the present application¹H NMR spectrum;

FIG. 28 is a drawing of compound II-5 in one embodiment of the present application¹³C NMR spectrum;

FIG. 29 is a drawing of compound II-6 in one embodiment of the present application¹H NMR spectrum;

FIG. 30 is a drawing of compound II-7 in one embodiment of the present application¹HNMR spectrogram;

FIG. 31 is a drawing of compound II-8 in one embodiment of the present application¹H NMR spectrum;

FIG. 32 is a drawing of compound II-9 in one embodiment of the present application¹H NMR spectrum;

FIG. 33 is a drawing of compound II-10 in one embodiment of the present application¹H NMR spectrum;

FIG. 34 is a drawing of compound II-11 in one embodiment of the present application¹H NMR spectrum;

FIG. 35 is a drawing of compound II-12 in one embodiment of the present application¹H NMR spectrum;

FIG. 36 is a drawing of compound II-13 in one embodiment of the present application¹H NMR spectrum;

FIG. 37 is a drawing of compound II-14 in one embodiment of the present application¹H NMR spectrum;

FIG. 38 is a drawing of compound II-15 according to one embodiment of the present application¹H NMR spectrum;

FIG. 39 is the EC for α -aryl- β -methoxy- γ -methoxy compounds (Ia, Ib, Id, If, Ig) in this application₅₀；

FIG. 40: a is EC of compound II-1-4₅₀(ii) a B is the virus inhibition rate of the compound II-3 which changes along with the concentration dependence;

FIG. 41 is a graph showing the inhibition rate of neuraminidase by Compound II-3;

FIGS. 42 and 43 show the results of molecular docking of Compound II-3 (Gray: oseltamivir, Violet: II-3), in which FIG. 42 shows the results of molecular docking of Compound II-3 (Gray: oseltamivir, Violet: II-3) and FIG. 43 shows the results of molecular docking of Compound II-3 (Violet: II-3).

FIG. 44 shows the results of in vivo experiments with II-3; wherein, the body temperature of the mice of the drug group and the control group is changed, and the body weight of the mice of the drug group and the control group is changed.

Detailed Description

The present application will be described in detail with reference to examples, but the present application is not limited to these examples.

Unless otherwise specified, the raw materials and catalysts in the examples of the present application were all purchased commercially.

The analysis method in the examples of the present application is as follows:

melting point analysis was performed using an X5 model microscopic digital melting point apparatus (Beijing Cork instruments electro-optical apparatus).

NMR analysis was carried out using a model DPX-400 superconducting NMR spectrometer (Bruker, Sweden).

In the examples said 0.3N represents 0.3 mol.

EXAMPLE 1 Synthesis of intermediates Ia' -i

2.00g (13.3mmol) of p-methylphenylacetic acid was weighed into a 50ml round-bottomed flask, and 10ml of Tetrahydrofuran (THF) was added to completely dissolve the p-methylphenylacetic acid, after which 1.956g (16mmol) of ethyl chloroacetate and 1.624g (16mmol) of triethylamine were added, and the mixture was heated under reflux at 60 ℃ for 6.5 hours. The reaction was monitored by TLC plate to completion and cooled to room temperature. And (3) carrying out suction filtration to remove triethylamine salt generated in the reaction, washing a filter cake with THF for three times, and carrying out reduced pressure concentration to obtain a light yellow transparent liquid. (3X 15) extraction with ethyl acetate, washing with saturated brine, drying over night with anhydrous sodium sulfate, suction filtration and concentration to give intermediate Ie'.

The intermediate Ie' in this example has the formula

Molecular formula C₁₃H₁₆O₄。

The p-methylphenylacetic acid was replaced with phenylacetic acid in the above process, and the remaining operations and conditions were the same as those of the preparation of Ie ', to give intermediate Ia'.

The p-methylphenylacetic acid in the method is replaced by p-fluorophenylacetic acid, and the rest of operations and conditions are the same as those of the preparation method of Ie, so that an intermediate Ib' is obtained.

The p-methylphenylacetic acid in the method is replaced by p-chlorophenylacetic acid, and the rest of operations and conditions are the same as those of the preparation method of Ie, so that the intermediate Ic' is obtained.

The p-methylphenylacetic acid in the method is replaced by p-bromophenylacetic acid, and the rest of the operation and conditions are the same as those of the preparation method of Ie, so as to obtain an intermediate Id'.

The p-methylphenylacetic acid in the method is replaced by p-methoxyphenylacetic acid, and the rest of operations and conditions are the same as those of the preparation method of Ie, so as to obtain an intermediate If'.

The p-methylphenylacetic acid in the method is replaced by 3-fluorophenylacetic acid, and the rest of operations and conditions are the same as those of the preparation method of Ie, so that an intermediate Ig' is obtained.

The p-methylphenylacetic acid in the method is replaced by 2-fluorophenylacetic acid, and the rest of operations and conditions are the same as those of the preparation method of Ie, so that an intermediate Ih' is obtained.

The above process was substituted for p-methylphenylacetic acid with 3, 4-difluorophenylacetic acid and the remaining operations and conditions were the same as those for the preparation of Ie, yielding intermediate Ii'.

EXAMPLE 2 Synthesis of intermediates IIa-i

Taking IIe as an example, 3.50g (18.3mmol) of intermediate I (Ia '-I') prepared in example 1 is weighed into a 50ml round-bottom flask, 15ml of N, N-Dimethylformamide (DMF) is added for dissolution, stirring is carried out under ice bath conditions, and 3.42g (36.6mmol) of potassium tert-butoxide is added in total in 6 portions in 30 min. The reaction was stirred for 30min under ice-bath conditions and then at room temperature for 3 h. TLC monitors the reaction to be complete, 20ml of water is added to dilute the reaction system, then the pH is adjusted to 2-3 by using 5 wt% of diluted HCl under the ice bath condition, and in the process of adjusting the pH, light yellow solid is continuously separated out from the system. Water was then added slowly to the system until no significant solids precipitated (100ml of water). The mixture was placed in a refrigerator overnight, filtered, and the filter cake was washed three times with water (3 × 10ml) to give a pale yellow solid, which was dried to give the crude compound. The crude compound was recrystallized by adding 20ml of dichloromethane and stirred for 15 min. And (5) carrying out suction filtration, and washing a filter cake by using dichloromethane to obtain a pure intermediate II.

Intermediates Ia '-i' obtained in the examples were each prepared as described above in the examples to give intermediates IIa-i.

Wherein the intermediate IIe has the structural formula

Molecular formula C₁₁H₁₀O₃。

Of the above intermediate IIe¹H NMR spectrum data: the doublet at δ 7.37-7.47(d, J ═ 8.2Hz,2H) is the two hydrogens at the 6 and 10 positions of the phenyl ring; the doublet at δ 7.18-7.26(d, J ═ 7.9Hz,2H) is two hydrogens at the 7 and 9 positions of the phenyl ring; δ 5.01-4.85(q, J ═ 6.7Hz,2H) is hydrogen on the carbon at position 3; δ 1.79(s) is hydrogen on the 11-carbon of the phenyl ring;¹³c NMR spectrum data: δ 184.7(s) is the peak at carbon 4; δ 168.5(s) is the peak at carbon 4; δ 137.4(s) is the peak at carbon 8; δ 134.5(s) is the peak at the carbon at the 6-and 10-positions of the phenyl ring; δ 131.5(s) is the peak at carbon 5; δ 128.2(s) is the peak at the carbon at the 7-and 9-positions of the phenyl ring; δ 102.8(s) is the peak at carbon 1; δ 68.4(s) is the peak at carbon 4; δ 22.6(s) is the peak at carbon position 11.

EXAMPLE 3 Synthesis of intermediate III-1a-i

In the case of III-1e, 1.50g (7.88mmol) of intermediate II (obtained in example 2) was weighed into a 50ml round-bottom flask, 15ml of acetone was added thereto and dissolved completely, and 1.525g (11.1mmol) of anhydrous potassium carbonate was added under stirring. 1.193g (9.5mmol) of dimethyl sulfate was dissolved in 10ml of acetone, and the solution was slowly dropped into the reaction system through a dropping funnel having a constant pressure for 30 min. The reaction is carried out for 10h at room temperature under the condition of keeping out of light. TLC monitors the reaction to be complete, decompression concentration is carried out to remove the reaction solvent, 25ml of water is added, anhydrous potassium carbonate is dissolved, meanwhile, a large amount of light yellow solid is separated out, suction filtration is carried out, and a filter cake is washed three times (3X 10ml) by water to obtain the light yellow solid. Recrystallizing petroleum ether and ethyl acetate (1:1) to obtain the pure intermediate III-1.

Intermediate III-1, corresponding to intermediate III-1a-i obtained, was prepared by the procedure described above in this example for intermediate IIa-i obtained in example 2, respectively.

Wherein the structural formula of the intermediate III-1e is shown as

Molecular formula C₁₂H₁₂O₃。

The intermediate III-1e in the example is subjected to nuclear magnetic hydrogen spectrum and nuclear magnetic carbon spectrum tests; wherein the nuclear magnetic hydrogen spectrum of the intermediate III-1 is the same as that of the intermediate II described in example 2¹Compared with an H NMR spectrum, the intermediate III-1e has a single peak at delta 3.86(s,3H), and the existence of the methoxyl at the gamma position of the butenolide ring is indicated.

EXAMPLE 4 Synthesis of Compounds Ia-I (formula I-5)

400mg (2.1mmol) of intermediate III-1 (prepared as described in example 3) are weighed into a 25ml round-bottomed flask, dissolved in 2ml of anhydrous methanol and stirred with 34mg (0.63mmol) of sodium methoxide and 37.8mg (1.26mmol) of paraformaldehyde. And refluxing and stirring at 75 ℃, adding 0.3N sodium methoxide and paraformaldehyde every 8 hours, and reacting for about 48 hours. TLC after completion of the reaction, the mixture was concentrated under reduced pressure to remove methanol, (3X 15ml) extracted with ethyl acetate, washed with 20ml of saturated brine, and washed with anhydrous Na₂SO₄And (5) drying. Filtering, concentrating, and purifying by column chromatography (petroleum ether: ethyl acetate: 4: 1).

Compounds I were prepared according to the above procedure in this example from intermediates III-1a-I prepared in example 3, respectively, to give compounds Ia-I.

The structural formula, yield, melting point and nuclear magnetic data of Ia-i are shown in the following, and see FIGS. 1-18.

Compound Ia as a white solid, 45.7%, m.p.185.7-186.8 ℃.¹H NMR(400MHz,CDCl₃)δ7.53(m,2H),7.41–7.33(m,3H),3.89(s,3H),3.34(s,3H),1.76(s,3H).¹³C NMR(100MHz,CDCl₃)δ169.86,169.68,129.90,128.86,128.39,128.21，105.71,103.24,60.08,51.00,22.80.

Compound Ib is light yellow solid, 50.9%，m.p.174.3-174.8℃。¹H NMR(400MHz,CDCl₃)δ7.59–7.56(m,2H),7.11–7.07(m,2H),3.95(s,3H),3.34(s,3H),1.78(s,3H).¹³C NMR(100MHz,CDCl₃)δ169.86,169.45,162.5(d,J＝247Hz),131.42(d,J＝8.1Hz),124.82(d,J＝3.4Hz),115.31(d,J＝22Hz),104.76,103.21,59.81,51.05,22.87.

Compound Ic white solid, 49.8%, m.p.189.6-190.7 ℃.¹H NMR(400MHz,DMSO)δ7.62(d,J＝8.1Hz,2H),δ7.49(d,J＝8.1Hz,2H),3.93(s,3H),3.24(s,3H),1.72(s,3H).13C NMR(100MHz,DMSO)δ170.11,168.52,132.73,131.25,128.05,127.86,103.42,103.00,60.10,50.65,22.60.

Compound Id is a pale yellow solid, 50.2%, m.p.251.9-253.3 ℃.¹H NMR(400MHz,CDCl₃)δ7.53(d,J＝8.1Hz,2H),δ7.49(d,J＝8.1Hz,2H),3.97(s,3H),3.33(s,3H),1.77(s,3H).¹³C NMR(100MHz,CDCl₃)δ170.21,169.07,131.43,131.06,127.79,122.55,104.62,103.19,59.85,51.09,22.88.

Compound Ie is a white solid, 52.4%, m.p.212.6-213.1 ℃.¹H NMR(400MHz，DMSO)δ7.42(d，J＝8.1Hz,2H),7.22(d,J＝8.1Hz,2H),3.86(s,3H),3.23(s,3H),2.33(s,3H),1.69(s,3H).¹³C NMR(100MHz,DMSO)δ169.30,168.97,137.45,129.62,128.52,125.97,104.62,102.86,59.97,50.48,22.61,20.79.

Wherein,¹h NMR spectrum data: the doublet at δ 7.37-7.47(d, J ═ 8.1Hz, 2H) is the two hydrogens at the 9 and 113 positions of the phenyl ring; the doublet at δ 7.18-7.26(d, J ═ 7.9Hz,2H) is two hydrogens at the 12 and 10 positions of the phenyl ring; the singlet at δ 3.86(3H, s) is hydrogen on carbon at position 7; the singlet at δ 3.23(3H, s) is hydrogen on carbon at position 6; the single peak at δ 2.33(3H, s) isHydrogen on carbon at position 14; the single peak at δ 1.69(3H, s) is hydrogen on 5 carbons.¹³C NMR spectrum data: δ 169.3(s) is the peak at carbon 1; δ 168.9(s) is the peak at carbon 3; δ 0137.4(s) is the peak at 11 carbons; δ 129.7(s) is the peak at carbons 10 and 12 of the phenyl rings, 128.5(s) is the peak at carbon 11; δ 125.9(s) is the peak for carbons at positions 10 and 12 of the phenyl ring; δ 104.6(s) is the peak at carbon 2; δ 102.8(s) is the peak at carbon 4; δ 59.9(s) is the peak at carbon 7; δ 50.5(s) is the peak at carbon 6; δ 22.6(s) is the peak at carbon 14; δ 20.79(s) is the peak at carbon 5.

Compound If is a pale yellow solid, 48.6%, m.p.165.8-166.4 ℃.¹H NMR(400MHz,CDCl₃)δ7.47(d,J＝8.7Hz,2H),6.93(d,J＝8.8Hz,2H),3.91(s,3H),3.82(s,3H),3.33(s,3H),1.75(s,3H).¹³C NMR(100MHz,CDCl₃)δ169.97,169.19,159.56,131.03,120.97,113.72,105.46,103.19,59.83,55.30,50.97,22.86.

Compound Ig was a pale yellow solid, 46.2%, m.p.198.1-198.8 ℃.¹H NMR(400MHz,CDCl3)δ7.43–7.35(m,3H),7.04(t,J＝8Hz,1H),4.00(s,3H),3.34(s,3H),1.79(s,3H).¹³CNMR(100MHz,CDCl3)δ170.41,168.97,162.40(d,J＝244Hz),130.85(d,J＝8.6Hz),129.73(d,J＝8.4Hz),125.23(d,J＝3Hz),116.47(d,J＝22Hz),115.32(d,J＝21Hz),104.59(d,J＝2Hz),103.12,59.82,51.10,22.93.

Compound Ih as a pale yellow solid, 47.5%, m.p.176.7-177.9 ℃.¹H NMR(400MHz,DMSO)δ7.52–7.47(m,2H),7.33–7.26(m,2H),3.77(s,3H),3.23(s,3H),1.66(s,3H).¹³C NMR(100MHz,DMSO)δ171.00,168.33,159.92(d,J＝244Hz),132.97(d,J＝2.2Hz),131.07(d,J＝8.1Hz),124.21(d,J＝3.4Hz),116.84(d,J＝16Hz),115.34(d,J＝21Hz),103.5,98.4,59.9,50.4,22.3.

Compound Ii is a pale yellow solid, 44.3%, m.p.187.3-188.6 ℃.¹HNMR(400MHzCDCl₃)δ7.60–7.55(m,1H),7.49–7.46(m,1H),7.21-7.15(m，1H),3.97(s,3H),3.36(s,3H),1.79(s,3H).¹³CNMR(100MHz,CDCl₃)δ170.30,168.75,151.39-148.62(overlap,2c),125.76-125.48(overlap,2c),118.31-117.01(overlap,2c),103.79,103.04,59.53,51.15,23.04.

EXAMPLE 5 Synthesis of intermediate III-2

1.50g (7.73mmol) of intermediate II (prepared as in example 2) are weighed into a 25ml round bottom flask, DMF and CH are added₂Cl₂10ml (1:1) of the mixed solution (9). 1.18g (6.44mmol) of oxalyl chloride are slowly added dropwise with stirring in an ice bath. The reaction was carried out for about 7h, and after completion of the reaction monitored by TLC, saturated NaHCO₃The pH was adjusted to neutral, (3X 15ml) extracted with ethyl acetate, washed with saturated brine and anhydrous Na₂SO₄And (5) drying. Filtering, concentrating, and separating by silica gel column chromatography (petroleum ether: ethyl acetate: 2:1) to obtain intermediate III-2.

Intermediate III-2 was prepared by the above-mentioned method in this example from intermediate IIb, intermediate IIc, intermediate IIf, intermediate IIe and intermediate IIg prepared in example 2, to give intermediate III-2b, intermediate III-2c, intermediate III-2f, intermediate III-2e and intermediate III-2 g.

EXAMPLE 6 Synthesis of intermediate IV-1

1.00g (4.71mmol) of intermediate III-2 (prepared as described in example 5) are weighed into a 25ml round-bottomed flask, dissolved in 2ml THF and 778mg (5.64mmol) of anhydrous K are added with stirring₂CO₃Thereafter, 492mg (5.64mmol) of morpholine were slowly added dropwise. During the dropping process, yellow solid is continuously generated, and after the dropping is finished, the mixture is stirred for 3 hours at room temperature. After TLC monitoring reaction is completed, the crude compound intermediate IV-1 is obtained by suction filtration.

Intermediate IV-1 was prepared by the above-mentioned method in this example from intermediate III-2b, intermediate III-2c, intermediate III-2f, intermediate III-2e and intermediate III-2g prepared in example 5, respectively, to give intermediate IV-1b, intermediate IV-1c, intermediate IV-1f, intermediate IV-1e and intermediate IV-1 g.

Wherein the intermediate IV-1b has a structural formula

Molecular formula C₁₄H₁₄FNO₃。

Of the above intermediate IV-1b¹H NMR spectrum data: the doublets at δ 7.35(d, J ═ 8.4Hz,2H) are two hydrogens on the carbons at the 7 and 9 positions of the phenyl ring, the doublets at δ 7.31(d, J ═ 8.4Hz,2H) are two hydrogens on the carbons at the 6 and 10 positions of the phenyl ring, δ 5.02(s,2H) is a hydrogen on the carbon at the 4 position on the butenolide ring, δ 3.69 to 3.63(m,4H) are four hydrogens on the carbons at the 12 and 14 positions on the morpholine ring, δ 3.52 to 3.46(m,2H) and δ 3.32 to 3.26(m,2H) are hydrogens on the 11 and 13 positions on the morpholine ring, respectively,¹³c NMR spectrum data: δ 168.72(s) is the peak at the carbonyl carbon at position 1, δ 161.55(d, J ═ 236Hz) is the doublet of the 8-position carbon split on the phenyl ring, δ 166.45(s) is the peak at the carbon of the ethylenic bond to the N atom at position 3, δ 130.83(d, J ═ 8.1Hz) is the peak at the carbon at positions 7 and 9 on the phenyl ring, δ 125.19(d, J ═ 3.2Hz) is the peak at the carbon at positions 6 and 10 on the phenyl ring, δ 113.21(d, J ═ 24Hz) is the peak at the carbon at position 5 on the phenyl ring, δ 67.12(s) is the peak at the carbon at positions 12 and 14 on morpholine, δ 64.23 is the peak at the carbon at position 4 on butenolide.

EXAMPLE 7 Synthesis of Compound II-1-5

Weighing 400mg (1.52mmol) of intermediate IV-1 (prepared in example 6) in a 25ml round bottom flask, adding 2ml of methanol, refluxing at 65-80 ℃, and then adding 0.6N paraformaldehyde and 0.3N sodium methoxide; 0.3N paraformaldehyde and sodium methoxide are added every 8 hours, and the reaction is carried out for about 72 hours. After completion of the reaction monitored by TLC, the mixture was concentrated under reduced pressure to remove methanol, dissolved in 15ml of ethyl acetate, extracted with (3X 15ml) saturated NaCl and dried over anhydrous sodium sulfate overnight. Suction filtration, decompression concentration and silica gel column chromatography separation (petroleum ether: ethyl acetate 4: 1).

Compound II was prepared according to the above-described method in this example from intermediate IV-1b, intermediate IV-1c, intermediate IV-1f, intermediate IV-1e and intermediate IV-1g obtained in example 6, to obtain compound II-1, compound II-2, compound II-3, compound II-4 and compound II-5.

Wherein the structural formulas, the yield, the melting point and the nuclear magnetic data of the compound II-1, the compound II-2, the compound II-3, the compound II-4 and the compound II-5 are shown in the specification, and particularly refer to the figures 19 to 28.

Compound II-1 as a yellow solid, 42.6%, m.p.196.4-196.8 ℃.¹H NMR(400MHz,CDCl₃)δ7.27(m,2H),7.08(t,J＝8.7Hz,2H),3.73–3.63(m,4H),3.53–3.45(m,2H),3.36(s,3H),3.34–3.25(m,2H),1.81(s,3H).¹³C NMR(100MHz,CDCl₃)δ170.33,162.17(d,J＝245Hz),159.60,132.13(d,J＝8.1Hz),127.83(d,J＝3.5Hz),115.35(d,J＝22Hz),103.61,98.45,66.69,50.89,48.39,24.90.

Wherein,¹h NMR spectrum data: the doublets at δ 7.35(d, J ═ 8.4Hz,2H) are two hydrogens on the 7 and 9 carbons of the phenyl ring, the doublets at δ 7.31(d, J ═ 8.4Hz,2H) are two hydrogens on the 6 and 10 carbons of the phenyl ring, δ 3.69-3.63(m,4H) are four hydrogens on the 12 and 14 carbons of the morpholine ring, δ 3.52-3.46(m,2H) and δ 3.32-3.26(m,2H) are the hydrogens on the 11 and 13 positions, respectively, on the morpholine ring, δ 3.36(s,3H) is three hydrogens on the 16 carbon, δ 1.80(s,3H) is three hydrogens on the 15 carbon.¹³C NMR spectrum data: δ 168.72(s) is the peak at the carbonyl carbon at position 4, δ 161.55(d, J ═ 236Hz) is the doublet of the 8-position carbon split on the benzene ring, δ 0166.45(s) is the peak at the carbon bond to the N atom at position 3, δ 1130.83(d, J ═ 8.1Hz) is the peaks at the carbon at positions 7 and 9 on the benzene ring, δ 125.19(d, J ═ 3.2Hz) is the peak at the carbon at positions 6 and 10 on the benzene ring, δ 113.21(d, J ═ 24Hz) is the peak at the carbon at position 5 on the benzene ring, δ 109.82(s) is the peak at the carbon at position 3, δ 101.95(s) is the peak at the carbon at position 2, δ 67.12(s) is the peaks at the carbon at positions 12 and 14 on morpholine, δ 52.81(s) is the peaks at the carbon at positions 11 and 13, δ 49.25(s) is the peak at the carbon at the methoxy carbon at position 16, δ 22.64(s) is the peak at the carbon at the methyl group 15.

Compound II-2Yellow solid, 35.2%, m.p.162.9-163.4 ℃.¹H NMR(400MHz,CDCl₃)δ7.35(d,J＝12Hz,2H),7.23(d,J＝8.4Hz,2H),3.73–3.63(m,4H),3.54–3.42(m,2H),3.36(s,3H),3.33–3.25(m,2H),1.80(s,3H).¹³C NMR(100MHz,CDCl₃)δ170.07,159.76,133.55,131.73,130.42,128.47,128.42,103.62,98.10,66.65,50.92,48.47,40.98 24.89.

Compound II-3 is a yellow solid, 38.9%, m.p.153.6-154.0 ℃.¹H NMR(400MHz,CDCl₃)δ7.20(d,J＝8.6Hz,2H),6.91(d,J＝8.7Hz,2H),3.82(s,3H),3.71–3.60(m,4H),3.54-3.45(m,2H),3.35(s,3H),3.32-3.27(m,2H),1.79(s,3H).¹³C NMR(100MHz,CDCl₃)δ170.74,159.08,159.04,131.58,123.98,113.78,103.56,99.24,66.75,55.29,50.83,48.26,29.83,24.93.

Compound II-4 as a yellow solid, 40.2%, m.p.149.5-149.7 ℃.¹H NMR(400MHz,CDCl₃)δ7.20-7.15(overlap,4H),3.72-3.62(m,4H),3.53-3.47(m,2H),3.35(s,3H),3.33-3.27(m,2H),2.37(s,3H),1.80(s,1H).¹³C NMR(100MHz,CDCl₃)δ170.74,159.04,137.47,130.30,128.98,128.83,103.56,99.23,66.75,50.83,48.26,24.93,21.23.

Compound II-5 is a yellow solid, 40.9%, m.p.168.3-169.4 ℃.¹H NMR(400MHz,CDCl₃)δ7.28-7.23(m,1H),6.99–6.88(m,3H),3.67–3.53(m,4H),3.45–3.36(m,2H),3.28(s,3H),3.25–3.16(m,2H),1.72(s,3H).¹³C NMR(100MHz,CDCl₃)δ169.90,162.43(d,J＝245Hz),159.93,134.07(d,J＝8Hz),129.68(d,J＝8.6Hz),126.26(d,J＝2.9Hz),117.40(d,J＝21Hz),114.63(d,J＝21Hz),103.59,98.30,66.64,50.92,48.51,24.89.

EXAMPLE 8 Synthesis of intermediate IV-2

1.00g (4.71mmol) of intermediate III-2g (prepared as in example 5) are weighed into a 25ml round bottom flask, dissolved by adding 2ml THF, and 778mg (5.64mmol) of anhydrous K are added with stirring₂CO₃After that, aniline (5.64mmol) was slowly added dropwise. During the dropping process, solid is continuously generated, and after the dropping is finished, the mixture is stirred for 3 hours at room temperature. After TLC monitoring reaction is completed, crude compound intermediate IV-2g is obtained by suction filtration.

In the method of the embodiment, aniline is replaced by 4-fluoroaniline, and the rest of operation and condition parameters are the same, so that the product obtained is an intermediate IV-2 h.

In the method of the embodiment, aniline is replaced by 4-chloroaniline, and the rest of operation and condition parameters are the same, so that the product obtained is an intermediate IV-2 i.

The product was prepared as intermediate IV-2j by replacing aniline in the above procedure with 4-bromoaniline, with the same operating and condition parameters.

In this example, aniline in the above process was replaced with 4-methylaniline, and the remaining operating and condition parameters were the same, to prepare the product as intermediate IV-2 k.

In the method of the embodiment, aniline is replaced by 4-methoxyaniline, and the rest operation and condition parameters are the same, so that the product is prepared to be an intermediate IV-2 l.

In the method of the embodiment, aniline is replaced by 2-fluoroaniline, and the rest of the operation and condition parameters are the same, so that the product obtained is an intermediate IV-2 m.

In the method of the embodiment, aniline is replaced by 3-fluoroaniline, and the rest of the operation and condition parameters are the same, so that the product obtained is an intermediate IV-2 n.

1.00g (4.71mmol) of intermediate III-2c (prepared as described in example 5) are weighed into a 25ml round-bottomed flask, 2ml THF are added and dissolved, 778mg (5.64mmol) of anhydrous K are added with stirring₂CO₃After that, p-methylaniline (5.64mmol) was slowly added dropwise. During the dropping process, solid is continuously generated, and after the dropping is finished, the mixture is stirred for 3 hours at room temperature. After TLC monitoring reaction is completed, crude compound intermediate IV-2c is obtained by suction filtration。

1.00g (4.71mmol) of intermediate III-2e (prepared as described in example 5) are weighed into a 25ml round-bottomed flask, dissolved in 2ml THF, and 778mg (5.64mmol) of anhydrous K are added with stirring₂CO₃After that, p-methylaniline (5.64mmol) was slowly added dropwise. During the dropping process, solid is continuously generated, and after the dropping is finished, the mixture is stirred for 3 hours at room temperature. After TLC monitoring reaction is completed, the crude compound intermediate IV-2e is obtained by suction filtration.

Example 9 Compound II6-15

400mg (1.52mmol) of intermediate IV-2 (prepared as described in example 8) are weighed into a 25ml round bottom flask, 2ml of methanol are added, reflux is carried out at 70 ℃ and then 0.6N paraformaldehyde and 0.3N sodium methoxide are added; 0.3N paraformaldehyde and sodium methoxide are added every 8 hours, and the reaction is carried out for about 48 hours. After completion of the reaction monitored by TLC, the mixture was concentrated under reduced pressure to remove methanol, dissolved in 15ml of ethyl acetate, extracted with (3X 15ml) saturated NaCl and dried over anhydrous sodium sulfate overnight. Suction filtration, decompression concentration and silica gel column chromatography separation (petroleum ether: ethyl acetate 4: 1).

Compound II was prepared from intermediate IV-2g, intermediate IV-2h, intermediate IV-2i, intermediate IV-2j, intermediate IV-2k, intermediate IV-2l, intermediate IV-2m and intermediate IV-2n obtained in example 8 by the method described above in this example to give compound II-6, compound II-7, compound II-8, compound II-9, compound II-10, compound II-11, compound II-12 and compound II-13.

Compound II was prepared from intermediate IV-2c and intermediate IV-2e obtained in example 8 by the method described above in this example, to give compound II-14 and compound II-15.

Wherein the structural formulas, the yields, the melting points and the nuclear magnetic data of the compound II-6, the compound II-7, the compound II-8, the compound II-9, the compound II-10, the compound II-11, the compound II-12, the compound II-13, the compound II-14 and the compound II-15 are shown in the following, and the specific reference is shown in FIGS. 29-38.

Compound II-6 white solid, 48.6%，m.p.191.2-191.5.1℃。¹H NMR(400MHz,DMSO)δ8.10(t,J＝6.4Hz,1H),7.35-7.28(m,1H),7.27–7.17(overlap,3H),7.11-7.08(m,1H),6.99-6.90(overlap,3H),4.23(s,2H),3.17(s,3H),1.68(s,3H).

Compound II-7 as a white solid, 42.3%, m.p.157.6-158.1 ℃.¹H NMR(400MHz,CDCl₃)δ7.39–7.31(m,1H),7.17–6.98(overlap,7H),5.03(s,1H),4.33(s,2H),3.32(s,3H),1.76(s,3H).

Compound II-8 as a white solid, 40.6%, m.p.119.8-120.3 ℃.¹H NMR(400MHz,CDCl₃)δ7.47(d,J＝8.4Hz,2H),7.38-7.28(m,1H),7.15–6.94(overlap,5H),5.07(s,1H),4.33(s,2H),3.30(s,3H),1.72(s,3H).

Compound II-9 as a white solid, 41.6%, m.p.208.3-208.5 ℃.¹H NMR(400MHz,CDCl₃)δ7.45(d,J＝8.3Hz,2H),7.35-7.29(m,1H),7.12–6.93(overlap,5H),5.02(s,1H),4.30(s,2H),3.30(s,3H),1.73(s,3H).

Compound II-10 as a white solid, 42.8%, m.p.135.3-135.7 ℃.¹H NMR(400MHz,CDCl₃)δ7.35-7.30(m,1H),7.14(d,J＝7.7Hz,3H),7.09(d,J＝9.7Hz,1H),7.01(d,J＝7.9Hz,3H),6.99–6.94(m,1H),4.98(s,1H),4.29(s,2H),3.29(s,3H),2.34(s,3H),1.74(s,3H).

Compound II-11 as a white solid, 44.9%, m.p.127.5-128.3 ℃.¹H NMR(400MHz,DMSO)8.03(t,J＝4Hz,1H),7.37-7.32(m,1H),7.12-7.07(m,1H),7.03-6.73(overlap,5H),4.15(s,2H),3.70(s,3H),3.15(s,3H),1.66(s,3H).

Compound II-12 is a white solid, 45.6%, m.p.155.9-156.2 ℃.¹H NMR(400MHz,CDCl₃)δ7.38-7.27(m,2H),7.14(d,J＝7.7Hz,1H),7.12–6.91(overlap,5H),5.09(s,1H),4.40(s,2H),3.25(s,3H),1.72(s,3H).

Compound II-13 as a white solid, 42.1%, m.p.200.8-201.9 ℃.¹H NMR(400MHz,DMSO)8.05(m,1H),7.30-7.25(m,2H),7.16-7.14(m,2H),7.09–7.00(m,2H),6.95(brs,1H),6.86(brs,1H),4.25(s,2H),3.20(s,3H),1.69(s,3H).

Compound II-14 as a white solid, 41.6%, m.p.188.2-190.0 ℃.¹H NMR(400MHz,CDCl₃)δ7.37-7.26(m,4H),7.14(d,J＝7.7Hz,2H),7.00(d,J＝9.7Hz,2H),4.90(s,1H),4.29(s,2H),3.29(s,3H),2.34(s,3H),1.74(s,3H).

Compound II-15 as a white solid, 44.3%, m.p.172.7-174.1 ℃.¹H NMR(400MHz,CDCl₃)δ7.26(d,J＝7.9Hz,2H),7.18(d,J＝7.9Hz,2H),7.05(d,J＝8.4Hz,2H),6.84(d,J＝8.6Hz,2H),4.84(s,1H),4.25(s,2H),3.80(s,3H),3.28(s,3H),2.35(s,3H),1.72(s,3H).

Example 10 evaluation of anti-influenza Virus Activity and inhibition of neuraminidase

The compounds Ia-i and II-1 to II-15 obtained in the examples were evaluated for anti-H1N 1 virus activity and for their inhibitory effect on neuraminidase as neuraminidase inhibitors.

The specific in vitro antiviral activity test procedure is as follows.

And (3) virus amplification:

1. cell transmission: after MDCK cells are digested, inoculating the MDCK cells into a large culture bottle with the size of 2 multiplied by 75 cm;

2.24h later inoculation: when the cells grow to a monolayer (about 70 percent), removing the culture medium, and washing with PBS for three times; adding virus solution (Influenza A virus, A/Weiss/43, Wuhan national culture Collection) and shaking once every 15min for 3 times; after 2h, the culture medium was discarded, and 12mL of DMEM containing 2% FBS (fetal bovine serum, GBICO, USA) was added thereto, followed by daily observation and recording by photographing;

3. toxic materials are collected when 70% -90% of pathological changes occur: placing the culture bottle to-80 deg.C, repeatedly freezing and thawing for 3 times, centrifuging at 4 deg.C and 4500r for 15min, collecting supernatant, subpackaging, labeling, and storing at-80 deg.C for use.

Virus TCID₅₀And (3) determination:

1. plate paving: MDCK cells were plated in 96-well plates at 2.5X 10/well⁴5% CO₂Incubating at 37 ℃;

2. infection with virus: after 24h, PBS was washed 3 times and 100. mu.L of virus solution diluted with pure DMEM was added to each well (serial log dilution of the expanded virus solution was made to 10%^-1、10^-2、10^-3、10^-3、10^-4、10^-5、10^-6、10^-7、10^-8、10^-9～10^-10) 8 multiple wells are arranged for each concentration, and a contrast is set;

after 3.2h, virus solution was removed and 100. mu.L of 2% FBS DMEM was added to each well;

4. the observation was made daily, and 72 hours later, the measurement was carried out with a Jiemsa staining solution. The degree of cytopathic effect was assessed according to the following criteria in Table 1:

TABLE 1

Calculation of Virus TCID by Reed-Muench formula₅₀

Distance ratio ═ (percentage of positivity greater than 50% — 50%)/(percentage of positivity greater than 50% — percentage of positivity less than 50%)

TCID₅₀High dilution log of percentage of positive greater than 50% + distance ratio x log of dilution factor

Cytotoxicity assay CC₅₀：

1. Plate paving: MDCK 96-well plate with 2.5 × 10 wells ⁴5% CO₂Incubating at 37 ℃;

2. adding medicine: after 24h, removing the culture solution, adding 200 mu L of the drug to be detected prepared by 2% FBS DMEM into each hole, diluting the drug to be detected into 9 concentrations of 200, 100 and 50-0.78 (mu M) in a multiple ratio, repeating the holes for 3 concentrations, and setting a reference;

3. and (3) detection: after 48h, cell survival was determined by MTT.

Detection of antiviral Activity EC₅₀：

1. Plate paving: MDCK 96-well plate with 2.5 × 10 wells⁴A plurality of;

2. infection with virus: after 24h, the medium was removed, washed 3 times with PBS and 100. mu.L of 100TCID in pure DMEM was added to each well₅₀A viral fluid;

3. adding medicine: after 2h, removing virus liquid, adding 200 μ L of the drug to be tested prepared by 2% FBS DMEM into each well, setting 6 concentrations of 50, 25, 12.5, 6.25, 3.12 and 1.56(μ M), and setting 3 multiple wells for each concentration;

4. and (3) detection: after 48h, the rate of virus inhibition was determined by MTT method.

The results of the antiviral activity and cytotoxicity tests are shown in fig. 39, fig. 40(a and B), table 2 and table 3.

TABLE 2 antiviral Activity and cytotoxicity of Compounds Ia-i

Com	EC₅₀(μM)	CC₅₀(μM)	SI
				Ia	26.30±4.44	168.12±9.65	6.4
Ib	16.17±1.648	736.01±69.57	46
				Ic	-	98.33±3.52	-
Id	61.06±5.01	265.96±18.39	13.9
				Ie	-	183.8±5.12	-
If	7.54±0.88	307.24±19.30	40.9
				Ig	9.38±0.82	19.80±0.96	2.1
Ih	-	204.34±3.16	-
				Ii	33.35±1.02	108.95±7.62	3.24
Ribavirin	105±51	532±20.27	5.06

"-" indicates no activity.

And (3) SI: the therapeutic index. Therapeutic index CC₅₀(μM)/EC₅₀(μM)。

EC from Table 1 above₅₀In the formula I, the synthesized compounds (Ia-I) in the series I have no obvious cytotoxicity, and most of the compounds have better inhibitory effect on influenza A virus H1N 1. EC for optimum activity of compound If₅₀7.54 ± 0.88 μ M. Overall analysis 1) regarding the substituents, when the substituent on the benzene ring is an electron-withdrawing group (CH)₃O, F) is superior to electron donating group (CH)₃) And unsubstituted benzene rings; 2) compared with compounds Ib, Ig and Ih, the activity of meta substitution is superior to that of para substitution and ortho substitution; 3) compared with the activity of the compounds Ib and Ii, the single substitution is superior to the double substitution.

The compounds of series I were found to act on the neuraminidase active pocket by computer modelling.

TABLE 3 antiviral Activity and cytotoxicity of Compounds II-1 to II-15

Com	EC₅₀(μM)	CC₅₀(μM)	SI
				II-1	14.30±1.718	776.23±63.15	54.2
II-2	34.71±5.86	209.37±14.09	6.0
				II-3	6.68±1.32	536.32±15.14	85.6
II-4	14.05±2.47	218.70±8.65	15.6
				II-5	-	146.57±8.09	-
II-6	-	309.80±3.98	-
				II-7	-	105.42±4.10	-
II-8	-	36.13±1.35	-
				II-9	-	294.5±9.38	-
II-10	-	57.4±3.48	-
				II-11	-	72.30±2.52	-
II-12	-	186.3±3.78	-
				II-13	-	124.97±5.42	-
II-14	23.17±1.03	90.82±3.42	12.0
				II-15	-	23.50±1.87	-
Ribavirin	105±51	532±20.27	5.06

"-" indicates no activity.

As can be seen from Table 2 above, the compounds have no significant cytotoxicity, with the activity of compound II-3 being optimal, EC₅₀6.68 +/-1.32 mu M, which is superior to the positive drug ribavirin (EC)₅₀105 ± 51 μ M) and also a higher therapeutic index SI ═ 85.6) morpholine substitution is generally the most active in the case of substituents; 2) when beta is modified with benzylamine, the compound has little antiviral activity, and only compound II-14 exhibits antiviral activity (EC)₅₀23.17 ± 1.03 μ M/L), but the therapeutic index is much lower than that of compound II-3; 3) comparing the compounds II-a and II-e, the para-substitution of the benzene ring is found to be superior to the meta-substitution; 4) comparing the activities of the compounds II-1, II-2, II-3 and II-5, the para-substituent CH of the benzene ring₃O>CH₃>F>Cl。

Example 11 targeting of Compounds to neuraminidase

The targeting of compounds Ia-i and II-1 to II-15 to neuraminidase, typically as If, Ig and II-3, was verified in this example as shown in Table 4.

The testing process comprises the following steps:

1. adding 70 mu L of neuraminidase detection buffer solution, 10 mu L of neuraminidase, 5 mu L of samples to be detected (blank group added with DMSO) and 5 mu L of LMilli-Q water in sequence according to the specification of a neuraminidase inhibitor screening kit (Biyun, Shanghai), vibrating and mixing uniformly for 1min, and incubating in an incubator for 2 min;

2. adding 10 μ L neuraminidase fluorescent substrate, shaking and mixing for 1min, incubating in incubator for 30min, and measuring fluorescence.

3. The emission wavelength is 450nm, and the excitation wavelength is 322 nm. The NA inhibitory amount X of the compound was calculated from a standard curve.

NA inhibition ratio (10-X)/10 × 100%.

TABLE 4

ID	MW	EC₅₀	CC₅₀	SI	tPSA	Log S
							If	264	7.54±0.88	307.24±19.30	40.9	53.99	-2.767
Ig	252	9.38±0.82	19.80±0.96	2.1	44.76	-2.99
							II-3	319	6.68±1.32	536.32±15.14	85.6	57.23	-3.233

Where tPSA is topologically polar surface area, a parameter commonly used in pharmaceutical chemistry, which is defined as the total surface area of polar molecules within a compound, mostly oxygen and nitrogen atoms, also including hydrogen atoms attached thereto.

Log S is the hydrophilicity index, S is the concentration of the compound in saturated aqueous solution, 85% drug: logs is between-1 and-5.

The experimental results show that the compound II-3 has concentration dependence on the inhibition effect of NA (neuraminidase), and the inhibition rate is equivalent to Oseltamivir (Oseltamivir) which is a positive drug, and the figure 41 (OSe in the figure is Oseltamivir).

EXAMPLE 12 Compound II-3 molecular docking results

The detection method of the molecular docking result of the compound II-3 is that a computer simulates molecular docking.

The molecular docking results of compound II-3 are shown in FIGS. 42 and 43. It can be seen from the figure that compound II-3 is able to act on the same active pocket as oseltamivir on neuraminidase, wherein the methoxy group at β position of butenolide is able to form hydrogen bond interaction with the Arg371, Arg292 amino acids in the active pocket.

EXAMPLE 13 inhibitory Effect of Compounds II-3 on different stages of viral replication

The specific experimental method comprises the following steps:

1. taking MDCK cell suspension in logarithmic growth phase, inoculating into 24-well plate, 40000/well, placing at 37 deg.C, and 5% CO₂Culturing in an incubator for 24 h. The compounds with different concentrations (50 mu M, 25 mu M, 12.5 mu M, 6.25 mu M, 3.12 mu M and 1.56 mu M) are added respectively 2h before, simultaneously with and 2h after the infection of the virus, and whether the compounds have the inhibiting, direct and therapeutic effects on the virus is detected.

2. Prevention effect: adding compounds with different concentrations (50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.12. mu.M, 1.56. mu.M) 2h before infecting the virus; remove the medium, wash 3 times with PBS, and use 100TCID₅₀Virus-infected cells, 5% CO at 37 ℃₂Incubating for 2h in an incubator; removing virus solution, adding 2% FBS culture medium, and measuring OD 72 hr later by MTT method_450nmThe value is obtained.

3. Direct action: washing with PBS 3 times before virus infection, adding 100TCID₅₀Incubation of different concentrations of compounds of the virus (diluted 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.12. mu.M, 1.56. mu.M in pure DMEM medium at fold-by-fold) for 2 h; removing supernatant, adding 2% FBS culture medium, and measuring OD 72 hr later by MTT method_450nmThe value is obtained.

4. The treatment effect is as follows: washing 3 times with PBS before infection with virus, and washing with 100TCID₅₀Virus-infected cells, 5% CO at 37 ℃₂Incubating for 1.5-2h in the incubator; the virus solution was removed, compounds prepared in 2% FBS medium at different concentrations (diluted to 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.12. mu.M, 1.56. mu.M in 2% FBS DMEM medium at double ratio) were added, and OD was measured by MTT method after 72 hours_450nmThe value is obtained.

5. Formula of virus inhibition rate

Viral inhibitory rate (drug treatment group OD)_450nmViral control group OD_450nm) /(Normal control group OD_450nm- -Virus control group OD_450nm)×100％。

To investigate at which stage of viral replication compound II-3 acts, compound II-3 was added before, during and after infection, respectively. The experimental result shows that the compound II-3 has little treatment effect on cells when added into the cells before H1N1 infection; and during and after infection, compound II-3 is added to have concentration-dependent therapeutic effect on cells. Indicating that compound II-3 does not act on the adsorption phase in the viral replication cycle, but on the replication phase after entry of the virus into the cell.

EXAMPLE 14 Compound II-3 in vivo animal experiments

The specific experimental method comprises the following steps:

SPF grade 7 week old Balb/c female mice, weighing 14 + -2g, were divided into 6 groups (normal control group, virus control group, ribavirin group, high dose group, medium dose group, low dose group) by completely random method, and 10 mice per group.

2. Infection with virus: mice were anesthetized with ether and infected with virus by nasal drip. Normal control group was given nasal equal amount of normal saline.

3. Administration: different amounts of the compounds were administered intragastrically 2h after viral infection for 5 consecutive days, once daily. Body temperature and body weight of the mice were monitored and recorded daily during the dosing period.

4. Material taking: on the sixth day of virus infection, mice were euthanized and lung tissue was harvested and stored at-80 ℃ for future use.

In vivo animal experiments show that the body weight of the compound II-3 mice is close to that of a normal group, and meanwhile, compared with a virus control group, the compound II-3 can reduce the expression of RIG-I. II-3 was able to increase the expression of IL-1 β compared to the control ribavirin group. Meanwhile, II-3 was able to reduce inflammation by inhibiting excessive secretion of TNF-. alpha.in lung tissue of infected mice, see FIG. 44(a) and FIG. 44 (b).

Although the present application has been described with reference to a few embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the application as defined by the appended claims.

Claims

1. A compound selected from any one of the compounds having the following structural formula:

2. a process for preparing a compound of claim 1, comprising:

step (I), (II), (III-1), (IV-1); or,

step (I), (II), (III-2), (IV-2), (V-2); or,

step (I), (II), (III-3), (IV-3), (V-3);

the individual steps are described below:

the substituted phenylacetic acid is selected from any one of the following compounds:

the halogenated ethyl acetate is selected from the compounds with the following structural formula:

wherein X is selected from halogen;

(IV-1) reacting a mixture III containing the intermediate III-1, sodium methoxide and paraformaldehyde to obtain a target product;

(V-2) reacting a mixture V containing the intermediate IV-1, sodium methoxide and paraformaldehyde to obtain a target product;

(III-3) carrying out chloro nucleophilic reaction on the raw material II containing the intermediate II to obtain an intermediate III-2; the structural formula of the intermediate III-2 is shown as a formula II-3-2:

(IV-3) carrying out nucleophilic substitution on the mixture VI containing the intermediate III-2 and the substituted benzylamine under alkaline conditions to obtain an intermediate IV-2; the structural formula of the intermediate IV-2 is shown as a formula II-4-2:

(V-3) reacting a mixture VII containing the intermediate IV-2, sodium methoxide and paraformaldehyde to obtain a target product;

the substituted benzylamine is selected from compounds of the following structural formula:

3. the preparation method according to claim 2, wherein the mixture I in step (I) further comprises a solvent I and an acid-binding agent;

4. The preparation method according to claim 3, wherein the solvent I is one selected from tetrahydrofuran, dichloromethane, DMF, ethyl acetate, acetone, methanol and ethanol;

the acid-binding agent is selected from at least one of triethylamine, pyridine, anhydrous potassium carbonate, sodium acetate and anhydrous sodium carbonate;

5. The method according to claim 2, wherein the raw material I in the step (II) further comprises a solvent II and a base I;

the molar ratio of the intermediate I to the base I is 1: 1.5 to 3;

6. The process according to claim 5, wherein the solvent II is selected from DMF, THF, t-BuOH, acetone, EtOAc, CH₂Cl₂At least one of DMSO and methanol;

the base I is selected from potassium tert-butoxide and anhydrous Na₂CO₃、CH₃ONa、C₂H₅ONa、NaOH、NaHCO₃NaH, anhydrous K₂CO₃At least one of;

the molar ratio of the intermediate I to the base I is 1: 2;

7. The method according to claim 2, wherein the mixture II in the step (III-1) further comprises a solvent III and a base II;

8. The process according to claim 7, wherein the solvent III is selected from acetone, methanol, CH₂Cl₂At least one of THF, DMF, EtOAc;

the base II is selected from anhydrous K₂CO₃Anhydrous Na₂CO₃、CH₃ONa、NaOH、NaHCO₃At least one of NaH;

9. The method according to claim 2, wherein the mixture III in the step (IV-1) further comprises a solvent IV;

the molar ratio of the intermediate III-1 to the sodium methoxide to the paraformaldehyde is 1: 0.1-1: 0.1 to 0.5;

the reaction conditions are as follows: reflux reaction at 60-100 ℃.

10. The process according to claim 9, wherein the solvent IV is selected from methanol, acetone, CH₂Cl₂At least one of THF, DMF, EtOAc;

11. The method of claim 10, wherein during the reaction: 0.3 mol of paraformaldehyde and sodium methoxide are added at intervals of 8 hours, and the reaction is carried out for 72 hours.

12. The method according to claim 2, wherein the raw material II in the step (III-2) further comprises: solvent V and nucleophile I;

the molar ratio of the intermediate II to the nucleophilic reagent I is 1: 1-4;

13. The process according to claim 12, wherein the solvent V is selected from the group consisting of solvents having a volume ratio of 1:1 of DMF and CH₂Cl₂At least one of THF, EtOAc, methanol and acetone;

the molar ratio of the intermediate II to the nucleophilic reagent I is 1: 2;

14. The method according to claim 2, wherein the mixture IV in the step (IV-2) further comprises a solvent VI and a base III;

the reaction time of the nucleophilic substitution is 3-6 hours.

15. The process according to claim 14, wherein the solvent VI is selected from THF, methanol, acetone, CH₂Cl₂At least one of DMF and EtOAc;

the base III is selected from: anhydrous K₂CO₃Anhydrous Na₂CO₃、CH₃ONa、NaOH、NaHCO₃At least one of NaH;

the molar ratio of the intermediate III-2 to the base III to the morpholine is 1:2: 2;

the reaction time for the nucleophilic substitution was 4 h.

16. The method according to claim 2, wherein the mixture V in the step (V-2) further comprises a solvent VII;

the reaction conditions are as follows: reflux reaction at 60-100 ℃.

17. The method according to claim 16, wherein the solvent VII is selected from methanol, acetone, CH₂Cl₂At least one of THF, DMF, EtOAc;

18. The method of claim 17, wherein during the reaction: 0.3 mol of paraformaldehyde and sodium methoxide are added at intervals of 8 hours, and the reaction is carried out for 72 hours.

19. The method according to claim 2, wherein the mixture VI in the step (IV-3) further comprises a solvent VIII and a base IV;

the molar ratio of the intermediate III-2 to the base IV to the substituted benzylamine is 1: 1-4: 1-4;

the reaction time of the nucleophilic substitution is 3-6 hours.

20. The process according to claim 19, wherein the solvent VIII is selected from THF, methanol, acetone, CH₂Cl₂At least one of DMF and EtOAc;

the base IV is selected from: anhydrous K₂CO₃Anhydrous Na₂CO₃、CH₃ONa、NaOH、NaHCO₃At least one of NaH;

the molar ratio of the intermediate III-2 to the base IV to the substituted benzylamine is 1:2: 2;

the reaction time for the nucleophilic substitution was 4 h.

21. The method according to claim 2, wherein the mixture VII in the step (V-3) further comprises a solvent IX;

the reaction conditions are as follows: reflux reaction at 60-100 ℃.

22. The process according to claim 21, wherein the solvent IX is selected from methanol, acetone, CH₂Cl₂At least one of THF, DMF, EtOAc;

23. The method of claim 22, wherein the reaction conditions further comprise: 0.3 mol of paraformaldehyde and sodium methoxide are added at intervals of 8 hours, and the reaction is carried out for 72 hours.

24. A neuraminidase inhibitor comprising a compound of claim 1.