阅读说明：本技术 一种新型3c样蛋白酶抑制剂及其制备方法和应用 (Novel 3C-like protease inhibitor and preparation method and application thereof ) 是由 鲍官虎 王威 刘士玉 于 2021-06-21 设计创作，主要内容包括：本发明提供了一种新型3C样蛋白酶抑制剂,该3C样蛋白酶抑制剂为具有特定结构的3”,4”-O-二乙酰基-表儿茶素反式咖啡酸酯。本发明还提供了上述3C样蛋白酶抑制剂的制备方法和应用。本发明的优点在于：本发明基于表儿茶素的结构,设计合成了上述化合物3”,4”-O-二乙酰基-表儿茶素反式咖啡酸酯,该化合物即使在浓度低时,仍能够在细胞内明显抑制新型冠状病毒中3C样蛋白酶的活性；本发明化合物可作为3C样蛋白酶抑制剂,且用于制备抗新型冠状病毒SARS-CoV-2感染的药物。(The present invention provides a novel 3C-like protease inhibitor, which is 3 ", 4" -O-diacetyl-epicatechin trans-caffeate having a specific structure. The invention also provides a preparation method and application of the 3C-like protease inhibitor. The invention has the advantages that: the invention designs and synthesizes the compound 3 ', 4' -O-diacetyl-epicatechin trans-caffeate based on the structure of epicatechin, and the compound can still obviously inhibit the activity of 3C-like protease in novel coronavirus in cells even when the concentration is low; the compound can be used as a 3C-like protease inhibitor and is used for preparing a medicine for resisting the infection of novel coronavirus SARS-CoV-2.)

1. A novel 3C-like protease inhibitor represented by the structure of formula i:

2. a process for the preparation of a novel 3C-like protease inhibitor according to claim 1, characterized in that the stepwise synthesis starting from epicatechin finally yields 3 ", 4" -O-diacetyl-epicatechin trans-caffeate having the structure shown in formula i.

3. The method of preparing a novel 3C-like protease inhibitor according to claim 2, comprising the steps of:

(1) synthesis of 3,5,7,3 ', 4' -O-pentaacetyl-epicatechin

Adding epicatechin into a flask, adding dried pyridine and acetic anhydride, and stirring at room temperature overnight under the protection of a drying tube; after TLC detection reaction is finished, adding water to stop reaction, and performing rotary evaporation to obtain a product 3,5,7,3 ', 4' -O-pentaacetyl-epicatechin;

(2) synthesis of 3-O-acetyl-epicatechin

Putting the compound 3,5,7,3 ', 4' -O-pentaacetyl-epicatechin prepared in the step (1) into a flask, adding methanol water solution and ammonium acetate, and stirring overnight at room temperature; after TLC detection reaction is finished, adding water to stop reaction, carrying out rotary concentration, and extracting for a plurality of times by using ethyl acetate with the same volume; mixing the ethyl acetate extractive solutions, concentrating, performing silica gel column chromatography, and eluting with ethyl acetate and methanol to obtain 3-O-acetyl-epicatechin;

(3) synthesis of 3-O-acetyl-5, 7,3 ', 4' -O-tetra-tert-butyldimethylsilyl-epicatechin

Putting the compound 3-O-acetyl-epicatechin prepared in the step (2) into a flask, adding dried dichloromethane, imidazole and 4-dimethylaminopyridine, slowly dropwise adding a dried dichloromethane solution containing tert-butyldimethylchlorosilane under an ice bath condition, stirring at room temperature overnight, and protecting by using a drying tube in the reaction process; after TLC detection reaction is finished, adding water to stop the reaction, and extracting for a plurality of times by using dichloromethane with the same volume; washing the dichloromethane extract with saturated sodium chloride solution, concentrating, performing silica gel column chromatography, eluting with petroleum ether and ethyl acetate to obtain product 3-O-acetyl-5, 7,3 ', 4' -O-tetra-tert-butyldimethylsilyl-epicatechin;

(4) synthesis of 5,7,3 ', 4' -O-tetra-tert-butyldimethylsilyl-epicatechin

Putting the compound 3-O-acetyl-5, 7,3 ', 4' -O-tetra-tert-butyldimethylsilyl-epicatechin prepared in the step (3) into a flask, dissolving the compound with dry dichloromethane, adding dry methanol and potassium carbonate, stirring the mixture at room temperature for reaction, and protecting the reaction by using a drying tube in the reaction process; after TLC detection reaction is finished, adding water to stop the reaction, and extracting for a plurality of times by using ethyl acetate with the same volume; drying the ethyl acetate extract with anhydrous sodium sulfate, concentrating, performing silica gel column chromatography, eluting with petroleum ether and ethyl acetate to obtain product 5,7,3 ', 4' -O-tetra-tert-butyl dimethyl silicon-epicatechin;

(5) synthesis of 3, 4-O-diacetyl caffeoyl chloride

Adding caffeic acid into a flask, adding dried pyridine and acetic anhydride, and stirring at room temperature overnight under the protection of a drying tube; after TLC detection reaction is finished, adding water to stop reaction, and rotationally evaporating to dryness to obtain a product 3, 4-O-diacetyl caffeic acid;

adding thionyl chloride into the synthesized 3, 4-O-diacetyl caffeic acid, carrying out heating reflux reaction in an oil bath, and protecting by using a drying tube in the reaction process; after the reaction is finished, the reaction solution is subjected to rotary evaporation to dryness to obtain a crude product of the 3, 4-O-diacetyl caffeic acid, which is directly used for the next reaction;

(6) synthesis of 3 ', 4' -O-diacetyl-5, 7,3 ', 4' -O-tetra-tert-butyldimethylsilyl-epicatechin trans-caffeate

Putting the compound 5,7,3 ', 4' -O-tetra-tert-butyldimethylsilyl-epicatechin prepared in the step (4) into a flask, adding dried pyridine and dried dichloromethane, slowly dropwise adding the dried dichloromethane containing the crude product of the 3, 4-O-diacetyl caffeic acid prepared in the step (5) under an ice bath condition, reacting at room temperature overnight, and protecting by using a drying tube in the reaction process; after TLC detection reaction is finished, adding water to stop the reaction, and extracting for a plurality of times by using dichloromethane with the same volume; drying the dichloromethane extract with anhydrous sodium sulfate, concentrating, performing silica gel column chromatography, eluting with petroleum ether and ethyl acetate to obtain product 3 ', 4' -O-diacetyl-5, 7,3 ', 4' -O-tetra-tert-butyldimethylsilyl-epicatechin trans-caffeate;

(7) synthesis of 3 ', 4' -O-diacetyl-epicatechin trans-caffeate

Taking the compound 3 ', 4' -O-diacetyl-5, 7,3 ', 4' -O-tetra-tert-butyldimethylsilyl-epicatechin trans-caffeate prepared in the step (6), adding potassium bifluoride and dry methanol, and stirring to fully react; then, adding water to terminate the reaction, extracting with ethyl acetate, and drying with anhydrous sodium sulfate; and finally, carrying out silica gel column chromatography on the concentrated ethyl acetate layer, and eluting by adopting petroleum ether and ethyl acetate to obtain the final product of 3 ', 4' -O-diacetyl-epicatechin trans-caffeate.

4. The method for preparing a novel 3C-like protease inhibitor according to claim 3, wherein in the step (2), the ratio of ethyl acetate: eluting with methanol 20:1 to obtain the product 3-O-acetyl-epicatechin.

5. The method for preparing a novel 3C-like protease inhibitor according to claim 3, wherein in the step (3), the ratio of petroleum ether: eluting with 100:1 ethyl acetate to obtain the product 3-O-acetyl-5, 7,3 ', 4' -O-tetra-tert-butyldimethylsilyl-epicatechin.

6. The method for preparing a novel 3C-like protease inhibitor according to claim 3, wherein in the step (4), the ratio of petroleum ether: eluting with 100:1 ethyl acetate to obtain the product 5,7,3 ', 4' -O-tetra-tert-butyldimethylsilyl-epicatechin.

7. The method for preparing a novel 3C-like protease inhibitor according to claim 3, wherein in the step (6), the ratio of petroleum ether: the product, 3 ", 4" -O-diacetyl-5, 7,3 ', 4' -O-tetra-tert-butyldimethylsilyl-epicatechin trans-caffeate, was obtained after elution with ethyl acetate 30: 1.

8. The process for preparing a novel 3C-like protease inhibitor according to claim 3, wherein the final product 3 ', 4' -O-diacetyl-epicatechin trans-caffeate is obtained by silica gel column chromatography, silica gel column chromatography and elution with petroleum ether and ethyl acetate 1:1 in the step (7).

9. Use of a novel 3C-like protease inhibitor according to claim 1 for the manufacture of a medicament against the infection with the novel coronavirus SARS-CoV-2.

10. A medicament against the infection of the novel coronavirus SARS-CoV-2, characterized in that it is prepared from the novel 3C-like protease inhibitor according to claim 1 and pharmaceutical excipients.

Technical Field

The invention relates to the technical field of medicinal chemistry, in particular to a novel 3C-like protease inhibitor and a preparation method and application thereof.

Background

In the novel coronavirus (SARS-CoV-2), 3C-like protease (3CL protease) plays a crucial role in the life cycle of coronavirus, its inactivation inhibits viral replication. Meanwhile, as the protease homologous with the 3C-like protease does not exist in the human body, the 3C-like protease becomes an ideal target for resisting the novel coronavirus.

Disclosure of Invention

The invention aims to provide a novel compound with the effect of inhibiting the activity of 3C-like protease in novel coronavirus, and a preparation method and application thereof.

The invention adopts the following technical scheme to solve the technical problems:

a novel 3C-like protease inhibitor represented by the structure of formula I:

a method for preparing the novel 3C-like protease inhibitor comprises gradually synthesizing epicatechin to obtain 3 ', 4' -O-diacetyl-epicatechin trans-caffeate with a structure shown in formula I.

As one of the preferable modes of the present invention, the method specifically comprises the following steps:

(1) synthesis of 3,5,7,3 ', 4' -O-pentaacetyl-epicatechin

Adding epicatechin into a flask, adding dried pyridine and acetic anhydride, and stirring at room temperature overnight under the protection of a drying tube; after TLC detection reaction is finished, adding water to stop reaction, and performing rotary evaporation to obtain a product 3,5,7,3 ', 4' -O-pentaacetyl-epicatechin;

(2) synthesis of 3-O-acetyl-epicatechin

Putting the compound 3,5,7,3 ', 4' -O-pentaacetyl-epicatechin prepared in the step (1) into a flask, adding methanol water solution and ammonium acetate, and stirring overnight at room temperature; after TLC detection reaction is finished, adding water to stop reaction, carrying out rotary concentration, and extracting for a plurality of times by using ethyl acetate with the same volume; mixing the ethyl acetate extractive solutions, concentrating, performing silica gel column chromatography, and eluting with ethyl acetate and methanol to obtain 3-O-acetyl-epicatechin;

(3) synthesis of 3-O-acetyl-5, 7,3 ', 4' -O-tetra-tert-butyldimethylsilyl-epicatechin

Putting the compound 3-O-acetyl-epicatechin prepared in the step (2) into a flask, adding dried dichloromethane, imidazole and 4-dimethylaminopyridine, slowly dropwise adding a dried dichloromethane solution containing tert-butyldimethylchlorosilane under an ice bath condition, stirring at room temperature overnight, and protecting by using a drying tube in the reaction process; after TLC detection reaction is finished, adding water to stop the reaction, and extracting for a plurality of times by using dichloromethane with the same volume; washing the dichloromethane extract with saturated sodium chloride solution, concentrating, performing silica gel column chromatography, eluting with petroleum ether and ethyl acetate to obtain product 3-O-acetyl-5, 7,3 ', 4' -O-tetra-tert-butyldimethylsilyl-epicatechin;

(4) synthesis of 5,7,3 ', 4' -O-tetra-tert-butyldimethylsilyl-epicatechin

Putting the compound 3-O-acetyl-5, 7,3 ', 4' -O-tetra-tert-butyldimethylsilyl-epicatechin prepared in the step (3) into a flask, dissolving the compound with dry dichloromethane, adding dry methanol and potassium carbonate, stirring the mixture at room temperature for reaction, and protecting the reaction by using a drying tube in the reaction process; after TLC detection reaction is finished, adding water to stop the reaction, and extracting for a plurality of times by using ethyl acetate with the same volume; drying the ethyl acetate extract with anhydrous sodium sulfate, concentrating, performing silica gel column chromatography, eluting with petroleum ether and ethyl acetate to obtain product 5,7,3 ', 4' -O-tetra-tert-butyl dimethyl silicon-epicatechin;

(5) synthesis of 3, 4-O-diacetyl caffeoyl chloride

Adding caffeic acid into a flask, adding dried pyridine and acetic anhydride, and stirring at room temperature overnight under the protection of a drying tube; after TLC detection reaction is finished, adding water to stop reaction, and rotationally evaporating to dryness to obtain a product 3, 4-O-diacetyl caffeic acid;

adding thionyl chloride into the synthesized 3, 4-O-diacetyl caffeic acid, carrying out heating reflux reaction in an oil bath, and protecting by using a drying tube in the reaction process; after the reaction is finished, the reaction solution is subjected to rotary evaporation to dryness to obtain a crude product of the 3, 4-O-diacetyl caffeic acid, which is directly used for the next reaction;

(6) synthesis of 3 ', 4' -O-diacetyl-5, 7,3 ', 4' -O-tetra-tert-butyldimethylsilyl-epicatechin trans-caffeate

Putting the compound 5,7,3 ', 4' -O-tetra-tert-butyldimethylsilyl-epicatechin prepared in the step (4) into a flask, adding dried pyridine and dried dichloromethane, slowly dropwise adding the dried dichloromethane containing the crude product of the 3, 4-O-diacetyl caffeic acid prepared in the step (5) under an ice bath condition, reacting at room temperature overnight, and protecting by using a drying tube in the reaction process; after TLC detection reaction is finished, adding water to stop the reaction, and extracting for a plurality of times by using dichloromethane with the same volume; drying the dichloromethane extract with anhydrous sodium sulfate, concentrating, performing silica gel column chromatography, eluting with petroleum ether and ethyl acetate to obtain product 3 ', 4' -O-diacetyl-5, 7,3 ', 4' -O-tetra-tert-butyldimethylsilyl-epicatechin trans-caffeate;

(7) synthesis of 3 ', 4' -O-diacetyl-epicatechin trans-caffeate

Taking the compound 3 ', 4' -O-diacetyl-5, 7,3 ', 4' -O-tetra-tert-butyldimethylsilyl-epicatechin trans-caffeate prepared in the step (6), adding potassium bifluoride and dry methanol, and stirring to fully react; then, adding water to terminate the reaction, extracting with ethyl acetate, and drying with anhydrous sodium sulfate; and finally, carrying out silica gel column chromatography on the concentrated ethyl acetate layer, and eluting by adopting petroleum ether and ethyl acetate to obtain the final product of 3 ', 4' -O-diacetyl-epicatechin trans-caffeate.

In a preferred embodiment of the present invention, in the step (2), the mixture is purified by silica gel column chromatography using ethyl acetate: eluting with methanol 20:1 to obtain the product 3-O-acetyl-epicatechin.

In a preferred embodiment of the present invention, in the step (3), the reaction mixture is purified by silica gel column chromatography using petroleum ether: eluting with 100:1 ethyl acetate to obtain the product 3-O-acetyl-5, 7,3 ', 4' -O-tetra-tert-butyldimethylsilyl-epicatechin.

In a preferred embodiment of the present invention, in the step (4), the silica gel column chromatography is performed to separate the petroleum ether: eluting with 100:1 ethyl acetate to obtain 5,7,3 ', 4' -O-tetra-tert-butyldimethylsilyl-epicatechin

In a preferred embodiment of the present invention, in the step (6), the reaction mixture is purified by silica gel column chromatography using petroleum ether: the product, 3 ", 4" -O-diacetyl-5, 7,3 ', 4' -O-tetra-tert-butyldimethylsilyl-epicatechin trans-caffeate, was obtained after elution with ethyl acetate 30: 1.

In a preferred embodiment of the present invention, the final product 3 ", 4" -O-diacetyl-epicatechin trans-caffeate is obtained in step (7) by silica gel column chromatography, silica gel column chromatography and elution with petroleum ether and ethyl acetate 1: 1.

An application of the above 3C-like protease inhibitor in preparing medicine for resisting SARS-CoV-2 infection is provided.

A medicine for resisting SARS-CoV-2 infection is prepared from the above 3C-like protease inhibitor and medicinal adjuvants.

Compared with the prior art, the invention has the advantages that: the invention designs and synthesizes a novel compound based on the structure of epicatechin, and the compound can still obviously inhibit the activity of 3C-like protease in novel coronavirus in cells even when the concentration is low; meanwhile, the IC50 value of in vitro test is 0.36 mu M, and the compound is also shown to have the effect of effectively inhibiting the activity of 3C-like protease in the novel coronavirus; therefore, the compound can be used as a 3C-like protease inhibitor for preparing a medicament for resisting the infection of novel coronavirus SARS-CoV-2.

Drawings

FIG. 1 is a scheme showing the synthesis of 3 ', 4' -diacetylyl-ECC in example 2;

FIG. 2 is a mass spectrum of 3 ', 4' -diacetyl-ECC from example 2;

FIG. 3 is a 3 ', 4' -diacetylyl-ECC of example 2¹H nuclear magnetic resonance spectrogram;

FIG. 4 is a 3 ', 4' -diacetylyl-ECC of example 2¹³C nuclear magnetic resonance spectrogram;

FIG. 5 is the DEPT-135 NMR spectrum of 3 ', 4' -diacetyl-ECC in example 2;

FIG. 6 is an HMBC NMR spectrum of 3 ', 4' -diacetyl-ECC from example 2;

FIG. 7 is a graph of the inhibitory activity of intracellular 3 ", 4" -diacetyl-ECC on 3C-like protease in example 3;

FIG. 8 is a graph of the extracellular inhibitory efficiency of the positive control drug Ebselen on 3C protease in example 4;

FIG. 9 is a graph showing the extracellular inhibition efficiency of 3 ', 4' -diacetyl-ECC on 3C protease in example 4.

Detailed Description

The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.

Example 1

A novel 3C-like protease inhibitor of this example.

The invention designs and synthesizes a novel compound based on the structure of epicatechin, the novel compound is 3 ', 4' -O-diacetyl-epicatechin trans-caffeate (3 ', 4' -diacetylyl-ECC), the structure is shown as formula I (the number in the formula I is the corresponding nuclear magnetic data attribution serial number):

the novel compound can still obviously inhibit the activity of 3C-like protease in novel coronavirus in cells even at low concentration, and the novel compound can be used as a novel 3C-like protease inhibitor.

Example 2

The preparation method of the above-mentioned novel 3C-like protease inhibitor (3 ', 4' -diacetyl-ECC) of this example, see FIG. 1, comprises the following steps:

(1) synthesis of 3,5,7,3 ', 4' -O-pentaacetyl-epicatechin (1a)

1.16g (4.0mmol) of Epicatechin (EC) was weighed accurately into a 50mL round-bottomed flask, 6.0mL of dried pyridine and 8.0mL of acetic anhydride (84.6mmol) were added, followed by stirring overnight (about 12 hours) at room temperature under the protection of a dry tube (containing anhydrous calcium chloride); and after TLC detection reaction is finished, adding water to stop the reaction, and performing rotary evaporation to obtain the product 3,5,7,3 ', 4' -O-pentaacetyl-epicatechin (1 a).

(2) Synthesis of 3-O-acetyl-epicatechin (2a)

Accurately weighing 500.0mg (1.0mmol) of the compound 3,5,7,3 ', 4' -O-pentaacetyl-epicatechin (1a) prepared in the step (1) into a 100mL round-bottomed flask, adding 50mL of 80% methanol aqueous solution and 7.8g (101.2mmol) of ammonium acetate, and then stirring at room temperature overnight; after TLC detection reaction is finished, adding a proper amount of water (about 200mL) to terminate the reaction, removing most of methanol through rotary concentration, and extracting for three times by using ethyl acetate with the same volume; combining the three ethyl acetate extracts, concentrating, and performing silica gel column chromatography to obtain a mixture of ethyl acetate: the product 3-O-acetyl-epicatechin (2a) was obtained after elution with methanol 20: 1.

(3) Synthesis of 3-O-acetyl-5, 7,3 ', 4' -O-tetra-tert-butyldimethylsilyl-epicatechin (3a)

200.0mg (0.6mmol) of the compound 3-O-acetyl-epicatechin (2a) obtained in step (2) was accurately weighed in a 100mL round-bottomed flask, 10mL of dried dichloromethane, 816.0mg (12.0 mmol) of imidazole and 40.0mg (0.33mmol) of 4-dimethylaminopyridine were added thereto, 10mL of a dried dichloromethane solution containing 723.4mg (4.8mmol) of t-butyldimethylchlorosilane was slowly dropped under ice bath conditions, and stirred at room temperature overnight, with a dry tube for protection during the reaction. After the TLC detection reaction is finished, adding a proper amount of water (about 100mL) to stop the reaction, and extracting three times by using dichloromethane with the same volume; washing the three dichloromethane extracts with saturated sodium chloride solution, concentrating, and performing silica gel column chromatography with petroleum ether: the product 3-O-acetyl-5, 7,3 ', 4' -O-tetra-tert-butyldimethylsilyl-epicatechin (3a) was obtained after elution with ethyl acetate 100: 1.

(4) Synthesis of 5,7,3 ', 4' -O-tetra-tert-butyldimethylsilyl-epicatechin (4a)

Accurately weighing 100.0mg (0.13mmol) of the compound 3-O-acetyl-5, 7,3 ', 4' -O-tetra-tert-butyldimethylsilyl-epicatechin (3a) prepared in the step (3) into a 50mL round-bottom flask, dissolving the compound with 100 mu L of dried dichloromethane, adding 10mL of dried methanol and 50.0mg (0.38 mmol) of potassium carbonate, stirring the mixture at room temperature for reacting for 2 hours, and protecting the mixture by using a drying tube in the reaction process; after TLC detection reaction is finished, adding a proper amount of water (100mL) to stop the reaction, and extracting for three times by using ethyl acetate with the same volume; the three ethyl acetate extracts were dried over anhydrous sodium sulfate and concentrated, and subjected to silica gel column chromatography with petroleum ether: the product 5,7,3 ', 4' -O-tetra-tert-butyldimethylsilyl-epicatechin (4a) was obtained after elution with ethyl acetate 100: 1.

(5) Synthesis of 3, 4-O-diacetyl caffeoyl chloride (5a)

Accurately weighing 180.1mg (1.0mmol) of caffeic acid into a 25mL round-bottom flask, adding 0.3mL of dried pyridine and 0.5mL of acetic anhydride (5.3mmol), and stirring at room temperature under the protection of a drying tube overnight; after TLC detection reaction is finished, adding a proper amount of water to stop the reaction, and rotationally evaporating to dryness to obtain 3, 4-O-diacetyl caffeic acid;

transferring the synthesized 3, 4-O-diacetyl caffeic acid into a 50mL round-bottom flask, adding 10mL thionyl chloride, heating and refluxing in an oil bath at 90 ℃ for reaction for 3 hours, and protecting by using a drying tube in the reaction process; and (3) rotationally evaporating the reaction solution to dryness (taking down the reaction solution after nitrogen protection) to obtain a crude product of the 3, 4-O-diacetyl caffeic acid (5a) (a small amount of sample is taken and added with anhydrous methanol, and TLC detection is compared with a substrate to find that a new main product point is generated, which proves that the reaction is successful), and directly using the crude product in the next reaction.

(6) Synthesis of 3 ', 4' -O-diacetyl-5, 7,3 ', 4' -O-tetra-tert-butyldimethylsilyl-epicatechin trans-caffeate (pro-ECC)

Accurately weighing 92.0mg (0.12mmol) of the compound 5,7,3 ', 4' -O-tetra-tert-butyldimethylsilyl-epicatechin (4a) prepared in the step (4) into a 25mL round-bottom flask, adding 0.2mL of dried pyridine and 2.0mL of dried dichloromethane, slowly dropwise adding 3.0mL of dried dichloromethane containing the crude product (0.18mmol) of the 3, 4-O-diacetyl caffeic acid (5a) prepared in the step (5) under the ice bath condition, reacting at room temperature overnight, and protecting by using a drying tube in the reaction process; after TLC detection reaction is finished, adding a proper amount of water (100mL) to stop the reaction, and extracting three times by using dichloromethane with the same volume; the three dichloromethane extracts were dried over anhydrous sodium sulfate and concentrated, chromatographed on silica gel column, purified with petroleum ether: the product 3 ", 4" -O-diacetyl-5, 7,3 ', 4' -O-tetra-tert-butyldimethylsilyl-epicatechin trans-caffeate (pro-ECC) was obtained after elution with ethyl acetate 30: 1.

(7) Synthesis of 3 ', 4' -O-diacetyl-epicatechin-trans-caffeate (3 ', 4' -diacetyl-ECC)

158.0mg of the compound 3 ', 4' -O-diacetyl-5, 7,3 ', 4' -O-tetra-tert-butyldimethylsilyl-epicatechin trans-caffeate (pro-ECC) obtained in step (6) was weighed out, 124.3mg of potassium bifluoride (KHF2) and 6mL of dried methanol were added, and the mixture was stirred at 20 ℃ for 3 hours; then, adding a large amount of water to terminate the reaction, extracting with ethyl acetate, and drying with anhydrous sodium sulfate; finally, the concentrated ethyl acetate layer is subjected to silica gel column chromatography, and eluted by petroleum ether and ethyl acetate which are 1:1 to obtain the final product 3 ', 4' -O-diacetyl-epicatechin trans-caffeate (3 ', 4' -diacetylyl-ECC), wherein the final yield is more than 76%.

In this example, mass spectrometry was performed on 3 ', 4' -diacetyl-ECC, and the results are shown in FIG. 2. [ M-H ]]^- m/z 535.1252(C₂₈H₂₃O₁₁Calcd 535.1246) (ESI-HRMS). Therefore, the measured m/z value of the compound under negative ions is 535.1252, the m/z value of the comparative standard negative ion is 535.1246, and the molecular formula is C₂₈H₂₃O₁₁With 17 unsaturations.

For 3 ', 4' -diacetylyl-ECC¹The results of H NMR spectroscopy are shown in FIG. 3.¹H NMR (600MHz,DMSO-d₆):9.31(1H,s,OH-5),9.03(1H,s,OH-7),8.86(1H,s,OH-3'), 8.82(1H,s,OH-4'),7.64(1H,d,J＝1.2Hz,H-2”),7.58(1H,dd,J＝8.4,1.2Hz, H-6”),7.44(1H,d,J＝16.2Hz,H-7”),7.23(1H,d,J＝8.4Hz；H-5”),6.83(1H,d,J ＝1.2Hz,H-2'),6.66(1H,dd,J＝8.4,1.2Hz,H-6'),6.62(1H,d,J＝8.4Hz,H-5'), 6.48(1H,d,J＝16.2Hz,H-8”),5.89(1H,d,J＝1.8Hz,H-6),5.74(1H,d,J＝ 1.8Hz,H-8),5.31(1H,br s,H-3),4.97(1H,s,H-2),2.90(1H,dd,J＝17.4,4.8Hz, H-4a),2.62(1H,br d,J＝17.4Hz,H-4b),2.22(6H,s,3”,4”-diacetyl).

For 3 ', 4' -diacetylyl-ECC¹³The results of C NMR spectroscopy and DEPT-135 NMR spectroscopy are shown in FIGS. 4 and 5, respectively.¹³C NMR (FIG. 4) + DEPT-135 (FIG. 5) (150MHz, DMSO-d₆):168.5(CH₃ C＝O),168.4(CH₃ C＝O),165.8(C＝O,C-9”), 157.1(s,C-7),156.9(s,C-5),155.9(s,C-9),145.2(s,C-3”),145.2(s,C-4”),144.0 (s,C-4'),143.5(d,C-7”),142.8(s,C-3'),133.2(s,C-1'),129.7(s,C-1”),127.4(d, C-6”),124.4(d,C-5”),123.7(d,C-2”),119.4(d,C-8”),118.0(d,C-6'),115.6(d, C-5'),114.7(d,C-2'),97.6(s,C-10),96.0(d,C-6),94.8(d,C-8),76.7(d,C-2),68.7 (d,C-3),25.9(t,C-4),20.8(CH₃C＝O),20.7q(CH₃C＝O).

Finally, HMBC NMR spectroscopy was performed on 3 ', 4' -diacetyl-ECC, the results are shown in FIG. 6. The¹H and ¹³C NMR were assigned by HMBC。

Example 3

Intracellular 3C-like protease inhibition assay:

measuring protease activity in living cells or organisms is a challenging task. Therefore, we used transgenically encoded biosensors to report 3C-like proteases in a highly sensitive and specific manner. The high sensitivity, signal-to-background ratio and specificity of the dual-luciferase biosensor system make it a useful tool for high-throughput study of human cellular proteolytic events and in vivo monitoring of human 3C-like protease activity. The research establishes a cell level screening model of a novel coronavirus 3C-like protease inhibitor by using a dual-luciferase biosensor technology, and performs primary application and evaluation on the screening model.

The specific operation is as follows:

first, day 1 cell inoculation:

293T/17 cells were seeded into 60 mm dishes 20-24 hours ago.

Second, day 2 cell inoculation:

1. 293T/17 cells were transiently transfected with three plasmids. The three plasmids were: a plasmid for expressing 3C protease substrate, wherein the plasmid is provided with renilla luciferase; ② a plasmid for expressing 3C protease; ③ luciferase plasmid of the fluorescent insect.

2. After 6-8 hours, the cells were centrifuged and suspended in growth medium and then counted in a cytometer.

3. The cell suspension in the growth medium is diluted to the desired density.

4. 90 μ L of the cell suspension was placed in a 96-well plate.

5. According to the plate map 10. mu.l of 10 Xcpds were added to 96-well plates with a final DMSO concentration of 0.5% in each well.

Third, measurement on day 3:

1. before measurement, the assay plates were equilibrated to room temperature.

2. Dual luciferase assay reagents were added to each well.

3. Mix on an orbital shaker for 2 minutes to induce cell lysis.

4. Incubate at room temperature for 5 minutes to stabilize the luminescence signal.

5. Luminescence was recorded on Paradigm.

Fourthly, data processing:

1. inhibition (%) was calculated using the following formula relative to vehicle (dmso) -treated control wells:

the suppression ratio (%). is 100- (RLU compound/Flu compound)/(RLU compound/Flu compound): 100%

2. Data were analyzed using Graphpad 7.0 and fitted to a four parameter equation to generate a concentration response curve.

Five results

The results are shown in FIG. 7. The results show that: 3 ', 4' -diacetylyl-EC has obvious inhibition effect on 3C-like protease in cells.

Example 4

Assay of half inhibitory concentration IC50 values for extracellular 3C-like proteases:

we also measured the IC50 value for the 3C-like protease in vitro, as follows:

first, 1. mu.l of 3C-like protease at 1.4. mu.g/. mu.l, 2. mu.l each of compounds at concentrations of 0.015, 0.046, 0.137, 0.412, 1.235, 3.704, 11.111, 33.333 and 100.000. mu.M, and 15. mu.l of Buffer (50mM Tris,1mM EDTA) solution were prepared.

And secondly, mixing and incubating for 30 minutes at normal temperature.

Third, 2. mu.l of substrate was added to the mixture.

Fourthly, mix and incubate for 20 minutes at room temperature.

Fifthly, finally recording luminescence on Paradigm.

And sixthly, processing data.

1. Reactivity (Reaction Activity) (%) was calculated relative to vehicle (dimethylsulfoxide) -treated control wells using the following equation:

Reaction Activity(％)＝RLU compound/RLUdmso control*100％

2. the data were analyzed using Graphpad 7.0, and a four parameter equation was fitted to generate a concentration response curve.

Seven, result in

The results are shown in FIG. 9 (see FIG. 8 for a comparison of the extracellular inhibition efficiency of 3C protease by Ebselen, a positive control drug).

The results show that: the 3 ', 4' -diacetylyl-ECC compound has stronger inhibitory activity to 3C-like protease in the extracellular environment, and IC₅₀The value of (d) is 0.36. mu.M; because the compound has a lower IC for 3C-like proteases₅₀The values indicate that the compound still has strong inhibition effect on 3C-like protease outside cells.

As can be seen from the above examples, the novel compound 3 ', 4' -diacetylyl-ECC, designed and synthesized based on the structure of epicatechin according to the present invention, shows strong inhibitory effect on 3C-like protease both inside and outside the cell, and can be used as a novel 3C-like protease inhibitor. When in specific application, the inhibitor can be used for preparing a medicine for resisting the infection of the novel coronavirus SARS-CoV-2 (the medicine for resisting the infection of the novel coronavirus SARS-CoV-2 is prepared from 3 ', 4' -diacetylyl-ECC and some conventional pharmaceutical auxiliary materials).

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.