阅读说明：本技术 3,4,6-三-o-没食子酰基-d-吡喃葡萄糖在制备抗冠状病毒药物方面的应用 (Application of 3,4, 6-tri-O-galloyl-D-glucopyranose in preparing anti-coronavirus medicine ) 是由 付军 于 2020-12-11 设计创作，主要内容包括：本申请是申请号为202011453321.9、申请日为2020年12月11日、发明名称为“没食子酸及其衍生物和结构类似物在制备抗冠状病毒药物方面的应用”的分案申请。本发明属于生物医药技术领域,具体涉及3,4,6-三-O-没食子酰基-D-吡喃葡萄糖在制备抗冠状病毒药物方面的应用。本发明研究显示,3,4,6-三-O-没食子酰基-D-吡喃葡萄糖对多种冠状病毒科病毒具有明显的抗病毒效果,包括2019新型冠状病毒(2019-nCoV或SARS-CoV-2)和中东呼吸综合征冠状病毒(MERS-CoV)等。而且此类物质来源天然,广泛存在于多种植物中,在食品、生物、医药领域有广泛的应用,十分安全,因此在制备开发针对冠状病毒科的抗病毒药物方面具有很好的应用价值。(The application is a divisional application with the application number of 202011453321.9, the application date of 2020, 12 months and 11 days, and the invention name of the application is the application of gallic acid and derivatives and structural analogues thereof in preparing anti-coronavirus medicines. The invention belongs to the technical field of biological medicines, and particularly relates to application of 3,4, 6-tri-O-galloyl-D-glucopyranose in preparation of an anti-coronavirus medicine. The research of the invention shows that the 3,4, 6-tri-O-galloyl-D-glucopyranose has obvious antiviral effect on various viruses of the family Coronaviridae, including 2019 novel coronavirus (2019-nCoV or SARS-CoV-2) and middle east respiratory syndrome coronavirus (MERS-CoV) and the like. The substances have natural sources, are widely present in various plants, have wide application in the fields of food, biology and medicine, and are very safe, so the substances have good application value in the aspect of preparing and developing antiviral drugs aiming at the coronavirus family.)

The application of 3,4, 6-tri-O-galloyl-D-glucopyranose in preparing anti-coronavirus medicines is characterized in that the 3,4, 6-tri-O-galloyl-D-glucopyranose has the following structural formula:

2. the use of claim 1, wherein the virus is a coronavirus.

3. The use of claim 2, wherein the coronaviruses are SARS-CoV, SARS-CoV-2, MERS-CoV, HCoV-229E, HCoV-NL63, HCoV-OC43 or HCoV-HKU 1.

4. A composition against a virus of the family coronaviridae, comprising 3,4, 6-tri-O-galloyl-D-glucopyranose and sialic acid.

5. The composition as claimed in claim 4, wherein the mass ratio of 3,4, 6-tri-O-galloyl-D-glucopyranose to sialic acid is 1 (0.01-20).

6. The composition according to claim 5, wherein the mass ratio of 3,4, 6-tri-O-galloyl-D-glucopyranose to sialic acid is 1: (0.1-15).

7. The composition of claim 6, wherein the mass ratio of 3,4, 6-tri-O-galloyl-D-glucopyranose to sialic acid is 1: 10.

8. Use of a composition according to any one of claims 4 to 7 for the manufacture of a medicament against a virus of the family Coronaviridae.

9. The use of claim 8, wherein the pharmaceutical dosage form is an injection, an oral agent, a spray, or an inhalant.

10. A medicament against a virus of Coronaviridae, comprising 3,4, 6-tri-O-galloyl-D-glucopyranose or a composition according to any one of claims 4 to 7.

Technical Field

The invention belongs to the technical field of biological medicines. More particularly, relates to the application of 3,4, 6-tri-O-galloyl-D-glucopyranose in preparing anti-coronavirus medicines.

Background

Coronaviruses belong to phylogenetic group of the order of the nested viruses (Nidovirales), the family of Coronaviridae (Coronaviridae), the genus coronaviruses (Coronavirus). Among them, viruses of the genus coronaviruses are enveloped (envelope) RNA viruses whose genome is a linear single-stranded positive strand, and are a large group of viruses widely existing in nature. The coronavirus has the diameter of about 80-120 nm, the 5 'end of the genome has a methylated cap structure, the 3' end has a poly (A) tail, and the total length of the genome is about 27-32 kb, so that the coronavirus is the largest virus in the genome of the currently known RNA viruses. Coronaviruses infect only vertebrates, including humans, and currently, coronaviruses known to infect humans and to bring about a very large epidemic situation are SARS-CoV, SARS-CoV-2, MERS-CoV, HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1, which cause diseases in the respiratory tract, digestive tract, liver, and nervous system. Human coronaviruses have been isolated in 1965, but until now their knowledge has been rather limited, and the serotype and antigenic variability of coronaviruses has not been clarified. Moreover, the coronavirus can be repeatedly infected, which shows that the coronavirus has a plurality of serotypes (at least 4 types are known) and antigenic variation, is difficult to immunize, and has no specific preventive and therapeutic medicine.

Only in experimental studies, it has been found that azauracil (α zauracil) and ribavirin (ribavirin, Virazole) and spiroadamantane (spiroadamantane) have significant inhibitory effects on coronavirus (Qian, Wang Rui. coronavirus infection characteristics and control [ J ] Chinesia Hospital J.Infectiong, 2003,013(011) (1097) -1100.).

However, the medicines are easy to cause adverse reactions such as diarrhea, anemia, dizziness, headache, weakness, hypodynamia and the like, and have certain potential safety hazards; moreover, with the variation of viruses and the appearance of new strains, the antiviral effect of the chemical drugs is greatly reduced, such as the new coronavirus 2019-nCoV appearing at the end of 2019, and until the drugs with exact curative effect are not found at present.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defects and shortcomings of the existing coronavirus control technology and provides a new choice of anti-coronavirus medicines, namely gallic acid and derivatives and structural analogues thereof. The inventor discovers through a great deal of creative work that the seed acid, the derivatives and the structural analogs thereof have obvious anti-coronavirus effects, and the substances are widely present in various plants, have natural sources, are widely applied in the fields of food and biomedicine, and are very safe, so the preparation method has good application prospects in the aspects of preparing and developing antiviral drugs aiming at the coronavirus family.

The invention aims to provide application of 3,4, 6-tri-O-galloyl-D-glucopyranose in preparing anti-coronavirus medicines.

It is another object of the present invention to provide a composition against viruses of the family Coronaviridae.

The invention also aims to provide the application of the composition for resisting the viruses of the family Coronaviridae in preparing anti-coronavirus medicaments.

The invention also aims to provide a medicament for resisting the coronavirus.

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

the research of the invention shows that the gallic acid and the derivatives and structural analogues thereof have obvious antiviral effect on the viruses of the family Coronaviridae.

Preferably, the coronavirus is a coronavirus genus virus.

Specifically, it has now been found that the identified viruses of the genus Coronaviridae include SARS-CoV, SARS-CoV-2 (or 2019-nCoV), MERS-CoV, HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU 1.

In addition, the invention verifies that dozens of reported gallic acid derivatives or gallic acid structural analogs have obvious antiviral effect on coronavirus viruses, the gallic acid derivatives or the gallic acid structural analogs have common galloyl and glycosyl, and experimental results prove that the compounds have good antiviral effect on the existing coronavirus. The mechanism of the correlation is further discussed.

We therefore believe that preferably the gallic acid derivatives are gallic acid derivatives containing galloyl groups and sugar groups and that the gallic acid and its derivatives and structural analogues contain galloyl groups and sugar groups.

Preferably, the gallic acid structural analog is a gallic acid structural analog containing galloyl and sugar groups.

Further, the galloyl is obtained by substituting a substitutable group on gallic acid; the glycosyl is obtained after the substitutable group on the six-carbon sugar is substituted.

Specifically, the gallic acid derivatives and structural analogs include the following (as shown in table 1):

ellagic acid, (-) -epigallocatechin gallate, 1,2,3,4, 6-O-pentagalloyl glucose, 1,2,3,4, 6-5-O-galloyl-beta-D-glucose, epicatechin gallate, quercetin 3-O- (6 "-galloyl) -beta-D-glucoside, kaempferol 3-O- (6" -galloyl) -beta-D-glucopyranoside, 2-O-cinnamoyl-1-O-galloyl-BETA-D-glucoside, 6-O-galloyl glucose, resveratrol-4' -O-BETA-D- (6 "-O-galloyl) glucoside, resveratrol-D, and its preparation method, 4' -hydroxyphenyl-2-butanone-4 ' -O-B-D- (2 ' -O-cinnamoyl-6 ' -O-galloyl glucoside, 4' -hydroxyphenyl-2-butanone-4 ' -O-B-D- (2-O-galloyl-6 ' -O-p-hydroxycinnamoyl) glucoside, 1,3, 6-tri-O-3, 4, 5-trihydroxybenzoyl-BETA-D-glucose, 1-galloylglucose, 1,4, 6-tri-O-galloyl-BETA-D-glucopyranose, resveratrol-4 ' -O- (6 ' -galloyl) glucopyranoside, BETA-glucosidase, BETA, 1,3,4, 6-tetra-O-galloyl-BETA-D-glucopyranose, quercetin 3-O- (6 "-galloyl) - β -D-galactoside, 3-O-galloyl- β -D-glucopyranose, 1, 4-di-O-galloyl- β -D-glucopyranose, 1,4, 6-tri-O-galloyl- β -D-glucopyranose, 1,2,3, 6-tetra-O-galloyl- β -D-glucopyranose, BETA, 1,2,4, 6-tetra-O-galloyl-beta-D-glucopyranose, 6-O-galloyl-D-glucopyranose, 3, 6-di-O-galloyl-D-glucopyranose, 3,4, 6-tri-O-galloyl-D-glucopyranose, 1,3, 4-tri-O-galloyl-6-O-caffeoyl-beta-D-glucopyranose, 1, 3-O-di-galloyl-6-O- (S) -caesalpinia-osa acyl-beta-D-glucopyranose, quercetin galloyl glucoside, galloyl paeoniflorin, galloyl glucose, 3,3' -O-dimethyl ellagic acid, 2,3, 8-tri-O-methyl ellagic acid, 3,4' -O-dimethyl ellagic acid, 4' -O-dimethyl ellagic acid, 3' -di-O-methyl-4-O- (BETA-D-xylopyranosyl) ellagic acid, epicatechin 3-O- (3-O-methyl) gallate, epigallocatechin 3-O- (3-O-methyl) gallate, 2 ' -O-galloylhyperoside, 5-galloylquinic acid, 3-galloylquinic acid, 4-galloylquinic acid, 3-O-galloylmucic acid, galloylquinic acid, galloylmucic acid, galloylquinic acid, galloylquini, 4-O-galloylbergenin.

TABLE 1 Gallic acid derivatives and detailed information

The invention is provided in table 1 as only one of the compounds, and the stereoisomers, geometric isomers, hydrates, solvates or pharmaceutically acceptable salts or prodrugs of the compounds in table 1 shall also fall within the scope of the invention.

The compounds and pharmaceutically acceptable salts of the present invention also include solvate or hydrate forms. In general, the solvate or hydrate forms are equivalent to the unsolvated or non-hydrated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the present invention may exist in polymorphic or amorphous forms. In general, all physical forms are equally useful and contemplated as within the scope of the present invention.

"solvate" of the present invention refers to an association of one or more solvent molecules with a compound of the present invention. Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and aminoethanol. The term "hydrate" refers to an association of solvent molecules that is water.

The compounds of the invention exist in free form or, where appropriate, as pharmaceutically acceptable derivatives. According to the present invention, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable prodrugs, salts, esters, salts of esters, or any other adduct or derivative that can be administered directly or indirectly in accordance with the needs of the patient, compounds described in other aspects of the invention, metabolites thereof, or residues thereof.

In the above pharmaceutical application of the invention, pharmaceutically acceptable adjuvants, carriers, excipients, diluents, vehicles and the like can be added to prepare different pharmaceutical dosage forms, such as injections, oral agents, sprays, inhalants and the like.

To prepare solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient (or carrier) to form a solid preformulation composition comprising a homogeneous mixture of a compound of the present invention. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.

The tablets or pills of the present invention may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action, or to protect the tablets or pills against the action of the acidic conditions found in the stomach. For example, a tablet or pill may include an inner dose and an outer dose component, the latter having the form of a sheath over the former. The two components may be separated by an enteric layer which serves to prevent disintegration in the stomach and to allow the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials may be used for such enteric layers or coatings, including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutical excipients. Preferably, these compositions are administered by the oral or nasal respiratory route to obtain a local or systemic effect. Compositions in preferred pharmaceutically acceptable solvents may be nebulized by the use of inert gases. The nebulized solution may be inhaled directly from the nebulizing device, or the nebulizing device may be attached to a mask tent, or an intermittent positive pressure ventilator. The solution, suspension, or powder compositions may be administered from a device that delivers the dosage form in a suitable manner, preferably by the oral or nasal route.

Based on the above research results, the present invention further provides a composition against viruses of the family coronaviridae, comprising any two or more of gallic acid, gallic acid derivatives, and gallic acid structural analogs.

Further, the composition further comprises sialic acid.

The sialic acid source can be mammal mandible extract, nidus Collocaliae, breast milk, egg, cheese, etc. The mammal may be derived from cattle, pig, etc.

Preferably, the mass ratio of the gallic acid and the derivatives and structural analogues thereof to the sialic acid is 1 (0.01-20).

More preferably, the mass ratio of the galloyl and the derivatives and structural analogs thereof to the sialic acid is 1 (0.1-15).

The compound or the composition claimed by the invention is applied to preparing anti-coronavirus medicines, but is not limited to the application of using an effective amount of the compound or the pharmaceutical composition provided by the invention to prepare medicines for preventing or treating diseases caused by coronavirus, relieving the symptoms of the diseases caused by coronavirus or delaying the development or the onset of the diseases caused by coronavirus.

In addition to being beneficial for human therapy, the presently claimed compounds or compositions may also find application in veterinary therapy for pets, animals of the introduced species and animals in farms, including mammals, rodents, and the like. Other examples of animals include horses, dogs, cats, and the like.

The invention has the following beneficial effects:

the research result of the invention shows that the 3,4, 6-tri-O-galloyl-D-glucopyranose has obvious effect of resisting coronavirus, including SARS-CoV, SARS-CoV-2, MERS-CoV, HCoV-229E, HCoV-NL63, HCoV-OC43 and HCoV-HKU1, and the curative effect is confirmed by live virus experiments, thus the invention has wide application prospect in the field of preparing anti-coronavirus medicines.

Furthermore, the composition consisting of the 3,4, 6-tri-O-galloyl-D-glucopyranose and the sialic acid has more obvious improvement on the effect of resisting the coronavirus and obvious synergistic effect.

In addition, the 3,4, 6-tri-O-galloyl-D-glucopyranose or sialic acid is natural in source, widely exists in various animals and plants, is a natural active compound existing in the nature, has wide application in the fields of food, biology and medicine, is very safe, and has good foundation for developing medicines.

Detailed Description

The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

The active virus experiments of the invention are all entrusted to the Guangdong province disease control center, and the microbial materials are provided by the Guangdong province disease control center.

EXAMPLE 1 half-infected cell number (TCID)₅₀) Measurement of (2)

1. Experimental Material

Virus: 2019-nCoV; cell lines: human embryonic kidney cells 293(HEK293 cells); DMEM medium; 96-well culture plates.

2. Experimental procedure

(1) The virus was serially diluted 10-fold in DMEM medium in a centrifuge tube from 10^-1To 10^-6Incubating for 1-2 hours on ice;

(2) adding virus solutions with different concentrations into a 96-well culture plate, inoculating one longitudinal row of 8 wells in total for each dilution, inoculating 100 mu l of virus solution into each well, and adding DMEM culture medium without virus into the two rows to serve as normal control;

(3) digesting, suspending and counting cultured human embryonic kidney cells 293(HEK293 cells), adding 100 mu l of cell suspension into each well of a 96-well plate containing viruses to ensure that the cell density is 2-3 multiplied by 10⁵Per mL;

(4)37℃、5％CO₂culturing in an incubator, observing cytopathic effect (CPE) until the lowest dilution is no longer diseased (generally, observing for 5-7 days), observing the cytopathic effect (CPE) under an inverted microscope, and recording cytopathic effect;

(5) calculating the half-infection amount (TCID) of the cells by a Reed-Muench method₅₀)。

3. Results of the experiment

According to the cytopathic condition caused by virus infection HEK293 cells, the TCID is calculated by a Reed-Muench method₅₀＝10^－4.7/0.1mL。

Example 2 Effect on the Activity of 2019-nCoV Virus to infect HEK293 cells

1. Experimental materials:

(1) medicine preparation: sialic acid and the compound of table 1.

(2) Other test materials

The virus is 2019-nCoV; the cell line is human embryonic kidney cell 293(HEK293 cell); DMEM medium; 96-well culture plates.

2. Experimental procedure

(1) Taking cell culture fluid, and diluting the medicine into series of mass concentrations (100, 33.33, 11.11, 3.70, 1.23, 0.41 and 0.00 mg/mL);

(2) get 50TCID again₅₀The 2019-nCoV virus solution and the medicine solution are neutralized in equal volume;

(3) incubating at 37 deg.C for 1h, inoculating into HEK293 cell at 100 μ L/well with each mass concentration of 4 multiple wells, simultaneously setting normal cell control group, virus control group and positive control group, incubating at 37 deg.C with 5% CO₂Culturing and observing cell morphology;

(4) after 48h, MT was usedT method, OD determination with microplate reader₄₉₀Value, calculate half Effective Concentration (EC) of drug₅₀)。

3. The experimental results are as follows:

as shown in Table 2, compounds 1-45 all showed higher antiviral activity, with EC₅₀The value range is 3 to 20. mu.g/ml.

TABLE 2 EC of compounds on 2019-nCoV infected human embryonic kidney cell 293(HEK293 cell) line₅₀Value of

Example 3 Effect on the Activity of SARS-CoV Virus infecting HEK293 cells

1. Experimental materials:

(1) medicine preparation: sialic acid and the compound of table 1.

(2) Other test materials

The virus is SARS-CoV; the cell line is human embryonic kidney cell 293(HEK293 cell); DMEM medium; 96-well culture plates.

2. The experimental steps are as follows: reference is made to example 2.

3. The experimental results are as follows:

as shown in Table 3, compounds 1-45 all showed higher antiviral activity, with EC₅₀The value range is 3 to 18. mu.g/ml.

TABLE 3 EC of Compounds on SARS-CoV Virus-infected human embryonic Kidney cell 293(HEK293 cells) line₅₀Value of

Example 4 Effect on the Activity of MERS-CoV Virus to infect HEK293 cells

1. Experimental materials:

(1) medicine preparation: sialic acid and the compound of table 1.

(2) Other test materials

The virus is MERS-CoV; the cell line is human embryonic kidney cell 293(HEK293 cell); DMEM medium; 96-well culture plates.

2. The experimental steps are as follows: reference is made to example 2.

3. The experimental results are as follows:

as shown in Table 4, compounds 1-45 all showed higher antiviral activity, with EC₅₀The value range is 3 to 18. mu.g/ml.

TABLE 4 EC of Compounds on MERS-CoV infected human embryonic Kidney cell 293(HEK 293) line₅₀Value of

EXAMPLE 5 Effect on HCoV-229E Virus Activity to infect HEK293 cells

1. Experimental materials:

(1) medicine preparation: sialic acid and the compound of table 1.

(2) Other test materials

The virus is HCoV-229E; the cell line is human embryonic kidney cell 293(HEK293 cell); DMEM medium; 96-well culture plates.

2. The experimental steps are as follows: reference is made to example 2.

3. The experimental results are as follows:

as shown in Table 5, compounds 1-45 all exhibited higher antiviral activity, with their EC₅₀The value range is 3 to 17. mu.g/ml.

TABLE 5 EC of Compounds on HCoV-229E infected human embryonic Kidney cell 293(HEK 293) line₅₀Value of

Example 6 Effect on the Activity of HCoV-NL63 Virus on infection of HEK293 cells

1. Experimental materials:

(1) medicine preparation: sialic acid and the compound of table 1.

(2) Other test materials

The virus is HCoV-NL 63; the cell line is human embryonic kidney cell 293(HEK293 cell); DMEM medium; 96-well culture plates.

2. The experimental steps are as follows: reference is made to example 2.

3. The experimental results are as follows:

as shown in Table 6, compounds 1-45 all exhibited higher antiviral activity, with their EC₅₀The value range is 3 to 16 mu g/ml.

TABLE 6 EC of Compounds on HCoV-NL63 Virus infected human embryonic Kidney cell 293(HEK293 cell) line₅₀Value of

Example 7 Effect on HCoV-OC43 Virus Activity in infecting HEK293 cells

1. Experimental materials:

(1) medicine preparation: sialic acid and the compound of table 1.

(2) Other test materials

The virus is HCoV-OC 43; the cell line is human embryonic kidney cell 293(HEK293 cell); DMEM medium; 96-well culture plates.

2. The experimental steps are as follows: reference is made to example 2.

3. The experimental results are as follows:

as shown in Table 7, the chemical compounds were converted toCompounds 1-45 all showed higher antiviral activity, with EC₅₀The value range is 3 to 18. mu.g/ml.

TABLE 7 EC of compounds on HCoV-OC43 virus-infected human embryonic kidney cell 293(HEK293 cell) line₅₀Value of

Example 8 Effect on HCoV-HKU1 Virus Activity to infect HEK293 cells

1. Experimental materials:

(1) medicine preparation: sialic acid and the compound of table 1.

(2) Other test materials

The virus is HCoV-HKU 1; the cell line is human embryonic kidney cell 293(HEK293 cell); DMEM medium; 96-well culture plates.

2. The experimental steps are as follows: reference is made to example 2.

3. The experimental results are as follows:

as shown in Table 8, compounds 1-45 all exhibited higher antiviral activity, with their EC₅₀The value range is 3 to 17. mu.g/ml.

TABLE 8 EC of compounds on HCoV-HKU1 virus-infected human embryonic kidney cell 293(HEK293 cells) line₅₀Value of

Example 9 Effect of sialic acid combinations on the Activity of coronavirus infection of HEK293 cells

1. Experimental materials:

(1) medicine preparation: compounds 1,3,4, 5, 14, 17, 18, 22, 23, 24, 25, 28, 29, 30, 39, 40 in table 1; sialic acid (CAS: 131-48-6), compound to sialic acid ratio was 1: 10.

(2) Other test materials

Virus: SARS-CoV, SARS-CoV-2, MERS-CoV, HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU 1; cell lines: human embryonic kidney cells 293(HEK293 cells); DMEM medium; 96-well culture plates.

2. The experimental steps are as follows: reference is made to example 2.

3. The experimental results are as follows:

as shown in tables 9-15, the combination of the compound and sialic acid shows higher antiviral activity, and shows obvious synergistic effect, and the EC of the compound and sialic acid shows₅₀The value range is 2 to 10 mu g/ml.

TABLE 9 EC of compositions on SARS-CoV coronavirus infected human embryonic kidney cell 293(HEK293 cells) lines₅₀Value of

TABLE 10 EC of compositions on SARS-CoV-2 coronavirus infected human embryonic kidney cell 293(HEK293 cells) line₅₀Value of

TABLE 11 EC of compositions on MERS-CoV coronavirus infected human embryonic kidney 293(HEK 293) cell line₅₀Value of

TABLE 12 EC of compositions on HCoV-229E coronavirus infected human embryonic kidney cell 293(HEK293 cells) line₅₀Value of

TABLE 13 EC of compositions on HCoV-NL63 coronavirus infected human embryonic kidney cell 293(HEK293 cell) line₅₀Value of

TABLE 14 EC of compositions on HCoV-OC43 coronavirus infected human embryonic kidney cell 293(HEK293 cells) line₅₀Value of

TABLE 15 EC of compositions on HCoV-HKU1 coronavirus infected human embryonic kidney cell 293(HEK293 cells) line₅₀Value of

EXAMPLE 10 synergistic Effect study with sialic acid compositions against coronavirus infection

1. Experimental materials:

(1) medicine preparation: compound 28 in table 1; sialic acid (CAS: 131-48-6), compound to sialic acid ratio was 1: 10.

(2) Other test materials

Virus: SARS-CoV, SARS-CoV-2, MERS-CoV, HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU 1; cell lines: human embryonic kidney cells 293(HEK293 cells); DMEM medium; 96-well culture plates.

2. The experimental steps are as follows:

diluting the medicine into a series of mass concentrations by taking cell culture solution; get 50TCID again₅₀The virus solution is respectively neutralized with the medicine solution in the same volume; incubating at 37 deg.C for 1h, inoculating into HEK293 cell at 100 μ L/well with each mass concentration of 4 multiple wells, simultaneously setting normal cell control group, virus control group and drug adding group, incubating at 37 deg.C with 5% CO₂Culturing and observing cell morphology; after 48h, the OD was measured by a microplate reader using MTT method₄₉₀Values, simultaneous calculation of cell viability and gold positive mean q-values (q)<0.85 is antagonistic, q is more than or equal to 0.85<1.15 is additive effect, q is more than or equal to 1.15 is synergistic effect).

3. The experimental results are as follows: see tables 16-22.

As can be seen from tables 16-22, the compound 28 of the present invention has a q value of not less than 1.15 for each virus when used in combination with sialic acid, and has an obvious synergistic effect.

TABLE 16 Effect of Compounds and sialic acid against SARS-CoV coronavirus

TABLE 17 Effect of Compounds and sialic acid against SARS-CoV-2 coronavirus

TABLE 18 Compound and sialic acid anti-MERS-CoV coronavirus Effect

TABLE 19 Compound and sialic acid anti-HCoV-229E Virus Effect

TABLE 20 Compounds and sialic acid anti-HCoV-NL 63 Virus Effect

TABLE 21 Compounds and sialic acid anti-HCoV-OC 43 Virus Effect

TABLE 22 Compound and sialic acid anti-HCoV-HKU Virus Effect

In addition, the invention also examines the dosage ratio of the compound 28 and the sialic acid, and the result shows that the mass ratio of the compound 28 and the sialic acid is controlled to be better within the range of 1 (0.01-20) (more preferably 1 (0.1-15), and most preferably 1:10), so that the compound can play a remarkable synergistic effect and can reduce the dosage.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.