PREPARATION METHOD FOR AND APPLICATION OF CLASS OF STELLATE BIFUNCTIONAL COMPOUNDS TARGETING SPIKE PROTEIN AGAINST RESPIRATORY TRACT INFECTION VIRUS AND SALT THEREOF

Abstract

Provided are stellate compounds that target spike proteins and have a significant anti-SARS-CoV-2 ability and a certain broad spectrum. Such molecules and salts thereof have at least one basic unit R(X).sub.n, which binds at an orthotopic binding site such as an MD domain or an allosteric site, to a virus containing spike proteins or a virus having spike proteins on its surface, thereby preventing coronavirus or other viruses which express spike proteins on their surfaces from invading host cells, and preventing the occurrence of viral infection. In addition, interaction of the molecules and salts thereof with vitamin K-dependent proteins in the human body inhibits the expression of vitamin K so as to inhibit blood coagulation in the human body, thereby treating thrombosis caused by coronavirus and producing a curative effect for pneumonia caused by a severe viral infection.

Claims

1. A compound, or a stereoisomer, a hydrate, a solvate, a pharmaceutically acceptable salt, a metabolite, or a prodrug thereof, the compound having at least one basic unit R(X).sub.n, wherein: R is a monosaccharide or a derivative thereof or an aromatic ring, wherein at least one of the monosaccharide or the derivative thereof and the aromatic ring is optionally substituted with at least one substituent selected from the group consisting of amino and carboxyl, n represents an integer selected from 1 to 6, each X is linked to a skeleton atom of R, and the n groups X are identical or different and each independently have a structure ∼QC(O)-R′((X′).sub.m), where: R′ represents an optionally substituted six-membered aromatic ring, coumarin, isocoumarin, flavone, isoflavone, or glucosamine, m represents an integer selected from 1 to 3, the m groups X′ are identical or different and each independently linked to a ring atom of R′, X′ represents QH or QC(O)R″, Q represents —O—, —N—, or —SO.sub.2—, and R″ represents a monosaccharide group.

2. The compound, or the stereoisomer, the hydrate, the solvate, the pharmaceutically acceptable salt, the metabolite, or the prodrug thereof according to claim 1, wherein when X′ represents QC(O)R″, the compound contains a plurality of basic units R(X).sub.n, wherein one of two adjacent basic units R(X).sub.n is linked to a skeleton atom of R in another one of the two adjacent basic units R(X).sub.n through QC(O) in X′, and a recursive hierarchy of the plurality of basic units R(X).sub.n in the compound comprises not more than 2 levels.

3. The compound, or the stereoisomer, the hydrate, the solvate, the pharmaceutically acceptable salt, the metabolite, or the prodrug thereof according to claim 1, wherein when X′ represents QC(O)R″, R″ represents the monosaccharide group, and the compound contains a plurality of basic units R(X).sub.n, wherein one of two adjacent basic units R(X).sub.n is linked to a skeleton atom of R in another one of the two adjacent basic units R(X).sub.n through the monosaccharide group in X′, and a recursive hierarchy of the plurality of basic units R(X).sub.n in the compound comprises not more than 2 levels.

4. The compound, or the stereoisomer, the hydrate, the solvate, the pharmaceutically acceptable salt, the metabolite, or the prodrug thereof according to claim 1, wherein R is the monosaccharide or the derivative thereof or the aromatic ring, the monosaccharide or the derivative thereof or the aromatic ring being optionally substituted with at least one substituent selected from the group consisting of amino and carboxyl; R′ represents the optionally substituted six-membered aromatic ring, coumarin, flavone, or isoflavone; and Q represents —O— or —N—.

5. The compound, or the stereoisomer, the hydrate, the solvate, the pharmaceutically acceptable salt, the metabolite, or the prodrug thereof according to claim 4, wherein the monosaccharide or the derivative thereof is selected from glucose, gluconic acid, or glucosaminic acid, and the aromatic ring is selected from C.sub.4-C.sub.20 aromatic rings.

6. The compound, or the stereoisomer, the hydrate, the solvate, the pharmaceutically acceptable salt, the metabolite, or the prodrug thereof according to claim 5, wherein the aromatic ring is selected from phenyl, chromone, or isoflavone.

7. The compound, or the stereoisomer, the hydrate, the solvate, the pharmaceutically acceptable salt, the metabolite, or the prodrug thereof according to claim 6, wherein R′ represents the optionally substituted six-membered aromatic ring, coumarin, or flavone, and Q represents —O—.

8. The compound, or the stereoisomer, the hydrate, the solvate, the pharmaceutically acceptable salt, the metabolite, or the prodrug thereof according to claim 1, wherein each of the at least one basic unit in the compound is one selected from the following structures: ##STR00016## ##STR00017## ##STR00018## ##STR00019## ##STR00020## ##STR00021## ##STR00022##

9. A pharmaceutical composition, comprising the compound, or the stereoisomer, the hydrate, the solvate, the pharmaceutically acceptable salt, the metabolite, or the prodrug thereof according to claim 1.

10. The pharmaceutical composition according to claim 9, wherein the pharmaceutical composition is in a dosage form selected from powders, tablets, granules, capsules, solutions, emulsions, suspensions, injections, sprays, aerosols, or dry powder inhalations, preferably nasal sprays.

11. A method for preventing and/or treating diseases caused by infections of viruses in host cells, wherein the viruses have spike proteins on surfaces thereof, the method comprising: administering the pharmaceutical composition according to claim 9 to a patient suffering from or suspected of suffering from the diseases caused by the infections of the viruses in the host cells, wherein: optionally, the diseases comprise COVID-19, influenza A, and influenza B; optionally, the viruses comprise coronaviruses and influenza viruses; optionally, the coronaviruses comprise SARS, MERS, and SARS-CoV-2.

12. The method according to claim 11, wherein the pharmaceutical composition is in a dosage form selected from powders, tablets, granules, capsules, solutions, emulsions, suspensions, injections, sprays, aerosols, or dry powder inhalations, preferably nasal sprays.

13. A method for reducing a vitamin K level, comprising administering the pharmaceutical composition according to claim 9 to a patient in need of reducing the vitamin K level.

14. The method according to claim 13, wherein the pharmaceutical composition is in a dosage form selected from powders, tablets, granules, capsules, solutions, emulsions, suspensions, injections, sprays, aerosols, or dry powder inhalations, preferably nasal sprays.

15. A method for preventing and/or treating pulmonary thrombosis, comprising administering the pharmaceutical composition according to claim 9 to a patient suffering from or suspected of suffering from the pulmonary thrombosis.

16. The method according to claim 15, wherein the pharmaceutical composition is in a dosage form selected from powders, tablets, granules, capsules, solutions, emulsions, suspensions, injections, sprays, aerosols, or dry powder inhalations, preferably nasal sprays.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0069] The foregoing and/or additional aspects and advantages of the present disclosure will become apparent and readily appreciated from the following description of the embodiments taken in conjunction with the accompanying drawings, in which:

[0070] FIGS. 1 to 6 show preparation schemes for the synthesis of compounds 1 to 6 in some embodiments provided in the present disclosure;

[0071] FIG. 7 shows the inhibitory activity of compounds 4 and 6 against virus infection of host cells at three different virus titers, wherein the bar graphs represent, from left to right, the percentage of infected cells after treatment with DMSO, 10 .Math.L of compound 4, 20 .Math.L of compound 4, 10 .Math.L of compound 5, and 20 .Math.L of compound 5, respectively, at each virus titer;

[0072] FIG. 8 shows the results of transmission electron microscopy experiments on compounds 4 and 6 inhibiting virus infection of host cells at three different virus titers. Experimental cells: A549; time of virus infection: 48 h; virus titer: 10^9 TU/mL; number of plated cells: 5*10^4/well; virus amount: MOI=20(1*10^6TU);

[0073] FIG. 9 shows changes in fluorescence intensity of virus within host cells with virus infection inhibited by compounds 4 and 6 at three different virus titers; and

[0074] FIG. 10 shows the anticoagulant activity of metabolite of compound 6.

DESCRIPTION OF EMBODIMENTS

[0075] The present disclosure will be further described with reference to specific examples and drawings, but should not be construed as being limited thereto. Without departing from the ideas and essence of the present disclosure, simple modifications or replacements to the methods, steps, or conditions of the present disclosure are within the scope of the present disclosure. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Terms

[0076] The term “include” is intended to be open-ended, i.e., to include what is specified in the present disclosure, without excluding contents of other aspects.

[0077] “Stereoisomer” refer to a compound that has the same chemical structure, but has a different spatial arrangement of atoms or groups. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotamers), geometric isomers, (cis/trans) isomers, atropisomers, and the like.

[0078] “Solvate” of the present disclosure refers to an association compound of one or more solvent molecules with a compound of the present disclosure. Solvents for the formation of 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 compound formed with water as the solvent.

[0079] As used herein, the term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of reasonable medical judgment, suitable for use in contact with the tissues of human and animals without excessive toxicity, irritation, allergic response, or the like, and are commensurate with a reasonable benefit/risk ratio. The pharmaceutically acceptable salts are well known in the art. For example, the pharmaceutically acceptable salts are described in detail by S.M.Berge, et al., J.Pharm.Sci.66:1-19, 1977. These salts can be prepared in situ during the final isolation and purification of the compounds of the disclosure, or prepared separately by reacting a free base group with a suitable organic acid. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzene sulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camsylate, carbonate, chloride, citrate, cypionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, caproate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, tosylate, undecanoate, valerate, etc. Representative alkali or alkaline earth metal salts include salts of sodium, lithium, potassium, calcium, magnesium, etc., as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc.

[0080] “Prodrug” is a pharmacological substance which is administered in an inactive (or significantly less active) form and is subsequently metabolized to an active metabolite in the body. The fundamental principles behind prodrugs is generally used to bring more drugs to the desired target at lower doses, which is generally attributed to better absorption, distribution, metabolism, and/or excretion (ADME) properties. The prodrug is often designed to improve oral bioavailability as poor gastrointestinal absorption is usually the limiting factor. In addition, the use of prodrug strategies may increase the selectivity of a drug for its intended target, thereby reducing the likelihood of off-target effects.

[0081] “One of two adjacent basic units is linked to a skeleton atom of R in another one of the two adjacent basic units through a monosaccharide group in X′” means that the two adjacent basic units are connected by esterification between the hydroxyl group in the monosaccharide group in X′ in one of the two adjacent basic units and the carboxyl group on the skeleton atom of R in the other basic unit.

[0082] “The recursive hierarchy of the basic units in the compound includes not more than 2 levels”.

[0083] The drug of the present disclosure may be prepared in various dosage forms including, but not limited to powders, tablets, granules, capsules, solutions, emulsions, suspensions, injections, sprays, aerosols, or dry powder inhalations, preferably nasal sprays.

[0084] The drug of the present disclosure may employ various pharmaceutically acceptable adjuvants/excipients, including but not limited to solvents, solubilizers, binders, stabilizers, antioxidants, pH modifiers, flavoring agents, etc., depending on the requirements and the common knowledge in the pharmaceutical field.

[0085] According to some specific embodiments of the present disclosure, provided is a class of stellate compounds and salts thereof targeting spike proteins, the compounds having s structure of:

[00001]$\text{R}{\left(\text{X}\right)}_{\text{n}},\text{where n =1 to 6,}$

wherein R is a monosaccharide or an aminomonosaccharide or a monosaccharide carboxyl, or a skeleton of an aromatic ring. X.sub.1-n is a plurality of substituents on the R skeleton, and X.sub.i may be the same as or different from X.sub.i+1, but the total number of substituents on R is not more than 6.

[0086] The structure of X is shown below:

[00002]$~\text{QC}\left(\text{O}\right)\text{-R'}\left({\left(\text{X'}\right)}_{\text{m}}\right),\text{where m =1 to 3}\text{.}$

[0087] R′= {a six-membered aromatic ring, coumarin, flavone, isoflavone, glucosamine}; [0088] Q={—O—, —N—, —SO.sub.2—}, X′={QH, QC(O)R″} [0089] R″= {R(X) .sub.n, a monosaccharide group}

[0090] These molecules and salts thereof bind to spike proteins on the surfaces of respiratory tract infection-causing virus particles containing spike proteins at orthotopic and allosteric sites, resulting in failure of the spike proteins on the surfaces of the virus particles in binding to receptor proteins of host cells (e.g., angiotensin-converting enzyme 2, or neuraminidase), thereby rendering the virus incapable of infecting humans.

[0091] According to some specific embodiments of the present disclosure, R is a monosaccharide or an aminomonosaccharide or a monosaccharide carboxyl, or a skeleton of an aromatic ring. X.sub.1-n is a plurality of substituents on the R skeleton, and X.sub.i may be the same as or different from X.sub.i+1, but the total number of substituents on R is not more than 6. The structure of X is defined as ~QC(O)-R′((X′).sub.m), where m = 1 to 3; R′ is defined as {a six-membered aromatic ring, coumarin, isocoumarin, flavone, and isoflavone}; Q={—O—, —N—}, X′={QH, QC(O)R″}; R″ is defined as {R(X).sub.n, a monosaccharide group}, n = 1 to 6.

[0092] Such molecules and salts thereof decompose into metabolites in the human body, including 3,4,5-trihydroxybenzoic acid glucoside polymers, polyhydroxy coumarin glycoside polymers, polyhydroxy flavone glycoside polymers; such metabolites inhibit the expression level of vitamin K in human body, prevent platelet aggregation, and thus treat thrombosis caused by coronaviruses.

[0093] According to some specific embodiments of the present disclosure, R is a monosaccharide or an aminomonosaccharide or a monosaccharide carboxyl, or a skeleton of an aromatic ring. X.sub.1-n is a plurality of substituents on the R skeleton, and X.sub.i may be the same as or different from X.sub.i+1, but the total number of substituents on R is not more than 6. The structure of X is defined as ~QC(O)-R′((X′).sub.m), where m = 1 to 3; R′ is defined as {a six-membered aromatic ring, coumarin, flavone}; Q={—O—, —N—}, X′={QH, QC(O)R″}; R″ is defined as {R(X).sub.n, a monosaccharide group}, n = 1 to 6. A method for the preparation of this class of compounds is characterized in that polyhydroxybenzoic acid is esterified with monosaccharide at room temperature, and then condensed with polyhydroxybenzoic acid, flavone, or coumarin to obtain stellate molecules. Such molecules and salts thereof bind at an orthotopic binding site, such as a RBD domain or an allosteric site, to SARS-CoV-2 or viruses with spike proteins on their surfaces, thereby preventing coronaviruses or other viruses which express spike proteins on their surfaces from invading host cells, and preventing the occurrence of viral infections.

[0094] According to some specific embodiments of the present disclosure, R is a monosaccharide or an aminomonosaccharide or a monosaccharide carboxyl, or a skeleton of an aromatic ring. X.sub.1-n is a plurality of substituents on the R skeleton, and X.sub.i may be the same as or different from X.sub.i+1, but the total number of substituents on R is not more than 6. The structure of X is defined as ~QC(O)-R′((X′).sub.m), where m = 1 to 3; R′ is defined as {a six-membered aromatic ring, coumarin, flavone}; Q={—O—, —N—}, X′={QH, QC(O)R″}; R″ is defined as {R(X).sub.n, a monosaccharide group}, where n = 1 to 6. A method for the preparation of this class of compounds is characterized in that polyhydroxybenzoic acid is esterified with monosaccharide at room temperature, and then condensed with polyhydroxybenzoic acid, flavone, or coumarin to obtain stellate molecules. Such molecules and salts thereof bind at an orthotopic binding site, such as a RBD domain or an allosteric site, to viruses containing spike proteins or viruses with spike proteins on their surface, thereby preventing coronaviruses or other viruses which express spike proteins on their surfaces from invading host cells, and preventing the occurrence of viral infections.

[0095] According to some specific embodiments of the present disclosure, R.sub.1 is a monosaccharide or an aminomonosaccharide or a monosaccharide carboxyl, or an aromatic ring. X.sub.1-n is a plurality of substituents on the R skeleton, and X.sub.i may be the same as or different from X.sub.i+1, but the total number of substituents on R is not more than 6. The structure of X is defined as ~QC(O)-R′((X′).sub.m), where m = 1 to 3; R′ is defined as {a six-membered aromatic ring, coumarin, flavone}; Q={—O—, —N—}, X′={QH, QC(O)R″}; R″ is defined as {R(X).sub.n, a monosaccharide radical}, where n = 1 to 6. According to a method for preparing this class of compounds, polyhydroxy benzoic acid is esterified with monosaccharide at room temperature, and then condensed with polyhydroxy benzoic acid, flavone, or coumarins to obtain stellate molecules. These molecules and salts thereof interact with vitamin K-dependent proteins in the human body to inhibit the expression of vitamin K so as to inhibit blood coagulation in the human body, thereby treating thrombosis caused by a coronavirus and producing a curative effect for pneumonia caused by a severe viral infection.

[0096] According to some specific examples of the present disclosure, typical stellate compounds are embodied as follows:

##STR00008##

##STR00009##

##STR00010##

##STR00011##

##STR00012##

##STR00013##

##STR00014##

[0097] According to some embodiments of the present disclosure, provided is a method for the preparation of a stellate bifunctional compound targeting spine protein-containing viruses causing pulmonary infections, including the following step:

##STR00015##

[0098] A synthetic scheme for the stellate bifunctional compounds targeting spine protein-containing viruses causing pulmonary infections is shown above. To dichloromethane (DMF, pyridine, tetrahydrofuran, etc. may be used instead) was added phenolic starting materials containing the carboxylic acid group (R.sub.1-COOH), and were added dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) simultaneously. The mixture was stirred at room temperature for 0.5 h. Saccharide with or without protecting groups, aromatic rings with hydroxyl groups, aliphatic chains with hydroxyl groups (R.sub.2-OH) were then added, and the mixture was stirred for 8 h at room temperature or under heating. The mixture was extracted with ethyl acetate or dichloromethane, and the organic layer was subjected to spin-drying to remove the organic solvent, and separation and purification by column chromatography to obtain the desired molecules.

[0099] The Examples of the present disclosure will be described in detail below. The examples described below are exemplary, and are only used to explain the present disclosure but should not be construed as a limitation to the present disclosure. Examples, where specific techniques or conditions are not specified, are implemented in accordance with techniques or conditions described in the literatures in the art or according to the product description. All of the used agents or instruments which are not specified with the manufacturer are conventional and commercially-available products.

Example 1 Synthesis of Compound 1

[0100] 3,4,5-tribenzyloxybenzoic acid (2.2 mmol), dicyclohexylcarbodiimide (DCC, 2.2 mmol), and 4-dimethylaminopyridine (DMAP, 0.2 mmol) were added to dichloromethane while being stirred for 0.5 h at room temperature; then 1,4-benzenediol (1 mmol) was added, and the mixture was stirred at room temperature for 8 h. Then, the mixture was extracted with ethyl acetate, and the organic layer was subjected to spin-drying to remove the organic solvent, and separation and purification by column chromatography to obtain an intermediate. The intermediate and palladium on carbon (60 mg) were added to methanol, air was suctioned out, and then hydrogen was filled; and the mixture was stirred at room temperature for 2 h. The palladium on carbon was removed by filtration and the organic layer was spin-dried. Column chromatography was conducted to give compound 1 with an overall yield of 62.4%. The synthesis scheme of compound 1 is shown in FIG. 1.

Example 2 Synthesis of Compound 2

[0101] 3,4,5-tribenzyloxybenzoic acid (3.3 mmol), dicyclohexylcarbodiimide (DCC, 3.3 mmol), and 4-dimethylaminopyridine (DMAP, 0.3 mmol) were added to dichloromethane while being stirred for 0.5 h at room temperature. Then 2-(6-(benzyloxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)acetamide (1 mmol) was added and the mixture was stirred at room temperature for 8 h. Then the mixture was extracted with ethyl acetate, the organic layer was subjected to spin-drying to remove the organic solvent, and separation and purification by column chromatography to obtain an intermediate. The intermediate and palladium on carbon (100 mg) were added to methanol, air was suctioned out and then hydrogen was filled; and the mixture was stirred at room temperature for 2 h. The palladium on carbon was removed by filtration and the organic layer was spin-dried. Column chromatography was conducted to give compound 2 with an overall yield of 53.5%. The synthesis scheme of compound 2 is shown in FIG. 2.

Example 3 Synthesis of Compound 3

[0102] 3,4,5-tribenzyloxybenzoic acid (3.3 mmol), dicyclohexylcarbodiimide (DCC, 3.3 mmol), and 4-dimethylaminopyridine (DMAP, 0.3 mmol) were added to dichloromethane while being stirred for 0.5 h at room temperature. Then 5,6,8-trihydroxy-4H-chromen-4-one (1 mmol) was added and the mixture was stirred at room temperature for 8 h. Then the mixture was extracted with ethyl acetate, and the organic layer was subjected to spin-drying to remove the organic solvent, and separation and purification by column chromatography to obtain an intermediate. The intermediate and palladium on carbon (100 mg) were added to methanol, air was suctioned out, and then hydrogen was filled; and the mixture was stirred at room temperature for 2 h. The palladium on carbon was removed by filtration and the organic layer was spin-dried. Column chromatography was conducted to give compound 3 with an overall yield of 61.2%. The synthesis scheme of compound 3 is shown in FIG. 3.

Example 4 Synthesis of Compound 4

[0103] 3,4,5-tribenzyloxybenzoic acid (4.4 mmol), dicyclohexylcarbodiimide (DCC, 4.4 mmol), and 4-dimethylaminopyridine (DMAP, 0.4 mmol) were added to dichloromethane while being stirred for 0.5 h at room temperature; Then 5,6,8-trihydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-one (1 mmol) was added and the mixture was stirred at room temperature for 8 h. Then the mixture was extracted with ethyl acetate, and the organic layer was subjected to spin-drying to remove the organic solvent, and separation and purification by column chromatography to obtain an intermediate. The intermediate and palladium on carbon (120 mg) were added to methanol, air was suctioned out, and then hydrogen was filled; and the mixture was stirred at room temperature for 2 h. The palladium on carbon was removed by filtration and the organic layer was spin-dried. Column chromatography was conducted to give compound 4 with an overall yield of 76.3%. The synthesis scheme of compound 4 is shown in FIG. 4.

Example 5 Synthesis of Compound 5

[0104] Compound 1 (1 mmol) and calcium bicarbonate (1 mmol) were added to tert-butanol and the mixture was stirred at room temperature for 12 h, followed by spin-drying to remove the solvent, to give the desired compound 5 with an overall yield of 98.9%. The synthesis scheme of compound 5 is shown in FIG. 5.

[Example 6 Synthesis of Compound 6

[0105] 3,4,5-tribenzyloxybenzoic acid (3.3 mmol), dicyclohexylcarbodiimide (DCC, 3.3 mmol), and 4-dimethylaminopyridine (DMAP, 0.3 mmol) were added to dichloromethane while being stirred for 0.5 h at room temperature. Then 2-(6-(benzyloxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)acetic acid (1 mmol) was added and the mixture was stirred at room temperature for 8 h. Then the mixture was extracted with ethyl acetate, and the organic layer was subjected to spin-drying to remove the organic solvent, and separation and purification by column chromatography to obtain an intermediate. The intermediate and palladium on carbon (100 mg) were added to methanol, air was suctioned out, and then hydrogen was filled; and the mixture was stirred at room temperature for 2 h. The palladium on carbon was removed by filtration and the organic layer was spin-dried. Column chromatography was conducted to give another intermediate b. The intermediate b and zinc hydroxide were added to tert-butanol, the mixture was stirred at room temperature for 2 h, followed by spin-drying to remove the solvent, to give compound 6 with an overall yield of 49.8%. The synthesis scheme of compound 6 is shown in FIG. 6.

Example 7 Anti-Coronavirus Invasion Experiment of Compounds Obtained in Examples 1 to 6

Experimental Materials and Reagents

[0106] H1299 Human lung cancer cells, cultured with RPMI-1640 10% fetal bovine serum in an incubator with 5% CO.sub.2 and saturated humidity at 37° C. [0107] RPMI-1640 + 10% Fetal Bovine Serum [0108] PBS (Life Science Products&Services) [0109] Trypsin-EDTA Solution (Gibco) [0110] 24-well plate (Corning) [0111] Lentivirus-NC virus solution (GenePharma, 1×10.sup.9 TU/mL)

[0112] Principles and procedures for virus infection experiment [0113] 1. H1299 cells in good condition were digested and then re-suspended, and an appropriate amount of cells were inoculated into a 24-well plate and was incubated in an incubator at 37° C. overnight; [0114] 2. negative control viruses were mixed and diluted with 1640 culture medium according to MOI of 5, 10, and 20, with a total volume of about 500 .Math.L, and Polybrene was added with a final concentration of 5 .Math.g/mL; [0115] 3. the original culture medium was pipetted away from the 24-well plate, the negative control virus gradient diluted solution was added instead, and the mixture was incubated in an incubator at 37° C.; [0116] 4. the negative control virus diluted solution was pipetted away after 24 h and replaced with 500 .Math.L of fresh culture medium, and the mixture was incubated in an incubator at 37° C.; [0117] 5. the plate was observed under a fluorescent Inverted microscope after 48 h and the results were recorded, with the results shown in FIGS. 7 to 9.

[0118] The results were shown in FIGS. 7 to 9: FIG. 7 shows the inhibitory activity of compounds 4 and 6 against virus infection of host cells at three different virus titers, wherein the bar graphs represent, from left to right, the percentage of infected cells after treatment with DMSO, 10 .Math.L of compound 4, 20 .Math.L of compound 4, 10 .Math.L of compound 6, and 20 .Math.L of compound 5, respectively, at each virus titer; FIG. 8 shows the results of transmission electron microscopy experiments on compounds 4 and 6 inhibiting virus infection of host cells at three different virus titers. Experimental cells: A549; time of virus infection: 48 h; virus titer: 10^9 TU/mL; number of plated cells: 5*10^4/well; virus amount: MOI=20(1*10^6TU); FIG. 9 shows changes in fluorescence intensity of virus within host cells with virus infection inhibited by compounds 4 and 6 at three different virus titers; and

[0119] FIGS. 7 and 9 show that at different virus titers, stellate compounds 4 and 6 at 20 .Math.M concentration could significantly inhibit virus infection in cells; and stellate compound 4 also significantly inhibited virus infection in cells at 10 .Math.M concentration.

Example 8 Anticoagulation Experiment Study of Metabolites of Compounds Obtained in Examples 1 to 6

1. Principle for Experiments

[0120] Coagulation is a complex process involving a series of coagulation factors, which essentially include two pathways, “intrinsic coagulation pathway” and “extrinsic coagulation pathway”. Activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) are adopted usually in the laboratory to evaluate the anticoagulant effect.

2. Preparation of Reagents

2.1 Preparation of Anticoagulation Experiment Reagents

[0121] APTT kit, PT kit, and TT kit, preheated for 2 min at 37° C. before use.

3. Procedures

3.1 Anticoagulation Experiment Procedures

3.1.1 Preparation of Plasma

[0122] New Zealand rabbits were anaesthetized with chloral hydrate, heart puncture was conducted to collect blood into an anticoagulant tube containing ⅒ vol 3.8% sodium citrate, followed by centrifuging at 3000 rpm for 15 min, supernatant was collected to obtain platelet poor plasma which was stored in a refrigerator for later use.

3.1.2 Preparation of Samples to Be Tested

[0123] The compounds were prepared with DMSO to obtain stock solutions with a concentration of 10 mM. Samples to be tested were diluted with normal saline during the test, with a final concentration of 50 .Math.M, 12.5 .Math.M, 3.13 .Math.M, 0.78 .Math.M, 0.20 .Math.M, and 0.05 .Math.M. The sample to be tested at each concentration had a volume of 50 .Math.L, with normal saline solution of the same volume as a negative control.

3.1.3 Test Method

[0124] APTT Assay: 100 .Math.L of plasma was mixed well with 50 .Math.L of samples to tested respectively, the mixture was pre-heated at 37° C. for 2 min, then 50 .Math.L of APTT reagent was added, followed by incubation at 37° C. for 3 min, addition of 50 .Math.L of 0.025 mol/L CaCl.sub.2, and detection on an automatic blood coagulation analyzer to record the coagulation time.

[0125] PT Assay: 50 .Math.L of plasma was mixed well with 20 .Math.L of samples to be tested respectively, the mixture was pre-heated at 37° C. for 2 min, followed by incubation at 37° C. for 3 min, and addition of 100 .Math.L of PT reagent. The mixture was mixed well and tested to record the coagulation time.

[0126] TT Assay: 100 .Math.L of prepared plasma was mixed well with 50 .Math.L of the sample mixture to be tested, and pre-heated at 37° C. for 2 min, followed by addition of 50 .Math.L of TT reagent to the mixture and detection to record results of the instrument.

3.1.4 Statistical Methods

[0127] One-way analysis of variance (One-Way ANOVA) was performed for multi-group comparison; pairwise comparison for means among groups was performed, with Dunnett’s multiple comparisons test in case of homogeneity of variance, and Dunnett’s T3 multiple comparisons test in case of heterogeneity of variance. P <0.05 means statistical significance.

[00003]$****p<0.0001,***p<0.001,**p<0.01,*p<\mathrm{0.05.}$

[0128] The in vitro effects of Compound 6 on the anti-coagulation indexes APTT, PT, and TT were tested using the above methods, and the results are shown in Table 1 and FIG. 10.

[0129] In the evaluation of coagulation indexes APTT, PT, and TT of compound 6 at different dose gradients of 50.00 .Math.L, 12.50 .Math.L, 3.13 .Math.L, 0.78 .Math.L, 0.20 .Math.L, and 0.05 .Math.L, stellate compound 6 could dose-dependently inhibit plasma coagulation.

TABLE-US-00001 Table 1 In vitro effect of compound 6 on APTT, PT, and TT ( x±s, n = 3) Group APTT/s PT/s TT/s Normal saline 21.24±1.04 10.96±0.39 23.00±0.10 Compound 6 50.00 (.Math.M) 106.51±0.94**** 16.66±0.45*** 38.87±0.77** Compound 6 12.50 (.Math.M) 75.11±2.58*** 13.73±0.17* 28.89±0.68* Compound 6 3.13 (.Math.M) 37.72±1.83** 12.83±0.19* 25.25±0.27** Compound 6 0.78 (.Math.M) 26.84±0.79* 10.54±0.37 23.95±0.07** Compound 6 0.20 (.Math.M) 24.03±1.05 10.11±0.30 22.90±0.09 Compound 6 0.05 (.Math.M) 20.12±0.09 9.76±0.85 23.35±0.31 [0138] Notes: compared to normal saline group, ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.

Example 9 External Pharmaceutics Experiment Study of Compounds Obtained in Examples 1 to 6

[0130] To 0.4 mg to 17.0 mg of the final products described in FIGS. 1 to 6 were conventionally added 0.85 g to 0.9 g of sodium chloride and 1.0 g to 1.9 mg of citric acid, and 100 ml of distilled water was injected conventionally to obtain a nasal spray according to a conventional nasal spray preparation method.

[0131] In addition, description with reference to the term “one embodiment”, “some embodiments”, “an example”, “a specific example”, “some examples” or the like means that a specific feature, structure, material, or characteristic described in combination with the embodiment(s) or example(s) are included in at least one embodiment or example of the present disclosure. In this specification, illustrative expressions of these terms do not necessarily refer to the same embodiment or example. Moreover, the specific feature, structure, material, or characteristic described may be combined in any suitable manner in any one or more embodiments or examples. In addition, without mutual contradiction, those skilled in the art may incorporate and combine different embodiments or examples and features of the different embodiments or examples described in this specification.

[0132] Although the embodiments of the present disclosure have been illustrated and described, it should be understood that the above embodiments are exemplary and should not be construed as limiting the present disclosure, and persons of ordinary skill in the art may make various changes, modifications, replacements, and variations to the above embodiments without departing from the scope of the present disclosure.