Cyano compounds, their preparation methods and uses

Abstract

This invention provides compounds of Formula I, their racemic forms, enantiomers, diastereomers, and pharmaceutically acceptable salts thereof, and their use in the prevention or treatment of related diseases caused by coronavirus and / or small RNA virus infections.

Description

Technical Field

[0001] This invention belongs to the fields of medicinal chemistry and chemical synthesis. Specifically, this invention relates to a class of cyano compounds, their preparation methods, and their uses. Prior Technology

[0002] Coronaviruses are single-stranded, positive-sense RNA viruses. Some coronaviruses can spread widely in the human population and cause severe symptoms. Currently, seven coronaviruses are known to infect humans: HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, and SARS-CoV-2. Most functional proteins of coronaviruses are encoded by the ORF1ab gene, which first translates into a polymer, then cleaves it into multiple active proteins by the 3CL and PL proteases. Therefore, inhibiting 3CL protease activity can effectively inhibit viral replication. Different coronaviruses share high structural homology in their 3CL proteases; therefore, 3CL protease inhibitors have broad-spectrum anti-coronavirus activity.

[0003] Besides coronaviruses, 3CL protease also plays an important role in the hydrolysis of polymers encoded by small RNA viruses, and 3CL protease inhibitors can effectively inhibit small RNA virus replication. Enterovirus 71 is a small RNA virus and one of the common viruses causing hand-foot-and-mouth disease. It can also cause meningitis, brainstem encephalitis, myocarditis, and other diseases. In recent years, enterovirus 71 has caused several outbreaks in infants and young children, and there is still a lack of highly effective treatments in clinical practice.

[0004] Therefore, there is still a need for compounds that can inhibit RNA / small RNA viruses, including coronaviruses and enterovirus 71. Summary of the Invention

[0005] Based on the crystal structure of the 3CL protease, the inventors rationally designed a class of cyano compounds. These compounds can effectively inhibit the 3CL protease activity of coronaviruses and / or small RNA viruses, effectively inhibit the 3CL protease activity of various small RNA viruses, including enterovirus 71, in vitro, and effectively inhibit the replication of small RNA viruses at the cellular level. They can be used to prepare drugs for treating diseases induced by coronaviruses and / or small RNA viruses. The inventors completed this disclosure based on this.

[0006] The primary objective of this invention is to provide cyano compounds as shown in Formula I, their racemic forms, enantiomers, diastereomers, and pharmaceutically acceptable salts thereof.

[0007] A second objective of this invention is to provide a method for preparing such compounds.

[0008] A third object of the present invention is to provide a pharmaceutical composition comprising such compounds.

[0009] A fourth objective of this invention is the use of these compounds in the preparation of 3CL protease inhibitors.

[0010] A fifth objective of this invention is the use of these compounds in the preparation of medicaments for the prevention or treatment of diseases induced by coronaviruses and / or small RNA viruses.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0012] On the one hand, the present invention provides a cyano compound as shown in Formula I, its racemic form, enantiomer, diastereomer and pharmaceutically acceptable salt thereof. Formula I

[0013] On the other hand, the present invention provides a method for preparing compounds of general formula I.

[0014] The technical solution of the present invention has at least the following technical effects:

[0015] The compounds of this invention have 3CL protease inhibitory activity, which can inhibit the hydrolysis of protein complexes involved in the gene expression of coronaviruses and small RNA viruses, thereby inhibiting viral replication and development. They can be used for the prevention and treatment of diseases caused by coronavirus or small RNA virus infections.

[0016] This invention relates to cyano compounds of formula I, their racemic forms, enantiomers, diastereomers, or pharmaceutically acceptable salts: Formula I in, R1 is selected from -COR 8 and -SO 2R 9; R2 and R3 are each independently selected from H, D, C1-C10 alkyl, adamantyl, and C3-C7 cycloalkyl, or R2 and R3 and the carbon atom attached thereto form a 3- to 8-membered carbon ring; X is selected from O, S, S(=O)², and S=O; Y does not exist, or Y is selected from O, S, S(=O)², and S=O; R4 is selected from H, C1~C10 alkyl, C3~C8 cycloalkyl, C6~C20 aryl, C1~C10 alkyl-substituted C6~C20 aryl, C1~C10 alkoxy-substituted C6~C20 aryl, and halo-C6~C20 aryl; R5 is selected from H, C1-C10 alkyl groups, and C3-C7 cycloalkyl groups; Alternatively, R4 and R5 may be linked together to form a C2-C6 alkylene group, thereby connecting X and Y; R 6 is selected from and ; R 7 is selected from H and D; R8 is selected from H, C1~C10 alkyl, C1~C10 alkoxy, C3~C7 cycloalkyl, halogenated C1~C10 alkyl, halogenated C3~C7 cycloalkyl, -NR13R14, C6~C20 aryl, halogenated C6~C20 aryl, C1~C10 alkyl-substituted C6~C20 aryl, halogenated C1~C10 alkyl-substituted C6~C20 aryl, 5~20-membered heteroaryl, and halogenated 5~20-membered heteroaryl; R 9 is selected from C1~C10 alkyl, C3~C7 cycloalkyl, halogenated C1~C10 alkyl, halogenated C3~C7 cycloalkyl, -NR 15R 16, C6~C20 aryl, halogenated C6~C20 aryl, C1~C10 alkyl-substituted C6~C20 aryl, halogenated C1~C10 alkyl-substituted C6~C20 aryl, 5~20-membered heteroaryl, and halogenated 5~20-membered heteroaryl; R13 and R14 are each independently selected from H and C1~C10 alkyl groups; R15 and R16 are each independently selected from H and C1~C10 alkyl groups.

[0017] In some embodiments, R2 and R3 are each independently selected from H, D, C1-C6 alkyl, adamantyl, and C3-C7 cycloalkyl, or R2 and R3 and the carbon atom attached thereto form a 3- to 8-membered carbon ring.

[0018] In some embodiments, R2 and R3 are each independently selected from H, isopropyl, tert-butyl, cyclopentyl, and adamantyl, or R2 and R3 and the carbon atom attached thereto form cyclopropyl and cyclopentyl.

[0019] In some embodiments, one of R2 and R3 is selected from H, and the other is selected from isopropyl, tert-butyl, cyclopentyl, and adamantyl; or, R2 and R3 and the carbon atom attached thereto form cyclopropyl and cyclopentyl.

[0020] In some embodiments, R4 is selected from H, C1-C6 alkyl, C3-C8 cycloalkyl, C6-C10 aryl, C1-C6 alkyl-substituted C6-C10 aryl, C1-C6 alkoxy-substituted C6-C10 aryl, and halogenated C6-C10 aryl; R5 is selected from H, C1-C6 alkyl, and C3-C7 cycloalkyl; Alternatively, R4 and R5 can be linked together to form a C2-C6 alkylene group, thereby connecting X and Y.

[0021] In some embodiments, X is selected from O, S, S(=O)2 and S=O; Y is absent, or Y is selected from O, S and S=O; R4 is selected from C1~C6 alkyl and C6~C10 aryl, and R5 is selected from H; or R4 and R5 are connected to each other to form C2~C6 alkylene, thereby connecting X and Y.

[0022] In some implementations, R4 and R5 are connected to each other to form CH2CH2 and CH2CH2CH2, thereby connecting X and Y.

[0023] In some implementations, X and Y are each independently selected from O, S, and S=O, and R4 and R5 are connected to each other to form CH2CH2 and CH2CH2CH2, thereby connecting X and Y.

[0024] In some implementations, X and Y are each independently selected from O and S, and R4 and R5 are connected to each other to form CH2CH2, thereby connecting X and Y.

[0025] In some implementations, X and Y are both selected from S, or X and Y are both selected from O, and R4 and R5 are connected to each other to form CH2CH2, thereby connecting X and Y.

[0026] In some embodiments, X is selected from S, Y is absent, R4 is selected from phenyl and isopropyl, and R5 is selected from H.

[0027] In some implementations, R6 is selected from... .

[0028] In some implementations, R7 is selected from H.

[0029] In some embodiments, R8 is selected from H, C1-C6 alkyl, C1-C6 alkoxy, C3-C7 cycloalkyl, halogenated C1-C6 alkyl, halogenated C3-C7 cycloalkyl, -NR13R14, C6-C10 aryl, halogenated C6-C10 aryl, C1-C6 alkyl-substituted C6-C10 aryl, halogenated C1-C6 alkyl-substituted C6-C10 aryl, 5-10 heteroaryl, and halogenated 5-10 heteroaryl.

[0030] In some embodiments, R8 is selected from C1-C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkoxy, -NR13R14, C3-C7 cycloalkyl, halogenated C3-C7 cycloalkyl, phenyl, halogenated phenyl, C1-C6 alkyl-substituted phenyl, halogenated C1-C6 alkyl-substituted phenyl, and 5-6 heteroaryl groups.

[0031] In some embodiments, R9 is selected from C1-C6 alkyl, C3-C7 cycloalkyl, halogenated C1-C6 alkyl, halogenated C3-C7 cycloalkyl, -NR15R16, C6-C10 aryl, halogenated C6-C10 aryl, C1-C6 alkyl-substituted C6-C10 aryl, halogenated C1-C6 alkyl-substituted C6-C10 aryl, 5-10 heteroaryl, and halogenated 5-10 heteroaryl.

[0032] In some embodiments, R9 is selected from C1-C6 alkyl, C3-C7 cycloalkyl, phenyl, C1-C6 alkyl-substituted phenyl and halogenated C1-C6 alkyl-substituted phenyl.

[0033] In some embodiments, R13 and R14 are each independently selected from H and C1-C6 alkyl groups.

[0034] In some embodiments, R15 and R16 are each independently selected from H and C1-C6 alkyl groups.

[0035] In some embodiments, R8 is selected from CH3, CF3, CH2CF3, CF2CF3, ... methoxy , Cyclopropyl , phenyl, , , And pyridin-3-yl.

[0036] In some embodiments, R9 is selected from CH3, cyclopropyl, phenyl, p-methylphenyl and p-trifluoromethylphenyl.

[0037] In some embodiments, the cyano compound represented by formula I is selected from the cyano compound represented by formula IA: Formula IA R1, R2, R3, R4, R5, X, and Y are as defined above.

[0038] In some embodiments, the cyano compound represented by Formula I is selected from the cyano compounds represented by Formula IB: Formula IB The definitions of each substituent are as described above.

[0039] In some embodiments, the cyano compound represented by Formula I is selected from any one of the cyano compounds represented by the following general formulas: Formula I-1, Formula I-2, Formula I-3, Formula I-4, Formula I-5, Formula I-6 or Formula I-7; R1, R2, R3 and R6 are as defined above.

[0040] In some embodiments, the compound represented by Formula I of the present invention is selected from the following compounds: Compound 1 Compound 2 Compound 3 Compound 4 Compound 5 Compound 6 Compound 7 Compound 8 Compound 9 Compound 10 Compound 11 Compound 12 Compound 13 Compound 14 Compound 15 Compound 16 Compound 17 Compound 18 Compound 19 Compound 20 Compound 21 Compound 22 Compound 23 Compound 24 Compound 25 Compound 26 Compound 27 Compound 28 Compound 29 Compound 30 Compound 31 Compound 32 Compound 33 Compound 34 Compound 35 Compound 36 Compound 37 Compound 38 Compound 39 Compound 40 Compound 41 Compound 42 Compound 43 Compound 44 Compound 45 or Compound 46.

[0041] This invention provides a method for preparing the compound shown in Formula I, wherein the method is one of the following:

[0042] Method i:

[0043] ia) The compound shown in Formula II and the compound shown in Formula III are reacted by a condensation reaction to obtain the compound shown in Formula IV; Preferably, step ia) is as follows: the compound shown in formula II is reacted with the compound shown in formula III in a solvent at a temperature of -20℃ to 50℃ for 0.1-12 hours by the action of a condensing agent and a base, thereby obtaining the compound shown in formula IV; The solvent is one or a mixture of the following: tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N,N-dimethylacetamide, ethyl acetate, and 1,4-dioxane; Optionally, the condensing agent is one or a mixture of the following: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, 2-hydroxypyridine-N-oxide, 1-propylphosphonic anhydride, 1H-benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate, N,N'-carbonyldiimidazole and O-benzotriazole-tetramethylurea hexafluorophosphate; Optionally, the base is N,N-diisopropylethylamine, triethylamine, and N-methylmorpholine;

[0044] The compound of formula IV (ib) is dehydrated to give the compound of general formula I; Preferably, step ib) is: reacting the compound of formula IV with a dehydrating agent in an anhydrous solvent at a temperature of -20℃ to 50℃ for 1-24 hours to obtain the compound of general formula I; The anhydrous solvent is one or a mixture of the following: tetrahydrofuran, dichloromethane, toluene, 1,4-dioxane, and pyridine; The dehydrating agent is trifluoroacetic anhydride and methyl N-(triethylammonium sulfonate)carbamate; or

[0045] Method ii:

[0046] (iia) The compound shown in formula V is condensed with the compound shown in formula III to obtain the compound shown in formula VI, wherein PG in the compound shown in formula V is a protecting group of the amine group; Preferably, step iia) is as follows: the compound shown in formula V reacts with the compound shown in formula III in a solvent at a temperature of -20℃ to 50℃ for 0.1-12 hours through the action of a condensing agent and a base, thereby obtaining the compound shown in formula VI. The solvent is one or a mixture of the following: tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N,N-dimethylacetamide, ethyl acetate, and 1,4-dioxane. The condensing agent is one or a mixture of the following: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, 2-hydroxypyridine-N-oxide, 1-propylphosphonic anhydride, 1H-benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate, N,N'-carbonyldiimidazole, and O-benzotriazole-tetramethylurea hexafluorophosphate; The base is N,N-diisopropylethylamine, triethylamine, and N-methylmorpholine; The protecting group PG of the amine group is tert-butyloxycarbonyl, benzyl, or p-methoxybenzyl;

[0047] The compound shown in formula VI was deprotected to obtain the compound shown in formula VII; Preferably, step iib) is: reacting the compound shown in formula VI with an organic solution of trifluoroacetic acid or hydrogen chloride or Pd / C / H2 at a temperature of -20℃ to 50℃ to obtain the compound shown in formula VII;

[0048] The compound shown in formula VII (iic) is obtained by amination, sulfonation or condensation reactions to yield the compound shown in formula IV; Preferably, step iic) is: At a temperature of -20℃ to 50℃, the compound shown in Formula VII is subjected to an amination reaction with acetic acid or anhydride under the condition of adding a base to obtain the compound shown in Formula IV; or, The compound of formula VII is sulfonated with sulfonyl chloride or sulfonic anhydride under alkaline conditions at a temperature of -20℃ to 50℃ to obtain the compound of formula IV; or, The compound shown in Formula VII was condensed with a carboxyl compound under the conditions of a condensing agent and a base at a temperature of -20℃ to 50℃ to obtain the compound shown in Formula IV. Optionally, the base is N,N-diisopropylethylamine, triethylamine, and N-methylmorpholine; Optionally, the condensing agent is one or a mixture of the following: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, 2-hydroxypyridine-N-oxide, 1-propylphosphonic anhydride, 1H-benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate, N,N'-carbonyldiimidazole, and O-benzotriazole-tetramethylurea hexafluorophosphate.

[0049] The compound of formula IV (iid) is dehydrated to give the compound of general formula I; Preferably, step iid) is: the compound shown in formula IV is reacted with a dehydrating agent in an anhydrous solvent at a temperature of -20℃ to 50℃ for 1-24 hours to obtain the compound shown in formula I; The anhydrous solvent is one or a mixture of the following: tetrahydrofuran, dichloromethane, toluene, 1,4-dioxane, and pyridine; Optionally, the dehydrating agent is trifluoroacetic anhydride and N-(triethylammonium sulfonate)carbamate. The definitions of each substituent are as described above.

[0050] Another aspect of the present invention provides a pharmaceutical composition comprising one or more of a cyano compound selected from general formula I, its racemic, enantiomer, and diastereomer, and pharmaceutically acceptable salts thereof. The pharmaceutical composition may further comprise one or more pharmaceutically acceptable excipients, diluents, carriers, excipients, or adjuvants.

[0051] Another aspect of the present invention provides a pharmaceutical composition comprising a cyano compound selected from general formula I, or a racemic, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient; optionally, the pharmaceutical composition further comprises ritonavir or a pharmaceutically acceptable salt thereof.

[0052] Another aspect of the present invention provides a pharmaceutical composition comprising a cyano compound selected from general formula I, its racemic, enantiomer, diastereomer or pharmaceutically acceptable salt, and ritonavir or a pharmaceutically acceptable salt thereof.

[0053] In another aspect, the present invention provides a pharmaceutical composition comprising a cyano compound selected from general formula I or a pharmaceutically acceptable salt thereof, ritonavir or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0054] Experiments have shown that the compounds of this invention have inhibitory activity against coronavirus 3CL protease and small RNA virus 3CL protease.

[0055] Therefore, in another aspect, the present invention provides a coronavirus 3CL protease inhibitor and / or a small RNA virus 3CL protease inhibitor, comprising one or more of a cyano compound selected from general formula I, its racemic, enantiomer and diastereomer, and pharmaceutically acceptable salts thereof, or the above pharmaceutical composition.

[0056] The present invention also provides the above-mentioned cyano compounds, their racemic, enantiomers, diastereomers, pharmaceutically acceptable salts thereof, or mixtures thereof, or the use of the above-mentioned pharmaceutical compositions in the preparation of a medicament, said medicament being selected from medicaments for inhibiting coronavirus 3CL protease activity, medicaments for preventing and / or treating coronavirus infection, medicaments for inhibiting small RNA virus 3CL protease activity, and medicaments for preventing and / or treating small RNA virus infection.

[0057] The present invention also provides a method for inhibiting 3CL protease, the method comprising applying to the object to be treated one or more of a cyano compound selected from general formula I of the present invention, its racemic, enantiomer and diastereomer, and pharmaceutically acceptable salts thereof, or a pharmaceutical composition according to the present invention.

[0058] The present invention also provides a method for preventing and / or treating a disease or condition, the method comprising administering to an object requiring treatment one or more of a cyano compound selected from general formula I of the present invention, its racemic, enantiomer and diastereomer, and pharmaceutically acceptable salts thereof, or a pharmaceutical composition according to the present invention, wherein the disease or condition is a 3CL protease-mediated disease or condition, particularly a disease or condition associated with coronavirus infection and / or small RNA virus infection.

[0059] The present invention also provides the use of one or more of the cyano compounds of the above general formula I, their racemic, enantiomers and diastereomers, and pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, in the preparation of medicaments for the prevention or treatment of diseases related to coronavirus and / or small RNA virus infection.

[0060] The present invention also provides a method for preventing or treating diseases associated with coronavirus and / or small RNA virus infection, the method comprising administering to a patient a therapeutically effective dose of one or more of the following pharmaceutical preparations: a cyano compound of general formula I of the present invention, its racemic, enantiomer and diastereomer, and pharmaceutically acceptable salts thereof.

[0061] In some embodiments, the coronavirus is selected from SARS-CoV, MERS-CoV, H229E-CoV, HKU1-CoV, NL63-CoV, OC43-CoV or SARS-CoV-2. In some embodiments, the related diseases caused by the coronavirus infection are selected from respiratory infections, pneumonia, or their complications.

[0062] Terminology Definitions and Explanations

[0063] "C1~C10 alkyl" refers to a straight-chain or branched saturated hydrocarbon group having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. Specific examples of the alkyl group include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, etc.; "C1~C6 alkyl" refers to a straight-chain or branched alkyl group containing 1-6 carbon atoms, including but not limited to methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, hexyl, etc.

[0064] "Alkylene" can be understood as having a straight-chain or branched saturated divalent hydrocarbon group. "C2~C6 alkylene" can be understood as having a straight-chain or branched saturated divalent hydrocarbon group with 2-6 carbon atoms, including but not limited to CH2CH2, CH2CH2CH2, CH2CH2CH2CH2, CH2CH2CH2CH2CH2 or CH2CH2CH2CH2CH2CH2, etc.

[0065] "C3~C8 cycloalkyl" refers to a cyclic alkyl group containing 3-8 cyclic carbon atoms. "C3~C7 cycloalkyl" refers to a cyclic alkyl group containing 3-7 cyclic carbon atoms. The cycloalkyl groups disclosed herein include, but are not limited to, cyclopropyl, methylcyclopropyl, ethylcyclopropyl, dimethylcyclopropyl, cyclobutyl, methylcyclobutyl, ethylcyclobutyl, cyclopentyl, cyclohexyl, etc.

[0066] "C1~C10 alkoxy" refers to a straight-chain, branched, or cyclic alkoxy group containing 1 to 10 carbon atoms. "C1~C6 alkoxy" refers to a straight-chain, branched, or cyclic alkoxy group containing 1 to 6 carbon atoms. The alkoxy groups disclosed herein include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentoxy, isopentoxy, cyclopentoxy, hexoxy, and cyclohexoxy.

[0067] "Aryl" refers to an aromatic ring group consisting of a single carbon ring or a fused polycyclic aromatic ring with a conjugated π-electron system. Aryl groups can have 6-20, 6-14, or 6-12 carbon atoms. "C 6~C 20 aryl" can be understood as aryl groups with 6-20 carbon atoms. Specifically, rings with 6 carbon atoms ("C 6 aryl"), such as phenyl; or rings with 9 carbon atoms ("C 9 aryl"), such as indenyl or indenyl; or rings with 10 carbon atoms ("C 10 aryl"), such as tetrahydronaphthyl, dihydronaphthyl, or naphthyl; or rings with 13 carbon atoms ("C 13 aryl"), such as fluorenyl; or rings with 14 carbon atoms ("C 14 aryl"), such as anthraceneyl. "C 6~C 10 aryl" can be understood as aryl groups with 6-10 carbon atoms. In particular, rings with 6 carbon atoms ("C 6 aryl"), such as phenyl; or rings with 9 carbon atoms ("C 9 aryl"), such as indenyl or indene; or rings with 10 carbon atoms ("C 10 aryl"), such as tetrahydronaphthyl, dihydronaphthyl, or naphthyl.

[0068] "Heteroaryl" is an aromatic cyclic group containing 5-20 ring atoms, one or more of which are heteroatoms selected from N, O, or S, and the remaining ring atoms are carbon. "5-20-membered heteroaryl" can be understood as a heteroaryl group having 5-20 ring atoms, particularly 5, 6, 9, 10, 13, or 14 ring atoms, and containing 1-7 heteroatoms independently selected from N, O, and S. "5-10-membered heteroaryl" can be understood as a heteroaryl group having 5-10 ring atoms, particularly 5, 6, 9, or 10 ring atoms, and containing 1-5 heteroatoms independently selected from N, O, and S. "5-6-membered heteroaryl" can be understood as a heteroaryl group having 5 or 6 ring atoms and containing 1-3 heteroatoms independently selected from N, O, and S. The heteroaryl group described in this invention may be selected from thienyl, furanyl, pyrroleyl, oxazolyl, thiazolyl, imidazoleyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, or thiadiazolyl, as well as their benzo[a] derivatives, such as benzofuranyl, benzothienyl, benzothiazolyl, benzoxazolyl, benzoisoxazolyl, benzoimidazolyl, benzotriazolyl, indazole, indolyl, or isindolyl; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, or triazinyl, as well as their benzo[a] derivatives, such as quinolinyl, quinazolinyl, or isoquinolinyl; or acryloxynyl, indazinyl, purinyl, as well as their benzo[a] derivatives; or terpineyl, phthalazinyl, quinazolinyl, quinoxolinyl, naphthidyl, pteridinyl, zoazolyl, acryloxynyl, phenazinyl, phenothiazinyl, or phenothiazinyl.

[0069] "Halogen" is selected from fluorine, chlorine, bromine, and iodine.

[0070] "Halogenation" includes monohalogenation, polyhalogenation, or full halogenation, which means that one, several, or all of the hydrogen atoms are replaced by halogens.

[0071] "Substitution" refers to the replacement of one or more hydrogen atoms on a group by one or more substituents.

[0072] The term "optional" or "optionally" means that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0073] "Therapeutic effective amount" means the amount of the compound of the present invention used to treat a particular disease, condition, or disorder; (ii) to reduce, improve, or eliminate one or more symptoms of a particular disease, condition, or disorder; or (iii) to delay the onset of one or more symptoms of a particular disease, condition, or disorder described herein. The amount of the compound of the present invention constituting a "therapeutic effective amount" varies depending on the compound, the disease state and its severity, the route of administration, and the age of the mammal to be treated, but may routinely be determined by a person skilled in the art based on their own knowledge and the contents of this disclosure.

[0074] "Pharmaceutically acceptable salts" refer to salts of pharmaceutically acceptable acids or bases, including salts formed by compounds with inorganic or organic acids, and salts formed by compounds with inorganic or organic bases.

[0075] The term "drug combination" refers to a combination containing two or more active ingredients or pharmaceutically acceptable salts thereof. In some embodiments of the present invention, the active ingredients or pharmaceutically acceptable salts thereof in the drug combination may be administered simultaneously; in other embodiments of the present invention, the active ingredients or pharmaceutically acceptable salts thereof in the drug combination may be administered separately or sequentially.

[0076] "Pharmaceutical composition" refers to a mixture of one or more compounds of the present invention or their salts with pharmaceutically acceptable excipients. The purpose of a pharmaceutical composition is to facilitate the administration of the compounds of the present invention to an organism.

[0077] "Pharmaceutical acceptable excipients" refer to those excipients that do not cause significant irritation to the organism and do not impair the biological activity and properties of the active compound. Suitable excipients are those well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc. Simple Explanation of the Diagram

[0078] Figure 1 is a graph showing the inhibition rate versus concentration of compound 2 against the WIV04 strain of SARS-CoV-2 in vitro. Figure 2 is a graph showing the inhibition rate versus concentration of compound 2 against the in vitro activity of SARS-CoV-2 strain B.1.351. Figures 3A and 3B show the inhibitory effect of compound 2 on the viral titer of mouse lungs after infection for 2 days (Figure 3A) and 4 days (Figure 3B), respectively, in Example 26. Figure 4 shows the changes in mouse weight in Example 26. Figure 5 shows the inhibitory effect of compound 2 on viral titer in mouse brains 4 days after infection in Example 26. Implementation

[0079] The present disclosure is further described below through examples. In the following examples, the raw materials are commercially available or can be prepared by methods described in the literature / organic synthesis methods known in the art.

[0080] Example 1

[0081] Preparation of compound 1-1:

[0082] Step 1: Starting materials SMA (2.74 g, 11.85 mmol), 35 mL dichloromethane, and 35 mL DMF were added to a reaction flask. The mixture was cooled to 0 °C, and then starting materials SMB (3.56 g, 11.86 mmol), benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP, 6.29 g, 14.22 mmol), and N-methylmorpholine (NMM, 3.91 mL, 35.56 mmol) were added sequentially. The mixture was heated to room temperature and reacted for 10 h. After the reaction was complete, an appropriate amount of dichloromethane was added. The organic phase was washed sequentially with 1 N hydrochloric acid aqueous solution and saturated brine. After washing, the organic phase was dried over anhydrous sodium sulfate. The organic phase was concentrated to dryness and column chromatography yielded 3.71 g of INT-1; ESI-MS: 433.2 m / z [M+H]+; 1H NMR (400 MHz, DMSO-d6): δH: 3.62 (s, 3H), 3.40-3.31 (m, 4H), 2.70 (dd, J = 13.1, 7.9 Hz, 1H), 2.37 (dd, J = 13.2, 8.4 Hz, 1H), 1.37 (s, 9H), 0.94 (s, 9H).

[0083] Step 2: Add INT-1 (3.71 g, 8.58 mmol), 37 ml of THF, 37 ml of purified water, and lithium hydroxide monohydrate (0.72 g, 17.16 mmol) to a reaction flask. Incubate at room temperature for 2 h. After the reaction is complete, adjust the pH to 4 with concentrated hydrochloric acid, filter, and obtain 3.4 g of compound 1-1. ESI-MS: 419.2 m / z [M+H]+; 1H NMR (400 MHz, DMSO-d6): δH: 12.68 (s, 1H), 6.71 (d, J = 9.4 Hz, 1H), 4.38–4.19 (m, 2H), 4.11 (d, J = 9.4 Hz, 1H), 3.88 (d, J = 10.9 Hz, 1H), 3.41–3.29 (m, 4H). 2.69 (dd, J = 13.1, 7.9 Hz, 1H), 2.34 (dd, J = 13.2, 8.9 Hz, 1H), 1.38 (s, 9H), 0.94 (s, 9H).

[0084] Preparation of compounds 1-2:

[0085] Ammonia-methanol solution (700 ml, 7 mol / L) and starting material SMD (100 g, 0.349 mol) were added to a reaction flask, stirred until dissolved, and kept at 25±5℃ for 36 h. After the reaction was completed, the reaction solution was concentrated to about 250 ml of remaining reaction solution. 300 ml of isopropanol was added and the solution was concentrated under reduced pressure to about 250 ml of remaining reaction solution (repeated three times). Nitrogen was used to purge the solution, and the temperature was lowered to 10±5℃. 500 ml of hydrogen chloride-isopropanol solution (4 mol / L) was added to the reaction vessel. After the addition was completed, the temperature was raised to 25±5℃ and kept at 25±5℃ for 9 h. After the reaction was complete, the reaction solution was concentrated under reduced pressure to approximately 250 ml. 300 ml of isopropanol was added, and the solution was further concentrated under reduced pressure to approximately 250 ml (repeated twice). 100 ml of isopropanol was added, and the mixture was stirred for 30 ± 5 min. The mixture was filtered, and the filter cake was washed with 50 ml of isopropanol to obtain a wet product. The product was dried under vacuum at 45 ± 5 °C to obtain 66.7 g of compound 1-2, yield: 92%. ¹H NMR (400 MHz, DMSO-d₆): δH: 8.45 (d, J = 5.1 Hz, 3H), 8.25 – 8.04 (m, 1H), 7.95 (s, 1H), 7.67 – 7.49 (m, 1H), 3.85–3.80 (m, 1H), 3.19–3.13 (m, 2H), 2.59 – 2.51 (m, 2H). 1H), 2.32-2.27 (m, 1H), 2.05-1.98 (m, 1H), 1.82 – 1.66 (m, 2H); ESI-MS: 172.1 m / z [M+H] +.

[0086] Preparation of Compound 1:

[0087] Compound 1-1 (419 mg, 1 mmol) was placed in a double-necked flask, and 5 mL of dichloromethane was added under nitrogen protection, followed by 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (400 mg, 1.1 mmol). The mixture was stirred at room temperature for 1 h. Compound 1-2 (1 mmol) was dissolved in 1 mL of dichloromethane and added to the above system. Then, N,N-diisopropylethylamine (0.5 mL, 1 mmol) was added under an ice-water bath. The ice-water bath was removed, and the system was stirred at room temperature overnight. For post-treatment, 50 mL of dichloromethane was added, followed by washing three times with 1M hydrochloric acid solution, three times with saturated sodium bicarbonate solution, and the organic phase was washed with saturated brine. The mixture was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain compound 1-3 (white solid, 469 mg, yield 82%). ESI-MS: m / z 572.3 [M+H]+.

[0088] Compounds 1-3 (114 mg, 0.2 mmol) and Burgess reagent (1.5 eq) were added to a two-necked flask. After purging with nitrogen three times, dichloromethane dried with molecular sieves was added. The mixture was stirred overnight at room temperature. Thin-layer chromatography showed that the starting material was essentially completely reacted. Post-treatment, column chromatography yielded compound 1 (white solid, 52 mg, 47% yield), ESI-MS: 554.3 m / z [M+H]+.

[0089] Example 2

[0090] Compounds 1-3 (572 mg, 1 mmol) were dissolved in 3 mL of 4M hydrogen chloride / 1,4-dioxane solution, or dissolved in 2 mL of dichloromethane followed by dropwise addition of 2 mL of trifluoroacetic acid. The mixture was stirred at ambient temperature, and thin-layer chromatography (TLC) showed that the starting material had essentially reacted completely. The solvent was then thoroughly evaporated. The crude product was dissolved in 2 mL of dichloromethane under nitrogen protection. Triethylamine (3 mmol) was added, and the system was placed in an ice-water bath. Trifluoroacetic anhydride (1.2 mmol) was added dropwise. TLC showed that the starting material had essentially reacted completely. 50 mL of dichloromethane was added, followed by washing three times with 1M hydrochloric acid aqueous solution, three times with saturated sodium bicarbonate aqueous solution, and the organic phase was washed with saturated brine. The solution was dried over anhydrous sodium sulfate and purified by column chromatography to give compound 2-1 (white solid, 265 mg, 46% yield). ESI-MS: m / z 568.3 [M+H]+.

[0091] Compound 2-1 (113 mg, 0.2 mmol) and Burgess reagent (1.5 eq) were added to a two-necked flask, and after purging with nitrogen three times, dichloromethane dried with molecular sieves was added. The mixture was stirred overnight at room temperature, and thin-layer chromatography showed that the starting material was basically completely reacted. Post-processing and column chromatography yielded compound 2 (white solid, 41 mg, 47% yield). ¹H NMR (400 MHz, DMSO-d6) δ 9.46 (d, J = 8.7 Hz, 1H), 9.05 (d, J = 8.6 Hz, 1H), 7.67 (s, 1H), 4.97 (ddd, J = 11.0, 8.5, 5.0 Hz, 1H), 4.53 (d, J = 8.7 Hz, 1H), 4.34 (dd, J = 9.9, 7.1 Hz, 1H), 4.26–4.14 (m, 1H), 3.92 (d, J = 10.9 Hz, 1H), 3.50–3.34 (m, 4H). 3.22–3.11 (m, 1H), 3.06 (td, J= 9.3, 7.1 Hz, 1H), 2.68–2.58 (m, 1H), 2.50–2.43 (m, 1H), 2.31 (dd, J= 13.0, 10.0 Hz, 1H), 2.23–2.07 (m, 2H), 1.71 (tdd, J= 14.9, 10.3, 7.4 Hz, 2H), 0.99 (s, 9H). ESI-MS: 550.3 m / z [M+H] +.

[0092] Example 3

[0093] Following the same method as in Example 2, except that acetic anhydride was used instead of trifluoroacetic anhydride, compound 3 (white solid, 45 mg, yield 45%) was obtained, ESI-MS: 496.3 m / z [M+H]+.

[0094] Example 4

[0095] Following the same method as in Example 2, except that cyclopropane carboxylic anhydride was used instead of trifluoroacetic anhydride, compound 4 (white solid, 32 mg, yield 31%) was obtained, ESI-MS: 522.3 m / z [M+H]+.

[0096] Example 5

[0097] Following the same method as in Example 1, except that 5-1 was used instead of 1-1, compound 5 (white solid, 22 mg, 20% yield) was obtained. ¹H NMR (400 MHz, DMSO-d6) δ 9.03 (d, J = 8.4 Hz, 1H), 7.67 (s, 1H), 7.41 (dd, J = 9.1, 2.6 Hz, 1H), 4.96 (ddd, J = 10.6, 8.4, 5.4 Hz, 1H), 4.58–4.50 (m, 1H), 4.34 (dd, J = 9.8, 7.2 Hz, 1H), 4.26–4.21 (m, 1H), 3.89 (d, J = 11.0 Hz, 1H), 3.45–3.33 (m, 4H), 3.22–3.03 (m, 2H), 2.64–2.57 (m, 1H), 2.45 (ddd, J= 10.1, 8.5, 4.5 Hz, 1H), 2.31 (dd, J= 12.9, 9.8 Hz, 1H), 2.20–2.10 (m, 2H), 1.84–1.67 (m, 2H), 1.39–1.31 (m, 2H), 1.24 (t, J= 9.5 Hz, 2H), 0.97 (s, 9H). ESI-MS: 540.2 m / z [M+H] +.

[0098] Example 6

[0099] Following the same method as in Example 1, except that 6-1 was used instead of 1-1, compound 6 (white solid, 38 mg, yield 37%) was obtained, ESI-MS: 512.2 m / z [M+H]+.

[0100] Example 7

[0101] Following the same method as in Example 1, except that 1-1 was replaced with 7-1, compound 7 (white solid, 49 mg, yield 44%) was obtained, ESI-MS: 553.3 m / z [M+H]+.

[0102] Example 8

[0103] Following the same method as in Example 2, except that benzoic anhydride was used instead of trifluoroacetic anhydride, compound 8 (white solid, 44 mg, yield 39%) was obtained, ESI-MS: 558.3 ​​m / z [M+H]+.

[0104] Example 9

[0105] Following the same method as in Example 2, except that 3,5-bis(trifluoromethyl)benzoyl chloride was used instead of trifluoroacetic anhydride to give compound 9 (white solid, 39 mg, yield 28%), ESI-MS: 694.2 m / z [M+H]+.

[0106] Example 10

[0107] Following the same method as in Example 2, except that 3,5-dimethylbenzoyl chloride was used instead of trifluoroacetic anhydride to give compound 10 (white solid, 31 mg, yield 26%), ESI-MS: 586.2 m / z [M+H]+.

[0108] Example 11

[0109] Following the same method as in Example 2, except that 3,3,3-trifluoropropionic anhydride was used instead of trifluoroacetic anhydride to obtain compound 11 (white solid, 28 mg, yield 25%), ESI-MS: 564.2 m / z [M+H]+.

[0110] Example 12

[0111] Following the same method as in Example 2, except that 3-pyridinemethyl chloride was used instead of trifluoroacetic anhydride, compound 12 (white solid, 39 mg, yield 35%) was obtained, ESI-MS: 559.2 m / z [M+H]+.

[0112] Example 13

[0113] Following the same method as in Example 2, except that pentafluoropropionic acid chloride was used instead of trifluoroacetic anhydride, compound 13 (white solid, 41 mg, yield 34%) was obtained, ESI-MS: 600.2 m / z [M+H]+.

[0114] Example 14

[0115] Following the same method as in Example 1, except that 1-1 was replaced with 14-1 to obtain compound 14 (white solid, 32 mg, yield 29%), ESI-MS: 550.2 m / z [M+H]+.

[0116] Example 15

[0117] Following the same method as in Example 2, except that methanesulfonic anhydride was used instead of trifluoroacetic anhydride, compound 15 (white solid, 38 mg, yield 36%) was obtained. ¹H NMR (400 MHz, Methanol-d⁴) δ 5.06 (dd, J = 11.5, 4.5 Hz, 1H), 4.46 (dd, J = 10.3, 7.1 Hz, 1H), 4.27 (dd, J = 11.0, 1.6 Hz, 1H), 4.02–3.89 (m, 2H), 3.45 (qt, J = 8.6, 4.7 Hz, 4H), 3.30–3.23 (m, 1H), 2.92 (s, 3H), 2.78 (tdd, J = 10.3, 8.5, 4.0 Hz, 1H). 1H), 2.68 (ddd, J= 13.0, 7.2, 1.6 Hz, 1H), 2.51 (dd, J= 13.0, 10.3 Hz, 1H), 2.45–2.27 (m, 2H), 1.94–1.77 (m, 2H), 1.05 (s, 9H). ESI-MS: 532.2 m / z [M+H] +.

[0118] Example 16

[0119] Following the same method as in Example 2, except that cyclopropanesulfonic acid chloride was used instead of trifluoroacetic anhydride, compound 16 (white solid, 29 mg, yield 26%) was obtained. ¹H NMR (400 MHz, Methanol-d⁴) δ 5.06 (dd, J = 11.5, 4.5 Hz, 1H), 4.44 (dd, J = 10.3, 7.0 Hz, 1H), 4.28 (dd, J = 11.0, 1.6 Hz, 1H), 4.00–3.94 (m, 2H), 3.52–3.38 (m, 4H), 3.31–3.21 (m, 1H), 2.79 (tdd, J = 10.4, 8.5, 4.1 Hz, 1H), 2.72–2.65 (m, 1H), 2.57 (ddd, J= 7.9, 6.2, 3.9 Hz, 1H), 2.51 (dd, J= 13.0, 10.4 Hz, 1H), 2.43–2.29 (m, 2H), 1.92–1.74 (m, 2H), 1.34–1.29 (m, 1H), 1.14 (qt, J= 6.4, 3.8 Hz, 1H), 1.06 (s, 9H), 1.01 (ddt, J= 7.9, 5.8, 2.7 Hz, 2H), 0.97–0.87 (m, 1H). ESI-MS: 558.2 m / z [M+H] +.

[0120] Example 17

[0121] Following the same method as in Example 2, except that benzenesulfonyl chloride was used instead of trifluoroacetic anhydride to give compound 17 (white solid, 35 mg, yield 29%), ESI-MS: 594.2 m / z [M+H]+.

[0122] Example 18

[0123] Following the same method as in Example 1, except that 1-1 was used instead of 1-1, compound 18 was obtained (white solid, 44 mg, yield 42%), ESI-MS: 518.2 m / z [M+H]+.

[0124] Example 19

[0125] Following the same method as in Example 1, except that 1-1 was used instead of 1-1, compound 19 (white solid, 41 mg, yield 36%) was obtained, ESI-MS: 568.2 m / z [M+H]+.

[0126] Example 20

[0127] Following the same method as in Example 1, except that 20-1 was used instead of 1-1 to obtain compound 20 (white solid, 45 mg, yield 35%), ESI-MS: 648.2 m / z [M+H]+.

[0128] Example 21

[0129] Following the same method as in Example 1, except that 21-1 was used instead of 1-1 to obtain compound 21 (white solid, 34 mg, yield 31%), ESI-MS: 640.2 m / z [M+H]+.

[0130] Example 22

[0131] Following the same method as in Example 1, except that 22-1 was used instead of 1-1 and 22-1 was used instead of 1-2, compound 22 (white solid, 22 mg, yield 18%) was obtained, ESI-MS: 614.2 m / z [M+H]+.

[0132] Example 23

[0133] Compound 23-1 (497 mg, 1 mmol) was placed in a double-necked flask, and 5 mL of dichloromethane was added under nitrogen protection, followed by 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (400 mg, 1.1 mmol). The mixture was stirred at room temperature for 1 h. Compound 1-2 (or its corresponding salt, 1 mmol) was dissolved in 1 mL of dichloromethane and added to the above system. Then, N,N-diisopropylethylamine (0.5 mL, 1 mmol) was added under an ice-water bath. The ice-water bath was removed, and the system was stirred at room temperature overnight. For post-treatment, 50 mL of dichloromethane was added, followed by washing three times with 1M hydrochloric acid solution, three times with saturated sodium bicarbonate solution, and the organic phase was washed with saturated brine. The mixture was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain compound 23-2 (white solid, 421 mg, yield 65%). ESI-MS: m / z 650.3[M+H]+.

[0134] Compound 23-2 (325 mg, 0.5 mmol) was dissolved in 1.5 mL of 4M hydrogen chloride / 1,4-dioxane solution, or dissolved in 1 mL of dichloromethane followed by dropwise addition of 1 mL of trifluoroacetic acid. The mixture was stirred at ambient temperature, and thin-layer chromatography (TLC) showed that the starting material had essentially reacted completely. The solvent was then thoroughly evaporated. The crude product was dissolved in 2 mL of dichloromethane under nitrogen protection. Triethylamine (1.5 mmol) was added, and the system was placed in an ice-water bath. Trifluoroacetic anhydride (0.6 mmol) was added dropwise. After TLC showed that the starting material had essentially reacted completely, 25 mL of dichloromethane was added, followed by washing three times with 1M hydrochloric acid aqueous solution, three times with saturated sodium bicarbonate aqueous solution, and the organic phase was washed with saturated brine. The solution was dried over anhydrous sodium sulfate and purified by column chromatography to give compound 23-3 (white solid, 167 mg, yield 52%). ESI-MS: m / z 646.3 [M+H]+.

[0135] Compound 23-3 (130 mg, 0.2 mmol) and Burgess reagent (1.5 eq) were added to a two-necked flask. After purging with nitrogen three times, dichloromethane dried with molecular sieves was added. The mixture was stirred overnight at room temperature. Thin-layer chromatography showed that the starting material was essentially completely reacted. Post-treatment, column chromatography yielded compound 23 (white solid, 37 mg, 29% yield), ESI-MS: 628.2 m / z [M+H]+.

[0136] Example 24: Assay on the inhibitory activity of the compound against SARS-CoV-2 3CLpro

[0137] The inhibitory activity of compounds against SARS-CoV-2 3CL pro enzyme activity was evaluated using fluorescence resonance energy transfer (FRET). The total volume of the enzymatic reaction system was 120 µL, with a final protease concentration of 30 nM and a final acceptor concentration of 20 µM. The reaction buffer consisted of 50 mM Tris (pH 7.3) and 1 mM EDTA. SARS-CoV-2 3CL protease and different concentrations of the compound were added to a 96-well plate and reacted at 30 °C for 10 min. The acceptor was then added and the plate was quickly placed in a microplate reader for reading. The excitation and emission wavelengths were 320 nM and 405 nM, respectively. The test duration was 3.5 min, with fluorescence readings taken every 35 s. The final result was calculated by fitting the reaction rate to the readings from the first 2 mins and comparing it with the control group (DMSO). The IC50 value and inhibition rate curve were obtained using GraphPad Prism 8 software.

[0138] The experimental results are shown in Table 1. The results indicate that the compounds of this invention have a potent inhibitory effect on SARS-CoV-2 3CL pro. The IC50 values ​​range as follows: A represents <0.1 μM, B represents 0.1-1 μM, and C represents 1-10 μM.

[0139] Table 1: Compounds [1]-

[23] Inhibitory effect on SARS-CoV-2 3CL pro Compound numbering Compound structural formula IC 50 (µM) 1 A 2 A 3 A 4 A 5 A 6 B 7 A 8 C 9 C 10 C 11 B 12 C 13 A 14 A 15 A 16 A 17 C 18 A 19 C 20 B 21 A 22 A 23 A

[0140] Example 24-2: Test of inhibitory activity of compound 2 against SARS-CoV-2 Omeprone mutant 3CL protease

[0141] Experimental principle: The inhibitory effect of the compound of this invention on the activity of the mutant 3CL protease (P132H) of Omecron strain was studied by using the method of fluorescence resonance energy transfer (FRET) generated by enzyme-substrate reaction.

[0142] The experimental materials are shown in the table below: Reagents and materials brand Item number 3CL protease-P132H mutant Shanghai Panchao / Dabcyl-KTSAVLQSGFRKME-Edans (Coronavirus main protease fluorescent receptor) Azure Sky P9733 DTT (dithiothreitol) Invitrogen P2325 BSA (Bovine Serum Albumin) Sigma V900933 EDTA (ethylenediaminetetraacetic acid) Invitrogen AM9260G Tris-HCl (tris-hydroxymethylaminomethane) Sangon Biotech B548127

[0143] Experimental instruments and equipment: Instrument Name brand model Echo Nanoscale Acoustic Plugging System Labcyte Echo 650 Flexstation 3 Micro-disc Inspection Instrument MolecMar Devices FLEX3 centrifuge Eppendorf 5810

[0144] Experimental steps:

[0145] Prepare reaction buffer containing 20 mM Tris-HCl, 1 mM EDTA, 0.01% BSA, 1 mM DTT, and 100 mM NaCl. Dilute the test compounds to different concentrations in dimethyl sulfoxide (DMSO) using an Echo pipetting system and transfer them to 384-well plates. Dilute the mutant 3CL protease with the reaction buffer and add 10 µL / well to each 384-well plate. Centrifuge at 1000 rpm for 1 min, then incubate at room temperature for 30 min. Add 10 µL / well of the acceptor and centrifuge at 1000 rpm for 30 s to initiate the enzyme reaction. The final enzyme concentration was 50 nM, the final acceptor concentration was 20 μM, and the compound concentration ranged from 10000 nM to 0.51 nM. Subsequently, using the Kinetic Reduction Vmax mode on the Flexstation 3 microplate reader, fluorescence values ​​at a wavelength of 490 nm were continuously read every 75 seconds for a total of 35 times to obtain the reaction rate value (V). The inhibition rate was calculated, and the half-maximum inhibitory concentration (IC50) was obtained by performing four-parameter fitting using XLfit software. The inhibition rate calculation method is as follows:

[0146] Inhibition rate = (V max - V compound) / (V max - V min) * 100%

[0147] Wherein, Vmax is the reaction rate value of a pore containing only the enzyme and the substrate, Vmin is the reaction rate value of a pore containing only the substrate, and VCompound is the reaction rate value of a pore containing the analyte compound, the enzyme, and the substrate.

[0148] Experimental results: Compound 2 still maintained significant inhibitory activity against the 3CL protease with the P132H mutation in the SARS-CoV-2 Omeprón strain (IC50 of 0.022 ± 0.00090 μM in three independent tests).

[0149] Table 2: Inhibitory effect of compound 2 on the 3CL protease activity of SARS-CoV-2 Omeprone strain 3CL protease Compound 2 IC50 (μM) (n=3*) SARS-CoV-2 (Omicron strain) 0.022±0.00090 *Refers to three independent replicate experiments.

[0150] Example 24-3: Test of inhibitory activity of compound 2 against coronavirus 3CL protease from different sources

[0151] Objective: To investigate the inhibitory effect of compound 2 on the 3CL protease activity of six other coronaviruses capable of infecting humans: SARS-CoV, MERS-CoV, OC43-CoV, H229E-CoV, NL63-CoV, and HKU1-CoV.

[0152] Experimental materials: 3CL protease: Recombinant full-length coronavirus 3CL protease was prepared based on coronavirus genome sequences. The GenBank numbers of the SARS-CoV, MERS-CoV, H229E-CoV, HKU1-CoV, NL63-CoV, and OC43-CoV genomes used were AAP13442.1, MT387202.1, AF304460.1, AY597011.2, AY567487.2, and AY903459.1, respectively. The DNA sequences required for the expression of the 3CL protease protein of the six coronaviruses were purchased from Nanjing GenScript Biotech Co., Ltd. The 3CL protease was purchased from Nanjing Genscript Biotech Co., Ltd. The chymotrypsin was purchased from Jier Biochemical Co., Ltd.

[0153] Other reagents are shown in the table below: Reagents and materials brand Item number Bovine pancreas-derived chymotrypsin Sigma C4129 Tris Sigma BCBX3837 EDTA Sigma 0001434776

[0154] Experimental steps:

[0155] Prepare reaction buffer (containing 50 mM Tris and 1 mM EDTA). Dissolve the analyte in DMSO to prepare a 100 mM stock solution, and then further serially dilute with reaction buffer to obtain 11 concentrations (2-fold serial dilutions). Add 3CL of protease and different concentrations of the compound to a 96-well plate, react at room temperature for 10 minutes, add the acceptor, and quickly transfer the plate to a microplate reader for reading. The total volume of the enzymatic reaction system was 120 µL. The final concentrations of the protease for SARS-CoV, MERS-CoV, H229E-CoV, HKU1-CoV, NL63-CoV, and OC43-CoV were 30 nM, 80 nM, 30 nM, 20 nM, 30 nM, and 10 nM, respectively, and the final acceptor concentration was 10 µM. The excitation and emission wavelengths for reading were 340 nm and 490 nm, respectively. The test lasted for 10 minutes, and the fluorescence value was read every minute. The final result was obtained by fitting the readings of the first 5 minutes to obtain the reaction rate and calculate the inhibition rate. The calculation formula is: Inhibition rate = 1 – (Reaction rate of test group / Reaction rate of control group).

[0156] Experimental results: As shown in Table 3, compound 2 showed a better inhibitory effect on 3CL proteases derived from six other coronaviruses, indicating that compound 2 may have broad-spectrum anti-coronavirus activity.

[0157] Table 3: Inhibitory effect of compound 2 on 3CL protease derived from other coronaviruses 3CL protease source Compound 2 IC50 (μM) HKU1-CoV 0.0049 OC43-CoV 0.010 SARS-CoV-1 0.024 MERS-CoV 0.060 H229E-CoV 0.13 NL63-CoV 0.85

[0158] Example 25-1: Inhibitory effect of compound 2 on SARS-CoV-2 WIV04 and B.1.351 cell lines.

[0159] Vero E6 cells were used in the experiment. 50,000 Vero E6 cells / well were added to 48-well plates, followed by 100 μL / well of culture medium containing gradient concentrations of the compound. One hour later, SARS-CoV-2 was added, with a multiplicity of infection (MOI) of 0.01. After reacting for one hour, the supernatant was aspirated, the cells were washed, and 200 μL / well of culture medium containing gradient concentrations of the compound was added again. The cells were incubated at 37°C for 24 hours. After 24 hours, the cell supernatant was collected, viral RNA was extracted, and the viral copy number was detected using real-time quantitative PCR. The inhibition rate of the compound was calculated based on the viral copy number, and the EC50 of the compound was calculated using Prism 6.0.

[0160] The experimental results show that the compound [2] The half-maximal effective concentration (EC50) for inhibiting SARS-CoV-2 WIV04 strain was 0.57 + / - 0.04 μM, and the EC50 for inhibiting SARS-CoV-2 B.1.351 strain was 0.73 + / - 0.06 μM. The EC50 curves are shown in Figures 1 and 2.

[0161] Example 25-2: Inhibitory effect of compound 2 on SARS-CoV-2 Vero E6 original strain (WIV04), Delta strain (B.1.617.2), and Omeprón strain (B.1.1.529) at the cellular level.

[0162] Objective: This experiment investigated the inhibitory effect of compound 2 on the replication of the original SARS-CoV-2 strain (WIV04), Delta strain (B.1.617.2), and Omeprone strain (B.1.1.529) in Vero E6 cells using real-time quantitative PCR to detect viral copy numbers in the culture supernatant. Because Vero E6 cells highly express the efflux transport protein P-gp, 0.5 μM of the P-gp inhibitor CP-100356 was added to co-react with the compound.

[0163] Experimental materials:

[0164] Vero E6 was purchased from ATCC (item number CRL-1586). The original SARS-CoV-2 strain (SARS-CoV-2-WIV04 strain), Delta strain (B.1.617.2), and Omicron strain (B.1.1.529) were derived from the Microbial Culture Collection Center of Wuhan Institute of Virology, Chinese Academy of Sciences.

[0165] Other reagents are shown in the table below: Reagent Name brand Item number TaKaRa MiniBEST Viral RNA / DNA Extraction Kit Ver.5.0 Takara 9766 TaKaRa PrimeScript™ RT reagent Kit with gDNA Eraser Takara RR047A TaKaRa SYBR® Premix Ex Taq™ II Takara RR820A Fetal Bovine Serum Gibco R2768 DMEM medium Gibco C11995500BT CCK8 Beyotime C0039 Chloroquine phosphate SIGMA C6628-50G pancreatic enzymes BIOSHARP BL512A

[0166] Experimental instruments: Biosafety cabinet (AC2-3S1, ESCO, Singapore) Carbon dioxide incubator (Thermo Scientific HERAcell 150i, Thermo Scientific, USA) Pure water system (SYS ultrapure water system, Chengdu) StepOne Plus Real-time PCR system (4376600, ABI, USA) TC20™ Automated Cell Counter (1450102, BIO-RAD, USA) T100™ Thermal Cycler (1861096, BIO-RAD, USA) Centrifuge (Micro21 / 21R Thermo Scientific, USA)

[0167] Experimental steps:

[0168] Vero E6 cells were digested with trypsin and seeded in 48-well plates (90% DMEM, 10% fetal bovine serum) at 50,000 cells per well, and cultured overnight. The test compound was dissolved in DMSO to prepare a 40 mM stock solution, which was then serially diluted with medium containing 0.5 μM Pgp inhibitor to obtain the required concentrations. The final concentration range of the test compound was 1 μM to 0.004 μM. The cell supernatant was removed, and the diluted compound (containing 0.5 μM Pgp inhibitor) was added to each well. The reaction was allowed to proceed for 1 hour. Different strains of SARS-CoV-2 were added in a biosafety level 3 (BSL-3) laboratory with a multiplicity of infection (MOI) of 0.01 or 0.001. After 1 hour of reaction, the supernatant was removed, the cells were washed with PBS, and 200 μL of the diluted compound (containing 0.5 μM Pgp inhibitor) was added to each well. The supernatant was collected 24 or 72 hours post-infection. Viral RNA was extracted from the supernatant and the viral copy number in the supernatant was detected by real-time quantitative PCR. The inhibition rate of the compound was calculated based on the viral copy number, and the IC50 of the compound was calculated using GraphPad Prism 8.

[0169] In the cytotoxicity assay, Vero E6 cells were digested and seeded in 96-well plates (90% DMEM, 10% fetal bovine serum) at 20,000 cells per well, and cultured overnight. The test compound was dissolved in DMSO to prepare a 40 mM stock solution, which was then serially diluted with medium or medium containing 0.5 μM Pgp inhibitor to obtain the desired concentration. The final concentration range of the test compound was 500 μM to 1.95 μM. The cell supernatant was removed from the 96-well plates, and 100 μL / well of the test compound (single drug or medium containing 0.5 μM Pgp inhibitor) was added. After 24 hours of reaction, cell viability was assessed using a CCK8 assay kit, and the inhibition rate and half-maximal cytotoxicity concentration (CC 50) were calculated.

[0170] Results: As shown in Table 4, compound 2, when combined with the P-gp inhibitor CP-100356, dose-dependently inhibited the replication of Delta strain in Vero E6 cells, with an IC50 of 0.040 μM. Compound 2, in combination with the P-gp inhibitor, also exhibited strong inhibitory activity in the original strain, with an IC50 of 0.027 μM. Furthermore, compound 2, in combination with the P-gp inhibitor, significantly inhibited the replication of Omeprone strain in Vero E6 cells, with an IC50 of 0.12 μM. Neither compound 2 alone nor in combination with the P-gp inhibitor showed significant cytotoxicity to Vero E6 cell proliferation, with a IC50 > 500 μM.

[0171] Table 4: Inhibitory effect of compound 2 in combination with P-gp inhibitor on SARS-CoV-2 replication in Vero E6 cells experiment Compound 2 + 0.5 μM CP-100356 IC 50 (μM) Select index SI SI = (CC 50 / IC 50) Vero E6 original strain (WIV04) 0.027 (Mean, n=2) >18519 Vero E6 Delta strain (B.1.617.2) 0.040 (Mean, n=2) >12500 Vero E6 Omeprone strain (B.1.1.529) 0.12 >4167 Vero E6 monotherapy CC 50 >500 µM Vero E6 combined with the P-gp inhibitor CC 50 >500 µM

[0172] Example 26: In vivo antiviral activity of compound 2 against SARS-CoV-2 delta strain in hACE2-K18 transgenic mice

[0173] Objective: This study evaluated the antiviral activity of compound 2 against SARS-CoV-2 delta strain in K18 transgenic mice (K18-hACE2) that stably express human angiotensin-converting enzyme 2 (ACE2).

[0174] Experimental materials: K18-hACE2 transgenic mice aged 7-8 weeks were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. The SARS-CoV-2 Delta strain virus was obtained from the Microbial Culture Collection Center of Wuhan Institute of Virology, Chinese Academy of Sciences. Ritonavir was purchased from Shanghai Disano Chemical Pharmaceutical Co., Ltd. Vero E6 cells were purchased from ATCC (catalog number CRL-1586).

[0175] Other reagents are shown in the table below: Reagent Name brand Item number Qiagen 74106 RNeasy Mini Kit Qiagen 74106 TaKaRa PrimeScript™ RT reagent Kit with gDNA Eraser Takara RR047A TaKaRa SYBR® Premix Ex Taq™ II Takara RR820A Fetal Bovine Serum Gibco 10099-141C DMEM medium Gibco C11995500BT tissue fixative BOSTER AR1068 Aquacide II (sodium methylcellulose) Millipore 17851 DMEM medium (powder, high sugar) Gibco 12100046 Crystal Violet Sinopharm Group 71012314

[0176] Experimental instruments: Biosafety cabinet (AC2-3S1, ESCO, Singapore) Carbon dioxide incubator (Thermo Scientific HERAcell 150i, Thermo Scientific, USA) Pure water system (SYS ultrapure water system, Chengdu) StepOne Plus Real-time PCR system (4376600, ABI, USA) TC20™ Automated Cell Counter (1450102, BIO-RAD, USA) T100™ Thermal Cycler (1861096, BIO-RAD, USA) Centrifuge (Micro21 / 21R Thermo Scientific, USA) Tissue homogenizer (JXFSTPRP-CL, Shanghai Jingxin, China)

[0177] Experimental Procedure: K18-hACE2 transgenic mice were infected with SARS-CoV-2 delta strain via intranasal instillation, designated as day 0. Two hours after infection, mice were administered either the solvent, 50 mg / kg or 200 mg / kg of compound 2 (combined with 50 mg / kg of the cytochrome P450 inhibitor ritonavir) via gavage, either twice daily for 2 days (once on day 0, twice on day 1, and once on day 2) or for 4 days (once on day 0, twice on days 1, 2, and 3). Mouse weight changes were recorded, and lung and brain tissues were collected at the endpoint. The left lung tissue was fixed in formaldehyde, embedded, sectioned, and stained with H&E for histopathological examination. The right lung and brain tissues were each divided into two portions. One portion was homogenized, and RNA was extracted and reverse transcribed for viral copy number detection using real-time quantitative PCR. The other portion was homogenized and used for viral titer detection via plaque assay. The plaque lysis assay was performed as follows: Vero E6 cells were seeded in 24-well plates at a density of 12,000 cells per well and cultured overnight. The original tissue homogenate solution was serially diluted 10-fold in DMEM medium. The cell supernatant was removed, and the diluted tissue homogenate solution was added and reacted for 1 hour. The supernatant was then removed, and medium containing 1% sodium methylcellulose and 2% FBS was added, followed by culturing for 4 days. The medium was then removed, and the cells were fixed with paraformaldehyde and stained with 1% (w / v) crystal violet. The number of empty plaques in each well was counted.

[0178] Results: As shown in Table 5, 2 days after infection, compared with the model group (mean viral copy number 9.19 ± 0.30 log10 copies / g), compound 2 at 50 mg / kg and 200 mg / kg significantly reduced viral load in the lungs when combined with ritonavir, with mean copy numbers of 7.66 ± 0.27 log10 copies / g and 6.79 ± 0.30 log10 copies / g, respectively. The reduction in viral copy number at the 200 mg / kg dose reached 2.4 log10 copies / g. 4 days after infection, compound 2 showed a sustained inhibitory effect on viral copy number. Regarding viral titer, as shown in Figure 3, compound 2 exhibited a significant inhibitory effect. 2 days after infection, the 200 mg / kg dose completely inhibited viral replication, with no detectable titer. At 50 mg / kg, compared with the model group, the viral titer decreased by more than 3 log10 PFU / g. 4 days after infection, compound 2 demonstrated a sustained inhibitory effect on viral titer. As shown in Figure 4, four days after infection, the model group mice lost approximately 10% of their body weight, while the compound 2 administration group did not show a significant decrease, indicating that compound 2 did not exhibit significant toxicity under continuous administration. We further examined the viral load in the mouse brain. Two days after infection, no significant infection was observed in any group. Four days after infection, compared to the model group, compound 2 significantly reduced viral copy number in the mouse brain at both 50 mg / kg and 200 mg / kg doses, especially at the 200 mg / kg dose, where the viral copy number was comparable to that of the uninfected normal group. We further examined the viral titer in the brain four days after infection, and the results are shown in Figure 5. Compared to the model group, no viral titer was detected at either dose of compound 2, demonstrating its strong inhibitory effect. Furthermore, histopathological analysis of the lungs showed that, compared to the model group, compound 2 at a dose of 200 mg / kg significantly improved lung damage, including reducing the degree of alveolar atrophy or dilation and the degree of alveolar membrane thickening.

[0179] Table 5. Viral load in the lungs and brain of mice 2 and 4 days after infection (mean ± SD) Group lung tissue (log10 copies / g) brain tissue (log10 copies / mg) Day 2 Day 4 Day 2 Day 4 Model group 9.19 ± 0.30 9.35 ± 0.30 1.34±0.78 7.17 ± 0.27 Compound 2 - 200 mg / kg + ritonavir - 50 mg / kg 6.79 ± 0.30 7.48 ± 0.63 1.04±0.24 0.99 ± 0.42 Compound 2 - 50 mg / kg + ritonavir - 50 mg / kg 7.66 ± 0.27 7.88 ± 0.74 0.77±0.20 2.88 ± 1.26 normal group 5.76 ± 0.32 6.27 ± 0.18 1.08±0.06 1.08 ± 0.06

[0180] Example 27: Selectivity of compound 2 for kinases

[0181] Experimental objective: To detect the inhibitory activity of compound 2 on 413 kinases on the KinaseProfile experimental platform and to study the selectivity of compound 2 for kinases.

[0182] Experimental materials: The Full Human Panel [10 uM ATP] KinaseProfiler is a test product provided by Eurofins, catalog number 50-005KP10. This product contains 413 kinases.

[0183] Experimental steps:

[0184] Compound analysis for each selected kinase was performed using the Eurofins standard Kinase Profiler assay, following relevant standard operating procedures. Protein kinases were detected using radiometric methods, while lipid kinases were detected using HTRF. The ATP concentration used in the experiments was 10 μM. Detailed information for each kinase is available on the Eurofins website at: https: / / www.eurofinsdiscoveryservices.com / catalogmanagement / viewItem / Full-Human-Panel-10-uM-ATP-KinaseProfiler / 50-005KP10.

[0185] Experimental results: For 413 kinases, compound 2 showed an inhibition rate of less than 30% at a concentration of 10 μM, indicating no significant inhibitory effect, suggesting that compound 2 has excellent selectivity.

[0186] Example 28: Selectivity of compound 2 for safety targets

[0187] Experimental objective: To investigate the effects of compound 2 on 47 safety-related targets using the Safetyscan experimental platform.

[0188] Experimental materials:

[0189] The Safety47 Panel Dose Response SAFETYscan is a testing product provided by Eurofins, part number 87-1003DR. This product contains 78 tests related to 47 safety targets.

[0190] Experimental steps:

[0191] The 78 tests related to 47 safety targets employed experimental methods including: cAMP assays, calcium flux assays, hormone nuclear receptor assays, kinase binding assays, enzyme activity assays, neurotransmitter transporter assays, ion channel assays, and transporter assays. Specific methodologies for each assay are available on the Eurofins website at: https: / / www.eurofinsdiscoveryservices.com / catalogmanagement / viewItem / Safety47-Panel-Dose-Response-SAFETYscan-DiscoverX / 87-1003DR.

[0192] Experimental results: For 47 safety-related targets, compound 2 showed no significant inhibition or initiation at a concentration of 100 μM (EC 50 values ​​were all greater than 100 μM), indicating that compound 2 has excellent selectivity.

[0193] Example 29: Human plasma protein binding assay of compound 2

[0194] Experimental materials

[0195] Human plasma was purchased from BioIVT, anticoagulated with EDTA K2, and stored at -80°C. 96-well equilibrium dialysis discs were purchased from HTDialysis LLC. Equilibrium dialysis membranes were purchased from Gales Ferry.

[0196] Experimental steps

[0197] An alkaline solution with a concentration of 14.2 g / L disodium hydrogen phosphate and 8.77 g / L sodium chloride was prepared using ultrapure water. This alkaline solution could be stored at 4°C for 7 days. An acidic solution with a concentration of 12.0 g / L disodium hydrogen phosphate and 8.77 g / L sodium chloride was also prepared using ultrapure water. This acidic solution could be stored at 4°C for 7 days. The alkaline solution was titrated with the acidic solution to a pH of 7.4. This buffer solution could be stored at 4°C for 7 days. The pH of the buffer solution was tested on the day of the experiment. If the pH exceeded the range of 7.4 ± 0.1, the pH was adjusted.

[0198] The dialysis membrane was immersed in ultrapure water for 60 minutes to separate into two pieces, then soaked in 20% ethanol for 20 minutes, and finally soaked in dialysis buffer for 20 minutes.

[0199] Thaw the frozen plasma rapidly at room temperature.

[0200] The plasma was centrifuged at 3,220 g for 10 minutes at 4°C to remove clots, and the supernatant was collected in a new centrifuge tube. The pH value of the plasma was measured and recorded.

[0201] Prepare a 10 mM DMSO stock solution of the analyte. Dilute 2 μL of the stock solution (10 mM) with 98 μL of DMSO to obtain a working solution (200 μM). Take 3 μL of the working solution and add it to 597 μL of human plasma to a final concentration of 1 μM (0.5% DMSO). Vortex thoroughly.

[0202] Add 120 μL of drug-treated plasma sample to one side of the dialysis membrane, and an equal volume of dialysis buffer (phosphate buffer) to the other side. The experiment was performed in duplicate. Seal the dialysis tray, place it in the reaction apparatus, and react for 6 hours at 37°C, 5% CO2, and approximately 100 rpm. After the reaction, remove the sealing membrane, and aspirate 50 μL from the buffer and plasma sides of each well into different wells of a new dialysis tray.

[0203] Add 50 μL of blank plasma to the phosphate-buffered saline (PBS) sample, and add an equal volume of blank PBS to the plasma sample. Add 300 μL of room temperature quencher (containing acetonitrile (550 nM labetalol, 100 nM alprazolam, and 2 µM ketoprofen)) to precipitate the protein. Vortex for 5 minutes. Centrifuge at 3220 g for 30 minutes at 4 °C. Transfer 100 μL of the supernatant to a new well plate. Dilute the supernatant with 100 μL or 200 μL of water according to the LC-MS response signal and peak shape of the analyte. Mix well and perform LC-MS analysis.

[0204] Perform all calculations using Microsoft Excel. Determine the peak areas of the analyte on the buffer and plasma sides. Calculate the plasma protein binding rates of the analyte and control drug using the following formulas: Free concentration = (Ratio of sample peak area to internal standard peak area (buffer side) / Ratio of sample peak area to internal standard peak area (plasma side)) * 100%, Binding concentration = 1 - Free concentration, Recovery rate = (Ratio of sample peak area to internal standard peak area (buffer side) + Ratio of sample peak area to internal standard peak area (plasma side)) / (Ratio of sample peak area to internal standard peak area (initial plasma sample)) * 100%. The Ratio of sample peak area to internal standard peak area (buffer side) represents the free concentration of the compound, the Ratio of sample peak area to internal standard peak area (plasma side) represents the sum of the free and bound concentrations of the compound, and the Ratio of sample peak area to internal standard peak area (initial plasma sample) represents the total concentration of the compound at the start of the reaction.

[0205] Experimental results:

[0206] See Table 6. Compound 2 at 1 μM reacted at 37 °C for 6 hours, with an average free fraction of 46.63%, a binding fraction of 53.37%, and a recovery rate of 88.02%.

[0207] Table 6 Results of human plasma protein binding assay for compound 2 compound Free rate (F%) Binding rate (%) Recovery rate (%) Repeat 1 Repeat 2 average value Compound 2 48.71 44.55 46.63 53.37 88.02

[0208] Example 30: Tissue distribution assay of compound 2 after a single gavage.

[0209] Experimental materials: Sixty Balb / c mice (purchased from Shanghai Minchang Biotechnology Co., Ltd.), half male and half female, weighing 18-25g.

[0210] Experimental steps:

[0211] Balb / c mice were given compound 2 via a single gavage at a dose of 100 mg / kg and a volume of 10 mL / kg.

[0212] Six mice (half male and half female) were sampled before and at 5 min, 0.25, 1.0, 2.0, 3.0, 5.0, 7.0, and 10 h after drug administration. At each time point, 0.2 ml of blood was collected via the retroocular venous plexus and placed in EDTA-K2 tubes. The tubes were centrifuged at 11000 rpm for 5 min to separate the plasma, which was then frozen at -70°C. Lung tissue was immediately collected after whole blood collection at 0.25, 1.0, 3.0, and 7.0 h. The tissue was rinsed with cold physiological saline to remove any residual blood and contents, dried, labeled, and stored at -70°C until analysis. The concentration of compound 2 in plasma and lung tissue was determined by LC / MS-MS, and the lung-to-blood ratio was calculated.

[0213] Experimental results:

[0214] After a single gavage administration of compound 2 to Balb / c mice, the ratio of lung tissue exposure to plasma exposure was 0.62, indicating that compound 2 had a high exposure level in lung tissue.

[0215] Example 31: Safety pharmacological study of the effects of gavage administration of compound 2 on the cardiovascular system of cynomolgus monkeys

[0216] In a 2-week repeated-dose toxicity study in cynomolgus monkeys, the effects of compound 2 on the cardiovascular system were also investigated.

[0217] Experimental materials:

[0218] Thirty-two cynomolgus monkeys, half male and half female, aged 2.5-5 years at the time of drug administration.

[0219] Animal sources: Yunnan Yingmao Biotechnology Co., Ltd.; Guangxi Xiongsen Primate Experimental Animal Breeding and Development Co., Ltd.; Zhongke Lingrui (Zhanjiang) Biotechnology Co., Ltd.

[0220] Systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MBP) were measured in all conscious animals using the BP-98E smart non-invasive blood pressure monitor, employing the Provantis / v10.2.3.1 electronic data acquisition system (PV-02).

[0221] Experimental Procedure: Thirty-two cynomolgus macaques (groups 1 and 4: 5 animals / sex / group; groups 2 and 3 animals / sex / group, totaling 4 groups) were randomly assigned to either group or administered compound 2 (40, 160, and 600 mg / kg / day) or the control formulation (98.9% solvent formulation + 1.1% MTBE, 0 mg / kg / day) via nasogastric tube, twice daily for 14 days, followed by a 14-day recovery period. All animals were included in this study to assess the effects of administration on ECG parameters (including heart rate, PR interval, QRS duration, QT interval, and QTcF) and on blood pressure during the pre-administration, administration, and recovery periods.

[0222] Results: Under the experimental conditions, cynomolgus monkeys were administered compound 2 (40, 160, and 600 mg / kg / day) via nasogastric gavage for 14 days, twice daily. No cardiovascular changes or arrhythmias were observed in the cynomolgus monkeys. No changes in ECG parameters or blood pressure were observed in the cynomolgus monkeys throughout the experiment.

[0223] none

[0224] none

Claims

1. A cyano compound of Formula I, or a racemic, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof: Formula I Wherein, R1 is selected from -COR8 and -SO2; R9; R2 and R3 are each independently selected from H, D, C1-C10 alkyl, adamantyl, and C3-C7 cycloalkyl, or R2 forms a 3-8 carbon ring with R3 and the carbon atom attached thereto; X is selected from O, S, S(=O)2, and S=O; Y is absent, or Y is selected from O, S, S(=O)2, and S=O; R4 is selected from H, C1-C10 alkyl, C3-C8 cycloalkyl, C6-C20 aryl, C1-C10 alkyl-substituted C6-C20 aryl, C1-C10 alkoxy-substituted C6-C20 aryl, and halogenated C6-C20 aryl; R5 is selected from H, C1-C10 alkyl, and C3-C7 cycloalkyl; Alternatively, R4 and R5 are linked together to form a C2-C6 alkylene group, thereby connecting X and Y; R6 is selected from H and D; R7 is selected from H and D; R8 is selected from H, C1-C10 alkyl, C1-C10 alkoxy, C3-C7 cycloalkyl, halo-C1-C10 alkyl, halo-C3-C7 cycloalkyl, -NR13R14, C6-C20 aryl, halo-C6-C20 aryl, C1-C10 alkyl-substituted C6-C20 aryl, halo-C1-C10 alkyl-substituted C6-C20 aryl, 5-20 heteroaryl and halo-5-20 heteroaryl; R9 is selected from C1-C10 alkyl, C3-C7 cycloalkyl, halogenated C1-C10 alkyl, halogenated C3-C7 cycloalkyl, -NR15R16, C6-C20 aryl, halogenated C6-C20 aryl, C1-C10 alkyl-substituted C6-C20 aryl, halogenated C1-C10 alkyl-substituted C6-C20 aryl, 5-20 heteroaryl, and halogenated 5-20 heteroaryl; R13 and R14 are each independently selected from H and C1-C10 alkyl; and R15 and R16 are each independently selected from H and C1-C10 alkyl.

2. The cyano compound as claimed in claim 1, or its racemic, enantiomer, diastereomer, or pharmaceutically acceptable salt, wherein, R2 and R3 are each independently selected from H, D, C1-C6 alkyl, adamantyl, and C3-C7 cycloalkyl, or R2 and R3 and the carbon atom attached thereto form a 3-8 member carbon ring.

3. The cyano compound as described in claim 2, or its racemic, enantiomer, diastereomer, or pharmaceutically acceptable salt, wherein, R2 and R3 are each independently selected from H, isopropyl, tert-butyl, cyclopentyl, and adamantyl, or R2 and R3 and the carbon atom attached thereto form cyclopropyl or cyclopentyl.

4. A cyano compound as described in claim 2, or a racemic, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof, wherein, One of R2 and R3 is selected from H, and the other is selected from isopropyl, tert-butyl, cyclopentyl, and adamantyl, or R2 and R3 and the carbon atom attached thereto form cyclopropyl or cyclopentyl.

5. A cyano compound as claimed in claim 1, or a racemic mixture, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof, wherein, R4 is selected from H, C1~C6 alkyl, C3~C8 cycloalkyl, C6~C10 aryl, C1~C6 alkyl-substituted C6~C10 aryl, C1~C6 alkoxy-substituted C6~C10 aryl, and halogenated C6~C10 aryl; R5 is selected from H, C1~C6 alkyl, and C3~C7 cycloalkyl; or, R4 and R5 are linked together to form CH2CH2 or CH2CH2CH2, thereby connecting X and Y.

6. A cyano compound as claimed in claim 1, or a racemic, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof, wherein, R8 is selected from H, C1-C6 alkyl, C1-C6 alkoxy, C3-C7 cycloalkyl, halogenated C1-C6 alkyl, halogenated C3-C7 cycloalkyl, -NR13R14, C6-C10 aryl, halogenated C6-C10 aryl, C1-C6 alkyl-substituted C6-C10 aryl, halogenated C1-C6 alkyl-substituted C6-C10 aryl, 5-10 member heteroaryl, and halogenated 5-10 member heteroaryl. R13 and R14 are each independently selected from H and C1-C6 alkyl. C6 alkyl; and R9 is selected from C1~C6 alkyl, C3~C7 cycloalkyl, halogenated C1~C6 alkyl, halogenated C3~C7 cycloalkyl, -NR15R16, C6~C10 aryl, halogenated C6~C10 aryl, C1~C6 alkyl-substituted C6~C10 aryl, halogenated C1~C6 alkyl-substituted C6~C10 aryl, 5~10-membered heteroaryl and halogenated 5~10-membered heteroaryl, R15 and R16 are each independently selected from H and C1~C6 alkyl.

7. A cyano compound as claimed in claim 6, or a racemic, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof, wherein, R8 is selected from C1-C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkoxy, -NR13R14, C3-C7 cycloalkyl, halogenated C3-C7 cycloalkyl, phenyl, halogenated phenyl, C1-C6 alkyl-substituted phenyl, halogenated C1-C6 alkyl-substituted phenyl and 5-6 heteroaryl, R13 and R14 are each independently selected from H and C1-C6 alkyl; and R9 is selected from C1-C6 alkyl, C3-C7 cycloalkyl, phenyl, C1-C6 alkyl-substituted phenyl and halogenated C1-C6 alkyl-substituted phenyl.

8. A cyano compound as claimed in claim 6, or a racemic mixture, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof, wherein, R8 is selected from CH3, CF3, CH2CF3, CF2CF3, methoxy, cyclopropyl, phenyl, and pyridin-3-yl; and R9 is selected from CH3, cyclopropyl, phenyl, p-methylphenyl and p-trifluoromethylphenyl.

9. A cyano compound as claimed in claim 1, or a racemic mixture, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof, wherein, X is selected from O, S, S(=O)2 and S=O; Y does not exist, or Y is selected from O, S and S=O; R4 is selected from C1~C6 alkyl and C6~C10 aryl, and R5 is selected from H; or R4 and R5 are connected to each other to form C2~C6 alkylene, thereby connecting X and Y.

10. A cyano compound as claimed in claim 9, or a racemic, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof, wherein, X and Y are each independently selected from O, S, and S=O. R4 and R5 are connected to each other to form CH2CH2 or CH2CH2CH2, thus connecting X and Y.

11. A cyano compound as claimed in claim 9, or a racemic mixture, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof, wherein, X and Y are each independently selected from O and S, and R4 and R5 are connected to each other to form CH2CH2, thus connecting X and Y.

12. A cyano compound as claimed in any one of claims 1 to 11, or a racemic, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof, wherein, The cyano compound represented by Formula I is selected from the cyano compound represented by Formula IA: Formula IA.

13. A cyano compound as claimed in any one of claims 1 to 11, or a racemic, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof, wherein, The cyano compounds represented by Formula I are selected from the cyano compounds represented by the following general formulas: Formula I-1, Formula I-2, Formula I-3, Formula I-4, Formula I-5, Formula I-6 or Formula I-7.

14. A cyano compound as claimed in claim 1, or a racemic, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof, wherein, The cyano compound represented by Formula I is selected from the following compounds:

15. A method for preparing a cyano compound of formula I, comprising: method i: ia) condensing a compound of formula II with a compound of formula III to obtain a compound of formula IV; ib) dehydrating the compound of formula IV to obtain a compound of general formula I; or method ii: iia) condensing a compound of formula V with a compound of formula III to obtain a compound of formula VI, wherein PG in the compound of formula V is a protecting group of an amino group; iib) deprotecting the compound of formula VI to obtain a compound of formula VII; iic) amination, sulfonation, or condensation of the compound of formula VII to obtain a compound of formula IV; iid) dehydrating the compound of formula IV to obtain a compound of formula I; wherein, The definitions of R1, R2, R3, R4, R5, R6, R7, X, and Y are as described in Request 1.

16. The method as described in claim 15, wherein in method i, step ia) is: reacting the compound of formula II with the compound of formula III in a solvent at a temperature of -20°C to 50°C for 0.1-12 h by the action of a condensing agent and a base to obtain the compound of formula IV; step ib) is: reacting the compound of formula IV with a dehydrating agent in an anhydrous solvent at a temperature of -20°C to 50°C for 1-24 h to obtain the compound of general formula I; in method ii, step iia) is: reacting the compound of formula V with the compound of formula III in a solvent at a temperature of -20°C to 50°C for 0.1-12 h by the action of a condensing agent and a base to obtain the compound of formula VI; step iib) is: reacting the compound of formula VI with an organic solution of trifluoroacetic acid or hydrogen chloride or Pd / C / H2 at a temperature of -20°C to 50°C to obtain the compound of formula VII; step iic) is: At a temperature of -20°C to 50°C, the compound shown in Formula VII is subjected to an amino acid reaction with acetic acid chloride or sulfonic acid anhydride under alkaline conditions to obtain the compound shown in Formula IV; or at a temperature of -20°C to 50°C, the compound shown in Formula VII is subjected to a sulfonation reaction with sulfonic acid chloride or sulfonic acid anhydride under alkaline conditions to obtain the compound shown in Formula IV; or at a temperature of -20°C to 50°C, the compound shown in Formula VII is subjected to a condensation reaction with a carboxyl compound under condensing agent and alkaline conditions to obtain the compound shown in Formula IV; and step iid) is: the compound shown in Formula IV is reacted with a dehydrating agent in an anhydrous solvent at a temperature of -20°C to 50°C for 1-24 hours to obtain the compound shown in General Formula I.

17. The method as claimed in claim 16, wherein in method i, in step ia), the solvent is one or a mixture of several selected from tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N,N-dimethylacetamide, ethyl acetate, and 1,4-dioxane; the condensing agent is one or a mixture of several selected from 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, 2-hydroxypyridine-N-oxide, 1-propylphosphonic anhydride, 1H-benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate, N,N'-carbonyldiimidazole, and O-benzotriazole-tetramethylurea hexafluorophosphate; and the base is N,N-diisopropylethylamine, triethylamine, or N-methylmorpholine. In step ib), the anhydrous solvent is one or a mixture of tetrahydrofuran, dichloromethane, toluene, and 1,4-dioxane and pyridine; the dehydrating agent is trifluoroacetic anhydride or methyl N-(triethylammonium sulfonyl)aminoformate; and in method ii), in step iia), the solvent is one or a mixture of tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N,N-dimethylacetamide, ethyl acetate, and 1,4-dioxane; the condensing agent is 2-(7-azabenzotriazole)-N,N,N',N'- One or a mixture of several of the following: tetramethylurea hexafluorophosphate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, 2-hydroxypyridine-N-oxide, 1-propylphosphonic anhydride, 1H-benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate, N,N'-carbonyldiimidazole, and O-benzotriazole-tetramethylurea hexafluorophosphate; wherein the base is N,N-diisopropylethylamine, triethylamine, or N-methylmorpholine; wherein the protecting group PG of the amine group is tert-butyloxycarbonyl, benzyl, or p-methoxybenzyl; in step iic), the base is N,N-diisopropylethylamine, triethylamine, or N-methylmorpholine; and the condensing agent is 2-(7-azabenzotriazole)-N,N,N',N'- One or a mixture of several of the following: tetramethylurea hexafluorophosphate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, 2-hydroxypyridine-N-oxide, 1-propylphosphonic anhydride, 1H-benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate, N,N'-carbonyldiimidazole, and O-benzotriazole-tetramethylurea hexafluorophosphate; In step iid), the anhydrous solvent is one or a mixture of several of tetrahydrofuran, dichloromethane, toluene, 1,4-dioxane, and pyridine; The dehydrating agent is trifluoroacetic anhydride or N-(triethylammonium sulfonyl)aminoformate.

18. A pharmaceutical composition comprising a cyano compound selected from any one of claims 1 to 14, or a racemic, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

19. The pharmaceutical composition as claimed in claim 18, wherein the pharmaceutical composition further comprises ritonavir or a pharmaceutically acceptable salt thereof.

20. A pharmaceutical combination comprising a cyano compound selected from any one of claims 1 to 14, or a racemic, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof, and ritonavir or a pharmaceutically acceptable salt thereof.

21. Use in the preparation of a cyano compound as claimed in any one of claims 1 to 14, or a racemic, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof, or a pharmaceutical composition as claimed in claim 18 or 19, or a pharmaceutical composition as claimed in claim 20, wherein the pharmaceutical composition is a medicament for inhibiting the activity of coronavirus 3CL protease.

22. Use of a cyano compound as described in any one of claims 1 to 14, or a racemic, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in claims 18 or 19, or a pharmaceutical composition as described in claim 20, in the preparation of a medicament, wherein the medicament is a medicament for the prevention and / or treatment of coronavirus infection.

23. The use as described in claim 21 or 22, wherein the coronavirus is selected from SARS-CoV, MERS-CoV, H229E-CoV, HKU1-CoV, NL63-CoV, OC43-CoV and SARS-CoV-2.

24. Use in the preparation of a cyano compound as claimed in any one of claims 1 to 14, or a racemic, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof, or a pharmaceutical composition as claimed in claim 18 or 19, or a pharmaceutical composition as claimed in claim 20, wherein the pharmaceutical is a medicament for inhibiting the 3CL protease activity of small RNA viruses.

25. Use of a cyano compound as claimed in any one of claims 1 to 14, or a racemic, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof, or a pharmaceutical composition as claimed in claim 18 or 19, or a pharmaceutical composition as claimed in claim 20, in the preparation of a medicament, wherein the medicament is a medicament for the prevention and / or treatment of small RNA virus infection.

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