Cyano compounds and their uses

Abstract

Provided are compounds of formula I, their racemates, enantiomers, diastereomers and pharma- ceutically acceptable salts, and their use in the prophylaxis or treatment of associated diseases resulting from coronavirus and / or picornavirus infection. JPEG2024534628000077.jpg38170

Description

[Technical Field]

[0001] The present invention claims priority to a Chinese patent application bearing application number 202111168232.4 and entitled "Cyano Compounds, Their Preparation and Use," filed with the State Intellectual Property Office of China on September 30, 2021, and application number 202210973184.4 and entitled "Cyano Compounds, Their Preparation and Use," filed with the State Intellectual Property Office of China on August 15, 2022. The complete disclosures of the above prior applications are incorporated herein by reference in their entirety.

[0002] The present invention is in the field of medicinal chemistry and chemical synthesis; specifically, the present invention relates to cyano compounds, their preparation and uses. [Background technology]

[0003] Coronaviruses are single-stranded, positive-stranded RNA viruses, some of which can spread widely and cause severe symptoms. There are currently seven known human coronaviruses: HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, and SARS-CoV-2. The majority of coronavirus functional proteins are encoded by the ORF 1ab gene, which is first translated into a single polyprotein and then cleaved into multiple active proteins by the 3CL protease and PL protease. Therefore, inhibiting 3CL protease activity can effectively inhibit viral replication. Because different coronavirus 3CL proteases share a high degree of structural homology, 3CL protease inhibitors possess broad-spectrum anticoronavirus activity.

[0004] In addition to coronaviruses, 3CL protease also plays an important role in the hydrolysis of picornavirus-encoded polyproteins, and 3CL protease inhibitors can effectively inhibit picornavirus replication. Enterovirus 71, a type of picornavirus, is one of the common viruses that causes hand, foot, and mouth disease and can also cause various diseases such as meningitis, brainstem encephalitis, and myocarditis. In recent years, enterovirus 71 has rapidly spread in multiple infant populations, and clinically effective therapeutic agents are yet to be developed. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, there remains a need for compounds that can inhibit RNA viruses / picornaviruses such as coronavirus / enterovirus 71. [Means for solving the problem]

[0006] The present invention is a cyano compound represented by general formula (I) or a racemate, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof. [ka] (where, R 1 -COR 8 and -SO2R 9 is selected from R 2 and R 3 are independently H, D, C1 to C 10 alkyl group, adamantyl group, and C3-C7 cycloalkyl group, or R 2 and R 3 together with the carbon atoms to which they are attached form a 3- to 8-membered carbocyclic ring, X is selected from O, S, S(=O)2 and S=O; Y is absent or selected from O, S, S(=O)2 and S=O; R 4H, C1~C 10 Alkyl groups, C3-C8 cycloalkyl groups, C6-C 20 Aryl groups, C1-C 10 C6-C substituted with alkyl groups 20 Aryl groups, C1-C 10 C6-C substituted with alkoxy groups 20 Aryl groups and halogenated C6-C 20 aryl groups; R 5 H, C1~C 10 selected from alkyl groups and C3 to C7 cycloalkyl groups; Or R 4 and R 5 are linked together to form a C2-C6 alkylene group, which links X and Y; R 6 teeth, [ka] is selected from R 7 is selected from H and D, R 8 H, C1~C 10 Alkyl groups, C1-C 10 Alkoxy groups, C3-C7 cycloalkyl groups, halogenated C1-C 10 Alkyl groups, halogenated C3-C7 cycloalkyl groups, -NR 13 R 14 , C6~C 20 Aryl groups, halogenated C6-C 20 Aryl groups, C1-C 10 C6-C substituted with alkyl groups 20 Aryl groups, halogenated C1-C 10 C6-C substituted with alkyl groups 20 selected from an aryl group, a 5- to 20-membered heteroaryl group, and a halogenated 5- to 20-membered heteroaryl group; R 9 is C1~C 10 Alkyl groups, C3-C7 cycloalkyl groups, halogenated C1-C 10 Alkyl groups, halogenated C3-C7 cycloalkyl groups, -NR 15R 16 , C6~C 20 Aryl groups, halogenated C6-C 20 Aryl groups, C1-C 10 C6-C substituted with alkyl groups 20 Aryl groups, halogenated C1-C 10 C6-C substituted with alkyl groups 20 selected from an aryl group, a 5- to 20-membered heteroaryl group, and a halogenated 5- to 20-membered heteroaryl group; R 13 and R 14 are each independently H, C1 to C 10 alkyl groups, R 15 and R 16 are each independently H, C1 to C 10 alkyl groups.

[0007] The present invention provides a method for preparing a compound of general formula I, which is one of method i and method ii, In the method i, [ka] ia) obtaining a compound represented by formula IV from a compound represented by formula II and a compound represented by formula III by a condensation reaction; Preferably, in step ia), the compound represented by formula II is reacted with the compound represented by formula III in the presence of a condensing agent and a base in a solvent at a temperature of −20° C. to 50° C. for 0.1 to 12 hours to obtain the compound represented by formula IV; Optionally, the solvent is a mixture of one or more of tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N,N-dimethylacetamide, ethyl acetate, and 1,4-dioxane; Optionally, the condensing agent is a mixture of one or more of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, 2-hydroxypyridine N-oxide, propylphosphonic anhydride, 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate, 1,1'-carbonyldiimidazole, and O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; Optionally, the base is N,N-diisopropylethylamine, triethylamine, N-methylmorpholine; ib) dehydrating the compound of formula IV to obtain the compound of general formula I, Preferably, in step ib), the compound represented by formula IV is reacted with a dehydrating agent in an anhydrous solvent at a temperature of −20° C. to 50° C. for 1 to 24 hours to obtain the compound represented by general formula I; Optionally, the anhydrous solvent is a mixture of one or more of tetrahydrofuran, dichloromethane, toluene, 1,4-dioxane, and pyridine; Optionally, the dehydrating agent is trifluoroacetic anhydride or methyl N-(triethylammoniosulfonyl)carbamate; In the method ii, [ka] 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 for an amino group; Preferably, in step iia), the compound represented by formula V is reacted with the compound represented by formula III in the presence of a condensing agent and a base in a solvent at a temperature of −20° C. to 50° C. for 0.1 to 12 hours to obtain the compound represented by formula VI; Optionally, the solvent is a mixture of one or more of tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N,N-dimethylacetamide, ethyl acetate, and 1,4-dioxane; Optionally, the condensing agent is a mixture of one or more of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, 2-hydroxypyridine N-oxide, propylphosphonic anhydride, 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate, 1,1'-carbonyldiimidazole, and O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; Optionally, the base is N,N-diisopropylethylamine, triethylamine, and N-methylmorpholine; Optionally, the amino-protecting group PG is a tert-butoxycarbonyl group, a benzyl group, and a p-methoxybenzyl group; iib) deprotecting a compound of formula VI to obtain a compound of formula VII; Preferably, in step iib), the compound of formula VI is reacted 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; iic) obtaining a compound of formula IV from a compound of formula VII by aminoacylation, sulfonylation or condensation reaction, Preferably, in step iic), At a temperature of -20°C to 50°C, the compound represented by formula VII is subjected to aminoacylation with an acyl chloride or an acid anhydride in the presence of a base to obtain a compound represented by formula IV; Sulfonylation of the compound of formula VII with a sulfonyl chloride or sulfonic acid anhydride under basic conditions at a temperature of -20°C to 50°C to obtain a compound of formula IV; The compound represented by formula VII is subjected to a condensation reaction with a carboxy compound under the conditions of a condensing agent and a base at a temperature of -20°C to 50°C to obtain a compound represented by formula IV; Optionally, the base is N,N-diisopropylethylamine, triethylamine, and N-methylmorpholine; Optionally, the condensing agent is a mixture of one or more of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, 2-hydroxypyridine N-oxide, propylphosphonic anhydride, 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate, 1,1'-carbonyldiimidazole, and O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; iid) dehydrating a compound of formula IV to obtain a compound of general formula I, Preferably, in step iid), the compound represented by formula IV is reacted with a dehydrating agent in an anhydrous solvent at a temperature of −20° C. to 50° C. for 1 to 24 hours to obtain the compound represented by general formula I; Optionally, the anhydrous solvent is a mixture of one or more of tetrahydrofuran, dichloromethane, toluene, 1,4-dioxane, and pyridine; Optionally, the dehydrating agent is trifluoroacetic anhydride or methyl N-(triethylammoniosulfonyl)carbamate; where R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 The definitions of X, Y and R are as described above.

[0008] The present invention is a pharmaceutical composition comprising the cyano compound described above, or a racemate, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof, and pharmaceutically acceptable auxiliary materials, and optionally further comprising ritonavir or a pharmaceutically acceptable salt thereof.

[0009] The present invention is a pharmaceutical combination comprising the above-described cyano compound, or a racemate, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof, and ritonavir or a pharmaceutically acceptable salt thereof.

[0010] The present invention relates to use of the above-described cyano compound, its racemate, enantiomer, diastereomer or pharmaceutically acceptable salt thereof, or the above-described pharmaceutical composition, or the above-described pharmaceutical composition or pharmaceutical combination, in the manufacture of a medicament, wherein the medicament is selected from a drug for inhibiting coronavirus 3CL protease activity, a drug for preventing and / or treating coronavirus infection, a drug for inhibiting picornavirus 3CL protease activity, and a drug for preventing and / or treating picornavirus infection, and optionally the coronavirus is selected from SARS-CoV, MERS-CoV, H229E-CoV, HKU1-CoV, NL63-CoV, OC43-CoV and SARS-CoV-2. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows the in vitro activity inhibition rate-concentration of Compound 2 against the WIV04 strain of SARS-CoV-2. [Figure 2] FIG. 1 shows the in vitro activity inhibition rate-concentration of Compound 2 against the B.1.351 strain of SARS-CoV-2. [Figure 3] FIG. 1 shows the inhibitory effect of Compound 2 on virus titers in mouse lungs 2 days (FIG. A) and 4 days (FIG. B) after infection in Example 26. [Figure 4] FIG. 10 is a graph showing changes in mouse body weight in Example 26. [Figure 5] FIG. 10 shows the inhibitory effect of Compound 2 on the viral titer in the brain of mice 4 days after infection in Example 26. DETAILED DESCRIPTION OF THE INVENTION

[0012] Based on the crystal structure of 3CL protease, the inventors rationally designed cyano compounds that can effectively inhibit the 3CL protease activity of coronaviruses and / or picornaviruses, can effectively inhibit the 3CL protease activity of multiple types of picornaviruses including enterovirus 71 in vitro, can effectively inhibit picornavirus replication at the cellular level, and can be used to manufacture drugs for treating coronavirus and / or picornavirus-induced diseases. Based on this, the inventors have completed the present disclosure.

[0013] A first object of the present invention is to provide a cyano compound represented by general formula I, its racemate, enantiomer, diastereomer and pharmaceutically acceptable salts thereof.

[0014] A second object of the present invention is to provide a method for preparing such compounds.

[0015] A third object of the present invention is to provide pharmaceutical compositions containing such compounds.

[0016] A fourth object of the present invention is the use of such compounds in the manufacture of 3CL protease inhibitors.

[0017] A fifth object of the invention is the use of such compounds in the manufacture of a medicament for the prevention or treatment of diseases induced by coronaviruses and / or picornaviruses.

[0018] To achieve the above objectives, the technical solutions of the present invention are as follows:

[0019] In one aspect, the present invention provides cyano compounds of general formula I, their racemates, enantiomers, diastereomers and pharmaceutically acceptable salts thereof: [ka]

[0020] In another aspect, the present invention provides a method for preparing compounds of general formula I.

[0021] The technical solution of the present invention has at least the following technical effects:

[0022] The compounds of the present invention have 3CL protease inhibitory activity, inhibit the hydrolysis of protein complexes expressed by coronavirus and picornavirus genes, and further inhibit viral replication and progression, and can be used for the prevention and treatment of diseases caused by coronavirus or picornavirus infection.

[0023] The present invention relates to cyano compounds of general formula I, their racemates, enantiomers, diastereomers or pharmaceutically acceptable salts: [ka] where: R 1 -COR 8 and -SO2R 9 is selected from R 2 and R 3 are independently H, D, C1 to C 10 alkyl group, adamantyl group, and C3-C7 cycloalkyl group, or R 2 and R 3 together with the carbon atoms to which they are attached form a 3- to 8-membered carbocyclic ring, X is selected from O, S, S(=O)2 and S=O; Y is absent or selected from O, S, S(=O)2 and S=O; R 4 H, C1~C 10Alkyl groups, C3-C8 cycloalkyl groups, C6-C 20 Aryl groups, C1-C 10 C6-C substituted with alkyl groups 20 Aryl groups, C1-C 10 C6-C substituted with alkoxy groups 20 Aryl groups and halogenated C6-C 20 aryl groups; R 5 H, C1~C 10 selected from alkyl groups and C3 to C7 cycloalkyl groups; Or R 4 and R 5 are linked together to form a C2-C6 alkylene group, thereby linking X and Y; R 6 teeth, [ka] is selected from R 7 is selected from H and D, R 8 H, C1~C 10 Alkyl groups, C1-C 10 Alkoxy groups, C3-C7 cycloalkyl groups, halogenated C1-C 10 Alkyl groups, halogenated C3-C7 cycloalkyl groups, -NR 13 R 14 , C6~C 20 Aryl groups, halogenated C6-C 20 Aryl groups, C1-C 10 C6-C substituted with alkyl groups 20 Aryl groups, halogenated C1-C 10 C6-C substituted with alkyl groups 20 selected from an aryl group, a 5- to 20-membered heteroaryl group, and a halogenated 5- to 20-membered heteroaryl group; R 9 is C1~C 10 Alkyl groups, C3-C7 cycloalkyl groups, halogenated C1-C 10 Alkyl groups, halogenated C3-C7 cycloalkyl groups, -NR 15 R 16 , C6~C20 Aryl groups, halogenated C6-C 20 Aryl groups, C1-C 10 C6-C substituted with alkyl groups 20 Aryl groups, halogenated C1-C 10 C6-C substituted with alkyl groups 20 selected from an aryl group, a 5- to 20-membered heteroaryl group, and a halogenated 5- to 20-membered heteroaryl group; R 13 and R 14 are each independently H, C1 to C 10 alkyl groups, R 15 and R 16 are each independently H, C1 to C 10 The alkyl group is selected from the group consisting of:

[0024] In some embodiments, R 2 and R 3 are each independently selected from H, D, a C1-C6 alkyl group, an adamantyl group, and a C3-C7 cycloalkyl group, or R 2 and R 3 together with the carbon atoms to which they are attached form a 3- to 8-membered carbocyclic ring.

[0025] In some embodiments, R 2 and R 3 are each independently selected from H, an isopropyl group, a tert-butyl group, a cyclopentyl group, and an adamantyl group, or R 2 and R 3 together with the carbon atoms to which they are attached form cyclopropyl and cyclopentyl groups.

[0026] In some embodiments, R 2 and R 3 one of which is selected from H and the other is selected from isopropyl, tert-butyl, cyclopentyl and adamantyl groups, or R 2 and R 3together with the carbon atoms to which they are attached form cyclopropyl and cyclopentyl groups.

[0027] In some embodiments, R 4 is H, C1-C6 alkyl group, C3-C8 cycloalkyl group, C6-C 10 C6-C substituted with aryl group, C1-C6 alkyl group 10 C6-C substituted with aryl group, C1-C6 alkoxy group 10 Aryl groups and halogenated C6-C 10 aryl groups; R 5 is selected from H, a C1-C6 alkyl group, and a C3-C7 cycloalkyl group; Or R 4 and R 5 are linked to each other to form a C2-C6 alkylene group, which links X and Y.

[0028] In some embodiments, X is selected from O, S, S(=O)2, and S=O; Y is absent or selected from O, S, and S=O; and R 4 is a C1 to C6 alkyl group and a C6 to C 10 aryl groups, R 5 is selected from H, or R 4 and R 5 are linked to each other to form a C2-C6 alkylene group, which links X and Y.

[0029] In some embodiments, R 4 and R 5 are linked together to form CH2CH2 and CH2CH2CH2, thereby linking X and Y.

[0030] In some embodiments, X and Y are each independently selected from O, S, and S=O; and R 4 and R 5 are linked together to form CH2CH2 and CH2CH2CH2, thereby linking X and Y.

[0031] In some embodiments, X and Y are each independently selected from O, S, and R 4 and R 5 are linked together to form CH2CH2, thereby linking X and Y.

[0032] In some embodiments, X and Y are both selected from S, or X and Y are both selected from O, and R 4 and R 5 are linked together to form CH2CH2, thereby linking X and Y.

[0033] In some embodiments, X is selected from S, Y is absent, and R 4 is selected from a phenyl group and an isopropyl group, and R 5 is selected from H.

[0034] In some embodiments, R 6 teeth [ka] is selected from.

[0035] In some embodiments, R 7 is selected from H.

[0036] In some embodiments, R 8 is H, C1-C6 alkyl group, C1-C6 alkoxy group, C3-C7 cycloalkyl group, halogenated C1-C6 alkyl group, halogenated C3-C7 cycloalkyl group, -NR 13 R 14 , C6~C 10 Aryl groups, halogenated C6-C 10 C6-C substituted with aryl group, C1-C6 alkyl group 10 C6-C substituted with aryl groups, halogenated C1-C6 alkyl groups 10 It is selected from an aryl group, a 5- to 10-membered heteroaryl group, and a halogenated 5- to 10-membered heteroaryl group.

[0037] In some embodiments, R 8 represents a C1-C6 alkyl group, a halogenated C1-C6 alkyl group, a C1-C6 alkoxy group, -NR 13 R 14 , a C3 to C7 cycloalkyl group, a halogenated C3 to C7 cycloalkyl group, a phenyl group, a halogenated phenyl group, a phenyl group substituted with a C1 to C6 alkyl group, a phenyl group substituted with a halogenated C1 to C6 alkyl group, and a 5- or 6-membered heteroaryl group.

[0038] In some embodiments, R 9 represents a C1-C6 alkyl group, a C3-C7 cycloalkyl group, a halogenated C1-C6 alkyl group, a halogenated C3-C7 cycloalkyl group, -NR 15 R 16 , C6~C 10 Aryl groups, halogenated C6-C 10 C6-C substituted with aryl group, C1-C6 alkyl group 10 C6-C substituted with aryl groups, halogenated C1-C6 alkyl groups 10 It is selected from an aryl group, a 5- to 10-membered heteroaryl group, and a halogenated 5- to 10-membered heteroaryl group.

[0039] In some embodiments, R 9 is selected from a C1 to C6 alkyl group, a C3 to C7 cycloalkyl group, a phenyl group, a phenyl group substituted with a C1 to C6 alkyl group, and a phenyl group substituted with a halogenated C1 to C6 alkyl group.

[0040] In some embodiments, R 13 and R 14 are each independently selected from H and a C1 to C6 alkyl group.

[0041] In some embodiments, R 15 and R 16 are each independently selected from H and a C1 to C6 alkyl group.

[0042] In some embodiments, R8 is CH3, CF3, CH2CF3, CF2CF3, methoxy group, [ka] a cyclopropyl group, [ka] phenyl group, [ka] and pyridin-3-yl.

[0043] In some embodiments, R 9 is selected from CH3, a cyclopropyl group, a phenyl group, a p-methylphenyl group, and a p-trifluoromethylphenyl group.

[0044] In some embodiments, the cyano compound of formula I is selected from cyano compounds of formula IA: [ka] where R 1 , R 2 , R 3 , R 4 , R 5 , X and Y are as defined above.

[0045] In some embodiments, the cyano compound of formula I is selected from the cyano compounds of formula IB: [ka] Here, the definitions of each substituent are as described above.

[0046] In some embodiments, the cyano compound of formula I is any one selected from the group consisting of cyano compounds of the following formulas: [ka] where R 1 , R 2 , R 3 and R 6 is as defined above.

[0047] In some embodiments, the compound of formula I of the present invention is selected from the following compounds: [ka] [ka] [ka] [ka]

[0048] The present invention provides a method for preparing a compound represented by general formula I, which is one of the following methods i and ii:

[0049] Method i: [ka] ia) obtaining a compound represented by formula IV from a compound represented by formula II and a compound represented by formula III by a condensation reaction; Preferably, in step ia), the compound represented by formula II is reacted with the compound represented by formula III in the presence of a condensing agent and a base in a solvent at a temperature of −20° C. to 50° C. for 0.1 to 12 hours to obtain the compound represented by formula IV; wherein the solvent is one or a mixture of two or more of tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N,N-dimethylacetamide, ethyl acetate, and 1,4-dioxane; Optionally, the condensing agent is a mixture of one or more of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, 2-hydroxypyridine N-oxide, propylphosphonic anhydride, 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate, 1,1'-carbonyldiimidazole, and O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; Optionally, the base is N,N-diisopropylethylamine, triethylamine, and N-methylmorpholine; ib) dehydrating the compound of formula IV to obtain the compound of general formula I, Preferably, in step ib), the compound represented by formula IV is reacted with a dehydrating agent in an anhydrous solvent at a temperature of −20° C. to 50° C. for 1 to 24 hours to obtain the compound represented by general formula I; wherein the anhydrous solvent is one or a mixture of two or more of tetrahydrofuran, dichloromethane, toluene, 1,4-dioxane, and pyridine; The dehydrating agent is trifluoroacetic anhydride, methyl N-(triethylammoniosulfonyl)carbamate, Method ii: [ka] 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 for an amino group; Preferably, in step iia, the compound represented by formula V is reacted with the compound represented by formula III in the presence of a condensing agent and a base in a solvent at a temperature of −20° C. to 50° C. for 0.1 to 12 hours to obtain the compound represented by formula VI; wherein the solvent is one or a mixture of two or more of tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N,N-dimethylacetamide, ethyl acetate, and 1,4-dioxane; the condensing agent is one or a mixture of more than one of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, 2-hydroxypyridine N-oxide, propylphosphonic anhydride, 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate, 1,1'-carbonyldiimidazole, and O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; the bases are N,N-diisopropylethylamine, triethylamine and N-methylmorpholine; the amino-protecting group PG is a tert-butoxycarbonyl group, a benzyl group, or a p-methoxybenzyl group; iib) deprotecting a compound of formula VI to obtain a compound of formula VII; Preferably, in step iib), the compound of formula VI is reacted 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; iic) obtaining a compound of formula IV from a compound of formula VII by aminoacylation, sulfonylation or condensation reaction, Preferably, in step iic), Aminoacylation of the compound of formula VII with an acyl chloride or an acid anhydride in the presence of a base at a temperature of -20°C to 50°C to obtain a compound of formula IV; or Sulfonylation of the compound of formula VII with a sulfonyl chloride or sulfonic acid anhydride under basic conditions at a temperature of -20°C to 50°C to obtain a compound of formula IV; or The compound represented by formula VII is subjected to a condensation reaction with a carboxy compound under the conditions of a condensing agent and a base at a temperature of -20°C to 50°C to obtain a compound represented by formula IV; Optionally, the base is N,N-diisopropylethylamine, triethylamine, and N-methylmorpholine; Optionally, the condensing agent is a mixture of one or more of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, 2-hydroxypyridine N-oxide, propylphosphonic anhydride, 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate, 1,1'-carbonyldiimidazole, and O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; iid) dehydrating a compound of formula IV to obtain a compound of general formula I, Preferably, in step iid), the compound represented by formula IV is reacted with a dehydrating agent in an anhydrous solvent at a temperature of −20° C. to 50° C. for 1 to 24 hours to obtain the compound represented by general formula I; wherein the anhydrous solvent is one or a mixture of two or more of tetrahydrofuran, dichloromethane, toluene, 1,4-dioxane, and pyridine; Optionally, the dehydrating agent is trifluoroacetic anhydride, methyl N-(triethylammoniosulfonyl)carbamate; Here, the definitions of each substituent are as described above.

[0050] Another aspect of the present invention further provides pharmaceutical compositions comprising one or more of the cyano compounds of general formula I, their racemates, enantiomers and diastereomers, and pharmaceutically acceptable salts thereof, which may further comprise one or more pharmaceutically acceptable auxiliary materials, diluents, carriers, excipients or adjuvants.

[0051] Another aspect of the present invention further provides a pharmaceutical composition comprising a cyano compound of general formula I, or a racemate, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable auxiliary material, optionally, said pharmaceutical composition further comprising ritonavir or a pharmaceutically acceptable salt thereof.

[0052] Another aspect of the present invention further provides a pharmaceutical combination comprising a cyano compound of general formula I, a racemate, enantiomer, diastereomer or pharmaceutically acceptable salt thereof, and ritonavir or a pharmaceutically acceptable salt thereof.

[0053] Another aspect of the present invention further provides a pharmaceutical composition comprising a cyano compound of general formula I or a pharmaceutically acceptable salt thereof, ritonavir or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable auxiliary materials.

[0054] Experiments have demonstrated that the compounds of the present invention have inhibitory activity against coronavirus 3CL protease and picornavirus 3CL protease.

[0055] Therefore, yet another aspect of the present invention provides a coronavirus 3CL protease inhibitor and / or picornavirus 3CL protease inhibitor comprising one or more selected from the group consisting of cyano compounds of general formula I, their racemates, enantiomers and diastereomers, and pharmaceutically acceptable salts thereof, or the pharmaceutical composition described above.

[0056] The present invention further provides use of the above-mentioned cyano compound, its racemate, enantiomer, diastereomer, pharmaceutically acceptable salt, or mixture thereof, or the above-mentioned pharmaceutical composition in the manufacture of a medicament for inhibiting coronavirus 3CL protease activity, a medicament for preventing and / or treating coronavirus infection, a medicament for inhibiting picornavirus 3CL protease activity, and a medicament for preventing and / or treating picornavirus infection.

[0057] The present invention further provides a method for inhibiting 3CL protease, which comprises administering to a subject in need thereof one or more selected from the cyano compounds of general formula I according to the present invention, their racemates, enantiomers and diastereomers, and pharmaceutically acceptable salts thereof, or a pharmaceutical composition according to the present invention.

[0058] The present invention further provides a method for preventing and / or treating a disease or condition, which comprises administering one or more selected from the cyano compounds of general formula I according to the present invention, their racemates, enantiomers and diastereomers, and pharmaceutically acceptable salts thereof, or a pharmaceutical composition according to the present invention, to a subject in need of treatment, wherein the disease or condition is a 3CL protease-mediated disease or condition, in particular a disease or condition associated with coronavirus infection and / or picornavirus infection.

[0059] The present invention further provides use of one or more of the cyano compounds represented by the above general formula I, their racemates, enantiomers and diastereomers, and pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, in the manufacture of a medicament for preventing or treating related diseases caused by coronavirus and / or picornavirus infection.

[0060] The present invention further provides a method for preventing or treating related diseases caused by coronavirus and / or picornavirus infection, which comprises administering to a patient a therapeutically effective amount of a pharmaceutical formulation containing one or more of the cyano compounds represented by general formula I of the present invention, their racemates, enantiomers and diastereomers, 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.

[0062] In some embodiments, the associated disease resulting from coronavirus infection is selected from a respiratory tract infection, pneumonia, or a complication thereof.

[0063] [Definition and explanation of terms] "C1~C 10 The term "alkyl group" refers to a linear 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, a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, an isopropyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an isopentyl group, a 2-methylbutyl group, a 1-methylbutyl group, a 1-ethylpropyl group, a 1,2-dimethylpropyl group, a neopentyl group, a 1,1-dimethylpropyl group, a 4-methylpentyl group, a 3-methylpentyl group, a 2-methylpentyl group, a 1-methylpentyl group, a 2-ethylbutyl group, a 1-ethylbutyl group, a 3,3-dimethylbutyl group, a 2,2-dimethylbutyl group, a 1,1-dimethylbutyl group, a 2,3-dimethylbutyl group, a 1,3-dimethylbutyl group, and a 1,2-dimethylbutyl group. The term "C1-C6 alkyl group" refers to a straight or branched chain alkyl group containing 1 to 6 carbon atoms, including, but not limited to, a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a tert-butyl group, a pentyl group, an isopentyl group, a hexyl group, and the like.

[0064] An "alkylene group" may be understood to represent a straight or branched chain divalent saturated hydrocarbon group. A "C2-C6 alkylene group" may be understood to represent a straight or branched chain divalent saturated hydrocarbon group having 2 to 6 carbon atoms, including, but not limited to, CH2CH2, CH2CH2CH2, CH2CH2CH2CH2, CH2CH2CH2CH2CH2 or CH2CH2CH2CH2CH2CH2.

[0065] A "C3-C8 cycloalkyl group" refers to a cyclic alkyl group containing 3 to 8 ring carbon atoms. A "C3-C7 cycloalkyl group" refers to a cyclic alkyl group containing 3 to 7 ring carbon atoms, and cycloalkyl groups of the present disclosure include, but are not limited to, cyclopropyl, methylcyclopropyl, ethylcyclopropyl, dimethylcyclopropyl, cyclobutyl, methylcyclobutyl, ethylcyclobutyl, cyclopentyl, cyclohexyl, and the like.

[0066] "C1~C 10 An "alkoxy group" refers to a straight-chain, branched-chain, or cyclic alkoxy group containing 1 to 10 carbon atoms. A "C1-C6 alkoxy group" refers to a straight-chain, branched-chain, or cyclic alkoxy group containing 1 to 6 carbon atoms, and alkoxy groups in the present disclosure include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentyloxy, isopentyloxy, cyclopentyloxy, hexyloxy, cyclohexyloxy, and the like.

[0067] The term "aryl group" refers to a monocyclic or fused polycyclic aromatic ring group having a conjugated pi-electron system and in which all ring atoms are carbon. An aryl group can have 6 to 20 carbon atoms, 6 to 14 carbon atoms, or 6 to 12 carbon atoms. "C6-C 20 An "aryl group" may be understood as an aryl group having 6 to 20 carbon atoms. In particular, a ring having 6 carbon atoms ("C6 aryl group"), such as a phenyl group, or a ring having 9 carbon atoms ("C9 aryl group"), such as an indanyl or indenyl group, or a ring having 10 carbon atoms ("C 10 aryl group), such as a tetralinyl group, a dihydronaphthyl group, or a naphthyl group, or a ring having 13 carbon atoms ("C 13 aryl group), such as a fluorenyl group, or a ring having 14 carbon atoms ("C 14 "aryl group"), for example, an anthryl group. 10An "aryl group" may be understood as an aryl group having 6 to 10 carbon atoms. In particular, a ring having 6 carbon atoms ("C6 aryl group"), such as a phenyl group, or a ring having 9 carbon atoms ("C9 aryl group"), such as an indanyl or indenyl group, or a ring having 10 carbon atoms ("C 10 aryl group), for example, a tetralinyl group, a dihydronaphthyl group, or a naphthyl group.

[0068] A "heteroaryl group" is an aromatic ring group containing 5 to 20 ring atoms, one or more of which are heteroatoms selected from N, O, or S, and the remaining ring atoms are carbon. A "5- to 20-membered heteroaryl group" may be understood as a heteroaryl group having 5 to 20 ring atoms, particularly 5, 6, 9, 10, 13, or 14 ring atoms, and containing 1 to 7 heteroatoms independently selected from N, O, and S. A "5- to 10-membered heteroaryl group" may be understood as a heteroaryl group having 5 to 10 ring atoms, particularly 5, 6, 9, or 10 ring atoms, and containing 1 to 5 heteroatoms independently selected from N, O, and S. A "5- to 6-membered heteroaryl group" may be understood as a heteroaryl group having 5 or 6 ring atoms and containing 1 to 3 heteroatoms independently selected from N, O, and S. The heteroaryl group according to the present invention is selected from thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl or thiadiazolyl groups and their benzo derivatives, such as benzofuryl, benzothienyl, benzothiazolyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzotriazolyl, indazolyl, indolyl or isoindolyl groups, or a pyridyl group , pyridazinyl group, pyrimidinyl group, pyrazinyl group, triazinyl group, etc., and benzo derivatives thereof, for example, quinolyl group, quinazolinyl group, or isoquinolyl group, or selected from azocinyl group, indolizinyl group, purinyl group, etc., and benzo derivatives thereof, or selected from cinnolinyl group, phthalazinyl group, quinazolinyl group, quinoxalinyl group, naphthyridinyl group, ptenidinyl group, carbazolyl group, acridinyl group, phenoxazinyl group, phenothiazinyl group, phenoxazinyl group, etc.

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

[0070] "Halogenated" includes monohalogenated, polyhalogenated or perhalogenated, i.e., one, more or all hydrogen atoms are replaced with halogen.

[0071] "Substituted" means that one or more hydrogen atoms on a group are replaced with one or more substituents.

[0072] The term "optionally" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes instances where the event or circumstance occurs and instances where the event or circumstance does not occur.

[0073] A "therapeutically effective amount" refers to the amount of a compound of the present invention used that will (i) treat a particular disease, condition, or disorder, (ii) reduce, ameliorate, or eliminate one or more symptoms of a particular disease, condition, or disorder, or (iii) delay the onset of one or more symptoms of a particular disease, condition, or disorder described herein. The amount of a compound of the present invention that constitutes a "therapeutically effective amount" will vary depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal being treated, but can be determined by one of ordinary skill in the art based on their own knowledge and the present disclosure, as is routine.

[0074] A "pharmaceutically acceptable salt" is a salt of a pharmaceutically acceptable acid or base, and includes salts formed between a compound and an inorganic acid or an organic acid, and salts formed between a compound and an inorganic base or an organic base.

[0075] The term "pharmaceutical combination" refers to a combination comprising 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 pharmaceutical combination may be administered simultaneously, and in some embodiments of the present invention, the active ingredients or pharmaceutically acceptable salts thereof in the pharmaceutical composition may be administered separately or sequentially.

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

[0077] "Pharmaceutically acceptable auxiliary material" refers to an auxiliary material that has no obvious irritating effect on the organism and does not impair the biological activity and performance of the active compound. Suitable auxiliary materials are 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. [Example]

[0078] The present disclosure is further illustrated by the following examples, in which raw materials are commercially available or can be prepared by literature methods / organic synthesis methods known in the art.

[0079] Example 1 Preparation of Compound 1-1: [ka]

[0080] Step 1: The starting material SMA (2.74 g, 11.85 mmol), 35 mL of dichloromethane, and 35 mL of DMF were placed in a reaction flask and cooled to 0 °C. The starting materials SMB (3.56 g, 11.86 mmol), benzotriazol-1-yloxytri(dimethylamino)phosphonium hexafluorophosphate (BOP, 6.29 g, 14.22 mmol), and N-methylmorpholine (NMM, 3.91 mL, 35.56 mmol) were added sequentially. The temperature was raised to room temperature and the reaction was allowed to proceed for 10 h. After the reaction was complete, an appropriate amount of dichloromethane was added. The organic phase was washed with 1N aqueous hydrochloric acid and saturated saline, followed by drying over anhydrous sodium sulfate. The organic phase was concentrated to dryness and purified by column chromatography to obtain 3.71 g of INT-1. ESI-MS: m / z [M+H] 433.2. + ; 1H NMR (400 MHz, DMSO-d): δ H :6.75(d,J=9.2Hz,1H),4.38(t,J=8.2Hz,1H),4.25(d,J=10.9Hz,1H),4.11(d,J=9.3Hz,1H),3.93(t,J=9.3Hz,1H),3 .62(s,3H),3.40-3.31(m,4H),2.70(dd,J=13.1,7.9Hz,1H),2.37(dd,J=13.2,8.4Hz,1H),1.37(s,9H),0.94(s,9H).

[0081] Step 2: 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) were added to a reaction flask and reacted at room temperature for 2 hours. After the reaction was completed, the pH was adjusted to 4 with concentrated hydrochloric acid, and the mixture was filtered to obtain 3.4 g of compound 1-1. ESI-MS: m / z 419.2 [M+H] + ; 1 H NMR (400 MHz, DMSO-d): δ H :12.68(s,1H),6.71(d,J=9.4Hz,1H),4.38-4.19(m,2H),4.11(d,J=9.4Hz,1H),3.88(d,J=10.9Hz,1H), 3.41-3.29(m,4H),2.69(dd,J=13.1,7.9Hz,1H),2.34(dd,J=13.2,8.9Hz,1H),1.38(s,9H),0.94(s,9H).

[0082] Preparation of Compound 1-2: [ka]

[0083] Ammonia-methanol solution (700 ml, 7 mol / L) and the starting material SMD (100 g, 0.349 mol) were placed in a reaction flask, stirred to dissolve until clear, and then kept at 25±5°C for 36 hours. After the reaction was complete, the reaction solution was concentrated until approximately 250 ml of reaction solution remained. 300 ml of isopropanol was added, and the reaction solution was further concentrated under reduced pressure until approximately 250 ml remained (repeated three times). The atmosphere was purged with nitrogen, the temperature was lowered to 10±5°C, and 500 ml of hydrogen chloride-isopropanol solution (4 mol / L) was added to the reaction vessel. After the addition was complete, the temperature was raised to 25±5°C, and the reaction was continued for 9 hours while keeping the temperature at 25±5°C. After the reaction was completed, the reaction mixture was concentrated under reduced pressure until approximately 250 ml of reaction mixture remained. 300 ml of isopropanol was added, and the mixture was further concentrated under reduced pressure until approximately 250 ml of reaction mixture remained (repeated twice). 100 ml of isopropanol was added, and the mixture was stirred for 30±5 minutes. The mixture was filtered, and the filter cake was rinsed with 50 ml of isopropanol to obtain a wet product. The wet product was dried under vacuum at 45±5°C to obtain 66.7 g of compound 1-2. Yield: 92%. 1 H NMR (400 MHz, DMSO-d): δ H :8.45(d,J=5.1Hz,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,1H),2.32-2.27(m,1H),2.05-1.98(m,1H),1.82-1.66(m,2H);ESI-MS:172.1m / z[M+H] + .

[0084] Preparation of Compound 1: [ka]

[0085] Compound 1-1 (419 mg, 1 mmol) was placed in a two-neck flask and 5 mL of dichloromethane was added under nitrogen gas protection. O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (400 mg, 1.1 mmol) was added and 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. N,N-diisopropylethylamine (0.5 mL, 1 mmol) was added in an ice-water bath. The ice-water bath was removed and the system was stirred at room temperature overnight. After workup, 50 mL of dichloromethane was added. The system was then washed three times with 1 M aqueous hydrochloric acid, three times with saturated aqueous sodium bicarbonate, and the organic phase was washed with saturated brine. The organic phase was then dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain compound 1-3 (white solid, 469 mg, 82% yield). ESI-MS: m / z 572.3 [M+H] + .

[0086] Compound 1-3 (114 mg, 0.2 mmol) and Burgess reagent (1.5 eq) were placed in a two-neck flask and filled with nitrogen gas three times. Dichloromethane dried over molecular sieves was added and the mixture was stirred at room temperature overnight. Thin layer chromatography showed that the starting materials had essentially reacted completely. After workup, compound 1 (white solid, 52 mg, 47% yield) was obtained by column chromatography. ESI-MS: 554.3 m / z [M+H] + .

[0087] <Example 2> [ka]

[0088] Compound 1-3 (572 mg, 1 mmol) was dissolved in 3 mL of 4 M hydrogen chloride / 1,4-dioxane solution or 2 mL of dichloromethane, followed by dropwise addition of 2 mL of trifluoroacetic acid and stirring at ambient temperature. After thin-layer chromatography showed that the starting materials were essentially completely reacted, the solvent was thoroughly spun. The resulting crude product was dissolved in 2 mL of dichloromethane, protected with nitrogen gas, and triethylamine (3 mmol) was added. The system was placed in an ice-water bath, and trifluoroacetic anhydride (1.2 mmol) was added dropwise. After thin-layer chromatography showed that the starting materials were essentially completely reacted, 50 mL of dichloromethane was added. The organic phase was then washed three times with 1 M aqueous hydrochloric acid, three times with saturated aqueous sodium bicarbonate, and saturated brine. The organic phase was then dried over anhydrous sodium sulfate and purified by column chromatography to obtain compound 2-1 (white solid, 265 mg, 46% yield). ESI-MS: m / z 568.3 [M+H] + .

[0089] Compound 2-1 (113 mg, 0.2 mmol) and Burgess reagent (1.5 eq) were placed in a two-necked flask and filled with nitrogen three times. Dichloromethane dried over molecular sieves was added and the mixture was stirred at room temperature overnight. Thin layer chromatography showed that the starting materials had essentially reacted completely. After workup, compound 2 (41 mg, 47% yield) was obtained by column chromatography. 1H NMR(400MHz,DMSO-d6)δ9.46(d,J=8.7Hz,1H),9.05(d,J=8.6Hz,1H),7.67(s,1H),4.97(ddd,J=11.0,8.5,5.0 Hz,1H),4.53(d,J=8.7Hz,1H),4.34(dd,J=9.9,7.1Hz,1H),4.26-4.14(m,1H),3.92(d,J=10.9Hz,1H),3.50-3. 34(m,4H),3.22-3.11(m,1H),3.06(td,J=9.3,7.1Hz,1H),2.68-2.58(m,1H),2.50-2.43(m,1H),2.31(dd,J=1 3.0,10.0Hz,1H),2.23-2.07(m,2H),1.71(tdd,J=14.9,10.3,7.4Hz,2H),0.99(s,9H).ESI-MS:550.3m / z[M+H] + .

[0090] Example 3 [ka]

[0091] Compound 3 (white solid, 45 mg, 45% yield) was obtained using the same method as in Example 2, except that acetic anhydride was used instead of trifluoroacetic anhydride. ESI-MS: 496.3 m / z [M+H] + .

[0092] Example 4 [ka]

[0093] Compound 4 (white solid, 32 mg, 31% yield) was obtained using the same method as in Example 2, except that cyclopropanecarboxylic anhydride was used instead of trifluoroacetic anhydride. ESI-MS: 522.3 m / z [M+H] + .

[0094] <Example 5> [ka]

[0095] Compound 5 (white solid, 22 mg, 20% yield) was obtained using the same method as in Example 1, except that 5-1 was used instead of 1-1. 1 H NMR(400MHz,DMSO-d6)δ9.03(d,J=8.4Hz,1H),7.67(s,1H),7.41(dd,J=9.1,2.6Hz,1H),4.96(ddd,J=10.6,8.4,5 .4Hz,1H),4.58-4.50(m,1H),4.34(dd,J=9.8,7.2Hz,1H),4.26-4.21(m,1H),3.89(d,J=11.0Hz,1H),3.45-3.33( m,4H),3.22-3.03(m,2H),2.64-2.57(m,1H),2.45(ddd,J=10.1,8.5,4.5Hz,1H),2.31(dd,J=12.9,9.8Hz,1H),2. 20-2.10(m,2H),1.84-1.67(m,2H),1.39-1.31(m,2H),1.24(t,J=9.5Hz,2H),0.97(s,9H).ESI-MS:540.2m / z[M+H] + .

[0096] Example 6 [ka]

[0097] Compound 6 (white solid, 38 mg, 37% yield) was obtained using the same method as in Example 1, except that 6-1 was used instead of 1-1. ESI-MS: 512.2 m / z [M+H] + .

[0098] Example 7 [ka]

[0099] Compound 7 (white solid, 49 mg, 44% yield) was obtained using the same method as in Example 1, except that 7-1 was used instead of 1-1. ESI-MS: 553.3 m / z [M+H] + .

[0100] Example 8 [ka]

[0101] Compound 8 (white solid, 44 mg, 39% yield) was obtained using the same method as in Example 2, except that benzoic anhydride was used instead of trifluoroacetic anhydride. ESI-MS: 558.3 ​​m / z [M+H] + .

[0102] Example 9 [ka]

[0103] Compound 9 (white solid, 39 mg, 28% yield) was obtained using the same method as in Example 2, except that 3,5-bis(trifluoromethyl)benzoyl chloride was used instead of trifluoroacetic anhydride. ESI-MS: 694.2 m / z [M+H] + .

[0104] Example 10 [ka]

[0105] Compound 10 (white solid, 31 mg, 26% yield) was obtained using the same method as in Example 2, except that 3,5-dimethylbenzoyl chloride was used instead of trifluoroacetic anhydride. ESI-MS: 586.2 m / z [M+H] + .

[0106] Example 11 [ka]

[0107] Compound 11 (white solid, 28 mg, 25% yield) was obtained using the same method as in Example 2, except that 3,3,3-trifluoropropionic anhydride was used instead of trifluoroacetic anhydride. ESI-MS: 564.2 m / z [M+H] + .

[0108] Example 12 [ka]

[0109] Compound 12 (white solid, 39 mg, 35% yield) was obtained using the same method as in Example 2, except that 3-pyridinecarboxylic acid chloride was used instead of trifluoroacetic anhydride. ESI-MS: 559.2 m / z [M+H] + .

[0110] Example 13 [ka]

[0111] Compound 13 (white solid, 41 mg, 34% yield) was obtained using the same method as in Example 2, except that pentafluoropropanoyl chloride was used instead of trifluoroacetic anhydride. ESI-MS: 600.2 m / z [M+H] + .

[0112] Example 14 [ka]

[0113] Compound 14 (white solid, 32 mg, 29% yield) was obtained using the same method as in Example 1, except that 14-1 was used instead of 1-1. ESI-MS: 550.2 m / z [M+H] + .

[0114] Example 15 [ka]

[0115] Using the same method as in Example 2, except that methanesulfonic anhydride was used instead of trifluoroacetic anhydride, compound 15 (white solid, 38 mg, 36% yield) was obtained. 1 H NMR(400MHz,Methanol-d4)δ5.06(dd,J=11.5,4.5Hz,1H),4.46(dd,J=10.3,7.1Hz,1H),4.27 (dd,J=11.0,1.6Hz,1H),4.02-3.89(m,2H),3.45(qt,J=8.6,4.7Hz,4H),3.30-3.23(m,1H),2. 92(s,3H),2.78(tdd,J=10.3,8.5,4.0Hz,1H),2.68(ddd,J=13.0,7.2,1.6Hz,1H),2.51(dd,J= 13.0,10.3Hz,1H),2.45-2.27(m,2H),1.94-1.77(m,2H),1.05(s,9H).ESI-MS:532.2m / z[M+H] + .

[0116] Example 16 [ka]

[0117] Using the same method as in Example 2, except that cyclopropanesulfonyl chloride was used instead of trifluoroacetic anhydride, compound 16 (white solid, 29 mg, 26% yield) was obtained using cyclopropanesulfonyl chloride instead of trifluoroacetic anhydride. 1H NMR(400MHz,Methanol-d4)δ5.06(dd,J=11.5,4.5Hz,1H),4.44(dd,J=10.3,7.0Hz,1H),4.28(dd,J=11.0,1.6Hz,1H), 4.00-3.94(m,2H),3.52-3.38(m,4H),3.31-3.21(m,1H),2.79(tdd,J=10.4,8.5,4.1Hz,1H),2.72-2.65(m,1H),2.57( ddd,J=7.9,6.2,3.9Hz,1H),2.51(dd,J=13.0,10.4Hz,1H),2.43-2.29(m,2H),1.92-1.74(m,2H),1.34-1.29(m,1H),1 .14(qt,J=6.4,3.8Hz,1H),1.06(s,9H),1.01(ddt,J=7.9,5.8,2.7Hz,2H),0.97-0.87(m,1H).ESI-MS:558.2m / z[M+H] + .

[0118] Example 17 [ka]

[0119] Compound 17 (white solid, 35 mg, 29% yield) was obtained using the same method as in Example 2, except that benzenesulfonyl chloride was used instead of trifluoroacetic anhydride. ESI-MS: 594.2 m / z [M+H] + .

[0120] Example 18 [ka]

[0121] Compound 18 (white solid, 44 mg, 42% yield) was obtained using the same method as in Example 1, except that 18-1 was used instead of 1-1. ESI-MS: 518.2 m / z [M+H] + .

[0122] Example 19 [ka]

[0123] Compound 19 (white solid, 41 mg, 36% yield) was obtained using the same method as in Example 1, except that 19-1 was used instead of 1-1. ESI-MS: 568.2 m / z [M+H] + .

[0124] Example 20 [ka]

[0125] Compound 20 (white solid, 45 mg, 35% yield) was obtained using the same method as in Example 1, except that 20-1 was used instead of 1-1. ESI-MS: 648.2 m / z [M+H] + .

[0126] Example 21 [ka]

[0127] Compound 21 (white solid, 34 mg, 31% yield) was obtained using the same method as in Example 1, except that 21-1 was used instead of 1-1. ESI-MS: 640.2 m / z [M+H] + .

[0128] <Example 22> [ka]

[0129] The same method as in Example 1 was used, except that 22-1 was used instead of 1-1 and 1-2, to obtain compound 22 (white solid, 22 mg, 18% yield). ESI-MS: 614.2 m / z [M+H] + .

[0130] Example 23 [ka]

[0131] Compound 23-1 (497 mg, 1 mmol) was placed in a two-neck flask and 5 mL of dichloromethane was added under nitrogen gas protection. O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (400 mg, 1.1 mmol) was added and 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. N,N-diisopropylethylamine (0.5 mL, 1 mmol) was added in an ice-water bath, the ice-water bath was removed, and the system was stirred at room temperature overnight. After workup, 50 mL of dichloromethane was added. The system was then washed three times with 1 M aqueous hydrochloric acid, three times with saturated aqueous sodium bicarbonate, and the organic phase was washed with saturated brine. The organic phase was then dried over anhydrous sodium sulfate, filtered, and rotary evaporated to give compound 23-2 (white solid, 421 mg, 65% yield). ESI-MS: m / z 650.3 [M+H] + .

[0132] Compound 23-2 (325 mg, 0.5 mmol) was dissolved in 1.5 mL of 4 M hydrogen chloride / 1,4-dioxane solution or 1 mL of dichloromethane, followed by dropwise addition of 1 mL of trifluoroacetic acid and stirring at ambient temperature. After thin-layer chromatography showed that the starting materials were essentially completely reacted, the solvent was thoroughly spun down. The resulting crude product was dissolved in 2 mL of dichloromethane, protected with nitrogen gas, and triethylamine (1.5 mmol) was added. The system was placed in an ice-water bath, and trifluoroacetic anhydride (0.6 mmol) was added dropwise. After thin-layer chromatography showed that the starting materials were essentially completely reacted, 25 mL of dichloromethane was added. The mixture was then washed three times with 1 M aqueous hydrochloric acid, three times with saturated aqueous sodium bicarbonate, and the organic phase was washed with saturated brine. The organic phase was then dried over anhydrous sodium sulfate and purified by column chromatography to obtain compound 23-3 (white solid, 167 mg, 52% yield). ESI-MS: m / z 646.3 [M+H] + .

[0133] Compound 23-3 (130 mg, 0.2 mmol) and Burgess reagent (1.5 eq) were placed in a two-neck flask and filled with nitrogen gas three times. Dichloromethane dried over molecular sieves was added and the mixture was stirred at room temperature overnight. Thin layer chromatography showed that the starting materials had essentially reacted completely. After workup, compound 23 (white solid, 37 mg, 29% yield) was obtained by column chromatography. ESI-MS: m / z [M+H] 628.2 + .

[0134] Example 24: Measurement of inhibitory activity of compounds against SARS-CoV-2 3CLpro SARS-CoV-2 3CL pro The inhibitory activity of the compounds against enzyme activity was evaluated by measuring fluorescence resonance energy transfer. The total volume of the enzyme reaction system was 120 μL, the final concentration of protease was 30 nM, and the final concentration of substrate was 20 μM. The reaction buffer contained 50 mM Tris pH 7.3 and 1 mM EDTA. SARS-CoV-2 3CL was placed in a 96-well plate. proProtease and different concentrations of compounds were added and incubated at 30°C for 10 minutes. Substrate was then added and the plate was quickly inserted into a microplate reader to read the data. The excitation and emission light were 320 nM and 405 nM, respectively. The measurement time was 3.5 minutes, and fluorescence values ​​were read every 35 seconds. The reaction rate was obtained by fitting the values ​​read in the previous 2 minutes from the final results, and the inhibition rate was calculated by comparing with the control group (DMSO). The IC was calculated by fitting using GraphPad Prism 8 software. 50 The values ​​and inhibition rate curves were obtained.

[0135] The experimental results are shown in Table 1. The results show that the compounds of the present invention inhibit SARS-CoV-2 3CL pro It has been shown that it has a strong inhibitory effect on IC 50 The value ranges are: A means <0.1 μM, B means 0.1-1 μM, and C means 1-10 μM.

[0136] [Table 1]

[0137] [Table 2]

[0138] [Table 3]

[0139] [Table 4]

[0140] <Example 24-2: Test of the inhibitory activity of Compound 2 against SARS-CoV-2 Omicron strain mutant 3CL protease> Test principle: The inhibitory effect of the compounds of the present invention on the activity of Omicron strain mutant 3CL protease (P132H) was studied by applying a method in which fluorescence resonance energy transfer (FRET) is generated by the reaction between the enzyme and the substrate.

[0141] The experimental materials are as shown in the table below.

[0142] [Table 5]

[0143] [Experimental equipment and devices]

[0144] [Table 6]

[0145] Experimental steps: A reaction buffer containing 20 mM Tris-HCl, 1 mM EDTA, 0.01% BSA, 1 mM DTT, and 100 mM NaCl was prepared. Using the Echo dispensing system, the target compounds were diluted to different concentrations with dimethyl sulfoxide (DMSO) and transferred to a 384-well plate. The mutant 3CL protease was diluted with the reaction buffer and added to the 384-well plate at 10 μL / well. The plate was centrifuged at 1000 rpm for 1 min and then incubated at room temperature for 30 min. The substrate was then added at 10 μL / well and centrifuged at 1000 rpm for 30 s to initiate the enzyme reaction. The final enzyme concentration in the reaction system was 50 nM, the final substrate concentration was 20 μM, and the compound concentrations ranged from 10,000 nM to 0.51 nM. Next, the Flexstation 3 microplate reader was set to Kinetic Reduction Vmax mode, and fluorescence readings at 490 nm wavelength were taken every 75 seconds for a total of 35 consecutive readings. The reaction velocity values ​​(V) were obtained, and the inhibition rates were calculated. The half maximal inhibitory concentrations (IC) were calculated using a four-parameter fitting using XLfit software. 50 The inhibition rate is calculated as follows:

[0146]

number

[0147] Experimental results: Compound 2 maintained significant inhibitory activity against the 3CL protease carrying the P132H mutation in the SARS-CoV-2 Omicron strain (IC in three independent experiments). 50 was 0.022±0.00090 μM).

[0148] [Table 7]

[0149] <Example 24-3: Experiment on the inhibitory activity of Compound 2 against 3CL proteases derived from different coronaviruses> Experimental Objective: To study the inhibitory effect of compound 2 on the activity of 3CL proteases derived from six other coronaviruses capable of infecting humans: SARS-CoV, MERS-CoV, OC43-CoV, H229E-CoV, NL63-CoV, and HKU1-CoV.

[0150] Test materials: Recombinant full-length coronavirus 3CL proteases were produced in-house based on the genome sequences of the coronaviruses. 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 protein expression of the six coronavirus 3CL proteases were purchased from Nanjing GenScript Biotechnology Co., Ltd.

[0151] The substrate for 3CL protease was purchased from Nanjing GenScript Biotechnology Co., Ltd.

[0152] Chymotrypsin substrate was purchased from GLBiochem.

[0153] Other reagents are as shown in the table below.

[0154] [Table 8]

[0155] Experimental steps: A reaction buffer solution (containing 50 mM Tris and 1 mM EDTA) was prepared. The target compounds were dissolved in DMSO to a 100 mM stock solution and then serially diluted two-fold with the reaction buffer to create a total of 11 concentrations. 3CL protease and different concentrations of compounds were placed in a 96-well plate and incubated at room temperature for 10 minutes. The substrate was then added and the plate was quickly loaded into a microplate reader for data reading. The total volume of the enzyme reaction system was 120 μL. The final concentrations of SARS-CoV, MERS-CoV, H229E-CoV, HKU1-CoV, NL63-CoV, and OC43-CoV proteases were 30 nM, 80 nM, 30 nM, 20 nM, 30 nM, and 10 nM, respectively, and the final substrate concentration was 10 μM. The excitation and emission wavelengths for data reading were 340 nm and 490 nm, respectively. The test lasted for 10 minutes, with the fluorescence readings taken every minute. The final result was fitted to the values ​​taken in the previous 5 minutes to obtain the reaction rate, and the inhibition rate was calculated using the following formula.

[0156]

number

[0157] Experimental results: As shown in Table 3, compound 2 exhibited good inhibitory effects against 3CL proteases derived from six other coronaviruses, demonstrating that compound 2 has broad-spectrum anti-coronavirus activity.

[0158] [Table 9]

[0159] Example 25-1: Cell-level inhibitory effect of Compound 2 against SARS-CoV-2 WIV04 and B.1.351 strains Vero E6 cells were used in the test. Vero E6 cells (50,000 cells / well) were plated in a 48-well plate, and 100 μL of medium containing gradient concentrations of compounds was added per well. After 1 hour, SARS-CoV-2 was added at a multiplicity of infection (MOI) of 0.01. After 1 hour of co-incubation, the supernatant was aspirated, washed, and 200 μL of medium containing gradient concentrations of compounds was added per well. The cells were then cultured at 37°C for 24 hours. After 24 hours, the cell supernatant was collected, viral RNA was extracted, and the viral copy number in the supernatant was measured by real-time fluorescent quantitative PCR. The compound's inhibition rate was calculated based on the viral copy number, and the EC value of the compound was calculated using Prism 6.0. 50 was calculated.

[0160] The test results showed that Compound 2 inhibits the SARS-CoV-2 WIV04 strain at a median effective concentration (EC 50 The EC 50 is 0.73 + / - 0.06 μM, and the EC 50 The curves are as shown in Figures 1 and 2.

[0161] Example 25-2: Cell-level inhibitory effect of Compound 2 on SARS-CoV-2 Vero E6 original strain (WIV04), Delta strain (B.1.617.2), and Omicron strain (B.1.1.529) Experimental Objective: This study investigated the inhibitory effect of compound 2 on the intracellular replication of SARS-CoV-2 original strain (WIV04 strain), Delta strain (B.1.617.2), and Omicron strain (B.1.1.529) in Vero E6 cells by detecting viral copy numbers in the culture supernatant using real-time fluorescent quantitative PCR. Because Vero E6 cells highly express the efflux transport protein P-gp, the compounds were co-incubated with 0.5 μM of the P-gp inhibitor CP-100356.

[0162] Test materials: Vero E6 was purchased from ATCC (product number CRL-1586), and the SARS-CoV-2 original strain (SARS-CoV-2-WIV04 strain), Delta strain (B.1.617.2), and Omicron strain (B.1.1.529) viruses were supplied by the Center for Microbial (Virulent) Seed Collection of Wuhan Institute of Virology, Chinese Academy of Sciences.

[0163] Other reagents are as shown in the table below.

[0164] [Table 10]

[0165] Lab equipment: Biohazard Safety Cabinet (AC2-3S1, ESCO, Singapore) Carbon dioxide incubator (Thermo Scientific HERAcell 150i, Thermo Scientific, USA) Pure water equipment (Yuyuan SYS ultra pure water equipment, Chengdu) StepOne Plus Real-time PCR system (4376600, ABI, USA) TC20 TM Automated cell counter (1450102, BIO-RAD, USA) T100 TM Thermal Cycler (1861096, BIO-RAD, USA) Centrifuge (Micro21 / 21R Thermo Fisher Scientific, USA)

[0166] Experimental steps: Vero E6 cells were digested with trypsin, placed in medium (90% DMEM, 10% fetal bovine serum), and seeded into a 48-well plate at 50,000 cells per well for overnight incubation. The target compounds were dissolved in DMSO to obtain a 40 mM stock solution, which was then serially diluted with medium containing 0.5 μM of a Pgp inhibitor to obtain the required concentrations for testing. The final concentrations of the target compounds in the experiments ranged from 1 μM to 0.004 μM. The cell supernatant was removed, and diluted compounds (containing 0.5 μM Pgp inhibitors) were added to each well and incubated for 1 hour. Different strains of SARS-CoV-2 were introduced into the wells at a multiplicity of infection (MOI) of 0.01 or 0.001 in a biosafety level 3 (BSL-3) laboratory. After 1 hour of incubation, the supernatant was removed and washed with PBS. 200 μL of diluted compounds (containing 0.5 μM Pgp inhibitors) were added to each well. The supernatant was collected 24 or 72 hours after infection. Viral RNA was extracted from the supernatant, and the viral copy number was measured using real-time fluorescent quantitative PCR. The compound inhibition rate was calculated based on the viral copy number, and the compound's IC was calculated using GraphPad Prism 8. 50 was calculated.

[0167] For the cytotoxicity test, Vero E6 cells were digested and placed in medium (90% DMEM, 10% fetal bovine serum), seeded into a 96-well plate, and cultured overnight at 20,000 cells per well. The target compound was dissolved in DMSO to obtain a 40 mM stock solution, which was then serially diluted with medium or medium containing 0.5 μM of a Pgp inhibitor to obtain the required concentration for the test. The final concentration range of the target compound in the experiment was 500 μM to 1.95 μM. The cell supernatant in the 96-well plate was removed, and 100 μL of medium containing the target compound (single agent or 0.5 μM of a Pgp inhibitor) was added to each well. After 24 hours of incubation, cell viability was measured using a CCK8 assay kit, and the inhibition rate and median cytotoxic concentration (CCT) were calculated. 50 ) was calculated.

[0168] Test results: As shown in Table 4, after combined use with the P-gp inhibitor CP-100356, compound 2 could dose-dependently inhibit the replication of Delta strain in Vero E6 cells, with an IC 50 The IC value was 0.040 μM. The combined use of Compound 2 and a P-gp inhibitor also exerted a high inhibitory effect on the original strain. 50 The IC value was 0.027 μM. Compound 2, a co-P-gp inhibitor, could significantly inhibit the replication of Omicron strains in Vero E6 cells. 50 The concentration of compound 2 was 0.12 μM. Neither compound 2 alone nor its combination with a P-gp inhibitor showed any obvious cytotoxicity to VeroE6 cells. 50 It was >500 μM.

[0169] [Table 11]

[0170] Example 26: In vivo antiviral activity of Compound 2 against SARS-CoV-2 Delta strain in hACE 2-K 18 transgenic mice Experimental Objective: This study evaluated the antiviral activity of compound 2 against the SARS-CoV-2 Delta strain in K18 transgenic mice stably expressing human angiotensin-converting enzyme 2 (ACE2) (K18-hACE2).

[0171] Test materials: Seven- to eight-week-old K18-hACE2 transgenic mice were purchased from Jiangsu Jixi Yaokang Biopharmaceutical Co., Ltd. SARS-CoV-2 Delta strain virus was provided by the Center for Microbial and Bacterial Species Collection, Wuhan Institute of Virology, Chinese Academy of Sciences.

[0172] Ritonavir was purchased from Shanghai Disinfectant Chemical Pharmaceutical Co., Ltd.

[0173] Vero E6 cells were purchased from ATCC (product number CRL-1586).

[0174] Other reagents are as shown in the table below.

[0175] [Table 12]

[0176] Lab equipment: Biohazard Safety Cabinet (AC2-3S1, ESCO, Singapore) Carbon dioxide incubator (Thermo Scientific HERAcell 150i, Thermo Scientific, USA) Pure water equipment (Yuyuan SYS ultrapure water equipment, Chengdu) StepOne Plus Real-time PCR system (4376600, ABI, USA) TC20 TM Automated cell counter (1450102, BIO-RAD, USA) T100 TM Thermal Cycler (1861096, BIO-RAD, USA) Centrifuge (Micro21 / 21R Thermo Fisher Scientific, USA) Tissue grinder (JXFSTPRP-CL, Shanghai Jingxin Co., Ltd., China)

[0177] Experimental Procedure: K18-hACE2 transgenic mice were infected with the SARS-CoV-2 delta strain via nasal instillation. The day of infection was designated day 0. Two hours after infection, mice were intragastrically administered either vehicle or 50 mg / kg or 200 mg / kg of compound 2 (combined with the cytochrome P450 inhibitor ritonavir at 50 mg / kg) twice daily for 2 days (once on day 0, twice on day 1, and once on day 2) or 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 end of the study. The left lung was fixed with formaldehyde, embedded, sliced, and stained with H&E for histopathological examination. The right lung and brain tissues were each divided into two parts. One part was crushed to obtain homogenates, and RNA was extracted and reverse-transcribed. Viral copy numbers were measured by real-time fluorescent quantitative PCR. The other portion was crushed and the homogenate was used to measure virus titer by the plaque assay. The plaque assay method was as follows: Vero E6 cells were seeded into a 24-well plate at 12,000 cells per well and cultured overnight. The stock tissue homogenate was serially diluted 10-fold with DMEM medium for use. The cell supernatant was removed, and the diluted tissue homogenate was added and incubated for 1 hour. The supernatant was then removed, and medium containing 1% sodium methylcellulose and 2% FBS was added and cultured 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 plaques in each well was counted.

[0178] Test results: As shown in Table 5, 2 days after infection, compared to the model group (mean viral copy number 9.19±0.30log10 copies / g), compound 2 at doses of 50mg / kg and 200mg / kg significantly reduced lung viral loads with ritonavir, with mean copy numbers of 7.66±0.27log10 copies / g and 6.79±0.30log10 copies / g, respectively, with the 200mg / kg dose reducing the viral copy number by 2.4log10 copies / g. Four days after infection, compound 2 demonstrated a sustained inhibitory effect on viral copy number. As shown in Figure 3, compound 2 exhibited a significant inhibitory effect on viral titer. At 2 days post-infection, a dose of 200 mg / kg completely inhibited viral replication, resulting in undetectable viral titers. At 50 mg / kg, viral titers were reduced by more than 3 log10 PFU / g compared to the model group. Four days post-infection, compound 2 demonstrated a sustained inhibitory effect on viral titers. As shown in Figure 4, four days post-infection, the model group lost approximately 10% of their body weight, while the compound 2-treated group showed no significant weight loss, demonstrating no obvious toxicity of compound 2 with continued administration. We further measured viral load in the brains of mice. Two days post-infection, there was no obvious infection in either group. Four days post-infection, compound 2 significantly reduced viral copy numbers in the brains of mice at both 50 mg / kg and 200 mg / kg doses compared to the model group. At 200 mg / kg, the viral copy numbers in the brains were comparable to those of the uninfected control group. We further measured the viral titer in the brain 4 days after infection. As shown in Figure 5, the viral titer was undetectable at both doses of Compound 2 compared to the model group, demonstrating the potent inhibitory effect of Compound 2. Furthermore, histopathological analysis of the lungs revealed that Compound 2 significantly improved lung injury at a dose of 200 mg / kg compared to the model group, including reducing the degree of alveolar collapse or expansion and the degree of alveolar membrane thickening.

[0179] [Table 13]

[0180] Example 27: Selectivity of Compound 2 against kinases Experimental Objective: To investigate the selectivity of compound 2 against kinases, the inhibitory activity of compound 2 against 413 kinases was measured using the KinaseProfile experimental platform.

[0181] Test materials: The Full Human Panel [10 μM ATP] KinaseProfiler is a test product provided by Eurofins, product number 50-005KP10, and contains 413 kinases.

[0182] Experimental steps: The compound assays for each selected kinase were performed using the Eurofins standard KinaseProfiler analytical method, with the operating process following the relevant standards. Protein kinases were detected by radioactivity, and lipid kinases by HTRF. The ATP concentration in the experiments was 10 μM. Detailed information on each kinase can be obtained on the Eurofins website at the following address: https: / / www.eurofinsdiscoveryservices.com / catalogmanagement / viewItem / Full-Human-Panel-10-uM-ATP-KinaseProfiler / 50-005KP10.

[0183] Experimental results: Compound 2 showed less than 30% inhibition against 413 kinases at a concentration of 10 μM, indicating no significant inhibitory effect and excellent selectivity.

[0184] Example 28: Selectivity of Compound 2 for safety targets Experimental Objective: The effects of Compound 2 on 47 safety-related targets were measured on the Safetyscan experimental platform.

[0185] Test materials: The Safety 47 Panel Dose Response SAFETYscan is a test product provided by Eurofins, part number 87-1003DR, which contains 78 tests on 47 safety targets.

[0186] Experimental steps: The experimental methods used in the 78 tests for the 47 safety targets include cAMP assay, calcium flux assay, hormone nuclear receptor assay, kinase binding assay, enzyme activity assay, neurotransmitter transporter assay, ion channel assay, and transporter assay. The specific methods for each assay can be obtained from the eurofins website at the following address: https: / / www.eurofinsdiscoveryservices.com / catalogmanagement / viewItem / Safety47-Panel-Dose-Response-SAFETYscan-DiscoverX / 87-1003DR

[0187] Experimental results: Compound 2 had no significant inhibitory or activating effects on 47 safe related targets at a concentration of 100 μM (EC 50 All of the agonist activity was greater than 100 μM, demonstrating that compound 2 has excellent selectivity.

[0188] Example 29: Human plasma protein binding test of compound 2 Test materials: Human plasma was purchased from BioIVT, anticoagulated with EDTA K2, and stored at −80° C. 96-well equilibrium dialysis plates were purchased from HTDialysis LLC. Equilibrium dialysis membranes were purchased from Gales Ferry.

[0189] Experimental steps: A basic solution of 14.2 g / L disodium hydrogen phosphate and 8.77 g / L sodium chloride is prepared in ultrapure water, and this basic solution can be stored at 4°C for 7 days. An acidic solution of 12.0 g / L sodium dihydrogen phosphate and 8.77 g / L sodium chloride is prepared in ultrapure water, and this acidic solution can be stored at 4°C for 7 days. The basic solution is titrated with the acidic solution to pH 7.4, and the resulting buffer solution can be stored at 4°C for 7 days. The pH of the buffer solution is measured on the day of the experiment, and if it exceeds the range of 7.4 ± 0.1, the pH is adjusted.

[0190] The dialysis membrane was immersed in ultrapure water for 60 minutes and separated into two pieces, which were then immersed in 20% ethanol for 20 minutes and finally in dialysis buffer for 20 minutes.

[0191] The frozen plasma was quickly thawed at room temperature.

[0192] 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.

[0193] A 10 mM DMSO stock solution of the target substance was prepared. 2 μL of the stock solution (10 mM) was diluted with 98 μL of DMSO to obtain a working solution (200 μM). 3 μL of the working solution was added to 597 μL of human plasma for a final concentration of 1 μM (0.5% DMSO). Mix thoroughly by swirling.

[0194] A 120 μL sample containing the drug was added to one side of the dialysis membrane, and an equal volume of dialysate (phosphate buffer) was added to the other side. The experiment was a parallel-group comparative study. The dialysis plate was sealed, placed in an incubator, and incubated at 37°C, 5% CO2, and approximately 100 rpm for 6 hours. After incubation, the blocking membrane was peeled off, and 50 μL of the solution was aspirated from the buffer and plasma side of each well and transferred to a different well on a new plate.

[0195] 50 μL of blank plasma was added to the phosphate buffer sample, and an equal volume of blank phosphate buffer was added to the plasma sample. 300 μL of room-temperature quencher (internal standard acetonitrile (IS), containing 500 nM labetalol, 100 nM alprazolam, and 2 μM ketoprofen)) was added to precipitate proteins. The mixture was vortexed for 5 minutes and centrifuged at 3220 g for 30 minutes at 4°C. 100 μL of the supernatant was transferred to a new plate. The supernatant was diluted with 100 μL or 200 μL of water based on the LC / MS response signal and peak shape of the analyte. After uniform mixing, the liquid was used for sample analysis.

[0196] All calculations were performed using Microsoft Excel. The peak areas of the test substance on the buffer side and plasma side were determined. The equations for calculating the plasma protein binding rates of the test substance and control drug are as follows:

[0197]

number

[0198] The buffer side of the ratio of the sample peak area to the internal standard peak area represents the free concentration of the compound, the plasma side of the ratio of the sample peak area to the internal standard peak area represents the sum of the free and bound concentrations of the compound, and the initial plasma sample of the ratio of the sample peak area to the internal standard peak area represents the total concentration of the compound when the sample incubation started.

[0199] Test results: The results are shown in Table 6. 1 μM Compound 2 was incubated at 37° C. for 6 hours, and the average release rate was 46.63%, the binding rate was 53.37%, and the recovery rate was 88.02%.

[0200] [Table 14]

[0201] Example 30: Tissue distribution test of compound 2 after single intragastric administration Test materials: Balb / c mice (purchased from Shanghai Minchang Biotechnology Co., Ltd.) were used, totaling 60 mice, 30 males and 30 females, weighing 18-25 g.

[0202] Experimental steps: Compound 2 was administered intragastrically to Balb / c mice in a single dose of 100 mg / kg in a volume of 10 mL / kg.

[0203] Before dosing and at 5 min, 0.25 h, 1.0 h, 2.0 h, 3.0 h, 5.0 h, 7.0 h, and 10 h (six mice, three males and three females per time point), 0.2 ml of blood was collected from the orbital venous plexus and placed in EDTA-K2 tubes. The blood was centrifuged at 11,000 rpm for 5 min to separate the plasma, which was then frozen in a -70°C refrigerator. After whole blood was collected at 0.25 h, 1.0 h, 3.0 h, and 7.0 h, the mice were immediately dissected and lung tissue was collected. The remaining blood and contents on the surface of the tissue were washed with cold saline, dried by suction, labeled, and stored at -70°C for further analysis. The concentrations of Compound 2 in plasma and lung tissue were measured by LC / MS-MS, and the ratio of the lung tissue content to the plasma content was calculated.

[0204] Test results: After a single intragastric administration of Compound 2 to Balb / c mice, the ratio of lung tissue exposure to plasma exposure was 0.62, indicating high lung tissue exposure of Compound 2.

[0205] <Example 31: Safety pharmacological study on the effect of intragastric administration of Compound 2 on the cardiovascular system in cynomolgus monkeys> The cardiovascular effects of Compound 2 were simultaneously investigated in a 2-week repeated dose toxicity study in cynomolgus monkeys.

[0206] Test materials: Thirty-two cynomolgus monkeys, 16 males and 16 females, were administered at the age of 2.5 to 5 years.

[0207] Animal sources: Yunnan Yingmao Biological Technology Co., Ltd., Guangxi Yusen Primate Experimental Animal Culture Development Co., Ltd., Zhongke Lingrui (Zhanjiang) Biotechnology Co., Ltd.

[0208] Systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MBP) were measured in all conscious animals using an intelligent noninvasive sphygmomanometer BP-98E and an electronic data acquisition system (PV-02) called Provantis / v10.2.3.1.

[0209] Experimental steps: Thirty-two cynomolgus monkeys (4 groups, 5 animals / sex / group in Groups 1 and 4, 3 animals / sex / group in Groups 2 and 3) were randomly assigned to each group and administered Compound 2 (40, 160, and 600 mg / kg / day) or a control formulation (98.9% vehicle formulation + 1.1% MTBE, 0 mg / kg / day) twice daily via intranasal administration for a total of 14 days, followed by a 14-day recovery period. All animals were included in this study, and the effects of drug administration on ECG parameters (including heart rate, PR interval, QRS duration, QT interval, and QTcF) and blood pressure before administration, during the administration period, and during the recovery period were evaluated.

[0210] Test results: Under the conditions of this test, Compound 2 was administered intranasally to cynomolgus monkeys twice daily for 14 days (40, 160, 600 mg / kg / day). As a result, no test product-related cardiovascular changes were observed, no test product-related arrhythmias were observed, and no test product-related changes in ECG parameters or blood pressure were observed throughout the entire test period.

Claims

1. A cyano compound represented by the general formula I-1, I-2, I-3, I-4, or I-7, or a racemate, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof. 【Chemical 1】 (where, R １ is -COR ８ and -SO ２ R ９ is selected from R ２ and R ３ are each independently H, D, C １ ~C １０ Alkyl group, adamantyl group and C ３ ~C ７ cycloalkyl groups, or R ２ and R ３ together with the carbon atoms to which they are attached form a 3- to 8-membered carbocyclic ring, R ６ teeth, 【Chemistry 2】 is selected from R ８ is H, C １ ~C １０ Alkyl group, C １ ~C １０ Alkoxy group, C ３ ~C ７ Cycloalkyl groups, halogenated C １ ~C １０ Alkyl group, halogenated C ３ ~C ７ cycloalkyl group, —NR １３ R １４ , C ６ ~C ２０ Aryl group, halogenated C ６ ~C ２０ Aryl group, C １ ~C １０ C substituted with alkyl group ６ ~C ２０ Aryl group, halogenated C １ ~C １０ C substituted with alkyl group ６ ~C ２０ selected from an aryl group, a 5- to 20-membered heteroaryl group, and a halogenated 5- to 20-membered heteroaryl group; R ９ is C １ ~C １０ Alkyl group, C ３ ~C ７ Cycloalkyl groups, halogenated C １ ~C １０ Alkyl group, halogenated C ３ ~C ７ cycloalkyl group, —NR １５ R １６ , C ６ ~C ２０ Aryl group, halogenated C ６ ~C ２０ Aryl group, C １ ~C １０ C substituted with alkyl group ６ ~C ２０ Aryl group, halogenated C １ ~C １０ C substituted with alkyl group ６ ~C ２０ selected from an aryl group, a 5- to 20-membered heteroaryl group, and a halogenated 5- to 20-membered heteroaryl group; R １３ and R １４ are each independently H, C １ ~C １０ alkyl groups, R １５ and R １６ are each independently H, C １ ~C １０ alkyl groups.)

2. R ２ and R ３ are each independently H, D, C １ ~C ６ Alkyl group, adamantyl group and C ３ ~C ７ cycloalkyl groups, or R ２ and R ３ together with the carbon atoms to which they are attached form a 3- to 8-membered carbocyclic ring, or R ２ and R ３ are each independently selected from H, an isopropyl group, a tert-butyl group, a cyclopentyl group, and an adamantyl group, or R ２ and R ３ together with the carbon atoms to which they are attached form cyclopropyl and cyclopentyl groups, or R ２ and R ３ one of which is selected from H and the other is selected from isopropyl, tert-butyl, cyclopentyl and adamantyl groups, or R ２ and R ３ and R 1 and R 2 together with the carbon atom to which they are attached form a cyclopropyl group and a cyclopentyl group, or a racemate, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof.

3. R ８ is H, C １ ~C ６ Alkyl group, C １ ~C ６ Alkoxy group, C ３ ~C ７ Cycloalkyl groups, halogenated C １ ~C ６ Alkyl group, halogenated C ３ ~C ７ cycloalkyl group, —NR １３ R １４ , C ６ ~C １０ Aryl group, halogenated C ６ ~C １０ Aryl group, C １ ~C ６ C substituted with alkyl group ６ ~C １０ Aryl group, halogenated C １ ~C ６ C substituted with alkyl group ６ ~C １０ R is selected from an aryl group, a 5- to 10-membered heteroaryl group, and a halogenated 5- to 10-membered heteroaryl group; １３ and R １４ are each independently H, C １ ~C ６ alkyl groups, or R ８ is C １ ~C ６ Alkyl group, halogenated C １ ~C ６ Alkyl group, C １ ~C ６ Alkoxy group, —NR １３ R １４ , C ３ ~C ７ Cycloalkyl groups, halogenated C ３ ~C ７ Cycloalkyl group, phenyl group, halogenated phenyl group, C １ ~C ６ Alkyl-substituted phenyl group, halogenated C １ ~C ６ R is selected from a phenyl group substituted with an alkyl group and a 5- to 6-membered heteroaryl group; １３ and R １４ are each independently H, C １ ~C ６ alkyl groups, or R ８ is CH ３ , C.F. ３ , C.H. ２ CF ３ , C.F. ２ CF ３ , methoxy group, 【Chemistry 3】 a cyclopropyl group, 【Chemistry 4】 phenyl group, 【Chemistry 5】 and pyridin-3-yl, or a racemate, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof, of claim 1 .

4. R 9 is selected from a C 1 -C 6 alkyl group, a C 3 -C 7 cycloalkyl group, a halogenated C 1 -C 6 alkyl group, a halogenated C 3 -C 7 cycloalkyl group, -NR 15 R 16 , a C 6 -C 10 aryl group, a halogenated C 6 -C 10 aryl group, a C 6 -C 10 aryl group substituted with a C 1 -C 6 alkyl group, a C 6 -C 10 aryl group substituted with a halogenated C 1 -C 6 alkyl group, a 5- to 10-membered heteroaryl group, and a halogenated 5- to 10-membered heteroaryl group; R 15 and R 16 are each independently selected from H, a C 1 -C 6 alkyl group, or R 9 is selected from a C 1 -C 6 alkyl group, a C 3 -C 7 cycloalkyl group, a phenyl group, a phenyl group substituted with a C 1 -C 6 alkyl group, and a phenyl group substituted with a halogenated C 1 -C 6 alkyl group; or The cyano compound or its racemate, enantiomer, diastereomer, or pharmaceutically acceptable salt according to any one of claims 1 to 3, wherein R 9 is selected from CH 3 , a cyclopropyl group, a phenyl group, a p-methylphenyl group, and a p-trifluoromethylphenyl group.

5. A cyano compound or a racemate, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof selected from the following compounds: 【Chemistry 6】 【Chemistry 7】 【Chemistry 8】 【Chemistry 9】

6. A pharmaceutical composition comprising the cyano compound according to any one of claims 1 to 5, or a racemate, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof, and pharmaceutically acceptable auxiliary materials.

7. A pharmaceutical composition according to claim 6, further comprising ritonavir or a pharmaceutically acceptable salt thereof.

8. A pharmaceutical combination comprising the cyano compound according to any one of claims 1 to 5, or a racemate, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof, and ritonavir or a pharmaceutically acceptable salt thereof.

9. Use of the cyano compound according to any one of claims 1 to 5, or a racemate, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof, in the manufacture of a drug, wherein the drug is selected from the group consisting of a drug for inhibiting coronavirus 3CL protease activity, a drug for preventing and / or treating coronavirus infection, a drug for inhibiting picornavirus 3CL protease activity, and a drug for preventing and / or treating picornavirus infection.

10. 10. The use according to claim 9, wherein the coronavirus is selected from SARS-CoV, MERS-CoV, H229E-CoV, HKU1-CoV, NL63-CoV, OC43-CoV and SARS-CoV-2.

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