Metabolically stable Anti-drug-resistant main protease inhibitor and use thereof in preparation of antiviral drug

Abstract

Disclosed in the present application are a metabolically stable anti-drug-resistant main protease inhibitor compound and the use thereof in preparation of an antiviral drug. In the compound, group C can effectively bind to the amino acid at position 163 of a main protease, and group C is difficult to oxidize, such that a virus will not develop drug resistance to the compound of the present application. Furthermore, the compound exhibits great anti-drug-resistant activity and metabolic stability, and does not need to be used with a hepatic enzyme inhibitor, thereby being conducive to mitigating liver function decline and toxic side effects caused by taking medication. In addition, group A can not only further improve the inhibitory activity of the compound against the main protease, but also enhance the anti-drug-resistant activity and metabolic stability of the compound. When R1 has a non-fluorine halogen substitution, the compound has better inhibitory activity against the main protease. Moreover, ring B further improves the inhibitory activity of the compound against the main protease; particularly, when ring B is substituted with trifluoromethyl or difluoride, the compound has better inhibitory activity against the main protease.

Description

A metabolically stable inhibitor of drug-resistant main protease and its application in the preparation of antiviral drugs Technical Field

[0001] This application belongs to the field of biomedical technology, specifically relating to a metabolically stable anti-drug-resistant main protease inhibitor and its application in the preparation of antiviral drugs. Background Technology

[0002] The main protease of coronaviruses (main protease M) pro M is a key enzyme in the viral replication process. Existing studies have found that the main protease M in different coronaviruses is... pro The substrate binding sites are the same and highly conserved, while M does not exist in the host. pro Homologous proteins, and none of the proteases exhibit substrate affinity similar to M. pro With the same preference for the substrate, M is inhibited by targeting. pro Its function can prevent the production of infectious viral particles, thereby alleviating the symptoms of the host after infection with the coronavirus.

[0003] According to different inhibition mechanisms, M pro Inhibitors can be divided into two categories: covalent inhibitors and non-covalent inhibitors. Non-covalent inhibitors generally bind to the active pocket of the main protease in a competitive manner, while covalent inhibitors bind covalently to the active site of the main protease. Obviously, the targeting inhibition ability of covalent inhibitors is theoretically relatively stronger.

[0004] For example, Chinese invention patent application CN 114681443A discloses a series of nitrile-containing antiviral compounds, the structural formulas of which are as follows:

[0005] In this series of compounds, the γ-lactam ring first binds to the active pocket of the main protease, and then its cyano group covalently binds to the active site of the main protease, thereby achieving targeted inhibition of the main protease activity. Among this series of nitrile-containing compounds, nirmatrelvir exhibits the best inhibitory activity against the novel coronavirus.

[0006] Although the inhibition constant (Ki) of nirmatrelvir against the novel coronavirus is as low as 5.10 nM, nirmatrelvir has two defects: (1) Nirmatrelvir relies on the binding of the gamma lactam ring to the active pocket of the novel coronavirus, but the gamma lactam ring is easily metabolized by the liver, resulting in poor pharmacokinetic activity of nirmatrelvir, so it is necessary to use a liver enzyme inhibitor (such as ritonavir) when using nirmatrelvir; this not only causes the liver function of the patient to decline during medication, but also the combination use of the two drugs may have potential mutual reactions, which may on the one hand cause the activity of the drug to decrease, and on the other hand, the reaction product may have toxic side effects on the human body; (2) Recent studies have found that after evolution, the main protease, which was originally thought to be highly conserved, has developed drug resistance mutations, and some drug resistance mutant strains have mutations at the E166 site of the main protease, and the binding of nirmatrelvir to the main protease is highly dependent on the E166 site; this results in a cliff-like decrease in the inhibitory activity of nirmatrelvir against the E166 mutant drug resistance mutant strain, which has lost its original high inhibitory activity.

[0007] Therefore, it is urgent to develop an antiviral drug that is metabolically stable and has certain drug resistance activity. SUMMARY

[0008] The purpose of the present application is to provide a metabolically stable anti-drug resistance main protease inhibitor and its application in the preparation of an antiviral drug, which not only has metabolic stability, but also has certain drug resistance activity.

[0009] To achieve the above-mentioned purpose of the application, the technical solution of the present application is as follows:

[0010] In one aspect, the present application provides a metabolically stable anti-drug resistance main protease inhibitor compound, or a stereoisomer, hydrate, deuterium, ester, solvate, metabolite, pharmaceutically acceptable salt or prodrug thereof, which comprises a structure represented by formula (I):

[0011] wherein:

[0012] Group A (i.e., group A) is selected from any one of the following: -CO- unsubstituted or substituted hydrocarbon group, hydrocarbon ether, indole, or -SO2- unsubstituted or substituted hydrocarbon group;

[0013] wherein R1 is selected from any one of the following: -CO- unsubstituted or substituted C1-C9 hydrocarbon group, hydrocarbon ether, 6-membered heterocyclic group, heteroatom-containing bicyclic or tricyclic structure, or -SO2- unsubstituted or substituted C1-C9 hydrocarbon group, 6-membered heterocyclic group, heteroatom-containing bicyclic or tricyclic structure;

[0014] R2 is selected from any of the following: unsubstituted or substituent C1-C 14 Hydrocarbon groups, 5-6 membered heterocyclic groups;

[0015] R3 and R4 can be independent of each other or connected to form a B ring. When R3 and R4 are independent of each other, R3 is selected from any of the following: hydrogen, methyl, halomethyl; R4 is selected from unsubstituted or substituent C1-C7 hydrocarbon groups; when R3 and R4 are connected to form a B ring, the B ring is selected from any of the following: unsubstituted or substituent 5-6 membered heterocyclic groups, bicyclic or tricyclic structures containing heteroatoms.

[0016] R5 is selected from any of the following: cyano, unsubstituted or substituted carbonyl;

[0017] Group C (i.e., group C) is selected from any of the following: -CH2- Unsubstituted or substituted pyridine ring, -CH2- Unsubstituted or substituted pyrrolidone;

[0018] Wherein, ring D is selected from any of the following: unsubstituted or substituent R7 benzene ring, pyridine ring, pyrimidine ring, pyrazine ring, imidazole ring, pyrrole ring, thiophene ring, furan ring;

[0019] The E ring is selected from any of the following: an unsubstituted benzene ring or a pyridine ring, a pyrimidine ring, a pyrazine ring, an imidazole ring, a pyrrole ring, a thiophene ring, or a furan ring;

[0020] R6, R7, R8 and R9 are each independently selected from any of the following: hydrogen, cyano, methyl, amino, halogen, halomethyl, unsubstituted or substituted alkyl, hydroxyalkyl, aminoalkyl, five-membered nitrogen heterocycle; X is selected from any of the following: -NH-, -O- or -S-.

[0021] In the main protease inhibitor of this application, the C group can not only effectively bind to the substrate recognition pocket of the main protease, but this binding is also mainly dependent on the 163rd amino acid site (H163) of the main protease. Existing studies have found that the H163 site of the main protease will not be mutated (once the H163 site is mutated, the main protease will be inactivated). Therefore, the main protease inhibitor of this application is not afraid of main protease mutations and can show basically consistent inhibitory activity (or a small decrease in inhibitory activity) against both mutant and wild-type strains. Viruses that rely on the main protease for replication will not develop resistance to the compound of this application.

[0022] Meanwhile, group C has the characteristic of "difficult to be oxidized"; therefore, the main protease inhibitor of the present application can resist the metabolism of liver enzymes, so that when the main protease inhibitor of the present application is used, not only the drug dosage can be reduced, but also the liver enzyme inhibitor does not need to be used in combination, which is beneficial to reduce the toxic side effects such as self-liver function reduction caused by drug taking.

[0023] On the basis of optimizing group C, the present application also optimizes groups A and B rings, wherein group A not only can further improve the inhibitory activity of the main protease inhibitor on the main protease, but also can enhance the anti-drug resistance activity and metabolic stability of the main protease inhibitor; and the B ring mainly plays a role in further improving the inhibitory activity of the main protease inhibitor on the main protease.

[0024] In some embodiments, in group A of the main protease inhibitor, the substituent on the C1-C9 hydrocarbon group is selected from one or more of the following: hydrogen, halogen, methyl, methoxy, halogenated methyl, and halogenated methoxy. 14 The substituent on the C1-C9 hydrocarbon group is selected from one or more of the following: hydrogen, halogen, methyl, methoxy, halogenated methyl, and halogenated methoxy.

[0025] In some embodiments, the substituent on the C1-C9 hydrocarbon group is at least one selected from non-fluorine halogen.

[0026] In some embodiments, group A is selected from any one of the following:

[0027] wherein R1 is selected from any one of the following:

[0028] R2 is selected from any one of the following:

[0029] In some embodiments, group A is selected from any one of the following:

[0030] wherein R1 is selected from any one of the following:

[0031] R2 is selected from

[0032] In some embodiments, R3 of the main protease inhibitor compound is selected from any one of the following: R4 is selected from any one of the following:

[0033] In some embodiments, the substituents on the B ring of the primary protease inhibitor compound are selected from any one or more of: hydrogen, halogen, hydroxyl, methyl, halomethyl, methoxy, halomethoxy, -C(CH3)3, -C(CD3)3.

[0034] In some embodiments, the B ring of the primary protease inhibitor compound is selected from any one of:

[0035] In some embodiments, the B ring of the primary protease inhibitor compound is selected from any one of:

[0036] In some embodiments, R5of the primary protease inhibitor compound is selected from any one of:

[0037] In some embodiments, R5of the primary protease inhibitor compound is

[0038] In some embodiments, the substituents on the pyridine ring of group C of the primary protease inhibitor compound are selected from any one or more of: hydrogen, halogen, methyl, halomethyl, cyano.

[0039] In some embodiments, group C of the primary protease inhibitor compound is selected from any one of: wherein the D ring and the E ring are each independently selected from any one of: X is selected from any one of: -NH-, -O-, or -S-;

[0040] R6, R7, R8, and R9are each independently selected from any one of:

[0041] In some embodiments, group C of the primary protease inhibitor compound is selected from any one of:

[0042] wherein the D ring is

[0043] R6, R7are

[0044] In some embodiments, group A of the primary protease inhibitor compound is

[0045] wherein R1is selected from any one of the following:

[0046] R2is selected from

[0047] In some embodiments, the B ring of the primary protease inhibitor compound is selected from any one of the following:

[0048] In some embodiments, R5of the primary protease inhibitor compound is

[0049] In some embodiments, the C group of the primary protease inhibitor compound is selected from any one of the following:

[0050] wherein the D ring is

[0051] R6, R7are

[0052] In some embodiments, the B ring of the primary protease inhibitor compound is

[0053] In some embodiments, the A group of the primary protease inhibitor compound is selected from any one of the following:

[0054] wherein R1is selected from any one of the following:

[0055] R2is selected from

[0056] In some embodiments, R5of the primary protease inhibitor compound is

[0057] In some embodiments, the C group of the primary protease inhibitor compound is

[0058] wherein the D ring is

[0059] R6, R7are

[0060] In some embodiments, the A group of the primary protease inhibitor compound is

[0061] wherein R1is selected from any one of the following:

[0062] R2is selected from

[0063] In some embodiments, R5of the primary protease inhibitor compound is

[0064] In some embodiments, group C of the primary protease inhibitor compound is

[0065] wherein ring D is

[0066] R6, R7are

[0067] In some embodiments, ring B of the primary protease inhibitor compound is

[0068] In some embodiments, group A of the primary protease inhibitor compound is selected from any one of:

[0069] wherein R1is selected from any one of:

[0070] R2is selected from

[0071] In some embodiments, group A of the primary protease inhibitor compound is

[0072] wherein R1is selected from any one of:

[0073] R2is selected from

[0074] In some embodiments, R5of the primary protease inhibitor compound is

[0075] In some embodiments, group C of the primary protease inhibitor compound is

[0076] wherein ring D is

[0077] R6, R7are

[0078] In some embodiments, the primary protease inhibitor compound has at least one of the following structural formulae:

[0079] In another aspect, the present application provides a method for preparing the compound.

[0080] The main protease inhibitors all exhibit target inhibition of main protease to varying degrees, wherein the inhibition constant for wild-type main protease of the novel coronavirus is as low as 0.583 nM (the inhibition constant of nirmatrelvir is 5.10 nM), the inhibition constant for main protease M49L / E166A drug-resistant mutant is as low as 1.38 nM (the inhibition constant of nirmatrelvir is 352 nM), the inhibition constant for main protease L50F / E166A / L167F drug-resistant mutant is as low as 41.8 nM (the inhibition constant of nirmatrelvir is 1799 nM), and the inhibition constant for wild-type main protease of feline coronavirus (FIPV) is as low as 0.044 nM (the inhibition constant of nirmatrelvir is 35.9 nM).

[0081] Based on this, in another aspect, the present application provides a pharmaceutical composition comprising the main protease inhibitor compound or its racemate, enantiomer, diastereomer or pharmaceutically acceptable salt described above, and a pharmaceutically acceptable excipient.

[0082] The available dosage forms of the pharmaceutical composition of the present application include oral preparations, injections, sprays, powders, emulsions, suspensions or transdermal administration preparations.

[0083] In another aspect, the present application provides the use of the main protease inhibitor compound or the pharmaceutical composition described above in the preparation of an antiviral drug.

[0084] The antiviral drug of the present application achieves the purpose of inhibiting viruses by inhibiting the activity of main protease, and therefore the present application has no special requirements for the type of virus and host (which can be human or animal), and is suitable for viruses that rely on main protease to complete replication; the viruses that rely on main protease for replication include wild-type or drug-resistant mutant strains of coronavirus, calicivirus, norovirus, hepatitis A virus, human rhinovirus and porcine transmissible gastroenteritis virus.

[0085] Compared with the prior art, the beneficial effects of the present application are reflected in:

[0086] (1) In the main protease inhibitors of the present application, the applicant found that when R1 in Group A has a non-fluorine halogen substitution, it is unexpectedly better than R1 being trifluorine-substituted, and this finding is true in different B rings.

[0087] (2) In the main protease inhibitor of this application, the group C can not only effectively bind to the substrate recognition pocket of the main protease, but also the binding mainly depends on the 163rd amino acid site (H163) of the main protease. Existing studies have found that the H163 site of the main protease will not be mutated (once the H163 site is mutated, the main protease will be inactivated). Therefore, the main protease inhibitor of this application can resist the mutation of the main protease and can show basically consistent inhibitory activity (or a small decrease in inhibitory activity) on both mutant and wild-type strains. Coronaviruses will not develop resistance to the main protease inhibitor of this application.

[0088] (3) In the main protease inhibitor of this application, the group C has the characteristic of being "difficult to oxidize"; therefore, the main protease inhibitor of this application can tolerate the metabolism of liver drug-metabolizing enzymes. Thus, when using the main protease inhibitor of this application, not only can the dosage of the drug be reduced, but there is also no need to use liver drug-metabolizing enzyme inhibitors in combination, which is beneficial to reduce the toxic side effects such as the decline in liver function caused by taking the drug.

[0089] (4) The main protease inhibitors of this application all exhibit targeted inhibitory effects on the main protease to varying degrees. Among them, the inhibition constant of the wild-type main protease of the novel coronavirus is as low as 0.583 nM (the inhibition constant of nematidine is 5.10 nM), the inhibition constant of the main protease M49L / E166A drug-resistant mutant is as low as 1.38 nM (the inhibition constant of nematidine is 352 nM), the inhibition constant of the main protease L50F / E166A / L167F drug-resistant mutant is as low as 41.8 nM (the inhibition constant of nematidine is 1799 nM), and the inhibition constant of the wild-type main protease of feline coronavirus (FIPV) is as low as 0.044 nM (the inhibition constant of nematidine is 35.9 nM).

[0090] (5) The antiviral drug of this application achieves the purpose of inhibiting coronavirus by inhibiting the activity of the main protease. Therefore, there are no special requirements for the type of coronavirus and the host (which can be human or animal). As long as the virus depends on the main protease to complete the replication, it is applicable to this application.

[0091] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. The detailed description below shows and describes only exemplary embodiments of this application. As those skilled in the art will recognize, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in this application are merely exemplary and not restrictive. Attached Figure Description

[0092] Figure 1 is a synthetic route chart of the main protease inhibitor Hit 3-1 of the present application;

[0093] Figure 2 is a mass spectrum analysis chart of Nirmatrelvir;

[0094] Figure 3 is a mass spectrum analysis chart of the main protease inhibitor Hit 3-1 of the present application;

[0095] Figure 4 and Figure 5 are respectively a synthetic route chart and a mass spectrum analysis chart of the main protease inhibitor Hit 3-2 of the present application;

[0096] Figure 6 and Figure 7 are respectively a synthetic route chart and a mass spectrum analysis chart of the main protease inhibitor Hit 3-3 of the present application;

[0097] Figure 8 and Figure 9 are respectively a synthetic route chart and a mass spectrum analysis chart of the main protease inhibitor Hit 3-4 of the present application;

[0098] Figure 10 and Figure 11 are respectively a synthetic route chart and a mass spectrum analysis chart of the main protease inhibitor Hit 3-5 of the present application;

[0099] Figure 12 and Figure 13 are respectively a synthetic route chart and a mass spectrum analysis chart of the main protease inhibitor Hit 3-6 of the present application;

[0100] Figure 14 and Figure 15 are respectively a synthetic route chart and a mass spectrum analysis chart of the main protease inhibitor Hit 3-7 of the present application;

[0101] Figure 16 and Figure 17 are respectively a synthetic route chart and a mass spectrum analysis chart of the main protease inhibitor Hit 3-8 of the present application;

[0102] Figure 18 and Figure 19 are respectively a synthetic route chart and a mass spectrum analysis chart of the main protease inhibitor Hit 3-11 of the present application;

[0103] Figure 20 and Figure 21 are respectively a synthetic route chart and a mass spectrum analysis chart of the main protease inhibitor Hit 3-16 of the present application;

[0104] Figure 22 is a general synthetic route chart of the main protease inhibitors Hit 3-15, 3-17 to 3-28 of the present application;

[0105] Figure 23 to Figure 35 are respectively mass spectrum analysis charts of the main protease inhibitors Hit 3-22, Hit 3-23, Hit 3-17, Hit 3-15, Hit 3-18, Hit 3-19, Hit 3-20, Hit 3-21, Hit 3-24, Hit 3-25, Hit 3-26, Hit 3-27 and Hit 3-28 of the present application;

[0106] Figure 36 is a general synthetic route chart of the main protease inhibitors Hit 3-9, Hit 3-30 and Hit 3-31 of the present application;

[0107] Figures 37 to 39 are, in sequence, mass spectrometry analysis charts of the main protease inhibitors Hit 3-9, Hit 3-30 and Hit 3-31 of the present application;

[0108] Figure 40 is a general synthetic route chart of the main protease inhibitors Hit 3-32, Hit 3-33 and Hit 3-34 of the present application;

[0109] Figures 41 to 44 are, in sequence, mass spectrometry analysis charts of the main protease inhibitors Hit 3-32, Hit 3-33, Hit 3-34 and Hit 3-36 of the present application;

[0110] Figure 45 is a general synthetic route chart of the main protease inhibitors FD7-29, FD7-1 and FD7-2 of the present application;

[0111] Figures 46 to 48 are, in sequence, mass spectrometry analysis charts of the main protease inhibitors FD7-29, FD7-1 and FD7-2 of the present application;

[0112] Figure 49 is a general synthetic route chart of the main protease inhibitors FD7-25, FD7-5, FD7-6, FD7-24, FD7-22 and FD7-23 of the present application;

[0113] Figures 50 to 55 are, in sequence, mass spectrometry analysis charts of the main protease inhibitors FD7-25, FD7-5, FD7-6, FD7-24, FD7-22 and FD7-23 of the present application;

[0114] Figures 56 and 57 are, respectively, a synthetic route chart and a mass spectrometry analysis chart of the main protease inhibitor FD7-3 of the present application;

[0115] Figures 58 and 59 are, respectively, a synthetic route chart and a mass spectrometry analysis chart of the main protease inhibitor FD7-32 of the present application;

[0116] Figure 60 is a cytotoxicity test result chart of the main protease inhibitors Hit 3-6, Hit 3-11, Hit 3-22, Hit 3-24, Hit 3-25, Hit 3-32, Hit 3-33 and Hit 3-34 of the present application;

[0117] Figure 61 is a stability test result chart of the main protease inhibitors Hit 3-2, Hit 3-3, Hit 3-6, Hit 3-32, Hit 3-33 and Hit 3-34 of the present application on liver phase I metabolism;

[0118] Figure 62 is the result of analysis of the inhibitory activity of the main protease inhibitors Hit 3-4, Hit 3-5, Hit 3-6, Hit 3-32, Hit 3-33 and Hit 3-34 of the mutant M ProM49L / E166A of the main protease of the novel coronavirus;

[0119] Figure 63 is the result of analysis of the inhibitory activity of the main protease inhibitors Hit 3-5, Hit 3-6, Hit 3-32, Hit 3-33 and Hit 3-34 of the mutant M ProL50F / E166A / L167F of the main protease of the novel coronavirus;

[0120] Figure 64 is the result of analysis of the inhibitory activity of the main protease inhibitors Hit 3-6, Hit 3-32, Hit 3-33 and Hit 3-34 of the main protease of the feline coronavirus;

[0121] Figure 65 is the result of detection of the pharmacokinetic properties of the main protease inhibitors Hit 3-32 and Hit 3-33;

[0122] Figure 66 is the result of acute toxicity experiment of the main protease inhibitors Hit 3-32 and Hit 3-33;

[0123] Figure 67 is the result of pharmacodynamics study of the main protease inhibitors Hit 3-32 and Hit 3-33 in combination with Ritonavir on viral nucleic acid load in vivo;

[0124] Figure 68 is the result of pharmacodynamics study of the main protease inhibitors Hit 3-32 and Hit 3-33 in combination with Ritonavir on viral nucleocapsid protein in vivo;

[0125] Figure 69 is the result of pharmacodynamics study of the main protease inhibitor Hit 3-32 as a single drug on viral nucleic acid load in vivo;

[0126] Figure 70 is the result of pharmacodynamics study of the main protease inhibitor Hit 3-32 as a single drug on viral nucleocapsid protein in vivo;

[0127] Figure 71 is the result of in vivo pharmacodynamics study of the main protease inhibitors Hit 3-36 and FD7-1 as single drugs;

[0128] Figure 72 is the result of detection of the pharmacokinetic properties of the main protease inhibitors Hit 3-6 and FD7-1. DETAILED DESCRIPTION

[0129] The following detailed description of the application is presented in order to better describe the best mode and / or a preferred embodiment of the application and to fully enable one to make and use at least one embodiment of the application. It will be appreciated that the detailed description is presented chiefly by way of example and that various modifications and alterations to the application can be made by persons having ordinary skill in the art without departing from the scope and spirit of the application as disclosed herein.

[0130] Definitions of Terms

[0131] The term "inhibitor" is known in the art and relates to a compound / substance that is capable of preventing or reducing, completely or partially, the physiological function (i.e. activity) of one or more specific proteins (e.g. the major protease). Inhibitors are also known as "antagonists". In the present application, the major protease inhibitor prevents or reduces or inhibits or inactivates the physiological activity of the major protease, e.g. after the compound / pharmaceutical composition binds to the major protease. In some embodiments, the major protease inhibitor can covalently bind to the active site of the major protease via a cyano group, thereby achieving the purpose of targeted inhibition of the major protease activity.

[0132] The term "compound" refers to the compounds of the present application, which term includes the various pharmaceutically acceptable salts, hydrates or solvates of the compounds of the present application.

[0133] The term "metabolically stable" refers to the ability of a compound to survive first-pass metabolism (gut and liver degradation or binding of an orally administered drug). This can be assessed in vitro, for example, by exposing the compound to mouse or human liver microsomes. In some embodiments, good metabolic stability refers to a t1 / 2>60 min, >90 min, >120 min, >150 min, preferably >180 min, when the compound is exposed to mouse or human liver microsomes.

[0134] The term "drug resistance" refers to the resistance or tolerance of microorganisms, parasites, tumor cells, etc. to the effect of a drug, at which point the efficacy of the drug in treating the disease or improving the patient's symptoms is reduced. In some embodiments, tumor cells develop resistance to the original drug due to mutations at different sites. The term "anti-drug resistance" refers to the ability of the compound to resist the resistance developed by tumor cells while maintaining an inhibitory effect substantially consistent with the wild type. In some embodiments, the compound binds to the amino acid site H163, which is not mutated in the major protease, thus being able to ignore the resistance developed by mutations in the major protease.

[0135] The term "main protease", also known as 3CLpro, 3C-like protease, coronavirus 3C-like protease, Mpro, SARS 3C-like protease, SARS coronavirus 3CL protease, SARS coronavirus main peptidase, SARS coronavirus main protease, SARS-CoV 3CLpro enzyme, SARS-CoV main protease, SARS-CoV Mpro, and severe acute respiratory syndrome coronavirus main protease, is the main protease found in coronaviruses. The 3C protease is a potential drug target for coronavirus infection because it plays an important role in processing the polyprotein translated from the viral RNA.

[0136] The term "stereoisomer" refers to a compound having the same chemical structure but differing in the spatial arrangement of atoms or groups.

[0137] The term "solvate" refers to an association or complex of one or more solvent molecules and a compound of the application. Examples of solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine. The term "hydrate" refers to a solvate wherein the solvent molecule is water.

[0138] The term "deuterated" refers to a compound in which 1H is replaced by D (2H).

[0139] The term "ester" refers to a -C-C(=0)-0-C bond.

[0140] The term "solvate" is a combination, physical association and / or solvation of a compound of the application with a solvent molecule, for example, a disolvate, monosolvate or hemisolvate, respectively, wherein the solvent molecule is about 2:1, about 1:1 or about 1:2, respectively, in proportion to the compound of the application. Such physical associations involve varying degrees of ionic and covalent bonding, including hydrogen bonding. Solvates typically are not highly soluble in nonpolar solvents. Solvates usually are not significantly more active or toxic than the base compounds and thus act as pharmacological equivalents to the base compounds. In certain instances, solvates can be isolated, such as when one or more solvent molecules are incorporated in the crystal lattice of the solid state form. Accordingly, "solvate" includes both solution-phase and isolatable solvates. The compounds disclosed herein can exist as solvated forms with pharmaceutically acceptable solvents such as water, methanol, ethanol, and the like, and it is intended that the application embrace both solvated and unsolvated forms.

[0141] The term "metabolite" generally refers to a substance produced during metabolism of a compound of the application after administration to a subject, and such derivatives including metabolites are encompassed within the scope of the application.

[0142] The term "pharmaceutically acceptable salt" generally refers to the common pharmaceutically acceptable salts. For example, when a compound has a hydroxy or acidic group such as carboxyl and tetrazolyl, it can form a base addition salt at the hydroxy or acidic group; or when a compound has an amino or basic heterocyclic group, it can form an acid addition salt at the amino or basic heterocyclic group.

[0143] The term "hydrocarbyl" includes any group containing carbon and hydrogen, including saturated, unsaturated, aromatic, straight chain or branched chain or cyclic (including polycyclic) groups. Hydrocarbyl groups include, but are not limited to, C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, C3-C10cycloalkyl, aryl such as phenyl and naphthyl, Ar(C1-C8)alkyl such as benzyl, which can be optionally substituted.

[0144] The term "hydrocarbyl ether" in the present application means that the -O- bond is attached to a hydrocarbyl group. In some embodiments, the -CO- is attached to a hydrocarbyl ether to form an ester bond.

[0145] The term "heterocyclyl" refers to a fully saturated or unsaturated aromatic or non-aromatic ring group, a 4-7 membered monocyclic, 7-11 membered bicyclic or 10-15 membered tricyclic ring system having at least one heteroatom in at least one of the carbon atom containing rings. Each ring of a heterocyclyl group containing a heteroatom can have 1, 2, 3 or 4 heteroatoms selected from nitrogen, oxygen and sulfur, wherein the nitrogen and sulfur heteroatoms are also optionally oxidized, and the nitrogen heteroatoms are also optionally quaternized. The heterocyclyl group can be attached at any heteroatom or carbon atom.

[0146] The term "ring structure" generally refers to a cyclic structure resulting from the bonding of two or more atoms by chemical bonds. Ring structures in the present application can be saturated, partially unsaturated, unsaturated or aromatic. It is noted that fused, spiro and bridged rings (e.g., a bridged ring can be formed when one or more C atoms link two non-adjacent atoms) are included in the definition of ring structure in the present application.

[0147] The term "halomethyl" refers to -CH3- in which the H is replaced by a halogen, which can be 1, 2 or 3 in number.

[0148] The term "cyano" refers to a -CN group.

[0149] The term "carbonyl" refers to -C(O)-.

[0150] A cyclic group can be bonded to another group in more than one way. If a particular bonding arrangement is not specified, then all possible arrangements are intended. For example, the term "pyridine ring" includes 2-pyridinyl, 3-pyridinyl or 4-pyridinyl. In some embodiments, the pyridine ring is fused to a heterocycle in Group C to form a fused ring structure. The term "indole" includes 2-indolyl or 3-indolyl.

[0151] The term "pyrrolidinone" means any one of the C atoms in pyrrolidine is replaced by an oxygen to form a carbonyl group. "Pyrrolidine" means pyrrole with H addition, changing from an unsaturated heterocycle to a saturated heterocycle.

[0152] The term "pyrimidine" means what it is generally accepted in the art. The term generally refers to conventional pyrimidine bases, including the standard pyrimidine bases uracil, thymine, and cytosine.

[0153] The term "halogen" generally refers to fluorine, chlorine, bromine, and iodine. The term "trifluoromethyl" generally refers to a "-CF3" group, and the term "hydroxyl" or "hydroxy" generally refers to a "-OH" group.

[0154] The term "halomethoxy" means the H in -O-CH3- is replaced by a halogen, which can be 1, 2, or 3.

[0155] The term "chemical means" means the use of principles and methods of chemistry to achieve a certain purpose or solve a certain problem. It involves various chemical operations, such as chemical reactions, chemical synthesis, chemical analysis, and the use of various chemical substances.

[0156] The term "pharmaceutical composition" means a composition for administration to a subject, preferably a human patient. The pharmaceutical compositions of the present application include any pharmaceutical dosage form established in the art, especially capsules, microcapsules, caplets, pills, tablets, powders, pellets, multiparticulate formulations (e.g., spheroids, granules, or crystals), aerosols, sprays, foams, solutions, dispersions, tinctures, syrups, elixirs, suspensions, water-in-oil emulsions (e.g., ointments), and oil-in-water emulsions (e.g., creams, lotions, and analgesics). The formulations can be packaged in separate dosage units or in a multiple-dose container.

[0157] The term "racemate" includes "racemic" and "meso". The term "meso" generally refers to an atom within a molecule that contains an asymmetry, but which has a total optical rotation of zero due to its having a symmetry factor. The term "racemic" or "racemic mixture" generally refers to a composition consisting of equal molar amounts of two enantiomeric species. Unless otherwise specified, all compounds appearing in the present application are intended to include all possible optical isomers, such as single-handed compounds, or mixtures of various chiral compounds (i.e., racemates). Within all compounds of the present application, each chiral carbon atom can optionally be in the R configuration or the S configuration, or a mixture of the R and S configurations.

[0158] The term "enantiomer" means two stereoisomers of a compound that are non-superimposable mirror images of one another.

[0159] The term "diastereomers" refers to stereoisomers that have two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, e.g., melting points, boiling points, spectral properties, and reactivities.

[0160] The term "pharmaceutically acceptable excipient" refers generally to a substance that is not biologically or otherwise undesirable, i.e., the substance can be administered to a subject, generally a human, without causing any significant adverse effects or undesirable physiological effects, or that does not substantially interfere with the biological activity or properties of the compounds. Such a substance can typically include salts, buffers, preservatives, compatible carriers, and optionally other formulation agents.

[0161] The term "dosage form" is a term used in the field of pharmaceutical formulation, also known as pharmaceutical dosage form or unit dose, refers to the form of a pharmaceutical product for sale. A dosage form dispenses a specific mixture of active and inactive ingredients (inactive ingredients are also known as excipients) into a certain dosage ratio, placed in a specific device (such as a capsule shell). In actual use, the commonly used classification method is to classify by administration route or administration method, such as: oral dosage form, eye medicine dosage form, injection dosage form, inhalation dosage form, etc. In some embodiments, the dosage form of the pharmaceutical composition is an oral dosage, an injection, a spray, a powder, an emulsion, a suspension, or a transdermal administration preparation.

[0162] The term "antiviral drug" is a class of drugs used to specifically treat viral infections. Specific antiviral drugs act on specific viruses to inhibit viruses in the body, inhibit the development of viruses. The mechanism of antiviral drugs is mainly to resist further infection of viruses by affecting some links that interfere with the replication cycle of viruses. For example, directly inhibiting or killing viruses, interfering with virus adsorption, preventing viruses from penetrating cells, inhibiting viral biosynthesis, inhibiting virus release, or enhancing the host's ability to resist viruses, etc. In some embodiments, the antiviral drug is used to inhibit viruses that rely on the main protease for replication.

[0163] The term "wild strain" refers to the wild type main protease of coronavirus. In some embodiments, the wild strain is the wild type main protease of the novel coronavirus. In some embodiments, the wild strain is the wild type main protease of the feline coronavirus (FIPV).

[0164] The term "drug-resistant mutant strain" refers to a main protease that has a mutation at a certain site and is resistant to existing drugs. In some embodiments, the drug-resistant mutant strain refers to the main protease M49L / E166A drug-resistant mutant. In some embodiments, the drug-resistant mutant strain refers to the main protease L50F / E166A / L167F drug-resistant mutant.

[0165] DETAILED DESCRIPTION

[0166] Compounds

[0167] In one aspect, the present application provides a metabolically stable anti-resistance proteasome inhibitor compound, or a stereoisomer, hydrate, deuteride, ester, solvate, metabolite, pharmaceutically acceptable salt or prodrug thereof, which can comprise a structure represented by Formula (I):

[0168] wherein:

[0169] Group A can be selected from any one of: -CO- unsubstituted or substituted hydrocarbyl, hydrocarbyl ether, indole, or -SO2- unsubstituted or substituted hydrocarbyl;

[0170] wherein R1may be selected from any one of: -CO- unsubstituted or substituted C1-C9 hydrocarbyl, hydrocarbyl ether, 6-membered heterocyclyl, heteroatom-containing bicyclic or tricyclic structure, or -SO2- unsubstituted or substituted C1-C9 hydrocarbyl, 6-membered heterocyclyl, heteroatom-containing bicyclic or tricyclic structure;

[0171] R2may be selected from any one of: unsubstituted or substituted C1-C 14 hydrocarbyl, 5-6 membered heterocyclyl;

[0172] R3and R4are independent of each other or connected to form B ring, when R3and R4are independent of each other, R3may be selected from any one of: hydrogen, methyl, halogenated methyl; R4may be selected from unsubstituted or substituted C1-C7 hydrocarbyl; when R3and R4are connected to form B ring, B ring can be selected from any one of: unsubstituted or substituted 5-6 membered heterocyclyl, heteroatom-containing bicyclic or tricyclic structure;

[0173] R5may be selected from any one of: cyano, unsubstituted or substituted carbonyl;

[0174] Group C can be selected from any one of: -CH2- unsubstituted or substituted pyridine ring, -CH2- unsubstituted or substituted pyrrolidone;

[0175] wherein D ring can be selected from any one of: unsubstituted or substituted R7phenyl ring, pyridine ring, pyrimidine ring, pyrazine ring, imidazole ring, pyrrole ring, thiophene ring, furan ring;

[0176] E ring can be selected from any one of: unsubstituted or substituted R9phenyl ring, pyridine ring, pyrimidine ring, pyrazine ring, imidazole ring, pyrrole ring, thiophene ring, furan ring;

[0177] R6, R7, R8, and R9may each independently be selected from any one of: hydrogen, cyano, methyl, amino, halogen, halogenated methyl, unsubstituted or substituted alkyl, hydroxyalkyl, aminoalkyl, five-membered nitrogen heterocycle; X can be selected from any one of: -NH-, -O-, or -S-.

[0178] In some embodiments, Group A can be

[0179] wherein R1may be selected from any one of: -CO-unsubstituted or substituted C1-C9alkyl, alkyl ether, 6-membered heterocycle, heteroatom-containing bicyclic or tricyclic structure, or -SO2-unsubstituted or substituted C1-C9alkyl, 6-membered heterocycle, heteroatom-containing bicyclic or tricyclic structure;

[0180] R2may be selected from any one of: unsubstituted or substituted C1-C 14 alkyl, 5-6 membered heterocycle.

[0181] For example, R1may be -CO-C1alkyl, -CO-C2alkyl, -CO-C3alkyl, -CO-C4alkyl, -CO-C5alkyl, -CO-C6alkyl, -CO-C7alkyl, -CO-C8alkyl, -CO-C9alkyl, -C(O)O-C1alkyl, -C(O)O-C2alkyl, -C(O)O-C3alkyl, -C(O)O-C4alkyl, -C(O)O-C5alkyl, -C(O)O-C6alkyl, -C(O)O-C7alkyl, -C(O)O-C8alkyl, -C(O)O-C9alkyl, -CO-tetrahydropyran, -CO-piperidine, -CO-pyridine, -CO-pyrazine, -CO-pyrimidine, -CO-pyridazine, -CO-C6fused heterocycle, -CO-C7fused heterocycle, -CO-C8fused heterocycle, -CO-C9fused heterocycle, -CO-C 10 fused heterocycle, -CO-C 11 fused heterocycle, -CO-C 12 fused heterocycle, -CO-C6spiro heterocycle, -CO-C7spiro heterocycle, -CO-C8spiro heterocycle, -CO-C9spiro heterocycle, -CO-C 10 spiro heterocycle, -CO-C 11 spiro heterocycle, -CO-C 12 spiro heterocycle, -CO-C6bridged heterocycle, -CO-C7bridged heterocycle, -CO-C8bridged heterocycle, -CO-C9bridged heterocycle, -CO-C 10 bridged heterocycle, -CO-C 11 bridged heterocycle, -CO-C 12C6spiro heterocycle, -SO2-C7spiro heterocycle, -SO2-C8spiro heterocycle, -SO2-C9spiro heterocycle, -SO2-C 10 C6spiro heterocycle, -SO2-C7spiro heterocycle, -SO2-C8spiro heterocycle, -SO2-C9spiro heterocycle, -SO2-C 11 C6spiro heterocycle, -SO2-C7spiro heterocycle, -SO2-C8spiro heterocycle, -SO2-C9spiro heterocycle, -SO2-C 12 C6spiro heterocycle, -SO2-C7spiro heterocycle, -SO2-C8spiro heterocycle, -SO2-C9spiro heterocycle, -SO2-C 10 C6spiro heterocycle, -SO2-C7spiro heterocycle, -SO2-C8spiro heterocycle, -SO2-C9spiro heterocycle, -SO2-C 11 C6spiro heterocycle, -SO2-C7spiro heterocycle, -SO2-C8spiro heterocycle, -SO2-C9spiro heterocycle, -SO2-C 12 C6spiro heterocycle, -SO2-C7spiro heterocycle, -SO2-C8spiro heterocycle, -SO2-C9spiro heterocycle, -SO2-C 10 C6spiro heterocycle, -SO2-C7spiro heterocycle, -SO2-C8spiro heterocycle, -SO2-C9spiro heterocycle, -SO2-C 11 C6spiro heterocycle, -SO2-C7spiro heterocycle, -SO2-C8spiro heterocycle, -SO2-C9spiro heterocycle, -SO2-C 12 C6spiro heterocycle.

[0182] For example, R2may be C1alkyl, C2alkyl, C3alkyl, C4alkyl, C5alkyl, C6alkyl, C7alkyl, C8alkyl, C9alkyl, C 10 alkyl, C 11 alkyl, C 12 alkyl, C 13 alkyl, C 14 alkyl, tetrahydrofuran, pyrrolidine, furan, thiophene, pyrrole, thiazole, imidazole, tetrahydropyran, piperidine, pyridine, pyrazine, pyrimidine, or pyridazine.

[0183] Both R1and R2groups described above can be unsubstituted or substituted with any substituents. The number of substituents can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Further, the substituents are selected from one or more of the following: hydrogen, halogen, methyl, methoxy, halogenated methyl, halogenated methoxy.

[0184] For example, R1may be

[0185] For example, R2may be

[0186] For example, R1may be -CO-C1alkyl substituted. For example, the substituent is one non-fluorine halogen. For example, the substituent can be chlorine, bromine, or iodine. For example, R1may be

[0187] In the above embodiments, when R3and R4are independent of each other, R3may be selected from any one of hydrogen, methyl, halomethyl; and R4may be selected from C1-C7alkyl unsubstituted or substituted with a substituent.

[0188] For example, R3may be hydrogen, methyl, fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, bromomethyl, dibromomethyl, tribromomethyl, iodomethyl, diiodomethyl, or triiodomethyl.

[0189] For example, R4may be C1alkyl, C2alkyl, C3alkyl, C4alkyl, C5alkyl, C6alkyl, or C7alkyl. The R4group can be unsubstituted or substituted with any number of substituents. The number of substituents can be 1, 2, 3, 4, 5, 6, 7, or 8. Further, the substituents can be selected from one or more of hydrogen, halogen, methyl, methoxy, halomethyl, halomethoxy, -C(CH3)3, -C(CD3)3.

[0190] For example, R3may be

[0191] For example, R4may be

[0192] In the above embodiments, when R3and R4are linked to form a B ring, the B ring can be selected from any one of 5-6 membered heterocyclyl unsubstituted or substituted with a substituent, heteroatom-containing bicyclic or tricyclic ring structure.

[0193] For example, the B ring can be tetrahydrofuran, pyrrolidine, furan, thiophene, pyrrole, thiazole, imidazole, tetrahydropyran, piperidine, pyridine, pyrazine, pyrimidine, pyridazine, C5annular heterocycle, C6annular heterocycle, C7annular heterocycle, C8annular heterocycle, C9annular heterocycle, C 10 annular heterocycle, C 11 annular heterocycle, C 12 annular heterocycle, C6spiro heterocycle, C7spiro heterocycle, C8spiro heterocycle, C9spiro heterocycle, C 10 spiro heterocycle, C 11 spiro heterocycle, C 12 spiro heterocycle, C6bridged heterocycle, C7bridged heterocycle, C8bridged heterocycle, C9bridged heterocycle, C 10 bridged heterocycle, C 11 bridged heterocycle, or C 12Bridged heterocyclic ring. The B ring may be unsubstituted or substituted with any substituent. The number of substituents may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Further, the substituents are selected from one or more of the following: hydrogen, halogen, methyl, methoxy, halomethyl, halomethoxy, -C(CH3)3, -C(CD3)3.

[0194] For example, ring B could be

[0195] For example, ring B can be pyrrolidine, with a substituent being a halomethyl group. For example, ring B can be...

[0196] For example, ring B can be a C5 heterocyclic ring with a methyl substituent.

[0197] For example, ring B can be a C6 spiroheterocycle with halogen substituents. For example, ring B can be...

[0198] In the above embodiments, R5 can be selected from any of the following: cyano, unsubstituted or substituted carbonyl. The substituent can be selected from any of the following: hydrogen, halogen, methyl, methoxy, halomethyl, halomethoxy.

[0199] For example, R5 could be

[0200] For example, R5 could be

[0201] In the above implementation scheme, Group C can be selected from any of the following: -CH2- Unsubstituted or substituted pyridine ring, -CH2- Unsubstituted or substituted pyrrolidone;

[0202] Wherein, ring D can be selected from any of the following: unsubstituted or substituent R7 benzene ring, pyridine ring, pyrimidine ring, pyrazine ring, imidazole ring, pyrrole ring, thiophene ring, furan ring;

[0203] The E ring can be selected from any of the following: an unsubstituted benzene ring or a pyridine ring, a pyrimidine ring, a pyrazine ring, an imidazole ring, a pyrrole ring, a thiophene ring, or a furan ring;

[0204] R6, R7, R8, and R9may each independently be selected from any one of: hydrogen, cyano, methyl, amino, halogen, halogenated methyl, unsubstituted or substituted alkyl, hydroxyalkyl, aminoalkyl, five-membered nitrogen heterocycle; X is selected from any one of: -NH-, -O-, or -S-.

[0205] For example, Group C can be

[0206] For example, D ring can be

[0207] For example, E ring can be

[0208] For example, R6, R7, R8, and R9may each independently be selected from any one of:

[0209] For example, Group C can be D ring can be

[0210] For example, Group C can be

[0211] In some embodiments, B ring can be

[0212] In the above embodiments, Group A can be selected from any one of: -CO-unsubstituted or substituted hydrocarbyl, hydrocarbyl ether, indole, or -SO2-unsubstituted or substituted hydrocarbyl;

[0213] wherein R1may be selected from any one of: -CO-unsubstituted or substituted C1-C9 hydrocarbyl, hydrocarbyl ether, 6-membered heterocyclyl, heteroatom-containing bicyclic or tricyclic structure, or -SO2-unsubstituted or substituted C1-C9 hydrocarbyl, 6-membered heterocyclyl, heteroatom-containing bicyclic or tricyclic structure;

[0214] R2may be selected from any one of: unsubstituted or substituted C1-C 14 hydrocarbyl, 5-6 membered heterocyclyl.

[0215] For example, Group A can be -CO-C1 hydrocarbyl, -CO-C2 hydrocarbyl, -CO-C3 hydrocarbyl, -CO-C4 hydrocarbyl, -CO-C5 hydrocarbyl, -CO-C6 hydrocarbyl, -CO-C7 hydrocarbyl, -CO-C8 hydrocarbyl, -CO-C9 hydrocarbyl, -CO-C 10 hydrocarbyl, -CO-C11 hydrocarbon group, -CO-C 12 hydrocarbon group, -CO-C 13 hydrocarbon group, -CO-C 14 Hydrocarbon group, -C(O)O-C1 hydrocarbon group, -C(O)O-C2 hydrocarbon group, -C(O)O-C3 hydrocarbon group, -C(O)O-C4 hydrocarbon group, -C(O)O-C5 hydrocarbon group, -C(O)O-C6 hydrocarbon group, -C(O)O-C7 hydrocarbon group, -C(O)O-C8 hydrocarbon group, -C(O)O-C9 hydrocarbon group, -C(O)OC 10 Hydrocarbon group, -C(O)OC 11 Hydrocarbon group, -C(O)OC 12 Hydrocarbon group, -C(O)OC 13 Hydrocarbon group, -C(O)OC 14 Hydrocarbon group, indole, -SO2-C1 hydrocarbon group, -SO2-C2 hydrocarbon group, -SO2-C3 hydrocarbon group, -SO2-C4 hydrocarbon group, -SO2-C5 hydrocarbon group, -SO2-C6 hydrocarbon group, -SO2-C7 hydrocarbon group, -SO2-C8 hydrocarbon group, -SO2-C9 hydrocarbon group, -SO2-C 10 hydrocarbon group, -SO2-C 11 hydrocarbon group, -SO2-C 12 hydrocarbon group, -SO2-C 13 Hydrocarbon group or -SO2-C 14 Hydrocarbon group.

[0216] For example, Group A could be

[0217] For example, R1 can be -CO-C1 hydrocarbon group, -CO-C2 hydrocarbon group, -CO-C3 hydrocarbon group, -CO-C4 hydrocarbon group, -CO-C5 hydrocarbon group, -CO-C6 hydrocarbon group, -CO-C7 hydrocarbon group, -CO-C8 hydrocarbon group, -CO-C9 hydrocarbon group, -C(O)O-C1 hydrocarbon group, -C(O)O-C2 hydrocarbon group, -C(O)O-C3 hydrocarbon group, -C(O)O-C4 hydrocarbon group, -C(O)O -C5 hydrocarbon group, -C(O)O-C6 hydrocarbon group, -C(O)O-C7 hydrocarbon group, -C(O)O-C8 hydrocarbon group, -C(O)O-C9 hydrocarbon group, -CO-tetrahydropyran, -CO-piperidine, -CO-pyridine, -CO-pyrazine, -CO-pyrimidine, -CO-pyridazine, -CO-C6 heterocyclic ring, -CO-C7 heterocyclic ring, -CO-C8 heterocyclic ring, -CO-C9 heterocyclic ring, -CO-C 10 Heterocyclic rings, -CO-C 11 Heterocyclic rings, -CO-C 12 Spirocyclic rings, -CO-C6 spirocyclic rings, -CO-C7 spirocyclic rings, -CO-C8 spirocyclic rings, -CO-C9 spirocyclic rings, -CO-C10 spiro heterocycle, -CO-C 11 spiro heterocycle, -CO-C 12 spiro heterocycle, -CO-C6bridged heterocycle, -CO-C7bridged heterocycle, -CO-C8bridged heterocycle, -CO-C9bridged heterocycle, -CO-C 10 bridged heterocycle, -CO-C 11 bridged heterocycle, -CO-C 12 bridged heterocycle, -SO2-C1alkyl, -SO2-C2alkyl, -SO2-C3alkyl, -SO2-C4alkyl, -SO2-C5alkyl, -SO2-C6alkyl, -SO2-C7alkyl, -SO2-C8alkyl, -SO2-C9alkyl, -SO2-tetrahydropyran, -SO2-piperidine, -SO2-pyridine, -SO2-pyrazine, -SO2-pyrimidine, -SO2-pyridazine, -SO2-C6fused heterocycle, -SO2-C7fused heterocycle, -SO2-C8fused heterocycle, -SO2-C9fused heterocycle, -SO2-C 10 fused heterocycle, -SO2-C 11 fused heterocycle, -SO2-C 12 fused heterocycle, -SO2-C6spiro heterocycle, -SO2-C7spiro heterocycle, -SO2-C8spiro heterocycle, -SO2-C9spiro heterocycle, -SO2-C 10 spiro heterocycle, -SO2-C 11 spiro heterocycle, -SO2-C 12 spiro heterocycle, -SO2-C6bridged heterocycle, -SO2-C7bridged heterocycle, -SO2-C8bridged heterocycle, -SO2-C9bridged heterocycle, -SO2-C 10 bridged heterocycle, -SO2-C 11 bridged heterocycle, or -SO2-C 12 bridged heterocycle.

[0218] For example, R2may be C1alkyl, C2alkyl, C3alkyl, C4alkyl, C5alkyl, C6alkyl, C7alkyl, C8alkyl, C9alkyl, C 10 alkyl, C 11 alkyl, C 12 alkyl, C 13 alkyl, C 14 alkyl, tetrahydrofuran, pyrrolidine, furan, thiophene, pyrrole, thiazole, imidazole, tetrahydropyran, piperidine, pyridine, pyrazine, pyrimidine, or pyridazine.

[0219] Each of the R1and R2groups described above can be unsubstituted or substituted with any number of substituents. The number of substituents can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Further, the substituents are selected from one or more of the following: hydrogen, halogen, methyl, methoxy, halogenated methyl, halogenated methoxy.

[0220] For example, R1may be

[0221] For example, R2may be

[0222] For example, Group A can be wherein R1may be R2may be

[0223] For example, Group A can be

[0224] For example, Group A can be

[0225] In the above embodiments, R5may be selected from any one of the following: cyano, carbonyl unsubstituted or having a substituent. The substituent is selected from any one of the following: hydrogen, halogen, methyl, methoxy, halogenated methyl, halogenated methoxy.

[0226] For example, R5may be

[0227] For example, R5may be

[0228] In the above embodiments, Group C can be selected from any one of the following: -CH2-pyridine ring unsubstituted or having a substituent, -CH2-pyrrolidone unsubstituted or having a substituent;

[0229] wherein D ring can be selected from any one of the following: benzene ring unsubstituted or having a substituent R7, pyridine ring, pyrimidine ring, pyrazine ring, imidazole ring, pyrrole ring, thiophene ring, furan ring;

[0230] E ring can be selected from any one of the following: benzene ring unsubstituted or having a substituent R9, pyridine ring, pyrimidine ring, pyrazine ring, imidazole ring, pyrrole ring, thiophene ring, furan ring;

[0231] R6, R7, R8and R9may each independently be selected from any one of the following: hydrogen, cyano, methyl, amino, halogen element, halogenated methyl, alkyl unsubstituted or having a substituent, hydroxyalkyl, aminoalkyl, five-membered nitrogen heterocycle; X can be selected from any one of the following: -NH-, -O- or -S-.

[0232] For example, Group C can be

[0233] For example, the D ring can be

[0234] For example, the E ring can be

[0235] For example, R6, R7, R8, and R9may each be independently selected from any one of the following:

[0236] For example, Group C can be The D ring can be

[0237] In some embodiments, the B ring can be

[0238] In the above embodiments, Group A can be selected from any one of the following: -CO- unsubstituted or substituted hydrocarbyl, hydrocarbyl ether, indole, or -SO2- unsubstituted or substituted hydrocarbyl;

[0239] wherein R1may be selected from any one of the following: -CO- unsubstituted or substituted C1-C9hydrocarbyl, hydrocarbyl ether, 6-membered heterocyclyl, heteroatom-containing bicyclic or tricyclic structure, or -SO2- unsubstituted or substituted C1-C9hydrocarbyl, 6-membered heterocyclyl, heteroatom-containing bicyclic or tricyclic structure;

[0240] R2may be selected from any one of the following: unsubstituted or substituted C1-C 14 hydrocarbyl, 5-6 membered heterocyclyl.

[0241] For example, Group A can be -CO-C1hydrocarbyl, -CO-C2hydrocarbyl, -CO-C3hydrocarbyl, -CO-C4hydrocarbyl, -CO-C5hydrocarbyl, -CO-C6hydrocarbyl, -CO-C7hydrocarbyl, -CO-C8hydrocarbyl, -CO-C9hydrocarbyl, -CO-C 10 hydrocarbyl, -CO-C 11 hydrocarbyl, -CO-C 12 hydrocarbyl, -CO-C 13 hydrocarbyl, -CO-C 14 hydrocarbyl, -C(O)O-C1hydrocarbyl, -C(O)O-C2hydrocarbyl, -C(O)O-C3hydrocarbyl, -C(O)O-C4hydrocarbyl, -C(O)O-C5hydrocarbyl, -C(O)O-C6hydrocarbyl, -C(O)O-C7hydrocarbyl, -C(O)O-C8hydrocarbyl, -C(O)O-C9hydrocarbyl, -C(O)O-C 10 hydrocarbyl, -C(O)O-C11 Hydrocarbon group, -C(O)OC 12 Hydrocarbon group, -C(O)OC 13 Hydrocarbon group, -C(O)OC 14 Hydrocarbon group, indole, -SO2-C1 hydrocarbon group, -SO2-C2 hydrocarbon group, -SO2-C3 hydrocarbon group, -SO2-C4 hydrocarbon group, -SO2-C5 hydrocarbon group, -SO2-C6 hydrocarbon group, -SO2-C7 hydrocarbon group, -SO2-C8 hydrocarbon group, -SO2-C9 hydrocarbon group, -SO2-C 10 Hydrocarbon group, -SO2-C 11 Hydrocarbon group, -SO2-C 12 Hydrocarbon group, -SO2-C 13 Hydrocarbon group or -SO2-C 14 Hydrocarbon group.

[0242] For example, Group A could be

[0243] For example, R1 can be -CO-C1 hydrocarbon group, -CO-C2 hydrocarbon group, -CO-C3 hydrocarbon group, -CO-C4 hydrocarbon group, -CO-C5 hydrocarbon group, -CO-C6 hydrocarbon group, -CO-C7 hydrocarbon group, -CO-C8 hydrocarbon group, -CO-C9 hydrocarbon group, -C(O)O-C1 hydrocarbon group, -C(O)O-C2 hydrocarbon group, -C(O)O-C3 hydrocarbon group, -C(O)O-C4 hydrocarbon group, -C(O)O -C5 hydrocarbon group, -C(O)O-C6 hydrocarbon group, -C(O)O-C7 hydrocarbon group, -C(O)O-C8 hydrocarbon group, -C(O)O-C9 hydrocarbon group, -CO-tetrahydropyran, -CO-piperidine, -CO-pyridine, -CO-pyrazine, -CO-pyrimidine, -CO-pyridazine, -CO-C6 heterocyclic ring, -CO-C7 heterocyclic ring, -CO-C8 heterocyclic ring, -CO-C9 heterocyclic ring, -CO-C 10 Heterocyclic rings, -CO-C 11 Heterocyclic rings, -CO-C 12 Spirohedromes, -CO-C6 spirohedromes, -CO-C7 spirohedromes, -CO-C8 spirohedromes, -CO-C9 spirohedromes, -CO-C 10 Spiroherocyclic, -CO-C 11 Spiroherocyclic, -CO-C 12 Spiral heterocycles, -CO-C6 bridged heterocycles, -CO-C7 bridged heterocycles, -CO-C8 bridged heterocycles, -CO-C9 bridged heterocycles, -CO-C 10 Bridged heterocyclic rings, -CO-C 11 Bridged heterocyclic rings, -CO-C 12Bridged heterocycles, -SO2-C1 hydrocarbon group, -SO2-C2 hydrocarbon group, -SO2-C3 hydrocarbon group, -SO2-C4 hydrocarbon group, -SO2-C5 hydrocarbon group, -SO2-C6 hydrocarbon group, -SO2-C7 hydrocarbon group, -SO2-C8 hydrocarbon group, -SO2-C9 hydrocarbon group, -SO2-tetrahydropyran, -SO2-piperidine, -SO2-pyridine, -SO2-pyrazine, -SO2-pyrimidine, -SO2-pyridazine, -SO2-C6 fused heterocycle, -SO2-C7 fused heterocycle, -SO2-C8 fused heterocycle, -SO2-C9 fused heterocycle, -SO2-C 10 Heterocyclic rings, -SO2-C 11 Heterocyclic rings, -SO2-C 12 Spirohedromes, -SO2-C6 spirohedromes, -SO2-C7 spirohedromes, -SO2-C8 spirohedromes, -SO2-C9 spirohedromes, -SO2-C 10 Spirohexane, -SO2-C 11 Spirohexane, -SO2-C 12 Spiral heterocyclic rings, -SO2-C6 bridged heterocyclic rings, -SO2-C7 bridged heterocyclic rings, -SO2-C8 bridged heterocyclic rings, -SO2-C9 bridged heterocyclic rings, -SO2-C 10 Bridged heterocyclic rings, -SO2-C 11 Bridged heterocyclic or -SO2-C 12 Bridge miscellaneous rings.

[0244] For example, R2 can be a C1 hydrocarbon group, C2 hydrocarbon group, C3 hydrocarbon group, C4 hydrocarbon group, C5 hydrocarbon group, C6 hydrocarbon group, C7 hydrocarbon group, C8 hydrocarbon group, C9 hydrocarbon group, C 10 hydrocarbon group, C 11 hydrocarbon group, C 12 hydrocarbon group, C 13 hydrocarbon group, C 14 Hydrocarbon group, tetrahydrofuran, pyrrolidine, furan, thiophene, pyrrole, thiazole, imidazole, tetrahydropyran, piperidine, pyridine, pyrazine, pyrimidine, or pyridazine.

[0245] Both R1 and R2 groups mentioned above can be unsubstituted or substituted with any substituent. The number of substituents can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Further, the substituents are selected from one or more of the following: hydrogen, halogen, methyl, methoxy, halomethyl, and halomethoxy.

[0246] For example, R1 could be

[0247] For example, R2 could be

[0248] For example, Group A could be wherein R1may be R2may be

[0249] In the above embodiments, R5is selected from any one of the following: cyano, carbonyl unsubstituted or having a substituent. The substituent is selected from any one of the following: hydrogen, halogen, methyl, methoxy, halogenated methyl, halogenated methoxy.

[0250] For example, R5may be

[0251] For example, R5may be

[0252] In the above embodiments, Group C can be selected from any one of the following: -CH2-pyridine ring unsubstituted or having a substituent, -CH2-pyrrolidone unsubstituted or having a substituent;

[0253] wherein D ring can be selected from any one of the following: benzene ring unsubstituted or having a substituent R7, pyridine ring, pyrimidine ring, pyrazine ring, imidazole ring, pyrrole ring, thiophene ring, furan ring;

[0254] E ring can be selected from any one of the following: benzene ring unsubstituted or having a substituent R9, pyridine ring, pyrimidine ring, pyrazine ring, imidazole ring, pyrrole ring, thiophene ring, furan ring;

[0255] R6, R7, R8and R9may each independently be selected from any one of the following: hydrogen, cyano, methyl, amino, halogen element, halogenated methyl, alkyl unsubstituted or having a substituent, hydroxyalkyl, aminoalkyl, five-membered nitrogen heterocycle; X is selected from any one of the following: -NH-, -O- or -S-.

[0256] For example, Group C can be

[0257] For example, D ring can be

[0258] For example, E ring can be

[0259] For example, R6, R7, R8and R9may each independently be selected from any one of the following:

[0260] For example, Group C can be D ring can be

[0261] Composition

[0262] In another aspect, the present application provides a pharmaceutical composition comprising a compound, or a racemate, an enantiomer, a diastereomer, or a pharmaceutically acceptable salt thereof, as described herein, and optionally a pharmaceutically acceptable carrier. For example, the pharmaceutical composition can be in the form of a solution, an aerosol, a gel, an ointment, a spray, or a suspension, which can be prepared by methods known and conventional in the art. For example, the pharmaceutically acceptable carrier can be selected from excipients for increasing solubility, adjuvants for enhancing viscosity, and adjuvants for enhancing permeability.

[0263] Applications

[0264] In another aspect, the present application provides the use of a compound as described herein and / or a pharmaceutical composition as described herein in the preparation of an antiviral drug, which can be used to inhibit viruses that replicate in dependence on the main protease.

[0265] In the above-mentioned applications, the viruses that replicate in dependence on the main protease can include wild-type or drug-resistant mutant strains of coronavirus, calicivirus, norovirus, hepatitis A virus, human rhinovirus, and porcine transmissible gastroenteritis virus. For example, wild-type or drug-resistant mutant strains of SARS-CoV-2, SARS-CoV, MERS-CoV viruses of the Coronaviridae family. For example, wild-type or drug-resistant mutant strains of poliovirus, coxsackievirus, rhinovirus of the Picornaviridae family. For example, wild-type or drug-resistant mutant strains of norovirus of the Caliciviridae family.

[0266] Without wishing to be bound by any theory, the examples below are merely intended to illustrate the compounds, the preparation methods, and the uses of the present application, and are not intended to limit the scope of the present application.

[0267] Examples

[0268] Example 1 - Synthesis of (1R,2S,5S)-N-(1-cyano-2-(pyridin-2-yl)ethyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido))butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Hit 3-1)

[0269] The present example is a main protease inhibitor Hit 3-1, which has the following structural formula:

[0270] The preparation method of the main protease inhibitor Hit 3-1 comprises the following steps (the reaction scheme is shown in Figure 1):

[0271] (1) First, (1R, 2S, 5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (50 mg, 0.1372 mmol, 1.0 eq.) and 2-amino-3-(pyridin-2-yl)propionic acid ethyl ester dihydrochloride (40.3 m, 0.1509, 1.1 eq.) were dissolved in dichloromethane, 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 37 mg, 0.1921 mmol, 1.4 eq.) and 4-dimethylaminopyridine (DMAP, 20 mg, 0.1647 mmol, 1.2 eq.) were added, and the reaction was stirred at room temperature overnight, and the reaction progress was monitored using a liquid chromatography-mass spectrometry system (LC-MS); after the reaction was completed, the DCM in the reaction liquid was evaporated, the residue was redissolved with 25% acetonitrile aqueous solution, and purified using a preparative high-performance liquid chromatography system (HPLC) through an SPX-C18 reversed-phase preparative column, the target product fraction was collected, and freeze-drying was performed to obtain intermediate ethyl 2-((1R, 2S, 5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamido)-3-(pyridin-2-yl)propanoate;

[0272] (2) The intermediate obtained in step (1) was dissolved in a 7M ammonium methylate solution, and stirred at room temperature for 48h, during which the amine ester exchange reaction was monitored using LC-MS, after the reaction was completed, the reaction liquid was evaporated, DCM was added for redissolution, and Burgess reagent (~2.5 eq.) was added, and stirred at room temperature for 3h, after the reaction was completed, the reaction liquid was evaporated, purified using a preparative high-performance liquid chromatography system (HPLC) through an SPX-C18 reversed-phase preparative column, the target product fraction was collected, and freeze-drying was performed, and finally the end product Hit3-1 was obtained (the mass spectrometry analysis result is shown in Figure 3).

[0273] The main protease inhibitor Hit3-1 appeared as a light brown solid, with a yield of 64%, and a purity of about 97.32%, RT = 3.775 min; HRMS: m / z = 494.23706 [M+H] + ; 1H NMR (600 MHz, Chloroform-d) δ 8.71 (d, J = 6.1 Hz, 1H), 8.28 (d, J = 8.1 Hz, 1H), 7.99 (td, J = 7.8, 1.7 Hz, 1H), 7.91 - 7.71 (m, 1H), 7.65 - 7.59 (m, 1H), 6.94 (d, J = 9.4 Hz, 1H), 5.27 (q, J = 6.8 Hz, 1H), 4.55 (d, J = 9.4 Hz, 1H), 4.38 (d, J = 12.9 Hz, 1H), 3.86 - 3.59 (m, 4H), 1.52 - 1.48 (m, 1H), 1.25 (s, 1H), 1.09 - 1.03 (m, 6H), 1.00 (s, 9H).

[0274] Example 2 - Synthesis of (1R,2S,5S)-N-((S)-1-cyano-2-(5-methylpyridin-2-yl)ethyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Hit 3-2)

[0275] This example is a primary protease inhibitor Hit 3-2, which has the following structural formula:

[0276] The method of preparing this primary protease inhibitor Hit 3-2 includes the following steps (synthetic route is shown in Figure 4):

[0277] (1) (1R, 2S, 5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (50 mg, 0.1372 mmol, 1.0 eq.) and (S)-2-amino-3-(5-methylpyridin-2-yl)propanoic acid methyl ester (35 mg, 0.1509, 1.1 eq.) were dissolved in dichloromethane, EDCI (37 mg, 0.1921 mmol, 1.4 eq.) and DMAP (20 mg, 0.1647 mmol, 1.2 eq.) were added, and the reaction was stirred at room temperature overnight. The progress of the reaction was monitored using a liquid chromatography-mass spectrometry (LC-MS) system. After the reaction was completed, the DCM in the reaction solution was evaporated, the residue was dissolved in 25% acetonitrile aqueous solution, and the target product fraction was collected and freeze-dried to obtain intermediate methyl (S)-2-((1R, 2S, 5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamido)-3-(5-methylpyridin-2-yl)propanoate;

[0278] (2) The intermediate obtained in step (1) was dissolved in 7M ammonium methylate solution and stirred at room temperature for 48 h. The progress of the reaction was monitored using LC-MS. After the reaction was completed, the reaction solution was evaporated, DCM was added to dissolve the residue, and Burgess reagent (~2.5 eq.) was added and stirred at room temperature for 3 h. After the reaction was completed, the reaction solution was evaporated, purified using a preparative high-performance liquid chromatography (HPLC) system on an SPX-C18 reversed-phase preparative column, the target product fraction was collected and freeze-dried, and finally the end product Hit3-2 was obtained (the mass spectral analysis results are shown in Figure 5).

[0279] The main protease inhibitor Hit3-2 was a light yellow solid with a yield of 72% and a purity of about 98.13%, and the RT was 3.790 min; HRMS: m / z = 508.25402 [M+H] + .

[0280] Example 3 - Synthesis of (1R, 2S, 5S)-N-(1-cyano-2-(5-methylpyridin-3-yl)ethyl)-3-((S)-3,3-dimethyl-2-(2,2,2)-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Hit 3-3)

[0281] This example is a main protease inhibitor Hit 3-3, and the structural formula is as follows:

[0282] The preparation method of the main protease inhibitor Hit 3-3 includes the following steps (the synthetic route is shown in Figure 6):

[0283] (1) 2-((diphenylmethylene)amino)acetonitrile (440 mg, 2.0 mmol, 1.0 eq.) was dissolved in dimethylformamide (DMF), and sodium hydride (60%, 280 mg, 7 mmol, 3.5 eq.) was slowly added step by step under nitrogen protection, and the reaction was stirred in an ice bath for 1 h. After the reaction was completed, 3-(chloromethyl)-5-methylpyridine (356 mg, 2 mmol, 1.0 eq.) and tetrabutylammonium iodide (TBAI, 75 mg, 0.2 mmol, 0.1 eq.) were added step by step to the reaction solution, and the reaction was carried out at 25°C overnight under nitrogen protection. After the reaction was completed, the reaction was quenched by adding an appropriate amount of water, ethyl acetate was added, and water and saturated brine were added in sequence to wash the organic layer. The organic layer was dried over anhydrous sodium sulfate and rotary evaporated. An appropriate amount of hydrochloric acid tetrahydrofuran solution was added, and the reaction was stirred at room temperature for 3 h. The reaction was neutralized with saturated sodium bicarbonate solution and hydrochloric acid. The product was extracted with ethyl acetate / water system and purified by silica gel column chromatography to obtain the intermediate 2-amino-3-(5-methylpyridin-3-yl)propanenitrile;

[0284] (2) The intermediate obtained in step (1) (50 mg, 0.3102, 1.0 eq.) and (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (124 mg, 0.3412 mmol, 1.1 eq.) were dissolved in DCM, EDCI (92 mg, 0.4776 mmol, 1.4 eq.) and DMAP (17 mg, 0.1365 mmol, 0.4 eq.) were added, and the reaction was stirred at room temperature overnight. The reaction progress was monitored using a liquid chromatography-mass spectrometry (LC-MS) system. After the reaction was completed, the DCM in the reaction solution was rotary evaporated, the residue was redissolved with 25% acetonitrile aqueous solution, and the product was purified by an SPX-C18 reversed-phase preparation column using a preparative high-performance liquid chromatography (HPLC) system. The target product fraction was collected and freeze-dried to obtain the final product Hit 3-3 (the mass spectrometry analysis result is shown in Figure 7).

[0285] The main protease inhibitor Hit 3-3 is a light yellow solid with a yield of 43% and a purity of about 91.31%, and the RT is 7.207 min; HRMS: m / z = 508.25579 [M+H] + ; 1H NMR (600 MHz, DMSO-d6) δ 9.39 (d, J = 8.3 Hz, 1H), 9.03 (d, J = 7.7 Hz, 1H), 8.46 (s, 1H), 7.77 - 7.70 (m, 1H), 7.41 (d, J = 8.1 Hz, 1H), 5.20 (q, J = 7.7 Hz, 1H), 4.39 (d, J = 8.4 Hz, 1H), 4.18 (s, 1H), 3.89 (dd, J = 10.4, 5.5 Hz, 1H), 3.68 (d, J = 10.5 Hz, 1H), 3.33 - 3.26 (m, 1H), 3.26 - 3.20 (m, 1H), 2.31 (s, 3H), 1.54 (dd, J = 7.5, 5.5 Hz, 1H), 1.20 (d, J = 7.6 Hz, 1H), 1.01 (s, 3H), 0.97 (s, 9H), 0.83 (s, 3H).

[0286] Example 4 - Synthesis of (1R,2S,5S)-N-((S)-2-(5-chloropyridin-2-yl)-1- cyanoethyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl- 3-azabicyclo[3.1.0]hexane-2-carboxamide (Hit 3-4)

[0287] This example is a primary protease inhibitor Hit 3-4, which has the following structure:

[0288] The method of making this primary protease inhibitor Hit 3-4 includes the following steps (synthetic route is shown in Figure 8):

[0289] (1) (1R, 2S, 5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6- dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (50 mg, 0.1372 mmol, 1.0 eq.) and (S)-2-amino-3-(5-chloropyridin-2-yl)propanoic acid methyl ester (41.35 mg, 0.1647, 1.2 eq.) were dissolved in dichloromethane, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 37 mg, 0.1921 mmol, 1.4 eq.) and 4-dimethylaminopyridine (DMAP, 20 mg, 0.1647 mmol, 1.2 eq.) were added, and the reaction was stirred at room temperature overnight, and the progress of the reaction was monitored using a liquid chromatography-mass spectrometry (LC-MS) system; after the reaction was completed, the DCM in the reaction solution was evaporated, the residue was redissolved in 40% acetonitrile aqueous solution, and purified using a preparative high performance liquid chromatography (HPLC) system on an SPX-C18 reversed-phase preparative column, the target product fractions were collected, and freeze-drying was performed to obtain intermediate methyl (S)-3-(5-chloropyridin-2-yl)-2-((1R, 2S, 5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-2-ylmethyl)propanoate;

[0290] (2) The intermediate obtained in step (1) was dissolved in a 7M ammonium methylate solution, and stirred at room temperature for 48 h in a sealed state, during which the amine ester exchange reaction was monitored using an LC-MS, after the reaction was completed, the reaction solution was evaporated, DCM was added for redissolution, and Burgess reagent (~2.5 eq.) was added, and stirred at room temperature for 3 h, after the reaction was completed, the reaction solution was evaporated, purified using a preparative high performance liquid chromatography (HPLC) system on an SPX-C18 reversed-phase preparative column, the target product fractions were collected, and freeze-drying was performed, and finally the end product Hit3-4 was obtained (the mass spectrometry analysis result is shown in FIG. 9).

[0291] The end product Hit3-4 was a yellow solid, the yield was 79%, the purity was about 97.42%, and RT = 4.642 min; HRMS: m / z = 528.19526 [M+H] + .

[0292] Example 5 - Synthesis of (1R, 2S, 5S)-N-(cyano(pyridin-3-yl)methyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-2- carboxamide (Hit 3-5)

[0293] The main protease inhibitor Hit 3-5 of this example has the following structural formula:

[0294] The preparation method of the main protease inhibitor Hit 3-5 includes the following steps (the synthetic route is shown in Figure 10):

[0295] (1) Dissolve nicotinaldehyde (1000 mg, 9.34 mmol, 1.0 eq.) in 10 mL of ammonium methanol solution, and add ammonium chloride (1018 mg, 18.67 mmol, 2.0 eq.). Stir at room temperature for 1 h, and then add trimethylsilyl cyanide (TMSCN, 2316 mg, 23.34 mmol, 2.5 eq.) dropwise into the reaction system under ice bath. After the dropwise addition is completed, place the reaction system in a room temperature water bath and stir overnight. Monitor the reaction progress by thin layer chromatography. After the reaction is completed, quench the reaction by adding 10 mL of water, extract with DCM, dry the organic layer over anhydrous sodium sulfate, and concentrate and dry. Purify the product by silica gel column chromatography using MeOH / DCM (1:10) as the developing agent to obtain the intermediate 2-amino-2-(pyridin-3-yl)acetonitrile in the form of yellow-brown oil;

[0296] (2) Dissolve the intermediate 2-amino-2-(pyridin-3-yl)acetonitrile (30 mg, 0.225 mmol, 1.0 eq.) obtained in step (1) and (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (91 mg, 0.248 mmol, 1.1 eq.) in DCM, and add EDCI (48 mg, 0.248 mmol, 1.1 eq.) and DMAP (11 mg, 0.0902 mmol, 0.4 eq.). Stir at room temperature overnight, and monitor the reaction progress by thin layer chromatography. After the reaction is completed, purify the product by preparative thin layer chromatography using MeOH / DCM (1:20) as the developing agent to obtain the final product Hit 3-5 (see Figure 11 for the mass spectral analysis results).

[0297] The final product Hit 3-5 is a light yellow solid with a yield of 62% and a purity of about 97.05%, and RT = 3.859 min; HRMS: m / z = 480.22977 [M+H] + ; 1H NMR (600 MHz, Chloroform-d) δ 8.72 (s, 1H), 8.63 (dd, J = 4.8, 1.6 Hz, 1H), 7.93 - 7.86 (m, 1H), 7.82 (dt, J = 7.7, 2.0 Hz, 1H), 7.33 (dd, J = 8.0, 4.8 Hz, 1H), 6.86 (d, J = 9.3 Hz, 1H), 6.20 (d, J = 8.6 Hz, 1H), 4.49 (d, J = 9.4 Hz, 1H), 4.40 (s, 1H), 3.89 - 3.82 (m, 2H), 1.65 - 1.57 (m, 2H), 0.84 (d, J = 1.4 Hz, 9H), 0.83 (d, J = 7.6 Hz, 6H).

[0298] Example 6 - Synthesis of (1R,2S,5S)-N-(cyano(isoquinolin-4-yl)methyl)-3-((S)-3,3- dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (Hit 3-6)

[0299] This example is a primary protease inhibitor Hit 3-6, which has the following structural formula:

[0300] The synthetic route of this primary protease inhibitor Hit 3-6 is shown in Figure 12, which specifically comprises the following steps:

[0301] (1) 4-Isoquinoline formaldehyde (500 mg, 6.36 mmol, 1.0 eq.) was dissolved in 10 mL of ammonium methanol solution, and ammonium chloride (347 mg, 12.7 mmol, 2.0 eq.) was added, stirred at room temperature for 1 h, and then TMSCN (789 mg, 15.9 mmol, 2.5 eq.) was added dropwise into the reaction system under ice bath, after the dropwise addition was completed, the reaction system was stirred at room temperature overnight, and the reaction progress was monitored by thin layer chromatography; after the reaction was completed, 10 mL of water was added to quench the reaction, extracted with DCM, and the organic layer was dried over anhydrous sodium sulfate and concentrated to dryness, and the intermediate 2-amino-2-(isoquinolin-4-yl)acetonitrile was purified by silica gel column chromatography with MeOH / DCM (1:10) as the developing agent, and the product was obtained as a yellow solid;

[0302] (2) 2-amino-2-(isoquinolin-4-yl)acetonitrile (40 mg, 0.218 mmol, 1.0 eq.) and (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxylic acid (88 mg, 0.240 mmol, 1.1 eq.) were dissolved in DCM, EDCI (47 mg, 0.240 mmol, 1.1 eq.) and DMAP (11 mg, 0.0874 mmol, 0.4 eq.) were added, and the reaction was stirred at room temperature overnight, using thin layer chromatography to monitor the progress of the reaction; after the reaction was completed, the end product Hit 3-6 (see Figure 13 for mass spectral analysis results) was obtained by purification on a preparative thin layer chromatography plate using MeOH / DCM (1:20) as the developing agent, and appeared as a white solid with a yield of 68% and a purity of about 99.38%, RT = 4.148 min; HRMS: m / z = 530.24543 [M+H] + ; 1 H NMR (600 MHz, Chloroform-d) δ 9.24 (s, 1H), 8.80 (s, 1H), 8.09 - 8.04 (m, 2H), 8.01 (d, J = 8.2 Hz, 1H), 7.98 (d, J = 8.5 Hz, 1H), 7.82 (ddd, J = 8.4, 7.0, 1.3 Hz, 1H), 7.70 (t, J = 7.5 Hz, 1H), 6.82 (d, J = 9.4 Hz, 1H), 6.75 (d, J = 8.7 Hz, 1H), 4.40 (d, J = 9.5 Hz, 1H), 3.77 (d, J = 3.0 Hz, 2H), 1.83 (d, J = 7.7 Hz, 1H), 1.62 - 1.59 (m, 1H), 0.80 (s, 6H), 0.54 (s, 9H).

[0303] Example 7 - Synthesis of Benzyl ((2S)-1-((cyano(pyridin-3-yl)methyl)amino)-4- methyl-1-oxopentan-2-yl)carbamate (Hit 3-7)

[0304] This example is a main protease inhibitor Hit 3-7, which has the following structural formula:

[0305] The synthetic route of this main protease inhibitor Hit 3-7 is shown in Figure 14, and specifically includes the following steps:

[0306] The intermediate 2-amino-2-(pyridin-3-yl)acetonitrile was synthesized according to the same method as in Example 5, step (1), and then 2-amino-2-(pyridin-3-yl)acetonitrile (30 mg, 0.225 mmol, 1.0 eq.) and ((benzyloxy)carbonyl)-L-leucine (66 mg, 0.248, 1.1 eq.) were dissolved in DCM, EDCI (48 mg, 0.248 mmol, 1.1 eq.) and DMAP (11 mg, 0.0902 mmol, 0.4 eq.) were added, and the reaction was stirred at room temperature overnight, with TLC monitoring of the reaction progress; after the reaction was completed, the final product Hit 3-7 (see Figure 15 for the mass spectral analysis results) was obtained by purification on a preparative TLC plate using MeOH / DCM (1:20) as the developing agent, and appeared as a white solid with a yield of 69% and a purity of about 99.11%, RT = 3.639 min; HRMS: m / z = 381.19810 [M+H] + ; 1 H NMR (600 MHz, Chloroform-d) δ 8.61 (d, J = 4.8 Hz, 1H), 8.58 (d, J = 4.8 Hz, 1H), 7.91 (d, J = 8.2 Hz, 2H), 7.75 (d, J = 8.5 Hz, 2H), 7.33 - 7.30 (m, 5H), 6.14 (d, J = 8.2 Hz, 1H), 5.47 (d, J = 8.3 Hz, 1H), 5.09 - 5.01 (m, 2H), 1.95 (s, 1H), 1.60 - 1.48 (m, 2H), 0.92 (d, J = 5.8 Hz, 3H), 0.87 (d, J = 6.1 Hz, 3H).

[0307] Example 8 - Synthesis of Benzyl ((2S)-1-((cyano(isoquinolin-4-yl)methyl)amino)-4- methyl-1-oxopentan-2-yl)carbamate (Hit 3-8)

[0308] This example is a proteasome inhibitor Hit 3-8, which has the following structural formula:

[0309] The synthetic route of this proteasome inhibitor Hit 3-8 is shown in Figure 16, and specifically comprises the following steps:

[0310] The intermediate 2-amino-2-(isoquinolin-4-yl)acetonitrile was synthesized according to the same method as in Example 6, step (1), and then 2-amino-2-(isoquinolin-4-yl)acetonitrile (50 mg, 0.273 mmol, 1.0 eq.) and ((benzyloxy)carbonyl)-L-leucine (80 mg, 0.300, 1.1 eq.) were dissolved in DCM, EDCI (58 mg, 0.300 mmol, 1.1 eq.) and DMAP (14 mg, 0.109 mmol, 0.4 eq.) were added, and the reaction was stirred at room temperature overnight, with TLC monitoring of the reaction progress; after the reaction was completed, the final product Hit 3-8 (see Figure 17 for the results of mass spectral analysis) was obtained by purification on a preparative TLC plate using MeOH / DCM (1:20) as the developing agent, and appeared as a white solid in a yield of 64% and a purity of about 99.46%, with an RT = 3.909 min; HRMS: m / z = 431.21404 [M+H] + ; 1 H NMR (600 MHz, Chloroform-d) δ 9.24 (s, 1H), 8.61 (s, 1H), 8.04 (d, J = 9.9 Hz, 1H), 7.97 (d, J = 8.2 Hz, 1H), 7.86 (d, J = 9.0 Hz, 2H), 7.77 (t, J = 7.7 Hz, 1H), 7.67 (t, J = 7.6 Hz, 1H), 7.32 - 7.26 (m, 5H), 6.70 (d, J = 8.8 Hz, 1H), 5.07 - 4.96 (m, 2H), 4.93 (d, J = 12.0 Hz, 1H), 1.84 (s, 1H), 1.73 - 1.63 (m, 2H), 0.83 (d, J = 6.3 Hz, 6H).

[0311] Example 9 - Synthesis of (6S)-N-(cyano(isoquinolin-4-yl)methyl)-5-((S)-3,3- dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-5-azaspiro[2.4]heptane-6-carboxamide (Hit 3-11)

[0312] This example is a main protease inhibitor Hit 3-11, which has the following structural formula:

[0313] The synthetic route of this main protease inhibitor Hit 3-11 is shown in Figure 18, and specifically comprises the following steps:

[0314] Intermediate 2-amino-2-(isoquinolin-4-yl)acetonitrile was synthesized according to the method in Example 6; then 2-amino-2-(isoquinolin-4-yl)acetonitrile (170 mg, 0.929 mmol, 1.0 eq.) and (S)-5-(tert-butoxycarbonyl)-5-azaspiro[2.4]heptane-6-carboxylic acid (247 mg, 1.02 mmol, 1.1 eq.) were dissolved in DCM, EDCI (200 mg, 1.02 mmol, 1.1 eq.) and DMAP (50 mg, 0.371 mmol, 0.4 eq.) were added, and the reaction was stirred at room temperature overnight, with TLC monitoring of the reaction progress; after the reaction was completed, the intermediate tert-butyl (6S)-6-((cyano(isoquinolin-4-yl)methyl)carbamoyl)-5-azaspiro[2.4]heptane-5-carboxylate was obtained by purification on a preparative TLC plate using MeOH / DCM (1:20) as the developing agent, with a yield of 81%; the intermediate was dissolved in 2 mL of DCM, 3 mL of a hydrochloric acid solution in dioxane (4 M, 15.0 eq.) was added dropwise slowly with stirring, and the reaction was stirred at room temperature for about 2 h; after the reaction was completed, the reaction solution was rotary evaporated under reduced pressure, the product was extracted with DCM, and washed with saturated aqueous sodium bicarbonate and saturated brine; the organic layer was dried over anhydrous sodium sulfate and concentrated and rotary evaporated to dryness to obtain the intermediate (6S)-N-(cyano(isoquinolin-4-yl)methyl)-5-azaspiro[2.4]heptane-6-carboxamide; (6S)-N-(cyano(isoquinolin-4-yl)methyl)-5-azaspiro[2.4]heptane-6-carboxamide (30 mg, 0.098 mmol, 1.0 eq.) and (S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoic acid (25 mg, 0.108 mmol, 1.1 eq.) were dissolved in DCM, EDCI (21 mg, 0.108 mmol, 1.1 eq.) and DMAP (12 mg, 0.098 mmol, 1.0 eq.) were added, and the reaction was stirred at room temperature overnight, with TLC monitoring of the reaction progress; after the reaction was completed, the final product Hit 3-11 (see Figure 19 for mass spectral analysis results) was obtained by purification on a preparative TLC plate using MeOH / DCM (1:20) as the developing agent; it was a white solid with a yield of 34%, a purity of about 98.44%, and an RT = 3.919 min; HRMS: m / z = 516.22919 [M+H] + ; 1H NMR (600 MHz, Chloroform-d) δ 9.27 (d, J = 6.1 Hz, 1H), 8.84 (d, J = 3.9 Hz, 1H), 8.18 - 8.07 (m, 1H), 8.03 (d, J = 8.1 Hz, 1H), 7.97 (t, J = 8.8 Hz, 1H), 7.92 (t, J = 8.9 Hz, 1H), 7.84 - 7.79 (m, 1H), 7.71 - 7.67 (m, 1H), 6.74 (dd, J = 8.8, 3.7 Hz, 1H), 4.78 - 4.72 (m, 1H), 4.60 - 4.30 (m, 1H), 3.67 (dd, J = 29.2, 9.4 Hz, 1H), 3.30 (dd, J = 27.8, 9.5 Hz, 1H), 2.37 - 2.25 (m, 1H), 2.13 - 2.03 (m, 1H), 1.03 (s, 6H), 0.74 - 0.53 (m, 3H).

[0315] Example 10 - Synthesis of (6S)-N-(cyano(isoquinolin-4-yl)methyl)-5-((S)-2- cyclohexyl-2-(2,2,2-trifluoroacetamido)acetyl)-5-azaspiro[2.4]heptane-6-carboxamide (Hit 3-16)

[0316] This example is a main protease inhibitor Hit 3-16, whose structural formula is as follows:

[0317] The synthetic route of this main protease inhibitor Hit 3-16 is shown in Figure 20, which specifically comprises the following steps:

[0318] First, the intermediate (6S)-N-(cyano(isoquinolin-4-yl)methyl)-5-azaspiro[2.4]heptane-6- carboxamide was synthesized according to the method in Example 9; then (6S)-N-(cyano(isoquinolin-4-yl)methyl)-5-azaspiro[2.4]heptane-6-carboxamide (30 mg, 0.098 mmol, 1.0 eq.) and (S)-2-cyclohexyl-2-(2,2,2-trifluoroacetamido)acetic acid (28 mg, 0.108 mmol, 1.1 eq.) were dissolved in DCM, EDCI (21 mg, 0.108 mmol, 1.1 eq.) and DMAP (12 mg, 0.098 mmol, 1.0 eq.) were added, and the reaction was stirred at room temperature overnight, with thin layer chromatography monitoring the progress of the reaction; after the reaction was completed, the final product Hit 3-16 was obtained by purification on a preparative thin layer chromatography plate with MeOH / DCM (1:20) as the developing agent (the mass spectral analysis result is shown in Figure 21), which was a white solid with a yield of 53% and a purity of about 99.78%, RT = 4.224 min; HRMS: m / z = 542.24554 [M+H]+ .

[0319] Examples 11-22 - Synthesis of protease inhibitor hits 3-15, 3-17-3-28

[0320] The structures of the protease inhibitor hits 3-15, 3-17-3-28 are shown in Table 1:

[0321] Table 1

[0322] As can be seen from Table 1, the protease inhibitor hits 3-15, 3-17-3-28 have the same skeleton, only the structure of the R1group is different, so the synthetic routes are similar. The general synthetic route of the protease inhibitor hits 3-15, 3-17-3-28 is shown in Figure 22, which specifically comprises the following steps:

[0323] (1) Synthesizing the intermediate (6S)-N-(cyano(isoquinolin-4-yl)methyl)-5- azaspiro[2.4]heptane-6-carboxamide by the method in Example 9;

[0324] (2) Dissolving (6S)-N-(cyano(isoquinolin-4-yl)methyl)-5-azaspiro[2.4]heptane-6- carboxamide (684 mg, 2.23 mmol, 1.0 eq.) and (S)-2-((tert-butoxycarbonyl)amino)- 3,3-dimethylbutanoic acid (775 mg, 3.35 mmol, 1.5 eq.) in DCM, adding EDCI (643 mg, 3.35 mmol, 1.5 eq.) and DMAP (273 mg, 2.23 mmol, 1.0 eq.), stirring the reaction at room temperature overnight, monitoring the progress of the reaction using thin layer chromatography; after the reaction is completed, purifying the product by silica gel column chromatography with MeOH / DCM (1:20) as the developing agent to obtain the intermediate tert-butyl ((2S)-1-((6S)-6-((cyano(isoquinolin-4-yl)methyl)amino carbonyl)-5-azaspiro[2.4]hept-5-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate with a yield of 64%; dissolving the intermediate in 6 mL of DCM, slowly adding 6 mL of a hydrochloric acid solution in dioxane (4 M, 15.0 eq.) dropwise with stirring, stirring the reaction at room temperature for about 2 h, after the reaction is completed, rotary evaporating the reaction solution under reduced pressure, extracting the product with DCM, washing with saturated aqueous sodium bicarbonate and saturated brine, drying the organic layer over anhydrous sodium sulfate and concentrating and rotary evaporating to obtain the intermediate (6S)-5-((S)-2-amino-3,3-dimethylbutanoyl)-N- (cyano(isoquinolin-4-yl)methyl)-5-azaspiro[2.4]heptane-6-carboxamide with a yield of 89%;

[0325] (3) According to the structure of the final product, an appropriate amount of (6S)-5-((S)-2-amino-3,3-dimethylbutanoyl)-N-(cyano(isoquinolin-4-yl)methyl)-5-azaspiro[2.4]heptan-6- formamide was weighed out separately from different substrates and subjected to condensation reaction under the catalysis of the corresponding catalyst to obtain the target product.

[0326] The specific implementation of step (3) will be described below in sequence, and steps (1) and (2) are general.

[0327] Example 11 - Synthesis of (6S)-5-((S)-2-(2-chloro-2,2-difluoroacetamido)-3,3- dimethylbutanoyl)-N-(cyano(isoquinolin-4-yl)methyl)-5-azaspiro[2.4]heptan-6- formamide (Hit 3-22)

[0328] In the synthesis route of the main protease inhibitor Hit 3-22 of this example, step (3) is specifically:

[0329] (6S)-5-((S)-2-amino-3,3-dimethylbutanoyl)-N-(cyano(isoquinolin-4-yl)methyl)-5- azaspiro[2.4]heptan-6-formamide (40 mg, 0.0954 mmol, 1.0 eq.) and methyl 2-chloro-2,2-difluoroacetate (22 mg, 0.153, 1.6 eq.) were dissolved in methanol, and triethylamine (40 mg, 0.382 mmol, 4.0 eq.) was added dropwise. The reaction was stirred at room temperature overnight, and the progress of the reaction was monitored using thin layer chromatography. After the reaction was completed, the final product Hit 3-22 was purified by preparative thin layer chromatography plate using MeOH / DCM (1:20) as the developing agent. The appearance of the final product was white solid, the yield was 45%, the purity was about 99.63%, and the RT was 4.128 min. The mass spectral analysis result is shown in Figure 23. HRMS: m / z = 532.19982 [M+H] + ; 1H NMR (600 MHz, Chloroform-d) δ 9.29 (s, 1H), 8.85 (s, 1H), 8.11 - 8.00 (m, 3H), 7.98 (d, J = 8.5 Hz, 1H), 7.82 (t, J = 8.1 Hz, 1H), 7.71 (t, J = 7.9 Hz, 1H), 6.74 (s, 1H), 4.76 (dd, J = 8.1, 3.4 Hz, 1H), 4.32 (d, J = 9.4 Hz, 1H), 3.67 (d, J = 9.6 Hz, 1H), 3.22 (d, J = 9.6 Hz, 1H), 2.39 (dd, J = 12.9, 3.4 Hz, 1H), 2.14 - 2.08 (m, 2H), 0.94 - 0.84 (m, 2H), 0.73 - 0.68 (m, 2H), 0.43 (s, 9H).

[0330] Example 12 - Synthesis of (6S)-N-(cyano(isoquinolin-4-yl)methyl)-5-((S)-3,3- dimethyl-2-(2,2,2-trichloroacetamido)butanoyl)-5-azaspiro[2.4]heptane-6-carboxamide (Hit 3-23)

[0331] In the synthesis route of the main protease inhibitor Hit 3-23 of this example, step (3) is specifically as follows:

[0332] (6S)-5-((S)-2-amino-3,3-dimethylbutanoyl)-N-(cyano(isoquinolin-4-yl)methyl)-5- azaspiro[2.4]heptane-6-carboxamide (40 mg, 0.0954 mmol, 1.0 eq.) and ethyl trichloroacetate (30 mg, 0.153, 1.6 eq.) were dissolved in methanol, triethylamine (40 mg, 0.382 mmol, 4.0 eq.) was added dropwise, and the reaction was stirred at room temperature overnight, with thin layer chromatography monitoring the progress of the reaction; after the reaction was completed, the final product Hit 3-23 was obtained by purification on a preparative thin layer chromatography plate with MeOH / DCM (1:20) as the developing agent, and the appearance was a white solid, the yield was 34%, the purity was about 98.50%, and the RT = 4.406 min; HRMS: m / z = 564.14137 [M+H] + .

[0333] Example 13 - Synthesis of (6S)-N-(cyano(isoquinolin-4-yl)methyl)-5-((S)-2-(2-(3,4- dichlorophenyl)acetamido)-3,3-dimethylbutanoyl)-5-azaspiro[2.4]heptane-6-carboxamide (Hit 3-17)

[0334] In the synthetic route of the main protease inhibitor Hit 3-17 of the present example, step (3) is specifically as follows:

[0335] (6S)-5-((S)-2-amino-3,3-dimethylbutanoyl)-N-(cyano(isoquinolin-4-yl)methyl)-5- azaspiro[2.4]heptane-6-carboxamide (30 mg, 0.0716 mmol, 1.0 eq.) and 2-(3,4- dichlorophenyl)acetic acid (17 mg, 0.0787, 1.1 eq.) were dissolved in DCM, EDCI (16 mg, 0.0787 mmol, 1.1 eq.) and DMAP (9 mg, 0.0716 mmol, 1.0 eq.) were added, and the reaction was stirred at room temperature overnight, with TLC monitoring of the reaction progress; after the reaction was completed, the final product Hit 3-17 (see Figure 25 for the mass spectral analysis result) was obtained by purification on a preparative thin layer chromatography plate with MeOH / DCM (1:20) as the developing agent, and had the appearance of a yellow solid, with a yield of 61% and a purity of about 98.86%, RT = 4.508 min; HRMS: m / z = 606.21196 [M+H] + .

[0336] Example 14 - Synthesis of (6S)-N-(cyano(isoquinolin-4-yl)methyl)-5-((S)-2-(((3,4- dichlorophenyl)methyl)sulfonamido)-3,3-dimethylbutanoyl)-5-azaspiro[2.4]heptane-6- carboxamide (Hit 3-15)

[0337] In the synthetic route of the main protease inhibitor Hit 3-15 of the present example, step (3) is specifically as follows:

[0338] (6S)-5-((S)-2-amino-3,3-dimethylbutanoyl)-N-(cyano(isoquinolin-4-yl)methyl)-5- azaspiro[2.4]heptane-6-carboxamide (31 mg, 0.0740 mmol, 1.1 eq.) and (3,4- dichlorophenyl)methanesulfonyl chloride (18 mg, 0.0673, 1.0 eq.) were dissolved in DCM, DMAP (2 mg, 0.0135 mmol, 0.2 eq.) and triethylamine (7 mg, 0.0673 mmol, 1.0 eq.) were added, and the reaction was stirred at room temperature overnight under nitrogen protection, with TLC monitoring of the reaction progress; after the reaction was completed, the final product Hit 3-15 (see Figure 26 for the mass spectral analysis result) was obtained by purification on a preparative thin layer chromatography plate with MeOH / DCM (1:20) as the developing agent, and had the appearance of a light yellow solid, with a yield of 63% and a purity of about 97.71%, RT = 4.597 min; HRMS: m / z = 642.17884 [M+H] + . 1H NMR (600 MHz, Chloroform-d) δ 9.23 (s, 1H), 8.89 (s, 1H), 8.44 (d, J = 8.9 Hz, 1H), 8.01 (dd, J = 8.3, 4.6 Hz, 2H), 7.79 (t, J = 7.7 Hz, 1H), 7.68 (t, J = 7.6 Hz, 1H), 7.45 - 7.37 (m, 3H), 7.16 (dd, J = 8.3, 2.1 Hz, 1H), 5.55 (d, J = 9.6 Hz, 1H), 4.77 (dd, J = 8.2, 3.9 Hz, 1H), 4.11 - 4.02 (m, 2H), 3.63 (d, J = 9.6 Hz, 1H), 3.37 (d, J = 9.5 Hz, 1H), 3.18 (d, J = 9.6 Hz, 1H), 2.38 (dd, J = 12.7, 4.0 Hz, 1H), 2.07 (dd, J = 12.8, 8.3 Hz, 1H), 0.91 - 0.77 (m, 4H), 0.42 (s, 9H).

[0339] Example 15 Synthesis of (6S)-N-(cyano(isoquinolin-4-yl)methyl)-5-((S)-2-(1-(3,4- dichlorophenyl)cyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-5-azaspiro[2.4]heptane- 6-carboxamide (Hit 3-18)

[0340] In the synthetic route of the main protease inhibitor Hit 3-18 of this example, step (3) is specifically as follows:

[0341] (6S)-5-((S)-2-amino-3,3-dimethylbutanoyl)-N-(cyano(isoquinolin-4-yl)methyl)-5- azaspiro[2.4]heptane-6-carboxamide (30 mg, 0.0716 mmol, 1.0 eq.) and 1-(3,4- dichlorophenyl)cyclopropane-1-carboxylic acid (19 mg, 0.0787, 1.1 eq.) were dissolved in DCM, EDCI (16 mg, 0.0787 mmol, 1.1 eq.) and DMAP (9 mg, 0.0716 mmol, 1.0 eq.) were added, and the reaction was stirred at room temperature overnight, with TLC monitoring of the reaction progress; after the reaction was completed, the final product Hit 3-18 was obtained by purification on a preparative TLC plate with MeOH / DCM (1:20) as the developing agent, and the appearance was yellow solid, the yield was 73%, the purity was about 99.13%, and the RT = 4.853 min; HRMS: m / z = 632.22742 [M+H] + .

[0342] Example 16 - Synthesis of (6S)-N-(cyano(isoquinolin-4-yl)methyl)-5-((S)-3,3- dimethyl-2-(l-(3-(trifluoromethyl)phenyl)cyclopropane-l-formamido)butanoyl)-5- azaspiro[2.4]heptane-6-carboxamide (Hit 3-19)

[0343] In the synthetic route of the main protease inhibitor Hit 3-19 of this example, step (3) is specifically:

[0344] (6S)-5-((S)-2-amino-3,3-dimethylbutanoyl)-N-(cyano(isoquinolin-4-yl)methyl)-5- azaspiro[2.4]heptane-6-carboxamide (30 mg, 0.0716 mmol, 1.0 eq.) and l-(3- (trifluoromethyl)phenyl)cyclopropane-l-carboxylic acid (20 mg, 0.0787, 1.1 eq.) were dissolved in DCM, EDCI (16 mg, 0.0787 mmol, 1.1 eq.) and DMAP (9 mg, 0.0716 mmol, 1.0 eq.) were added, and the reaction was stirred at room temperature overnight, with TLC monitoring of the reaction progress; after the reaction was completed, the final product Hit 3-19 was obtained by purification on a preparative TLC plate using MeOH / DCM (1:20) as the developing agent, and had a mass spectral analysis result shown in Figure 28; it was a yellow solid with a yield of 75% and a purity of about 96.60%, and had an RT = 4.732 min; HRMS: m / z = 632.29300 [M+H] + .

[0345] Example 17 - Synthesis of (6S)-N-(cyano(isoquinolin-4-yl)methyl)-5-((2S)-2-(2,2- difluoro-l-phenylcyclopropane-l-formamido)-3,3-dimethylbutanoyl)-5-azaspiro[2.4]heptane- 6-carboxamide (Hit 3-20)

[0346] In the synthetic route of the main protease inhibitor Hit 3-20 of this example, step (3) is specifically:

[0347] (6S)-5-((S)-2-amino-3,3-dimethylbutanoyl)-N-(cyano(isoquinolin-4-yl)methyl)-5- azaspiro[2.4]heptane-6-carboxamide (30 mg, 0.0716 mmol, 1.0 eq.) and 2,2-difluoro-1- phenylcyclopropane-1 -carboxylic acid (16 mg, 0.0787, 1.1 eq.) were dissolved in DCM, EDCI (16 mg, 0.0787 mmol, 1.1 eq.) and DMAP (9 mg, 0.0716 mmol, 1.0 eq.) were added, the reaction was stirred at room temperature overnight, the reaction progress was monitored using thin layer chromatography; after the reaction was completed, the final product Hit 3-20 (see Figure 29 for mass spectral analysis results) was obtained by purification on a preparative thin layer chromatography plate with MeOH / DCM (1 :20) as the developing agent, it was a yellow solid with a yield of 77% and a purity of about 97.91%, RT = 4.442 min; HRMS: m / z = 600.28617 [M+H] + .

[0348] Example 18 - Synthesis of (6S)-N-(cyano(isoquinolin-4-yl)methyl)-5-((S)-2-(1-(3,4- dichlorophenyl)cyclobutane-1-carboxamido)-3,3-dimethylbutanoyl)-5-azaspiro[2.4]heptane-6- carboxamide (Hit 3-21)

[0349] In the synthesis route of the main protease inhibitor Hit 3-21 of this example, step (3) is in particular:

[0350] (6S)-5-((S)-2-amino-3,3-dimethylbutanoyl)-N-(cyano(isoquinolin-4-yl)methyl)-5- azaspiro[2.4]heptane-6-carboxamide (30 mg, 0.0716 mmol, 1.0 eq.) and 1-(3,4- dichlorophenyl)cyclobutane-1-carboxylic acid (20 mg, 0.0787, 1.1 eq.) were dissolved in DCM, EDCI (16 mg, 0.0787 mmol, 1.1 eq.) and DMAP (9 mg, 0.0716 mmol, 1.0 eq.) were added, the reaction was stirred at room temperature overnight, the reaction progress was monitored using thin layer chromatography; after the reaction was completed, the final product Hit 3-21 (see Figure 30 for mass spectral analysis results) was obtained by purification on a preparative thin layer chromatography plate with MeOH / DCM (1 :20) as the developing agent, it was a yellow solid with a yield of 76% and a purity of about 96.79%, RT = 4.989 min; HRMS: m / z = 646.24303 [M+H] + .

[0351] Example 19 - Synthesis of (6S)-N-(cyano(isoquinolin-4-yl)methyl)-5-((S)-2- (cyclopropanecarboxamido)-3,3-dimethylbutanoyl)-5-azaspiro[2.4]heptane-6- carboxamide (Hit 3-24)

[0352] In the synthesis route of the main protease inhibitor Hit 3-24 of this example, step (3) is specifically:

[0353] (6S)-5-((S)-2-amino-3,3-dimethylbutanoyl)-N-(cyano(isoquinolin-4-yl)methyl)-5- azaspiro[2.4]heptane-6-carboxamide (40 mg, 0.0954 mmol, 1.0 eq.) and cyclopropane carboxylic acid (10 mg, 0.105, 1.1 eq.) were dissolved in DCM, EDCI (22 mg, 0.105 mmol, 1.1 eq.) and DMAP (12 mg, 0.0954 mmol, 1.0 eq.) were added, and the reaction was stirred at room temperature overnight, with TLC monitoring of the reaction progress; after the reaction was completed, the final product Hit 3-24 was obtained by purification on a preparative TLC plate with MeOH / DCM (1:20) as the developing agent, and the appearance was a yellow solid, with a yield of 73%, a purity of about 95.68%, and an RT = 3.594 min; HRMS: m / z = 488.27203 [M+H] + ; 1 H NMR (600 MHz, Chloroform-d) δ 9.29 (s, 1H), 8.84 (s, 1H), 8.38 (d, J = 8.8 Hz, 1H), 8.06 (dd, J = 18.5, 8.1 Hz, 2H), 7.98 (d, J = 8.5 Hz, 1H), 7.81 (t, J = 8.4, 6.9, 1.4 Hz, 1H), 7.70 (t, J = 7.6 Hz, 1H), 6.73 (d, J = 8.8 Hz, 1H), 6.23 (d, J = 9.4 Hz, 1H), 4.76 (dd, J = 8.2, 2.9 Hz, 1H), 4.35 (d, J = 9.4 Hz, 1H), 3.77 (d, J = 9.7 Hz, 1H), 3.12 (d, J = 9.7 Hz, 1H), 2.33 (dd, J = 12.7, 3.1 Hz, 1H), 2.27 - 2.17 (m, 1H), 0.74 - 0.58 (m, 8H), 0.37 (s, 9H).

[0354] Example 20 - Synthesis of (6S)-N-(cyano(isoquinolin-4-yl)methyl)-5-((2S)-2-(2,2- difluorocyclopropane-1 -carboxamido)-3,3-dimethylbutanoyl)-5-azaspiro[2.4]heptane-6- carboxamide (Hit 3-25)

[0355] In the synthetic route of the main protease inhibitor Hit 3-25 of this example, step (3) is specifically:

[0356] (6S)-5-((S)-2-amino-3,3-dimethylbutanoyl)-N-(cyano(isoquinolin-4-yl)methyl)-5- azaspiro[2.4]heptane-6-carboxamide (40 mg, 0.0954 mmol, 1.0 eq.) and 2,2- difluorocyclopropane-1 -carboxylic acid (13 mg, 0.105, 1.1 eq.) were dissolved in DCM, EDCI (22 mg, 0.105 mmol, 1.1 eq.) and DMAP (12 mg, 0.0954 mmol, 1.0 eq.) were added, and the reaction was stirred at room temperature overnight, with TLC monitoring of the reaction progress; after the reaction was completed, the final product Hit 3-25 (see Figure 32 for mass spectral analysis results) was obtained by purification on a preparative TLC plate using MeOH / DCM (1 :20) as the developing agent, and appeared as a light yellow solid with a yield of 98% and a purity of about 98.67%, RT = 3.781 min; HRMS: m / z = 524.25396 [M+H] + .

[0357] Example 21 - Synthesis of (6S)-N-(cyano(isoquinolin-4-yl)methyl)-5-((S)-2-(1- cyano cyclopropane-1 -carboxamido)-3,3-dimethylbutanoyl)-5-azaspiro[2.4]heptane-6- carboxamide (Hit 3-26)

[0358] In the synthetic route of the main protease inhibitor Hit 3-26 of this example, step (3) is specifically:

[0359] (6S)-5-((S)-2-amino-3,3-dimethylbutanoyl)-N-(cyano(isoquinolin-4-yl)methyl)-5- azaspiro[2.4]heptane-6-carboxamide (40 mg, 0.0954 mmol, 1.0 eq.) and 1- cyanocyclopropane-1-carboxylic acid (12 mg, 0.105, 1.1 eq.) were dissolved in DCM, EDCI (22 mg, 0.105 mmol, 1.1 eq.) and DMAP (12 mg, 0.0954 mmol, 1.0 eq.) were added, the reaction was stirred at room temperature overnight, the reaction progress was monitored using thin layer chromatography; after the reaction was completed, the final product Hit 3-26 (see Figure 33 for mass spectral analysis results) was obtained by purification on a preparative thin layer chromatography plate with MeOH / DCM (1:20) as the developing agent, it was a light yellow solid with a yield of 55% and a purity of about 91.86%, RT = 3.861 min; HRMS: m / z = 513.26923 [M+H] + .

[0360] Example 22 - Synthesis of (6S)-N-(cyano(isoquinolin-4-yl)methyl)-5-((S)-3,3-dimethyl- 2-(1-(trifluoromethyl)cyclopropane-1-carboxamido)butanoyl)-5-azaspiro[2.4]heptane-6- carboxamide (Hit 3-27)

[0361] In the synthesis route of the main protease inhibitor Hit 3-27 of this example, step (3) is specifically:

[0362] (6S)-5-((S)-2-amino-3,3-dimethylbutanoyl)-N-(cyano(isoquinolin-4-yl)methyl)-5- azaspiro[2.4]heptane-6-carboxamide (40 mg, 0.0954 mmol, 1.0 eq.) and 1- (trifluoromethyl)cyclopropane-1-carboxylic acid (17 mg, 0.105, 1.1 eq.) were dissolved in DCM, EDCI (22 mg, 0.105 mmol, 1.1 eq.) and DMAP (12 mg, 0.0954 mmol, 1.0 eq.) were added, the reaction was stirred at room temperature overnight, the reaction progress was monitored using thin layer chromatography; after the reaction was completed, the final product Hit 3-27 (see Figure 34 for mass spectral analysis results) was obtained by purification on a preparative thin layer chromatography plate with MeOH / DCM (1:20) as the developing agent, it was a light yellow solid with a yield of 65% and a purity of about 99.31%, RT = 4.238 min; HRMS: m / z = 556.26093 [M+H] + .

[0363] Example 23 - Synthesis of (6S)-N-(cyano(isoquinolin-4-yl)methyl)-5-((S)-2-(3,3- difluoro-1-(trifluoromethyl)cyclobutane-1-carboxamido)-3,3-dimethylbutanoyl)-5- azaspiro[2.4]heptane-6-carboxamide (Hit 3-28)

[0364] In the synthetic route of the main protease inhibitor Hit 3-28 of this example, step (3) is specifically as follows:

[0365] (6S)-5-((S)-2-amino-3,3-dimethylbutanoyl)-N-(cyano(isoquinolin-4-yl)methyl)-5- azaspiro[2.4]heptane-6-carboxamide (40 mg, 0.0954 mmol, 1.0 eq.) and 3,3-difluoro-1- (trifluoromethyl)cyclobutane-1-carboxylic acid (22 mg, 0.105, 1.1 eq.) were dissolved in DCM, EDCI (22 mg, 0.105 mmol, 1.1 eq.) and DMAP (12 mg, 0.0954 mmol, 1.0 eq.) were added, and the reaction was stirred at room temperature overnight, with TLC monitoring of the reaction progress; after the reaction was completed, the final product Hit 3-28 was obtained by purification on a preparative TLC plate with MeOH / DCM (1:20) as the developing agent, and the appearance was a light yellow solid, the yield was 51%, the purity was about 98.76%, and the RT was 4.457 min; HRMS: m / z = 606.26005 [M+H] + .

[0366] Examples 24-26 - Synthesis of Hit 3-9, 3-30-3-31

[0367] The structural formulas of the main protease inhibitors Hit 3-9, 3-30-3-31 are shown in Table 2.

[0368] Table 2

[0369] As can be seen from Table 2, the main protease inhibitors Hit 3-9, 3-30-3-31 have the same skeleton, only the structure of group A is different, and even only the difference between the substituents of group A, so the synthetic routes are similar.

[0370] The general synthetic route of Hit 3-9, 3-30-3-31 is shown in Figure 36, which specifically includes the following steps:

[0371] (1) (tert-butoxycarbonyl)-L-leucine (274 mg, 1.18 mmol, 1.1 eq.) and 2-amino-2-(isoquinolin-4-yl)acetonitrile (197 mg, 1.08 mmol, 1.0 eq.) were dissolved in DCM, EDCI (227 mg, 1.18 mmol, 1.1 eq.) and DMAP (53 mg, 0.430 mmol, 0.4 eq.) were added, and the reaction was stirred at room temperature overnight, with TLC monitoring of the reaction progress; after the reaction was completed, the intermediate tert-butyl ((2S)-1-((cyano(isoquinolin-4-yl)methyl)amino)-4-methyl-1-oxopentan-2-yl)carbamate was obtained by silica gel column chromatography with MeOH / DCM (1:10) as the developing agent, with a yield of 67%;

[0372] (2) The intermediate was dissolved in 5 mL of DCM, 5 mL of a hydrochloric acid solution in dioxane (4 M, 15.0 eq.) was added dropwise with stirring, and the reaction was stirred at room temperature for about 2 h; after the reaction was completed, the reaction solution was rotary evaporated under reduced pressure, the product was extracted with DCM, and washed with saturated aqueous sodium bicarbonate and saturated brine; the organic layer was dried over anhydrous sodium sulfate and concentrated and rotary evaporated to obtain the intermediate (2S)-2-amino-N-(cyano(isoquinolin-4-yl)methyl)-4-methylpentanamide, with a yield of 95%;

[0373] (3) According to the structure of the final product, an appropriate amount of (2S)-2-amino-N-(cyano(isoquinolin-4-yl)methyl)-4-methylpentanamide was weighed and subjected to amide condensation reaction with different carboxylates (RCOOH) under the catalysis of EDCI and DMAP to obtain the target product.

[0374] Hereinafter, step (3) in the synthesis route of Hit 3-9, 3-30-3-31 will be described in detail in sequence, and steps (1) and (2) are common.

[0375] Example 24 - Synthesis of N-((2S)-1-((cyano(isoquinolin-4-yl)methyl)amino)-4-methyl-1-oxopentan-2-yl)-4-methoxy-1H-indole-2-carboxamide (Hit 3-9)

[0376] In the synthesis route of the main protease inhibitor Hit 3-9 of this example, step (3) is specifically:

[0377] (2S)-2-amino-N-(cyano(isoquinolin-4-yl)methyl)-4-methylpentanamide (50 mg, 0.169 mmol, 1.0 eq.) and 4-methoxy-1H-indole-2-carboxylic acid (36 mg, 0.186, 1.1 eq.) were dissolved in DCM, EDCI (36 mg, 0.186 mmol, 1.1 eq.) and DMAP (20 mg, 0.169 mmol, 1.0 eq.) were added, the reaction was stirred at room temperature overnight, the reaction progress was monitored using thin layer chromatography; after the reaction was completed, the final product Hit 3-9 (see Figure 37 for mass spectral analysis results) was obtained by purification on a preparative thin layer chromatography plate using MeOH / DCM (1 :20) as the developing agent, it was a dark yellow solid with a yield of 33% and a purity of about 92.35%, RT = 3.968 min; HRMS: m / z = 470.21938 [M+H] + .

[0378] Example 25 - Synthesis of N-((2S)-1-((cyano(isoquinolin-4-yl)methyl)amino)-4- methyl-1-oxopentan-2-yl)-5-(trifluoromethoxy)-1H-indole-2-carboxamide (Hit 3-30)

[0379] In the synthesis route of the main protease inhibitor Hit 3-30 of this example, step (3) is specifically:

[0380] (2S)-2-amino-N-(cyano(isoquinolin-4-yl)methyl)-4-methylpentanamide (50 mg, 0.169 mmol, 1.0 eq.) and 5-(trifluoromethoxy)-1H-indole-2-carboxylic acid (46 mg, 0.186, 1.1 eq.) were dissolved in DCM, EDCI (36 mg, 0.186 mmol, 1.1 eq.) and DMAP (20 mg, 0.169 mmol, 1.0 eq.) were added, the reaction was stirred at room temperature overnight, the reaction progress was monitored using thin layer chromatography; after the reaction was completed, the final product Hit 3-30 (see Figure 38 for mass spectral analysis results) was obtained by purification on a preparative thin layer chromatography plate using MeOH / DCM (1 :20) as the developing agent, it was a yellow-brown solid with a yield of 28% and a purity of about 97.63%, RT = 4.503 min; HRMS: m / z = 524.19302 [M+H] + .

[0381] Example 26 - Synthesis of 4-chloro-N-((2S)-1-((cyano(isoquinolin-4-yl)methyl)amino)- 4-methyl-1-oxopentan-2-yl)-1H-indole-2-carboxamide (Hit 3-31)

[0382] In the synthetic route of the main protease inhibitor Hit 3-31 of the present embodiment, step (3) is specifically as follows:

[0383] (2S)-2-amino-N-(cyano(isoquinolin-4-yl)methyl)-4-methylpentanamide (50 mg, 0.169 mmol, 1.0 eq.) and 4-chloro-1H-indole-2-carboxylic acid (37 mg, 0.186, 1.1 eq.) were dissolved in DCM, EDCI (36 mg, 0.186 mmol, 1.1 eq.) and DMAP (20 mg, 0.169 mmol, 1.0 eq.) were added, and the reaction was stirred at room temperature overnight, with TLC monitoring of the reaction progress; after the reaction was completed, the final product Hit 3-31 (see Figure 39 for the mass spectral analysis result) was obtained by purification on a preparative TLC plate with MeOH / DCM (1:20) as the developing agent, and had a yellow solid appearance, a yield of 29%, and a purity of about 93.05%, with RT = 4.277 min; HRMS: m / z = 474.17207 [M+H] + .

[0384] Examples 27-29 - Synthesis of Hits 3-32-3-34

[0385] The structural formulas of the main protease inhibitors Hits 3-32-3-34 are shown in Table 3 below.

[0386] Table 3

[0387] As can be seen from Table 3, the main protease inhibitors Hits 3-32-34 and Hit 3-36 have the same skeleton, and only the structure of R1 in group A is different, so the synthetic routes are similar; their general synthetic route is shown in Figure 40, and specifically includes the following steps:

[0388] (1) (S)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutanoic acid (2474 mg, 10.7 mmol, 1.1 eq.) and (1R,2S,5S)-methyl 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate hydrochloride (2000 mg, 9.72 mmol, 1.0 eq.) were dissolved in DCM, EDCI (2054 mg, 10.7 mmol, 1.1 eq.) and DMAP (949 mg, 7.78 mmol, 0.8 eq.) were added, the reaction was stirred at room temperature overnight, the reaction progress was monitored by thin layer chromatography; after the reaction was completed, the intermediate methyl (1R,2S,5S)-3-((S)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate was obtained by silica gel column chromatography with ethyl acetate / petroleum ether (1:5) as the developing agent, with a yield of 46%;

[0389] (2) The intermediate was dissolved in 10 mL of tetrahydrofuran (THF), 10 mL of methanol and 10 mL of aqueous lithium hydroxide solution (1 M, 3 eq.) were added dropwise with stirring, the reaction was carried out at room temperature for 48 h, after the reaction was completed, the pH of the reaction solution was adjusted to acidic by adding an appropriate amount of 1 M aqueous hydrochloric acid solution, the product was extracted with DCM, and washed with saturated brine, the organic layer was dried over anhydrous sodium sulfate and concentrated to dryness, the intermediate (1R,2S,5S)-3-((S)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid was obtained in the form of colorless transparent needle-like crystals;

[0390] (3) The intermediate was dissolved in DCM, and 1.1 eq. of 2-amino-2-(isoquinolin-4-yl)acetonitrile was added, along with EDCI and DMAP. The reaction was stirred at room temperature overnight. Purification by column chromatography on silica gel using MeOH / DCM (1 :20) as the eluent gave the intermediate tert-butyl ((2S)-1-((1R,2S,5S)-2-((cyano(isoquinolin-4-yl)methyl)amino carbonyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hex-3-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate as a white solid in 73% yield. The intermediate was dissolved in 10 mL of DCM, and 10 mL of a hydrochloric acid solution in dioxane (4 M, 15.0 eq.) was added dropwise slowly with stirring. The reaction was stirred at room temperature for about 2 h. After the reaction was completed, the reaction solution was rotary evaporated under reduced pressure. The product was extracted with DCM and washed with saturated aqueous sodium bicarbonate and saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated and rotary evaporated to give the intermediate (1R,2S,5S)-3-((S)-2-amino-3,3-dimethylbutanoyl)-N-(cyano(isoquinolin-4-yl)methyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide in 98% yield;

[0391] (4) Depending on the structure of the final product to be prepared, an appropriate amount of (1R,2S,5S)-3-((S)-2-amino-3,3-dimethylbutanoyl)-N-(cyano(isoquinolin-4-yl)methyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide and different carboxylic acid (RCOOH) were weighed out separately and subjected to amide condensation reaction under catalysis of EDCI and DMAP to obtain the target product.

[0392] The step (4) in the synthesis route of Hit 3-32~34 and Hit 3-36 will be described in detail below in sequence, and steps (1), (2) and (3) are common.

[0393] Example 27 - Synthesis of (1R,2S,5S)-3-((S)-2-(2-chloro-2,2-difluoroacetamido)-3,3-dimethylbutanoyl)-N-(cyano(isoquinolin-4-yl)methyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Hit 3-32)

[0394] In the synthesis route of the main protease inhibitor Hit 3-32 of the present example, step (4) is specifically:

[0395] (1R,2S,5S)-3-((S)-2-amino-3,3-dimethylbutanoyl)-N-(cyano(isoquinolin-4- yl)methyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (40 mg, 0.0923 mmol, 1.0 eq.) and 2-chloro-2,2-difluoroacetic acid (15 mg, 0.111, 1.2 eq.) were dissolved in DCM, EDCI (22 mg, 0.111 mmol, 1.2 eq.) and DMAP (11 mg, 0.0923 mmol, 1.0 eq.) were added, the reaction was stirred at room temperature overnight, the reaction progress was monitored using thin layer chromatography; after the reaction was completed, the final product Hit 3-32 (see Figure 41 for mass spectral analysis results) was obtained by purification on a preparative thin layer chromatography plate with MeOH / DCM (1:20) as the developing agent, it was a light yellow solid with a yield of 36% and a purity of about 98.96%, RT = 4.316 min; HRMS: m / z = 546.20875 [M+H] + .

[0396] Example 28 - Synthesis of (1R,2S,5S)-N-(cyano(isoquinolin-4-yl)methyl)-3-((2S)-2-(2,2- difluorocyclopropane-1-carboxamido)-3,3-dimethylbutanoyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Hit 3-33)

[0397] In the synthesis route of the main protease inhibitor Hit 3-33 of this example, step (4) is specifically:

[0398] (1R,2S,5S)-3-((S)-2-amino-3,3-dimethylbutanoyl)-N-(cyano(isoquinolin-4- yl)methyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (30 mg, 0.0692 mmol, 1.0 eq.) and 2,2-difluorocyclopropane-1-carboxylic acid (11 mg, 0.0830, 1.2 eq.) were dissolved in DCM, EDCI (16 mg, 0.0830 mmol, 1.2 eq.) and DMAP (9 mg, 0.0692 mmol, 1.0 eq.) were added, the reaction was stirred at room temperature overnight, the reaction progress was monitored using thin layer chromatography; after the reaction was completed, the final product Hit 3-33 (see Figure 42 for mass spectral analysis results) was obtained by purification on a preparative thin layer chromatography plate with MeOH / DCM (1:20) as the developing agent, it was a light yellow solid with a yield of 63% and a purity of about 99.63%, RT = 4.000 min; HRMS: m / z = 538.26631 [M+H] + .

[0399] Example 29 - Synthesis of (1R,2S,5S)-N-(cyano(isoquinolin-4-yl)methyl)-3-((S)-2-(3,3- difluorocyclobutane-1 -carboxamido)-3,3-dimethylbutanoyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (Hit 3-34)

[0400] In the synthesis route of the main protease inhibitor Hit 3-34 of the present example, step (4) is specifically as follows:

[0401] (1R,2S,5S)-3-((S)-2-amino-3,3-dimethylbutanoyl)-N-(cyano(isoquinolin-4-yl)methyl)- 6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (40 mg, 0.0923 mmol, 1.0 eq.) and 3,3-difluorocyclobutane-1 -carboxylic acid (16 mg, 0.111, 1.2 eq.) were dissolved in DCM, EDCI (22 mg, 0.111 mmol, 1.2 eq.) and DMAP (11 mg, 0.0923 mmol, 1.0 eq.) were added, and the reaction was stirred at room temperature overnight, with TLC monitoring of the reaction progress; after the reaction was completed, the final product Hit 3-34 (see Figure 43 for the mass spectral analysis result) was obtained by purification on a preparative thin layer chromatography plate with MeOH / DCM (1 :20) as the developing agent, and was a light yellow solid with a yield of 37% and a purity of about 99.62%, RT = 4.112 min; HRMS: m / z = 552.27931 [M+H] + .

[0402] Example 30 - Synthesis of (1R,2S,5S)-3-((S)-2-(2-bromo-2,2-difluoroacetamido)-3,3- dimethylbutanoyl)-N-(cyano(isoquinolin-4-yl)methyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide (Hit 3-36)

[0403] In the synthesis route of the main protease inhibitor Hit 3-36 of the present example, step (4) is specifically as follows:

[0404] (1R,2S,5S)-3-((S)-2-amino-3,3-dimethylbutanoyl)-N-(cyano(isoquinolin-4- yl)methyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (86 mg, 0.198 mmol, 1.0 eq.) and 2-bromo-2,2-difluoroacetic acid (42 mg, 0.238 mmol, 1.2 eq.) were dissolved in DCM, EDCI (46 mg, 0.238 mmol, 1.2 eq.) and DMAP (10 mg, 0.0793 mmol, 0.4 eq.) were added, the reaction was stirred at room temperature overnight, the reaction progress was monitored by thin layer chromatography; after the reaction was completed, the final product Hit 3-36 (see Figure 44 for mass spectral analysis results) was obtained by purification on a preparative thin layer chromatography plate with MeOH / DCM (1:20) as the developing agent, it was a yellow solid with a yield of 21% and a purity of about 99.05%, RT = 4.323 min; HRMS: m / z = 590.16092; 592.15943 [M+H] + .

[0405] Examples 31-33 - Synthesis of FD7-29, FD7-1 and FD7-2

[0406] Table 4

[0407] As can be seen from Table 4, the main protease inhibitors FD7-29, 7-1-7-2 have the same skeleton, only the structure of the R1 group is different, so the synthesis routes are similar. The general synthetic route of the main protease inhibitors FD7-29, 7-1-7-2 is shown in Figure 45, which specifically includes the following steps:

[0408] (1) First, synthesize the intermediate 2-amino-2-(isoquinolin-4-yl)acetonitrile according to the method in Example 6;

[0409] (2) (2S, 4R)-1-(tert-butoxycarbonyl)-4-(trifluoromethyl)pyrrolidine-2- carboxylic acid (1000 mg, 3.53 mmol, 1.0 eq.) and 2-amino-2-(isoquinolin-4- yl)acetonitrile (776 mg, 4.24 mmol, 1.2 eq.) were dissolved in DCM, EDCI (815 mg, 4.24 mmol, 1.2 eq.) and HOPO (98 mg, 0.883 mmol, 0.25 eq.) were added, after fully dissolved, DIEA (1369 mg, 10.59 mmol, 3.0 eq.) was added dropwise, the reaction was stirred at room temperature overnight, the reaction progress was monitored by thin layer chromatography; after the reaction was completed, the intermediate tert-butyl (2S, 4R)-2-((cyano(isoquinolin-4-yl)methyl)carbamoyl)-4- (trifluoromethyl)pyrrolidine-1-carboxylate was obtained by silica gel column chromatography with MeOH / DCM (1:20) as the developing agent, the yield was 85%; the intermediate was dissolved in 10 mL DCM, 10 mL hydrochloric acid in dioxane (4M, 15.0 eq.) was added dropwise slowly under stirring, the reaction was stirred at room temperature for about 2 h, after the reaction was completed, the reaction solution was rotary evaporated under reduced pressure, the product was extracted with DCM, and washed with saturated aqueous sodium bicarbonate solution and saturated brine, the organic layer was dried over anhydrous sodium sulfate and concentrated and rotary evaporated to dryness, to obtain the intermediate (2S, 4R)-N-(cyano(isoquinolin-4-yl)methyl)-4- (trifluoromethyl)pyrrolidine-2-carboxamide, the yield was 78%;

[0410] (3) According to the structure of the final product, an appropriate amount of (2S, 4R)-N-(cyano(isoquinolin-4-yl)methyl)-4-(trifluoromethyl)pyrrolidine-2-carboxamide was weighed and subjected to condensation reaction with different substrates under the catalysis of corresponding catalysts to obtain the target product.

[0411] The specific implementation of step (3) will be described in sequence below, and steps (1) and (2) are general.

[0412] Example 31 - Synthesis of (2S, 4R)-N-(cyano(isoquinolin-4-yl)methyl)-1-((S)-3,3- dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-4-(trifluoromethyl)pyrrolidine-2- carboxamide (FD7-29)

[0413] In the synthesis route of the main protease inhibitor FD7-29 of the present example, step (3) is specifically:

[0414] (2S,4R)-N-(cyano(isoquinolin-4-yl)methyl)-4-(trifluoromethyl)pyrrolidine-2- carboxamide (100 mg, 0.287 mmol, 1.0 eq.) and (S)-3,3-dimethyl-2-(2,2,2- trifluoroacetamido)butanoic acid (98 mg, 0.431 mmol, 1.5 eq.) were dissolved in DCM, EDCI (83 mg, 0.431 mmol, 1.5 eq.) and DMAP (14 mg, 0.115 mmol, 0.4 eq.) were added, the reaction was stirred at room temperature overnight, the reaction progress was monitored using thin layer chromatography; after the reaction was completed, the final product FD7-29 (see Figure 46 for mass spectral analysis results) was obtained by purification on a preparative thin layer chromatography plate using MeOH / DCM (1 :20) as the developing agent, it was a light yellow solid with a yield of 74% and a purity of about 100%, RT = 4.360 min; HRMS: m / z = 558.14684 [M+H] + .

[0415] Example 32 - Synthesis of (2S,4R)-1-((S)-2-(2-chloro-2,2-difluoroacetamido)-3,3- dimethylbutanoyl)-N-(cyano(isoquinolin-4-yl)methyl)-4-(trifluoromethyl)pyrrolidine-2- carboxamide (FD7-1)

[0416] In the synthesis route of the main protease inhibitor FD7-1 of this example, step (3) is specifically:

[0417] (2S,4R)-N-(cyano(isoquinolin-4-yl)methyl)-4-(trifluoromethyl)pyrrolidine-2- carboxamide (100 mg, 0.287 mmol, 1.0 eq.) and (S)-2-(2-chloro-2,2-difluoroacetamido)- 3,3-dimethylbutanoic acid (105 mg, 0.431 mmol, 1.5 eq.) were dissolved in DCM, EDCI (83 mg, 0.431 mmol, 1.5 eq.) and DMAP (14 mg, 0.115 mmol, 0.4 eq.) were added, the reaction was stirred at room temperature overnight, the reaction progress was monitored using thin layer chromatography; after the reaction was completed, the final product FD7-1 (see Figure 47 for mass spectral analysis results) was obtained by purification on a preparative thin layer chromatography plate using MeOH / DCM (1 :20) as the developing agent, it was a light yellow solid with a yield of 62% and a purity of about 98.43%, RT = 4.421 min; HRMS: m / z = 574.10918 [M+H] + .

[0418] Example 33 - Synthesis of (2S,4R)-1-((S)-2-(2-bromo-2,2-difluoroacetamido)-3,3- dimethylbutanoyl)-N-(cyano(isoquinolin-4-yl)methyl)-4-(trifluoromethyl)pyrrolidine-2- carboxamide (FD7-2)

[0419] In the synthetic route of the main protease inhibitor FD7-2 of this example, step (3) is specifically as follows:

[0420] (2S,4R)-N-(cyano(isoquinolin-4-yl)methyl)-4-(trifluoromethyl)pyrrolidine-2-carboxamide (100 mg, 0.287 mmol, 1.0 eq.) and (S)-2-(2-bromo-2,2-difluoroacetamido)-3,3- dimethylbutanoic acid (124 mg, 0.431 mmol, 1.5 eq.) were dissolved in DCM, EDCI (83 mg, 0.431 mmol, 1.5 eq.) and DMAP (14 mg, 0.115 mmol, 0.4 eq.) were added, and the reaction was stirred at room temperature overnight, with TLC monitoring of the reaction progress; after the reaction was completed, the final product FD7-2 (see Figure 48 for the mass spectral analysis result) was obtained by purification on a preparative thin layer chromatography plate using MeOH / DCM (1:20) as the developing agent, and was a light yellow solid with a yield of 69% and a purity of about 98.09%, RT = 4.469 min; HRMS: m / z = 618.22563 [M+H] + .

[0421] Examples 34-39 - Synthesis of FD7-5, FD7-6 and FD7-22-7-25

[0422] Table 5

[0423] As can be seen from Table 5, the main protease inhibitors FD7-5-7-6 and FD7-22-7-25 have the same skeleton, only the structure of the R1 group is different, and therefore the synthetic routes are similar. The general synthetic route of the main protease inhibitors FD7-5-7-6 and FD7-22-7-25 is shown in Figure 49, and specifically includes the following steps:

[0424] (1) First, the intermediate 2-amino-2-(isoquinolin-4-yl)acetonitrile is synthesized according to the method in Example 6;

[0425] (2) (3S,6S)-5-(tert-butoxycarbonyl)-1,1-difluoro-5-azaspiro[2.4]heptane-6- carboxylic acid (1000 mg, 3.61 mmol, 1.0 eq.) and 2-amino-2-(isoquinolin-4- yl)acetonitrile (793 mg, 4.33 mmol, 1.2 eq.) were dissolved in DCM, EDCI (832 mg, 4.33 mmol, 1.2 eq.) and HOPO (100 mg, 0.903 mmol, 0.25 eq.) were added, after fully dissolved, DIEA (1400 mg, 10.83 mmol, 3.0 eq.) was added dropwise, the reaction was stirred at room temperature overnight, the reaction progress was monitored by thin layer chromatography; after the reaction was completed, the intermediate tert-butyl tert-butyl (3S,6S)-6-((cyano(isoquinolin-4-yl)methyl)carbonyl)-1,1-difluoro-5-azaspiro[2.4]heptane-5-carboxylate was obtained by silica gel column chromatography with MeOH / DCM (1:20) as the developing agent, the yield was 88%; the intermediate was dissolved in 10 mL DCM, 10 mL hydrochloric acid in dioxane (4M, 15.0 eq.) was added dropwise slowly under stirring, the reaction was stirred at room temperature for about 2 h, after the reaction was completed, the reaction solution was rotary evaporated under reduced pressure, the product was extracted with DCM, and washed with saturated aqueous sodium bicarbonate solution and saturated brine, the organic layer was dried over anhydrous sodium sulfate and concentrated and rotary evaporated to dryness, to obtain the intermediate (3S,6S)-N-(cyano(isoquinolin-4-yl)methyl)-1,1-difluoro-5-azaspiro[2.4]heptane-6- amide, the yield was 75%;

[0426] (3) According to the structure of the final product, an appropriate amount of (3S,6S)-N-(cyano(isoquinolin-4-yl)methyl)-1,1-difluoro-5-azaspiro[2.4]heptane-6-amide was weighed and subjected to condensation reaction with different substrates under the catalysis of corresponding catalysts to obtain the target product.

[0427] The specific implementation of step (3) will be described in sequence below, and steps (1) and (2) are general.

[0428] Example 34 - Synthesis of (3S,6S)-N-(cyano(isoquinolin-4-yl)methyl)-5-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-1,1-difluoro-5-azaspiro[2.4]heptane-6- amide (FD7-25)

[0429] In the synthesis route of the main protease inhibitor FD7-25 of this example, step (3) is specifically:

[0430] (3S,6S)-N-(cyano(isoquinolin-4-yl)methyl)-1,1-difluoro-5-azaspiro[2.4]heptane-6- carboxamide (100 mg, 0.292 mmol, 1.0 eq.) and (S)-3,3-dimethyl-2-(2,2,2- trifluoroacetamido)butanoic acid (100 mg, 0.438 mmol, 1.5 eq.) were dissolved in DCM, EDCI (84 mg, 0.438 mmol, 1.5 eq.) and DMAP (15 mg, 0.117 mmol, 0.4 eq.) were added, the reaction was stirred at room temperature overnight, the reaction progress was monitored using thin layer chromatography; after the reaction was completed, the final product FD7-25 (mass spectral analysis results are shown in Figure 50) was obtained by purification on a preparative thin layer chromatography plate with MeOH / DCM (1 :20) as the developing agent, it was a light yellow solid with a yield of 82% and a purity of about 94.60%, RT = 4.245 min; HRMS: m / z = 552.161 1 1 [M+H] + .

[0431] Example 35 - Synthesis of (3S,6S)-5-((S)-2-(2-chloro-2,2-difluoroacetamido)-3,3- dimethylbutanoyl)-N-(cyano(isoquinolin-4-yl)methyl)-1,1-difluoro-5-azaspiro[2.4]heptane- 6-carboxamide (FD7-5)

[0432] In the synthesis route of the main protease inhibitor FD7-5 of this example, step (3) is specifically as follows:

[0433] (3S,6S)-N-(cyano(isoquinolin-4-yl)methyl)-1,1-difluoro-5-azaspiro[2.4]heptane-6- carboxamide (100 mg, 0.292 mmol, 1.0 eq.) and (S)-2-(2-chloro-2,2-difluoroacetamido)- 3,3-dimethylbutanoic acid (100 mg, 0.438 mmol, 1.5 eq.) were dissolved in DCM, EDCI (106 mg, 0.438 mmol, 1.5 eq.) and DMAP (15 mg, 0.117 mmol, 0.4 eq.) were added, the reaction was stirred at room temperature overnight, the reaction progress was monitored using thin layer chromatography; after the reaction was completed, the final product FD7-5 (mass spectral analysis results are shown in Figure 51 ) was obtained by purification using a reverse-phase high-performance liquid chromatography system, it was a light yellow solid with a yield of 64% and a purity of about 100%, RT = 4.444 min; HRMS: m / z = 568.13217 [M+H] + .

[0434] Example 36 - Synthesis of (3S,6S)-5-((S)-2-(2-bromo-2,2-difluoroacetamido)-3,3- dimethylbutanoyl)-N-(cyano(isoquinolin-4-yl)methyl)-1,1-difluoro-5-azaspiro[2.4]heptane-6- amide (FD7-6)

[0435] In the synthetic route of the main protease inhibitor FD7-6 of this example, step (3) is specifically:

[0436] (3S,6S)-N-(cyano(isoquinolin-4-yl)methyl)-1,1-difluoro-5-azaspiro[2.4]heptane-6- amide (100 mg, 0.292 mmol, 1.0 eq.) and (S)-2-(2-bromo-2,2-difluoroacetamido)-3,3- dimethylbutanoic acid (126 mg, 0.438 mmol, 1.5 eq.) were dissolved in DCM, EDCI (106 mg, 0.438 mmol, 1.5 eq.) and DMAP (15 mg, 0.117 mmol, 0.4 eq.) were added, and the reaction was stirred at room temperature overnight, with the progress of the reaction monitored using thin layer chromatography; after the reaction was completed, the end product FD7-6 was purified using a reverse phase high performance liquid chromatography system (mass spectral analysis results are shown in Figure 52), and was a light yellow solid with a yield of 71% and a purity of about 100% (RT = 4.470 min); HRMS: m / z = 612.08119 [M+H] + .

[0437] Example 37 - Synthesis of methyl ((2S)-1-((3S,6S)-6-((cyano(isoquinolin-4-yl)methyl) aminocarbamoyl)-1,1-difluoro-5-azaspiro[2.4]heptan-5-yl)-3,3-dimethyl-1-oxobutan-2- yl)carbamate (FD7-24)

[0438] In the synthetic route of the main protease inhibitor FD7-24 of this example, step (3) is specifically:

[0439] (3S,6S)-N-(cyano(isoquinolin-4-yl)methyl)-1,1-difluoro-5-azaspiro[2.4]heptane-6- carboxamide (100 mg, 0.292 mmol, 1.0 eq.) and (S)-2-((methoxycarbonyl)amino)-3,3- dimethylbutanoic acid (83 mg, 0.438 mmol, 1.5 eq.) were dissolved in DCM, EDCI (106 mg, 0.438 mmol, 1.5 eq.) and DMAP (15 mg, 0.117 mmol, 0.4 eq.) were added and the reaction was stirred at room temperature overnight, the reaction progress was monitored using thin layer chromatography; after completion of the reaction, the final product FD7-24 (mass spectral analysis results shown in Figure 53) was obtained by purification on a preparative thin layer chromatography plate using MeOH / DCM (1 :20) as the developing agent, it was a light yellow solid with a yield of 72% and a purity of about 93.76%, RT = 3.806 min; HRMS: m / z = 514.18042 [M+H] + .

[0440] Example 38 - Synthesis of (3S,6S)-N-(cyano(isoquinolin-4-yl)methyl)-1,1-difluoro-5-(4- methoxy-1 H-indole-2-carbonyl)-5-azaspiro[2.4]heptane-6-carboxamide (FD7-22)

[0441] In the synthesis route of the main protease inhibitor FD7-22 of this example, step (3) is specifically as follows:

[0442] (3S,6S)-N-(cyano(isoquinolin-4-yl)methyl)-1,1-difluoro-5-azaspiro[2.4]heptane-6- carboxamide (100 mg, 0.292 mmol, 1.0 eq.) and 4-methoxy-1 H-indole-2-carboxylic acid (84 mg, 0.438 mmol, 1.5 eq.) were dissolved in DCM, EDCI (106 mg, 0.438 mmol, 1.5 eq.) and DMAP (15 mg, 0.117 mmol, 0.4 eq.) were added and the reaction was stirred at room temperature overnight, the reaction progress was monitored using thin layer chromatography; after completion of the reaction, the final product FD7-22 (mass spectral analysis results shown in Figure 54) was obtained by purification on a preparative thin layer chromatography plate using MeOH / DCM (1 :20) as the developing agent, it was a light yellow solid with a yield of 59% and a purity of about 91.05%, RT = 3.899 min; HRMS: m / z = 516.13012 [M+H] + .

[0443] Example 39 - Synthesis of (3S,6S)-N-(cyano(isoquinolin-4-yl)methyl)-5-((3,4- dichlorobenzyl)sulfonyl)-1,1-difluoro-5-azaspiro[2.4]heptane-6-carboxamide (FD7-23)

[0444] In the synthetic route of the main protease inhibitor FD7-23 of this example, step (3) is specifically as follows:

[0445] (3S,6S)-N-(cyano(isoquinolin-4-yl)methyl)-1,1-difluoro-5-azaspiro[2.4]heptane-6- carboxamide (100 mg, 0.292 mmol, 1.0 eq.), (3,4-dichlorophenyl)methanesulfonyl chloride (75 mg, 0.292 mmol, 1.0 eq.) and DMAP (15 mg, 0.117 mmol, 0.4 eq.) were dissolved in DCM, triethylamine (89 mg, 0.876 mmol, 3.0 eq.) was added dropwise, and the reaction was stirred at room temperature overnight, with the progress of the reaction being monitored using thin layer chromatography; after the reaction was completed, the end product FD7-23 was purified using a reverse phase high performance liquid chromatography system (see Figure 55 for the mass spectral analysis results), and was a light yellow solid with a yield of 55% and a purity of about 96.56%, RT = 4.566 min; HRMS: m / z = 565.02221 [M+H] + .

[0446] Example 40 - Synthesis of (3R,6S)-5-((S)-2-(2-chloro-2,2-difluoroacetamido)-3,3- dimethylbutanoyl)-N-(cyano(isoquinolin-4-yl)methyl)-1,1-difluoro-5-azaspiro[2.4]heptane- 6-carboxamide (FD7-3)

[0447] This example is a main protease inhibitor FD7-3, and its structural formula is as follows:

[0448] The synthetic route of the main protease inhibitor FD7-3 is shown in Figure 56, and specifically comprises the following steps:

[0449] (1) First, the intermediate 2-amino-2-(isoquinolin-4-yl)acetonitrile was synthesized according to the method in Example 6;

[0450] (2) (3R, 6S)-5-(tert-butoxycarbonyl)-1, 1-difluoro-5-azaspiro[2.4]heptane-6- carboxylic acid (1000 mg, 3.61 mmol, 1.0 eq.) and 2-amino-2-(isoquinolin-4- yl)acetonitrile (793 mg, 4.33 mmol, 1.2 eq.) were dissolved in DCM, EDCI (832 mg, 4.33 mmol, 1.2 eq.) and HOPO (100 mg, 0.903 mmol, 0.25 eq.) were added, after fully dissolved, DIEA (1400 mg, 10.83 mmol, 3.0 eq.) was added dropwise, the reaction was stirred at room temperature overnight, the reaction progress was monitored by thin layer chromatography; after the reaction was completed, the intermediate tert-butyl (3R, 6S)-6-((cyano(isoquinolin-4-yl)methyl)carbonyl)-1, 1-difluoro-5- azaspiro[2.4]heptane-5-carboxylate was obtained by silica gel column chromatography with MeOH / DCM (1:20) as developing agent, the yield was 79%; the intermediate was dissolved in 10 mL DCM, 10 mL hydrochloric acid in dioxane (4M, 15.0 eq.) was added dropwise slowly under stirring, the reaction was stirred at room temperature for about 2 h, after the reaction was completed, the reaction solution was rotary evaporated under reduced pressure, the product was extracted with DCM, and washed with saturated aqueous sodium bicarbonate solution and saturated brine, the organic layer was dried over anhydrous sodium sulfate and concentrated and rotary evaporated to dryness, to obtain the intermediate (3R, 6S)-N-(cyano(isoquinolin-4-yl)methyl)-1, 1-difluoro-5-azaspiro[2.4]heptane-6- carboxamide, the yield was 80%;

[0451] (3) (3R, 6S)-N-(cyano(isoquinolin-4-yl)methyl)-1, 1-difluoro-5-azaspiro[2.4]heptane-6- carboxamide (100 mg, 0.292 mmol, 1.0 eq.) and (S)-2-(2-chloro-2, 2-difluoroacetamido)-3, 3- dimethylbutanoic acid (100 mg, 0.438 mmol, 1.5 eq.) were dissolved in DCM, EDCI (106 mg, 0.438 mmol, 1.5 eq.) and DMAP (15 mg, 0.117 mmol, 0.4 eq.) were added, the reaction was stirred at room temperature overnight, the reaction progress was monitored by thin layer chromatography; after the reaction was completed, the final product FD7-3 (see Figure 57 for mass spectral analysis results) was obtained by purification using a reverse phase high performance liquid chromatography system, the appearance was a light yellow solid, the yield was 55%, the purity was about 92.09%, RT = 4.367 min; HRMS: m / z = 568.26785 [M+H] + .

[0452] Example 41 — Synthesis of (1R,5S)-3-((S)-2-(2-chloro-2,2-difluoroacetamido)-3,3- dimethylbutanoyl)-N-((1S)-1-cyano-2-(2-oxopyrrolidin-3-yl)ethyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (FD7-32)

[0453] The main protease inhibitor FD7-32 of this example has the following structural formula:

[0454] The synthetic route of the main protease inhibitor FD7-32 is shown in Figure 58, which specifically comprises the following steps:

[0455] (1R,5S)-3-((S)-2-(2-chloro-2,2-difluoroacetamido)-3,3-dimethylbutanoyl)-6,6-dimethyl- 3-azabicyclo[3.1.0]hexane-2-carboxylic acid (1000 mg, 2.63 mmol, 1.0 eq.) and (2S)-2- amino-3-(2-oxopyrrolidin-3-yl)propanenitrile (483.15 mg, 3.16 mmol, 1.2 eq.) were dissolved in DCM, EDCI (607 mg, 3.16 mmol, 1.2 eq.) and HOPO (73 mg, 0.658 mmol, 0.25 eq.) were added, after fully dissolved, DIEA (1017 mg, 7.89 mmol, 3.0 eq.) was added dropwise, and the reaction was stirred at room temperature overnight, the reaction progress was monitored by thin layer chromatography; after the reaction was completed, the end product FD7-32 (mass spectrum analysis results are shown in Figure 59) was purified by silica gel column chromatography with MeOH / DCM (1:20) as the developing agent, which was a white solid with a yield of 92% and a purity of about 99.03%, RT = 4.551 min; HRMS: m / z = 516.17266 [M+H] + .

[0456] Example 42 — Determination of inhibition constants (K i ) of each main protease inhibitor on coronavirus main protease

[0457] The main protease of the new coronavirus (SARS-CoV-2 Mpro) was used as the test object, and the main protease inhibitors synthesized in Examples 1-41 and the positive controls Nirmatrelvir and Ibuzatrelvir were tested for their inhibitory activity on coronavirus main protease, and the test method was as follows:

[0458] The main protease is added in a 96-well plate to make the final concentration of the main protease 100 nM, and a series of concentration gradients of the main protease inhibitor to be tested are added (the concentration gradient is adjusted according to the activity of each main protease inhibitor, 4-fold dilution), 1 μl of the fluorescent polypeptide substrate with a concentration of 1 mM is added to each well after incubation at 37 °C for 5 min, and then immediately placed in a Tecan enzyme reader controlled at 37 °C, with the excitation wave set at 340 nm and the emission wave set at 490 nm, and the fluorescence value (RFU) of each well is dynamically detected with time, reading once every 30 s, a total of 120 times, and finally the enzyme activity kinetic curve of each well can be measured, and the slope (Slope) of the linear region of the initial time period of the curve is used to represent the enzyme activity in each well, and the greater the slope, the stronger the enzyme catalytic activity; and the inhibition activity of the main protease inhibitor to be tested on the main protease is quantified by the following formula: Enzyme activity percentage (%) = (Slope 实验孔 -Slope 溶剂背景孔 ) / (Slope 对照孔 -Slope 溶剂背景孔 );

[0459] In the formula, the control well refers to the well containing only buffer, main protease and substrate but no drug; the experimental well refers to the well containing buffer, main protease, substrate and drug; and the solvent background well refers to the well containing only buffer and substrate.

[0460] The enzyme activity percentage is used as the vertical coordinate, and the concentration of the drug to be tested is used as the horizontal coordinate, and the GraphPad software is used to plot the inhibition curve of the drug to be tested on the main protease, and the Morrison equation is used for fitting to measure the K i value of the main protease inhibitor to be tested on the enzyme, and the test results are shown in Table 6. The lower the K i value, the stronger the inhibition activity of the main protease inhibitor to be tested on the enzyme.

[0461] Table 6

[0462] From the test data in Table 6, it can be seen that the main protease inhibitors prepared in Examples 1-41 exhibit inhibition activity on the main protease to different extents. Among them, Hit 3-6, Hit 3-22, Hit 3-24, Hit 3-25, Hit 3-32, Hit 3-33, Hit 3-34, Hit 3-36, FD7-1, FD7-2, FD7-5, FD7-6, FD7-24, FD7-25 and FD7-32 have stronger inhibition activity on the main protease than other main protease inhibitors, and the K i values of Hit 3-6, Hit 3-22, Hit 3-24, Hit 3-25, Hit 3-33, Hit 3-36, FD7-1, FD7-24 and FD7-25 are all less than 100 nM level, which is comparable to the positive control Nirmatrelvir and significantly better than the positive control Ibuzatrelvir; while the K i values of Hit 3-32, Hit 3-34, FD7-2, FD7-5, FD7-6 and FD7-32 are all at the level of 10 -1 nM or below, even 10 -3 nM level, which is significantly better than the positive controls Nirmatrelvir and Ibuzatrelvir.

[0463] In addition, it can be seen that Hit 3-1, Hit 3-2, Hit 3-3, Hit 3-4, Hit 3-5 and Hit 3-6 have basically the same structural skeleton, and the only difference is that the group C is different; wherein the group C on Hit 3-1~3-4 is -CH2- unsubstituted or substituted pyridine ring, the group C on Hit 3-5 is pyridine ring, and the group C on Hit 3-6 is quinoline ring. It can be seen that the inhibition activity of Hit 3-5 on main protease is improved by two orders of magnitude compared with Hit 3-1~3-4, indicating that the presence of methylene in group C will affect the binding of the compound to the main protease. However, although Hit 3-5 also shows good inhibition activity on the main protease, it is still inferior to Hit 3-6. It shows that quinoline ring is easier to bind to the substrate recognition pocket of the main protease than pyridine ring.

[0464] It can also be seen from Tables 3 and 6 that Hit 3-6, Hit 3-32, Hit 3-33, Hit 3-34 and Hit 3-36 have basically the same structural skeleton, and the only difference is that R1 is different, but the K i values of the four main protease inhibitors on the main protease are different; and FD7-1, FD7-2 and FD7-29 also have basically the same structural skeleton, and the only difference is that R1 is different, but the K iThe values are significantly lower than those of FD7-29, that is, the inhibitory activity on the main protease is significantly better than that of FD7-29; similarly, the inhibitory activity of FD7-5 and FD7-6 is better than that of FD7-25, the inhibitory activity of FD7-32 is better than that of nirmatrelvir, and the inhibitory activity of Hit 3-22 is better than that of Hit 3-11. It is shown that not only the change of group A will affect the inhibitory activity of the main protease inhibitor, but also R1 in group A will affect the inhibitory activity of the main protease inhibitor. When R1 is replaced by trifluoro instead of perfluoro halogen substitution, the inhibitory activity of the main protease inhibitor is significantly improved, especially difluoro-monochloro substitution or difluoro-monobromo substitution. In addition, Hit 3-22, Hit 3-32 and FD7-1 also have basically the same structural skeleton, the only difference is the B ring, but the inhibitory activity of Hit 3-32 and FD7-1 on the main protease is significantly better than that of Hit 3-22; and Hit 3-6 and FD7-25 also have basically the same structural skeleton, the only difference is the B ring, but the inhibitory activity of FD7-25 on the main protease is better than that of Hit 3-6, which shows that the B ring also contributes to the inhibitory activity of the main protease inhibitor.

[0465] Example 43 - Cytotoxicity test of the main protease inhibitors of the present application

[0466] In this example, the cytotoxicity of Hit 3-6, Hit 3-11, Hit 3-22, Hit 3-24, Hit 3-25, Hit 3-32, Hit 3-33 and Hit 3-34 was tested using human liver cell line THLE-2, and the test method was as follows:

[0467] The human liver cell line THLE-2 was recovered and cultured, and THLE-2 cells were seeded into a transparent 96-well plate at a cell density of 5×10 3 cells / well; after 24h of adhesion at 37℃, different concentrations of the tested drugs were added; at the same time, negative control wells and zero adjustment wells were set, wherein the negative control wells were wells without drugs and containing only cells, and the zero adjustment wells were wells containing only culture medium; the plate was incubated in a 37℃ incubator for 24h; finally, 10μl of WST reagent was added to each well, and after incubation at 37℃ for 60min, the plate was placed in a microplate reader to detect the OD value at 460nm wavelength, and the higher the OD value, the more the number of surviving cells, and the cell survival rate was calculated according to the following formula:

[0468] Cell survival rate (%) = (OD 实验孔 - OD 调零孔 ) / (OD 阴性对照孔 - OD 调零孔 ).

[0469] The test results are shown in Figure 60.

[0470] As shown in FIG. 60, Hit 3-6, Hit 3-11, Hit 3-22, Hit 3-24, Hit 3-25, Hit 3-32, Hit 3-33 and Hit 3-34 all showed no cytotoxicity at 50 mM, indicating that these main protease inhibitors have high biological safety.

[0471] Example 44 - Detection of stability of main protease inhibitors of the application to liver phase I metabolism

[0472] In this example, mouse liver microsomes were used as the test object to detect the tolerance of Hit 3-2, Hit 3-3, Hit 3-6, Hit 3-32, Hit 3-33 and Ht3-34, Nirmatrelvir and the positive control Testosterone to the phase I metabolic reaction mediated by rat liver microsomes. The test method is as follows:

[0473] According to the experimental steps described in the instructions for use of the phase I metabolism stability kit (Beijing Huizhiheyuan Biotechnology Co., Ltd.), liver microsomes and NADPH were sequentially added to the main protease inhibitors to be tested, which were placed in a 37°C water bath, and samples were taken at specific time points. The remaining main protease inhibitors at each time point were quantified using a liquid chromatography-mass spectrometry (LC-MS) system. The peak area was used as the calculation data. The zero-time concentration of the drug to be tested was taken as 100%. The concentration at each time point was compared with the zero-time concentration to obtain the remaining percentage of the main protease inhibitor. The degradation curve of the main protease inhibitor to be tested was obtained by plotting the time. By comparing the degradation curves, the tolerance of each drug to be tested to liver microsomal phase I metabolism was compared. The test results are shown in FIG. 61.

[0474] As shown in FIG. 61, after 60 min of degradation, the effective amount (the part not degraded) of Nirmatrelvir decreased to about 60%, while the effective amount of Hit 3-2 and Hit 3-3 remained above 90%. After 180 min of degradation, the effective amount of Nirmatrelvir decreased to about 10%, while the effective amount of Hit 3-6 was about 40%, the effective amount of Hit 3-32 and Hit 3-34 was about 50%, and the effective amount of Hit 3-33 was about 70%. It can be seen that compared with Nirmatrelvir, the main protease inhibitors of the application have stronger tolerance to liver microsomal phase I metabolism and are less susceptible to liver metabolism.

[0475] Example 45 - Determination of the inhibition constant (K proM49L / E166A ) of main protease inhibitors of the application to coronavirus main protease drug-resistant mutant M i

[0476] ​The present example is based on a main protease drug-resistant mutant M proM49L / E166A As test objects, Hit 3-4, Hit 3-5, Hit 3-6, Hit 3-32, Hit 3-33 and Hit 3-34 and the positive control Nirmatrelvir were tested for their inhibitory activity against the main protease of the coronavirus, and the test method was as follows:

[0477] The main protease was added in a 96-well plate to a final concentration of 200 nM, and a series of concentration gradients of the main protease inhibitor to be tested were added (the concentration gradient was adjusted according to the activity of each main protease inhibitor, 4-fold dilution), and 1 μl of a fluorescent polypeptide substrate with a concentration of 1 mM was added to each well after incubation at 37°C for 5 min, and immediately placed in a Tecan enzyme reader controlled at 37°C, with an excitation wave of 340 nm and an emission wave of 490 nm, and the fluorescence value (RFU) of each well was dynamically detected over time, with one reading every 30 s, for a total of 120 readings. The final enzyme activity kinetic curve of each well could be measured, and the slope (Slope) of the linear region of the initial time period of the curve represented the enzyme activity in each well, and the greater the slope, the stronger the enzyme catalytic activity; and the inhibitory activity of the main protease inhibitor to be tested against the main protease was quantified by the following formula:

[0478] Enzyme activity percentage (%) = (Slope 实验孔 -Slope 溶剂背景孔 ) / (Slope 对照孔 -Slope 溶剂背景孔 );

[0479] In the formula, the control well refers to the well containing only buffer, main protease and substrate but no drug; the experimental well refers to the well containing buffer, main protease, substrate and drug; and the solvent background well refers to the well containing only buffer and substrate.

[0480] Using the enzyme activity percentage as the vertical coordinate and the concentration of the drug to be tested as the horizontal coordinate, the inhibition curve of the drug to be tested against the main protease was obtained by plotting using GraphPad software, and the K i value of the main protease inhibitor to be tested against the enzyme was measured by fitting using the Morrison equation, and the lower the K i value, the stronger the inhibitory activity of the main protease inhibitor to be tested against the enzyme; and the test results are shown in Figure 62.

[0481] As can be seen from Figure 62, the K proM49L / E166A value of Nirmatrelvir against the main protease drug-resistant mutant M i is only 352 nM, which is about 70 times higher than the K i value against the wild-type main protease (5.10 nM), and the difference is two orders of magnitude.

[0482] Hit 3-4 against main protease drug-resistant mutant M proM49L / E166A K i value of 30.3 μM, which is equivalent to its K i value (2708 nM) against wild-type main protease; Hit 3-5 against main protease drug-resistant mutant M proM49L / E166A K i value of 542 nM, which is one order of magnitude higher than its K i value (29.0 nM) against wild-type main protease; indicating that the pyridine ring and the -CH2-pyridine ring both contribute to the anti-drug resistance activity of main protease, and the -CH2-pyridine ring contributes more.

[0483] While Hit 3-6 against main protease drug-resistant mutant M proM49L / E166A K i value of 8.62 nM (K i value of 3.08 nM) against wild-type main protease, Hit 3-32 against main protease drug-resistant mutant M proM49L / E166A K i value of 2.18 nM (K i value of 0.513 nM) against wild-type main protease, Hit 3-33 against main protease drug-resistant mutant M proM49L / E166A K i value of 1.76 nM (K i value of 1.23 nM) against wild-type main protease, Hit 3-34 against main protease drug-resistant mutant M proM49L / E166A K i value of 10.6 nM (K i value of 0.983 nM) against wild-type main protease.

[0484] It can be seen that when the main protease has M49L / E166A drug resistance mutation, the inhibitory activity of Nirmatrelvir against main protease drops sharply, and the inhibitory activity against the drug-resistant mutant is poor; while the inhibitory activities of Hit 3-6, Hit 3-32, Hit 3-33 and Hit 3-34 against wild-type main protease and main protease drug-resistant mutant M proM49L / E166A are all maintained at a very effective level.

[0485] Among them, compared with the inhibitory activity against wild-type main protease, in addition to the inhibitory activity of Hit 3-32 and Hit 3-34 against main protease drug-resistant mutant M proM49L / E166A decreased by one order of magnitude, the inhibitory activity of Hit 3-6 against drug-resistant mutant M proM49L / E166A decreased slightly, while the K proM49L / E166A value of Hit 3-33 against main protease drug-resistant mutant M iThe values are very close, almost unchanged; indicating that compared with pyridine ring, -CH2-pyridine ring, quinoline ring is more conducive to improve the main protease resistance activity, in addition, R1 in group A also affects the main protease inhibitor resistance activity.

[0486] Example 46 - Main protease inhibitor of the application against coronavirus main protease resistant mutant M proL50F / E166A / L167F inhibitory constant (K i ) determination

[0487] This example takes the main protease resistant mutant M proL50F / E166A / L167F of the new coronavirus as the test object, and tests the inhibition activity of Hit3-5, Hit 3-6, Hit 3-32, Hit 3-33 and Hit 3-34 and positive control Nirmatrelvir on coronavirus main protease, and the test method is as follows:

[0488] Add main protease in a 96-well plate to make the final concentration of main protease 400nM, and add a series of concentration gradients of the main protease inhibitor to be tested (the concentration gradient is adjusted according to the activity of each main protease inhibitor, 4-fold dilution), 37℃ incubate for 5min, then add 1ul of fluorescent polypeptide substrate with a concentration of 1mM per well, immediately place in a Tecan enzyme reader with 37℃ temperature control, set the excitation wave to 340nm and the emission wave to 490nm, dynamically detect the change of fluorescence value (RFU) of each well with time, read once every 30s, a total of 120 times, and finally the enzyme activity kinetic curve of each well can be measured, the slope of the linear region of the initial time period of the curve represents the enzyme activity in each well, the greater the slope, the stronger the enzyme catalytic activity; and the inhibition activity of the main protease inhibitor to be tested on the main protease is quantified by the following formula: Enzyme activity percentage (%) = (Slope 实验孔 -Slope 溶剂背景孔 ) / (Slope 对照孔 -Slope 溶剂背景孔 );

[0489] In the formula, the control well refers to the well containing only buffer, main protease and substrate but no drug; the experimental well refers to the well containing buffer, main protease, substrate and drug; and the solvent background well refers to the well containing only buffer and substrate.

[0490] The test results are shown in Figure 63.

[0491] As can be seen from Figure 63, the K proL50F / E166A / L167F value of Nirmatrelvir for main protease resistant mutant M i is only 1799nM, which is about 353 times higher than the K i value for wild type main protease (5.10nM), and the difference is three orders of magnitude.

[0492] Hit 3-5 on main protease drug-resistant mutant M proL50F / E166A / L167F i The K i value of Hit 3-6 on main protease drug-resistant mutant M proL50F / E166A / L167F i is 41.8 nM (the K i value of wild-type main protease is 3.08 nM), the K proL50F / E166A / L167F value of Hit 3-32 on main protease drug-resistant mutant M i i is 43.1 nM (the K proL50F / E166A / L167F value of wild-type main protease is 0.513 nM), the K i value of Hit 3-33 on main protease drug-resistant mutant M i proL50F / E166A / L167F is 75.8 nM (the K i value of wild-type main protease is 1.23 nM), the K i value of Hit 3-34 on main protease drug-resistant mutant M proL50F / E166A / L167F

[0493] It can be seen that when the main protease has L50F / E166A / L167F drug-resistant mutations, the inhibitory activity of Nirmatrelvir on main protease drops sharply, and the inhibitory activity on drug-resistant mutants is poor; while Hit 3-6, Hit 3-32, Hit 3-33 and Hit 3-34, although the inhibitory activity on M i

[0494] Example 47 - Determination of the inhibition constant (K pro ) of the main protease inhibitors of the present application on the main protease of feline coronavirus

[0495] In this example, the main protease of feline coronavirus (FIPV M pro ) was used as the test object, and the inhibitory activities of Hit 3-6, Hit 3-32, Hit 3-33 and Hit 3-34 and the positive control Nirmatrelvir on the main protease of coronavirus were tested, and the test method was as follows:

[0496] ​​​​The main protease was added in a 96-well plate to make the final concentration of the main protease 100 nM, and a series of concentration gradients of the main protease inhibitor to be tested were added (the concentration gradient was adjusted according to the activity of each main protease inhibitor, 4-fold dilution), 1 μl of the fluorescent polypeptide substrate with a concentration of 1 mM was added to each well after incubation at 37°C for 5 min, and immediately placed in a Tecan enzyme reader controlled at 37°C, with an excitation wave of 340 nm and an emission wave of 490 nm, and the fluorescence value (RFU) of each well was dynamically detected with time, reading once every 30 s, a total of 120 times, and finally the enzyme activity kinetic curve of each well was measured, and the slope (Slope) of the linear region of the initial time period of the curve was used to represent the activity of the enzyme in each well, and the greater the slope, the stronger the catalytic activity of the enzyme; and the inhibition activity of the main protease inhibitor to be tested on the main protease was quantified by the following formula:

[0497] Enzyme activity percentage (%) = (Slope 实验孔 -Slope 溶剂背景孔 ) / (Slope 对照孔 -Slope 溶剂背景孔 );

[0498] In the formula, the control well refers to the well containing only buffer, main protease and substrate but no drug; the experimental well refers to the well containing buffer, main protease, substrate and drug; and the solvent background well refers to the well containing only buffer and substrate.

[0499] The test results are shown in FIG. 64.

[0500] As can be seen from FIG. 64, the K i value of Nirmatrelvir for the main protease of feline coronavirus is 35.9 nM; the K i value of Hit 3-6 for the main protease of feline coronavirus is 0.273 nM, the K i value of Hit 3-32 for the main protease of feline coronavirus is 0.0939 nM, the K i value of Hit 3-33 for the main protease of feline coronavirus is 0.0712 nM, and the K i value of Hit 3-34 for the main protease of feline coronavirus is 0.0440 nM. It can be seen that the inhibition activity of the main protease inhibitor of the present application on the main protease of feline coronavirus is 10 2 or 10 3 times that of Nirmatrelvir; indicating that the main protease inhibitor of the present application has a broad inhibition activity on coronaviruses.

[0501] Example 48 - In vitro anti-viral activity detection of the main protease inhibitor of the present application

[0502] The present example takes the SARS-CoV-2 Omicron BA.5.2 strain as the test object, determines the in vitro antiviral activity of a plurality of main protease inhibitors of the application, and takes Nirmatrelvir and Ibuzatrelvir as positive controls, and the test scheme is as follows: Test steps carried out by P2 laboratory:

[0503] ①Vero E6 cells were cultured in a 96-well plate (cell density = 20000 cells / well) containing 100 μL of culture medium (high-sugar DMEM + 10% FBS + 1% penicillin / streptomycin) per well, and were cultured overnight;

[0504] ②Prepare a new 96-well plate, add 147 μL of culture medium (high-sugar DMEM + 2% FBS + 1% penicillin / streptomycin) to B2 well, and add 3 μL of drug mother liquor (1 mM) to be tested, so that the final concentration is 20 μM, and the final volume is 150 μL;

[0505] ③Add 120 μL of culture medium containing 2% DMSO (high-sugar DMEM + 2% FBS + 1% penicillin / streptomycin) to B3-B11 wells;

[0506] ④From B2 well, 30 μL of culture medium was aspirated and added to B3 well for mixing, and the remaining wells were gradient diluted in the same way. After dilution was completed in B10 well, 30 μL of culture medium was discarded, and finally the volume of culture medium in each well was ensured to be 120 μL.

[0507] 2) Test steps carried out by P3 laboratory:

[0508] ①Dilute the cultured virus in culture medium (high-sugar DMEM + 2% FBS + 1% penicillin / streptomycin) to prepare virus-containing culture medium;

[0509] ②Use the gun to add 120 μL of virus-containing culture medium (100 TCID50) to each well of the previously prepared drug-containing 96-well plate, so that the final concentration of DMSO is 1%, the final concentration of drug is 10000 nM, 2000 nM, 400 nM, 80 nM, 16 nM, 3.2 nM, 0.64 nM, 0.128 nM, 0.0256 nM and 0 nM, and the final volume is 240 μL / well;

[0510] ③Use the gun to aspirate the liquid from the previously prepared 96-well cell plate and discard it into a waste box containing 75% alcohol;

[0511] ④Use the gun to aspirate 200 μL of the mixed drug and virus liquid from the 96-well U-shaped plate and add it to the 96-well cell culture plate. Place the culture plate in a latch box, tighten the latch box, and fully spray the outside of the box with alcohol in the biosafety cabinet;

[0512] ⑤Incubate at 37℃, 5% CO2 for 48h, and observe cytopathic effect.

[0513] 3) Sample collection and detection:

[0514] ① Virus quantification: Collect the cell culture supernatant, and quantitatively analyze the SARS-CoV-2 nucleocapsid protein gene expression by RT-qPCR.

[0515] ② Immunofluorescence staining:

[0516] A. Fixation: Reserve the cell culture supernatant, add an appropriate amount of PBS to each well, and wash for 5 min on a horizontal shaker at room temperature, repeat for 3 times, and fix with 4% paraformaldehyde for 10 min;

[0517] B. Permeation: Add an appropriate amount of PBS to each well, and wash for 5 min on a horizontal shaker at room temperature, repeat for 3 times, and permeate with 0.2% Triton X-100 for 30 min;

[0518] C. Blocking: Add an appropriate amount of 1% BSA PBS solution to each well, and slowly shake on a horizontal shaker at room temperature for 2h;

[0519] D. Antibody incubation: Add an appropriate amount of PBS to each well, and wash for 5 min on a horizontal shaker at room temperature, repeat for 3 times, add an appropriate amount of SARS-CoV-2 nucleocapsid protein antibody (rabbit source, primary antibody, dilution ratio 1:2000) to each well, and incubate overnight at 4℃; recover the primary antibody, add an appropriate amount of PBS to each well, and wash for 5 min on a horizontal shaker at room temperature, repeat for 3 times, add an appropriate amount of rhodamine-labeled goat anti-rabbit secondary antibody (dilution ratio 1:80) to each well, and slowly shake on a horizontal shaker at room temperature for 1h;

[0520] E. Nucleus staining: Recover the secondary antibody, add an appropriate amount of PBS to each well, and wash for 5 min on a horizontal shaker at room temperature, repeat for 3 times, add DAPI working solution to each well, and stain for 3 min, then wash with PBS for 3 times;

[0521] F. Observation and analysis: After immunofluorescence staining, observe the expression and distribution of viral nucleocapsid protein in Vero E6 cells after infection by using BZ-X810 fluorescence microscope (KEYENCE). Semi-quantify the immunofluorescence images by using Image J.

[0522] The test results are shown in Table 7.

[0523] As can be seen from Table 7, the tested compounds of the present application can strongly inhibit the infection of the new coronavirus, and the inhibition activity is 1-4 orders of magnitude stronger than the positive control. As can be seen from the test data in Table 7, the main protease inhibitors of the present application have strong inhibition activity on the N pro The expression and nucleic acid levels all showed inhibition activity on the main protease to different degrees. The inhibition activity on the Npro In the expression test, compared with other main protease inhibitors, the inhibition activities of Hit 3-6, Hit 3-32, Hit 3-33, Hit 3-34, Hit 3-36, FD7-1, FD7-2, FD7-24 and FD7-25 on main protease were stronger, the EC50 values of Hit 3-6, Hit 3-33, Hit 3-34, FD7-2, FD7-24 and FD7-25 were all in the order of 10 nM, which was obviously better than the positive controls Nirmatrelvir and Ibuzatrelvir; and the K values of Hit 3-32, Hit 3-36 and FD7-1 were all in the order of 10 nM, which was more superior to the positive controls Nirmatrelvir and Ibuzatrelvir. 0-1 i -1 In the inhibition viral nucleic acid test, compared with other main protease inhibitors, the EC50 values of Hit 3-32 and Hit 3-33 were all in the order of 10 nM, which was obviously better than the positive controls Nirmatrelvir and Ibuzatrelvir. It was shown that the compounds of the present application could effectively inhibit the new coronavirus at both the protein level and the nucleic acid level, especially the compounds with non-fluorine halogen substitution in Group A. -1

[0524] Table 7

[0525] Example 49 - Detection of pharmacokinetic properties of main protease inhibitors Hit 3-32 and Hit 3-33 of the present application

[0526] In this example, healthy BALB / c mice were used as test objects to determine the in vivo pharmacokinetic properties of Nirmatrelvir, Hit 3-32 and Hit 3-33, and the test scheme was as follows:

[0527] 1) Dosing scheme: healthy BALB / c male mice (17-20 g) were selected and orally administered (p.o.) with Nirmatrelvir, Hit 3-32 or Hit 3-33 at a dose of 20 mg / kg or intravenously injected (i.v.) with Nirmatrelvir, Hit 3-32 or Hit 3-33 at a dose of 2 mg / kg; all three drugs were suspended in the formulation A solution containing 5% DMSO, 20% HP-β-CD and 15% HS-15.

[0528] 2) Sample collection: at the designated time points (0.083, 0.25, 0.5, 1, 2, 4, 8 and 24 h for intravenous injection; 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h for oral administration), blood samples of each group of mice were collected using an anticoagulant tube, and plasma (supernatant) was extracted by centrifugation at 3000 g for 10 min.​​​

[0529] 3) Detection analysis:

[0530] ① Preparation of standard curve: dilute healthy mouse plasma with physiological saline, and add test compounds at specified concentrations (0, 20, 50, 200, 1000, 2000, 5000, 10000 ng / mL) to prepare a standard curve.

[0531] ② Concentration determination: determine the concentration of the compound in the plasma by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The equipment parameters are as follows:

[0532] A. Analysis column: ACQUITY Premier BEH C18 column (2.1 x 50 mm, 1.7 μm);

[0533] B. Analysis system: Waters Acquity Premier UPLC-Xevo TQ Absolute;

[0534] C. Flow rate: 0.5 mL / min;

[0535] D. Mobile phase A: 0.2% formic acid in water;

[0536] E. Mobile phase B: acetonitrile;

[0537] F. Gradient

[0538] Time period (min) Concentration Solvent

[0539] 0~0.5 10% mobile phase B

[0540] 0.5~1.2 10%~90%

[0541] 1.2~1.7 90%

[0542] 1.7~1.8 90%~10%

[0543] 1.8~2.5 10%

[0544] ③ Data analysis: calculate the average plasma concentration and standard deviation of each drug at each time point, and use DAS 3.2.8 software to fit the pharmacokinetic parameters by non-compartment model.

[0545] The test results are shown in FIG. 65.

[0546] As can be seen from Figure 65, the intravenous half-life (t1 / 2) of Nirmatrelvir is 0.238 h, and the intravenous t1 / 2 of Hit 3-32 is 0.859 h, which is ~3.6 times slower than NTV. Although both of them have isoquinoline ring as P1 group, the intravenous t1 / 2 of Hit 3-33 is only 0.150 h, which is ~1.6 times and ~5.7 times faster than NTV and Hit 3-32, respectively. In oral administration, we also found that the elimination rate of Hit 3-32 is slower than NTV, but there is no significant difference in t1 / 2 between them, which are ~2.70 h (Nirmatrelvir) and 2.81 h (Hit 3-32), respectively. In contrast, the oral elimination rate of Hit 3-33 is still stronger than Nirmatrelvir and Hit 3-32, and its oral t1 / 2 is only 0.469 h. Interestingly, we found that the oral bioavailability of Hit 3-32 and Hit 3-33 can reach 37.8% and 62.8%, respectively, while the oral bioavailability of Nirmatrelvir is only 25.6%, and the peak concentration (Cmax) is also ~2.5 times higher than Nirmatrelvir, indicating that our compounds have stronger oral administration advantages.

[0547] Example 50 - Acute toxicity experiment of the main protease inhibitor Hit 3-32 and Hit 3-33 of the application

[0548] This example uses healthy BALB / c mice as test objects to determine the in vivo acute toxicity of Nirmatrelvir, Hit 3-32 and Hit 3-33, and the test scheme is as follows:

[0549] 1) Dosing: Healthy BALB / c male mice (17-20 g) were selected and orally administered with low dose (100 mg / kg), medium dose (300 mg / kg) and high dose (900 mg / kg) of NTV, Hit 3-32 or Hit 3-33, respectively;

[0550] 2) Sampling: After 7 days of administration, samples of blood, liver and kidney were collected from each group of mice after anesthesia;

[0551] 3) Detection: The specific indicators are as follows

[0552] ① Body weight monitoring: The clinical status of each group of mice within 7 days after administration was observed and the daily body weight was recorded;

[0553] ② Biochemical indicators: mouse serum was separated for detection of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA) and blood urea nitrogen (BUN);

[0554] The test results are shown in Figure 66.

[0555] As can be seen from Figure 66, under the three dosages, neither oral Nirmatrelvir nor Hit 3-32 / Hit 3-33 caused the death of mice; and within 7 days after administration, the body weight of all mice did not decrease significantly, and the body weight change trend was consistent with that of the mice in the oral solvent group, indicating that the three compounds did not produce systemic toxicological reactions. On the 7th day, we sacrificed all the mice and detected the content of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the blood of each mouse, and the results showed that oral administration of the three compounds did not significantly increase the two indicators, and they were all within the normal threshold range, proving that the three compounds did not exhibit significant liver toxicity. In addition, we also detected the content of creatinine (CREA) and blood urea nitrogen (BUN) in the blood of mice, and the results also showed that the three compounds did not significantly increase the two indicators, and they were all within the normal threshold range, indicating that the three compounds did not exhibit significant kidney toxicity. The above in vivo acute toxicity study showed that Hit 3-32 and Hit 3-33 have good oral safety, which is consistent with the clinical drug Nirmatrelvir, and meet the basic requirements of oral pharmacodynamic studies.

[0556] Example 51 - In vivo pharmacodynamic study of the main protease inhibitor Hit 3-32 and Hit 3-33 of the present application in combination with Ritonavir

[0557] This example uses mice infected with the Omicron BA.5.2 strain as test objects to determine the in vivo efficacy of Nirmatrelvir, Hit 3-32 and Hit 3-33 in combination with Ritonavir, and the test scheme is as follows:

[0558] All animal experiments involving SARS-CoV-2 infection were carried out in the BSL3 laboratory of Fudan University, and were authorized by the Animal Ethics Committee of Fudan University School of Medicine. The specific experimental scheme is as follows:

[0559] 1) Infection: C57BL6 / S hACE2 transgenic mice CAG-hACE2-IRES-Luc-Tg male mice (10-14 weeks) with genetic background were randomly divided into 4 groups (6 in each group), and 20 μL / g of avertin (ready-to-use tribromoethanol) was injected to anesthetize the mice, and then the mice were infected with ~5000 PFUs of SARS-CoV-2 Omicron BA.5.2 strain (DMEM system, 40 μL) by nasal drops.

[0560] 2) Drug administration: 2 h after infection, the test drugs NTV, Hit 3-32 and Hit 3-33 suspended in 2% Tween-80 and 0.5% methyl cellulose preparation solution were respectively administered to the infected mice by gavage, and RTV was used to improve the pharmacokinetic properties at the same time to simulate the clinical use scenario of NTV. The specific grouping and dosage are as follows:

[0561] ① Healthy group (Mock): uninfected and undosed group, as a healthy control group;

[0562] ② Nirmatrelvir (NTV) group: 200 mg / kg NTV + 50 mg / kg Ritonavir (RTV), twice a day (b.i.d.);

[0563] ③ Hit 3-32 group: 200 mg / kg Hit 3-32 + 50 mg / kg RTV, once a day (q.d.);

[0564] ④ Hit 3-33 group: 200 mg / kg Hit 3-32 + 50 mg / kg RTV, once a day (q.d.).

[0565] 3) Detection: 3 days after drug administration, the mice were sacrificed after anesthesia and sampling, and the following detection was performed:

[0566] ① Nucleic acid load: RT-qPCR detection was performed on lung and brain tissues to quantitatively detect viral nucleic acid load;

[0567] ② Pathological analysis: part of the lung and brain tissues were fixed with 4% paraformaldehyde for H&E staining analysis to characterize the pathological changes of the mouse lung tissue;

[0568] ③ Immunohistochemistry / immunofluorescence analysis: visualization / quantification of the distribution and expression of SARS-CoV-2 nucleocapsid protein in lung tissue.

[0569] The test results are shown in Figures 67 and 68.

[0570] As shown in FIG. 67, both Hit 3-32 and Hit 3-33 significantly reduced viral nucleic acid load in the bronchi and lungs (P<0.0001), with at least 2 orders of magnitude reduction in copy number. However, the NTV-treated group showed a trend of reduction in viral nucleic acid load in the bronchi and lungs, but there was no statistical difference (P=0.8215). In contrast, the viral nucleic acid load in the bronchi and lungs of mice treated with Hit 3-32 and Hit 3-33 was significantly lower than that in the NTV group, indicating that the in vivo inhibitory activity of our compounds against the virus was still significantly stronger than that of NTV. In addition to the bronchi and lung tissues, we found that a high level of viral nucleic acid could be detected in the brain tissue of mice after infection, with a copy number of ~ 10 8 copies / g, while the viral nucleic acid load in the brain tissue was reduced by ~ 5 orders of magnitude after treatment with NTV, Hit 3-32 and Hit 3-33, which also indicated that Hit 3-32 / Hit 3-33 and NTV had a prominent brain protective effect.

[0571] As shown in FIG. 68, Hit 3-32 and Hit 3-33 significantly reduced the expression of viral nucleocapsid protein in lung tissue, almost consistent with the uninfected group, while the viral nucleocapsid protein in the lung tissue of the NTV group was lower than that in the solvent-treated group, which was consistent with the results of immunohistochemical staining. The semi-quantitative results showed that the fluorescent positive area in the lung tissue of the Hit 3-32 and Hit 3-33 treatment groups was significantly lower than that in the solvent treatment group (P<0.0001), almost similar to the uninfected group. Although there was no significant difference in viral nucleic acid load, NTV treatment could significantly reduce the fluorescent positive area in the lung tissue (P<0.0001). Consistent with the results of viral nucleic acid load, the expression of viral nucleocapsid protein in the lung tissue after treatment with Hit 3-32 or Hit 3-33 was significantly lower than that in the NTV group, fully demonstrating that our inhibitors had stronger in vivo antiviral activity than NTV. And under the treatment of Hit 3-32 and Hit 3-33, the lung injury of mice was significantly reduced, with less alveolar exudation and no obvious thickening of the alveolar membrane, almost similar to the lung tissue morphology of the uninfected group of mice. In contrast, the treatment effect of NTV was far inferior to that of Hit 3-32 and Hit 3-33, although it was improved compared to the solvent treatment, but there was still a large amount of exudation in the alveoli of the NTV-treated group of mice, and the alveolar membrane was also thickened with immune cell infiltration.

[0572] Example 52 - In vivo pharmacodynamic study of the main protease inhibitor Hit 3-32 monotherapy

[0573] This example uses mice infected with the Omicron BA.5.2 strain as test objects to determine the in vivo efficacy of Hit 3-32 monotherapy and NTV combined with RTV, and the test scheme is as follows:

[0574] All animal experiments involving SARS-CoV-2 infection were performed in the BSL3 laboratory of Fudan University and authorized by the Animal Ethics Committee of Fudan University School of Medicine. The specific experimental protocol is as follows:

[0575] 1) Infection: C57BL6 / S genetic background hACE2 transgenic mice CAG-hACE2-IRES-Luc-Tg male mice (10-14 weeks) were randomly divided into 4 groups (6 mice in each group), and the mice were injected with 20 μL / g of Avertin (ready-to-use tribromoethanol) anesthesia, and then the mice were infected with ~5000 PFUs of SARS-CoV-2 Omicron BA.5.2 strain (DMEM system, 40 μL) by nasal drops.

[0576] 2) Drug administration: 2h after infection, the test drugs NTV and Hit 3-32 suspended in 2% Tween-80 and 0.5% methyl cellulose preparation solution were administered to the infected mice by gavage, among which Hit 3-32 was only administered as a single drug, while NTV was administered in combination with RTV. The specific grouping and dosage are as follows:

[0577] ① Healthy group (Mock): uninfected and undosed group, as a healthy control group;

[0578] ③ NTV+RTV group: 200mg / kg NTV+50mg / kg RTV, twice a day (b.i.d.);

[0579] ③ Hit 3-32 group: 200mg / kg Hit 3-32, twice a day (b.i.d.);

[0580] 3) Detection: 3 days after drug administration, the mice were sacrificed after anesthesia and the samples were taken for the following detection:

[0581] ① Nucleic acid load: RT-qPCR detection was performed on lung and brain tissues to quantitatively detect viral nucleic acid load;

[0582] ② Pathological analysis: part of the lung and brain tissues were fixed with 4% paraformaldehyde for H&E staining analysis to characterize the pathological changes of the mouse lung tissue;

[0583] ③ Immunohistochemistry / immunofluorescence analysis: visualization / quantification of the distribution and expression of SARS-CoV-2 nucleocapsid protein in lung tissue.

[0584] The test results are shown in Figures 69 and 70.

[0585] As shown in FIG. 69, Hit 3-32 alone can effectively reduce viral nucleic acid load in the bronchus and brain tissue, with statistical differences (P = 0.0114; P = 0.0019), and also has a trend of reducing viral nucleic acid load in lung tissue, but without statistical difference (P = 0.2337); while the viral nucleic acid copy number in lung tissue and brain tissue of NTV + RTV treatment group mice is lower than that of solvent treatment group mice, with statistical differences (P = 0.0126; P = 0.0006), while there is no significant difference in the copy number in the bronchus (P = 0.5698). The above results show that the antiviral activity of Hit 3-32 as a single drug is almost the same as that of NTV combined with RTV.

[0586] As shown in FIG. 70, both Hit 3-32 and NTV + RTV can significantly reduce the expression of viral nucleocapsid protein in lung tissue. The semi-quantitative results show that the fluorescent positive area in the lung tissue of the NTV + RTV treatment group is significantly lower than that of the solvent treatment group (P = 0.0009), although there is no significant difference in viral nucleic acid load, Hit 3-32 alone can also effectively reduce the fluorescent positive area in the lung tissue (P < 0.0002), which fully shows that Hit 3-32 as a single drug can achieve a therapeutic effect comparable to NTV + RTV. Although NTV + RTV can effectively inhibit viral infection in lung tissue, there is still a considerable amount of exudation in the alveoli and the alveolar membrane is thickened in the NTV + RTV treatment group mice. Interestingly, although the in vivo antiviral activity of Hit 3-32 as a single drug is comparable to that of NTV + RTV, the degree of alveolar exudation and alveolar membrane thickening in the Hit 3-32 single drug treatment group mice is much smaller than that in the NTV + RTV treatment group, indicating that Hit 3-32 has better lung protection function. The above results all show that Hit 3-32 has the potential to be used as a single drug.

[0587] Example 53 - In vivo pharmacodynamic study of the main protease inhibitor Hit 3-36 and FD7-1 as a single drug

[0588] This example uses mice infected with the Omicron BA.5.2 strain as test objects to determine the in vivo efficacy of Hit 3-36 and FD7-1 as single drugs and NTV combined with RTV, and the test scheme is as follows:

[0589] All animal experiments involving SARS-CoV-2 infection were carried out in the BSL3 laboratory of Fudan University, and were authorized by the Animal Ethics Committee of Fudan University School of Medicine. The specific experimental scheme is as follows:

[0590] 1) Infection: C57BL6 / S hACE2 transgenic mice CAG-hACE2-IRES-Luc-Tg male mice (10-14 weeks) with genetic background were randomly divided into 4 groups (6 in each group), and 20 μL / g of avertin (ready-to-use tribromoethanol) was injected to anesthetize the mice, and then the mice were infected with ~5000 PFUs of SARS-CoV-2 Omicron BA.5.2 strain (DMEM system, 40 μL) by nasal drops.

[0591] 2) Drug administration: 2 h after infection, the test drugs NTV, Hit 3-36 and FD7-1 suspended in 2% Tween-80 and 0.5% methyl cellulose preparation solution were respectively administered to the infected mice by gavage, wherein Hit 3-36 and FD7-1 were administered as single drugs, and NTV was administered in combination with RTV. The specific grouping and doses are as follows:

[0592] ① Healthy group (Mock): uninfected and undosed group, as a healthy control group;

[0593] ② NTV+RTV group: 200 mg / kg NTV+50 mg / kg RTV, twice a day (b.i.d.);

[0594] ④ Hit 3-36 group: 200 mg / kg Hit 3-36, twice a day (b.i.d.);

[0595] ④ FD7-1 group: 200 mg / kg FD7-1, twice a day (b.i.d.).

[0596] 3) Detection: 3 days after drug administration, the mice were sacrificed after anesthesia and sampling, and the following detection was performed:

[0597] ① Nucleic acid load: RT-qPCR detection was performed on lung and brain tissues to quantitatively detect viral nucleic acid load;

[0598] ② Pathological analysis: part of the lung and brain tissues were fixed with 4% paraformaldehyde for H&E staining analysis to characterize the pathological changes of the lung tissues of the mice;

[0599] ④ Immunohistochemistry / immunofluorescence analysis: visualization / quantification of the distribution and expression of SARS-CoV-2 nucleocapsid protein in lung tissues.

[0600] The test results are shown in FIG. 71.

[0601] As shown in FIG. 71, Hit 3-36 and FD7-1 alone can effectively reduce the viral nucleic acid load in the bronchus, lung and brain tissues, with statistical differences, and also have a trend of reducing the viral nucleic acid load in the intestinal tissue, but without statistical differences; the effects of NTV+RTV and FD7-1 groups are similar but weaker than Hit 3-36, and can also make the viral nucleic acid copy number in the bronchus, lung and brain tissues of mice lower than that of the solvent treatment group, with statistical differences, but the control effect on the intestinal viral nucleic acid load is not obvious. The above results show that Hit 3-36 and FD7-1 as single drugs can effectively treat COVID-19 infection.

[0602] Example 54 - Detection of pharmacokinetic properties of the main protease inhibitor Hit 3-6 and FD7-1 of the application

[0603] In this example, healthy BALB / c mice were used as test objects to determine the in vivo pharmacokinetic properties of Ibuzatrelvir (ITV), Hit 3-36 and FD7-1, and the test scheme was as follows:

[0604] 1) Dosing scheme: healthy BALB / c male mice (17-20 g) were orally (p.o.) administered with 20 mg / kg or intravenously (i.v.) administered with 2 mg / kg of ITV, Hit 3-36 or FD7-1, respectively; all three drugs were suspended in the formulation A solution containing 5% DMSO, 20% HP-β-CD and 15% HS-15.

[0605] 2) Sample collection: blood samples of each group of mice were collected using an anticoagulant tube at the specified time points (0.083, 0.25, 0.5, 1, 2, 4, 8 and 24 h for intravenous injection; 0.083, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h for oral administration), and the plasma (supernatant) was extracted by centrifugation at 3000 g for 10 min.

[0606] 3) Detection analysis:

[0607] ① Preparation of standard curve: physiological saline was used to dilute the plasma of healthy mice, and standard curves were prepared by adding the test compounds at specified concentrations (0, 20, 50, 200, 1000, 2000, 5000, 10000 ng / mL).

[0608] ② Concentration determination: the concentration of the compound in the plasma was determined by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The equipment parameters were as follows:

[0609] A. Analysis column: ACQUITY Premier BEH C18 column (2.1 x 50 mm, 1.7 μm);

[0610] B. Analysis system: Waters Acquity Premier UPLC-Xevo TQ Absolute;

[0611] C. Flow rate: 0.5 mL / min;

[0612] D. Mobile phase A: 0.2% formic acid in water;

[0613] E. Mobile phase B: acetonitrile;

[0614] F. Gradient

[0615] Time period (min) Concentration Solvent

[0616] 0-0.5 10% mobile phase B

[0617] 0.5-1.2 10%-90%

[0618] 1.2-1.7 90%

[0619] 1.7-1.8 90%-10%

[0620] 1.8-2.5 10%

[0621] 3. Data analysis: The mean plasma concentration and standard deviation of each drug at each time point were calculated, and the pharmacokinetic parameters were fitted by non-compartment model using DAS 3.2.8 software.

[0622] The test results are shown in FIG. 72.

[0623] As shown in FIG. 72, the intravenous half-life (t1 / 2) of ITV is 0.23 h, the intravenous t1 / 2 of Hit 3-36 is 1.41 h, the metabolic speed is slowed down by ~5 times compared with ITV, and the intravenous t1 / 2 of FD7-1 is 0.57 h, the metabolic speed is slowed down by ~2.5 times compared with ITV. The oral t1 / 2 of ITV is 0.233 h, the oral t1 / 2 of Hit 3-36 is 3.51 h, the metabolic speed is slowed down by ~1.5 times compared with ITV, and the oral t1 / 2 of FD7-1 is 1.00 h. Interestingly, we found that the oral bioavailability of Hit 3-36 and FD7-1 can reach 72.9% and 70.6%, respectively, while the oral bioavailability of ITV is only 7.1%, and the in vivo exposure, such as the peak concentration (Cmax), is at least ~30 times higher than that of ITV, indicating that our compounds have stronger oral administration advantages.

[0624] Examples 55-123 - Synthesis of primary protease inhibitor hits 3-10, 3-12, 3-14, 3-29, 3-35-38, 3-41-3-98

[0625] The chemical structures of primary protease inhibitor hits 3-10, 3-12-3-14, 3-29, 3-35-3-98 are shown in Table 8. As can be seen from Table 8, the structures of the listed primary protease inhibitors can be found in Examples 1-41 and thus the synthesis methods are generally the same. The differences, including starting materials and amounts, are discernible and implementable by one of ordinary skill in the art without any doubt and thus are not described herein.

[0626] Table 8

Claims

1. A metabolically stable inhibitory compound against drug-resistant main protease, or its stereoisomers, hydrates, deuterated derivatives, esters, solvates, metabolites, pharmaceutically acceptable salts, or prodrugs, said compound comprising the structure shown in formula (I): wherein: Group A is selected from any one of: -CO-unsubstituted or substituted hydrocarbyl, hydrocarbyl ether, indole, or -SO2-unsubstituted or substituted hydrocarbyl; wherein R1is selected from any one of the following: -CO-unsubstituted or substituted C1-C9hydrocarbyl, hydrocarbyl ether, 6-membered heterocyclyl, heteroatom-containing bicyclic or tricyclic structure, or -SO2-unsubstituted or substituted C1-C9hydrocarbyl, 6-membered heterocyclyl, heteroatom-containing bicyclic or tricyclic structure; R2is selected from any one of the following: unsubstituted or substituted C1-C 14 hydrocarbyl, 5-6 membered heterocyclyl; R3and R4are independently or connected to form ring B, when R3and R4are independent, R3is selected from any one of the following: hydrogen, methyl, halogenated methyl; R4is selected from unsubstituted or substituted C1-C7hydrocarbyl; when R3and R4are connected to form ring B, ring B is selected from any one of the following: unsubstituted or substituted 5-6 membered heterocyclyl, heteroatom-containing bicyclic or tricyclic structure; R5is selected from any one of the following: cyano, unsubstituted or substituted carbonyl; Group C is selected from any one of: -CH2-unsubstituted or substituted pyridine ring, -CH2-unsubstituted or substituted pyrrolidone; wherein ring D is selected from any one of the following: unsubstituted or substituted with R7benzene ring, pyridine ring, pyrimidine ring, pyrazine ring, imidazole ring, pyrrole ring, thiophene ring, furan ring; ring E is selected from any one of the following: unsubstituted or substituted with R9benzene ring, pyridine ring, pyrimidine ring, pyrazine ring, imidazole ring, pyrrole ring, thiophene ring, furan ring; R6, R7, R8and R9are each independently selected from any one of the following: hydrogen, cyano, methyl, amino, halogen, halogenated methyl, unsubstituted or substituted alkyl, hydroxyalkyl, aminoalkyl, five-membered nitrogen heterocycle; X is selected from any one of the following: -NH-, -O- or -S-.

2. The compound of claim 1, wherein the hydrocarbyl, hydrocarbyl ether, indole, C1-C9 hydrocarbyl, 6-membered heterocyclyl, C1-C 14 the substituents on the hydrocarbyl or 5-6 membered heterocyclyl are selected from one or more of: hydrogen, halogen, methyl, methoxy, halogenated methyl, halogenated methoxy.

3. The compound of claim 1 or 2, wherein at least one of the substituents on the C1-C9hydrocarbyl is selected from non-fluorine halogen.

4. The compound of any one of claims 1-3, wherein the Group A is selected from any one of the following: wherein, R1is selected from any one of: R2is selected from any one of the following:

5. The compound of any one of claims 1-4, wherein the Group A is selected from any one of the following: wherein, R1is selected from any one of: R2is selected from 6. The compound according to any one of claims 1-5, wherein R3 is selected from any one of the following: R4 is selected from any one of the following:

7. The compound of any one of claims 1-5, wherein the substituents on the ring B are selected from any one or more of the following: hydrogen, halogen, hydroxyl, methyl, halogenated methyl, methoxy, halogenated methoxy, -C(CH3)3, -C(CD3)3.

8. The compound of claim 7, wherein the B ring is selected from any one of the following:

9. The compound of claim 8, wherein the B ring is selected from any one of the following:

10. The compound according to any one of claims 1-9, wherein the R5 is selected from any one of the following:

11. The compound of claim 10, wherein R5 is 12. The compound of any one of claims 1-11, wherein in Group C, the substituents on the pyridine ring are selected from any one or more of the following: hydrogen, halogen, methyl, halogenated methyl, cyano.

13. The compound of claim 12, wherein the Group C is selected from any one of the following: wherein D and E rings are each independently selected from any one of: X is selected from any one of the following: -NH-, -O- or -S-; R6, R7, R8, and R9 are each independently selected from any one of the following:

14. The compound of claim 13, wherein the Group C is selected from any one of the following: wherein, D ring is R6, R7are 15. The compound according to any one of claims 1-14, wherein the Group A is wherein, R1is selected from any one of: R2is selected from 16. The compound of claim 15, wherein the B ring is selected from any one of the following:

17. The compound of claim 15 or 16, wherein R5 is 18. The compound of any one of claims 15-17, wherein the Group C is selected from any one of the following: wherein, D ring is R6, R7are 19. The compound according to any one of claims 1-14, wherein the B ring is 20. The compound of claim 19, wherein the Group A is selected from any one of the following: wherein, R1is selected from any one of: R2is selected from 21. The compound of claim 19 or 20, wherein the Group A is wherein, R1is selected from any one of: R2is selected from 22. The compound of any one of claims 19-21, wherein R5 is 23. The compound of any one of claims 19-22, wherein the Group C is wherein, D ring is R6, R7are 24. The compound according to any one of claims 1-14, wherein the B ring is 25. The compound of claim 24, wherein the Group A is selected from any one of the following: wherein R1is selected from any one of: R2is selected from 26. The compound of claim 24 or 25, wherein the Group A is wherein R1is selected from any one of: R2is selected from 27. The compound of any one of claims 24-26, wherein R5 is 28. The compound of any one of claims 24-27, wherein the Group C is wherein D ring is R6, R7are 29. The compound of any one of claims 1-28, wherein the compound has at least one of the following structural formulae:

30. A method of preparing the compound of any one of claims 1-29, comprising synthesizing the compound by chemical means.

31. A pharmaceutical composition, wherein the pharmaceutical composition comprises the compound of any one of claims 1-29 or a racemate, enantiomer, diastereomer, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

32. The pharmaceutical composition of claim 31, wherein the pharmaceutical composition is in the form of an oral dosage, an injection, a spray, a powder, an emulsion, a suspension, or a transdermal preparation.

33. Use of a compound of any one of claims 1-29 or a pharmaceutical composition of any one of claims 31-32 in the manufacture of an antiviral medicament for inhibiting a virus that replicates in dependence on a main protease.

34. The use of claim 33, wherein the virus that replicates in dependence on a main protease comprises a wild-type or drug-resistant mutant strain of a coronavirus, a calicivirus, a norovirus, a hepatitis A virus, a human rhinovirus, and a porcine transmissible gastroenteritis virus.

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