Metabolism-stable anti-drug-resistant main protease inhibitor and application thereof in preparation of antiviral drugs

Abstract

The invention discloses a drug-resistant main protease inhibitor with stable metabolism and application thereof in preparation of antiviral drugs. In the main protease inhibitor, a group C not only can be effectively combined with an active pocket of main protease, but also the combination mainly depends on the 163rd amino acid site of the main protease; meanwhile, the group C is relatively difficult to oxidize, so that the main protease inhibitor can show basically consistent inhibitory activity to both mutant strains and wild strains, viruses do not generate drug resistance to the main protease inhibitor, and the main protease inhibitor can tolerate liver drug enzyme metabolism, has excellent drug-resistant activity and metabolic stability, and can be used for preparing the main protease inhibitor. In use, a liver drug enzyme inhibitor does not need to be used cooperatively, and reduction of self liver function decline and toxic and side effects caused by medicine taking is facilitated. In addition, the 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 ring B mainly plays a role in further improving the inhibitory activity of the main protease inhibitor on the main protease.

Description

[0001] This invention is a divisional application of application number 2024106609591, filed on May 24, 2024, entitled "A metabolically stable anti-drug-resistant main protease inhibitor and its application in the preparation of antiviral drugs". Technical Field

[0002] This invention 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

[0003] 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.

[0004] 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.

[0005] For example, Chinese invention patent application CN 114681443A discloses a series of nitrile-containing antiviral compounds, the structural formulas of which are as follows: .

[0006] 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. .

[0007] Although the inhibition constant (Ki) of nematidine against the novel coronavirus is as low as 5.10 nM, nematidine has two defects: (1) It depends on the binding of the γ-lactam ring to the active pocket of the novel coronavirus, but the γ-lactam ring is easily metabolized by the liver, resulting in poor pharmacokinetic activity of nematidine. Therefore, when using nematidine, it is necessary to use liver enzyme inhibitors (such as ritonavir). This will not only lead to the decline of liver function in patients during medication, but also the two drugs may have potential mutual reactions. On the one hand, it may lead to a decrease in drug activity, and on the other hand, the reaction products may produce toxic side effects on the human body. (2) The latest research found that, through evolution, the main protease, which was originally thought to be highly conserved, has developed drug resistance mutations. Among them, some drug resistance mutants have mutations that occur precisely at the E166 site of the main protease, and the binding of nematidine to the main protease is highly dependent on the E166 site. This leads to a cliff-like drop in the inhibitory activity of nematidine against drug resistance mutants with E166 mutations, and it can no longer show the original high-efficiency inhibitory activity.

[0008] Therefore, there is an urgent need to develop antiviral drugs that are metabolically stable and have certain anti-drug resistance activity. Summary of the Invention

[0009] The purpose of this invention is to provide a metabolically stable anti-drug-resistant main protease inhibitor and its application in the preparation of antiviral drugs. This main protease inhibitor is not only metabolically stable, but also has certain anti-drug-resistant activity.

[0010] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows: A metabolically stable inhibitor of a drug-resistant main protease, the general structural formula of which is as follows: (I); In formula (Ⅰ), Group A (i.e., group A) is selected from... R1 is selected from unsubstituted or substituent C1-C9 hydrocarbon groups, 6-membered heterocyclic groups, bicyclic or tricyclic structures containing heteroatoms, or unsubstituted or substituent C1-C9 hydrocarbon groups, 6-membered heterocyclic groups, bicyclic or tricyclic structures containing heteroatoms; R2 is selected from unsubstituted or substituent C1-C9 hydrocarbon groups, 6-membered heterocyclic groups, bicyclic or tricyclic structures containing heteroatoms. 14 Hydrocarbon groups, 5-6 membered heterocyclic groups; 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 hydrogen, methyl, or 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 unsubstituted or substituent 5-6 membered heterocyclic groups, bicyclic or tricyclic structures containing heteroatoms. R5 is selected from cyano, unsubstituted or substituted carbonyl groups; Group C (i.e., group C) is selected from -CH2- Unsubstituted or substituent pyridine ring; wherein, ring D is selected from unsubstituted or substituent R7 benzene ring, pyridine ring, pyrimidine ring, pyrazine ring, imidazole ring, pyrrole ring, thiophene ring, furan ring; R6, R7, R8 and R9 are independently selected from hydrogen, cyano, methyl, amino, halogen, halomethyl, unsubstituted or substituent alkyl, hydroxyalkyl, aminoalkyl, five-membered nitrogen heterocycle.

[0011] In the main protease inhibitor of the present invention, the group C 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 the present invention 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 the present invention.

[0012] Meanwhile, the group C has the characteristic of being "difficult to oxidize"; therefore, the main protease inhibitor of the present invention can tolerate the metabolism of hepatic drug-metabolizing enzymes. Thus, when using the main protease inhibitor of the present invention, not only can the dosage of the drug be reduced, but there is also no need to use hepatic drug-metabolizing enzyme inhibitors in combination, which is beneficial to reduce the toxic side effects such as the decline in liver function caused by medication.

[0013] Based on the optimization of group C, this invention also optimizes group A and ring B. Group A can not only further enhance the inhibitory activity of the main protease inhibitor on the main protease, but also enhance the anti-drug resistance activity and metabolic stability of the main protease inhibitor; while ring B mainly plays the role of further enhancing the inhibitory activity of the main protease inhibitor on the main protease.

[0014] Preferably, in group A of the aforementioned main protease inhibitor, the hydrocarbon group, hydrocarbon ether, indole, C1-C7 hydrocarbon group, 6-membered heterocyclic group, C1-C 14 The substituents on the hydrocarbon group or the 5-6 membered heterocyclic group are selected from hydrogen, halogen, methyl, methoxy, halomethyl, and halomethoxy.

[0015] As a further preferred option, group A is selected. , ; Among them, R1 is selected from ; R2 is selected from .

[0016] Preferably, among the above-mentioned main protease inhibitors, R3 is selected from... R4 is selected from .

[0017] Preferably, in the above-mentioned main protease inhibitors, the substituents on the B ring are selected from hydrogen, halogen, hydroxyl, methyl, halomethyl, methoxy, halomethoxy, -C(CH3)3, and -C(CD3)3.

[0018] As a further preferred option, among the aforementioned main protease inhibitors, ring B is selected from... .

[0019] Preferably, among the above-mentioned main protease inhibitors, R5 is selected from... .

[0020] Preferably, in the group C of the main protease inhibitor, the substituent on the pyridine ring is selected from hydrogen, halogen, methyl, halomethyl, or cyano.

[0021] As a further preferred embodiment, in the above-mentioned main protease inhibitors, the C group is selected from... ; Among them, rings D and E are independently selected. ; R6, R7, R8, and R9 are selected independently. .

[0022] As a further preferred embodiment, the above-mentioned main protease inhibitor has at least one of the following structural formulas:

[0023]

[0024]

[0025] The aforementioned main protease inhibitors all exhibited targeted inhibitory effects on the main protease to varying degrees. Specifically, the inhibition constant against the wild-type main protease of the novel coronavirus was as low as 0.583 nM (the inhibition constant of nematidine was 5.10 nM), the inhibition constant against the M49L / E166A resistant mutant of the main protease was as low as 1.38 nM (the inhibition constant of nematidine was 352 nM), the inhibition constant against the L50F / E166A / L167F resistant mutant of the main protease was as low as 41.8 nM (the inhibition constant of nematidine was 1799 nM), and the inhibition constant against the wild-type main protease of feline coronavirus (FIPV) was as low as 0.044 nM (the inhibition constant of nematidine was 35.9 nM).

[0026] Based on this, the present invention also provides the application of the above-mentioned main protease inhibitor in the preparation of antiviral drugs.

[0027] The present invention also provides an antiviral drug comprising the above-mentioned main protease inhibitor or its racemic form, enantiomer, non-corresponding isomer or pharmaceutically acceptable salt and pharmaceutically acceptable excipients.

[0028] The antiviral drug of the present invention inhibits the virus by inhibiting the activity of the main protease. Therefore, the present invention has no special requirements for the type of virus and the host (which can be human or animal). As long as the virus depends on the main protease to complete replication, it is applicable to the present invention. The viruses that depend on the main protease to replicate include wild-type or drug-resistant mutant strains of coronavirus, calicivirus, norovirus, hepatitis A virus, human rhinovirus and porcine transmissible gastroenteritis virus.

[0029] Available dosage forms of the antiviral drugs of the present invention include oral, injectable, spray, powder, emulsion, suspension or transdermal formulations.

[0030] Compared with the prior art, the beneficial effects of the present invention are reflected in: (1) In the main protease inhibitor of the present invention, the group C can not only effectively bind to the substrate recognition pocket of the main protease, but 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 the present invention is not afraid of main protease mutation and can show basically consistent inhibitory activity (or a small decrease in inhibitory activity) to both mutant and wild-type strains. Coronaviruses will not develop resistance to the main protease inhibitor of the present invention.

[0031] (2) In the main protease inhibitor of the present invention, the group C has the characteristic of being "difficult to oxidize"; therefore, the main protease inhibitor of the present invention can tolerate the metabolism of liver drug-metabolizing enzymes. Thus, when using the main protease inhibitor of the present invention, 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.

[0032] (3) The main protease inhibitors of the present invention all exhibit targeted inhibition of 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).

[0033] (4) The antiviral drug of the present invention achieves the purpose of inhibiting the virus by inhibiting the activity of the main protease. Therefore, there are no special requirements for the type of virus 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 the present invention. Attached Figure Description

[0034] Figure 1 This is a synthetic route diagram for Hit 3-1, the main protease inhibitor of this invention; Figure 2 Mass spectrometry analysis of Nirmatrelvir; Figure 3 This is a mass spectrometry analysis of Hit 3-1, the main protease inhibitor of this invention. Figure 4 and Figure 5 The images show the synthetic route and mass spectrometry analysis of Hit 3-2, the main protease inhibitor of this invention. Figure 6 and Figure 7 The images show the synthetic route and mass spectrometry analysis of Hit 3-3, the main protease inhibitor of this invention. Figure 8 and Figure 9 The images show the synthetic route and mass spectrometry analysis of Hit 3-4, the main protease inhibitor of this invention. Figure 10 and Figure 11 The images show the synthetic route and mass spectrometry analysis of Hit 3-5, the main protease inhibitor of this invention. Figure 12 and Figure 13 The images show the synthetic route and mass spectrometry analysis of Hit 3-6, the main protease inhibitor of this invention. Figure 14 and Figure 15 The images show the synthetic route and mass spectrometry analysis of Hit 3-7, the main protease inhibitor of this invention. Figure 16 and Figure 17 The images show the synthetic route and mass spectrometry analysis of Hit 3-8, the main protease inhibitor of this invention. Figure 18 and Figure 19 The images show the synthetic route and mass spectrometry analysis of Hit 3-11, the main protease inhibitor of this invention. Figure 20 and Figure 21 The images show the synthetic route and mass spectrometry analysis of Hit 3-16, the main protease inhibitor of this invention. Figure 22 This is a general synthetic route diagram for the main protease inhibitors Hit 3-15, 3-17~3-28 of this invention; Figures 23 to 35 The mass spectrometry chromatograms 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 this invention are shown in sequence. Figure 36 This is a general synthetic route diagram for the main protease inhibitors Hit 3-9, Hit 3-30 and Hit 3-31 of this invention; Figures 37 to 39 The mass spectrometry results for the main protease inhibitors Hit 3-9, Hit 3-30, and Hit 3-31 of this invention are shown in sequence. Figure 40 This is a general synthetic route diagram for the main protease inhibitors Hit 3-32, Hit 3-33 and Hit 3-34 of this invention; Figures 41 to 44 The mass spectrometry results are shown in order for the main protease inhibitors Hit 3-32, Hit 3-33, Hit 3-34 and Hit 3-36 of this invention. Figure 45The figure shows the cytotoxicity test results 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 this invention; Wherein, Concentration represents concentration, and Cell viability represents cell viability; the same applies below. Figure 46 The figure shows the stability test results of the main protease inhibitors Hit 3-2, Hit 3-3, Hit 3-6, Hit 3-32, Hit 3-33 and Hit 3-34 in liver phase I metabolism. Wherein, Time represents degradation time, Remaining Compound represents the remaining main protease inhibitor, and Testosterone represents testosterone; Figure 47 The main protease inhibitors Hit 3-4, Hit 3-5, Hit 3-6, Hit 3-32, Hit 3-33, and Hit 3-34 of this invention are for the action against the main protease mutant M of the novel coronavirus. ProM49L / E166A The results of the inhibitory activity analysis; Wherein, Percent velocity represents the percentage of enzyme catalytic rate, and the same applies below; Figure 48 The main protease inhibitors Hit 3-5, Hit 3-6, Hit 3-32, Hit 3-33, and Hit 3-34 of this invention are effective against the main protease mutant M of the novel coronavirus. ProL50F / E166A / L167F The results of the inhibitory activity analysis; Figure 49 The results show the inhibitory activity of the main protease inhibitors Hit 3-6, Hit 3-32, Hit 3-33 and Hit 3-34 of this invention against the main protease of feline coronavirus. Detailed Implementation

[0035] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0036] The main protease inhibitors involved in the embodiments of the present invention have the following general structural formula: (I); In formula (Ⅰ), Group A (i.e., group A) is selected from... R1 is selected from unsubstituted or substituent C1-C9 hydrocarbon groups, 6-membered heterocyclic groups, bicyclic or tricyclic structures containing heteroatoms, or unsubstituted or substituent C1-C9 hydrocarbon groups, 6-membered heterocyclic groups, bicyclic or tricyclic structures containing heteroatoms; R2 is selected from unsubstituted or substituent C1-C9 hydrocarbon groups, 6-membered heterocyclic groups, bicyclic or tricyclic structures containing heteroatoms. 14 Hydrocarbon groups, 5-6 membered heterocyclic groups; 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 hydrogen, methyl, or 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 unsubstituted or substituent 5-6 membered heterocyclic groups, bicyclic or tricyclic structures containing heteroatoms. R5 is selected from cyano, unsubstituted or substituted carbonyl groups; Group C (i.e., group C) is selected from -CH2- Unsubstituted or substituent pyridine ring; wherein, ring D is selected from unsubstituted or substituent R7 benzene ring, pyridine ring, pyrimidine ring, pyrazine ring, imidazole ring, pyrrole ring, thiophene ring, furan ring; R6, R7, R8 and R9 are independently selected from hydrogen, cyano, methyl, amino, halogen, halomethyl, unsubstituted or substituent alkyl, hydroxyalkyl, aminoalkyl, five-membered nitrogen heterocycle.

[0037] The examples listed below will illustrate the specific structures and synthesis methods of each main protease inhibitor that conforms to this general formula.

[0038] 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))butyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Hit3-1) This embodiment describes a main protease inhibitor, Hit 3-1, whose structural formula is as follows: (1) The preparation method of the main protease inhibitor Hit 3-1 includes the following steps (reaction route as follows): Figure 1 (as shown) (1) First, dissolve (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (50 mg, 0.1372 mmol, 1.0 eq.) and ethyl 2-amino-3-(pyridin-2-yl)propionate dihydrochloride (40.3 mg, 0.1509 mg, 1.1 eq.) in dichloromethane, then add 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.) was stirred overnight at room temperature, and 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 evaporated to dryness, and the residue was reconstituted with 25% acetonitrile aqueous solution. The residue was purified by preparative high performance liquid chromatography (HPLC) using an SPX-C18 reversed-phase column. The target product fraction was collected and lyophilized to obtain the intermediate ethyl 2-((1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-formamido)-3-(pyridin-2-yl)propionate. (2) The intermediate obtained in step (1) was dissolved in 7M ammonia-methanol solution and stirred under sealed conditions at room temperature for 48 hours. During this period, the amine-ester exchange reaction was monitored by LC-MS. After the reaction was complete, the reaction solution was evaporated to dryness, redissolved in DCM, and then Burgess reagent (~2.5 eq.) was added and stirred at room temperature for 3 hours. After the reaction was complete, the reaction solution was evaporated to dryness and purified by preparative high performance liquid chromatography (HPLC) using an SPX-C18 reversed-phase column. The target product fraction was collected and lyophilized to obtain the final product Hit3-1 (mass spectrometry analysis results are shown in Figure 1). Figure 3 ).

[0039] The main protease inhibitor Hit3-1 appeared as a light brown solid, with a yield of 64% and a purity of approximately 97.32%. RT = 3.775 min; HRMS: m / z = 494.23706 [M+H] + ; 1 H 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).

[0040] 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)butyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Hit 3-2) This embodiment describes a main protease inhibitor, Hit 3-2, whose structural formula is as follows: (2); The preparation method of the main protease inhibitor Hit 3-2 includes the following steps (synthetic route as follows): Figure 4 (as shown) (1) Dissolve (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butyryl)-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)propionate methyl ester (35 mg, 0.1509, 1.1 eq.) in dichloromethane, and add EDCI (37 mg, 0.1921 mmol, 1.4 eq.) and DMAP (20 mg, 0. 1647 mmol (1.2 eq.) was stirred overnight at room temperature, and 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 evaporated to dryness, and the residue was reconstituted with 25% acetonitrile aqueous solution. The residue was purified by preparative high performance liquid chromatography (HPLC) using an SPX-C18 reversed-phase column. The target product fraction was collected and lyophilized to obtain the intermediate methyl(S)-2-((1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamido)-3-(5-methylpyridin-2-yl)propionate. (2) The intermediate obtained in step (1) was dissolved in 7M ammonia-methanol solution and stirred in a sealed container at room temperature for 48h. During this period, the amine-ester exchange reaction was monitored by LC-MS. After the reaction was complete, the reaction solution was evaporated to dryness, DCM was added to redissolve the solution, and Burgess reagent (~2.5 eq.) was added and stirred at room temperature for 3h. After the reaction was complete, the reaction solution was evaporated to dryness and purified by preparative high performance liquid chromatography (HPLC) using an SPX-C18 reversed-phase preparative column. The target product fraction was collected and lyophilized to finally obtain the final product Hit3-2 (mass spectrometry analysis results are shown in 5).

[0041] The main protease inhibitor Hit3-2 appeared as a light yellow solid, with a yield of 72% and a purity of approximately 98.13%. RT = 3.790 min; HRMS: m / z = 508.25402 [M+H] + .

[0042] 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)butyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Hit 3-3) This embodiment describes a main protease inhibitor, Hit 3-3, with the following structural formula: (3).

[0043] The preparation method of the main protease inhibitor Hit 3-3 includes the following steps (synthetic route as follows): Figure 6 (as shown) (1) Dissolve 2-((diphenylmethylene)amino)acetonitrile (440 mg, 2.0 mmol, 1.0 eq.) in dimethylformamide (DMF), and slowly add sodium hydride (60%, 280 mg, 7 mmol, 3.5 eq.) under nitrogen protection. Stir the mixture in an ice bath for 1 h. After the reaction is complete, gradually add 3-(chloromethyl)-5-methylpyridine (356 mg, 2 mmol, 1.0 eq.) and tetrabutylammonium iodide (TBAI, 75 mg) to the reaction mixture. 0.2 mmol (0.1 eq.) was added and the reaction was carried out overnight at 25 °C under nitrogen protection. After the reaction was completed, an appropriate amount of water was added to quench the reaction, ethyl acetate was added, and water and saturated brine were added successively for washing. The organic layer was dried with anhydrous sodium sulfate and then evaporated to dryness. An appropriate amount of tetrahydrofuran hydrochloride solution was added, and the reaction was stirred at room temperature for 3 h. The hydrochloric acid was neutralized with saturated sodium bicarbonate solution. 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)propionitrile. (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)butyryl)-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 mixture was stirred overnight at room temperature. The reaction progress was monitored using liquid chromatography-mass spectrometry (LC-MS). After the reaction was complete, the DCM in the reaction solution was evaporated to dryness, and the residue was redissolved in 25% acetonitrile aqueous solution. The residue was purified by preparative high performance liquid chromatography (HPLC) using an SPX-C18 reversed-phase column. The target product fraction was collected and lyophilized to obtain the final product Hit3-3 (mass spectrometry analysis results are shown in [link to product description]). Figure 7 ).

[0044] The main protease inhibitor Hit3-3 appeared as a light yellow solid, with a yield of 43% and a purity of approximately 91.31%. RT = 7.207 min; HRMS: m / z = 508.25579 [M+H] + ; 1 H NMR (600 MHz, DMSO- d 6) δ 9.39 (d, J = 8.3Hz, 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).

[0045] 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)butyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Hit 3-4) This embodiment describes a main protease inhibitor, Hit 3-4, with the following structural formula: (4).

[0046] The preparation method of the main protease inhibitor Hit 3-4 includes the following steps (synthetic route as follows): Figure 8 (as shown) (1) Dissolve (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butyryl)-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)propionate methyl ester (41.35 mg, 0.1647 mmol, 1.2 eq.) in dichloromethane, and add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 37 mg, 0.1921 mmol, 1.4 eq.) and 4-dimethylaminopyridine (DMAP, 20 mg, 0.164 mmol, 1.4 eq.). 7 mmol (1.2 eq.) was stirred overnight at room temperature, and 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 evaporated to dryness, and the residue was reconstituted with 40% acetonitrile aqueous solution. The residue was purified by preparative high performance liquid chromatography (HPLC) using an SPX-C18 reversed-phase column. The target product fraction was collected and lyophilized to obtain the intermediate methyl(S)-3-(5-chloropyridin-2-yl)-2-((1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carbamate)propionate. (2) The intermediate obtained in step (1) was dissolved in 7M ammonia-methanol solution and stirred under sealed conditions at room temperature for 48 hours. During this period, the amine-ester exchange reaction was monitored by LC-MS. After the reaction was complete, the reaction solution was evaporated to dryness, reconstituted with DCM, and Burgess reagent (~2.5 eq.) was added and stirred at room temperature for 3 hours. After the reaction was complete, the reaction solution was evaporated to dryness and purified by preparative high performance liquid chromatography (HPLC) using an SPX-C18 reversed-phase column. The target product fraction was collected and lyophilized to obtain the final product Hit3-4 (mass spectrometry analysis results are shown in [link to mass spectrometry analysis]). Figure 9 ).

[0047] The final product, Hit3-4, was a yellow solid with a yield of 79% and a purity of approximately 97.42%. The reaction time (RT) was 4.642 min, and the HRMS result was m / z = 528.19526 [M+H]. + .

[0048] Example 5 – Synthesis of (1R,2S,5S)-N-(cyano(pyridin-3-yl)methyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Hit 3-5) This embodiment describes a main protease inhibitor, Hit 3-5, with the following structural formula: (5).

[0049] The preparation method of the main protease inhibitor Hit 3-5 includes the following steps (synthetic route as follows): Figure 10 (as shown) (1) Nicotinaldehyde (1000 mg, 9.34 mmol, 1.0 eq.) was dissolved in 10 mL of ammonia methanol solution, and ammonium chloride (1018 mg, 18.67 mmol, 2.0 eq.) was added. The mixture was stirred at room temperature for 1 h. Trimethylsilyl cyanide (TMSCN, 2316 mg, 23.34 mmol, 2.5 eq.) was added dropwise to the reaction system under ice bath. After the addition was complete, the reaction system was stirred at room temperature overnight. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, 10 mL of water was added to quench the reaction. The mixture was extracted with DCM. The organic layer was dried with anhydrous sodium sulfate and concentrated by rotary evaporation. The intermediate 2-amino-2-(pyridin-3-yl)acetonitrile was purified by silica gel column chromatography with MeOH / DCM (1:10) as the developing solvent. The intermediate was a yellowish-brown oil. (2) The intermediates 2-amino-2-(pyridin-3-yl)acetonitrile (30 mg, 0.225 mmol, 1.0 eq.) and (1R,2S,5S)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (91 mg, 0.248 mmol, 1.1 eq.) obtained in step (1) were dissolved in DCM, and EDCI (48 mg, 0.248 mmol, 1.1 eq.) and DMAP (11 mg, 0.0902 mmol, 0.4 eq.) were added. The mixture was stirred overnight at room temperature, and the reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the final product Hit 3-5 was obtained by preparative thin-layer chromatography using MeOH / DCM (1:20) as the developing solvent (mass spectrometry analysis results are shown in [link to mass spectrometry analysis]). Figure 11 ).

[0050] The final product, Hit 3-5, was a pale yellow solid with a yield of 62% and a purity of approximately 97.05%. The reaction time (RT) was 3.859 min. HRMS showed m / z = 480.22977 [M+H]. + ; 1 H 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).

[0051] Example 6 – Synthesis of (1R,2S,5S)-N-(cyano(isoquinoline-4-yl)methyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Hit 3-6) This embodiment describes a main protease inhibitor, Hit 3-6, with the following structural formula: (6).

[0052] The synthetic route for the main protease inhibitor Hit 3-6 is shown below. Figure 12 Specifically, it includes the following steps: (1) Dissolve 4-isoquinoline formaldehyde (500 mg, 6.36 mmol, 1.0 eq.) in 10 mL of ammonia methanol solution and add ammonium chloride (347 mg, 12.7 mmol, 2.0 eq.). Stir at room temperature for 1 h. Add TMSCN (789 mg, 15.9 mmol, 2.5 eq.) dropwise to the reaction system under ice bath. After the addition is complete, stir the reaction system at room temperature overnight. Monitor the reaction progress by thin-layer chromatography. After the reaction is complete, add 10 mL of water to quench the reaction. Extract with DCM. Dry the organic layer with anhydrous sodium sulfate and concentrate it to dryness. Purify the intermediate 2-amino-2-(isoquinoline-4-yl)acetonitrile by silica gel column chromatography with MeOH / DCM (1:10) as the developing solvent. The solid is yellow. (2) 2-Amino-2-(isoquinoline-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)butyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid (88 mg, 0.240 mmol, 1.1 eq.) were dissolved in DCM, and EDCI (47 mg, 0.240 mmol, 1.1 eq.) and DMAP (11 mg, 0.0874 mmol, 0.4 eq.) were added. The mixture was stirred overnight at room temperature, and the reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the final product Hit3-6 was obtained by preparative thin-layer chromatography using MeOH / DCM (1:20) as the developing solvent (mass spectrometry analysis results are shown in [link to mass spectrometry analysis]). Figure 13 It appears as a white solid with a yield of 68% and a purity of approximately 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 = 9.4Hz, 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).

[0053] Example 7 – Synthesis of ((2S)-1-((cyano(pyridin-3-yl)methyl)amino)-4-methyl-1-oxopentane-2-yl)carbamate (Hit 3-7) This embodiment describes a main protease inhibitor, Hit 3-7, with the following structural formula: (7).

[0054] The synthetic route for the main protease inhibitor Hit 3-7 is shown below. Figure 14 Specifically, it includes the following steps: First, the intermediate 2-amino-2-(pyridin-3-yl)acetonitrile was synthesized using the same method as in step (1) of Example 5. 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 mmol, 1.1 eq.) were dissolved in DCM, and EDCI (48 mg, 0.248 mmol, 1.1 eq.) and DMAP (11 mg, 0.0902 mmol, 0.4 eq.) were added. The mixture was stirred overnight at room temperature, and the reaction progress was monitored using thin-layer chromatography. After the reaction was completed, the final product Hit 3-7 was obtained by purification using a preparative thin-layer chromatography plate with MeOH / DCM (1:20) as the developing solvent (mass spectrometry analysis results are shown in [link to mass spectrometry analysis]). Figure 15 It appears as a white solid with a yield of 69% and a purity of approximately 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).

[0055] Example 8 – Synthesis of ((2S)-1-((cyano(isoquinoline-4-yl)methyl)amino)-4-methyl-1-oxopentane-2-yl)carbamate (Hit 3-8) This embodiment describes a main protease inhibitor, Hit 3-8, with the following structural formula: (8).

[0056] The synthetic route for the main protease inhibitor Hit 3-8 is shown below. Figure 16 Specifically, it includes the following steps: First, the intermediate 2-amino-2-(isoquinoline-4-yl)acetonitrile was synthesized using the same method as in step (1) of Example 6. Then, 2-amino-2-(isoquinoline-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, and EDCI (58 mg, 0.300 mmol, 1.1 eq.) and DMAP (14 mg, 0.109 mmol, 0.4 eq.) were added. The mixture was stirred overnight at room temperature, and the reaction progress was monitored using thin-layer chromatography. After the reaction was completed, the final product Hit 3-8 was obtained by purification using a preparative thin-layer chromatography plate with MeOH / DCM (1:20) as the developing solvent (mass spectrometry analysis results are shown in [link to mass spectrometry analysis]). Figure 17 It appears as a white solid with a yield of 64% and a purity of approximately 99.46%. 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).

[0057] Example 9 – Synthesis of (6S)-N-(cyano(isoquinoline-4-yl)methyl)-5-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butyryl)-5-azaspiro[2,4]heptane-6-carboxamide (Hit 3-11) This embodiment describes a main protease inhibitor, Hit 3-11, with the following structural formula: (11).

[0058] The synthetic route for the main protease inhibitor Hit 3-11 is shown below. Figure 18 Specifically, it includes the following steps: First, the intermediate 2-amino-2-(isoquinoline-4-yl)acetonitrile was synthesized according to the method in Example 6; then, 2-amino-2-(isoquinoline-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, and EDCI (200 mg, 1.02 mmol, 1.1 eq.) and DMAP (50 mg, 0.371 mmol, 1.02 mmol, 1.1 eq.) were added. 0.4 eq.), and the reaction was stirred overnight at room temperature. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the intermediate (6S)-6-((cyano(isoquinoline-4-yl)methyl)carbamoyl)-5-azaspiro[2.4]heptane-5-carboxylic acid tert-butyl ester was obtained by preparative thin-layer chromatography with MeOH / DCM (1:20) as the developing solvent, with a yield of 81%. The intermediate was dissolved in 2 mL of DCM, and 3 mL of dioxane solution (4 M, 4 M) of hydrochloric acid was slowly added dropwise with stirring. 15.0 eq.), the reaction was stirred at room temperature for about 2 h. After the reaction was completed, the reaction solution was evaporated under reduced pressure. The product was extracted with DCM and washed with saturated sodium bicarbonate aqueous solution and saturated brine. The organic layer was dried with anhydrous sodium sulfate and concentrated to obtain the intermediate (6S)-N-(cyano(isoquinoline-4-yl)methyl)-5-azaspiro[2.4]heptane-6-carboxamide; (6S)-N-(cyano(isoquinoline-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)butyric acid (25 mg, 0.108 mmol, 1.1 eq.) were dissolved in DCM, and EDCI (21 mg, 0.108 mmol, 1.1 eq.) and DMAP (12 mg, 0.098 mmol, 1.0 eq.), and stirred overnight at room temperature. The reaction progress was monitored using thin-layer chromatography. After the reaction was completed, the final product Hit 3-11 was purified by preparative thin-layer chromatography using MeOH / DCM (1:20) as the developing solvent (mass spectrometry analysis results are shown in [link to mass spectrometry analysis]). Figure 19 It appears as a white solid with a yield of 34% and a purity of approximately 98.44%. RT = 3.919 min; HRMS: m / z = 516.22919 [M+H] + ; 1 H 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).

[0059] Example 10 – Synthesis of (6S)-N-(cyano(isoquinoline-4-yl)methyl)-5-((S)-2-cyclohexyl-2-(2,2,2-trifluoroacetamido)acetyl)-5-azaspiro[2.4]heptane-6-carboxamide (Hit 3-16) This embodiment describes a main protease inhibitor, Hit 3-16, with the following structural formula: (12).

[0060] The synthetic route for the main protease inhibitor Hit 3-16 is shown below. Figure 20 Specifically, it includes the following steps: The intermediate (6S)-N-(cyano(isoquinoline-4-yl)methyl)-5-azaspiro[2.4]heptane-6-carboxamide was first synthesized according to the method in Example 9; then, (6S)-N-(cyano(isoquinoline-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, and EDCI (21 mg, 0.108 mmol, 1.1 eq.) and DMAP (12 mg, 0.098 mmol, 1.1 eq.) were added. 1.0 eq. was stirred overnight at room temperature, and the reaction progress was monitored using thin-layer chromatography. After the reaction was completed, the final product Hit 3-16 was purified by preparative thin-layer chromatography using MeOH / DCM (1:20) as the developing solvent (mass spectrometry analysis results are shown in [link to mass spectrometry analysis]). Figure 21 It appears as a white solid with a yield of 53% and a purity of approximately 99.78%. RT = 4.224 min; HRMS: m / z = 542.24554 [M+H] + .

[0061] Examples 11-22 – Synthesis of main protease inhibitors Hit 3-15, 3-17-3-28 The structural formulas of the main protease inhibitors Hit 3-15, 3-17~3-28 are shown in Table 1.

[0062] Table 1

[0063] As shown in Table 1, the main protease inhibitors Hit 3-15 and 3-17~3-28 share the same backbone, differing only in the structure of the R1 group; therefore, their synthetic routes are also similar. The general synthetic routes for the main protease inhibitors Hit 3-15 and 3-17~3-28 are as follows: Figure 22 As shown, the specific steps include: (1) The intermediate (6S)-N-(cyano(isoquinoline-4-yl)methyl)-5-azaspiro[2.4]heptane-6-carboxamide was synthesized according to the method in Example 9; (2) Dissolve (6S)-N-(cyano(isoquinoline-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-dimethylbutyric acid (775 mg, 3.35 mmol, 1.5 eq.) in DCM, and add EDCI (643 mg, 3.35 mmol, 1.5 eq.) and DMAP (273 mg, 2.23 mmol, 1.0 eq.), and stirred overnight at room temperature. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the intermediate tert-butyl((2S)-1-((6S)-6-((cyano(isoquinoline-4-yl)methyl)carbamoyl)-5-azaspiro[2.4]hept-5-yl)-3,3-dimethyl-1-oxobut-2-yl)carbamate was purified by silica gel column chromatography with MeOH / DCM (1:20) as the developing solvent, with a yield of 64%. The intermediate was dissolved in 6 mL of DCM, and 6 mL of dioxane solution (4 M, 2.23 mmol, 1.0 eq.) of hydrochloric acid was slowly added dropwise with stirring. 15.0 eq.), the reaction was stirred at room temperature for about 2 h. After the reaction was completed, the reaction solution was evaporated under reduced pressure. The product was extracted with DCM and washed with saturated sodium bicarbonate aqueous solution and saturated brine. The organic layer was dried with anhydrous sodium sulfate and concentrated to obtain the intermediate (6S)-5-((S)-2-amino-3,3-dimethylbutyryl)-N-(cyano(isoquinoline-4-yl)methyl)-5-azaspiro[2.4]heptane-6-carboxamide, with a yield of 89%; (3) Based on the structure of the final product, weigh appropriate amounts of (6S)-5-((S)-2-amino-3,3-dimethylbutyryl)-N-(cyano(isoquinoline-4-yl)methyl)-5-azaspiro[2.4]heptane-6-carboxamide and carry out condensation reactions with different substrates under the catalysis of the corresponding catalysts to obtain the target product.

[0064] The specific implementation of step (3) above will be described in the following text, while steps (1) and (2) are general.

[0065] Example 11 – Synthesis of (6S)-5-((S)-2-(2-chloro-2,2-difluoroacetamyl)-3,3-dimethylbutyryl)-N-(cyano(isoquinoline-4-yl)methyl)-5-azaspiro[2.4]heptane-6-carboxamide (Hit 3-22) In the synthetic route of the main protease inhibitor Hit 3-22 in this embodiment, step (3) specifically includes: (6S)-5-((S)-2-amino-3,3-dimethylbutyryl)-N-(cyano(isoquinoline-4-yl)methyl)-5-azaspiro[2.4]heptane-6-carboxamide (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 mixture was stirred overnight at room temperature, and the reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the final product Hit 3-22 was obtained by preparative thin-layer chromatography using MeOH / DCM (1:20) as the developing solvent (mass spectrometry analysis results are shown in [link to mass spectrometry analysis]). Figure 23 It appears as a white solid with a yield of 45% and a purity of approximately 99.63%. RT = 4.128 min; HRMS: m / z = 532.19982 [M+H]. + ; 1 H 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).

[0066] Example 12 – Synthesis of (6S)-N-(cyano(isoquinoline-4-yl)methyl)-5-((S)-3,3-dimethyl-2-(2,2,2-trichloroacetamido)butyryl)-5-azaspiro[2,4]heptane-6-carboxamide (Hit 3-23) In the synthetic route of the main protease inhibitor Hit 3-23 in this embodiment, step (3) specifically includes: (6S)-5-((S)-2-amino-3,3-dimethylbutyryl)-N-(cyano(isoquinoline-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, and triethylamine (40 mg, 0.382 mmol, 4.0 eq.) was added dropwise. The mixture was stirred overnight at room temperature, and the reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the final product Hit 3-23 was obtained by preparative thin-layer chromatography using MeOH / DCM (1:20) as the developing solvent (mass spectrometry analysis results are shown in [link to mass spectrometry analysis]). Figure 24 It appears as a white solid with a yield of 34% and a purity of approximately 98.50%. RT = 4.406 min; HRMS: m / z = 564.14137 [M+H] + .

[0067] Example 13 – Synthesis of (6S)-N-(cyano(isoquinoline-4-yl)methyl)-5-((S)-2-(2-(3,4-dichlorophenyl)acetamyl)-3,3-dimethylbutyryl)-5-azaspiro[2.4]heptane-6-carboxamide (Hit 3-17) In the synthetic route of the main protease inhibitor Hit 3-17 in this embodiment, step (3) specifically includes: (6S)-5-((S)-2-amino-3,3-dimethylbutyryl)-N-(cyano(isoquinoline-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, and EDCI (16 mg, 0.0787 mmol, 1.1 eq.) and DMAP (9 mg, 0.0716 mmol, 1.0 eq.) were added. The mixture was stirred overnight at room temperature, and the reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the final product Hit 3-17 was obtained by preparative thin-layer chromatography using MeOH / DCM (1:20) as the developing solvent (mass spectrometry analysis results are shown in [link to mass spectrometry analysis]). Figure 25 It appears as a yellow solid, with a yield of 61% and a purity of approximately 98.86%. RT = 4.508 min; HRMS: m / z = 606.21196 [M+H] + .

[0068] Example 14 – Synthesis of (6S)-N-(cyano(isoquinoline-4-yl)methyl)-5-((S)-2-(((3,4-dichlorophenyl)methyl)sulfonamido)-3,3-dimethylbutyryl)-5-azaspiro[2.4]heptane-6-carboxamide (Hit 3-15) In the synthetic route of the main protease inhibitor Hit 3-15 in this embodiment, step (3) specifically includes: (6S)-5-((S)-2-amino-3,3-dimethylbutyryl)-N-(cyano(isoquinoline-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, and DMAP (2 mg, 0.0135 mmol, 0.2 eq.) and triethylamine (7 mg, 0.0673 mmol, 1.0 eq.) were added. The mixture was stirred overnight at room temperature under nitrogen protection, and the reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the final product Hit 3-15 was obtained by preparative thin-layer chromatography using MeOH / DCM (1:20) as the developing solvent (mass spectrometry analysis results are shown in [link to mass spectrometry analysis]). Figure 26 The product was a light yellow solid with a yield of 63% and a purity of approximately 97.71%. The reaction time (RT) was 4.597 min, and the HRMS result was m / z = 642.17884 [M+H]. + . 1 H 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 = 7.7Hz, 1H), 7.68 (t, J = 7.6 Hz, 1H), 7.45 – 7.37 (m, 3H), 7.16 (dd, J = 8.3, 2.1Hz, 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).

[0069] Example 15 Synthesis of (6S)-N-(cyano(isoquinoline-4-yl)methyl)-5-((S)-2-(1-(3,4-dichlorophenyl)cyclopropane-1-formamide)-3,3-dimethylbutyryl)-5-azaspiro[2.4]heptane-6-carboxamide (Hit 3-18) In the synthetic route of the main protease inhibitor Hit 3-18 in this embodiment, step (3) specifically includes: (6S)-5-((S)-2-amino-3,3-dimethylbutyryl)-N-(cyano(isoquinoline-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, and EDCI (16 mg, 0.0787 mmol, 1.1 eq.) and DMAP (9 mg, 0.0716 mmol, 1.0 eq.) were added. The mixture was stirred overnight at room temperature, and the reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the final product Hit 3-18 was obtained by preparative thin-layer chromatography using MeOH / DCM (1:20) as the developing solvent (mass spectrometry analysis results are shown in [link to mass spectrometry analysis]). Figure 27 It appears as a yellow solid with a yield of 73% and a purity of approximately 99.13%. RT = 4.853 min; HRMS: m / z = 632.22742 [M+H] + .

[0070] Example 16 – Synthesis of (6S)-N-(cyano(isoquinoline-4-yl)methyl)-5-((S)-3,3-dimethyl-2-(1-(3-(trifluoromethyl)phenyl)cyclopropane-1-carboxyamino)butyryl)-5-azaspiro[2.4]heptane-6-carboxamide (Hit 3-19) In the synthetic route of the main protease inhibitor Hit 3-19 in this embodiment, step (3) specifically includes: (6S)-5-((S)-2-amino-3,3-dimethylbutyryl)-N-(cyano(isoquinoline-4-yl)methyl)-5-azaspiro[2.4]heptane-6-carboxamide (30 mg, 0.0716 mmol, 1.0 eq.) and 1-(3-(trifluoromethyl)phenyl)cyclopropane-1-carboxylic acid (20 mg, 0.0787, 1.1 eq.) were dissolved in DCM, and EDCI (16 mg, 0.0787 mmol, 1.1 eq.) and DMAP (9 mg, 0.0716 mmol, 1.0 eq.) were added. The mixture was stirred overnight at room temperature, and the reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the final product Hit 3-19 was obtained by preparative thin-layer chromatography using MeOH / DCM (1:20) as the developing solvent (mass spectrometry analysis results are shown in [link to mass spectrometry analysis]). Figure 28 It appears as a yellow solid with a yield of 75% and a purity of approximately 96.60%. RT = 4.732 min; HRMS: m / z = 632.29300 [M+H] + .

[0071] Example 17 – Synthesis of (6S)-N-(cyano(isoquinoline-4-yl)methyl)-5-((2S)-2-(2,2-difluoro-1-phenylcyclopropane-1-carboxamido)-3,3-dimethylbutyryl)-5-azaspiro[2.4]heptane-6-carboxamide (Hit 3-20) In the synthetic route of the main protease inhibitor Hit 3-20 in this embodiment, step (3) specifically includes: (6S)-5-((S)-2-amino-3,3-dimethylbutyryl)-N-(cyano(isoquinoline-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, and EDCI (16 mg, 0.0787 mmol, 1.1 eq.) and DMAP (9 mg, 0.0716 mmol, 1.0 eq.) were added. The mixture was stirred overnight at room temperature, and the reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the final product Hit 3-20 was obtained by preparative thin-layer chromatography using MeOH / DCM (1:20) as the developing solvent (mass spectrometry analysis results are shown in [link to mass spectrometry analysis]). Figure 29 It appears as a yellow solid with a yield of 77% and a purity of approximately 97.91%. RT = 4.442 min; HRMS: m / z = 600.28617 [M+H] + .

[0072] Example 18 – Synthesis of (6S)-N-(cyano(isoquinoline-4-yl)methyl)-5-((S)-2-(1-(3,4-dichlorophenyl)cyclobutane-1-formamide)-3,3-dimethylbutyryl)-5-azaspiro[2.4]heptane-6-carboxamide (Hit 3-21) In the synthetic route of the main protease inhibitor Hit 3-21 in this embodiment, step (3) specifically includes: (6S)-5-((S)-2-amino-3,3-dimethylbutyryl)-N-(cyano(isoquinoline-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, and EDCI (16 mg, 0.0787 mmol, 1.1 eq.) and DMAP (9 mg, 0.0716 mmol, 1.0 eq.) were added. The mixture was stirred overnight at room temperature, and the reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the final product Hit 3-21 was obtained by preparative thin-layer chromatography using MeOH / DCM (1:20) as the developing solvent (mass spectrometry analysis results are shown in [link to mass spectrometry analysis]). Figure 30 The product was a yellow solid with a yield of 76% and a purity of approximately 96.79%. RT = 4.989 min; HRMS: m / z = 646.24303 [M+H]. + .

[0073] Example 19 – Synthesis of (6S)-N-(cyano(isoquinoline-4-yl)methyl)-5-((S)-2-(cyclopropanecarbamoyl)-3,3-dimethylbutyryl)-5-azaspiro[2.4]heptane-6-carboxamide (Hit 3-24) In the synthetic route of the main protease inhibitor Hit 3-24 in this embodiment, step (3) specifically includes: (6S)-5-((S)-2-amino-3,3-dimethylbutyryl)-N-(cyano(isoquinoline-4-yl)methyl)-5-azaspiro[2.4]heptane-6-carboxamide (40 mg, 0.0954 mmol, 1.0 eq.) and cyclopropanecarboxylic acid (10 mg, 0.105, 1.1 eq.) were dissolved in DCM, and EDCI (22 mg, 0.105 mmol, 1.1 eq.) and DMAP (12 mg, 0.0954 mmol, 1.0 eq.) were added. The mixture was stirred overnight at room temperature, and the reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the final product Hit 3-24 was obtained by preparative thin-layer chromatography using MeOH / DCM (1:20) as the developing solvent (mass spectrometry analysis results are shown in [link to mass spectrometry analysis]). Figure 31 It appears as a yellow solid, with a yield of 73% and a purity of approximately 95.68%. RT = 3.594 min; HRMS: m / z = 488.27203 [M+H] + ; 1 HNMR (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).

[0074] Example 20 – Synthesis of (6S)-N-(cyano(isoquinoline-4-yl)methyl)-5-((2S)-2-(2,2-difluorocyclopropane-1-formamide)-3,3-dimethylbutyryl)-5-azaspiro[2.4]heptane-6-carboxamide (Hit 3-25) In the synthetic route of the main protease inhibitor Hit 3-25 in this embodiment, step (3) specifically includes: (6S)-5-((S)-2-amino-3,3-dimethylbutyryl)-N-(cyano(isoquinoline-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, and EDCI (22 mg, 0.105 mmol, 1.1 eq.) and DMAP (12 mg, 0.0954 mmol, 1.0 eq.) were added. The mixture was stirred overnight at room temperature, and the reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the final product Hit 3-25 was obtained by preparative thin-layer chromatography using MeOH / DCM (1:20) as the developing solvent (mass spectrometry analysis results are shown in [link to mass spectrometry analysis]). Figure 32 It appears as a light yellow solid, with a yield of 98% and a purity of approximately 98.67%. RT = 3.781 min; HRMS: m / z = 524.25396 [M+H] + .

[0075] Example 21 – Synthesis of (6S)-N-(cyano(isoquinoline-4-yl)methyl)-5-((S)-2-(1-cyanocyclopropane-1-formamido)-3,3-dimethylbutyryl)-5-azaspiro[2.4]heptane-6-carboxamide (Hit 3-26) In the synthetic route of the main protease inhibitor Hit 3-26 in this embodiment, step (3) specifically includes: (6S)-5-((S)-2-amino-3,3-dimethylbutyryl)-N-(cyano(isoquinoline-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, and EDCI (22 mg, 0.105 mmol, 1.1 eq.) and DMAP (12 mg, 0.0954 mmol, 1.0 eq.) were added. The mixture was stirred overnight at room temperature, and the reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the final product Hit 3-26 was obtained by preparative thin-layer chromatography using MeOH / DCM (1:20) as the developing solvent (mass spectrometry analysis results are shown in [link to mass spectrometry analysis]). Figure 33 The product was a light yellow solid with a yield of 55% and a purity of approximately 91.86%. The reaction time (RT) was 3.861 min, and the HRMS result was m / z = 513.26923 [M+H]. + .

[0076] Example 22 – Synthesis of (6S)-N-(cyano(isoquinoline-4-yl)methyl)-5-((S)-3,3-dimethyl-2-(1-(trifluoromethyl)cyclopropane-1-formamide)butyryl)-5-azaspiro[2.4]heptane-6-carboxamide (Hit 3-27) In the synthetic route of the main protease inhibitor Hit 3-27 in this embodiment, step (3) specifically includes: (6S)-5-((S)-2-amino-3,3-dimethylbutyryl)-N-(cyano(isoquinoline-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, and EDCI (22 mg, 0.105 mmol, 1.1 eq.) and DMAP (12 mg, 0.0954 mmol, 1.0 eq.) were added. The mixture was stirred overnight at room temperature, and the reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the final product Hit 3-27 was obtained by preparative thin-layer chromatography using MeOH / DCM (1:20) as the developing solvent (mass spectrometry analysis results are shown in [link to mass spectrometry analysis]). Figure 34 It appears as a light yellow solid, with a yield of 65% and a purity of approximately 99.31%. RT = 4.238 min; HRMS: m / z = 556.26093 [M+H] + .

[0077] Example 23 – Synthesis of (6S)-N-(cyano(isoquinoline-4-yl)methyl)-5-((S)-2-(3,3-difluoro-1-(trifluoromethyl)cyclobutane-1-formamide)-3,3-dimethylbutyryl)-5-azaspiro[2.4]heptane-6-carboxamide (Hit 3-28) In the synthetic route of the main protease inhibitor Hit 3-28 in this embodiment, step (3) specifically includes: (6S)-5-((S)-2-amino-3,3-dimethylbutyryl)-N-(cyano(isoquinoline-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, and EDCI (22 mg, 0.105 mmol, 1.1 eq.) and DMAP (12 mg, 0.0954 mmol, 1.0 eq.) were added. The mixture was stirred overnight at room temperature, and the reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the final product Hit 3-28 was obtained by preparative thin-layer chromatography using MeOH / DCM (1:20) as the developing solvent (mass spectrometry analysis results are shown in [link to mass spectrometry analysis]). Figure 35 The product was a light yellow solid with a yield of 51% and a purity of approximately 98.76%. The reaction time (RT) was 4.457 min. HRMS: m / z = 606.26005 [M+H]. + .

[0078] Examples 24-26 – Synthesis of Hit 3-9, 3-30-3-31 The structural formulas of the main protease inhibitors Hit 3-9, 3-30~3-31 are shown in Table 2.

[0079] Table 2

[0080] As can be seen from Table 2, the main protease inhibitors Hit 3-9, 3-30~3-31 have the same skeleton, with only the structure of group A being different. Even among groups A, there are only differences in the substituents, so the synthetic routes are also similar.

[0081] The general synthetic routes for Hit 3-9, 3-30~3-31 are as follows: Figure 36 As shown, the specific steps include: (1) Dissolve (tert-butoxycarbonyl)-L-leucine (274 mg, 1.18 mmol, 1.1 eq.) and 2-amino-2-(isoquinoline-4-yl)acetonitrile (197 mg, 1.08 mmol, 1.0 eq.) in DCM, add EDCI (227 mg, 1.18 mmol, 1.1 eq.) and DMAP (53 mg, 0.430 mmol, 0.4 eq.), stir overnight at room temperature, and monitor the reaction progress using thin-layer chromatography; after the reaction is complete, use MeOH / DCM (1:10) as the developing solvent and purify by silica gel column chromatography to obtain the intermediate ((2S)-1-((cyano(isoquinoline-4-yl)methyl)amino)-4-methyl-1-oxopentane-2-yl)carbamate tert-butyl ester, yield 67%; (2) The intermediate was dissolved in 5 mL of DCM, and 5 mL of dioxane hydrochloric acid solution (4 M, 15.0 eq.) was slowly added dropwise with stirring. The reaction was stirred at room temperature for about 2 h. After the reaction was completed, the reaction solution was evaporated under reduced pressure. The product was extracted with DCM and washed with saturated sodium bicarbonate aqueous solution and saturated brine. The organic layer was dried with anhydrous sodium sulfate and concentrated to obtain the intermediate (2S)-2-amino-N-(cyano(isoquinoline-4-yl)methyl)-4-methylpentanamide, with a yield of 95%. (3) Based on the structure of the final product, weigh an appropriate amount of (2S)-2-amino-N-(cyano(isoquinoline-4-yl)methyl)-4-methylpentanamide and different carboxylates (RCOOH) and carry out amide condensation reaction under EDCI and DMAP catalysis to obtain the target product.

[0082] The following will describe in detail step (3) of the synthesis route of Hit 3-9, 3-30~3-31 in sequence. Steps (1) and (2) are common.

[0083] Example 24 – Synthesis of N-((2S)-1-((cyano(isoquinoline-4-yl)methyl)amino)-4-methyl-1-oxopentane-2-yl)-4-methoxy-1H-indole-2-carboxamide (Hit 3-9) In the synthetic route of the main protease inhibitor Hit 3-9 in this embodiment, step (3) specifically includes: (2S)-2-amino-N-(cyano(isoquinoline-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 mmol, 1.1 eq.) were dissolved in DCM, and EDCI (36 mg, 0.186 mmol, 1.1 eq.) and DMAP (20 mg, 0.169 mmol, 1.0 eq.) were added. The mixture was stirred overnight at room temperature, and the reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the final product Hit 3-9 was obtained by preparative thin-layer chromatography using MeOH / DCM (1:20) as the developing solvent (mass spectrometry analysis results are shown in [link to mass spectrometry analysis]). Figure 37 The product was a dark yellow solid with a yield of 33% and a purity of approximately 92.35%. RT = 3.968 min; HRMS: m / z = 470.21938 [M+H]. + .

[0084] Example 25 – Synthesis of N-((2S)-1-((cyano(isoquinoline-4-yl)methyl)amino)-4-methyl-1-oxopentane-2-yl)-5-(trifluoromethoxy)-1H-indole-2-carboxamide (Hit 3-30) In the synthetic route of the main protease inhibitor Hit 3-30 in this embodiment, step (3) specifically includes: (2S)-2-amino-N-(cyano(isoquinoline-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 mmol, 1.1 eq.) were dissolved in DCM, and EDCI (36 mg, 0.186 mmol, 1.1 eq.) and DMAP (20 mg, 0.169 mmol, 1.0 eq.) were added. The mixture was stirred overnight at room temperature, and the reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the final product Hit 3-30 was obtained by preparative thin-layer chromatography using MeOH / DCM (1:20) as the developing solvent (mass spectrometry analysis results are shown in [link to mass spectrometry analysis]). Figure 38 The product was a yellowish-brown solid with a yield of 28% and a purity of approximately 97.63%. RT = 4.503 min; HRMS: m / z = 524.19302 [M+H]. + .

[0085] Example 26 – Synthesis of 4-chloro-N-((2S)-1-((cyano(isoquinoline-4-yl)methyl)amino)-4-methyl-1-oxopentane-2-yl)-1H-indole-2-carboxamide (Hit 3-31) In the synthetic route of the main protease inhibitor Hit 3-31 in this embodiment, step (3) specifically includes: (2S)-2-amino-N-(cyano(isoquinoline-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 mmol, 1.1 eq.) were dissolved in DCM, and EDCI (36 mg, 0.186 mmol, 1.1 eq.) and DMAP (20 mg, 0.169 mmol, 1.0 eq.) were added. The mixture was stirred overnight at room temperature, and the reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the final product Hit 3-31 was obtained by preparative thin-layer chromatography using MeOH / DCM (1:20) as the developing solvent (mass spectrometry analysis results are shown in [link to mass spectrometry analysis]). Figure 39 It appears as a yellow solid with a yield of 29% and a purity of approximately 93.05%. RT = 4.277 min; HRMS: m / z = 474.17207 [M+H] + .

[0086] Examples 27-29 – Synthesis of Hit 3-32-3-34 The structural formulas of the main protease inhibitors Hit 3-32~3-34 are shown in Table 3 below.

[0087] Table 3

[0088] As shown in Table 3, the main protease inhibitors Hit 3-32~34 and Hit 3-36 have the same skeleton, differing only in the structure of R1 in group A; therefore, their synthetic routes are also similar. Their general synthetic routes are as follows: Figure 40 As shown, the specific steps include: (1) Dissolve (S)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutyric acid (2474 mg, 10.7 mmol, 1.1 eq.) and (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid methyl ester hydrochloride (2000 mg, 9.72 mmol, 1.0 eq.) in DCM, and add EDCI (2054 mg, 10.7 mmol, 1.1 eq.) and DMAP (949 mg, 7.78 mmol, 1.1 eq.). 0.8 eq.), the reaction was stirred overnight at room temperature, and 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-dimethylbutyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate was purified by silica gel column chromatography with ethyl acetate / petroleum ether (1:5) as the developing solvent, with a yield of 46%; (2) The intermediate was dissolved in 10 mL of tetrahydrofuran (THF), and 10 mL of methanol and 10 mL of lithium hydroxide aqueous solution (1 M, 3 eq.) were slowly added dropwise under stirring. The reaction was carried out at room temperature for 48 h. After the reaction was completed, an appropriate amount of 1 M hydrochloric acid aqueous solution was added to adjust the pH of the reaction solution to acidic. The product was extracted with DCM and washed with saturated brine. The organic layer was dried with anhydrous sodium sulfate and concentrated to obtain the intermediate (1R,2S,5S)-3-((S)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid, which is a colorless transparent needle-like crystal. (3) The intermediate was dissolved in DCM, and 1.1 eq. of 2-amino-2-(isoquinoline-4-yl)acetonitrile was added, along with EDCI and DMAP. The mixture was stirred overnight at room temperature. The intermediate was purified by silica gel column chromatography using MeOH / DCM (1:20) as the developing solvent to obtain tert-butyl((2S)-1-((1R,2S,5S)-2-((cyano(isoquinoline-4-yl)methyl)carbamoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hex-3-yl)-3,3-dimethyl-1-oxobut-2-yl)carbamate, which was a white solid with a yield of 73%. The intermediate was dissolved in 10 mL of DCM, and 10 mL of dioxane solution (4M) of hydrochloric acid was slowly added dropwise with stirring. 15.0 eq.), the reaction was stirred at room temperature for about 2 h. After the reaction was completed, the reaction solution was evaporated under reduced pressure. The product was extracted with DCM and washed with saturated sodium bicarbonate aqueous solution and saturated brine. The organic layer was dried with anhydrous sodium sulfate and concentrated to obtain the intermediate (1R,2S,5S)-3-((S)-2-amino-3,3-dimethylbutyryl)-N-(cyano(isoquinoline-4-yl)methyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide, with a yield of 98%; (4) According to the desired final product structure, weigh appropriate amounts of (1R,2S,5S)-3-((S)-2-amino-3,3-dimethylbutyryl)-N-(cyano(isoquinoline-4-yl)methyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide and different carboxylates (RCOOH) for amide condensation reaction under EDCI and DMAP catalysis to obtain the target product.

[0089] The following will describe in detail step (4) of the synthesis route of Hit 3-32~34 and Hit 3-36 in sequence. Steps (1), (2) and (3) are universal.

[0090] Example 27 – Synthesis of (1R,2S,5S)-3-((S)-2-(2-chloro-2,2-difluoroacetamyl)-3,3-dimethylbutyryl)-N-(cyano(isoquinoline-4-yl)methyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Hit 3-32) In the synthetic route of the main protease inhibitor Hit 3-32 in this embodiment, step (4) specifically involves: (1R,2S,5S)-3-((S)-2-amino-3,3-dimethylbutyryl)-N-(cyano(isoquinoline-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, and EDCI (22 mg, 0.111 mmol, 1.2 eq.) and DMAP (11 mg, 0.0923 mmol, 1.0 eq.) were added. The mixture was stirred overnight at room temperature, and the reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the final product Hit 3-32 was obtained by preparative thin-layer chromatography using MeOH / DCM (1:20) as the developing solvent (mass spectrometry analysis results are shown in [link to mass spectrometry analysis]). Figure 41 The product was a light yellow solid with a yield of 36% and a purity of approximately 98.96%. RT = 4.316 min; HRMS: m / z = 546.20875 [M+H]. + .

[0091] Example 28 – Synthesis of (1R,2S,5S)-N-(cyano(isoquinoline-4-yl)methyl)-3-((2S)-2-(2,2-difluorocyclopropane-1-carboxamido)-3,3-dimethylbutyryl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Hit 3-33) In the synthetic route of the main protease inhibitor Hit 3-33 in this embodiment, step (4) specifically involves: (1R,2S,5S)-3-((S)-2-amino-3,3-dimethylbutyryl)-N-(cyano(isoquinoline-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, and EDCI (16 mg, 0.0830 mmol, 1.2 eq.) and DMAP (9 mg, 0.0692 mmol, 1.0 eq.) were added. The mixture was stirred overnight at room temperature, and the reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the final product Hit 3-33 was obtained by preparative thin-layer chromatography using MeOH / DCM (1:20) as the developing solvent (mass spectrometry analysis results are shown in [link to mass spectrometry analysis]). Figure 42 The product was a light yellow solid with a yield of 63% and a purity of approximately 99.63%. The reaction time (RT) was 4.000 min, and the HRMS reading was m / z = 538.26631 [M+H]. + .

[0093] In the synthetic route of the main protease inhibitor Hit 3-34 in this embodiment, step (4) specifically involves: (1R,2S,5S)-3-((S)-2-amino-3,3-dimethylbutyryl)-N-(cyano(isoquinoline-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, and EDCI (22 mg, 0.111 mmol, 1.2 eq.) and DMAP (11 mg, 0.0923 mmol, 1.0 eq.) were added. The mixture was stirred overnight at room temperature, and the reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the final product Hit 3-34 was obtained by preparative thin-layer chromatography using MeOH / DCM (1:20) as the developing solvent (mass spectrometry analysis results are shown in [link to mass spectrometry analysis]). Figure 43 It appears as a light yellow solid, with a yield of 37% and a purity of approximately 99.62%. RT = 4.112 min; HRMS: m / z = 552.27931 [M+H]. + .

[0094] Example 30 – Synthesis of (1R,2S,5S)-3-((S)-2-(2-bromo-2,2-difluoroacetamyl)-3,3-dimethylbutyryl)-N-(cyano(isoquinoline-4-yl)methyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (Hit 3-36) In the synthetic route of the main protease inhibitor Hit 3-36 in this embodiment, step (4) specifically involves: (1R,2S,5S)-3-((S)-2-amino-3,3-dimethylbutyryl)-N-(cyano(isoquinoline-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, and EDCI (46 mg, 0.238 mmol, 1.2 eq.) and DMAP (10 mg, 0.0793 mmol, 0.4 eq.) were added. The mixture was stirred overnight at room temperature, and the reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the final product Hit 3-36 was obtained by preparative thin-layer chromatography using MeOH / DCM (1:20) as the developing solvent (mass spectrometry analysis results are shown in [link to mass spectrometry analysis]). Figure 44The product was a yellow solid with a yield of 21% and a purity of approximately 99.05%. The reaction time (RT) was 4.323 min. HRMS values ​​were: m / z = 590.16092; 592.15943 [M+H]. + .

[0095] Example 31 – Inhibition constants (K) of various main protease inhibitors against coronavirus main protease i ) Measurement Using the main protease of the novel coronavirus (SARS-CoV-2 Mpro) as the test target, the inhibitory activity of the main protease inhibitors synthesized in Examples 1-30 and the positive control Nirmatrelvir against the coronavirus main protease was tested. The test methods are as follows: The main protease was added to a 96-well plate to achieve a final concentration of 100 nM. A series of main protease inhibitors at varying concentrations (adjusted for each inhibitor's activity, 4-fold dilution) were then added. After incubation at 37°C for 5 min, 1 μl of a 1 mM fluorescent peptide substrate was added to each well. The plate was immediately placed in a Tecan microplate reader at 37°C with an excitation wavelength of 340 nm and an emission wavelength of 490 nm. The fluorescence value (RFU) of each well was dynamically monitored over time, with readings every 30 s for a total of 120 readings. The enzyme activity kinetic curve for each well was then obtained. The slope of the linear region in the initial time period of the curve characterizes the enzyme activity in each well; a larger slope indicates stronger catalytic activity. The inhibitory activity of the main protease inhibitors on the main protease was quantified using 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 ); In the formula, control wells refer to wells containing only buffer, main protease, and substrate but no drug; experimental wells refer to wells containing buffer, main protease, substrate, and drug; and solvent background wells refer to wells containing only buffer and substrate.

[0096] Plotting enzyme activity percentage on the ordinate and drug concentration on the x-axis using GraphPad software yields the inhibition curve of the drug on the main protease. Fitting this curve using the Morrison equation allows for the determination of the Kelvin (K) effect of the main protease inhibitor on the enzyme. i The values ​​and test results are shown in Table 4. K i The lower the value, the stronger the inhibitory activity of the main protease inhibitor on the enzyme.

[0097] Table 4

[0098] As can be seen from the test data in Table 4, the main protease inhibitors prepared in Examples 1-30 exhibited inhibitory activity against the main protease to varying degrees. Among them, compared with other main protease inhibitors, Hit 3-6, Hit 3-22, Hit 3-24, Hit 3-25, Hit 3-32, Hit 3-33, Hit 3-34, and Hit 3-36 showed stronger inhibitory activity against the main protease. The K values ​​of Hit 3-6, Hit 3-22, Hit 3-24, Hit 3-25, Hit 3-33, and Hit 3-36 were also significant. i The values ​​are all above 10 0 The nM level was comparable to that of the positive control Nirmatrelvir; while the K levels of Hit 3-32 and Hit 3-34 were... i The values ​​are all above 10 -1 At the nM level, it was significantly superior to the positive control Nirmatrelvir.

[0099] Furthermore, 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 essentially the same structural skeleton, differing only in the C group. Specifically, the C group on Hit 3-1 to 3-4 is an unsubstituted or substituent-containing pyridine ring (-CH2-), while the C group on Hit 3-5 is a pyridine ring, and the C group on Hit 3-6 is a quinoline ring. It can be seen that compared to Hit 3-1 to 3-4, Hit 3-5 exhibits two orders of magnitude higher inhibitory activity against the main protease, indicating that the presence of the methylene group in the C group affects the binding of the compound to the main protease. However, although Hit 3-5 also shows good inhibitory activity against the main protease, it is still inferior to Hit 3-6. This suggests that the quinoline ring binds more readily to the substrate recognition pocket of the main protease than the pyridine ring.

[0100] Combining Tables 3 and 4, it can also be seen that Hit 3-6, Hit 3-32, Hit 3-33, Hit 3-34, and Hit 3-36 have essentially the same structural framework, differing only in R1. However, these four main protease inhibitors exhibit different K+ kinase activity against the main protease. i However, the values ​​differ. This indicates that not only changes in group A affect the inhibitory activity of the main protease inhibitor, but the R1 group in group A also influences its inhibitory activity. Furthermore, Hit 3-22 and Hit 3-32 have essentially the same structural framework, differing only in ring B, yet Hit 3-32 exhibits significantly better inhibitory activity against the main protease than Hit 3-22. This suggests that ring B also contributes to the inhibitory activity of the main protease inhibitor.

[0101] Example 32 – Cytotoxicity assay of the main protease inhibitor of the present invention This embodiment uses the human hepatocyte cell line THLE-2 as an example to test 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. The test method is as follows: Resuscitate and culture the human hepatocyte line THLE-2 at 5 × 10⁻⁶ cells / year. 3 THLE-2 cells were seeded into clear 96-well plates at a cell density of [number] cells / well. After adhesion at 37°C for 24 hours, different concentrations of the test drug were added. Negative control wells and zero-adjustment wells were also included. The negative control wells contained only cells and no drug, while the zero-adjustment wells contained only culture medium. The plates were incubated at 37°C for 24 hours. Finally, 10 μl of WST reagent was added to each well, and the plates were incubated at 37°C for 60 minutes. The OD value was measured at 460 nm using a microplate reader. A higher OD value indicates more surviving cells. Cell viability was calculated using the following formula: Cell viability (%) = (OD) 实验孔 -OD 调零孔 ) / (OD 阴性对照孔 -OD 调零孔 ).

[0102] Test results as follows Figure 45 As shown.

[0103] Depend on Figure 45 As can be seen, 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 did not exhibit cytotoxicity at 50 μM, indicating that these main protease inhibitors have high biocompatibility.

[0104] Example 33 – Stability test of the main protease inhibitor of the present invention on liver phase I metabolism This embodiment uses mouse liver microsomes as the test subject to detect the tolerance of Hit 3-2, Hit 3-3, Hit 3-6, Hit 3-32, Hit 3-33, and Ht3-34, as well as Nirmatrelvir and the positive control testosterone to the phase I metabolic response mediated by mouse liver microsomes. The test method is as follows: Following the experimental procedures described in the instructions for the Phase I metabolic stability kit (Beijing Huizhi Heyuan Biotechnology Co., Ltd.), liver microsomes and NADPH were added sequentially to the main protease inhibitor to be tested. The kit was then placed in a 37°C water bath, and samples were taken at specific time points. The remaining main protease inhibitor at each time point was quantified using a liquid chromatography-mass spectrometry (LC-MS) system. The peak area was used as the calculation data, with the zero-time concentration of the test drug taken as 100%. The percentage of remaining main protease inhibitor (Remaining compound (%)) was obtained by comparing the concentration at each time point with the zero-time concentration. A degradation curve of the main protease inhibitor was obtained by plotting the degradation curves over time. By comparing the degradation curves, the tolerance of each test drug to phase I metabolism of liver microsomes can be compared. The test results are shown in [Figure 1]. Figure 46 .

[0105] Depend on Figure 46 As can be seen, after 60 minutes of degradation, the effective amount of Nirmatrelvir (the undegraded portion) decreased to approximately 60%, while the effective amounts of Hit 3-2 and Hit 3-3 remained above 90%. After 180 minutes of degradation, the effective amount of Nirmatrelvir decreased to approximately 10%, while the effective amount of Hit 3-6 was approximately 40%, the effective amounts of Hit 3-32 and Hit 3-34 were approximately 50%, and the effective amount of Hit 3-33 was approximately 70%. It can be observed that, compared to Nirmatrelvir, the main protease inhibitor of this invention exhibits stronger tolerance to hepatic microsomal phase I metabolism and is less easily metabolized by the liver.

[0106] Example 34—The main protease inhibitor of the present invention is resistant to the coronavirus main protease mutant M. proM49L / E166A The inhibition constant (K) i ) Measurement This embodiment uses the drug-resistant mutant M of the main protease of the novel coronavirus. proM49L / E166A As test subjects, the inhibitory activity of Hit 3-4, Hit 3-5, Hit 3-6, Hit 3-32, Hit 3-33, and Hit 3-34, as well as the positive control Nirmatrelvir, against the main protease of coronavirus was tested. The test method is as follows: The main protease was added to a 96-well plate to achieve a final concentration of 200 nM. A series of main protease inhibitors at varying concentrations (adjusted for each inhibitor's activity, 4-fold dilution) were then added. After incubation at 37°C for 5 min, 1 μl of a 1 mM fluorescent peptide substrate was added to each well. The plate was immediately placed in a Tecan microplate reader at 37°C with an excitation wavelength of 340 nm and an emission wavelength of 490 nm. The fluorescence value (RFU) of each well was dynamically monitored over time, with readings every 30 s for a total of 120 readings. The enzyme activity kinetic curve for each well was then obtained. The slope of the linear region in the initial time period of the curve characterizes the enzyme activity in each well; a larger slope indicates stronger catalytic activity. The inhibitory activity of the main protease inhibitors on the main protease was quantified using 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 ); In the formula, control wells refer to wells containing only buffer, main protease, and substrate but no drug; experimental wells refer to wells containing buffer, main protease, substrate, and drug; and solvent background wells refer to wells containing only buffer and substrate.

[0107] Plotting enzyme activity percentage on the ordinate and drug concentration on the x-axis using GraphPad software yields the inhibition curve of the drug on the main protease. Fitting this curve using the Morrison equation allows for the determination of the Kelvin (K) effect of the main protease inhibitor on the enzyme. i Value, K i The lower the value, the stronger the inhibitory activity of the main protease inhibitor; test results are shown in [link to results]. Figure 47 .

[0108] Depend on Figure 47 It is evident that Nirmatrelvir is resistant to the main protease mutant M. proM49L / E166A K i The value is only 352 nM, which is similar to the K value for the wild-type main protease. i The value (5.10 nM) is about 70 times higher, which is two orders of magnitude higher.

[0109] Hit 3-4 main protease resistant mutant M proM49L / E166A K i The value was 30.3 μM, which is related to its K value against the wild-type main protease. i The value (2708 nM) is comparable; Hit 3-5 is resistant to the main protease mutant M. proM49L / E166A K i The value is 542 nM, compared to its K value for the wild-type main protease.i The value (29.0 nM) increased by an order of magnitude, indicating that both the pyridine ring and the -CH2-pyridine ring contribute to improving the anti-drug resistance activity of the main protease, with the -CH2-pyridine ring contributing more.

[0110] Hit 3-6, a main protease-resistant mutant M... proM49L / E166A K i The value was 8.62 nM (K value against wild-type main protease). i The value is 3.08 nM), Hit 3-32 main protease resistant mutant M proM49L / E166A K i The value is 2.18 nM (K value against wild-type main protease). i The value was 0.513 nM), and Hit 3-33 was a main protease-resistant mutant M. proM49L / E166A K i The value is 1.76 nM (K value against wild-type main protease). i The value is 1.23 nM), and the Hit3-34 main protease resistant mutant M proM49L / E166A K i The value is 10.6 nM (K value against wild-type main protease). i The value is 0.983 nM.

[0111] It can be seen that when the main protease undergoes the M49L / E166A resistance mutation, the inhibitory activity of Nirmatrelvir against the main protease drops sharply, and its inhibitory activity against the resistant mutant is poor; while Hit 3-6, Hit 3-32, Hit 3-33, and Hit 3-34 inhibit both the wild-type main protease and the main protease resistance mutant M49L / E166A. proM49L / E166A The inhibitory activity remained at a very effective level.

[0112] Among them, compared with the inhibitory activity against the wild-type main protease, except for Hit 3-32 and Hit3-34 against the main protease resistant mutant M... proM49L / E166A The inhibitory activity of Hit 3-6 decreased by an order of magnitude compared to the drug-resistant mutant M. proM49L / E166A The inhibitory activity was slightly reduced, while Hit 3-33 showed resistance to the main protease mutant M. proM49L / E166A K i The values ​​are very close and almost unchanged, indicating that the quinoline ring is more conducive to improving the anti-drug resistance activity of the main protease than the pyridine ring and the -CH2-pyridine ring. In addition, R1 in group A also affects the anti-drug resistance activity of the main protease inhibitor.

[0113] Example 35—The main protease inhibitor of the present invention resists coronavirus main protease mutant M proL50F / E166A / L167FThe inhibition constant (K) i ) Measurement This embodiment uses the drug-resistant mutant M of the main protease of the novel coronavirus. proL50F / E166A / L167F As test subjects, the inhibitory activity of Hit 3-5, Hit 3-6, Hit 3-32, Hit 3-33, and Hit 3-34, as well as the positive control Nirmatrelvir, against the main protease of coronavirus was tested. The test method is as follows: The main protease was added to a 96-well plate to achieve a final concentration of 400 nM. A series of main protease inhibitors at varying concentrations (adjusted for each inhibitor's activity, 4-fold dilution) were then added. After incubation at 37°C for 5 min, 1 μl of a 1 mM fluorescent peptide substrate was added to each well. The plate was immediately placed in a Tecan microplate reader at 37°C with an excitation wavelength of 340 nm and an emission wavelength of 490 nm. The fluorescence value (RFU) of each well was dynamically monitored over time, with readings every 30 s for a total of 120 readings. The enzyme activity kinetic curve for each well was then obtained. The slope of the linear region in the initial time period of the curve characterizes the enzyme activity in each well; a larger slope indicates stronger catalytic activity. The inhibitory activity of the main protease inhibitors on the main protease was quantified using 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 ); In the formula, control wells refer to wells containing only buffer, main protease, and substrate but no drug; experimental wells refer to wells containing buffer, main protease, substrate, and drug; and solvent background wells refer to wells containing only buffer and substrate.

[0114] Test results are available Figure 48 .

[0115] Depend on Figure 48 It is evident that Nirmatrelvir is resistant to the main protease mutant M. proL50F / E166A / L167F K i The value is only 1799 nM, which is similar to the K value for the wild-type main protease. i The value (5.10 nM) is about 353 times higher, which is three orders of magnitude higher.

[0116] Hit 3-5 main protease resistant mutant M proL50F / E166A / L167F K i The value was 2.16 μM, which is consistent with the K value of the wild-type main protease. i The value (29.0 nM) differed by two orders of magnitude; while the Hit 3-6 main protease resistance mutant M...proM49L / E166A K i The value was 41.8 nM (K value against wild-type main protease). i The value is 3.08 nM), Hit 3-32 main protease resistant mutant M proM49L / E166A K i The value was 43.1 nM (K value against wild-type main protease). i The value was 0.513 nM), and Hit 3-33 was a main protease-resistant mutant M. proM49L / E166A K i The value was 75.8 nM (K value against wild-type main protease). i The value is 1.23 nM), and the Hit3-34 main protease resistant mutant M proM49L / E166A K i The value was 99.6 nM (K value against wild-type main protease). i The value is 0.983 nM.

[0117] It can be seen that when the main protease undergoes the M49L / E166A resistance mutation, the inhibitory activity of Nirmatrelvir against the main protease drops sharply, and its inhibitory activity against the resistant mutant is poor; while Hit 3-6, Hit 3-32, Hit 3-33, and Hit 3-34, although inhibiting the M49L / E166A resistance mutation, show a more pronounced effect. proL50F / E166A / L167F The inhibitory activity decreased, but it is worth noting that it remained at a relatively effective level; this again shows that both the pyridine ring and the quinoline ring contribute to improving the anti-drug resistance activity of the main protease inhibitor.

[0118] Example 36—The inhibition constant (K) of the main protease inhibitor of the present invention against the feline coronavirus main protease i ) Measurement This embodiment uses the main protease of feline coronavirus (FIPV M) pro Using Hit 3-6, Hit 3-32, Hit 3-33, and Hit 3-34, as well as the positive control Nirmatrelvir, as test subjects, the inhibitory activity against the coronavirus main protease was tested. The test method is as follows: The main protease was added to a 96-well plate to achieve a final concentration of 100 nM. A series of main protease inhibitors at varying concentrations (adjusted for each inhibitor's activity, 4-fold dilution) were then added. After incubation at 37°C for 5 min, 1 μl of a 1 mM fluorescent peptide substrate was added to each well. The plate was immediately placed in a Tecan microplate reader at 37°C with an excitation wavelength of 340 nm and an emission wavelength of 490 nm. The fluorescence value (RFU) of each well was dynamically monitored over time, with readings every 30 s for a total of 120 readings. The enzyme activity kinetic curve for each well was then obtained. The slope of the linear region in the initial time period of the curve characterizes the enzyme activity in each well; a larger slope indicates stronger catalytic activity. The inhibitory activity of the main protease inhibitors on the main protease was quantified using 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 ); In the formula, control wells refer to wells containing only buffer, main protease, and substrate but no drug; experimental wells refer to wells containing buffer, main protease, substrate, and drug; and solvent background wells refer to wells containing only buffer and substrate.

[0119] Test results are available Figure 49 .

[0120] Depend on Figure 49 It is evident that Nirmatrelvir inhibits the K+ of the main protease of feline coronavirus. i The value was 35.9 nM; while Hit 3-6 showed a K value of 35.9 nM against the main protease of feline coronavirus. i The value was 0.273 nM, and the K value of Hit 3-32 against the main protease of feline coronavirus was... i The value was 0.0939 nM, and the K value of Hit 3-33 against the main protease of feline coronavirus was... i The value is 0.0712 nM, and the K value of Hit3-34 against the main protease of feline coronavirus is... i The value is 0.0440 nM. It can be seen that the inhibitory activity of the main protease inhibitor of this invention against the feline coronavirus main protease is 10 times that of Nirmatrelvir. 2 Or 10 3 The result shows that the main protease inhibitor of the present invention has broad inhibitory activity against coronaviruses.

[0121] Example 37-105 – Synthesis of main protease inhibitors Hit 3-10, 3-12~3-14, 3-29, 3-35~3-98 The chemical structures of the main protease inhibitors Hit 3-10, 3-12~3-14, 3-29, 3-35~3-98 are shown in Table 5.

[0122] As can be seen from Table 5, the structures of the listed main protease inhibitors can all be found in similar structures in Examples 1-30, so the synthesis methods are also roughly the same. The differences (including raw materials and dosages) are obvious to those skilled in the art and can be implemented without any doubt, so they will not be described in detail in this example.

[0123] Table 5

[0124]

[0125]

[0127]

[0128]

[0129]

Claims

1. A metabolically stable inhibitor of drug-resistant main protease, characterized in that, The structural formula is as follows: 。 2. The application of the metabolically stable anti-drug-resistant main protease inhibitor as described in claim 1 in the preparation of antiviral drugs, characterized in that, The antiviral drug described herein is used to inhibit wild-type or drug-resistant mutant strains of coronavirus.

3. An antiviral drug, characterized in that, It includes the metabolically stable, drug-resistant main protease inhibitor or pharmaceutically acceptable salt as described in claim 1, and pharmaceutically acceptable excipients.

4. The antiviral drug as described in claim 3, characterized in that, Dosage forms include oral preparations, injections, sprays, powders, emulsions, suspensions, or transdermal preparations.

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