A compound, composition and use thereof for viral 3cl protease inhibitors

Abstract

The application discloses a compound with a structural formula I, wherein the compound has good 3CL protease inhibitory activity, has significant proliferation inhibitory activity on virus-infected cells, and has potential application value in treating diseases related to virus infection. The compound has good solubility and permeability, good in-vivo metabolic stability, high in-vivo exposure, and high bioavailability, and is a potential drug compound.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of drug molecule research and development, and particularly relates to a compound for a viral 3CL protease inhibitor and application thereof. BACKGROUND

[0002] The Orthocoronavirinae, commonly known as coronaviruses, are a group of viruses that can infect mammals and birds, belonging to the order Nidovirales and family Coronaviridae, and are positive-sense single-stranded RNA viruses with an envelope. For a long time, coronaviruses have been considered as important pathogens causing respiratory and gastrointestinal diseases in birds and mammals. Before SARS-CoV-2, six coronaviruses have been found to infect humans: HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). The first four are restrictive endemic strains that cause common cold, while the latter two can cause severe respiratory epidemics. SARS is a non-typical viral respiratory disease that caused 8098 cases and 774 deaths during a large-scale outbreak in 2003 in 17 countries (9.6% mortality rate). MERS is a zoonotic respiratory infection that initially broke out in the Middle East in 2012. According to the records of the World Health Organization, MERS coronavirus has so far caused 2494 cases of infection and 858 deaths. Based on the high prevalence and wide distribution of coronaviruses, as well as their genetic diversity and frequent genome recombination, they pose a constant threat to humans.

[0003] Due to the current public health emergency caused by COVID-19, the US Food and Drug Administration (FDA) has issued an emergency use authorization (EUA) for the experimental drug remdesivir. However, the effectiveness and safety of remdesivir are still controversial. The Japanese Ministry of Health, Labor and Welfare has also approved remdesivir as a treatment for COVID-19. In addition, α-interferon combined with anti-human immunodeficiency virus (HIV) drugs lopinavir / ritonavir Combination of Remdesivir and Baricitinib has been used to treat COVID-19, but the evidence of its effectiveness is still limited, and the drug can have toxic side effects. Recently, Merck reported interim data from its Phase III clinical trial of Molnupiravir, the first oral drug it developed to treat patients with mild to moderate COVID-19 pneumonia. The treatment group had a hospitalization or mortality rate of 7.3% (28 / 385), while the control group had a hospitalization or mortality rate of 7.3% (28 / 385), and the mortality rate was 14.1% (53 / 377). The latest report shows that the drug is only 30% effective, much lower than the 50% reported in the interim. Therefore, there is an urgent need to develop highly specific and effective anti-coronavirus drugs targeting key viral targets, especially SARS-CoV-2, which is of great significance for the prevention and treatment of future recurrence of coronavirus epidemics.

[0004] 3CL protease (also known as main protease) plays a crucial role in the life cycle of coronavirus and is highly conserved, and inhibition of 3CL protease can effectively block viral RNA replication and transcription, further blocking viral proliferation, which is an important target for the development of antiviral drugs. An oral 3CL protease inhibitor PF-07321332 (Comparative Compound 1) developed by Pfizer is currently in clinical trials for the treatment of COVID-19 in combination with ritonavir. The currently published interim data from the Phase II / III clinical trial shows that it can reduce the risk of hospitalization or death by 89%, but in view of the experience of Molnupiravir, further investigation of its effectiveness is needed, and in order to slow down the metabolism and decomposition of the small molecule viral inhibitor PF-07321332 in the body when administered orally, low-dose ritonavir needs to be used in combination for better efficacy in clinical treatment. Therefore, there is an urgent need to develop new viral 3CL protease inhibitors with stronger efficacy, better drugability, and the ability to overcome drug resistance after long-term administration, as a new effective treatment for anti-coronavirus therapy. SUMMARY

[0005] The present application relates to pharmaceutically active compounds and their pharmaceutically acceptable salts, which can be used for the treatment of respiratory diseases caused by viral infections.

[0006] The present application provides a compound having structural formula I, an isomer thereof, or a pharmaceutically acceptable salt thereof.

[0007]

[0008] wherein:

[0009] Ring A is selected from

[0010] L1 is selected from

[0011] L2 is selected from 5, 6 and 7-membered cyclic lactam;

[0012] R1is selected from alkynyl, C 1-3 alkyl, C 3-7 cycloalkyl, 4- to 7-membered heterocycloalkyl, 5- to 7-membered heteroaryl, which alkynyl, alkyl, cycloalkyl, heterocycloalkyl, heteroaryl can be optionally substituted by one or more C 1-3 alkyl, C 1-3 haloalkyl, halogen, amino, hydroxy, cyano, nitro, phenyl ring;

[0013] R2is selected from hydrogen, C 1-3 alkyl;

[0014] R3and R4are each independently selected from hydrogen, C 1-5 alkyl, C 3-7 cycloalkyl;

[0015] R2and R3may together with the atoms to which they are attached form a 4- or 5- membered heterocycloalkyl, which heterocycloalkyl can be optionally substituted by one or more halogen;

[0016] R3and R4may together with the atoms to which they are attached form a C 3-5 cycloalkyl;

[0017] R5, R6and R7are each independently selected from hydrogen, C 1-5 alkyl, C 1-5 alkoxy, which alkyl, alkoxy can be optionally substituted by one or more halogen;

[0018] R8and R9are each independently selected from hydrogen, C 1-3 alkyl;

[0019] R5and R7may together with the atoms to which they are attached form a 5- or 6- membered ring;

[0020] R6and R8may together with the atoms to which they are attached form a C 3-5 cycloalkyl;

[0021] R7and R8may together with the atoms to which they are attached form a C 3-5 cycloalkyl, which cycloalkyl can be optionally substituted by one or more C 1-3 alkyl, halogen;

[0022] R8and R9may together with the atoms to which they are attached form a 5- or 6- membered ring;

[0023] when ring A is R3and R4are each hydrogen, and the other is and the other is hydrogen, R1is not CF3.

[0024] In some embodiments of the application, the compound has the structure of Formula II, an isomer thereof, or a pharmaceutically acceptable salt thereof:

[0025]

[0026]

[0027] wherein:

[0028]

[0029] In some embodiments of the application, the compound has the structure of Formula II, an isomer thereof, or a pharmaceutically acceptable salt thereof:

[0030] R5, R6, and R7 are each independently selected from hydrogen, C 1-5 alkyl, C 1-5 alkoxy, the alkyl, alkoxy groups are optionally substituted with one or more halogens;

[0031] R8 is selected from hydrogen;

[0032] R9 is selected from hydrogen, C 1-3 alkyl;

[0033] In some embodiments of the application, the compound has the structure of Formula II, an isomer thereof, or a pharmaceutically acceptable salt thereof:

[0034] R5 and R7, together with the atoms to which they are attached, form a 5- or 6-membered ring;

[0035] R6 is selected from hydrogen, C 1-5 alkyl, C 1-5 alkoxy, the alkyl, alkoxy groups are optionally substituted with one or more halogens;

[0036] R8 is selected from hydrogen;

[0037] R9 is selected from hydrogen, C 1-3 alkyl;

[0038] In some embodiments of the application, the compound has the structure of Formula II, an isomer thereof, or a pharmaceutically acceptable salt thereof:

[0039] R5 and R7 are each independently selected from hydrogen, C 1-5 alkyl, C 1-5 alkoxy, the alkyl, alkoxy groups are optionally substituted with one or more halogens;

[0040] R6 and R8, together with the atoms to which they are attached, form a C 3-5 cycloalkyl group;

[0041] R9 is selected from hydrogen, C 1-3 alkyl;

[0042] In some embodiments of the application, the compounds are characterized in that:

[0043] R5is selected from hydrogen, C 1-5 alkyl, C 1-5 alkoxy, said alkyl, alkoxy groups can optionally be substituted by one or more halogen;

[0044] R6is selected from hydrogen;

[0045] R7and R8, together with the atoms to which they are attached, form a C 3-5 cycloalkyl group, which can optionally be substituted by one or more C 1-3 alkyl, halogen;

[0046] R9is selected from hydrogen, C 1-3 alkyl;

[0047] In some embodiments of the application, the compounds are characterized in that:

[0048] R5, R6and R7are each independently selected from hydrogen, C 1-5 alkyl, C 1-5 alkoxy, said alkyl, alkoxy groups can optionally be substituted by one or more halogen;

[0049] R8and R9, together with the atoms to which they are attached, can form a 5- or 6- membered ring.

[0050] In some embodiments of the application, the compounds are characterized in that ring A is selected from:

[0051]

[0052] In some embodiments of the application, the compounds are characterized in that L1is selected from

[0053] In some embodiments of the application, the compounds are characterized in that L2is selected from

[0054] In some embodiments of the application, the compounds are characterized in that R1is selected from -CF3,

[0055] In some embodiments of the application, the compounds have the following structure:

[0056]

[0057] R1is preferably fluorinated cyclopropyl (mono-fluoro substitution, di-fluoro substitution, which can occur on the same carbon atom or on different carbon atoms), trifluoromethyl.

[0058] In some embodiments of the present application, the compound is:

[0059]

[0060]

[0061] In some embodiments of the present application, a pharmaceutical composition is provided comprising the compound, isomer thereof, pharmaceutically acceptable salt, solvate or prodrug thereof of any one of the above.

[0062] In some embodiments of the present application, the use of the compound, isomer thereof, pharmaceutically acceptable salt, solvate or prodrug thereof of any one of the above, is characterized in that it is for the preparation of an inhibitor or a medicament for inhibiting viral 3CL protease.

[0063] In some embodiments of the present application, the use of the compound, isomer thereof, pharmaceutically acceptable salt, solvate or prodrug thereof of any one of the above, is characterized in that the medicament is for the treatment of a disease caused by viral infection.

[0064] In some embodiments of the present application, the use of the compound, isomer thereof, pharmaceutically acceptable salt, solvate or prodrug thereof of any one of the above, is characterized in that the disease is selected from pneumonia or asymptomatic infection caused by SARS-CoV-2 coronavirus infection.

[0065] Definitions and Descriptions

[0066] The following terms and phrases used herein are intended to have the following meanings unless otherwise indicated. A particular term or phrase should not be considered indefinite or unclear if not specifically defined, but should be understood according to the ordinary meaning.

[0067] The term "pharmaceutically acceptable" pertains to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0068] The term "isomers" means that the compounds of the present application can exist in specific geometric or stereoisomeric forms. The present application contemplates all such compounds, including cis- and trans-forms, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)- isomers, (L)-isomers, as well as the racemic mixtures and other mixtures of the enantiomers or diastereomers, and all such isomers, whether free or in salts, or hydrates, or solvates, are included within the scope of the present application.

[0069] The term "enantiomers" or "optical isomers" means stereoisomers that are mirror images of one another unless otherwise specified.

[0070] The term "cis-trans isomers" or "geometric isomers" means stereoisomers that arise from the restriction on free rotation about a single bond or ring-forming carbon atom single bond.

[0071] The term "diastereomers" means stereoisomers that have two or more chiral centers and are not mirror images of one another unless otherwise specified.

[0072] "(D)" or "(+)" means dextrorotary and "(L)" or "(-)" means levorotary, "(DL)" or "(rac)" means racemic unless otherwise noted.

[0073] Unless otherwise indicated, the absolute configuration of a stereogenic center is indicated by a wedged and dashed line The relative configuration of a stereogenic center is indicated by a straight and dashed line A wavy line indicates a wedged or dashed line or a wavy line indicates a straight and dashed line

[0074] The term "cis-trans isomers" means configurations that arise from the restriction on free rotation about a double bond or ring-forming carbon atom single bond.

[0075] The term "enantiomers" means stereoisomers that are mirror images of one another.

[0076] The term "diastereomers" means stereoisomers that have two or more chiral centers and are not mirror images of one another.

[0077] The term "pharmaceutically acceptable salt" means a salt of a compound of this application that is found to be pharmaceutically acceptable, i.e., water or oil-soluble or dispersible, non-toxic, and efficacious, etc. Alkali addition salts can be prepared from the neutral forms of the compounds of the present application by contacting the neutral forms with a sufficient amount of the desired alkali base in pure solution or in a suitable inert diluent. Pharmaceutically acceptable alkali addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. Acid addition salts are prepared by contacting the neutral form of the compound of the present application with a sufficient amount of the desired acid in pure solution or in a suitable inert diluent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts selected from hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, nitrate, carbonate, bicarbonate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate; organic acid salts selected from formate, acetate, octanoate, isobutyrate, oxalate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, succinate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, salicylate, picrate, glutamate, ascorbate, camphorate, camphorsulfonate, and the like.

[0078] "Optional" or "optionally" means that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0079] The term "substituted" means that any one or more hydrogen atoms on the designated atom is replaced with a substituent group, which can include deuterium and variations of hydrogen, provided that the valency of the designated atom is not exceeded and that the substituted compound is stable. When the substituent is oxygen (i.e., =0), it means that two hydrogen atoms are replaced. Oxygen substitution does not occur on aromatic groups. The term "optionally substituted" means that the group can or can not be substituted, and unless otherwise specified, the kinds and number of substituents are any that are chemically feasible.

[0080] When any variable (e.g., R) occurs more than one time in a compound, its definition in each instance is independent of the definition of the other occurrences. Thus, for example, if a group is substituted with 0-2 R groups, then the group is optionally substituted with up to two R groups, and at each occurrence R is selected independently. In addition, combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0081] Unless otherwise specified, the term "alkyl" is used to denote a straight-chain or branched saturated hydrocarbon group, which may be monosubstituted (e.g., -CH₂F) or polysubstituted (e.g., -CF₃), and may be monovalent (e.g., methyl), divalent (e.g., methylene), or polyvalent (e.g., methine). Unless otherwise specified, C 1-3 Alkyl groups include C1, C2, and C3 alkyl groups; C 1-5 Alkyl groups include C1, C2, C3, C4, and C5 alkyl groups. Examples of alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, s-butyl, t-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), etc.

[0082] Unless otherwise specified, the term "cycloalkyl" includes any stable cyclic or polycyclic hydrocarbon group in which all carbon atoms are saturated, and which may be monosubstituted or polysubstituted, and may be divalent or polyvalent. Unless otherwise specified, C 3-7 Cycloalkyl groups include C3, C4, C5, C6, and C7 cycloalkyl groups; C 3-5 Cycloalkyl groups include C3, C4, and C5 cycloalkyl groups. Examples of these cycloalkyl groups include, but are not limited to, cyclopropyl, norbornel, [2.2.2]bicyclooctane, [4.4.0]bicyclodecane, etc.

[0083] Unless otherwise specified, the term "heterocyclic alkyl" means a cycloalkyl group containing 1 to 4 heteroatoms selected from N, O and S.

[0084] Unless otherwise specified, the term "cyclic lactam" is meant as The structures shown are for 5-, 6-, and 7-membered lactams, where n is a positive integer. Unless otherwise specified, lactams with 5, 6, and 7-membered rings are represented as follows: The structure shown.

[0085] Unless otherwise specified, the term "halogen" itself or as part of another substituent means a fluorine (F), chlorine (Cl), bromine (Br) or iodine (I) atom.

[0086] Unless otherwise specified, the term "alkoxy" means an alkyl group attached to the remainder of the molecule via an oxygen atom, wherein the alkyl group has the meaning as described herein. Unless otherwise specified, C 1-5 Alkoxy groups include C1, C2, C3, C4, and C5 alkoxy groups. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and S-pentoxy. The alkoxy group may optionally be substituted by one or more substituents described in this invention.

[0087] Unless otherwise specified, the term "amino" refers to -NH2, -NH (alkyl) or -N (alkyl) (alkyl).

[0088] Unless otherwise specified, the term "aryl" means a polyunsaturated, aromatic hydrocarbon monocyclic ring, which can be mono- or poly-substituted.

[0089] Unless otherwise specified, the term "heteroaryl" means an aryl group containing 1 to 4 heteroatoms selected from N, O and S.

[0090] Unless otherwise specified, the term "ring" includes any chemically stable existing cycloalkyl, heterocycloalkyl, aryl, heteroaryl.

[0091] The compound of the present application has good 3CL protease inhibitory activity, significant proliferation inhibitory activity on virus-infected cells, and potential application value in the treatment of diseases related to viral infection. The compound of the present application has good solubility and permeability, good in vivo metabolic stability, high in vivo exposure and high bioavailability, and is a potential drug compound. DETAILED DESCRIPTION

[0092] The present application is described in detail below by way of examples, but does not mean any unfavorable limitation of the present application. The present application has been described in detail herein, and specific implementation manners thereof have also been disclosed. It will be obvious to those skilled in the art to make various changes and improvements to the specific implementation manners of the present application without departing from the spirit and scope of the present application.

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099] Example 1. (2S,4R)-4-(tert-Butoxy)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-1-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)pyrrolidine-2-carboxamide (A1)

[0100]

[0101]

[0102] Step 1: (2S,4S)-2-((tert-Butoxycarbonyl)amino)-4-(cyanomethyl)glutarate dimethyl ester (compounds 1-3)

[0103] Dimethyl(tert-butyloxycarbonyl)-L-glutamic acid (1-2) (6 g, 21.8 mmol) was dissolved in 60 mL of anhydrous tetrahydrofuran. Under nitrogen protection, a tetrahydrofuran solution (47 mL, 1 mol / L) of lithium hexamethyldisilamide (LHDMS) was slowly added dropwise. After the addition was complete, the mixture was stirred at -78 °C for 1 hour. Then, 2-bromoacetonitrile (1.62 mL, 23.3 mmol) was slowly added dropwise. After the addition was complete, the reaction was continued at -78 °C for 1-2 hours. After the reaction was complete, pre-cooled methanol (3 mL) and pre-cooled tetrahydrofuran acetate solution (3 mL acetic acid dissolved in 20 mL tetrahydrofuran) were added dropwise to quench the reaction. The mixture was stirred for 30 minutes and then moved to room temperature with stirring. After the system was restored to room temperature, the solvent was removed by vacuum distillation. The residue was dissolved in ethyl acetate, washed with saturated brine, and the resulting organic phase was dried over anhydrous Na₂SO₄ and concentrated. The residue was purified by column chromatography to give 6 g of a pale yellow oil, with a yield of 88%. 1 H NMR(400MHz, CDCl3)δ5.05-5.17(1H),4.31-4.44(1H),3.76-3.76(3H),3.75-3.75(3H), 2.81-2.90(1H),2.76-2.81(1H),2.10-2.22(2H),1.43-1.45(9H); ESI-MS: m / z=315[M+1] + .

[0104] Step 2: Methyl (S)-2-(Boc-amino)-3-[(S)-2-oxo-3-pyrrolidinyl]propionate (intermediates 1-4)

[0105] Intermediate 1-3 (6 g, 19 mmol) was dissolved in 90 mL of methanol, and CHCl3 (8 mL) and PtO2 (345 mg, 1.52 mmol) were added. The mixture was stirred for 24 hours under a hydrogen atmosphere. Platinum dioxide solid was filtered off, and AcONa (3.1 g, 38 mmol) was added. The mixture was heated under reflux for 6 hours. After the reaction was complete, the solvent was removed by vacuum distillation. The residue was extracted with ethyl acetate and saturated brine. The organic phase was dried over anhydrous Na2SO4 and concentrated. The residue was purified by column chromatography to give 2.5 g of compound 1-4, yield 46%, ESI-MS: m / z = 287 [M+1]. + .

[0106] Step 3: (S)-2-(Boc-amino)-3-[(S)-2-oxo-3-pyrrolidinyl]propionic acid formamide (1-5)

[0107] Compound 1-4 (2.5 g, 8.74 mmol) was dissolved in ammonia methanol solution (7M; 150 mL, 1.05 mol) and stirred at room temperature for 48 hours. It was concentrated under reduced pressure to obtain 2.36 g of yellow solid with a yield of 100%, ESI-MS: m / z = 272 [M+1] + .

[0108] Step 4: tert-Butyl ((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)carbamate (Intermediate 1-6)

[0109] Compound 1-5 (2.36 g, 8.71 mmol) was dissolved in 20 mL of dichloromethane and 4.16 g of Burgess reagent (17.42 mmol) was added and stirred for 6 hours. It was extracted with dichloromethane and saturated brine. The organic phase was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 2.5 g of compound 1-4 with a yield of 46%, ESI-MS: m / z = 254 [M+1] + .

[0110] Step 5: (S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoic acid (Intermediate 1-9)

[0111] (S)-2-amino-3,3-dimethylbutanoic acid (1-8) (1.3 g, 1 mmol) was dissolved in 20 mL of methanol and cooled to 0°C. 3 mL of triethylamine was added dropwise. After the reaction system was slowly raised to room temperature, 1.7 g of ethyl trifluoroacetate (1-7) was added and stirred overnight. After the reaction was completed, the reaction liquid was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain 1.9 g of compound 1-9 with a yield of 86%. 1 HNMR (400 MHz, CDCl3) δ 7.32-7.42 (1H), 4.13-4.20 (1H), 0.99-1.01 (9H), ESI-MS: m / z = 228 [M+1] + .

[0112] Step 6: (S)-2-amino-3-((S)-2-oxo-3-pyrrolidinyl)methylaminopropanenitrile (Intermediate 1-10)

[0113] Intermediate 1-6 (506 mg, 2 mmol) was dissolved in 20 mL of dichloromethane and 5 mL of trifluoroacetic acid was slowly added dropwise under ice bath conditions. After the addition was completed, it was concentrated under reduced pressure after stirring at room temperature for 30 minutes to obtain crude compound 1-10, which was used in the next step without further purification.

[0114] Step 7: tert-butyl (2S,4R)-4-(tert-butoxy)-2-(((S)-1-cyano-2-((S)-2-oxopyrrolidin-3- yl)ethyl)carbamoyl)pyrrolidine-1-carboxylate (Intermediate 1-12)

[0115] (2S,3S)-4-(tert-butoxy)-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylic acid (1-11) (574 mg, 2 mmol), 1-hydroxybenzotriazole (HOBt) (297 mg, 2.2 mmol) and 1-ethyl-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDC-HCl) (420 mg, 2.2 mmol) were dissolved in 30 mL of dichloromethane, stirred in ice bath for 10 min, 0.98 mL of diisopropylethylamine was added, after continued stirring in ice bath for 15 min, a solution of Intermediate 1-10 in dichloromethane was added slowly, stirred at room temperature overnight. After the reaction was completed, extraction was performed with dichloromethane and saturated brine, the organic phase was combined, dried over anhydrous Na2SO4, concentrated under reduced pressure, the residue was separated by column chromatography to obtain 350 mg of Intermediate 1-12, yield 41%, ESI-MS: m / z = 423 [M+1] + .

[0116] Step 8: Intermediate 1-13

[0117] The synthesis step was referred to Example 1, Step 6, and compound 1-6 was replaced by compound 1-12 to obtain compound 1-13. After being concentrated under reduced pressure, it was used in the next step without further purification.

[0118] Step 9: (2S,4R)-4-(tert-butoxy)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-1- ((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)pyrrolidine-2-carboxamide (A1)

[0119] The synthesis step was referred to Example 1, Step 7, and compound 1-11 was replaced by 1-9 and compound 1-10 was replaced by 1-13 to obtain compound A1, 1H NMR (400 MHz, Chloroform-d) δ 8.32 (d, J = 6.6 Hz, 1H), 7.15 (d, J = 8.8 Hz, 1H), 5.92 (s, 1H), 4.86 - 4.77 (m, 1H), 4.60 - 4.55 (m, 1H), 4.51 - 4.46 (m, 1H), 4.42 - 4.36 (m, 1H), 3.92 - 3.86 (m, 1H), 3.44 - 3.39 (m, 1H), 3.37 - 3.31 (m, 2H), 2.56 - 2.46 (m, 1H), 2.43 - 2.35 (m, 1H), 2.34 - 2.27 (m, 1H), 2.03 - 1.93 (m, 3H), 1.18 (s, 9H), 1.02 (s, 9H). Yield 42%, ESI-MS: m / z = 532 [M+1] + .

[0120] Example 2: (2S,4R)-4-phenyl-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-1- ((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)pyrrolidine-2-carboxamide (A2)

[0121]

[0122] Following the synthetic procedure of Example 1, Step 7 to 9, substituting (2S,4R)-1- (tert-butoxycarbonyl)-4-(4-fluorobenzyl)pyrrolidine-2-carboxylic acid 2-1 for 1-11 to give intermediate 2-2; substituting intermediate 2-2 for 1-12 to give intermediate 2-3; substituting intermediate 2-3 for 1-13 to give compound A2, 1 H NMR (400 MHz, Chloroform-d) δ 8.32 (d, J = 6.6 Hz, 1H), 7.15 (d, J = 8.8 Hz, 1H), 5.92 (s, 1H), 4.86 - 4.77 (m, 1H), 4.60 - 4.55 (m, 1H), 4.51 - 4.46 (m, 1H), 4.42 - 4.36 (m, 1H), 3.92 - 3.86 (m, 1H), 3.44 - 3.39 (m, 1H), 3.37 - 3.31 (m, 2H), 2.56 - 2.46 (m, 1H), 2.43 - 2.35 (m, 1H), 2.34 - 2.27 (m, 1H), 2.03 - 1.93 (m, 3H), 1.18 (s, 9H), 1.02 (s, 9H). Yield 42%, ESI-MS: m / z = 532 [M+1]

[0120] Example 2: (2S,4R)-4-phenyl-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-1- ((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)pyrrolidine-2-carboxamide (A2)

[0121]

[0122] Following the synthetic procedure of Example 1, Step 7 to 9, substituting (2S,4R)-1- (tert-butoxycarbonyl)-4-(4-fluorobenzyl)pyrrolidine-2-carboxylic acid 2-1 for 1-11 to give intermediate 2-2; substituting intermediate 2-2 for 1-12 to give intermediate 2-3; substituting intermediate 2-3 for 1-13 to give compound A2,+ .

[0123] Example 3: (S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-5-((S)-3,3- dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-5-azaspiro[2.4]heptane-6-carboxamide (A3)

[0124]

[0125] The synthetic procedures were referred to Example 1, steps 7-9, and (S)-5-(tert- butyloxycarbonyl)-5-azaspiro[2.4]heptane-6-carboxylic acid 3-1 was replaced by 1-11 to give intermediate 3-2; intermediate 3-2 was replaced by 1-12 to give intermediate 3-3; intermediate 3-3 was replaced by 1-13 to give compound A3, 1 H NMR (400 MHz, CDC13) δ 8.33 (d, J = 7.1 Hz, 1H), 7.11 (d, J = 9.3 Hz, 1H), 6.10 (s, 1H), 4.92 - 4.80 (m, 1H), 4.56 - 4.48 (m, 2H), 3.74 (d, J = 9.6 Hz, 1H), 3.54 - 3.49 (m, 1H), 3.36 - 3.31 (m, 2H), 2.67 - 2.58 (m, 1H), 2.42 - 2.33 (m, 2H), 2.24 - 2.16 (m, 2H), 2.02 - 1.96 (m, 2H), 1.03 (s, 9H), 0.73 - 0.68 (m, 2H), 0.63 - 0.57 (m, 2H). ESI-MS: m / z = 486 [M+1] + .

[0126] Example 4: (3S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-2-((S)-3,3- dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-2-azabicyclo[2.2.1]heptane-3- carboxamide (A4)

[0127]

[0128] The synthetic procedures were referred to Example 1, steps 7-9, and (3S)-2-(tert- butyloxycarbonyl)-2-azabicyclo[2.2.1]heptane-3-carboxylic acid 4-1 was replaced by 1-11 to give intermediate 4-2; intermediate 4-2 was replaced by 1-12 to give intermediate 4-3; intermediate 4-3 was replaced by 1-13 to give compound A4, 1H NMR (400 MHz, CDC13) δ 8.38 (d, J = 6.8 Hz, 1H), 7.87 (d, J = 9.4 Hz, 1H), 6.26 (s, 1H), 4.75 - 4.67 (m, 1H), 4.61 (d, J = 9.4 Hz, 1H), 4.50 (s, 1H), 3.89 (s, 1H), 3.36 - 3.31 (m, 2H), 2.81 - 2.78 (m, 1H), 2.47 - 2.31 (m, 3H), 2.27 - 2.19 (m, 2H), 2.19 - 2.15 (m, 3H), 2.02 - 1.92 (m, 3H), 1.03 (s, 9H). ESI-MS: m / z = 486 [M+1] + .

[0129] Example 5: (2S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-1-((S)-3,3- dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-4-(trifluoromethyl)pyrrolidine-2- carboxamide (A5)

[0130]

[0131] The synthetic steps were referred to Example 1, steps 7-9, (2S,4R)-N-Boc-4- trifluoromethyl proline 5-1 was replaced by 1-11 to give intermediate 5-2; intermediate 5-2 was replaced by 1-12 to give intermediate 5-3; intermediate 5-3 was replaced by 1-13 to give compound A5, ESI-MS: m / z = 528 [M+1] + .

[0132] Example 6: N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-2-((S)-3,3-dimethyl-2- (2,2,2-trifluoroacetamido)butanoyl)-2-azabicyclo[2.1.1]hexane-1-carboxamide

[0133]

[0134] The synthetic steps were referred to Example 1, steps 7-9, (3S)-2-(tert- butyloxycarbonyl)-2-azabicyclo[2.1.1]hexane-3-carboxylic acid 6-1 was replaced by 1-11 to give intermediate 6-2; intermediate 6-2 was replaced by 1-12 to give intermediate 6-3; intermediate 6-3 was replaced by 1-13 to give compound A6, ESI-MS: m / z = 472 [M+1] + .

[0135] Example 7: (2S)-6,6-dichloro-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-3- ((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-3-azabicyclo[3.1.0]hexane-2- carboxamide The synthetic procedure is referred to Example 1, steps 7-9, substituting (1S,2S,5R)-3- (tert-butoxycarbonyl)-6,6-dichloro-3-azabicyclo[3.1.0]hexane-2-carboxylic acid 7-1 for 1-11 to give intermediate 7-2; substituting intermediate 7-2 for 1-12 to give intermediate 7-3; substituting intermediate 7-3 for 1-13 to give compound A7, ESI-MS: m / z = 540 [M+1] + .

[0136] Example 8: (1S,3aR,6aS)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-2-((S)-3,3- dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)octahydrocyclopenta[C]pyrrole-1-carboxamide (A8)

[0137]

[0138] The synthetic procedure is referred to Example 1, steps 7-9, substituting (1S,3aR,6aS)-2- (tert-butoxycarbonyl)octahydrocyclopenta[C]pyrrole-1-carboxylic acid 8-1 for 1-11 to give intermediate 8-2; substituting intermediate 8-2 for 1-12 to give intermediate 8-3; substituting intermediate 8-3 for 1-13 to give compound A8, ESI-MS: m / z = 500 [M+1] + .

[0139] Example 9: (S)-N-((S)-1-cyano-2-oxopyrrolidin-3-yl)ethyl)-2-((S)-3,3-dimethyl-2-(2,2,2- trifluoroacetamido)butanoyl)-4,4-dimethylpyrrole-2-carboxamide (A9)

[0140]

[0141] The synthetic procedure is referred to Example 1, steps 7-9, substituting (S)-1-tert- butyloxycarbonyl-4,4-dimethylpyrrolidine-2-carboxylic acid 9-1 for 1-11 to give intermediate 9-2; substituting intermediate 9-2 for 1-12 to give intermediate 9-3; substituting intermediate 9-3 for 1-13 to give compound A9, ESI-MS: m / z = 488 [M+1] + .

[0142] Example 10: (2S)-4-(Bicyclo[l. l. l]pentan- 1 -yl)-N-((S)- 1 -cyano-2-((S)-2- oxopyrrolidin-3-yl)ethyl)- 1 -((S)-3,3 -dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)pyrrole- 2-carboxamide (A10)

[0143]

[0144] The synthetic procedure was referred to Example 1, step 7-9, (2S)-4-(bicyclo[l. l. l]pentan- 1 -yl)- 1 -(tert-butoxycarbonyl)pyrrole-2-carboxylic acid 10-1 was used to replace 1-11 to get intermediate 10-2; intermediate 10-2 was used to replace 1-12 to get intermediate 10-3; intermediate 10-3 was used to replace 1-13 to get compound A10, ESI-MS: m / z = 526 [M+l] + .

[0145] Example 11 : (S)-N-((S)- 1 -cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)- 1 -((S)-3,3 -dimethyl-2- (spiro[3.3]heptane-2-carboxamide)butanoyl)pyrrole-2-carboxamide (A11)

[0146]

[0147] Step 1 : tert-Butyl ((S)- 1 -((S)-2-(((S)- 1 -cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)pyrrolidin- 1 -yl)-3,3 -dimethyl- 1 -acetylacet-2-yl)carbamate (intermediate 11-5)

[0148] The synthetic procedure was referred to Example 1, step 7-9, (tert-butoxycarbonyl)-L-proline 11-1 was used to replace 1-11 to get intermediate 11-2; intermediate 11-2 was used to replace 1-12 to get intermediate 11-3; intermediate 11-3 was used to replace 1-13, N-Boc-L-tert-leucine was used to replace 1-9 to get intermediate 11-5, ESI-MS: m / z = 464 [M+l]+.

[0149] Step 2: tert-Butyl ((S)- 1 -((S)-2-(((S)- 1 -cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)pyrrolidin- 1 -yl)-3,3 -dimethyl- 1 -acetylacet-2-yl)carbamate (A11)

[0150] The synthesis was performed according to reference example 1 step 8 to 9, replacing 1-12 with 11-5 to give intermediate 11-6; replacing intermediate 11-6 for 1-13, replacing 1-9 with spiro[3.3]heptane-2-carboxylic acid to give compound A11, ESI-MS: m / z = 486 [M+1] + .

[0151] Example 12: N-((S)-1-((1R,2S,5S)-2-(((S)-1-cyano-2-((S)-2-oxopyrrolidin-3- yl)ethyl)carbamoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hex-3-yl)-3,3-dimethyl-1- oxobutan-2-yl)-5-azaspiro[2.4]heptane-6-carboxamide (A12)

[0152]

[0153] Step 1: Intermediate 12-2

[0154] Reference example 1 step 7, replacing 1-10 with intermediate 11-6, replacing 1-11 with (S)-5-(tert-butoxycarbonyl)-5-azaspiro[2.4]heptane-6-carboxylic acid (intermediate 12-1) to give intermediate 12-2, ESI-MS: m / z = 587 [M+1] + .

[0155] Step 2: N-((S)-1-((1R,2S,5S)-2-(((S)-1-cyano-2-((S)-2-oxopyrrolidin-3- yl)ethyl)carbamoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hex-3-yl)-3,3-dimethyl-1- oxobutan-2-yl)-5-azaspiro[2.4]heptane-6-carboxamide (A12)

[0156] To the ethyl acetate solution of compound 12-2, slowly added saturated HCl in ethyl acetate solution, after stirring at room temperature for 30 minutes, concentrated under reduced pressure to give white powder. The target compound A12 was obtained, ESI-MS: m / z = 486 [M+1] + .

[0157] Example 13: (1R,2S,5S)-N-(1-cyano-2-(1H-imidazol-4-yl)ethyl)-3-((S)-3,3-dimethyl- 2-(2,2,2)-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (A13)

[0158]

[0159] Step 1: (S)-2-amino-3-(lH-imidazol-4-yl)propanenitrile (Intermediate 13-2)

[0160] The synthetic procedure was referred to Example 1, step 7, with compound 13-1 instead of compound 1-6 to give intermediate 13-2. Step 2: (lR,2S,5S)-N-(l-cyano-2-(lH-imidazol-4-yl)ethyl)-3-((S)-3,3-dimethyl-2-(2,2,2)- trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide (A13)

[0161] The synthetic procedure was referred to Example 1, steps 7 to 9, with (tert- butoxycarbonyl)-L-proline 11-1 instead of 1-11, intermediate 13-2 instead of intermediate 1-10 to give intermediate 13-3; intermediate 13-3 instead of 1-12 to give intermediate 13-4; intermediate 13-4 instead of 1-13 to give compound A13, ESI-MS: m / z = 443 [M+1] + .

[0162] Example 14: (S)-N-((S)-l-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-l-((S)-3,3-dimethyl- 2-(spiro[3.3]heptane-2-carboxamide)butanoyl)-5-azaspiro[2.4]heptane-6-carboxamide (A14)

[0163]

[0164] The synthetic procedure was referred to Example 1, steps 7 to 9, with intermediate 11-4 instead of 1-11 to give intermediate 14-1; intermediate 14-1 instead of 1-12 to give intermediate 14-2; intermediate 14-2 instead of 1-13, with spiro[3.3]heptane-2-carboxylic acid instead of 1-9 to give compound A14, ESI-MS: m / z = 512 [M+1] + .

[0165] Example 15: (S)-N-((S)-l-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-5-(l-(2,2,2- trifluoroacetamido)cyclopropyl-l-carbonyl)-5-azaspiro[2.4]heptane-6-carboxamide (A15)

[0166]

[0167] Step 1: l-(2,2,2-trifluoroacetamido)cyclopropyl-l-carboxylic acid (Intermediate 15-2)

[0168] The synthetic procedure was referred to example 1 step 5, replacing compound 1-6 with compound 15-1 to give intermediate 15-2, ESI-MS: m / z = 198 [M+1] + .

[0169] Step 2: (S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-5-(1-(2,2,2- trifluoroacetamido)cyclopropyl-1-carbonyl)-5-azaspiro[2.4]heptane-6-carboxamide (A15)

[0170] The synthetic procedure was referred to example 1 step 7-9, replacing intermediate 1-11 with intermediate 15-3 to give intermediate 15-4; replacing 1-12 with intermediate 15-4 to give intermediate 15-5; replacing 1-13 with intermediate 15-5, replacing 1-9 with 15-2 to give compound A15, ESI-MS: m / z = 470 [M+1] + .

[0171] Example 16: (1R,2S,5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-3-(4- fluoro-1-(2,2,2-trifluoroacetylpyrrolidine-2-carbonyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (A16)

[0172]

[0173] Step 1: 1-(2,2,2-trifluoroacetamido)cyclopropyl-1-carboxylic acid (intermediate 16-2)

[0174] The synthetic procedure was referred to example 1 step 5, replacing compound 1-6 with compound 16-1 to give intermediate 16-2, ESI-MS: m / z = 258 [M+1] + .

[0175] Step 2: (1R,2S,5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-3-(4-fluoro-1- (2,2,2-trifluoroacetylpyrrolidine-2-carbonyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide (A16)

[0176] The synthetic procedure was referred to example 1 step 7, replacing 1-11 with compound 15-5, replacing intermediate 1-10 with intermediate 16-2 to give compound A16, ESI-MS: m / z = 502 [M+1] + .

[0177] Example 17: (6S)-N-((S)-1-cyano-2-oxopyrrolidin-3-yl)ethyl)-3-(4-fluoro-1-(2,2,2- trifluoroacetylpyrrolidine-2-carbonyl)-5-azabicyclo[2.4]heptane-6-carboxamide (A17)

[0178]

[0179] The synthetic procedure was referred to Example 1, step 7, with compound 3-3 instead of 1-11, and intermediate 16-2 instead of intermediate 1-10, to give compound A17, ESI-MS: m / z = 488 [M+1] + .

[0180] Example 18: (1R,2S,5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-6,6-dimethyl- 3-(1-(2,2,2-trifluoroacetyl)azetidine-2-carbonyl)-3-azabicyclo[3.1.0]hexane-2-carboxamide (A18)

[0181]

[0182] Step 1: synthesis of intermediate 18-2

[0183] The synthetic procedure was referred to Example 1, step 5, with compound 18-1 instead of compound 1-6, to give intermediate 18-2, ESI-MS: m / z = 226 [M+1] + .

[0184] Step 2: (1R,2S,5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-6,6-dimethyl-3-(1-(2,2,2- trifluoroacetyl)azetidine-2-carbonyl)-3-azabicyclo[3.1.0]hexane-2-carboxamide (A18)

[0185] The synthetic procedure was referred to Example 1, step 7, with compound 15-5 instead of 1-11, and intermediate 18-2 instead of intermediate 1-10, to give compound A18, ESI-MS: m / z = 470 [M+1] + .

[0186] Example 19: (6S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-5-(1-(2,2,2- trifluoroacetyl)azetidine-2-carbonyl)-5-azabicyclo[2.4]heptane-6-carboxamide (A19)

[0187]

[0188] The synthetic procedure was referred to example 1 step 7, replacing compound 3-3 with 1-11, intermediate 18-2 with intermediate 1-10 to give compound A19, ESI-MS: m / z = 456 [M+1] + .

[0189] Example 20: (1R,2S,5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-3-((2S)-2- (2,2-difluorocyclopropyl-1-formamido)-3,3-dimethylbutanoyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (A20)

[0190]

[0191] The synthetic procedure was referred to example 1 step 7 to 9, replacing intermediate 1-11 with intermediate 11-4 to give intermediate 20-1; replacing intermediate 1-12 with intermediate 20-1 to give intermediate 20-2; replacing intermediate 1-13 with intermediate 20-2, replacing 1-9 with compound 20-3 to give compound A20, ESI-MS: m / z = 508 [M+1] + .

[0192] Example 21: (6S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-5-((2S)-2-(2,2- difluorocyclopropyl-1-formamido)-3,3-dimethylbutanoyl)-5-azabicyclo[2.4]heptane-6- carboxamide (A21)

[0193]

[0194] The synthetic procedure was referred to example 1 step 7, replacing intermediate 1-11 with intermediate 20-3, intermediate 1-10 with intermediate 14-2 to give compound A21, 1H NMR (400 MHz, Chloroform-d) δ 8.18 (d, J = 7.6 Hz, 1H), 6.77 (d, J = 9.2 Hz, 1H), 6.36 (s, 1H), 5.00 - 4.85 (m, 1H), 4.60 - 4.55 (m, 2H), 3.72 - 3.64 (m, 2H), 3.38 - 3.31 (m, 2H), 2.66 - 2.60 (m, 1H), 2.43 - 2.33 (m, 4H), 2.21 - 2.15 (m, 1H), 2.09 - 1.99 (m, 2H), 1.96 - 1.91 (m, 1H), 1.65 - 1.59 (m, 1H), 0.99 (s, 9H), 0.71 - 0.66 (m, 2H), 0.64 - 0.61 (m, 2H). ESI-MS: m / z = 494 [M+1] + .

[0195] Example 22: (1R,2S,5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-6,6- dimethyl-3-((2,2,2-trifluoroacetyl)-L-leucyl)-3-azabicyclo[3.1.0]hexane-2-carboxamide (A22)

[0196]

[0197]

[0198] The synthesis step is referred to Example 16, compound 22-2 is used instead of compound 16-1 to give compound A22, ESI-MS: m / z = 500 [M+1] + .

[0199] Example 23: (S)-N-((S)-1-cyano-2-oxopyrrolidin-3-yl)ethyl)-5-((2,2,2- trifluoroacetyl)-L-leucyl)-5-azabicyclo[2.4]heptane-6-carboxamide (A23)

[0200]

[0201] The synthesis step is referred to Example 1 step 7, intermediate 22-3 is used instead of 1-11, intermediate 3-3 instead of 1-10 to give compound A23, ESI-MS: m / z = 486 [M+1] + .

[0202] Example 24: (1R,2S,5S)-N-((S)-1-cyano-2-((S)-2-oxopiperidin-3-yl)ethyl)-6,6- dimethyl-3-((2,2,2-trifluoroacetyl)-L-leucinyl)-3-azabicyclo[3.1.0]hexane-2- carboxamide (A24)

[0203]

[0204] Step 1. Synthesis of intermediate 24-3

[0205] The synthetic procedure in steps was referred to Example 1 step 3-4, replacing 1-4 with 24-1 to afford intermediate 24-3, ESI-MS: m / z = 196 [M+1]. + .

[0206] Step 2. Synthesis of (1R,2S,5S)-N-((S)-1-cyano-2-((S)-2-oxopiperidin-3-yl)ethyl)-6,6- dimethyl-3-((2,2,2-trifluoroacetyl)-L-leucinyl)-3-azabicyclo[3.1.0]hexane-2- carboxamide (A24)

[0207] The synthetic procedure in steps was referred to Example 1 step 7-9, replacing 1-11 with intermediate 24-3 to afford intermediate 24-4; replacing 1-12 with intermediate 24-4 to afford intermediate 24-5; replacing 1-13 with intermediate 24-5, replacing 1-9 with compound 22-2 to afford compound A24, ESI-MS: m / z = 514 [M+1]. + .

[0208] Example 25: (1R,2S,5S)-N-((S)-1-cyano-2-((S)-2-oxopiperidin-3-yl)ethyl)-6,6- dimethyl-3-((2,2,2-trifluoroacetyl)-L-leucinyl)-3-azabicyclo[3.1.0]hexane-2- carboxamide (A25)

[0209]

[0210] The synthetic procedure in steps was referred to Example 1 step 7-9, replacing 1-11 with intermediate 24-3 to afford intermediate 25-1; replacing 1-12 with intermediate 25-1 to afford intermediate 25-2; replacing 1-13 with intermediate 25-2, replacing 1-9 with compound 22-2 to afford compound A24, ESI-MS: m / z = 500 [M+1]. + .

[0211] Example 26: (1R,2S,5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-3- ((S)-2-(1-fluorocyclopropyl-1-carbonylamino)-3,3-dimethylbutanoyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (A26)

[0212]

[0213] The synthetic procedure was referred to Example 1, step 7, with intermediate 14-2 instead of 1-11, 1-fluorocyclopropane carboxylic acid instead of 1-10 to give compound A26, ESI-MS: m / z = 490 [M+1] + .

[0214] Example 27: (S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-5-((S)-2-(1- fluorocyclopropyl-1-carbonylamino)-3,3-dimethylbutanoyl)-5-azabicyclo[2.4]heptane-6- carboxamide (A27)

[0215]

[0216] The synthetic procedure was referred to Example 1, step 7, with intermediate 20-2 instead of 1-11, 1-fluorocyclopropane carboxylic acid instead of 1-10 to give compound A27, 1 H NMR (400 MHz, Chloroform-d) δ 8.24 (d, J = 6.9 Hz, 1H), 7.03 (dd, J = 9.3, 3.7 Hz, 1H), 5.92 (s, 1H), 4.94 - 4.85 (m, 1H), 4.62 - 4.51 (m, 2H), 3.70 - 3.61 (m, 2H), 3.38 - 3.34 (m, 2H), 2.70 - 2.58 (m, 1H), 2.48 - 2.30 (m, 2H), 2.20 (dd, J = 12.6, 6.2 Hz, 1H), 2.02 - 1.82 (m, 3H), 1.35 - 1.29 (m, 4H), 1.04 (s, 9H), 0.69 - 0.54 (m, 4H). ESI-MS: m / z = 476 [M+1] + .

[0217] Example 28: (1R,2S,5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-3- ((S)-3,3-dimethyl-2-(1-methyl-1H-pyrazole-4-carbonylamino)butanoyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (A28)

[0218]

[0219] The synthesis was performed according to the procedure described in Example 1, Step 7, substituting intermediate 14-2 for 1-11 and 1-fluorocyclopropanecarboxylic acid for 1-10 to give compound A28, ESI-MS: m / z = 512 [M+1] + .

[0220] Example 29: (1R,2S,5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-3-((S)-3,3- dimethyl-2-(1-methyl-1H-pyrazole-4-carboxamido)butanoyl)-5-azabicyclo[2.4]heptane-6- carboxamide (A29)

[0221]

[0222] The synthesis was performed according to the procedure described in Example 1, Step 7, substituting intermediate 20-2 for 1-11 and 1-fluorocyclopropanecarboxylic acid for 1-10 to give compound A29, 1 H NMR (400 MHz, Chloroform-d) δ 8.22 (d, J = 7.3 Hz, 1H), 7.79 (d, J = 17.1 Hz, 2H), 6.52 (d, J = 9.6 Hz, 1H), 5.97 (s, 1H), 4.91 (ddd, J = 10.7, 7.2, 5.4 Hz, 1H), 4.72 (d, J = 9.5 Hz, 1H), 4.51 (dd, J = 7.9, 6.4 Hz, 1H), 3.89 (s, 3H), 3.70 (s, 2H), 3.37 - 3.27 (m, 2H), 2.68 - 2.55 (m, 1H), 2.49 - 2.38 (m, 1H), 2.37 - 2.28 (m, 1H), 2.21 - 2.14 (m, 1H), 1.99 - 1.83 (m, 3H), 1.04 (s, 9H), 0.89 - 0.80 (m, 2H), 0.68 - 0.62 (m, 2H). ESI-MS: m / z = 498 [M+1] + .

[0223] Example 30: (1R,2S,5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-3-((S)-3,3- dimethyl-2-(6-methylpyrazine-2-carboxamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0] hexane-2-carboxamide (A30)

[0224]

[0225] The synthetic procedure was referred to example 1 step 7, replacing intermediate 14-2 with 1-11, 1-fluorocyclopropane carboxylic acid with 1-10 to give compound A30, ESI-MS: m / z = 524 [M+1] + .

[0226] Example 31: (S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-5-((S)-3,3- dimethyl-2-(6-methylpyrazine-2-carboxamido)butanoyl)-5-azaspiro[2.4]heptane-6- carboxamide (A31)

[0227]

[0228] The synthetic procedure was referred to example 1 step 7, replacing intermediate 14-2 with 1-11, 1-fluorocyclopropane carboxylic acid with 1-10 to give compound A30, ESI-MS: m / z = 524 [M+1] + .

[0229] Example 32: (1R,2S,5S)-3-((S)-2-(4-amino-3-chlorobenzamide)-3,3-dimethylbutanoyl)- N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide (A32)

[0230]

[0231] Step 1: 4-((tert-butoxycarbonyl)amino)-3-chlorobenzoic acid (32-2)

[0232] 4-Amino-3-chlorobenzoic acid 32-1 (342 mg, 2 mmol) was dissolved in dichloromethane, sodium bicarbonate (504 mg, 6 mmol), di-tert-butyl dicarbonate (654 mg, 3 mmol) were added under ice bath, stirred overnight. After the reaction was completed, it was washed with dilute hydrochloric acid and saturated brine, and the organic layer was concentrated under reduced pressure to give compound 32-2, ESI-MS: m / z = 272 [M+1] + .

[0233] Step 2: tert-butyl (2-chloro-4-((S)-1-((1R,2S,5S)-2-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3- yl)ethyl)carbamoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-yl)-3,3-dimethyl-1- oxobutan-2-yl)carbamoyl)phenylcarbamate (32-3)

[0234] The synthetic procedure is referred to Example 1, Step 7 with compound 32-2 replacing 1-11 and intermediate 13-2 replacing intermediate 1-10 to give compound A32, ESI-MS: m / z = 657 [M+1] + .

[0235] Step 3: (1R,2S,5S)-3-((S)-2-(4-amino-3-chlorobenzamide)-3,3-dimethylbutanoyl)-N- ((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide (A32)

[0236] The synthetic procedure is referred to Example 12, Step 2 with compound 32-3 replacing 12-2 to give compound A32, ESI-MS: m / z = 557 [M+1] + .

[0237] Example 33: (S)-5-((S)-2-(4-amino-3-chlorobenzamide)-3,3-dimethylbutanoyl)-N- ((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-5-azaspiro[2.4]heptan-6-carboxamide (A33)

[0238]

[0239] Step 1: 4-((tert-butoxycarbonyl)amino)-3-chlorobenzoic acid (32-2)

[0240] The synthetic procedure is referred to Example 32, Step 1

[0241] Step 2: tert-butyl (2-chloro-4-((S)-1-((S)-6-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3- yl)ethyl)carbamoyl)-5-azaspiro[2.4]heptan-5-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (33-1)

[0242] The synthetic procedure is referred to Example 1, Step 7 with compound 32-2 replacing 1-11 and intermediate 20-2 replacing intermediate 1-10 to give compound A33, ESI-MS: m / z = 643 [M+1] + .

[0243] Step 3: (1R, 2S, 5S)-3-((S)-2-(4-amino-3-chlorobenzamide)-3,3-dimethylbutanoyl)-N- ((S)-1 -cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (A32)

[0244] The synthetic procedure in Step 2 of Reference Example 12 was followed using the compound 33-1 from the previous step in place of 12-2 to give compound A33, ESI-MS: m / z = 543 [M+1 ] + .

[0245] Example 34: (1R, 2S, 5S)-N-((S)-1 -cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-3-((S)-2- (2,5-dichlorobenzamide)-3,3-dimethylbutanoyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (A34)

[0246]

[0247] The synthetic procedure in Step 7 of Reference Example 1 was followed using intermediate 14-2 in place of 1-11 and 2,5-dichlorobenzoic acid 34-1 in place of 1-10 to give compound A34, ESI-MS: m / z = 576 [M+1 ] + .

[0248] Example 35: (S)-N-((S)-1 -cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-5-((S)-2-(2,5- dichlorobenzamide)-3,3-dimethylbutanoyl)-5-azaspiro[2.4]heptane-6- carboxamide (A35)

[0249]

[0250] The synthetic procedure in Step 7 of Reference Example 1 was followed using 2,5- dichlorobenzoic acid 35-1 in place of 1-11 and intermediate 20-1 in place of intermediate 1-10 to give compound A35, ESI-MS: m / z = 562 [M+1 ] + .

[0251] Example 36: (1R, 2S, 5S)-N-((S)-1 -cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-3-((S)-3,3- dimethyl-2-(2-morpholinoacetamido)butanoyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (A36)

[0252]

[0253] The synthetic procedure is referred to example 1, step 7, replacing 1-11 with 2- morpholinoacetic acid 36-1, replacing intermediate 1-10 with intermediate 14-2, to give compound A36, ESI-MS: m / z = 531 [M+1] + .

[0254] Example 37: ((S)-N-((S)-l-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-5-((S)-3,3- dimethyl-2-(2-morpholinoacetamido)butanoyl)-5-azaspiro[2.4]heptane-6- carboxamide (A37)

[0255]

[0256] The synthetic procedure is referred to example 1, step 7, replacing 1-11 with 2- morpholinoacetic acid 36-1, replacing intermediate 1-10 with intermediate 14-2, to give compound A36, ESI-MS: m / z = 531 [M+1] + .

[0257] Example 38: N-((S)-l-((lR,2S,5S)-2-((S)-l-cyano-2-((S)-2-oxopyrrolidin-3- yl)ethyl)carbamoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexan-3-yl)-3,3-dimethyl-l- oxobutan-2-yl)-2-methylthiazole-5-carboxamide (A38)

[0258]

[0259] The synthetic procedure is referred to example 1, step 7, replacing 1-11 with 2- morpholinoacetic acid 36-1, replacing intermediate 1-10 with intermediate 14-2, to give compound A36, ESI-MS: m / z = 531 [M+1] + .

[0260] Example 39: N-((S)-l-((S)-6-((S)-l-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)carbamoyl)- 5-azaspiro[2.4]heptan-5-yl)-3,3-dimethyl-l-oxobutan-2-yl)-2-methylthiazole-5- carboxamide (A39)

[0261]

[0262] The synthetic procedure is referred to example 1, step 7, replacing 1-11 with 2- morpholinoacetic acid 36-1, replacing intermediate 1-10 with intermediate 14-2, to give compound A36, ESI-MS: m / z = 531 [M+1]+ .

[0263] Example 40: (1R, 2S, 5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-3- ((S)-3,3-dimethyl-2-((trifluoromethyl)sulfonamido)butanoyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (A40)

[0264]

[0265] Step 1: (1R, 2S, 5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-3- ((S)-3,3-dimethyl-2-((trifluoromethyl)sulfonamido)butanoyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (A40)

[0266] Compound 14-2 (403 mg, 1 mmol) was dissolved in 20 mL of dry dichloromethane, 0.43 mL of triethylamine was added dropwise at 0 °C, after 20 min, trifluoromethanesulfonyl chloride (169 mg, 1 mmol) was added, and stirred overnight. After the reaction was completed, extraction was carried out with dichloromethane and saturated brine, the combined organic phase was dried over anhydrous Na2SO4, concentrated under reduced pressure, and the obtained residue was separated by column chromatography to obtain 250 mg of compound A40, yield 47%, ESI-MS: m / z = 536 [M+1] + .

[0267] Example 41: (S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-5-((S)-3,3- dimethyl-2-((trifluoromethyl)sulfonamido)butanoyl)-5-azaspiro[2.4]heptane-6- carboxamide (A41)

[0268]

[0269] Step 1: (S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-5-((S)-3,3-dimethyl- 2-((trifluoromethyl)sulfonamido)butanoyl)-5-azaspiro[2.4]heptane-6-carboxamide (A41) Synthesis step reference Example 40 step 1, intermediate 20-1 instead of intermediate 14-2 to obtain compound A41, 1HNMR (400 MHz, Chloroform-d) δ 8.25 (d, J = 8.0 Hz, 1H), 7.48 (d, J = 13.9 Hz, 1H), 6.42 (s, 1H), 4.97 (ddd, J = 10.7, 8.0, 5.2 Hz, 1H), 4.51 (t, J = 7.8 Hz, 1H), 3.94 (d, J = 9.8 Hz, 1H), 3.74 (d, J = 9.6 Hz, 1H), 3.30 (dd, J = 9.9, 5.7 Hz, 3H), 2.72 - 2.61 (m, 1H), 2.47 - 2.31 (m, 2H), 2.26 - 2.17 (m, 1H), 1.98 - 1.88 (m, 3H), 1.04 (s, 9H), 0.74 - 0.66 (m, 2H), 0.64 - 0.53 (m, 2H). ESI-MS: m / z = 522 [M+1] + .

[0270] Example 42: (1R,2S,5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-3-((S)-2- (cyclopropanesulfonamido)-3,3-dimethylbutanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane- 2-carboxamide (A42)

[0271]

[0272] The synthetic procedure was referred to Example 40, Step 1, with cyclopropane sulfonyl chloride 42-1 replacing 40-1, and intermediate 20-1 replacing intermediate 14-2, to give compound A43, ESI-MS: m / z = 494 [M+1] + .

[0273] Example 43: (S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-5-((S)-2- (cyclopropanesulfonamido)-3,3-dimethylbutanoyl)-5-azaspiro[2.4]heptane-6-carboxamide (A43)

[0274]

[0275] The synthetic procedure was referred to Example 40, Step 1, with cyclopropane sulfonyl chloride 42-1 replacing 40-1, and intermediate 20-1 replacing intermediate 14-2, to give compound A43, ESI-MS: m / z = 494 [M+1] + .

[0276] Example 44: (1R, 2S, 5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-3- ((S)-3, 3-dimethyl-2-((4-methylphenyl)sulfonamido)butanoyl)-6, 6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (A44)

[0277]

[0278] The synthetic procedure was referred to Example 40, Step 1, with p-toluenesulfonyl chloride 44-1 replacing 40-1, and intermediate 20-1 replacing intermediate 14-2, to give compound A45, ESI-MS: m / z = 544 [M+1]

[0279] Example 45: (S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-5-((S)-3, 3- dimethyl-2-((4-methylphenyl)sulfonamido)butanoyl)-5-azaspiro[2.4]heptane-6- carboxamide (A45)

[0280]

[0281] The synthetic procedure was referred to Example 40, Step 1, with p-toluenesulfonyl chloride 44-1 replacing 40-1, and intermediate 20-1 replacing intermediate 14-2, to give compound A45, ESI-MS: m / z = 544 [M+1] +

[0282] Example 46: (1R, 2S, 5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-3- ((S)-3, 3-dimethyl-2-(6-phenylpyridinecarboxamido)butanoyl)-6, 6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (A46)

[0283]

[0284] The synthetic procedure was referred to Example 1, Step 7, with 6-phenylpicolinic acid 46-1 replacing 1-11, and intermediate 14-2 replacing intermediate 1-10, to give compound A46, ESI-MS: m / z = 585 [M+1] + .

[0285] Example 47: (S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-5-((S)-3, 3- dimethyl-2-(6-phenylpyridinecarboxamido)butanoyl)-5-azaspiro[2.4]heptane-6- carboxamide (A47)

[0286]

[0287] The synthetic procedure was referred to example 1 step 7, replacing 1-11 with 6- phenylpicolinic acid 46-1, replacing intermediate 1-10 with intermediate 20-1, to give compound A47, ESI-MS: m / z = 571 [M+1] + .

[0288] Example 48: (1R,2S,5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-3- ((S)-2-(2-cyanoacetamido)-3,3-dimethylbutanoyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (A48)

[0289]

[0290] The synthetic procedure was referred to example 1 step 7, replacing 1-11 with 2- cyanoacetic acid 48-1, replacing intermediate 1-10 with intermediate 14-2, to give compound A48, ESI-MS: m / z = 471 [M+1] + .

[0291] Example 49: (S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-5-((S)-2-(2- cyanoacetamido)-3,3-dimethylbutanoyl)-5-azaspiro[2.4]heptane-6-carboxamide (A49)

[0292]

[0293] The synthetic procedure was referred to example 1 step 7, replacing 1-11 with 2- cyanoacetic acid 48-1, replacing intermediate 1-10 with intermediate 20-1, to give compound A49, ESI-MS: m / z = 457 [M+1] + .

[0294] Example 50: (1R,2S,5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-3- ((S)-3,3-dimethyl-2-(N-methylcyclopropanesulfonamido)butanoyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (A50)

[0295]

[0296] Step 1: (1R, 2S, 5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-3-((S)-3,3- dimethyl-2-(methylamino)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (50-1)

[0297] Compound 14-2 (403 mg, 1 mmol), potassium carbonate (276 mg, 2 mmol) were dissolved in 20 mL of acetone, after stirring for 20 minutes, iodomethane (170 mg, 1.2 mmol) was added, and the reaction was allowed to proceed overnight. After the reaction was completed, it was concentrated under reduced pressure, extracted with dichloromethane and saturated brine, the organic phase was combined and concentrated under reduced pressure, and purified by column chromatography to obtain 380 mg of the target compound 50-1, with a yield of 91%, ESI-MS: m / z = 418 [M+1]+.

[0298] Step 2: (1R, 2S, 5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-3-((S)-3,3- dimethyl-2-(N-methylcyclopropanesulfonamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0] hexane-2-carboxamide (A50)

[0299] The synthesis step was referred to Example 40 Step 1, 50-1 was replaced by 14-2, and cyclopropanesulfonyl chloride 42-1 was replaced by 40-1, to obtain compound A50, ESI-MS: m / z = 522 [M+1] + .

[0300] Example 51: (S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-5-((S)-3,3-dimethyl- 2-(N-methylcyclopropanesulfonamido)butanoyl)-5-azaspiro[2.4]heptane-6-carboxamide (A51)

[0301]

[0302] The synthesis step was referred to Example 50 Step 1-3, 20-1 was replaced by 14-2, to obtain compound A51, ESI-MS: m / z = 508 [M+1] + .

[0303] Example 52: (1R, 2S, 5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-3-((S)-3,3- dimethyl-2-((5-(trifluoromethyl)pyridine)-2-sulfonamido)butanoyl)-6,6-dimethyl-3- azabicyclo[3.1.0]hexane-2-carboxamide (A52)

[0304]

[0305] The synthetic procedure is referred to Example 40, Step 1 with substitution of 5- (trifluoromethyl)pyridine-2-sulfonyl chloride 52-1 for 40-1 to give Compound A52, ESI-MS: m / z = 613 [M+1] + .

[0306] Example 53: (S)-N-((S)-l-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-5-((S)-3,3- dimethyl-2-((5-(trifluoromethyl)pyridin)-2-sulfonamido)butanoyl)-5- azaspiro[2.4]heptane-6-carboxamide (A53)

[0307]

[0308] The synthetic procedure is referred to Example 40, Step 1 with substitution of compound 20-1 for 14-2 and 5-(trifluoromethyl)pyridine-2-sulfonyl chloride 52-1 for 40-1 to give Compound A53, ESI-MS: m / z = 599 [M+1] + .

[0309] Example 54: (lR,2S,5S)-3-((S)-2-(but-2-ynoylamino)-3,3-dimethylbutanoyl)-N-((S)-l- cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2- carboxamide (A54)

[0310]

[0311] The synthetic procedure is referred to Example 40, Step 1 with substitution of 2- butynoic acid 54-1 for 40-1 to give Compound A54, ESI-MS: m / z = 470 [M+1] + .

[0312] Example 55: (S)-5-((S)-2-(but-2-ynoylamino)-3,3-dimethylbutanoyl)-N-((S)-l-cyano-2- ((S)-2-oxopyrrolidin-3-yl)ethyl)-5-azaspiro[2.4]heptane-6-carboxamide (A55)

[0313]

[0314] The synthetic procedure is referred to Example 1, Step 7 with substitution of intermediate 20-1 for 14-2 and 2-butyneoic acid 54-1 for 1-11 to give Compound A55, ESI-MS: m / z = 456 [M+1] + .

[0315] Example 56: (1R,2S,5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-3- ((S)-3,3-dimethyl-2-(4-(trifluoromethyl)pyridinecarboxamido)butanoyl)-6,6-dimethyl- 3-azabicyclo[3.1.0]hexane-2-carboxamide (A56)

[0316]

[0317] The synthetic procedure was referred to Example 1, step 7, with 4- (trifluoromethyl)picolinic acid 56-1 instead of 1-11, and intermediate 14-2 instead of intermediate 1-10, to afford compound A56, ESI-MS: m / z = 577 [M+1] + .

[0318] Example 57: (S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-5-((S)-3,3- dimethyl-2-(4-(trifluoromethyl)pyridinecarboxamido)butanoyl)-5-azaspiro[2.4]heptane-6- carboxamide (A57)

[0319]

[0320] The synthetic procedure was referred to Example 1, step 7, with 4- (trifluoromethyl)picolinic acid 56-1 instead of 1-11, and intermediate 20-1 instead of intermediate 1-10, to afford compound A57, 1 H NMR (400 MHz, Chloroform-d) δ 8.83 (dq, J = 5.0, 0.7 Hz, 1H), 8.65 (d, J = 9.8 Hz, 1H), 8.42 (ddt, J = 21.2, 1.6, 0.8 Hz, 1H), 8.23 (d, J = 7.2 Hz, 1H), 7.72 (ddd, J = 5.0, 1.8, 0.7 Hz, 1H), 6.10 (s, 1H), 4.99 (ddd, J = 10.3, 7.2, 5.9 Hz, 1H), 4.81 (d, J = 9.8 Hz, 1H), 4.61 (dd, J = 8.1, 5.9 Hz, 1H), 3.85 - 3.67 (m, 2H), 3.44 - 3.37 (m, 2H), 2.75 - 2.60 (m, 1H), 2.55 - 2.33 (m, 2H), 2.09 - 1.96 (m, 2H), 1.93 - 1.82 (m, 2H), 1.16 (s, 9H), 0.82 - 0.73 (m, 2H), 0.71 - 0.57 (m, 2H). ESI-MS: m / z = 563 [M+1] + .

[0321] Example 58 Evaluation of 2019 Novel Coronavirus 3CL Protease Inhibitory Activity

[0322] Determination of the inhibitory activity of compounds on 2019 Novel Coronavirus 3CL protease (2019-nCoV 3CL pro ) : The enzyme level inhibitory activity of the inhibitors against 3CL protease was determined by fluorescence resonance energy transfer (FRET) technology. The Assay Reagent was prepared according to the ratio of 92 μL of Assay Buffer, 2019-nCoV 3CL pro 1 μL, 93 μL of buffer Assay Reagent was added to each well in a 96-well black plate, 5 μL of compound (final concentration 0.5 μM) and 2 μL of Substrate were added at the same time. The fluorescence parameters were determined by Tecan multifunctional microplate reader, the excitation wavelength and emission wavelength were 325 nm and 393 nm respectively, the temperature was maintained at 37℃, the data was read after 10 min. Negative control and positive control were used, the negative control did not add compound, the positive control was PF-07321332, and the rest was the same. The obtained data was processed by software GraphPad Prism 7, and the experimental results are shown in Table 1.

[0323] Table 1 2019 Novel Coronavirus 3CL Protease Inhibitory Activity

[0324]

[0325]

[0326] The experimental results show that the compounds of the present application have certain 3CL protease inhibitory activity, among them, the activities of compounds A5, A10, A11, A23, A40, A41 and A43 are comparable or superior to those of the comparative compounds.

[0327] Example 59 Evaluation of Compound 2019 Novel Coronavirus Replication Inhibitory Activity

[0328] Vero cells were inoculated in batches with SARS-CoV-2 (Delta strain) at a multiplicity of infection (MOI) of 0.01 in a BSL-3 laboratory. The virus-inoculated cells were then added to ready-made compound plates at a density of 4000 cells / well in DMEM (dulbecco's modified eagle medium) containing 2% heat-inactivated fetal bovine serum. The cells were incubated at 37°C and 5% CO2for 3 days, at which time the virus-induced CPE (cytopathic effect) was 90% under untreated, infected control conditions. Cell viability was assessed using CCK8. Test compounds were tested alone or in the presence of the P-glycoprotein (P-gp) inhibitor CP-100356 at the indicated concentrations.

[0329] Table 2 CPE of Vero cells after infection with 2019 novel coronavirus using compounds

[0330] Compound CPE at 1 μM Comparative Compound (+ 1 μM CP-100356) <10％ A1 (+ 1 μM CP-100356) 40-50％ A3 (+ 1 μM CP-100356) <10％ A4 (+ 1 μM CP-100356) 80％ A21 (+ 1 μM CP-100356) <10％ A27 (+ 1 μM CP-100356) <10％ A41 (+ 1 μM CP-100356) <10％ Comparative Compound 1 >80％ A1 >80％ A3 >80％ A4 >80％

[0331] The experimental results show that the compounds of the present application (with the addition of a P-gp inhibitor) have certain anti-viral effects, wherein compounds A3, A21, A27, A41 have in vitro drug efficacy comparable to that of the comparative compound 1 at a drug concentration of 1 μM.

[0332] Example 60 Cytotoxicity test:

[0333] In a BSL-2 laboratory, the cytotoxicity of the compounds was evaluated in parallel with uninfected cells vero using ready-made compound plates. The drug concentrations were set at 1 μM and 10 μM. The non-toxicity indicator was a cell count of 90% to 110% of that before administration of the drug.

[0334] Table 3 Toxicity of compounds to normal cells

[0335]

[0336]

[0337] From the experiments, for normal VERO cells, the toxicity of compounds A3, A4, A21, A27, A41 is less than that of the comparative compound, and the safety window is greater than that of the comparative compound.

[0338] Example 61 Plasma stability test:

[0339] 1. Pre-warm 0.1 M potassium phosphate buffer, pH 7.4 ± 0.1, with 5 mM MgCl2(K / Mg-buffer).

[0340] 2. Plasma preparation: Frozen human plasma was thawed rapidly at 37°C.

[0341] 3. Test compound and internal standard compound solutions:

[0342] 0.5 mM solution A: 5 μL of a 10 mM stock solution was added to 95 μL ACN. 0.01 mM solution B: 20 μL of solution A was added to 980 μL of 0.1 M K / Mg-buffer buffer.

[0343] 4. Plasma was pre-warmed with solution B for 5 min at 37°C.

[0344] 5. 90 μL of pre-warmed plasma was added to each well at time points 0, 5, 15, 30, 60, 120 min, respectively.

[0345] 6. 10 μL of solution B and 400 μL of ACN containing internal standard (IS) were added to each well at time point 0 min.

[0346] 7. For the other time points, 10 μL of pre-warmed solution B was added to the designated wells at time points 5, 15, 30, 60, 120 min, respectively.

[0347] 8. The reaction was stopped by adding 400 μL of ACN containing IS to the respective wells at time points 5, 15, 30, 60, 120 min, respectively.

[0348] 9. After quenching, the samples were shaken for 5 min (600 rpm) and stored at -20°C if necessary.

[0349] 10. Before LC / MS / MS analysis, the samples were thawed at room temperature and centrifuged for 20 min at 6000 rpm.

[0350] 11. 100 μL of supernatant from each well was transferred to a 96-well sample plate containing 100 μL of ultrapure water for LC / MS analysis.

[0351] Table 4 Plasma stability of compounds

[0352]

[0353] The results show that the compounds of the present application have good plasma stability with a half-life (T 1 / 2 ) of more than 289.1 min.

[0354] Example 6 Liver microsomal stability test:

[0355] 1. 100 mM K-buffer was pre-warmed with 5 mM MgCl2 at pH 7.41.

[0356] 2. Test solution preparation

[0357] 0.5 mM A solution: 10 mM stock solution of compound + 5 μL, 95 μL of ACN.

[0358] Microsomes 1.5 μM solution (0.75 mg / mL): 1.5 μL of 500 μM solution and 18.75 μL of 20 mg / mL liver microsomes were added to 479.75 μL of K / Mg-buffer.

[0359] 3. K / Mg-buffer NADPH solution (6 mM, 5 mg / mL) was prepared.

[0360] 4. 30 μM containing 0.75 mg / mL human liver microsomes were added to the assay plate at different time points (0, 15, 30, 45, 60 min) respectively.

[0361] 5. Incubate for 5 min at 37 °C.

[0362] 6. At 0 min, add 150 μL of IS-containing ACN before adding 15 μL of NADPH stock solution (6 mM).

[0363] NADPH was dissolved in K / mg buffer to prepare a 6 mm, 5 mg / mL NADPH stock solution.

[0364] 7. At other time points, 15 μL of NADPH stock solution (6 mM) was added to the wells, the reaction was started and timed.

[0365] 8. At 15 min, 30 min, 45 min, 60 min, 150 μL of IS-containing ACN was added to the corresponding plate wells to stop the reaction.

[0366] 9. After quenching, shake for 10 min (600 rpm) and then centrifuge at 6000 rpm for 15 min.

[0367] 10. Transfer 80 μL of supernatant from each well to a 96-well sample plate (containing 140 μL of pure water) for LC / MS analysis.

[0368] Table 5 Compound liver microsome stability

[0369]

[0370] The results show that the compound of the present application has moderate liver microsome stability, with a half-life (T 1 / 2 ) of 46.4 min and a clearance (Cl int ) of 29.9 mL / min / kg.

[0371] Example 63 Pharmacokinetic test

[0372] The solvent used for the sample was 2% (v / v) Tween 80 / 98% 0.5% (w / v) methyl cellulose. After a single dose of 10 mg / kg oral administration in SD rats, blood samples were collected at different time points.

[0373] Preparation of standard curve and quality control sample: The compound mixed stock solution was diluted with 50% methanol water to obtain a standard working solution containing 20, 40, 100, 200, 400, 1000, 2000, 4000, 10000 ng / mL of each compound, and a quality control working solution containing 60, 600, and 8000 ng / mL. 2.50 μL of the standard curve working solution and the quality control working solution were added to 47.5 μL of blank rat plasma, respectively, to prepare a standard curve containing 1.00, 2.00, 5.00, 10.00, 20.00, 50.00, 100.00, 200.00, and 500.00 ng / mL of each compound, and a quality control sample containing 3.00, 30.00, and 400.00 ng / mL. 200 μL of acetonitrile (containing 2 ng / mL of internal standard verapamil) was added, vortexed for 3 min, centrifuged at 20000 rcf and 4°C for 10 min, and the supernatant was analyzed by LC-MS / MS.

[0374] Preparation of unknown sample: 50 μL of plasma sample was taken, 200 μL of acetonitrile (containing 2 ng / mL of internal standard verapamil) was added, vortexed for 3 min, centrifuged at 20000 rcf and 4°C for 10 min, and the supernatant was analyzed by LC-MS / MS.

[0375] Preparation of unknown sample diluted 10 times: 45 μL of blank plasma was taken, 5 μL of plasma sample was added, 200 μL of acetonitrile (containing 2 ng / mL of internal standard verapamil) was added, vortexed for 3 min, centrifuged at 20000 rcf and 4°C for 10 min, and the supernatant was analyzed by LC-MS / MS.

[0376] Table 6. Pharmacokinetic properties of the compounds of the present application in rats

[0377]

[0378] Conclusion: The compound of the present application is well absorbed in rats, has pharmacokinetic advantages, and the Cmax and AUC of compound A3 are superior to those of the comparative compound 1.

[0379] Example 64 Tissue distribution test

[0380] Drug lung tissue distribution: SD rats were orally administered with the compound of the example at a dose of 30 mg / kg, and the administration solution was 2% (v / v) Tween 80 in 0.5% (w / v) MC solution. At 0.25, 1, 2, 4, and 6 hours after administration, lung tissue of the SD rats was taken, homogenized, and added with 200 μL of acetonitrile (containing 2 ng / mL of internal standard verapamil) together with blood samples. After protein precipitation with a methanol solution, vortex oscillation was performed for 3 min, 20000 rcf centrifugation was performed at 4°C for 10 min, and the supernatant was subjected to LC-MS / MS analysis.

[0381] Table 7. Tissue distribution of the compound of the application in rats

[0382] Compound Lung / Plasma (ng) Ratio A3 4886 / 1357 3.6 A21 4924 / 1201 4.1 A41 6973 / 1516 4.6 Comparative Compound 1 1007 / 1157 0.87

[0383] Conclusion: The compound of the application is well absorbed in rats, and has higher exposure and higher lung targeting.

Claims

1. A compound, characterized in that, The compound is selected from: ； Or its pharmaceutically acceptable salt.

2. A pharmaceutical composition, characterized in that, It comprises the compound of claim 1.

3. The use of the compound of claim 1 or the composition of claim 2, characterized in that, Used to prepare drugs for treating pneumonia or asymptomatic infections caused by SARS-CoV-2 coronavirus infection.

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