WO2022021841A1 - Novel coronavirus main protease inhibitor, and preparation method therefor and use thereof - Google Patents

Abstract

Disclosed are a novel coronavirus main protease inhibitor, and a preparation method therefor and the use thereof. Specifically provided are a compound represented by formula (I), a pharmaceutically acceptable salt, a stereoisomer, an optical isomer, or an isotopically substituted form thereof. The compound can effectively inhibit the activity of SARS-CoV-2 M pro, can be used to prepare a SARS-CoV-2 M pro inhibitor, and block the replication and transcription of SARS-CoV-2 viruses in a patient. The compound has very good application prospects in the preparation of a SARS-CoV-2 M pro inhibitor, an anti-SARS-CoV-2 drug, and a drug for preventing and/or treating novel coronavirus pneumonia.

Description

A kind of inhibitor of novel coronavirus main protease and preparation method and use thereof technical field

The invention belongs to the technical field of organic synthetic medicines, and in particular relates to an inhibitor of novel coronavirus main protease, a preparation method and pharmaceutical use thereof.

Background technique

The 2019 coronavirus pneumonia (COVID-19, also known as novel coronavirus pneumonia) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, also known as the new coronavirus) is a global health emergency. Although alpha-interferon and anti-HIV drugs lopinavir/ritonavir have been used clinically

combination, but still has very limited efficacy and may have toxic side effects. Remdesivir, a broad-spectrum antiviral drug developed by Gilead Sciences, Inc., is also being explored to treat COVID-19, but more data is needed to prove its efficacy. Therefore, there is still an urgent need to develop safe and effective anti-SARS-CoV-2 drugs.

The genomic RNA of coronavirus is about 30knt long, has a 5' cap structure and a 3'-poly-a tail, and contains at least 6 open reading frames (ORFs). The first ORF (ORF1a/b) occupies approximately two-thirds of the genome length and directly translates two polyproteins: pp1a and pp1ab, with an a-1 frameshift between ORF1a and ORF1b. These polyproteins are processed by a major protease ( ^Mpro for short; also known as 3C-like protease ( ^3CLpro )) and one or two papain-like proteases (PLPs) into 16 nonstructural proteins. These nonstructural proteins are involved in the production of subgenomic RNAs, encoding the four major structural proteins (envelope (E), membrane (M), spine (S), and nucleocapsid (N) proteins) and other auxiliary proteins to complete the Virus replication and invasion process.

M ^pro proteolytically cleaves overlapping pp1a and pp1ab polysomes into functional proteins, a critical step in viral replication. Enzymes necessary for viral replication such as RdRp or nsp13 cannot fully function to complete replication without prior proteolytic release. Thus, virus-suppressing M ^pro can prevent the production of infectious virus particles, thereby reducing disease symptoms.

M ^pro is conserved among coronaviruses, and the substrates of M ^pro in different coronaviruses have some common features: amino acids from N-terminal to C-terminal are numbered in a paired form (-P4-P3-P2-P1↓P1 '-P2'-P3'), the cleavage site is between P1 and P1'. In particular, ^Mpro has a unique substrate preference for glutamine at the P1 site (Leu-Gln↓(Ser, Ala, Gly)), which is absent in host proteases, suggesting that by targeting the virus It is feasible to achieve high selectivity with M ^pro . Thus, the absolute dependence of the virus on the correct function of this protease, coupled with the lack of a homologous human protease, makes ^Mpro an ideal antiviral target.

Therefore, there is an urgent need to develop a drug that can effectively inhibit the M ^pro activity of SARS-CoV-2 virus.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide the inhibitor of novel coronavirus main protease and its preparation method and pharmaceutical use.

The present invention provides the compound shown in formula I, or its pharmaceutically acceptable salt, or its stereoisomer, or its optical isomer, or its isotopic substitution form:

Wherein, X is O or S;

Ring A is selected from the following groups unsubstituted or substituted by one or more R ₆ : 5-6 membered saturated heterocyclyl, 5-6 membered unsaturated heterocyclyl, saturated heterofused ring, unsaturated heterofused ring group; R ₆ is independently selected from C _1-6 alkyl, C _1-6 alkoxy, halogen, hydroxyl, cyano, amino, and carboxyl;

R ₃ is L ₃ M ₀ L ₄ R _3a ; wherein L ₃ is selected from none, C _1-4 alkylene, halogenated C _1-4 alkylene, C _2-4 alkenylene, halogenated C _{2- 4} alkenylene, L ₄ is selected from none, C _1-4 alkylene, halogenated C _1-4 alkylene, M ₀ is selected from none, O, S, NH, CO, CONH, NHCO, R _3a is The following groups are unsubstituted or substituted by one or more R _3b : 5-6 membered aryl, 5-6 membered heteroaryl, unsaturated heterofused ring group, unsaturated fused ring alkyl; R _3b are each independently Selected from C _1-5 alkyl substituted or unsubstituted by R _3c , C _1-5 alkoxy substituted or unsubstituted by R _3c , halogen, phenyl substituted or unsubstituted by R _3c , NR ₁₄ R ₁₅ , substituted or unsubstituted naphthyl and hydroxyl by R _3c ; R ₁₄ and R ₁₅ are each independently selected from hydrogen or C _1-5 alkyl, and R _3c is independently selected from halogen, deuterium, cyano, hydroxyl, amino ,carboxyl;

R ₄ is selected from the following groups unsubstituted or substituted by one or more substituents: 5-6 membered aryl, 5-6 membered heteroaryl, C _1-5 alkyl, COOR ₁₀ ; the substituents are each independently selected from =O, hydroxyl, nitro, amino, carboxyl, halogen, C _1-5 alkyl; R ₁₀ is C _1-5 alkyl;

R ₅ is selected from COR ₈ or WCOOR ₇ ; wherein, R ₈ is selected from hydrogen or

W is selected from none, C _1-4 alkylene, C _2-4 alkenylene, C _2-4 alkynylene, R ₇ is selected from C _1-6 alkyl; M is selected from none, CO, NH, CONH , NHCO, COO or OCO, L ₀ is selected from none, C _1-4 alkylene, C _2-4 alkenylene, L ₁ is selected from none, C _1-4 alkylene, C _2-4 alkenylene , R ^8a is selected from C _1-5 alkyl, halogenated C _1-5 alkyl, 3-6 membered saturated cycloalkyl, 3-6 membered saturated heterocyclic group, 5-6 membered aryl or 5-6 membered aryl Yuan Heteroaryl.

Further, the structure of the compound is shown in formula II, formula III or formula IV:

Wherein, X is O or S;

n is selected from an integer of 0-3, preferably an integer of 0-2;

R ₁ and R ₂ are each independently selected from hydrogen, C _1-5 alkyl, C _1-5 alkoxy, halogen, hydroxyl, cyano, amino, and carboxyl;

R ₃ is L ₃ M ₀ L ₄ R _3a ; wherein L ₃ is selected from none, C _1-4 alkylene, halogenated C _1-4 alkylene, C _2-3 alkenylene, and L ₄ is selected from none , C _1-4 alkylene, halogenated C _1-4 alkylene, M ₀ is selected from none, O, S, NH, CO, CONH, NHCO, R _3a is unsubstituted or by one or more R _3b Substituted the following groups: phenyl,

R _3b are each independently selected from C _1-4 alkyl, halogen-substituted C _1-4 alkyl, deuterated C _1-4 alkyl, cyano-substituted C _1-4 alkyl, C _1-4 alkyl Oxyl, halogen-substituted C _1-4 alkoxy, deuterated C _1-4 alkoxy, cyano-substituted C _1-4 alkoxy, halogen, phenyl, halogenated phenyl, NR ₁₄ R ₁₅ ,

Hydroxyl, R ₁₄ and R ₁₅ are each independently selected from hydrogen or C _1-4 alkyl;

R ₄ is selected from the following groups unsubstituted or substituted by one or more substituents: 5-6 membered aryl, 5-6 membered heteroaryl, C _1-5 alkyl, COOR ₁₀ ; the substituents are each independently selected from =O, hydroxyl, nitro, amino, carboxyl, halogen, C _1-5 alkyl; R ₁₀ is C _1-5 alkyl;

R ₈ is selected from hydrogen or

M is selected from None, CO, NH, CONH, NHCO, COO or OCO, L ₀ is selected from None, C _1-3 alkylene, C _2-4 alkenylene, L ₁ is selected from None, C _1-3 alkylene Alkyl, C _2-4 alkenylene, R ^8a is selected from C _1-4 alkyl, halogenated C _1-4 alkyl, 3-6 membered saturated cycloalkyl, 3-6 membered saturated heterocyclic group, A 5- to 6-membered aryl group or a 5- to 6-membered heteroaryl group.

Further, R ₁ and R ₂ are each independently selected from hydrogen, C _1-4 alkyl, C _1-4 alkoxy, halogen, and hydroxyl;

R ₃ is selected from

L ₃ M ₀ L ₄ R _3a ; L ₃ is selected from none, C _1-3 alkylene, halogenated C _1-3 alkylene, C _2-3 alkenylene, L ₄ is selected from none, C _{1-3 3} alkylene, halogenated C _1-3 alkylene, M ₀ is selected from none, O, NH, CO, CONH, R _3a is phenyl, phenyl substituted by one or more R _3b , R _3b Each independently selected from C _1-4 alkyl, halogen-substituted C _1-4 alkyl, deuterated C _1-4 alkyl, cyano-substituted C _1-4 alkyl, C _1-4 alkoxy , halogen-substituted C _1-4 alkoxy, deuterated C _1-4 alkoxy, cyano-substituted C _1-4 alkoxy, halogen, phenyl, halogenated phenyl, NR ₁₄ R ₁₅ ,

Hydroxyl, R ₁₄ and R ₁₅ are each independently selected from hydrogen or C _1-3 alkyl;

R ₄ is selected from

C _1-2 alkyl, COOR ₁₀ , substituted or unsubstituted phenyl; the substituent is selected from hydroxyl, nitro; R _a1 and R _a2 are independently selected from hydrogen, C _1-3 alkyl, halogen; R ₁₀ is C _1-3 alkyl;

R ₈ is selected from hydrogen, CONHR ₁₁ , L ₂ COOR ₁₂ , C _1-4 alkyl, halogenated C _1-4 alkyl; R ₁₁ is selected from 3-6 membered saturated cycloalkyl, C _1-4 alkyl , benzyl,

L ₂ is a C _1-2 alkylene group, a C _2-3 alkenylene group, and R ₁₂ is a C _1-3 alkyl group.

Further, described formula II is shown as formula II-1 or formula II-2:

Wherein, X is O or S, preferably O;

R ₁ and R ₂ are each independently selected from hydrogen, C _1-3 alkyl, preferably methyl;

m is selected from an integer from 0 to 3, and R _3b is independently selected from phenyl, halogenated phenyl, halogen, C _1-3 alkyl, halogenated or deuterated C _1-3 alkyl, C _{1- 3} alkoxy, halogenated or deuterated C _1-3 alkoxy, hydroxyl;

R _a1 and R _a2 are independently selected from hydrogen, C _1-3 alkyl, and halogen;

R _b is selected from hydrogen, C _1-3 alkyl, halogenated C _1-3 alkyl;

L ₃ is selected from none, C _1-2 alkylene, halogenated C _1-2 alkylene, C ₂ alkenylene, L ₄ is selected from none, C _1-3 alkylene, halogenated C _1-3 Alkylene, M ₀ is selected from none, O, NH, CO, CONH;

The halogen is preferably chlorine or fluorine.

Further, the structure of the compound is one of the following structures:

The present invention also provides a pharmaceutical composition, wherein the pharmaceutical composition is the above compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer, or an isotopic substitution form thereof. Active ingredient, plus pharmaceutically acceptable excipients.

The present invention also provides the use of the above-mentioned compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer, or an isotopic substituted form thereof, in the preparation of a coronavirus proteolytic enzyme inhibitor; preferably Preferably, the coronavirus proteolytic enzyme is a coronavirus main protease; more preferably, the coronavirus proteolytic enzyme is SARS-COV-2M ^pro .

The present invention also provides the use of the above-mentioned compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer, or an isotopic substituted form thereof, in the preparation of an anti-coronavirus drug, preferably, The coronavirus is a novel coronavirus SARS-CoV-2.

The present invention also provides the above-mentioned compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or an isotopic substituted form thereof in the preparation of prevention and/or treatment of SARS-COV-2M ^pro Use in medicines for related diseases, preferably, the disease related to SARS-COV-2M ^pro is novel coronavirus pneumonia COVID-19.

Further, the medicine of described coronavirus proteolytic enzyme inhibitor, anti-coronavirus or the medicine of prevention and/or treatment of viral pneumonia can suppress the activity of SARS-COV-2M ^pro and/or can suppress SARS-COV-2 infection cell.

Definitions of terms used in the present invention: Unless otherwise specified, the initial definitions of groups or terms provided herein apply to the groups or terms throughout the specification; for terms that are not specifically defined herein, they should be based on the disclosure and context. , give their meanings that those skilled in the art can give them.

Minimum and maximum carbon content in a hydrocarbon group are indicated by prefixes, eg, the prefix C _a-b alkyl denotes any alkyl group containing "a" to "b" carbon atoms. For example, C _1-6 alkyl refers to a straight or branched chain alkyl group containing 1 to 6 carbon atoms.

"Substituted" herein refers to the replacement of 1, 2 or more hydrogen atoms in a molecule by a different atom or molecule, including 1, 2 or more substitutions on isotopic or isotopic atoms in the molecule .

"Isotopic substitution form" refers to a compound obtained by replacing one or more than two atoms in a compound with its corresponding isotope, for example, hydrogen in the compound is replaced by protium, deuterium or tritium.

"Pharmaceutically acceptable" means that a carrier, vehicle, diluent, adjuvant, and/or salt formed is generally chemically or physically compatible with the other ingredients that make up a pharmaceutical dosage form and physiologically compatible with receptor compatible.

"Salt" is an acid and/or base salt of a compound or its stereoisomer with inorganic and/or organic acids and/or bases, including zwitterionic salts (inner salts), and quaternary ammonium salts , such as alkylammonium salts. These salts can be obtained directly in the final isolation and purification of the compounds. It can also be obtained by mixing a compound, or a stereoisomer thereof, with a certain amount of acid or base as appropriate (eg, equivalent). These salts may form a precipitate in solution and be collected by filtration, or recovered after evaporation of the solvent, or obtained by lyophilization after reaction in an aqueous medium.

"Pharmaceutically acceptable salts" may be hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, Succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate.

Halogen is fluorine, chlorine, bromine or iodine.

"Aryl" refers to an all-carbon monocyclic or fused polycyclic (ie, rings sharing adjacent pairs of carbon atoms) groups having a conjugated pi-electron system, such as phenyl. The aryl group contains no heteroatoms, such as nitrogen, oxygen, or sulfur, while the point of attachment to the parent must be on a carbon atom on the ring with a conjugated pi-electron system. Aryl groups can be substituted or unsubstituted. The "5- to 6-membered aryl group" refers to an aryl group having 5 or 6 ring carbon atoms.

"Heteroaryl" refers to a heteroaromatic group containing one to more heteroatoms. The heteroatoms referred to herein include oxygen, sulfur and nitrogen. For example, furanyl, thienyl, pyridyl, pyrazolyl and the like. The heteroaryl group can be optionally substituted or unsubstituted. The "5- to 6-membered heteroaryl group" refers to a heteroaryl group having 5 or 6 ring atoms.

"Cycloalkyl" refers to a saturated or unsaturated cyclic hydrocarbon substituent; the cyclic hydrocarbon may be monocyclic or polycyclic. For example, "a 3- to 6-membered saturated cycloalkyl group" refers to a saturated cycloalkyl group having 3 to 6 ring carbon atoms.

"Heterocyclyl" refers to a saturated or unsaturated cyclic hydrocarbon substituent; cyclic hydrocarbons may be monocyclic or polycyclic and carry at least one ring heteroatom (including but not limited to O, S or N). For example, the "3- to 6-membered saturated heterocyclic group" refers to a saturated heterocyclic group having 3 to 6 ring atoms.

"Fused cycloalkyl" refers to a polycyclic cycloalkyl in which two rings share two adjacent carbon atoms.

"Hetero-fused ring group" refers to a polycyclic heterocyclic group, which contains at least one heteroatom, and two rings in the polycyclic heterocyclic group share two adjacent carbon atoms or heteroatoms.

"Alkylene" refers to a group in which an alkyl group has lost one atom. For example C ₁ alkylene:

C ₂ alkylene:

"Alkenylene" refers to a group in which an alkenyl group has lost one atom. For example C ₂ alkenyl:

"Alkynylene" refers to a group in which an alkynyl group has lost one atom. For example C ₂ alkynyl:

The experimental results show that the present invention provides a compound that can effectively inhibit the activity of the new coronavirus main protease M ^pro , the compound can effectively inhibit the replication of SARS-COV-2 virus in cells, and inhibit the SARS-COV-2 virus in cells. Infection, resists in vivo SARS-COV-2 infection in transgenic mice; reduces viral load in the lungs of SARS-COV-2-infected transgenic mice, and reduces lung chemokine ligand 10 (CXCL10) and beta-type in mice The gene expression level of interferon (IFN-β) reduces the number of neutrophils (NEU) and macrophages (MAC) in the lungs of mice, and improves the pathological damage of the lungs of mice. Meanwhile, the compounds provided by the present invention also have good in vivo safety and pharmacokinetic properties. The compounds of the present invention have very good application prospects in the preparation of SARS-CoV-2 M ^pro inhibitors, anti-SARS-CoV-2 drugs, and drugs for preventing and/or treating novel coronavirus pneumonia.

Obviously, according to the above-mentioned content of the present invention, according to the common technical knowledge and conventional means in the field, without departing from the above-mentioned basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

The above content of the present invention will be further described in detail below through the specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention.

Description of drawings

Fig. 1 is the inhibitory activity curve of compound 26 to SARS-COV-2M ^pro .

Fig. 2 is the inhibitory activity curve of compound 33 on SARS-COV-2M ^pro .

Figure 3 is the inhibitory activity curve of compound 37 against SARS-COV-2M ^pro .

Figure 4. Inhibition experiments of compounds on the replication of SARS-COV-2 in human alveolar epithelial cells.

Figure 5 Pulmonary viral load in SARS-CoV-2-infected mice.

Figure 6 Lung histopathological sections (3dpi) of SARS-CoV-2-infected mice.

Figure 7. Representative cytokine expression levels (3dpi) in the lungs of SARS-CoV-2-infected mice.

Figure 8 Pulmonary neutrophil and macrophage counts (3 dpi) in SARS-CoV-2-infected mice.

detailed description

The raw materials and equipment used in the present invention are all known products, obtained by purchasing commercially available products.

Example 1: Preparation of Compound 1

The compound 1 of the present invention is prepared according to the above-mentioned preparation route, and the reaction conditions of each step in the route are as follows:

i, a, dimethyl 2-fluoromalonate, benzyl alcohol, toluene, p-toluenesulfonic acid, 110°C; b, isopropanol, n-hexane, -10°C;

ii, isopropanol, sodium hydroxide, water, 45 ℃;

iii. Anhydrous tetrahydrofuran, isopropylmagnesium chloride tetrahydrofuran solution, Ar, 0°C;

iv. Anhydrous tetrahydrofuran, N,N'-carbonyldiimidazole, Ar, 0°C;

v, ethyl acetate, 10% palladium carbon, hydrogen, room temperature;

vi, dichloromethane, hydrochloric acid dioxane solution;

vii, dichloromethane, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea, N,N-diisopropylethyl Amine, -20°C;

viii, dichloromethane, trifluoroacetic acid;

ix, dichloromethane, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea, N,N-diisopropylethyl Amine, -20°C;

The following are the specific synthesis steps:

Intermediate 2: Preparation of dibenzyl 2-fluoromalonate

Dimethyl 2-fluoromalonate (10g, 66.6mmol, 1.0eq) and benzyl alcohol (35mL, 338.2mmol, 5.0eq) were dissolved in 100mL of toluene, 1.15g of p-toluenesulfonic acid (6.7mmol, 0.1eq) was added, The reaction was refluxed, monitored by TLC, and the reaction was completed after about 8 hours. Cool to room temperature, evaporate toluene under reduced pressure, add 15 mL of isopropanol, stir evenly, slowly add 30 mL of n-hexane with stirring, place in a -10°C cold trap and continue stirring for 2 hours, a large amount of white solids are precipitated. After suction filtration, the filter cake was washed twice with 10 mL×2 frozen n-hexane, and the filter cake was dried under vacuum at 30° C. to obtain 18.4 g of product with a yield of 91.4%. ¹ H NMR (400MHz, DMSO-d6)δ7.64-6.91(m,10H),6.00(d,J=46.3Hz,1H),5.30-5.20(m,4H).

Intermediate 3: Preparation of monobenzyl 2-fluoromalonate

Dibenzyl 2-fluoromalonate (18.4g, 60.9mmol, 1.0eq) was dissolved in 100mL of isopropanol, the temperature was raised to 45°C, sodium hydroxide (2.55g, 63.9mmol, 1.05eq) was dissolved in 60mL of water and then slowly Slowly drip, dripping time > 1 hour. After the dropwise addition, the reaction was continued for 30 minutes, the isopropanol was evaporated under reduced pressure, 50 mL of water was added, and the pH value was adjusted to about 9 with saturated sodium bicarbonate solution. The aqueous phase was extracted twice with dichloromethane 20mL×2 dichloromethane, the pH value of the aqueous phase was adjusted to 1-2 with 6mol/L hydrochloric acid, extracted three times with 40mL×3 isopropyl ether, the organic phases were combined and washed with 30mL saturated brine once. The organic phase was dried by adding anhydrous magnesium sulfate, filtered, and concentrated to obtain a viscous residue, which was added with 60 mL of n-hexane and stirred overnight. A white solid was precipitated, filtered, and the filter cake was dried under vacuum at 40 °C to obtain 6.5 g of the product with a yield of 50.3%. ¹ H NMR (400MHz, Chloroform-d) δ7.41-7.32(m, 5H), 5.87(s, 2H), 5.39(d, J=47.9Hz, 1H), 5.31(s, 1H).

Preparation of Intermediate 4

Monobenzyl 2-fluoromalonate was dissolved in anhydrous tetrahydrofuran (2 mL/mmol), protected by argon replacement, cooled to 0 °C, and isopropylmagnesium chloride tetrahydrofuran solution (2M tetrahydrofuran solution, 2.0eq) was slowly added dropwise to obtain White suspension. Stirring was continued for 1 hour at 0°C, and the product suspension was directly used for the next reaction.

Intermediate 6: Preparation of 1-benzyl 6-methyl(4S)-4-(((tert-butoxycarbonyl)amino)-2-fluoro-3-oxoadipate

Boc-L-aspartate 4-methyl ester (2.2g, 8.8mmol, 1.0eq) was dissolved in 50mL of anhydrous tetrahydrofuran, protected by argon replacement, cooled to 0°C, added CDI (1.5g, 9.3mmol, 1.05eq) ) and incubated for 1 hour. The reaction solution was cooled to -20°C, 1.5 eq of intermediate 4 was slowly added, and the reaction was incubated for 1 hour, and then the temperature was raised to room temperature for 6 hours. The reaction solution was slowly poured into 300 mL of 2M dilute hydrochloric acid under an ice-water bath, extracted three times with 100 mL × 3 ethyl acetate, the organic phases were combined, washed with saturated sodium bicarbonate solution until weakly alkaline, and washed once with 50 mL of saturated brine. Anhydrous magnesium sulfate was added to dry, filtered and concentrated, and the obtained crude product was directly used in the next reaction.

Intermediate 7: Preparation of (S)-methyl 3-(((tert-butoxycarbonyl)amino)methyl-5-fluoro-4-oxopentanoate

Add 50 mL of ethyl acetate to the crude intermediate 6 obtained in the previous step, add 200 mg of 10% palladium on carbon, replace with hydrogen, react at room temperature overnight under hydrogen, filter and concentrate, and use petroleum ether: ethyl acetate = 10:1 mobile phase column layer for the crude product obtained 1.5g of colorless oily substance was obtained by precipitation, and the yield was 65%. ¹ H NMR (400MHz, Chloroform-d)δ5.51(d,J=8.0Hz,1H),5.28-5.06(m,2H),4.73-4.52(m,1H),3.70(s,3H),3.08 (dd, J=17.2, 4.6Hz, 1H), 2.84 (dd, J=17.2, 5.0Hz, 1H), 1.46 (s, 9H).

Intermediate 8: Preparation of (S)-methyl 3-amino-5-fluoro-4-oxopentanoate

500 mg of intermediate 7 was dissolved in 5 mL of dichloromethane, and then 5 mL of dioxane hydrochloride was added. After the reaction was completed, the mixture was spin-dried to obtain intermediate 8 with a yield of 91.2%. ¹ H NMR (400MHz, Chloroform-d) δ5.25-5.10(m, 2H), 4.53(dd, J=8.7, 1.0Hz, 2H), 4.44(d, J=7.9Hz, 1H), 3.69(s ,3H),2.83–2.71(m,2H).

Intermediate 11: Preparation of methyl(1H-indole-2-carbonyl)-L-proline

1H-Indole-2-carboxylic acid (1g, 6.21mmol, 1.0eq) was dissolved in dichloromethane, then HATU (2.81g, 7.40mmol, 1.2eq) was added at -20°C and L-proline methyl ester was added The hydrochloride salt (1.03 g, 6.21 mmol, 1.0 eq), finally DIEA (3 mL, 18.51 mmol, 3.0 eq) was added and the reaction was monitored by TLC. After completion of the reaction, extraction was performed with aqueous solution and DCM, and the organic layer was concentrated and separated by column chromatography to obtain Intermediate 11 (1.53 g) with a yield of 75.2%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.68 (dt, J=7.4, 1.5 Hz, 1H), 7.43 (dd, J=7.4, 1.6 Hz, 1H), 7.26 (td, J=7.5, 1.7 Hz, 1H), 7.19–7.14 (m, 2H), 4.31 (t, J=7.0Hz, 1H), 3.72 (td, J=7.1, 2.3Hz, 2H), 3.68 (s, 3H), 2.11–2.00 (m, 2H), 1.93–1.81 (m, 2H).

Intermediate 12: Preparation of (1H-indole-2-carbonyl)-L-proline

500 mg of intermediate 11 was dissolved in 10 mL of dichloromethane, then 5 mL of trifluoroacetic acid was added, and after the reaction was completed, it was spin-dried to obtain 378 mg of intermediate 12, which was directly used as the first step reaction. The yield was 91.2%.

Compound 1: Preparation of methyl (S)-3-((S)-1-(1H-indole-2-carbonyl)pyrrolidine-2-carboxamide)-5-fluoro-4-oxopentanoic acid

Intermediate 12 (168mg, 0.61mmol, 1.0eq) was dissolved in dichloromethane, HATU (280mg, 0.73mmol, 1.2eq) was added at -20°C, followed by intermediate 8 (100mg, 0.61mmol, 1.0eq), and finally DIEA (301 μL, 1.83 mmol, 3.0 eq) was added and the reaction was monitored by TLC. After the completion of the reaction, extraction was performed with aqueous solution and DCM, the organic layer was concentrated, and the compound 1 was obtained by column chromatography with a yield of 34%. ¹ H NMR(400MHz, DMSO)δ11.55(s,1H),8.69(s,1H),7.65(d,J=7.6Hz,1H),7.46(d,J=8.3Hz,1H),7.20( m,1H),7.06(d,J=7.8Hz,2H),5.26(m,2H),4.60(m,1H),4.49(m,1H),3.96(dd,J=15.0,7.4Hz,2H ),3.61(s,3H),2.86(m,1H),2.60(dd,J=15.9,7.7Hz,1H),2.02(m,2H),1.82(m,2H).HRMS m/z(ESI )calcd for C ₂₀ H ₂₅ FN ₄ O ₅ [M+H] ⁺ 403.1543 found:404.1476.

Example 2: Preparation of Compound 3

The compound 3 of the present invention is prepared according to the above-mentioned preparation route, and the reaction conditions of each step in the route are as follows:

i. Boc-L-dimethyl glutamate, LiHMDS tetrahydrofuran solution, argon, anhydrous tetrahydrofuran, -78°C,;

ii, (2S,4R)-dimethyl 2-(tert-butoxycarbonylamino)-4-(cyanomethyl)glutarate, anhydrous methanol, cobalt chloride hexahydrate, sodium borohydride;

iii, (S)-methyl 2-(tert-butoxycarbonylamino)-3-((S)-2-carbonylpyrrolidin-3-yl)propionate, lithium hydroxide monohydrate, tetrahydrofuran, 0 °C;

iv. Anhydrous tetrahydrofuran, Ar, N,N'-carbonyldiimidazole, 0°C;

v, ethyl acetate, 10% palladium carbon, hydrogen, room temperature;

vi, dichloromethane, hydrochloric acid dioxane solution;

vii, dichloromethane, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea, N,N-diisopropylethyl Amine, -20°C;

viii, dichloromethane, trifluoroacetic acid;

ix, dichloromethane, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea, N,N-diisopropylethyl Amine, -20°C;

The following are the specific synthesis steps:

Intermediate 14: Preparation of (2S,4R)-2-((tert-butoxycarbonyl)amino)-4-(cyanomethyl)glutaric acid dimethyl ester

Boc-L-dimethyl glutamate (12 g, 43.6 mmol, 1.0 eq) was dissolved in 100 mL of anhydrous tetrahydrofuran, protected by argon replacement, cooled to -78°C, and slowly added dropwise 94 mL of LiHMDS tetrahydrofuran solution (1M tetrahydrofuran solution, 94mmol, 2.2eq), the reaction was incubated for 1 hour after the dropwise addition. 3.24 mL of bromoacetonitrile (46.6 mmol, 1.1 eq) was slowly added dropwise to the reaction solution, and the reaction was incubated for 6 hours and then quenched with 50 mL of saturated ammonium chloride solution. The quenched reaction solution was warmed to room temperature, extracted three times with 60 mL × 3 ethyl acetate, the organic phases were combined, washed with 50 mL of saturated brine, dried over anhydrous magnesium sulfate, filtered, and concentrated, the obtained crude product was mixed with petroleum ether:ethyl acetate Ester=4:1 mobile phase column chromatography to obtain 9.36 g of pale yellow oil, yield 68.3%. ¹ H NMR (400MHz, Chloroform-d) δ5.11(d, J=8.6Hz, 1H), 4.39(s, 1H), 3.77(s, 3H), 3.76(s, 3H), 2.90-2.82(m ,1H),2.82–2.74(m,2H),2.28–2.06(m,2H),1.45(s,9H).

Intermediate 15: Preparation of (S)-methyl 2-(((tert-butoxycarbonyl)amino)methyl-3-((S)-2-oxopyrrolidin-3-yl)propanoate

(2S,4R)-dimethyl 2-(tert-butoxycarbonylamino)-4-(cyanomethyl)glutarate (9.36 g, 29.8 mmol, 1.0 eq) was dissolved in 150 mL of anhydrous methanol, Cool to 0°C. Cobalt chloride hexahydrate (4.25 g, 18 mmol, 0.6 eq) was added, and sodium borohydride (6.76 g, 180 mmol, 6.0 eq) was added in batches. After the addition, the temperature was raised to room temperature, and the reaction was performed overnight. TLC monitored the completion of the reaction. 50 mL of saturated ammonium chloride solution was added to quench the reaction, methanol was evaporated under reduced pressure, extracted three times with 100 mL × 3 ethyl acetate, the organic phases were combined, washed three times with 200 mL × 3 saturated ammonium chloride solution, and 200 mL × 3 saturated brine successively Three times, the organic phase was dried by adding anhydrous magnesium sulfate, filtered and concentrated to obtain 3.94 g of white solid with a mobile phase column chromatography of petroleum ether:ethyl acetate=1:1 for the crude product obtained, with a yield of 46.2%. ¹ H NMR (400MHz, Chloroform-d) δ5.92(s, 1H), 5.49(d, J=8.4Hz, 1H), 4.41-4.26(m, 1H), 3.74(s, 3H), 3.45-3.26 (m, 2H), 2.58–2.39 (m, 2H), 2.27–2.07 (m, 1H), 1.98–1.78 (m, 2H), 1.44 (s, 9H).

Intermediate 16: Preparation of (S)-2-((tert-butoxycarbonyl)amino)-3-((S)-2-oxopyrrolidin-3-yl)propionic acid

(S)-Methyl 2-(tert-butoxycarbonylamino)-3-((S)-2-carbonylpyrrolidin-3-yl)propanoate (0.88 g, 3.1 mmol, 1.0 eq) was dissolved in 10 mL of tetrahydrofuran, cooled to 0 °C. Lithium hydroxide monohydrate (0.64 g, 15.4 mmol, 5.0 eq) was dissolved in 10 mL of water and slowly added dropwise. After dropping, the reaction was incubated for 4 hours, and the reaction was monitored by TLC. The pH value of saturated citric acid aqueous solution was adjusted to neutral, the tetrahydrofuran was evaporated under reduced pressure, extracted once with 10 mL of ethyl acetate, the aqueous phase was adjusted to pH 3-4 with saturated aqueous citric acid solution, extracted three times with 20 mL × 3 ethyl acetate, and the organic The phase was washed with 20 mL of saturated brine, dried over anhydrous magnesium sulfate, filtered, and concentrated to obtain 0.78 g of an off-white solid with a yield of 93.2%. ¹ H NMR (400MHz, Chloroform-d)δ7.19(s,1H),5.69(d,J=7.9Hz,1H),4.35(q,J=7.6Hz,1H),3.48-3.31(m,2H) ), 2.70–2.55 (m, 1H), 2.51–2.36 (m, 1H), 2.27–2.12 (m, 1H), 1.99–1.80 (m, 2H), 1.44 (s, 9H).

Intermediate 17: Benzyl (4S)-4-((tert-butoxycarbonyl)amino)-2-fluoro-3-oxo-5-((S)-2-oxopyrrolidin-3-yl)pentanoate preparation

(S)-2-((tert-butoxycarbonyl)amino)-3-((S)-2-carbonylpyrrolidin-3-yl)propanoic acid (2.4 g, 8.8 mmol, 1.0 eq) was dissolved in 50 mL Anhydrous tetrahydrofuran, argon replacement protection, cooled to 0 °C, CDI (1.5 g, 9.3 mmol, 1.05 eq) was added, and the reaction was maintained for 1 hour. The reaction solution was cooled to -20°C, 1.5 eq of intermediate 4 was slowly added, and the reaction was incubated for 1 hour, and then the temperature was raised to room temperature for 6 hours. The reaction solution was slowly poured into 300 mL of 2M dilute hydrochloric acid under an ice-water bath, extracted three times with 100 mL × 3 ethyl acetate, the organic phases were combined, washed with saturated sodium bicarbonate solution until weakly alkaline, and washed once with 50 mL of saturated brine. Anhydrous magnesium sulfate was added to dry, filtered and concentrated, and the obtained crude product was directly used in the next reaction.

Intermediate 18: Preparation of tert-butyl ((S)-4-fluoro-3-oxo-1-((S)-2-oxopyrrolidin-3-yl)butan-2-yl)carbamate

The crude intermediate 17 obtained in the previous step was added with 50 mL of ethyl acetate, added with 200 mg of 10% palladium carbon, replaced with hydrogen, reacted at room temperature overnight under hydrogen, filtered, and concentrated. 1.3 g of white solid were obtained, with a yield of 50%. ¹ H NMR (400MHz, Chloroform-d) δ5.99(d, J=7.5Hz, 1H), 5.91(s, 1H), 5.31-4.95(m, 2H), 4.56(s, 1H), 3.42-3.32 (m, 2H), 2.56–2.42 (m, 2H), 2.10–1.97 (m, 1H), 1.96–1.81 (m, 2H), 1.45 (s, 9H).

Intermediate 19: Preparation of (S)-3-((S)-2-amino-4-fluoro-3-oxobutyl)pyrrolidin-2-one

500 mg of intermediate 18 was dissolved in 5 mL of dichloromethane, and then 5 mL of dioxane hydrochloride was added. After the reaction was completed, the mixture was spin-dried to obtain intermediate 19 with a yield of 85%.

Intermediate 22: Preparation of ethyl(1S,3aR,6aS)-2-(2-(2,4-dichlorophenoxy)acetyl)octahydrocyclopenteno[c]pyrrole-1-carboxylate .

2,4-Dichlorophenoxyacetic acid (Intermediate 21, 0.58g, 2.62mmol), 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylureahexa Fluorophosphate (1.2 g, 3.14 mmol), N,N-diisopropylethylamine (1.3 mL, 7.86 mmol), and (1S,3aR,6aS)-octahydrocyclopenteno[c]pyrrole-1 - Ethyl carboxylate hydrochloride (Intermediate 20, 0.58 g, 2.62 mmol) was dissolved in 15 mL of ultra-dry N,N-dimethylformamide, and the reaction was carried out at 25 ° C under argon protection for 12 hours, d reaction 4 times the volume of water was added, extracted three times with dichloromethane, the combined organic phases were washed with saturated ammonium chloride solution and saturated sodium carbonate solution, dried over anhydrous sodium sulfate, filtered, and subjected to silica gel column chromatography (petroleum ether/ethyl acetate) = 1 : 1) to yield intermediate 22 (0.60 g, 59%). ¹ H NMR (400MHz, MeOD) δ 7.42 (d, J=5.4Hz, 1H), 7.26-7.19 (m, 1H), 6.97 (d, J=8.9Hz, 1H), 4.80-7.72 (m, 2H) ), 4.28(d, J=3.6Hz, 1H), 4.22-4.10(m, 2H), 3.87(d, J=10.6Hz, 1H), 3.63-3.48(m, 1H), 3.57(d, J= 10.5Hz, 2H), 2.71–2.61 (m, 1H), 2.08–1.84 (m, 1H), 1.83–1.46 (m, 4H), 1.31–1.17 (m, 3H). ESI-MS (m/z) :386.02(M+H) ⁺ .

Intermediate 23: Preparation of (1S,3aR,6aS)-2-(2-(2,4-dichlorophenoxy)acetyl)octahydrocyclopenteno[c]pyrrole-1-carboxylic acid

Ethyl (1S,3aR,6aS)-2-(2-(2,4-dichlorophenoxy)acetyl)octahydrocyclopenteno[c]pyrrole-1-carboxylate (Intermediate 22, 200 mg, 0.52 mmol) was dissolved in 20 mL of methanol, then 2M sodium hydroxide solution (10 mL) was added, and the reaction was stirred at 25 ° C for 4 hours. After the reaction was monitored by TLC, the methanol was spin-dried, and the pH was adjusted to weakly acidic with hydrochloric acid. Methyl chloride was extracted three times, and the organic phases were combined and dried over anhydrous sodium sulfate, and then the crude product was selected and dried, and the crude product was directly carried out to the next step.

Compound 3: (1S,3aR,6aS)-2-(2-(2,4-dichloro)acetyl)-N-((S)-4-fluoro-3-oxo-1-((S)- Preparation of 2-carbonyl-3-yl)butan-2-yl)octahydrocyclopenteno[c]pyrrole-1-carboxamide

Intermediate 23 (168mg, 0.61mmol, 1.0eq) was dissolved in dichloromethane, HATU (280mg, 0.73mmol, 1.2eq) was added at -20°C, followed by intermediate 19 (100mg, 0.61mmol, 1.0eq), and finally DIEA (301 μL, 1.83 mmol, 3.0 eq) was added and the reaction was monitored by TLC. After the completion of the reaction, extraction was performed with aqueous solution and DCM, and the organic layer was concentrated and separated by column chromatography to obtain compound 3 with a yield of 34%. ¹ H NMR(400MHz, DMSO)δ8.61(d,J=7.4Hz,2H),8.29(d,J=7.7Hz,1H),8.15(d,J=8.5Hz,1H),7.65(s, 1H), 7.56(d, J＝7.0Hz, 2H), 7.41(td, J＝11.1, 6.0Hz, 4H), 6.75(dd, J＝15.9, 6.1Hz, 1H), 5.15(m, 2H), 4.39(s, 1H), 3.62(d, J=4.1Hz, 2H), 3.16(m, 1H), 3.11(m, 2H), 2.28(d, J=36.4Hz, 1H), 2.12(s, 1H) ),1.96(m,1H),1.62(m,2H),1.50(dd,J=15.5,8.6Hz,2H),0.88(m,6H).HRMS m/z(ESI)calcd for C ₂₃ H ₃₀ FN ₃ O ₄ [M+H] ⁺ 432.2293 found: 432.2291.

Example 3: Preparation of Compound 9

The compound 9 of the present invention is prepared according to the above-mentioned preparation route, and the reaction conditions of each step in the route are as follows:

i. 1-Hydroxybenzotriazole, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N-diisopropylethylamine, N,N-diisopropylethylamine Methylformamide, room temperature.

ii. Sodium hydroxide, methanol, water, 55 degrees

iii, 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate, N,N-diisopropylethylamine, N,N-diisopropylethylamine Methylformamide, 0°C

iv, sodium borohydride, methanol, room temperature

v. Dess Martin oxidant, ultra-dry dichloromethane, room temperature

The following are the specific synthesis steps:

Intermediate 25: Methyl(1R,2S,5S)-6,6-dimethyl 3-(quinoline-2-carbonyl)-3-azabicyclo[3.1.0]hexane-2-carboxylic acid preparation

The raw material 23 (quinoline 2-carboxylic acid 1.0 g, 11.6 mmol), 1-hydroxybenzotriazole (2.03 g, 15.08 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbon two Imine hydrochloride (4.43g, 23.3mmol), 30mL of N,N-dimethylformamide was placed in a round-bottomed flask and stirred at room temperature for 0.5 hours, 2.5mL of N,N-diisopropylethylamine was added, and 0.59 mL of N,N-diisopropylethylamine was added. g Intermediate 24, after reacting for 8 hours, the solvent was distilled off under reduced pressure, extracted with dichloromethane, ammonium chloride solution and sodium bicarbonate solution, washed with water and saturated sodium chloride solution, finally dried with sodium sulfate, and filtered with suction. , and the organic phase was chromatographed to obtain a white solid. The yield was 85%. ¹ H NMR(400MHz,DMSO)δ8.12-8.03(m,1H),8.00-7.90(m,2H),7.66-7.48(m,3H),4.54(s,1H),4.03(q,J= 7.1Hz, 1H), 3.75(d, J=6.7Hz, 3H), 3.47(d, J=5.4Hz 1H), 3.39(d, J=5.7Hz 1H), 1.99(t, J=6.2Hz, 1H) ), 1.54 (t, J=6.8 Hz, 1H), 1.03 (s, 3H), 0.97 (s, 3H). MS (ESI, positive ion) m/z: 325.04.87 [M+H] ⁺ .

Intermediate 26: Preparation of (1R,2S,5S)-6,6-dimethyl 3-(quinoline-2-carbonyl)-3-azabicyclo[3.1.0]hexane-2-carboxylic acid

Intermediate 25 was dissolved in 20mL of tetrahydrofuran, slowly added 10mL of 2mol/L sodium hydroxide solution, the reaction solution was gradually warming up to 55 degrees after stirring for 3 hours and terminated and cooled to normal temperature, concentrated reaction solution, add water to adjust pH to be weakly acidic , a white solid was precipitated, and the intermediate 5 was obtained by suction filtration, which was used in the next step without further purification.

Intermediate 28: Methyl((1R,5S)-6,6-dimethyl-3-(quinoline-2-carbonyl)-3-azabicyclo[3.1.0]hexane-2-carbonyl)- Preparation of L-phenylalanine

1.0 g of intermediate 26, 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate 1.93g were added to 20mL of N,N-dimethylmethane In the amide, stir at 0°C for 0.5 hours, add 2.0 mL of N,N-diisopropylethylamine, and then add 0.89 g of intermediate 27, react under argon protection at 0°C for 12h, add 4 times the volume of water, and use Ethyl acetate was extracted three times. The organic phases were combined, extracted with ammonium chloride solution and sodium bicarbonate solution, washed with water and saturated sodium chloride solution, finally dried with sodium sulfate, suction filtered, and the organic phase was subjected to column chromatography to obtain a white solid. The yield was 65%. ¹ H NMR (400MHz, DMSO) δ 8.53 (d, J=7.5Hz, 1H), 8.04 (d, J=6.4Hz, 1H), 7.99 (d, J=6.4Hz, 1H), 7.94 (d, J=3.7Hz, 1H), 7.83(d, J=8.5Hz, 1H), 7.61–7.57(m, 1H), 7.33(dd, J=8.4, 1.5Hz, 1H), 7.28(s, 2H), 7.23–7.18 (m, 1H), 7.13 (d, J=1.6Hz, 1H), 6.95 (d, J=2.5Hz, 1H), 4.55–4.44 (m, 1H), 3.95 (d, J=5.2Hz) ,1H),3.81(t,J＝11.3Hz,1H),3.61(s,3H),3.05(d,J＝13.8,7.5Hz,1H),2.84(dd,J＝13.8,5.6Hz,1H) ,2.82(d,J＝5.6Hz,1H),1.42-1.37(m,1H),1.36-1.32(m,1H),0.96(s,3H),0.91(s,3H).

Intermediate 29: (1R,5S)-N-((S)-1-hydroxy-3-phenylpropan-2-yl)-6,6-dimethyl-3-(quinoline-2-carbonyl)- Preparation of 3-azabicyclo[3.1.0]hexane-2-carboxamide

The intermediate 28(((1R,5S)-6,6-dimethyl-3-(quinoline-2-carbonyl)-3-azabicyclo[3.1.0]hexane-2-carbonyl)-L -Phenylalanine) 500 mg, dissolved in 30 mL of dry methanol, sodium borohydride was added at room temperature, stirred for 3 hours, quenched by adding water, spin-dried methanol, and the aqueous phase was extracted with ethyl acetate (50 mL × 3), The collected organic phase was dried with sodium sulfate, and after suction filtration, the organic phase was spin-dried to obtain intermediate 29 with a yield of 80%.

Compound 9: (1R,5S)-6,6-dimethyl-N-((S)-1-oxo-3-phenylpropan-2-yl)-3-(quinoline-2-carbonyl)-3 - Preparation of azabicyclo[3.1.0]hexane-2-carboxamide

The intermediate 29(1R,5S)-N-((S)-1-hydroxy-3-phenylpropan-2-yl)-6,6-dimethyl-3-(quinoline-2-carbonyl)- 200 mg of 3-azabicyclo[3.1.0]hexane-2-carboxamide was dissolved in 10 mL of dry dichloromethane, and Dess Martin oxidant was added under stirring at room temperature. TLC monitored the completion of the reaction, and the oxidant was removed by suction filtration. The filtrate was Column chromatography gave compound 9 in 65% yield. ¹ H NMR (400MHz, DMSO) δ 9.55(s, 1H), 9.06(s, 1H), 8.56(d, J=7.3Hz, 1H), 8.44(dd, J=8.5, 2.4Hz, 1H), 7.86(d,J＝8.5Hz,1H),7.76(m,1H),7.32-7.21(m,5H),7.14(d,J＝2.9Hz,,1H),7.08(d,J＝5.7Hz, 1H), 5.33(s, 1H), 4.44(d, J=5.6Hz, 1H), 4.33(m, 2H), 4.13(m, 2H), 1.82(m, 1H), 1.50(m, 1H), 1.02(s, 3H), 0.87(s, 3H).

Example 4: Preparation of Compound 14

The compound 14 of the present invention is prepared according to the above-mentioned preparation route, and the reaction conditions of each step in the route are as follows:

i, trifluoroacetic acid, dichloromethane, 25℃

ii, 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate, N,N-diisopropylethylamine, N,N-diisopropylethylamine Methylformamide, room temperature.

iii. Sodium hydroxide, methanol, water, 55 degrees

iv, 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate, N,N-diisopropylethylamine, N,N-diisopropylethylamine Methylformamide, 0°C

v, sodium borohydride, methanol, room temperature

vi. Dess Martin oxidizer, ultra-dry dichloromethane, room temperature.

The following are the specific synthesis steps:

Intermediate 30: Preparation of methyl(S)-2-amino-3-((S)-2-carbonyl-3-yl)propionate trifluoroacetate

(S)-Methyl 2-(tert-butoxycarbonylamino)-3-((S)-2-carbonylpyrrolidin-3-yl)propanoate (Intermediate 14, 2.5 g) was dissolved in 30 mL In the dichloromethane, 20 mL of trifluoroacetic acid was added, stirred at room temperature for 14 hours, and then directly spin-dried to obtain the crude product which was directly used in the next reaction.

Intermediate 32: Methyl(1R,2S,5S)-6,6-dimethyl-3-(2-(4-(trifluoromethoxy)phenoxy)acetyl)-3-azabicyclo[ 3.1.0] Preparation of hexane-2-carboxylic acid

2-(4-(Trifluoromethoxy)phenoxy)acetic acid (commercially available Intermediate 31, 0.24 g, 1.0 mmol), 2-(7-benzotriazole oxide)-N,N,N ',N'-tetramethylurea hexafluorophosphate (0.49g, 1.2mmol) and N,N-diisopropylethylamine (494μL, 3mmol) were dissolved in a certain amount of N,N-dimethylformamide, Then (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylate methyl ester hydrochloride (commercially available Intermediate 24, 0.21 g) was added , 1.0 mmol). The reaction was carried out under argon protection at room temperature for 12 hours. The reaction was added with 4 times the volume of water, extracted three times with dichloromethane, and the combined organic phases were washed with saturated ammonium chloride solution and saturated sodium carbonate solution, dried over anhydrous sodium sulfate and filtered. , and silica gel column chromatography (petroleum ether/ethyl acetate=1:1) gave intermediate 32 as a white solid (0.34 g, 88%). ¹ H NMR(400MHz,MeOD)δ7.19(d,J=8.9Hz,2H),7.00(d,J=8.7Hz,2H),4.80-4.71(m,2H),4.78-4.72(m,1H) ) 3.89-3.72(m, 1H), 3.73(s, 3H), 3.67-3.60(m, 1H), 1.61-1.55(m, 1H), 1.49(d, J=7.4Hz, 1H), 1.08(s ,3H),0.97(s,3H).ESI-MS(m/z):389.08(M+H) ⁺ .

Intermediate 33: (1R,2S,5S)-6,6-dimethyl-3-(2-(4-(trifluoromethoxy)phenoxy)acetyl)-3-azabicyclo[3.1. Preparation of 0] Hexane-2-carboxylic acid

Methyl(1R,2S,5S)-6,6-dimethyl-3-(2-(4-(trifluoromethoxy)phenoxy)acetyl)-3-azabicyclo[3.1 .0] Hexane-2-carboxylic acid (intermediate 32, 200mg), then add 2M NaOH solution 20mL. Stir at room temperature for 2.5 hours. After the TLC monitoring reaction finishes, spin dry methanol, adjust pH to weak acidity with hydrochloric acid, dichloromethane Extract three times, combine the organic phases and dry with anhydrous sodium sulfate and select the dry crude product to directly carry out the next step of the reaction.

Intermediate 34: Methyl(1R,2S,5S)-6,6-dimethyl-3-(2-(4-(trifluoromethoxy)phenoxy)acetyl)-3-azabicyclo[ 3.1.0] Preparation of hexane--2-amide)-3-((S)-2-carbonyl-3-yl)propionate

Under the reaction conditions of 0 °C, to (1R,2S,5S)-6,6-dimethyl-3-(2-(4-(trifluoromethoxy)phenoxy)acetyl)-3-azabicyclo [3.1.0] Hexane-2-carboxylic acid (Intermediate 33, 0.45g, 1.2mmol) was added to the ultra-dry DMF solution, 2-(7-benzotriazole oxide)-N,N,N', N'-tetramethylurea hexafluorophosphate (0.61 g, 1.6 mmol), after stirring for 30 minutes, was added NN-diisopropylethylamine (0.59 mL, 3.6 mmol). Then, the crude product of Intermediate 14 (0.27 g 1.45 mmol) was added to the reaction system. The reaction was stirred at 0°C under argon for 12 hours. After the reaction was monitored by TLC, 4 times the volume of water was added, extracted three times with ethyl acetate, the combined organic phases were washed with saturated ammonium chloride solution and saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, filtered, and subjected to column chromatography with mixed sample (Ethyl acetate/methanol=10:1) The white solid was obtained as intermediate 34. ¹ H NMR (400MHz, MeOD) δ 7.17 (d, J=8.0 Hz, 2H), 6.97 (d, J=8.9 Hz) ,2H),4.80–4.67(m,2H),4.55(d,J=11.8Hz,1H),3.94-3.83(m,1H),3.72(s,3H),3.68–3.57(m,1H), 3.23–3.12 (m, 1H), 3.11–3.00 (m, 2H), 2.58 (d, J=8.8Hz, 1H), 2.26–2.04 (m, 2H), 1.73–1.40 (m, 4H), 1.10 ( s,3H),0.92(s,3H).ESI-MS(m/z):542.13(M+H) ⁺ .

Intermediate 35: (1R,2S,5S)-N-((S)-1-hydroxy-3-((S)-2-carbonyl-3-yl)propanoate-2-yl)-3-( Preparation of 2-(4-(trifluoromethoxy)phenoxy)acetyl)-3-azabicyclo[3.1.0]hexane-2-carboxamide

Methyl(1R,2S,5S)-6,6-dimethyl-3-(2-(4-(trifluoromethoxy)phenoxy)acetyl)-3-azabicyclo[3.1.0] Hexane--2-amide)-3-((S)-2-carbonyl-3-yl)propionate (intermediate 340.56 mg, 1.1 mmol) was added to 50 mL, and sodium borohydride was added in portions at low temperature (0.14g, 8.8mmol). The reaction was stirred at room temperature for 2 hours, quenched by adding water, spin-dried methanol, and the remaining aqueous phase was extracted with ethyl acetate (50mL×3). The organic phases were combined with anhydrous sodium sulfate. Dry, filter and spin dry to obtain a white solid as a crude product, which is directly used in the next reaction.

Compound 14: (1R,2S,5S)-6,6-dimethyl-N-((S)-1-aldol-3-((S)-2-carbonyl-3-yl)propanoate- Preparation of 2-yl)-3-(2-(4-(trifluoromethoxy)phenoxy)acetyl)-3-azabicyclo[3.1.0]hexane-2-carboxamide

(1R,2S,5S)-N-((S)-1-hydroxy-3-((S)-2-carbonyl-3-yl)propanoate-2-yl)-3-(2-( 4-(Trifluoromethoxy)phenoxy)acetyl)-3-azabicyclo[3.1.0]hexane-2-carboxamide (Intermediate 36, (0.38 g, 0.75 mmol) was dissolved in ultra-dry dicyclomine In methyl chloride, then Dess Martin oxidant (0.95mg, 0.79mmol) was added in batches, and the reaction was carried out for 3.5 hours at room temperature. The reaction was monitored by TLC, and the reaction system was filtered. phase, concentrated and separated by preparative chromatography (acetonitrile/water=30:70) to obtain compound 14 (0.28 g, 45%) as a white solid. ¹ H NMR (400 MHz, MeOD) δ 7.24–7.13 (m, 2H), 7.04-6.92(m, 2H), 4.49(d, J=9.1Hz, 1H), 4.37-4.30(m, 1H), 4.06-3.89(m, 1H), 3.65-3.49(m, 1H) , 3.11–2.98 (m, 1H), 2.55 (d, J=9.5Hz, 1H), 2.29–2.10 (m, 1H), 2.06–1.99 (m, 1H), 1.72–1.44 (m, 4H), 1.13 (s, 3H), 1.00 (s, 3H). ¹³ C NMR (101MHz, MeOD) δ 181.60, 172.58, 167.18, 156.90, 142.99, 121.99, 115.47 (d, J=7.7Hz), 66.06, 61.05, 60.14, 51.26 ,46.00,39.94,37.75,30.87(d,J=7.8Hz),29.88(d,J=18.5Hz),27.66(d,J=17.9Hz),25.03,19.04,11.63.HRMS(m/z): calculated for C ₂₄ H ₂₈ F ₃ N ₃ O ₆ ⁺ [M+H] ⁺ 512.1964; found, 512.2137.

Example 5: Preparation of Compound 15

The compound 15 of the present invention is prepared according to the above-mentioned preparation route, and the reaction conditions of each step in the route are as follows:

i, 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate, N,N-diisopropylethylamine, N,N-diisopropylethylamine Methylformamide, 0°C

ii. Sodium borohydride, methanol, room temperature

iii. Dess Martin oxidant, ultra-dry dichloromethane, room temperature.

The specific synthesis steps are as follows:

Intermediate 36: Methyl(S)-2-((1S,3aR,6aS)-2-(2-(2,4-dichlorophenoxy)acetyl)octahydrocyclopenteno[c]pyrrole- Preparation of 1-Carboxamide)-3-((S)-2-carbonyl-3-yl)propionate

First, 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate (0.099 g, 0.26 mmol) was added to (1S,3aR,6aS)- Ultradry N,N of 2-(2-(2,4-dichlorophenoxy)acetyl)octahydrocyclopenteno[c]pyrrole-1-carboxylic acid (Intermediate 23, 0.071 g, 0.20 mmol) -In dimethylformamide solution, the reaction system was first stirred for 30 minutes, N,N-diisopropylethylamine (100 μL, 0.60 mmol), methyl (S)-2-amino-3-((S)- 2-Carbonyl-3-yl) propionate trifluoroacetate (intermediate 30, 0.060 g, 0.32 mmol) was added to the reaction system in turn. The reaction was carried out at 0 ° C under argon protection for 12 hours, and TLC monitoring was completed. , 4 times the volume of water was added, extracted three times with ethyl acetate, the combined organic phases were washed with saturated ammonium chloride solution and saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, filtered, and subjected to mixed sample column chromatography (ethyl acetate/ Methanol=10:1) to obtain intermediate 36 (0.063g, 59%). ¹ H NMR (400MHz, MeOD) δ 7.40 (d, J=2.5Hz, 1H), 7.26–7.19 (m, 1H), 6.95 (d, J=8.9Hz, 1H), 4.79-4.71 (m, 2H), 4.54 (d, J=3.7Hz, 1H), 4.25 (t, J=4.3Hz, 1H), 3.72 (s, 3H) ,3.54-3.46(m,2H),3.21-3.12(m,1H),3.10-2.99(m,1H),2.86-2.70(m,2H),2.04-1.97(m,2H),1.96-1- 85(m, 3H), 1.83–1.46(m, 6H). ESI-MS(m/z): 526.03(M+H) ⁺ .

Intermediate 37: (1S,3aR,6aS)-2-(2-(2,4-dichlorophenoxy)acetyl)-N-((S)-1-hydroxy-3-((S)-2 Preparation of -carbonyl-3-yl)propionate-2-yl)octahydrocyclopenteno[c]pyrrole-1-carboxamide

Methyl(S)-2-((1S,3aR,6aS)-2-(2-(2,4-dichlorophenoxy)acetyl)octahydrocyclopenteno[c]pyrrole-1-methyl Amide)-3-((S)-2-carbonyl-3-yl)propionate (Intermediate 36, 1,00 g, 2.0 mmol) was dissolved in anhydrous methanol, and then boron was added in batches at 0 °C Sodium hydride (0.6 g, 16 mmol), then the temperature was raised to room temperature, and stirring was continued for 2 hours. TLC detected the end of the reaction, quenched by adding water, spin-dried methanol, and extracted the remaining aqueous phase with ethyl acetate (50 mL×3). The organic phases were combined, dried with anhydrous sodium sulfate, filtered and spin-dried to obtain a white solid as a crude product, which was directly used in the next reaction.

Compound 15: (1S,3aR,6aS)-2-(2-(2,4-dichlorophenoxy)acetyl)-N-((S)-1-aldehyde-3-((S)-2 Preparation of -carbonyl-3-yl)propionate-2-yl)octahydrocyclopenteno[c]pyrrole-1-carboxamide

In (1S,3aR,6aS)-2-(2-(2,4-dichlorophenoxy)acetyl)-N-((S)-1-hydroxy-3-((S)-2-carbonyl- 3-yl)propionate-2-yl)octahydrocyclopenteno[c]pyrrole-1-carboxamide (Intermediate 37, 0.50 g, 1.0 mmol) in ultra-dry dichloromethane, slowly in batches Dess Martin oxidant (0.55g, 1.3mmol) was added, reacted at room temperature for 3.5 hours, TLC monitored the reaction, the reaction system was filtered, the organic phase was washed with sodium thiosulfate solution and saturated sodium bicarbonate solution, concentrated and then used for preparative chromatography. The system was separated (acetonitrile/water=45:55) to give compound 30 as a white solid (0.21 g, 42%). ¹ H NMR (400MHz, MeOD) δ 7.42 (t, J=4.3Hz, 1H), 7.28-7.17 (m, 1H), 7.03-6.90 (m, 1H), 4.47 (dd, J=9.6, 6.1 Hz) ,1H),4.26(t,J＝5.8Hz,1H),4.03–3.86(m,2H),3.51(dd,J＝10.4,4.0Hz,1H),3.15(t,J＝8.4Hz,1H) , 3.04–2.82 (m, 2H), 2.75–2.50 (m, 2H), 2.18 (dd, J=13.2, 7.0Hz, 1H), 2.18 (dd, J=13.2, 7.0Hz, 1H), 2.08–1.77 (m, 5H), 1.76–1.43 (m, 5H). ¹³ C NMR (101MHz, MeOD) δ 181.64, 173.39, 167.10 (d, J=2.9Hz), 152.81, 129.30, 127.35, 125.73, 123.12, 114.81, 66.81 ,60.14,53.90(d,J＝35.6Hz),52.20,51.20(d,J＝18.6Hz),43.34,40.00,37.73,31.69(d,J＝4.2Hz),31.14(d,J＝2.6Hz) ,29.62(d,J＝24.1Hz),27.67,24.69,19.48,13.09.HRMS(m/z):calculated for C ₂₃ H ₂₇ Cl ₂ N ₃ O ₅ ⁺ [M+H] ⁺ 496.1361; found, 496.0842 .

Example 6: Preparation of Compound 42

The compound 42 of the present invention was prepared according to the above-mentioned preparation route, and the reaction conditions of each step in the route were as follows:

i, LDA, ClCH ₂ I, THF

ii. HCl dioxane solution

iii, 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate, N,N-diisopropylethylamine, N,N-diisopropylethylamine Methylformamide, 0°C.

The specific synthesis steps are as follows:

Intermediate 38: Preparation of tert-butyl ((S)-4-chloro-3-oxo-1-((S)-2-carbonyl-3-yl)butan-2-yl)carbamate

Choose a dry three-necked flask, prepare argon protection and a thermometer respectively, add (S)-methyl 2-(tert-butoxycarbonylamino)-3-((S)-2-carbonylpyrrolidin-3-yl)propane Acid ester (Intermediate 16, 5 g, 17.5 mmol), tetrahydrofuran (50 mL), chloroiodomethane (5 mL, 68 mmol) were stirred at -77°C. Lithium diisopropylamide (70 mL, 105 mmol) was further added dropwise. After the addition was completed, the reaction was further carried out for 2 hours, and then acetic acid and tetrahydrofuran were added under low temperature conditions for quenching, and the resulting black suspension was further stirred for 10 minutes while warming to room temperature. The reaction was further diluted with ethyl acetate, washed with water, saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated by column chromatography to obtain a pale yellow solid as intermediate 38. ¹ H NMR (400MHz, DMSO-d ₆ ) δ 7.89(s, 1H), 7.72(d, J=7.5Hz, 1H), 4.72-4.94(m, 2H), 4.35(m, 1H), 3.26- 3.40(m, 2H), 2.45(m, 1H), 2.32-2.42(m, 1H), 2.00-2.14(m, 1H), 1.79-1.99(m, 2H), 1.61(s, 9H).

Intermediate 39: Preparation of (S)-3-((S)-2-amino-4-chloro-3-oxobutyl)pyrrolidin-2-one hydrochloride

250 mg of tert-butyl ((S)-4-chloro-3-oxo-1-((S)-2-carbonyl-3-yl)butan-2-yl)carbamate (Intermediate 38) was added In 20 mL of dioxane, 20 mL of 20 mL of hydrogen chloride dioxane solution was then added, and after stirring at room temperature for 4 hours, TLC detected the completion of the reaction, and the reaction solution was spin-dried to obtain a crude product for the next reaction.

Compound 42: (1S,3aR,6aS)-N-((S)-4-chloro-3-oxo-1-((S)-2-carbonyl-3-yl)butan-2-yl)-2 Preparation of -(2-(2,4-Dichlorophenoxy)acetyl)octahydrocyclopenteno[c]pyrrole-1-carboxamide

2-(7-Benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate (1 g, 2.6 mmol) was added to (1S,3aR,6aS)-2-( Ultradry N,N-dimethylform of 2-(2,4-dichlorophenoxy)acetyl)octahydrocyclopenteno[c]pyrrole-1-carboxylic acid (Intermediate 23, 0.71 g, 2.0 mmol) In the methylformamide solution, the reaction system was first stirred for 30 minutes, N,N-diisopropylethylamine (1 mL, 6.0 mmol), (S)-3-((S)-2-amino-4-chloro-3 -Oxybutyl) pyrrolidin-2-one hydrochloride (intermediate 39, 0.652g, 3.2mmol) was added to the reaction system successively. The reaction was reacted under 0 ° C of argon protection for 16 hours, and the TLC monitoring reaction was completed, adding 3 times the volume of water, extracted three times with ethyl acetate, the combined organic phases were washed with saturated ammonium chloride solution and saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate and filtered to prepare a purification system (acetonitrile/water=30:70) Compound 42 was isolated and obtained. ¹ H NMR(400MHz, MeOD)δ7.40(s,1H),7.20(dd,J=8.7,2.4Hz,1H),7.05-6.91(m,1H),5.00-4.89(m ,1H),4.79(d,J＝16.1Hz,1H),4.69-4.35(m,2H),4.25(m,1H),3.99-3.85(m,1H),3.58-3.45(m,1H), 3.25–3.03 (m, 1H), 2.93–2.77 (m, 1H), 2.76–2.50 (m, 2H), 2.45–2.24 (m, 1H), 2.18 (d, J=10.9Hz, 1H), 2.11– 1.47(m, 9H), 1.39–1.27(m, 1H). ESI-MS(m/z): 544.07(M+H) ⁺ .

Example 7: Preparation of Compound 50

The compound 50 of the present invention is prepared according to the above-mentioned preparation route, and the reaction conditions of each step in the route are as follows:

i, tert-butylacetonitrile, acetic acid, ultra-dry dichloromethane;

ii, 1M sodium hydroxide solution, methanol;

iii. Dess Martin oxidant, ultra-dry dichloromethane.

The specific synthesis steps are as follows:

Intermediate 40: (3S)-1-(tert-Butylamino)-3-((1S,3aR,6aS)-2-(2-(2,4-dichloro)acetyl)octahydrocyclopenteno[ Preparation of c]pyrrole-1-carboxamide)-1-oxo-4-((S)-2-carbonyl-3-yl)butan-2-ylcarboxylic acid

First, compound 15 (0.40 mmol) was dissolved in ultra-dry dichloromethane, then acetic acid (0.028 g, 0.47 mmol), tert-butylisonitrile (0.43 mmol) were added sequentially. The reaction was stirred at room temperature for 24 hours, and the intermediate 40 was obtained by distillation column chromatography under reduced pressure (dichloromethane/methanol=15:1). ¹ H NMR (400MHz, DMSO-d ₆ ) δ 7.87 (d, J=12.1 Hz, 1H), 7.46 (s, 1H), 7.44 (d, J=1.4 Hz, 1H), 7.37 (t, J= 4.6Hz, 1H), 7.27 (dd, J=7.5, 1.5Hz, 1H), 7.11 (d, J=7.5Hz, 1H), 5.09 (d, J=7.1Hz, 1H), 4.82 (s, 2H) ,4.36–4.28(m,2H),3.64(ddd,J=59.5,12.4,7.0Hz,2H),3.22(td,J=7.1,4.6Hz,2H),2.67–2.44(m,3H),2.09 (s, 3H), 1.99–1.51 (m, 10H), 1.27 (s, 9H).

Intermediate 41: (1S,3aR,6aS)-N-((2S)-4-(tert-butylamino)-3-hydroxy-4-oxo-1-((S)-2-carbonyl-3-yl ) butan-2-yl)-2-(2-(2,4-dichloro)acetyl)octahydrocyclopenteno[c]pyrrole-1-carboxamide

1M sodium hydroxide solution was added (0.5mL) to the methanol solution of intermediate 40 (0.164mmol), the reaction was stirred at room temperature for 2 hours, and the pH was adjusted to neutrality with 1M hydrochloric acid. The methyl chloride was dissolved, extracted with water, and the reaction solution was spin-dried to obtain the crude product 41, which was directly used in the next reaction.

Compound 50: (1S,3aR,6aS)-N-((S)-4-(tert-butylamino)-3,4-oxo-1-((S)-2-carbonyl-3-yl)butane Preparation of -2-yl)-2-(2-2,4-dichloro)acetyl)octahydrocyclopenteno[c]pyrrole-1-carboxamide

In the ultra-dry dichloromethane solution of intermediate 41, Dess Martin oxidant was slowly added in batches, and the reaction was carried out at room temperature for 4 hours. TLC monitored the end of the reaction, and the reaction system was filtered, using sodium thiosulfate solution and saturated sodium bicarbonate solution. The organic phase was washed, concentrated and separated by preparative chromatography (acetonitrile/water=50:50) to obtain compound 50 as a white solid. ¹ H NMR (400MHz, MEOD) δ 7.42 (d, J=1.4Hz, 1H), 7.26 (dd, J=7.5, 1.5Hz, 1H), 7.12 (d, J=7.5Hz, 1H), 4.84 ( s, 2H), 4.67 (dt, J=11.9, 7.0Hz, 1H), 4.36 (dd, J=7.0, 0.7Hz, 1H), 3.74–3.57 (m, 2H), 3.23–3.13 (m, 2H) , 2.70–2.46 (m, 3H), 2.15–1.54 (m, 10H), 1.43 (s, 9H). ESI-MS (m/z): 595.07 (M+H) ⁺ .

According to the preparation routes in Examples 1 to 7, the raw materials were changed to obtain the remaining compounds shown in Table 1 of the present invention.

Table 1 Structure and Characterization Data of Compounds of the Invention

The pharmacological effects of the compounds of the present invention are demonstrated below through experimental examples.

Experimental Example 1: Test of the Inhibitory Level of the Compounds of the Invention on ^Mpro Enzyme Activity

(1) Experimental method

Recombinant SARS-CoV-2M ^pro (750 nM final concentration) was mixed with serial dilutions of each compound in 25 μL of assay buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2 mM DTT) and incubated 10 minutes. The reaction was initiated by adding 25 μL of fluorogenic substrate (MCA-AVLQ↓SGFR-Lys(Dnp)-Lys-NH2) at a final concentration of 20 μM, and the fluorescence signal at 320nm (excitation)/405nm (emission) was measured with a microplate reader. The Vmax of the reaction to which different concentrations of compound were added and the Vmax of the reaction to which DMSO was added was calculated and used to generate an _IC50 curve. For each compound, half inhibitory concentration ( _IC50 ) values against SARS-CoV-2M ^pro were measured at 9 concentrations and 3 independent replicates. All experimental data were analyzed using GraphPad Prism software.

(2) Experimental results

The enzymatic activity inhibition effect of table 2 compounds on SARS-COV-2M ^pro

compound	Anti-M pro IC 50 (μM)
1	1.1
2	0.11
3	0.018
4	0.012
5	0.011
6	0.009
7	0.015
8	0.017
9	24.24
10	0.585
11	0.960
12	16.93

13	82.74
14	0.014
15	0.016
16	0.343
17	0.020
18	0.028
19	0.011
20	0.012
twenty one	0.073
twenty two	0.093
twenty three	0.037
twenty four	0.022
25	0.016
26	0.009
27	0.054
28	0.047
29	0.078
30	0.014
31	0.015
32	0.036
33	0.013
34	0.020
35	0.051
36	0.016
37	0.007
38	0.091
39	0.033
40	0.017
41	0.056
42	0.190
43	0.009
44	0.010
45	0.019
46	0.010
47	0.012
48	0.011
49	0.022
50	1.190
51	50.39
52	0.875
53	63.09

54	0.024
55	0.031
56	0.033
57	0.026
58	0.030
59	0.037
60	0.033
61	0.039
62	0.040
63	0.026
64	0.033
65	0.044
66	0.036
67	0.040
68	0.035
69	0.107
70	0.081
71	0.040
72	0.031
73	0.031
74	0.051
75	0.041
76	0.051
77	0.056
78	0.068
79	0.065
80	0.023

It can be seen from Table 2 and Figure 1, Figure 2 and Figure 3 that the compounds of the present invention can effectively inhibit the activity of SARS-CoV-2M ^pro , and can be used to prepare SARS-CoV-2M ^pro inhibitors and anti-new coronavirus medicines, and the preparation of medicines for the prevention and/or treatment of novel coronavirus pneumonia.

Experimental Example 2: Inhibition experiment of the compounds of the present invention on cell death caused by SARS-COV-2 infection of Vero E6 cells

(1) Experimental method

The antiviral activity of the compounds was preliminarily evaluated by detecting the inhibitory effect of the compounds on cell death caused by SARS-COV-2 infection of Vero E6 cells. The specific experimental protocol is as follows: Vero E6 cells were seeded in a 96-well plate at a cell density of 2×10 ⁴ cells/well, 100 μL/well, and incubated overnight at 37° C. in a 5% CO ₂ incubator. The next day, 100 μL of drug and 100 μL of virus diluent (MOI= ₁ ) were added to each well at the same time, and a drug-free positive control and a virus-free negative control were set up. 8 kits were used to detect cell viability, and the inhibition rate and half-effect concentration (EC ₅₀ ) value of drugs on virus replication were calculated. All experiments were set up as 3 independent replicates, and all experimental data were analyzed by GraphPad Prism software.

(2) Experimental results

Table 3 Inhibitory activity of the compounds of the present invention to cell death caused by SARS-COV-2 infection of Vero E6 cells

compound	EC50 (μM)
1	NT
2	NT
3	NT
4	NT
5	NT
6	NT
7	NT
8	NT
9	NT
10	NT
11	NT
12	NT
13	NT
14	0.86
15	0.54
16	NT
17	24.72
18	NT
19	5.63
20	1.73
twenty one	NT
twenty two	NT
twenty three	NT
twenty four	1.50
25	NT
26	0.67
27	1.27
28	4.68
29	NT
30	1.15
31	1.18

32	NT
33	5.57
34	1.64
35	NT
36	2.23
37	2.97
38	NT
39	NT
40	NT
41	NT
42	NT
43	0.53
44	0.66
45	0.83
46	NT
47	NT
48	NT
49	NT
50	NT
51	NT
52	NT
53	NT
55	NT
56	NT
57	NT
58	NT
59	NT
60	NT
61	NT
62	NT
63	NT
64	NT
65	NT
66	NT
67	NT
68	NT
69	NT
70	NT
71	NT
72	NT
73	NT

74	NT
75	NT
76	NT
77	NT
78	NT

Note: NT stands for untested cell viability

As can be seen from Table 3, the compound of the present invention can effectively inhibit the cell death caused by SARS-COV-2 infection of Vero E6 cells, indicating that the compound of the present invention can effectively inhibit the replication of SARS-COV-2 virus in cells.

Experimental Example 3: Toxicity test of compounds on Vero E6 cells

(1) Experimental method

Cytotoxicity assessment of compounds was performed using Vero E6 cells. The specific experimental protocol is as follows: Vero E6 cells were seeded in a 96-well plate at a cell density of 2×10 ⁴ cells/well, 100 μL/well, and incubated overnight at 37° C. in a 5% CO2 incubator. The next day, 200 μL of drug-containing medium was added to each well. The compound was initially diluted at 200 μM, with a 5-fold gradient dilution. There were 6 gradients in total. Three replicate wells were set for each concentration. Each group of experiments set a negative control and blank without drug. control. After 72 hours of drug treatment, the cell viability was detected by using the CCK-8 kit, and the toxicity of the compound to Vero E6 cells and the cytotoxicity concentration (CC ₅₀ ) value were calculated. All experimental data were analyzed using GraphPad Prism software.

(2) Experimental results

Table 4 Toxicity of the compounds of the present invention to Vero E6 cells

compound	CC 50 (μM)
1	>200
2	>200
3	>200
4	>200
5	>200
6	>200
7	>200
8	>200
9	>200
10	>200
11	>200
12	>200
13	>200
14	>200
15	>200
16	>200
17	>200

18	>200
19	>200
20	>200
twenty one	>200
twenty two	>200
twenty three	>200
twenty four	>200
25	>200
26	>200
27	>200
28	>200
29	>200
30	>200
31	>200
32	>200
33	>200
34	>200
35	>200
36	>200
37	>200
38	>200
39	>200
40	>200
41	>200
42	>200
43	>200
44	>200
46	>200
47	>200
48	>200
49	>200
50	>200
51	>200
52	>200
53	>200
54	>200
55	>200
56	>200
57	>200
58	>200
59	>200

60	>200
61	>200
62	>200
63	>200
64	>200
65	>200
66	>200
67	>200
68	>200
69	>200
70	>200
71	>200
72	>200
73	>200
74	>200
75	>200
76	>200
77	>200
78	>200

As can be seen from Table 4, the compounds of the present invention have very low toxicity to Vero E6 cells.

Experimental Example 4: Inhibition experiment of compounds on the replication of SARS-COV-2 in human alveolar epithelial cells

(1) Experimental method

For the RT-qPCR method, human alveolar epithelial cells were seeded into 48-well plates (200 μL/well) at a density of 8×10 ⁵ cells/well and grown overnight. Cells were then treated with virus infection (MOI=0.01) and various concentrations of compound. After 1 hour incubation at 37°C, the medium containing the virus-drug mixture was removed and replaced with fresh medium containing the compound. After 48 hours of incubation, the cell supernatant was collected to extract viral RNA, which was quantitatively analyzed by RT-qPCR and the inhibitory rate and EC ₅₀ value of the drug on viral replication were calculated. _EC50 values were calculated using a dose-response model in GraphPad Prism 8.0 software, and the experiment was set up with 2 independent replicates.

(2) Experimental results

The test results are shown in Figure 4. Compounds 14, 15, 26, 43, 44, and 45 all exhibited nanomolar activity in inhibiting the replication of SARS-COV-2 in human alveolar epithelial cells, which was better than the highest reported activity. SARS-COV-2M ^pro inhibitor 11b (Dai et al., 2020, Science. 368(6497): 1331-1335) and GC376 (Ma et al., 2020, Cell Res. 30(8): 678-692) Antiviral activity under the same test conditions (11b, _EC50 =23.6 nM; GC376, _EC50 =151.3 nM).

Experimental Example 5: Plaque assay to evaluate compound anti-SARS-COV-2 activity (Vero E6 cells)

(1) Experimental method

The anti-SARS-COV-2 activity of compounds 3 and 39 in Vero E6 cells was evaluated using the plaque assay. Vero E6 was seeded in a 24-well cell culture plate at 1.0×10 ⁵ cells per well, and incubated overnight at 37°C for use. After adding the serially diluted drug, SARS-CoV-2 infected cells were added, and the MOI was about 0.002. After culturing in a 37°C cell incubator for 1 hour, the drug-containing infection supernatant was removed, washed with PBS, and 0.5 mL of sodium carboxymethyl cellulose containing different concentrations of drugs was added, and the final concentration of sodium carboxymethyl cellulose was 0.9%. Placed in a 37°C cell incubator for 72 hours. Fix with 20% formaldehyde for 2 hours, add 0.5% crystal violet to stain for 20 minutes, dry and take pictures, observe the size of plaques and record the number of plaques. The experiment set blank control wells (normal cells), virus control wells, and positive drug control wells.

Calculation formula: inhibition rate (%) = (number of plaques in virus control wells - number of plaques in sample wells)/number of plaques in virus control wells*100

The resulting cell viability and inhibition rates were calculated, and _EC50 (half effective concentration) values were calculated using Graphpad Prism8.

(2) Experimental results

Table 5 Inhibitory effects of small molecule compounds on SARS-CoV-2

Compound name	EC50 (μM)
3	0.24
39	1.20
Remdesivir (Remdesivir)	0.69

The experimental results are shown in Table 5. The compounds of the present invention can effectively inhibit SARS-COV-2 infection in Vero E6 cells; in particular, compound 3 has an EC ₅₀ of 0.2373 μM, and its activity is better than that of the positive control Remdesivir (EC ₅₀ of 0.692 μM).

Experimental Example 6: Evaluation of in vivo pharmacokinetic properties of compounds in rats

(1) Dosing schedule

60 male Sprague-Dawley (SD) rats, weighing 200-230 g, were randomly divided into 3 groups with 3 rats in each group. The series of test compounds were administered by gavage (p.o.), intravenous (i.v.) and intraperitoneal (i.p.), respectively, according to the protocol in Table 6 below. They were fasted for 12 h before the experiment and had free access to water. 2h after the administration of unified food.

Gavage, intravenous and intraperitoneal administration solutions were formulated in DMSO/HS15/NaCl (5/3/92, v/v/v). Administer the drug according to the dosage shown in Table 6, record the administration time, and collect blood through the jugular vein at the time set above or other suitable methods. Each sample is collected about 0.20mL, heparin sodium is anticoagulated, and placed on ice after collection. superior. Plasma was centrifuged within 1 hour (centrifugation conditions: 6800g, 6 minutes, 2-8°C). Plasma samples were stored in a -80°C freezer prior to analysis. The grouping and blood collection time points are shown in Table 6, with 3 animals at each time point.

Table 6 Experimental protocol for evaluating the in vivo pharmacokinetic properties of compounds in rats

(2) Experimental results

The main pharmacokinetic parameters of the compounds in Table 7

The results are shown in Table 7. Pharmacokinetic studies of compounds 3, 14, 15, 26, 39, 40, 43, 44 and 45 were carried out in the present invention. Among them, the oral exposure of compound 3 was 2293h*ng/mL, and the bioavailability was 55.1%. The intraperitoneal exposure of compound 14 was 11581h*ng/mL, and the bioavailability was 78.0%; the oral exposure was 1665h*ng/mL, and the bioavailability was 11.2%. The exposure of compound 15 by intraperitoneal injection was 12166h*ng/mL, and the bioavailability was 62.3%; the oral exposure was 2843h*ng/mL, and the bioavailability was 14.6%. The oral exposure of compound 26 was 842 h*ng/mL, and the bioavailability was 7.2%. The oral exposure of compound 39 was 14586 h*ng/mL, and the bioavailability was 14.7%. The oral exposure of compound 40 was 2888h*ng/mL, and the bioavailability was 22.1%. The oral exposure of compound 43 was 258 h*ng/mL, and the bioavailability was 4.8%. The oral exposure of compound 44 was 381 h*ng/mL and the bioavailability was 4.1%. The oral exposure of compound 45 was 968 h*ng/mL, and the bioavailability was 5.1%.

The experimental results show that the compounds of the present invention have good pharmacokinetic properties in rats.

Experimental Example 7: Preliminary evaluation of the in vivo safety of the compound in rats

(1) Experimental method

Compounds were dissolved in 5% (v/v) DMSO (Sigma-Aldrich), 3% (v/v) HS15 (GLPBIO) and 92% saline. SPF SD rats (age: 7-11 weeks) were divided into half females (190-220 g) and males (weight 200-230 g). Experiments were carried out according to the dosing schedule in Table 8, and clinical observations were performed on all animals. And at the end of the experiment, samples of heart, liver, spleen, lung, kidney and administration site were collected. The test results are shown in Table 8.

(2) Experimental results

Table 8 Preliminary evaluation of in vivo safety in rats

The experimental results show that the compounds of the present invention have good in vivo safety in rats.

Experimental Example 8: Study on the activity of compounds against SARS-COV-2 infection in vivo in transgenic mice

(1) Experimental scheme

Humanized angiotensin-converting enzyme 2 (ACE2) transgenic mice (age: 8-10 weeks) were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. (#T037659. Compounds were dissolved in 5% (v/v) DMSO ( Sigma-Aldrich), 3% (v/v) HS15 (GLPBIO) and 92% saline. SARS-CoV-2 (stain107) intranasal infection and administration were performed according to the experimental protocol in Table 9. All mice were observed and Body weights were monitored daily until sacrificed. On days 1 (1 dpi), 3 (3 dpi) and 5 (5 dpi) after viral infection, lung tissues (n=3, each dpi group) were collected for viral load detection, H&E histopathology Physical analysis, representative inflammatory cytokine and chemokine assays and inflammatory cell (neutrophil and macrophage) counts.

Table 9 Study protocol for the activity of compounds against SARS-COV-2 infection in transgenic mice

The specific experimental protocol for lung viral load detection was: RNA was extracted from lung tissue using TRIzol ^™ reagent (Invitrogen), and the

Viral RNA was quantified by a one-step qRT-PCR kit (Toyobo) and the results were expressed as copies of viral RNA per microgram of tissue.

The specific experimental protocol for lung histopathological analysis was: lung tissue was fixed with 4% paraformaldehyde for at least 7 days, embedded in paraffin and cut into 3 μm sections. Sections were stained with hematoxylin and eosin (H&E) and analyzed by light microscopy. Lung injury was assessed according to histological features (thickening of alveolar septa, hemorrhage, inflammatory cell infiltration, etc.).

A specific embodiment of a representative inflammatory cytokine and chemokine assay in the lung is: using the PrimeScript ^™ RT kit (Takara), RNA extracted from the lungs was reverse transcribed into cDNA and then analyzed by

Ex Taq ^™ II (TliRNaseH Plus) (Takara) and ViiA ^™ quantified gene expression. Primer sequences used to quantify inflammatory gene expression are shown in Table 10.

Table 10 Primer sequences for the determination of representative inflammatory cytokines and chemokines

A specific embodiment for the determination of inflammatory cells (neutrophils and macrophages) in the lungs is performed by fixing mouse lung tissue in 4% paraformaldehyde for at least 7 days, then paraffin-embedding according to standard procedures. Cut into 4 μm sections. After deparaffinization in xylene, antigen retrieval and blocking, lung sections were incubated with rat monoclonal antibody F4/80 (Huabio, 1:100) or rabbit polyclonal antibody Ly6G (Servicebio, 1:300) at 4°C Overnight, then reacted with horseradish peroxidase (HRP)-conjugated goat anti-rat secondary antibody or HRP-conjugated goat anti-rabbit secondary antibody for 1 hour at room temperature to catalyze Cy3 according to tyramide signal amplification (TSA) -Tyramine and Cy5-tyramine and amplify the staining signal. After nuclei were stained with DAPI, all sections were photographed using a LEICA DMI 4000B microscope (Germany) and analyzed by ImageJ software (NIH, USA) and FlowJo software (BD, USA). To semiquantitatively measure macrophage and neutrophil infiltration, 5 randomly selected areas of lung parenchyma in each lung section were examined by light microscopy for the presence of neutrophils or macrophages. This assessment was performed in a blinded fashion. The cumulative score for each animal is expressed as the number (%) of positive fields per 100 fields.

The above experiments used a placebo as a control. The placebo is a formulation in the same dosage form as the test drug, but without the active pharmaceutical ingredient.

(2) Experimental results

The results of the lung viral load detection experiment are shown in Figure 5. Both oral and intraperitoneal administration of compound 14 and intraperitoneal administration of compound 15 can effectively reduce the viral load in the lungs of transgenic mice infected with SARS-COV-2.

The experimental results of lung histopathological analysis are shown in Figure 6. Oral and intraperitoneal administration of compound 14 and intraperitoneal administration of compound 15 can effectively improve the pathological damage of the lungs of SARS-COV-2 infected transgenic mice.

The representative inflammatory cytokine and chemokine assay results in the lungs are shown in Figure 7. Oral and intraperitoneal administration of compound 14 and intraperitoneal administration of compound 15 can effectively reduce lung chemokine ligand 10 (CXCL10) and β-type interference Gene expression level of IFN-β.

The results of the experiment to determine the number of inflammatory cells (neutrophils and macrophages) in the lungs are shown in Figure 8. Both oral and intraperitoneal administration of compound 14 and intraperitoneal administration of compound 15 can effectively reduce the transgenic cells infected by SARS-COV-2. Number of neutrophils (NEU) and macrophages (MAC) in murine lungs.

The experimental results show that the compounds of the present invention can effectively resist SARS-COV-2 infection in vivo in transgenic mice.

To sum up, the present invention provides a novel coronavirus main protease inhibitor represented by formula I and a preparation method and application thereof. The compound represented by formula I can effectively inhibit the activity of SARS-CoV-2M ^pro , and can be used to prepare a SARS-CoV-2M ^pro inhibitor to block the replication and transcription of SARS-CoV-2 virus in patients. The compounds of the present invention have very good application prospects in the preparation of SARS-CoV-2 M ^pro inhibitors, anti-SARS-CoV-2 drugs, and drugs for preventing and/or treating novel coronavirus pneumonia.

Claims (10)

1. The compound shown in formula I, or its pharmaceutically acceptable salt, or its stereoisomer, or its optical isomer, or its isotopic substitution form:

   

   Wherein, X is O or S;

   Ring A is selected from the following groups unsubstituted or substituted by one or more R ₆ : 5-6 membered saturated heterocyclyl, 5-6 membered unsaturated heterocyclyl, saturated heterofused ring, unsaturated heterofused ring group; R ₆ is independently selected from C _1-6 alkyl, C _1-6 alkoxy, halogen, hydroxyl, cyano, amino, and carboxyl;

   R ₃ is L ₃ M ₀ L ₄ R _3a ; wherein L ₃ is selected from none, C _1-4 alkylene, halogenated C _1-4 alkylene, C _2-4 alkenylene, halogenated C _{2- 4} alkenylene, L ₄ is selected from none, C _1-4 alkylene, halogenated C _1-4 alkylene, M ₀ is selected from none, O, S, NH, CO, CONH, NHCO, R _3a is The following groups are unsubstituted or substituted by one or more R _3b : 5-6 membered aryl, 5-6 membered heteroaryl, unsaturated heterofused ring group, unsaturated fused ring alkyl; R _3b are each independently Selected from C _1-5 alkyl substituted or unsubstituted by R _3c , C _1-5 alkoxy substituted or unsubstituted by R _3c , halogen, phenyl substituted or unsubstituted by R _3c , NR ₁₄ R ₁₅ , substituted or unsubstituted naphthyl and hydroxyl by R _3c ; R ₁₄ and R ₁₅ are each independently selected from hydrogen or C _1-5 alkyl, and R _3c is independently selected from halogen, deuterium, cyano, hydroxyl, amino ,carboxyl;

   R ₄ is selected from the following groups unsubstituted or substituted by one or more substituents: 5-6 membered aryl, 5-6 membered heteroaryl, C _1-5 alkyl, COOR ₁₀ ; the substituents are each independently selected from =O, hydroxyl, nitro, amino, carboxyl, halogen, C _1-5 alkyl; R ₁₀ is C _1-5 alkyl;

   R ₅ is selected from COR ₈ or WCOOR ₇ ; wherein, R ₈ is selected from hydrogen or

   

   W is selected from none, C _1-4 alkylene, C _2-4 alkenylene, C _2-4 alkynylene, R ₇ is selected from C _1-6 alkyl; M is selected from none, CO, NH, CONH , NHCO, COO or OCO, L ₀ is selected from none, C _1-4 alkylene, C _2-4 alkenylene, L ₁ is selected from none, C _1-4 alkylene, C _2-4 alkenylene , R ^8a is selected from C _1-5 alkyl, halogenated C _1-5 alkyl, 3-6 membered saturated cycloalkyl, 3-6 membered saturated heterocyclic group, 5-6 membered aryl or 5-6 membered aryl Yuan Heteroaryl.
2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer, or an isotopic substitution form thereof, characterized in that: the structure of the compound is as shown in formula II , formula III or formula IV:

   

   Wherein, X is O or S;

   n is selected from an integer of 0-3, preferably an integer of 0-2;

   R ₁ and R ₂ are each independently selected from hydrogen, C _1-5 alkyl, C _1-5 alkoxy, halogen, hydroxyl, cyano, amino, and carboxyl;

   R ₃ is L ₃ M ₀ L ₄ R _3a ; wherein L ₃ is selected from none, C _1-4 alkylene, halogenated C _1-4 alkylene, C _2-3 alkenylene, and L ₄ is selected from none , C _1-4 alkylene, halogenated C _1-4 alkylene, M ₀ is selected from none, O, S, NH, CO, CONH, NHCO, R _3a is unsubstituted or by one or more R _3b Substituted the following groups: phenyl,

   

   

   R _3b are each independently selected from C _1-4 alkyl, halogen-substituted C _1-4 alkyl, deuterated C _1-4 alkyl, cyano-substituted C _1-4 alkyl, C _1-4 alkyl Oxyl, halogen-substituted C _1-4 alkoxy, deuterated C _1-4 alkoxy, cyano-substituted C _1-4 alkoxy, halogen, phenyl, halogenated phenyl, NR ₁₄ R ₁₅ ,

   

   Hydroxyl, R ₁₄ and R ₁₅ are each independently selected from hydrogen or C _1-4 alkyl;

   R ₄ is selected from the following groups unsubstituted or substituted by one or more substituents: 5-6 membered aryl, 5-6 membered heteroaryl, C _1-5 alkyl, COOR ₁₀ ; the substituents are each independently selected from =O, hydroxyl, nitro, amino, carboxyl, halogen, C _1-5 alkyl; R ₁₀ is C _1-5 alkyl;

   R ₈ is selected from hydrogen or

   

   M is selected from None, CO, NH, CONH, NHCO, COO or OCO, L ₀ is selected from None, C _1-3 alkylene, C _2-4 alkenylene, L ₁ is selected from None, C _1-3 alkylene Alkyl, C _2-4 alkenylene, R ^8a is selected from C _1-4 alkyl, halogenated C _1-4 alkyl, 3-6 membered saturated cycloalkyl, 3-6 membered saturated heterocyclic group, A 5- to 6-membered aryl group or a 5- to 6-membered heteroaryl group.
3. The compound according to claim 2, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer, or an isotopic substituted form thereof, is characterized in that:

   R ₁ and R ₂ are each independently selected from hydrogen, C _1-4 alkyl, C _1-4 alkoxy, halogen, and hydroxyl;

   R ₃ is selected from

   

   

   L ₃ M ₀ L ₄ R _3a ; L ₃ is selected from none, C _1-3 alkylene, halogenated C _1-3 alkylene, C _2-3 alkenylene, L ₄ is selected from none, C _{1-3 3} alkylene, halogenated C _1-3 alkylene, M ₀ is selected from none, O, NH, CO, CONH, R _3a is phenyl, phenyl substituted by one or more R _3b , R _3b Each independently selected from C _1-4 alkyl, halogen-substituted C _1-4 alkyl, deuterated C _1-4 alkyl, cyano-substituted C _1-4 alkyl, C _1-4 alkoxy , halogen-substituted C _1-4 alkoxy, deuterated C _1-4 alkoxy, cyano-substituted C _1-4 alkoxy, halogen, phenyl, halogenated phenyl, NR ₁₄ R ₁₅ ,

   

   Hydroxyl, R ₁₄ and R ₁₅ are each independently selected from hydrogen or C _1-3 alkyl;

   R ₄ is selected from

   

   C _1-2 alkyl, COOR ₁₀ , substituted or unsubstituted phenyl; the substituent is selected from hydroxyl, nitro; R _a1 and R _a2 are independently selected from hydrogen, C _1-3 alkyl, halogen; R ₁₀ is C _1-3 alkyl;

   R ₈ is selected from hydrogen, CONHR ₁₁ , L ₂ COOR ₁₂ , C _1-4 alkyl, halogenated C _1-4 alkyl;

   R ₁₁ is selected from 3-6 membered saturated cycloalkyl, C _1-4 alkyl, benzyl,

   

   L ₂ is a C _1-2 alkylene group, a C _2-3 alkenylene group, and R ₁₂ is a C _1-3 alkyl group.
4. The compound according to claim 3, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer, or an isotopic substitution form thereof, wherein the formula II is such as formula II- 1 or as shown in formula II-2:

   

   Wherein, X is O or S, preferably O;

   R ₁ and R ₂ are each independently selected from hydrogen, C _1-3 alkyl, preferably methyl;

   m is selected from an integer from 0 to 3, and R _3b is independently selected from phenyl, halogenated phenyl, halogen, C _1-3 alkyl, halogenated or deuterated C _1-3 alkyl, C _{1- 3} alkoxy, halogenated or deuterated C _1-3 alkoxy, hydroxyl;

   R _a1 and R _a2 are each independently selected from hydrogen, C _1-3 alkyl, and halogen;

   R _b is selected from hydrogen, C _1-3 alkyl, halogenated C _1-3 alkyl;

   L ₃ is selected from none, C _1-2 alkylene, halogenated C _1-2 alkylene, C ₂ alkenylene, L ₄ is selected from none, C _1-3 alkylene, halogenated C _1-3 Alkylene, M ₀ is selected from none, O, NH, CO, CONH;

   The halogen is preferably chlorine or fluorine.
5. The compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or an isotopic substitution form thereof, wherein the compound is characterized in that: is one of the following structures:

   

   

   

   

   

   
6. A pharmaceutical composition, characterized in that: the pharmaceutical composition is the compound described in any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof The active ingredient, or its isotopic substitute form, is a preparation prepared by adding pharmaceutically acceptable excipients.
7. The compound described in any one of claims 1 to 5, or its pharmaceutically acceptable salt, or its stereoisomer, or its optical isomer, or its isotopic substitution form in the preparation of a coronavirus proteolytic enzyme inhibitor Preferably, the coronavirus proteolytic enzyme is coronavirus main protease; more preferably, the coronavirus proteolytic enzyme is SARS-COV-2M ^pro .
8. Use of the compound described in any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or an isotopic substitution form thereof, in the preparation of an anti-coronavirus medicament , preferably, the coronavirus is a novel coronavirus SARS-CoV-2.
9. The compound described in any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or an isotopic substituted form thereof, is used in the preparation of prevention and/or treatment and SARS- The use in medicine for a disease related to COV-2M ^pro , preferably, the disease related to SARS-COV-2M ^pro is a new type of coronavirus pneumonia COVID-19.
10. The use according to any one of claims 7 to 9, wherein the coronavirus proteolytic enzyme inhibitor, anti-coronavirus drug or drug for preventing and/or treating viral pneumonia can inhibit SARS-COV- 2M ^pro is active and/or capable of inhibiting SARS-COV-2 infection of cells.

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