CN114057702A - Novel inhibitor of coronavirus main protease and preparation method and application thereof - Google Patents

Abstract

The invention provides an inhibitor of novel coronavirus main protease and a preparation method and application thereof. Specifically, the compound represented by formula I, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or an isotopic substitution form thereof is provided. The compound can effectively inhibit the activity of SARS-CoV-2 M ^pro , and can be used to prepare a SARS-CoV-2 M ^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.

Description

Novel inhibitor of coronavirus main protease and preparation method and application thereof

Technical Field

The invention belongs to the technical field of organic synthetic drugs, and particularly relates to a novel inhibitor of coronavirus main protease, a preparation method and pharmaceutical application thereof.

Background

Coronavirus pneumonia (COVID-19, also known as novel coronavirus pneumonia) caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), and lopinavir/ritonavir which are anti-HIV drugs and interferon-alpha have been used clinically

The efficacy is still very limited and toxic side effects may occur. The broad spectrum antiviral drug developed by Gilead Sciences, Inc, also explored the treatment of COVID-19, but more data was needed to demonstrate its efficacy. Therefore, there is still an urgent need to develop a safe and effective anti-SARS-CoV-2 drug.

The genomic RNA of coronaviruses 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 about two thirds of the genome length, directly translating two polyproteins: an a-1 frameshift exists between pp1a and pp1ab, ORF1a and ORF1 b. These polyproteins consist of a main protease (abbreviated as M)^pro(ii) a Also known as 3C-like proteases (3 CL)^pro) And one or two papain-like proteases (PLPs) to convert into 16 non-structural proteins. These non-structural proteins are involved in the production of subgenomic RNA, encoding four major structural proteins (envelope (E), membrane (M), spinous process (S), and nucleocapsid (N) proteins) and other accessory proteins to complete the viral replication and invasion process.

M^proThe proteolytic cleavage of the overlapping pp1a and pp1ab into functional proteins is a key step in the viral replication process. Enzymes necessary for viral replication, such as RdRp or nsp13, do not function fully to complete replication without prior proteolytic release. Thus, inhibition of viral M^proCan prevent the generation of infectious virus particles, thereby alleviating the symptoms of the disease。

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

Therefore, there is a need to develop an M that can effectively inhibit SARS-CoV-2 virus^proAn active drug.

Disclosure of Invention

The invention aims to provide a novel inhibitor of coronavirus main protease, a preparation method and a pharmaceutical application thereof.

The invention provides a compound shown as a formula I, or a pharmaceutically acceptable salt, a stereo isomer, an optical isomer or an isotope substitution form thereof:

wherein X is O or S;

a ring is selected from unsubstituted or substituted by one or more R₆Substituted of the following groups: 5-6-membered saturated heterocyclic group, 5-6-membered unsaturated heterocyclic group, saturated hetero-condensed ring group, unsaturated hetero-condensed ring group; r₆Each independently selected from C_1～6Alkyl radical, C_1～6Alkoxy, halogen, hydroxy, cyano, amino, carboxy;

R₃is L₃M₀L₄R_3a(ii) a Wherein L is₃Selected from the group consisting of_1～4Alkylene, halogeno C_1～4Alkylene radical, C_2～4Alkenylene, halogeno C_2～4Alkenylene radical, L₄Selected from the group consisting of_1～4Alkylene, halogeno C_1～4Alkylene radical, M₀Selected from among none, O, S, NH, CO, CONH, NHCO, R_3aIs unsubstituted or substituted by one or more R_3bSubstituted of the following groups: 5-6-membered aryl, 5-6-membered heteroaryl, unsaturated hetero-condensed ring group, unsaturated condensed ring alkyl; r_3bEach independently selected from R_3cSubstituted or unsubstituted C_1～5Alkyl radical, by R_3cSubstituted or unsubstituted C_1～5Alkoxy, halogen, by R_3cSubstituted or unsubstituted phenyl, NR₁₄R₁₅Quilt R_3cSubstituted or unsubstituted naphthyl, hydroxy; r₁₄、R₁₅Each independently selected from hydrogen or C_1～5Alkyl radical, R_3cEach independently selected from halogen, deuterium, cyano, hydroxyl, amino, carboxyl;

R₄selected from the following groups unsubstituted or substituted with one or more substituents: 5-6 membered aryl, 5-6 membered heteroaryl, C_1～5Alkyl, COOR₁₀(ii) a The substituents are independently selected from ═ O, hydroxyl, nitro, amino, carboxyl, halogen and C_1～5An alkyl group; r₁₀Is C_1～5An alkyl group;

R₅is selected from COR₈Or WCOOR₇(ii) a Wherein R is₈Selected from hydrogen or

W is selected from the group consisting of_1～4Alkylene radical, C_2～4Alkenylene radical, C_2～4Alkynylene, R₇Is selected from C_1～6An alkyl group; m is selected from among nothing, CO, NH, CONH, NHCO, COO or OCO, L₀Selected from the group consisting of_1～4Alkylene radical, C_2～4Alkenylene radical, L₁Selected from the group consisting of_1～4Alkylene radical, C_2～4Alkenylene radical, R^8aIs selected from C_1～5Alkyl, halogenated C_1～5An alkyl group, a 3-to 6-membered saturated cycloalkyl group, a 3-to 6-membered saturated heterocyclic group, a 5-to 6-membered aryl group or a 5-to 6-membered heteroaryl group.

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

wherein X is O or S;

n is an integer of 0 to 3, preferably 0 to 2;

R₁、R₂each independently selected from hydrogen, C_1～5Alkyl radical, C_1～5Alkoxy, halogen, hydroxy, cyano, amino, carboxy;

R₃is L₃M₀L₄R_3a(ii) a Wherein L is₃Selected from the group consisting of_1～4Alkylene, halogeno C_1～4Alkylene radical, C_2～3Alkenylene radical, L₄Selected from the group consisting of_1～4Alkylene, halogeno C_1～4Alkylene radical, M₀Selected from among none, O, S, NH, CO, CONH, NHCO, R_3aIs unsubstituted or substituted by one or more R_3bSubstituted of the following groups: phenyl, phenyl,

R_3bEach independently selected from C_1～4Alkyl, halogen substituted C_1～4Alkyl, deuterated C_1～4Alkyl, cyano-substituted C_1～4Alkyl radical, C_1～4Alkoxy, halogen substituted C_1～4Alkoxy, deuterated C_1～4Alkoxy, cyano-substituted C_1～4Alkoxy, halogen, phenyl, halogenated phenyl, NR₁₄R₁₅、

Hydroxy radical, R₁₄、R₁₅Each independently selected from hydrogen or C_1～4An alkyl group;

R₄selected from unsubstituted or substituted by oneOr the following groups substituted with a plurality of substituents: 5-6 membered aryl, 5-6 membered heteroaryl, C_1～5Alkyl, COOR₁₀(ii) a The substituents are independently selected from ═ O, hydroxyl, nitro, amino, carboxyl, halogen and C_1～5An alkyl group; r₁₀Is C_1～5An alkyl group;

R₈selected from hydrogen or

M is selected from among nothing, CO, NH, CONH, NHCO, COO or OCO, L₀Selected from the group consisting of_1～3Alkylene radical, C_2～4Alkenylene radical, L₁Selected from the group consisting of_1～3Alkylene radical, C_2～4Alkenylene radical, R^8aIs selected from C_1～4Alkyl, halogenated C_1～4An alkyl group, a 3-6-membered saturated cycloalkyl group, a 3-6-membered saturated heterocyclic group, a 5-6-membered aryl group or a 5-6-membered heteroaryl group.

Further, R₁、R₂Each independently selected from hydrogen, C_1～4Alkyl radical, C_1～4Alkoxy, halogen, hydroxy;

R₃is selected from

L₃M₀L₄R_3a； L₃Selected from the group consisting of_1～3Alkylene, halogeno C_1～3Alkylene radical, C_2～3Alkenylene radical, L₄Selected from the group consisting of_1～3Alkylene, halogeno C_1～3Alkylene, M₀Selected from among O, NH, CO, CONH, R_3aIs phenyl, substituted by one or more R_3bSubstituted phenyl radicals, R_3bEach independently selected from C_1～4Alkyl, halogen substituted C_1～4Alkyl, deuterated C_1～4Alkyl, cyano-substituted C_1～4Alkyl radical, C_1～4Alkoxy, halogen substituted C_1～4Alkoxy, deuterated C_1～4Alkoxy radical,Cyano-substituted C_1～4Alkoxy, halogen, phenyl, halogenated phenyl, NR₁₄R₁₅、

Hydroxy radical, R₁₄、R₁₅Each independently selected from hydrogen or C_1～3An alkyl group;

R₄is selected from

C_1～2Alkyl, COOR₁₀Substituted or unsubstituted phenyl; the substituent is selected from hydroxyl and nitro; r_a1、R_a2Each independently selected from hydrogen, C_1～3Alkyl, halogen; r₁₀Is C_1～3An alkyl group;

R₈selected from hydrogen, CONHR₁₁、L₂COOR₁₂、C_1～4Alkyl, halogenated C_1～4An alkyl group; r₁₁Selected from 3-6 membered saturated cycloalkyl, C_1～4Alkyl, benzyl, or a salt thereof,

L₂Is C_1～2Alkylene radical, C_2～3Alkenylene radical, R₁₂Is C_1～3An alkyl group.

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

wherein X is O or S, preferably O;

R₁、R₂each independently selected from hydrogen, C_1～3Alkyl, preferably methyl;

m is an integer of 0 to 3, R_3bEach independently selected from phenyl, halogenated phenyl, halogen, C_1～3Alkyl, halo or deuterated C_1～3Alkyl radical, C_1～3Alkoxy, halo or deuterated C_1～3Alkoxy radicalA hydroxyl group;

R_a1、R_a2each independently selected from hydrogen, C_1～3Alkyl, halogen;

R_bselected from hydrogen, C_1～3Alkyl, halogenated C_1～3An alkyl group;

L₃selected from the group consisting of_1～2Alkylene, halogeno C_1～2Alkylene radical, C₂Alkenylene radical, L₄Selected from the group consisting of_1～3Alkylene, halogeno C_1～3Alkylene radical, M₀Selected 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 invention also provides a pharmaceutical composition, which is a preparation prepared by taking the compound, or pharmaceutically acceptable salt thereof, or stereoisomer thereof, or optical isomer thereof, or isotope substitution form thereof as an active ingredient and adding pharmaceutically acceptable auxiliary materials.

The invention also provides the use of the above compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or an isotopic substitution form thereof, for the preparation of inhibitors of coronavirus proteolytic enzyme; preferably, the coronavirus proteolytic enzyme is coronavirus main protease; more preferably, the coronavirus proteolytic enzyme is SARS-COV-2M^pro。

The invention also provides the application of the compound or the pharmaceutically acceptable salt thereof, or the stereoisomer thereof, or the optical isomer thereof, or the isotope substitution form thereof in preparing the anti-coronavirus medicament, preferably, the coronavirus is novel coronavirus SARS-CoV-2.

The invention also provides the application of the compound, or the pharmaceutically acceptable salt, the stereoisomer, the optical isomer or the isotope substitution form thereof in the preparation of the medicines for preventing and/or treating SARS-COV-2M^proUse in medicine of related diseases, preferably, the SARS-COV-2M^proThe related disease is the novel coronavirus pneumonia COVID-19.

Further, the coronavirus proteolytic enzyme inhibitor, the anti-coronavirus drug or the drug for preventing and/or treating viral pneumonia can inhibit SARS-COV-2M^proAnd/or can inhibit SARS-COV-2 infection of cells.

Definitions of terms used in connection with the present invention: the initial definitions provided herein for a group or term apply to that group or term throughout the specification unless otherwise indicated; terms not specifically defined herein should be given the meanings that would be afforded to them by a person skilled in the art in light of the disclosure and the context.

The minimum and maximum values of the carbon atom content in the hydrocarbon group are indicated by a prefix, e.g. prefix C_a～bAlkyl represents any alkyl group containing from "a" to "b" carbon atoms. E.g. C_1～6Alkyl means containing 1E to EA linear or branched alkyl group of 6 carbon atoms.

By "substituted" herein is meant that 1, 2 or more hydrogen atoms in the molecule are replaced by other different atoms or molecules, including 1, 2 or more substitutions on the same or different atoms in the molecule.

"isotopically substituted forms" refer to compounds wherein one or more than two atoms are replaced by their corresponding isotopes, for example compounds wherein hydrogen is replaced by protium, deuterium or tritium.

By "pharmaceutically acceptable" is meant that the carrier, cargo, diluent, excipient, and/or salt formed is generally chemically or physically compatible with the other ingredients comprising a pharmaceutical dosage form and physiologically compatible with the recipient.

"salts" are acidic and/or basic salts formed from a compound or stereoisomer thereof with inorganic and/or organic acids and/or bases, and also include zwitterionic (inner) salts, and also quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly in the final isolation and purification of the compounds. The compound, or a stereoisomer thereof, may be obtained by appropriately (e.g., equivalentlymixing) a certain amount of an acid or a base. These salts may form precipitates in the solution and be collected by filtration, or recovered after evaporation of the solvent, or prepared by reaction in an aqueous medium followed by lyophilization.

The "pharmaceutically acceptable salt" may be a hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate salt of the compound.

Halogen is fluorine, chlorine, bromine or iodine.

"aryl" refers to an all-carbon monocyclic or fused polycyclic (i.e., rings which share adjacent pairs of carbon atoms) group having a conjugated pi-electron system, such as phenyl. The aryl group does not contain heteroatoms such as nitrogen, oxygen, or sulfur, and the point of attachment to the parent must be at a carbon atom in the ring that has a conjugated pi-electron system. The aryl group may be substituted or unsubstituted. The term "5-to 6-membered aryl" refers to an aryl group having 5 or 6 carbon atoms in the ring.

"heteroaryl" refers to a heteroaromatic group containing one to more heteroatoms. The hetero atoms referred to herein include oxygen, sulfur and nitrogen. Such as furyl, thienyl, pyridyl, pyrazolyl, and the like. The heteroaryl group can be optionally substituted or unsubstituted. "5-to 6-membered heteroaryl" refers to heteroaryl 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, "3-to 6-membered saturated cycloalkyl" refers to a saturated cycloalkyl group having 3 to 6 carbon atoms in the ring.

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

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

"Heterofused cyclic" refers to polycyclic heterocyclic groups containing at least 1 heteroatom, and wherein two rings of the polycyclic heterocyclic group share two adjacent carbon or heteroatoms.

"alkylene" refers to a group in which one atom of an alkyl group has been removed. E.g. C₁Alkylene group:

C₂alkylene group:

"alkenylene" refers to a group resulting from the loss of one atom from an alkenyl group. E.g. C₂Alkenyl:

"alkynylene" refers to a group in which one atom of the alkynyl group has been lost. E.g. C₂Alkynyl:

the experimental result shows that the invention provides a new type coronavirus main protease M which can be effectively inhibited^proAn active compound which can effectively inhibit the replication of SARS-COV-2 virus in cells, inhibit the infection of SARS-COV-2 in cells and resist the infection of SARS-COV-2 in vivo of transgenic mice; reducing virus load of lung of transgenic mouse infected by SARS-COV-2, reducing gene expression level of chemokine ligand 10(CXCL10) and beta-type interferon (IFN-beta) in mouse lung, reducing quantity of neutrophilic granulocyte (NEU) and Macrophage (MAC) in mouse lung, and improving pathological injury of mouse lung. Meanwhile, the compound provided by the invention also has good in-vivo safety and pharmacokinetic properties. Application of compound in preparing SARS-CoV-2M^proThe inhibitor, the medicine for resisting SARS-CoV-2 and the medicine for preventing and/or treating the novel coronavirus pneumonia have very good application prospects.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 shows that compound 26 is against SARS-COV-2M^proInhibitory activity curve of (1).

FIG. 2 shows that compound 33 is against SARS-COV-2M^proInhibitory activity curve of (1).

FIG. 3 is a drawing showingCompound 37 for SARS-COV-2M^proInhibitory activity curve of (1).

FIG. 4 shows the inhibition of SARS-COV-2 replication in human alveolar epithelial cells by the compound.

FIG. 5 pulmonary viral load of SARS-CoV-2 infected mice.

FIG. 6 Lung pathological tissue section (3dpi) of SARS-CoV-2 infected mouse.

FIG. 7 lung representative cytokine expression levels (3dpi) of SARS-CoV-2 infected mice.

FIG. 8 Lung neutrophil and macrophage counts (3dpi) of SARS-CoV-2 infected mice.

Detailed Description

The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.

Example 1: preparation of Compound 1

Compound 1 of the present invention is prepared according to the above preparation route, wherein the reaction conditions of the steps are as follows:

i. a, 2-fluoro malonic acid dimethyl ester, benzyl alcohol, toluene, p-toluenesulfonic acid, 110 ℃; b, isopropanol, n-hexane, -10 ℃;

ii. Isopropanol, sodium hydroxide, water, 45 ℃;

iii, anhydrous tetrahydrofuran, isopropyl magnesium chloride tetrahydrofuran solution, Ar, 0 ℃;

iv, anhydrous tetrahydrofuran, N' -carbonyldiimidazole, Ar, 0 ℃;

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

vi, dichloromethane and dioxane hydrochloride solution;

vii, dichloromethane, N' -tetramethyl-O- (7-azabenzotriazol-1-yl) urea hexafluorophosphate, N-diisopropylethylamine, -20 ℃;

viii, dichloromethane, trifluoroacetic acid;

ix, dichloromethane, N' -tetramethyl-O- (7-azabenzotriazol-1-yl) urea hexafluorophosphate, N-diisopropylethylamine-20 ℃;

the specific synthesis steps are as follows:

intermediate 2: preparation of dibenzyl 2-fluoromalonate

Dimethyl 2-fluoropropanoate (10g, 66.6mmol, 1.0eq) and benzyl alcohol (35mL, 338.2mmol, 5.0eq) were dissolved in 100mL toluene, 1.15g p-toluenesulfonic acid (6.7 mmol, 0.1eq) was added, the reaction was refluxed, monitored by TLC, and after about 8 hours the reaction was complete. And cooling to room temperature, evaporating toluene under reduced pressure, adding 15mL of isopropanol, uniformly stirring, slowly adding 30mL of n-hexane under stirring, placing in a cold trap at the temperature of-10 ℃, and continuously stirring for 2 hours to separate out a large amount of white solid. Suction filtration is carried out, the filter cake is washed twice by 10mL multiplied by 2 frozen n-hexane, and the filter cake is dried under vacuum and reduced pressure at 30 ℃ to obtain 18.4g of product with the yield of 91.4 percent.¹H 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 2-fluoro-malonic acid monobenzyl ester

Dibenzyl 2-fluoropropanedioate (18.4g, 60.9mmol, 1.0eq) was dissolved in 100mL of isopropanol, the temperature was raised to 45 ℃ and sodium hydroxide (2.55g, 63.9mmol, 1.05eq) was dissolved in 60mL of water and then added dropwise over 1 hour. After the dropwise addition, the reaction was continued for 30 minutes, isopropanol was distilled off under reduced pressure, 50mL of water was added, and the pH was adjusted to about 9 with a saturated sodium bicarbonate solution. The aqueous phase is extracted twice with dichloromethane 20mL × 2 dichloromethane, the pH of the aqueous phase is adjusted to 1-2 with 6 mol/L hydrochloric acid, extracted three times with 40mL × 3 isopropyl ether, the organic phases are combined and washed once with 30mL saturated brine. Adding anhydrous magnesium sulfate into the organic phase, drying, filtering, concentrating to obtain viscous residue, adding 60mL of n-hexane, stirring overnight to separate out white solid, filtering, and vacuum drying the filter cake at 40 ℃ under reduced pressure to obtain 6.5g of product with the yield of 50.3%.¹H 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

2-Fluoromalonic acid monobenzyl ester is dissolved in anhydrous tetrahydrofuran (2mL/mmol), replaced by argon for protection, cooled to 0 ℃, and slowly added with isopropyl magnesium chloride tetrahydrofuran solution (2M tetrahydrofuran solution, 2.0eq) to obtain white suspension. Stirring was continued at 0 ℃ for 1 hour and the product suspension was used directly in the next reaction.

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

Boc-L-aspartic acid 4-methyl ester (2.2g, 8.8mmol, 1.0eq) was dissolved in 50mL of anhydrous tetrahydrofuran, replaced with argon, cooled to 0 deg.C, CDI (1.5g, 9.3mmol, 1.05eq) was added, and the reaction was incubated for 1 hour. The reaction solution is cooled to-20 ℃,1.5 eq of the intermediate 4 is slowly added, the reaction is kept for 1 hour, and then the temperature is raised to room temperature for reaction for 6 hours. Slowly pouring the reaction solution into 300mL of 2M diluted hydrochloric acid in an ice water bath, extracting with 100mL of multiplied by 3 ethyl acetate for three times, combining organic phases, washing with saturated sodium bicarbonate solution to be alkalescent, washing with 50mL of saturated saline once, adding anhydrous magnesium sulfate for drying, filtering, concentrating, and directly using the obtained crude product for the next reaction.

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

Adding 50mL of ethyl acetate into the crude intermediate 6 obtained in the previous step, adding 200mg of 10% palladium carbon, replacing with hydrogen, reacting at room temperature under hydrogen overnight, filtering, concentrating, and reacting the obtained crude product with petroleum ether: ethyl acetate 10: 1.5g of colorless oil was obtained by mobile phase column chromatography in 65% yield.¹H 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) -3-amino-5-fluoro-4-oxopentanoic acid methyl ester

500mg of intermediate 7 was dissolved in 5mL of dichloromethane, then 5mL of dioxane hydrochloride was added, and after completion of the reaction, spin-dried to give intermediate 8 in 91.2% yield.¹H 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-After 2-carboxylic acid (1g, 6.21mmol, 1.0eq) was dissolved in dichloromethane, HATU (2.81g, 7.40mmol, 1.2eq) was added at-20 deg.C followed by L-proline methyl ester hydrochloride (1.03g, 6.21mmol, 1.0eq) and finally DIEA (3mL, 18.51mmol, 3.0eq) was added and the reaction was monitored by TLC. After the reaction was completed, extraction was performed with an aqueous solution and DCM, and the organic layer was concentrated and separated by column chromatography to give intermediate 11(1.53g) with a yield of 75.2%.¹H NMR(400MHz,DMSO-d₆)δ7.68(dt,J＝7.4,1.5Hz, 1H),7.43(dd,J＝7.4,1.6Hz,1H),7.26(td,J＝7.5,1.7Hz,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

500mg of intermediate 11 was dissolved in10 mL of dichloromethane, then 5mL of trifluoroacetic acid was added, and after completion of the reaction, 378mg of intermediate 12 was obtained by spin-drying, which was used directly as the first reaction. The yield thereof was found to be 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, followed by the addition of HATU (280mg, 0.73mmol, 1.2eq) at-20 ℃, followed by the addition of intermediate 8(100 mg, 0.61mmol, 1.0eq), and finally DIEA (301 μ L, 1.83mmol, 3.0eq), and the reaction monitored by TLC. After the reaction, the mixture was extracted with an aqueous solution and DCM, the organic layer was concentrated and separated by column chromatography to give compound 1 with a yield of 34%.¹H 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 C₂₀H₂₅FN₄O₅[M+H]⁺403.1543found:404.1476。

Example 2: preparation of Compound 3

Compound 3 of the present invention is prepared according to the above preparation route, wherein the reaction conditions of the steps are as follows:

i. Boc-L-glutamic acid dimethyl ester, LiHMDS tetrahydrofuran solution, argon, anhydrous tetrahydrofuran, at-78 ℃;

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 ℃;

iv, anhydrous tetrahydrofuran, Ar, N' -carbonyldiimidazole, 0 ℃;

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

vi, dichloromethane and dioxane hydrochloride solution;

vii, dichloromethane, N' -tetramethyl-O- (7-azabenzotriazol-1-yl) urea hexafluorophosphate, N-diisopropylethylamine, -20 ℃;

viii, dichloromethane, trifluoroacetic acid;

ix, dichloromethane, N' -tetramethyl-O- (7-azabenzotriazol-1-yl) urea hexafluorophosphate, N-diisopropylethylamine-20 ℃;

the specific synthesis steps are as follows:

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

Boc-L-glutamic acid dimethyl ester (12g, 43.6mmol, 1.0eq) was dissolved in 100mL of anhydrous tetrahydrofuran, replaced with argon for protection, cooled to-78 deg.C, 94mL of LiHMDS tetrahydrofuran solution (1M tetrahydrofuran solution, 94mmol, 2.2eq) was slowly added dropwise, and after the addition was complete, the reaction was allowed to warm for 1 hour. 3.24mL bromoacetonitrile (46.6mmol, 1.1eq) was slowly added dropwise to the reaction mixture, the reaction was incubated for 6 hours and then quenched with 50mL saturated ammonium chloride solution. The quenched reaction solution was warmed to room temperature, extracted three times with 60mL of X3 ethyl acetate, the organic phases were combined, washed with 50mL of saturated brine, and then anhydrous sulfuric acid was addedMagnesium drying, filtering, concentrating, and purifying the crude product with petroleum ether: ethyl acetate 4: 1 mobile phase column chromatography gave 9.36g of pale yellow oil, yield 68.3%.¹H 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 methyl (S) -2- (((tert-butoxycarbonyl) amino) methyl-3- ((S) -2-oxopyrrolidin-3-yl) propionate

(2S,4R) -dimethyl 2- (tert-butoxycarbonylamino) -4- (cyanomethyl) glutarate (9.36g, 29.8mmol, 1.0eq) was dissolved in 150mL of anhydrous methanol and cooled to 0 ℃. Cobalt chloride hexahydrate (4.25g, 18mmol, 0.6eq) was added, sodium borohydride (6.76 g, 180mmol, 6.0eq) was added in portions, after the addition was completed, the temperature was raised to room temperature, the reaction was allowed to stand overnight, and the completion of the reaction was monitored by TLC. Adding 50mL of saturated ammonium chloride solution to quench the reaction, evaporating methanol under reduced pressure, extracting with 100mL of multiplied by 3 ethyl acetate for three times, combining organic phases, washing with 200mL of multiplied by 3 saturated ammonium chloride solution for three times and 200mL of multiplied by 3 saturated saline for three times in sequence, adding anhydrous magnesium sulfate to the organic phase, drying, filtering and concentrating to obtain a crude product, and adding petroleum ether: ethyl acetate ═ 1:1 mobile phase column chromatography gave 3.94g of white solid with a yield of 46.2%.¹H 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 (0.88g, 3.1mmol, 1.0eq) was dissolved in10 mL tetrahydrofuran and cooled to 0 ℃. Lithium hydroxide monohydrate (0.64g, 15.4mmol, 5.0eq) was dissolved in10 mL of water and slowly added dropwise, after completion of the addition, the reaction was kept for 4 hours, and the completion of the reaction was monitored by TLC. Adjusting pH value of saturated citric acid aqueous solution to neutrality, evaporating tetrahydrofuran under reduced pressure, extracting with 10mL ethyl acetate once, adjusting pH value of water phase to 3-4 with saturated citric acid aqueous solution, extracting with 20mL × 3 ethyl acetate three times, mixing organic phases, washing with 20mL saturated saline, addingDried over anhydrous magnesium sulfate, filtered, and concentrated to give 0.78g of off-white solid with a yield of 93.2%.¹H NMR(400MHz, Chloroform-d)δ7.19(s,1H),5.69(d,J＝7.9Hz,1H),4.35(q,J＝7.6 Hz,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: preparation of benzyl (4S) -4- ((tert-butoxycarbonyl) amino) -2-fluoro-3-oxo-5- ((S) -2-oxopyrrolidin-3-yl) pentanoate

(S) -2- ((tert-butoxycarbonyl) amino) -3- ((S) -2-carbonylpyrrolidin-3-yl) propionic acid (2.4 g,8.8mmol, 1.0eq) was dissolved in 50mL of anhydrous tetrahydrofuran, purged with argon, cooled to 0 deg.C, CDI (1.5g, 9.3mmol, 1.05eq) was added and the reaction was allowed to incubate for 1 hour. The reaction solution is cooled to-20 ℃,1.5 eq of the intermediate 4 is slowly added, the temperature is kept for reaction for 1 hour, and then the temperature is raised to room temperature for reaction for 6 hours. The reaction solution is slowly poured into 300mL of 2M diluted hydrochloric acid in an ice water bath, extracted with 100mL of multiplied by 3 ethyl acetate for three times, the organic phases are combined, washed to be alkalescent by saturated sodium bicarbonate solution, washed once by 50mL of saturated saline, dried by adding anhydrous magnesium sulfate, filtered and concentrated, and the obtained crude product is directly used for the next reaction.

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

And adding 50mL of ethyl acetate into the crude intermediate 17 obtained in the previous step, adding 200mg of 10% palladium carbon, replacing with hydrogen, reacting at room temperature under hydrogen overnight, filtering, concentrating, and reacting the obtained crude product with petroleum ether: ethyl acetate ═ 1:1 mobile phase column chromatography gave 1.3g of white solid with a yield of 50%.¹H 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

500mg of intermediate 18 was dissolved in 5mL of dichloromethane, then 5mL of dioxane hydrochloride was added, and after completion of the reaction, the intermediate 19 was obtained by spin-drying, the yield was 85%.

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

2, 4-Dichlorophenoxyacetic acid (intermediate 21, 0.58g,2.62mmol),2- (7-benzotriazol oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (1.2g,3.14mmol), N, N-diisopropylethylamine (1.3mL,7.86mmol), and (1S,3aR,6aS) -octahydrocyclopenta [ c ] ester]Pyrrole-1-carboxylic acid ethyl ester hydrochloride (intermediate 20, 0.58g,2.62mmol) was dissolved in 15mL of ultra dry N, N-dimethylformamide and reacted at 25 ℃ under argon protection for 12 hours, d reaction was added with 4 times volume of water, extracted three times with dichloromethane, combined organic phases were washed with saturated ammonium chloride solution, saturated sodium carbonate solution, dried over anhydrous sodium sulfate and filtered, silica gel column chromatography with stirring (petroleum ether/ethyl acetate 1:1) gave intermediate 22(0.60g, 59%).¹H NMR(400MHz,MeOD)δ7.42(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＝3.6 Hz,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) octahydrocyclopenta [ c ] pyrrole-1-carboxylic acid

Dissolving ethyl (1S,3aR,6aS) -2- (2- (2, 4-dichlorophenoxy) acetyl) octahydro cyclopenteno [ c ] pyrrole-1-carboxylate (intermediate 22, 200mg,0.52mmol) in 20mL of methanol, adding 2M sodium hydroxide solution (10mL), stirring for 4 hours at 25 ℃, after TLC monitoring reaction is finished, spin-drying the methanol, adjusting the pH to weak acidity with hydrochloric acid, extracting with dichloromethane for three times, combining organic phases, drying with anhydrous sodium sulfate, and selecting to dry to obtain a crude product to be directly subjected to next reaction.

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

Intermediate 23(168mg, 0.61mmol, 1.0eq) was dissolved in dichloromethane and HATU (280mg, 0.73mmol, 1.2eq) was added at-20 ℃ followed by intermediate 19(100 mg, 0.61mmol, 1.0eq)) DIEA (301. mu.L, 1.83mmol, 3.0eq) was added and the reaction monitored by TLC. After the reaction, the mixture was extracted with aqueous solution and DCM, the organic layer was concentrated and separated by column chromatography to give compound 3 with a yield of 34%.¹H 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＝11.1,6.0 Hz,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 C₂₃H₃₀FN₃O₄[M+H]⁺432.2293 found:432.2291。

Example 3: preparation of Compound 9

Compound 9 of the present invention is prepared according to the above preparation route, wherein the reaction conditions of the steps are as follows:

i. 1-hydroxybenzotriazole, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N, N-diisopropylethylamine, N, N-dimethylformamide, room temperature.

ii. Sodium hydroxide, methanol, water, 55 degree

iii 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate, N, N-diisopropylethylamine, N, N-dimethylformamide, 0 deg.C

iv, sodium borohydride, methanol, room temperature

v. dessimutan oxidant, ultra-dry dichloromethane, room temperature

The specific synthesis steps are as follows:

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

Starting material 23 (quinoline 2-carboxylic acid 1.0g, 11.6mmoL), 1-hydroxybenzotriazole (2.03g, 15.08mmoL), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (4.43g,23.3 mmoL), N, N-dimethyl-carbodiimidePlacing 30mL of benzamide into a round bottom flask, stirring at normal temperature for 0.5 hour, adding 2.5mL of N, N-diisopropylethylamine, adding 0.59g of intermediate 24, reacting for 8 hours, distilling under reduced pressure to remove the solvent, extracting with dichloromethane, ammonium chloride solution and sodium bicarbonate solution, washing with water and saturated sodium chloride solution, drying with sodium sulfate, performing suction filtration, and performing organic phase column chromatography to obtain a white solid. The yield thereof was found to be 85%.¹H NMR (400MHz, DMSO) δ 8.12-8.03 (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 ═ 5.4 Hz 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 ], [ M,1H ], (M,1H) ]]⁺。

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

Dissolving the intermediate 25 in 20mL of tetrahydrofuran, slowly adding 10mL of 2mol/L sodium hydroxide solution, gradually heating the reaction solution to 55 ℃, stirring for 3 hours, stopping and cooling to normal temperature, concentrating the reaction solution, adding water to adjust the pH value to be weak acid, separating out a white solid, and performing suction filtration to obtain an intermediate 5 which is used for the next reaction without further purification

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

261.0 g of intermediate and 1.93g of 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethylurea hexafluorophosphate are added into 20mL of N, N-dimethylformamide, 2.0mL of N, N-diisopropylethylamine is added after stirring for 0.5 hour at 0 ℃, 270.89 g of intermediate is added, after reaction is carried out for 12 hours at 0 ℃ under the protection of argon, 4 times of volume of water is added, and extraction is carried out for three times by using ethyl acetate. And combining organic phases, extracting by using an ammonium chloride solution and a sodium bicarbonate solution, washing by using water and a saturated sodium chloride solution, drying by using sodium sulfate, performing suction filtration, and performing column chromatography on the organic phases to obtain white solids. The yield thereof was found to be 65%.¹H NMR(400MHz,DMSO)δ8.53(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: preparation of (1R,5S) -N- ((S) -1-hydroxy-3-phenylpropan-2-yl) -6, 6-dimethyl-3- (quinoline-2-carbonyl) -3-azabicyclo [3.1.0] hexane-2-carboxamide

500mg of intermediate 28(((1R,5S) -6, 6-dimethyl-3- (quinoline-2-carbonyl) -3-azabicyclo [3.1.0] hexane-2-carbonyl) -L-phenylalanine) was dissolved in 30mL of dry methanol, sodium borohydride was added at room temperature, after stirring for 3 hours, water was added thereto for quenching, methanol was dried by spinning, the aqueous phase (50 mL. times.3) was extracted with ethyl acetate, the organic phase was collected and dried over sodium sulfate, and the organic phase was intermediate 29 after suction filtration, showing a yield of 80%.

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

Intermediate 29(1R,5S) -N- ((S) -1-hydroxy-3-phenylprop-2-yl) -6, 6-dimethyl-3- (quinoline-2-carbonyl) -3-azabicyclo [3.1.0]Dissolving hexane-2-formamide 200mg in10 mL of dry dichloromethane, adding dessimutan oxidant under the condition of stirring at normal temperature, monitoring the reaction by TLC to be finished, filtering to remove the oxidant, and performing column chromatography on the filtrate to obtain a compound 9 with the yield of 65%.¹H NMR (400MHz,DMSO)δ9.55(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

Compound 14 of the present invention is prepared according to the above preparation route, wherein the reaction conditions of the steps are as follows:

i. trifluoroacetic acid, dichloromethane, 25 deg.C

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

iii, sodium hydroxide, methanol, water, 55 degree

iv, 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate, N, N-diisopropylethylamine, N, N-dimethylformamide, 0 deg.C

v, sodium borohydride, methanol, room temperature

vi, dessimutan oxidizer, ultra-dry dichloromethane, room temperature.

The specific synthesis steps are as follows:

intermediate 30: preparation of methyl (S) -2-amino-3- ((S) -2-oxo-3-yl) propionate trifluoroacetate salt

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

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

2- (4- (trifluoromethoxy) phenoxy) acetic acid (commercially available intermediate 31, 0.24g,1.0 mmol),2- (7-benzotriazol oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (0.49g, 1.2mmol) and N, N-diisopropylethylamine (494. mu.L, 3mmol) were dissolved in N, N-dimethylformamide and (1R,2S,5S) -6, 6-dimethyl-3-azabicyclo [3.1.0 mmol ] was added]Hexane-2-carboxylic acid methyl ester hydrochloride (commercially available intermediate 24, 0.21g,1.0 mmol.) argon protected reaction at ambient temperature for 12 hours reaction 4 times volume of water was added, extracted three times with dichloromethane, combined organic phases washed with saturated ammonium chloride solution, saturated sodium carbonate solution, dried over anhydrous sodium sulfate and filtered, silica gel stirred column chromatography (petroleum ether/ethyl acetate 1:1) gave intermediate 32 as a white solid (0.34g, 88%).¹H 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: preparation of (1R,2S,5S) -6, 6-dimethyl-3- (2- (4- (trifluoromethoxy) phenoxy) acetyl) -3-azabicyclo [3.1.0] hexane-2-carboxylic acid

Methyl (1R,2S,5S) -6, 6-dimethyl-3- (2- (4- (trifluoromethyl oxy) phenoxy) acetyl) -3-azabicyclo [3.1.0] hexane-2-formic acid (intermediate 32, 200mg) is dissolved in 30mL of methanol, then 2M NaOH solution is added for 20mL, stirring is carried out at normal temperature for 2.5 hours, after TLC monitoring reaction is finished, methanol is dried in a spinning mode, pH is adjusted to weak acidity by hydrochloric acid, dichloromethane is extracted for three times, and after the combined organic phase anhydrous sodium sulfate is dried, the crude product is selected to be dried to directly carry out next reaction.

Intermediate 34: preparation of methyl (1R,2S,5S) -6, 6-dimethyl-3- (2- (4- (trifluoromethoxy) phenoxy) acetyl) -3-azabicyclo [3.1.0] hexane-2-amide) -3- ((S) -2-oxo-3-yl) propanoate

To (1R,2S,5S) -6, 6-dimethyl-3- (2- (4- (trifluoromethoxy) phenoxy) acetyl) -3-azabicyclo [3.1.0] at 0 deg.C]To a solution of hexane-2-carboxylic acid (intermediate 33, 0.45g,1.2 mmol) in ultra-dry DMF was added 2- (7-benzotriazole oxide) -N, N' -tetramethyluronium hexafluorophosphate (0.61g,1.6mmol), and after stirring for 30 minutes, n.n-diisopropylethylamine (0.59mL,3.6mmol) was added, and then, the crude intermediate 14 (0.27g, 1.45mmol) was added to the reaction system. The reaction was stirred for 12 hours at 0 ℃ under argon protection. After TLC monitoring the reaction was complete, 4 volumes of water were 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 column chromatographed with stirring (ethyl acetate/methanol 10:1) to give intermediate 34 as a white solid.¹H NMR(400MHz,MeOD)δ7.17(d,J＝8.0Hz,2H), 6.97(d,J＝8.9Hz,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: preparation of (1R,2S,5S) -N- ((S) -1-hydroxy-3- ((S) -2-oxo-3-yl) propanoate-2-yl) -3- (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,1.1mmol) was added to 50mL, sodium borohydride (0.14g,8.8mmol) was added in portions at low temperature, after stirring for 2 hours at normal temperature, water was added for quenching, methanol was spin-dried, the remaining aqueous phase was extracted with ethyl acetate (50mL × 3), the organic phases were combined and dried over anhydrous sodium sulfate, and the spin-dried by filtration to obtain a white solid as a crude product, which was directly used in the next reaction step.

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

Mixing (1R,2S,5S) -N- ((S) -1-hydroxy-3- ((S) -2-carbonyl-3-yl) propionate-2-yl) -3- (2- (4- (trifluoromethoxy) phenoxy) acetyl) -3-azabicyclo [ 3.1.0%]Hexane-2-carboxamide (intermediate 36, (0.38g,0.75mmol) was dissolved in ultra dry dichloromethane and dessimutan oxidant (0.95mg,0.79mmol) was added in portions and reacted at room temperature for 3.5 hours, TLC monitored for the completion of the reaction, the reaction system was filtered, the organic phase was washed with sodium thiosulfate solution and saturated sodium bicarbonate solution, concentrated and isolated with preparative chromatography (acetonitrile/water 30:70) to give compound 14(0.28g, 45%) as a white solid.¹H NMR(400 MHz,MeOD)δ7.24–7.13(m,2H),7.04–6.92(m,2H),4.49(d,J＝9.1 Hz,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).¹³C NMR(101 MHz,MeOD)δ181.60,172.58,167.18,156.90,142.99,121.99,115.47 (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 C₂₄H₂₈F₃N₃O₆ ⁺[M+H]⁺512.1964； found,512.2137.

Example 5: preparation of Compound 15

Compound 15 of the present invention was prepared according to the above preparation route, wherein the reaction conditions for the steps are as follows:

i. 2- (7-Benzotriazole oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate, N, N-diisopropylethylamine, N, N-dimethylformamide, 0 deg.C

ii. Sodium borohydride, methanol, room temperature

iii, dessimutane oxidizer, ultra-dry dichloromethane, room temperature.

The specific synthesis steps are as follows:

intermediate 36: preparation of methyl (S) -2- ((1S,3aR,6aS) -2- (2- (2, 4-dichlorophenoxy) acetyl) octahydro cyclopenteno [ c ] pyrrole-1-carboxamide) -3- ((S) -2-carbonyl-3-yl) propionate

First, 2- (7-Benzotolyltriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (0.099g,0.26mmol) was added to (1S,3aR,6aS) -2- (2- (2, 4-dichlorophenoxy) acetyl) octahydrocyclopenta [ c ] ester]Pyrrole-1-carboxylic acid (intermediate 23, 0.071g,0.20mmol) in ultra dry N, N-dimethylformamide, the reaction stirred for 30 minutes, N-diisopropylethylamine (100 μ L,0.60mmol), methyl (S) -2-amino-3- ((S) -2-carbonyl-3-yl) propionate trifluoroacetate (intermediate 30, 0.060g,0.32mmol) were added to the reaction in sequence, the reaction was reacted at 0 ℃ under argon protection for 12 hours, TLC monitored for the end of the reaction, 4 volumes of water were added, extracted three times with ethyl acetate, the combined organic phases were washed with saturated ammonium chloride solution, saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate and filtered, column chromatography (ethyl acetate/methanol ═ 10:1) afforded intermediate 36(0.063g, 59%).¹H NMR(400 MHz,MeOD)δ7.40(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: preparation of (1S,3aR,6aS) -2- (2- (2, 4-dichlorophenoxy) acetyl) -N- ((S) -1-hydroxy-3- ((S) -2-oxo-3-yl) propanoate-2-yl) octahydrocyclopenta [ c ] pyrrole-1-carboxamide

Methyl (S) -2- ((1S,3aR,6aS) -2- (2- (2, 4-dichlorophenoxy) acetyl) octahydrocyclopenta [ c ] pyrrole-1-carboxamide) -3- ((S) -2-carbonyl-3-yl) propionate (intermediate 36, 1,00g,2.0mmol) was dissolved in anhydrous methanol, then sodium borohydride (0.6g,16mmol) was added in portions at 0 ℃, the temperature was then raised to room temperature, stirring was continued for 2 hours, the reaction was detected by TLC, water was added to quench, methanol was spin-dried, the remaining aqueous phase was extracted with ethyl acetate (50 mL. times.3), the organic phases were combined and dried over anhydrous sodium sulfate, filtered to give a white solid aS a crude product, which was used directly in the next reaction.

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

In (1S,3aR,6aS) -2- (2- (2, 4-dichlorophenoxy) acetyl) -N- ((S) -1-hydroxy-3- ((S) -2-carbonyl-3-yl) propanoate-2-yl) octahydro-cyclopenteno [ c]Pyrrole-1-carboxamide (intermediate 37, 0.50g,1.0mmol) in ultra-dry dichloromethane was added slowly in portions to bosentan oxidant (0.55g,1.3mmol), reacted at room temperature for 3.5 hours, monitored by TLC for reaction completion, the reaction was filtered, the organic phase was washed with sodium thiosulfate solution and saturated sodium bicarbonate solution, concentrated and chromatographed using preparative chromatography (acetonitrile/water 45:55) to give compound 30(0.21g, 42%) as a white solid.¹H NMR(400MHz,MeOD)δ7.42 (t,J＝4.3Hz,1H),7.28–7.17(m,1H),7.03–6.90(m,1H),4.47(dd,J ＝9.6,6.1Hz,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).¹³C NMR(101MHz, MeOD)δ181.64,173.39,167.10(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 C₂₃H₂₇Cl₂N₃O₅ ⁺[M+H]⁺496.1361；found,496.0842.

Example 6: preparation of Compound 42

Compound 42 of the present invention is prepared according to the above preparation route, wherein the reaction conditions of the steps are as follows:

i、LDA,ClCH₂I,THF

ii. HCl dioxane solution

iii, 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethylurea hexafluorophosphate, N, N-diisopropylethylamine, N, N-dimethylformamide, 0 ℃.

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

A dry three-necked flask was selected, separately prepared for argon shielding and a thermometer, and (S) -methyl 2- (tert-butoxycarbonylamino) -3- ((S) -2-carbonylpyrrolidin-3-yl) propionate (intermediate 16, 5g,17.5mmol), tetrahydrofuran (50mL), chloroiodomethane (5mL,68mmol) was added and stirred at-77 deg.C. lithium diisopropylamide (70mL,105mmol) was further added dropwise. After the addition was completed, the reaction was further carried out for 2 hours, and then quenched by adding acetic acid and tetrahydrofuran at a low temperature, 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, concentrated, sample-mixed and chromatographed to give pale yellow solidAn intermediate 38.¹H NMR(400MHz,DMSO-d₆)δ7.89(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,2 H),1.61(s,9H)。

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

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

Compound 42: preparation of (1S,3aR,6aS) -N- ((S) -4-chloro-3-oxo-1- ((S) -2-oxo-3-yl) butan-2-yl) -2- (2- (2, 4-dichlorophenoxy) acetyl) octahydrocyclopenta [ c ] pyrrole-1-carboxamide

2- (7-Benzotolyltriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (1g, 2.6mmol) was added to (1S,3aR,6aS) -2- (2- (2, 4-dichlorophenoxy) acetyl) octahydrocyclopenta [ c ] c]Pyrrole-1-carboxylic acid (intermediate 23, 0.71g,2.0mmol) in ultra dry N, N-dimethylformamide, the reaction was stirred for 30 minutes, N, N-diisopropylethylamine (1mL,6.0 mmol), (S) -3- ((S) -2-amino-4-chloro-3-oxobutyl) pyrrolidin-2-one hydrochloride (intermediate 39, 0.652g,3.2mmol) was added to the reaction in sequence, the reaction was carried out at 0 ℃ under argon protection for 16 hours, TLC, after the reaction was completed, 3 volumes of water were added, extraction was performed 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 obtain compound 42, which was isolated from preparative purification system (acetonitrile/water 30: 70).¹H 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

Compound 50 of the present invention was prepared according to the above preparation route, wherein the reaction conditions for the steps are as follows:

i. tert-butyl isobutyronitrile, acetic acid, ultra-dry dichloromethane;

ii. 1M sodium hydroxide solution, methanol;

iii, dessimutan oxidizer, ultra-dry dichloromethane.

The specific synthesis steps are as follows:

intermediate 40: preparation of (3S) -1- (tert-butylamino) -3- ((1S,3aR,6aS) -2- (2- (2, 4-dichloro) ethanoyl) octahydrocyclopenta [ c ] pyrrole-1-carboxamide) -1-oxo-4- ((S) -2-oxo-3-yl) butan-2-ylcarboxylic acid

First, compound 15(0.40mmol) was dissolved in ultra dry dichloromethane, followed by the addition of acetic acid (0.028g,0.47mmol), tert-butylisonitrile (0.43mmol) in that order. The reaction was stirred at room temperature for 24 hours, and column chromatography was performed under reduced pressure (dichloromethane/methanol 15: 1) to give intermediate 40.¹H NMR(400MHz,DMSO-d₆)δ7.87(d,J＝12.1Hz,1H), 7.46(s,1H),7.44(d,J＝1.4Hz,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: preparation of (1S,3aR,6aS) -N- ((2S) -4- (tert-butylamino) -3-hydroxy-4-oxo-1- ((S) -2-oxo-3-yl) butan-2-yl) -2- (2- (2, 4-dichloro) acetyl) octahydro-cyclopenta [ c ] pyrrole-1-carboxamide

1M sodium hydroxide solution was added (0.5mL) to a solution of intermediate 40(0.164mmol) in methanol, the reaction was stirred at room temperature for 2 hours, the pH was adjusted to neutral with 1M hydrochloric acid, the reaction solution was spin-dried, the residue was dissolved in dichloromethane, and the reaction solution was spin-dried after extraction with water to give crude product 41 which was used directly in the next reaction.

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

To the ultra-dry dichloromethane solution of intermediate 41, desmartin was added slowly in portions, the reaction was carried out at room temperature for 4 hours, TLC monitored for completion of the reaction, the reaction system was filtered, the organic phase was washed with a sodium thiosulfate solution and a saturated sodium bicarbonate solution, and after concentration, a preparative chromatography system (acetonitrile/water 50:50) was used to obtain compound 50 as a white solid.¹H NMR (400MHz,MEOD)δ7.42(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)⁺。

The remaining compounds of the invention shown in Table 1 were prepared according to the preparation routes of examples 1-7, with changing the starting materials.

Table 1 structure and characterization data for compounds of the invention

The pharmacological effects of the compounds of the present invention are demonstrated by the following experimental examples.

Experimental example 1: compound pair M of the present invention^proAssay for level of inhibition of enzyme Activity

(1) Experimental methods

Recombinant SARS-CoV-2M^pro(final concentration 750nM) was mixed with serial dilutions of each compound in 25. mu.L assay buffer (20mM Tris-HCl, pH 7.5, 150mM NaCl, 1mM EDTA, 2mM DTT) and incubated for 10 min. The reaction was initiated by adding 25. mu.L of fluorogenic substrate (MCA-AVLQ ↓: SGFR-Lys (Dnp) -Lys-NH2) at a final concentration of 20. mu.M, and the fluorescence signal at 320nm (excitation)/405 nm (emission) was measured with a microplate reader. Vmax of reactions with different concentrations of compound added and Vmax of reactions with DMSO added were calculated and used to generate IC₅₀Curve line. anti-SARS-CoV-2M measurements at 9 concentrations and 3 independent replicates for each compound^prosemi-Inhibitory Concentration (IC)₅₀) The value is obtained. All experimental data were analyzed using GraphPad Prism software.

(2) Results of the experiment

TABLE 2 Compound vs SARS-COV-2M^proEnzyme activity inhibiting effect of

As can be seen from Table 2 and FIGS. 1, 2 and 3, the compounds of the present invention are effective in inhibiting SARS-CoV-2M^proCan be used for preparing SARS-CoV-2M^proAn inhibitor, a medicament for preparing anti-novel coronavirus and a medicament for preventing and/or treating novel coronavirus pneumonia.

Experimental example 2: inhibition experiment of SARS-COV-2 infection Vero E6 cell death

(1) Experimental methods

The antiviral activity of the compound is preliminarily evaluated by detecting the inhibition effect of the compound on cell death caused by SARS-COV-2 infection of Vero E6 cells. The specific experimental scheme is as follows: vero E6 cells were cultured at a cell density of 2X 10⁴Cells/well, 100. mu.L/well in 96-well plates, 5% CO at 37 ℃₂Incubate overnight in an incubator. The following day, 100 μ L of drug and 100 μ L of virus diluent (MOI ═ 1) were added simultaneously to each well, and a positive control containing no drug and a negative control containing no virus were set at 37 ℃ with 5% CO₂Culturing for 72h, detecting cell survival rate with CCK-8 kit, and calculating the inhibition rate and half-Effective Concentration (EC) of the drug on virus replication₅₀) Values, 3 independent replicates were set for all experiments and all experimental data were analyzed using GraphPad Prism software.

(2) Results of the experiment

TABLE 3 inhibitory Activity of the Compounds of the present invention against cell death caused by SARS-COV-2 infection of Vero E6 cells

Note: NT stands for untested cell Activity

As can be seen from Table 3, the compound of the present invention can effectively inhibit cell death caused by SARS-COV-2 infecting Vero E6 cells, which shows 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 methods

Cytotoxicity assessment of compounds was performed using Vero E6 cells. The specific experimental scheme is as follows: vero E6 cells were cultured at a cell density of 2X 10⁴Cells/well, 100. mu.L/well were seeded in 96-well plates and incubated overnight at 37 ℃ in a 5% CO2 incubator. The next day, 200 μ L of drug-containing medium was added to each well, and the compound was diluted in 5-fold gradients at 200 μ M initial concentration for 6 gradients, with 3 replicate wells per concentration, and each set of experiments was set with negative and blank controls without drug. After 72h of drug treatment, cell viability is detected by using a CCK-8 kit, and the toxicity and half-cell toxicity concentration (CC) of the compound on Vero E6 cells are calculated₅₀) The value is obtained. All experimental data were analyzed using GraphPad Prism software.

(2) Results of the experiment

TABLE 4 toxicity of the Compounds of the invention on Vero E6 cells

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 SARS-COV-2 replication in human alveolar epithelial cells

(1) Experimental methods

For the RT-qPCR method, human alveolar epithelial cells were cultured at 8X 10⁵The density of individual cells/well was seeded into 48-well plates (200. mu.L/well) and grown overnight. Cells were then treated with virus infection (MOI ═ 0.01) and varying concentrations of compound. After 1 hour incubation at 37 ℃, the medium containing the virus-drug mixture was removed and replaced with fresh medium containing the compound. After a further 48 hours of incubation, cell supernatants were collected and extractedTaking virus RNA, carrying out RT-qPCR quantitative analysis on the virus RNA, and calculating the inhibition rate and EC of the drug on virus replication₅₀The value is obtained. EC (EC)₅₀Values were calculated using a dose response model in GraphPad Prism 8.0 software, experiment set up for 2 independent replicates.

(2) Results of the experiment

The test results are shown in FIG. 4, and all the compounds 14, 15, 26, 43, 44 and 45 show nanomolar activity of inhibiting SARS-COV-2 replication in human alveolar epithelial cells, which is superior to the reported SARS-COV-2M with the highest activity^proAntiviral activity of inhibitors 11b (Dai et al, 2020, science.368(6497):1331-1335) and GC376(Ma et al, 2020, Cell Res.30(8):678-692) under the same test conditions (11b, EC)₅₀＝23.6 nM；GC376，EC₅₀＝151.3nM)。

Experimental example 5: plaque assay for evaluating Compounds against SARS-COV-2 Activity (Vero E6 cells)

(1) Experimental methods

Compounds 3 and 39 were evaluated for anti-SARS-COV-2 activity in Vero E6 cells using the plaque method. Vero E6 at 1.0X 10 per well⁵One was inoculated into a 24-well cell culture plate and cultured overnight at 37 ℃ for use. After addition of the serially diluted drug, SARS-CoV-2 was added to infect the cells with an MOI of about 0.002. After culturing in a 37 ℃ cell culture box for 1 hour, the drug-containing infection supernatant is removed, PBS is washed once, 0.5mL of sodium carboxymethylcellulose containing drugs with different concentrations and the final concentration of the sodium carboxymethylcellulose is 0.9 percent are added, and the mixture is cultured in the 37 ℃ cell culture box for 72 hours. Fixing with 20% formaldehyde for 2 hours, adding 0.5% crystal violet for dyeing for 20 minutes, drying, taking pictures, observing the size of the plaques and recording the number of the plaques. The experiment was performed with a blank control well (normal cells), a virus control well, and a positive drug control well.

Calculating the formula: inhibition (%) - (number of plaques in virus control well-number of plaques in sample well)/number of plaques in virus control well × (100)

The resulting cell activity and inhibition rate were calculated, and EC was calculated using Graphpad Prism8₅₀(median effective concentration) value.

(2) Results of the experiment

TABLE 5 inhibitory Effect of Small molecule Compounds on SARS-CoV-2

Name of Compound	EC50(μM)
		3	0.24
39	1.20
		Remdesivir (Ruidexiwei)	0.69

The experimental result is shown in Table 5, the compound of the invention can effectively inhibit SARS-COV-2 infection in Vero E6 cells; in particular the compound 3, EC₅₀0.2373 μ M, the activity was better than that of the positive control Reidesvir (EC)₅₀0.692 μ M).

Experimental example 6: evaluation of in vivo pharmacokinetic Properties of Compounds on rats

(1) Dosing regimens

Male Sprague-Dawley (SD) rats 60, weighing 200-230g, were randomly divided into 3 groups of 3 rats each. The test compounds were administered intragastrically (p.o.), intravenously (i.v.) and intraperitoneally (i.p.) respectively according to the protocol of table 6 below. Fasting was performed for 12h before the experiment, and water was freely drunk. The food is taken 2h after administration.

The gavage, intravenous and intraperitoneal solutions were formulated in DMSO/HS15/NaCl (5/3/92, v/v/v). The drugs were administered at the doses indicated in table 6, the time of administration was recorded, and approximately 0.20mL of each sample was collected via jugular vein blood sampling or other suitable means at the time points set forth above, anticoagulated with heparin sodium, and placed on ice after collection. And the plasma was centrifuged within 1 hour (centrifugation conditions: 6800g, 6 minutes, 2-8 ℃ C.). Plasma samples were stored in a-80 ℃ freezer prior to analysis. Grouping and blood sampling time points are shown in table 6, 3 animals per time point.

Table 6 evaluation of in vivo pharmacokinetic Properties of Compounds on rats

(2) Results of the experiment

Table 7 major pharmacokinetic parameters of the compounds

The results are shown in Table 7. The present inventors have conducted pharmacokinetic studies on compounds 3, 14, 15, 26, 39, 40, 43, 44 and 45. Wherein the oral exposure of compound 3 was 2293 h ng/mL, and the bioavailability was 55.1%. Compound 14 was exposed to 11581 h ng/mL in the peritoneal cavity with a bioavailability of 78.0%; the oral exposure was 1665h ng/mL, and the bioavailability was 11.2%. The intraperitoneal administration exposure of the compound 15 is 12166h ng/mL, and the bioavailability is 62.3%; the oral exposure was 2843h ng/mL, and the bioavailability was 14.6%. The oral exposure of compound 26 was 842h ng/mL, with a bioavailability of 7.2%. Compound 39 was orally exposed to 14586h ng/mL with a bioavailability of 14.7%. Compound 40 had an oral exposure of 2888h ng/mL and a bioavailability of 22.1%. Compound 43 had an oral exposure of 258h ng/mL and a bioavailability of 4.8%. The oral exposure of compound 44 was 381h ng/mL, with a bioavailability of 4.1%. Compound 45 had an oral exposure of 968h ng/mL and a bioavailability of 5.1%.

The experimental result shows that the compound has good pharmacokinetic property in the body of a rat.

Experimental example 7: preliminary evaluation of in vivo safety of Compounds in rats

(1) Experimental methods

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) female 190-. The test was performed according to the dosing schedule of table 8 and clinical observations were made for all animals. And at the end of the experiment, samples of the heart, liver, spleen, lung, kidney and administration site were collected. The test results are shown in Table 8.

(2) Results of the experiment

TABLE 8 preliminary evaluation of in vivo safety in rats

The experimental result shows that the compound of the invention has good in-vivo safety to rats.

Experimental example 8: activity study of Compounds against SARS-COV-2 infection in vivo in transgenic mice

(1) Experimental protocol

Humanized angiotensin converting enzyme 2(ACE2) transgenic mice (age: 8-10 weeks) were purchased from Jiangsu Jiejieyaokang biotechnology limited (# T037659. compounds were dissolved in 5% (v/v) DMSO (Sigma-Aldrich), 3% (v/v) HS15(GLPBIO) and 92% saline. SARS-CoV-2(stain107) nasal drip infection and dosing were performed according to the test protocol of table 9. all mice were observed and their body weights were monitored daily until sacrificed. day 1(1dpi), 3(3dpi) and 5(5dpi) after viral infection, lung tissue (n ═ 3, each dpi group) was collected for viral load detection, H & E histopathological analysis, representative inflammatory cytokine and chemokine assays, and inflammatory cell (neutrophil and macrophage) counts.

TABLE 9 Activity Studies of Compounds against SARS-COV-2 infection in transgenic mice

The specific experimental scheme for detecting the lung viral load is as follows: using TRIzol^TMReagent (Invitrogen) for extraction of RNA from lung tissue and use

The Probe one-step method qRT-PCR kit (Toyobo) quantitated viral RNA and 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 threonin and eosin (H & E) and analyzed by light microscopy. Lung injury was assessed by histological features (thickening of alveolar spaces, bleeding, inflammatory cell infiltration, etc.).

Specific embodiments of lung representative inflammatory cytokine and chemokine assays are: using PrimeScript^TMRT kit (Takara) reverse transcribing RNA extracted from lung into cDNA, followed by

Premix Ex Taq^TMII (TliRNaseH plus) (Takara) and ViiA^TMGene expression was quantified. Primer sequences for quantifying inflammatory gene expression are shown in table 10.

TABLE 10 determination of primer sequences for representative inflammatory cytokines and chemokines

Specific embodiments for pulmonary determination of the number of inflammatory cells (neutrophils and macrophages) are: mouse lung tissue was fixed in 4% paraformaldehyde for at least 7 days, then paraffin embedded and cut into 4 μm sections according to standard procedures. After deparaffinization in xylene, antigen recovery and blocking, lung sections were incubated with either rat monoclonal antibody F4/80(Huabio, 1: 100) or rabbit polyclonal antibody Ly6G (Servicebio, 1: 300) overnight at 4 ℃ and then reacted with either horseradish peroxidase (HRP) -conjugated goat anti-rat secondary antibody or HRP-conjugated goat anti-rabbit secondary antibody for 1 hour at room temperature to amplify (TSA) the Cy 3-tyramine and Cy 5-tyramine according to tyramide signals and to amplify the staining signals. After staining the nuclei with DAPI, all sections were photographed using a LEICA DMI 4000B microscope (germany) and analyzed by ImageJ software (NIH us) and FlowJo software (BD us). To semi-quantitatively measure macrophage and neutrophil infiltration, 5 arbitrarily selected lung parenchymal areas in each lung section were examined by light microscopy to observe the presence of neutrophils or macrophages. The evaluation was performed in a blind manner. The cumulative score for each animal was expressed as the number of positive fields (%) per 100 fields.

The above experiment was controlled with placebo. The placebo is the same formulation as the test drug formulation, but does not contain a pharmaceutically active ingredient.

(2) Results of the experiment

The lung viral load test experiment results are shown in fig. 5, and the lung viral load of the transgenic mice infected by SARS-COV-2 can be effectively reduced by orally and intraperitoneally administering the compound 14 and the compound 15.

The lung histopathological analysis experiment result is shown in figure 6, and the pathological damage of the lung of transgenic mice infected by SARS-COV-2 can be effectively improved by orally taking and intraperitoneally taking the compound 14 and the compound 15.

The results of lung representative inflammatory cytokine and chemokine assay experiments are shown in fig. 7, and oral and intraperitoneal administration of compound 14 and compound 15 can effectively reduce the gene expression levels of lung chemotactic factor ligand 10(CXCL10) and interferon-beta (IFN- β).

Experimental results for pulmonary determination of the number of inflammatory cells (neutrophils and macrophages) As shown in FIG. 8, both oral and intraperitoneal administration of Compound 14 and Compound 15 were effective in reducing the number of Neutrophils (NEU) and Macrophages (MAC) in the lung of transgenic mice infected with SARS-COV-2.

Experimental results show that the compound can effectively resist SARS-COV-2 infection in vivo of transgenic mice.

In conclusion, the invention provides a novel coronavirus main protease inhibitor shown as a formula I, and a preparation method and application thereof. The compound shown in the formula I can effectively inhibit SARS-CoV-2M^proActive, can be used for preparing SARS-CoV-2M^proInhibitor for blocking the replication and transcription of SARS-CoV-2 virus in a patient. Application of compound of the invention in preparation of SARS-CoV-2M^proThe inhibitor, the medicine for resisting SARS-CoV-2 and the medicine for preventing and/or treating the novel coronary virus pneumonia have good application prospect.

SEQUENCE LISTING

<110> Sichuan university

<120> novel inhibitor of coronavirus main protease, preparation method and application thereof

<130> GYKH1218-2020P0112407CCZ

<150> 2020107612837

<151> 2020-07-31

<150> 2020107613026

<151> 2020-07-31

<150> 2020109063980

<151> 2020-09-01

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 40

<212> DNA

<213> Artificial sequence

<400> 1

agaacatcat ccctgcatcc cacattgggg gtaggaacac 40

<210> 2

<211> 44

<212> DNA

<213> Artificial sequence

<400> 2

aaccaagaga taagctggag tcacaacgca ctaggtttgc cgag 44

<210> 3

<211> 42

<212> DNA

<213> Artificial sequence

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tgccaccttt tgacagtgat gaatcaggac agcccaggtc aa 42

<210> 4

<211> 40

<212> DNA

<213> Artificial sequence

<400> 4

tgcaggatga tggtcaagcc ccacttgagc gaggactcag 40

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<211> 41

<212> DNA

<213> Artificial sequence

<400> 5

cagcaaggcg aaaaaggatg ccttcctgag gctggattcc g 41

<210> 6

<211> 41

<212> DNA

<213> Artificial sequence

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gtgggagatg tcctcaactg ctctctgctc ggaccaccat c 41

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 its pharmaceutically acceptable salt, or its stereoisomer, or its optical isomer, or its isotopic substitution form, wherein the compound has a structure such 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 substitution form thereof, 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 as shown in the 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. 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, or an isotopic substituted form thereof, characterized in that: The structure of the compound 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 optically active compound thereof. Isomers, or their isotopic substitution forms, are preparations of active ingredients plus pharmaceutically acceptable excipients. 7. 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, or an isotopic surrogate form thereof, inhibits the preparation of coronavirus proteolytic enzymes. Preferably, the coronavirus proteolytic enzyme is coronavirus main protease; more preferably, the coronavirus proteolytic enzyme is SARS-COV-2M ^pro . 8. 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, or an isotopic substitution form thereof, in the preparation of an anti-coronavirus drug Preferably, the coronavirus is a novel coronavirus SARS-CoV-2. 9. The compound of any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer, or an isotopic substituted form thereof, in the preparation of prophylactic and/or therapeutic and Use in medicine for SARS-COV-2M ^pro -related diseases, preferably, the SARS-COV-2M ^pro -related diseases are novel coronavirus pneumonia COVID-19. 10. purposes according to any one of claim 7～9, it is characterized in that: described coronavirus proteolytic enzyme inhibitor, the medicine of anti-coronavirus or the medicine of prevention and/or treatment of viral pneumonia can suppress SARS- The activity of COV-2M ^pro and/or the ability to inhibit SARS-COV-2 infection of cells.

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