CN116239649A

CN116239649A - Compound and composition for virus 3CL protease inhibitor and application thereof

Info

Publication number: CN116239649A
Application number: CN202211569646.2A
Authority: CN
Inventors: 杨波; 童乐仙; 董晓武; 何俏军; 袁梦; 曹戟; 车金鑫
Original assignee: Institute Of Intelligent Innovative Medicine Zhejiang University
Current assignee: Institute Of Intelligent Innovative Medicine Zhejiang University
Priority date: 2021-12-08
Filing date: 2022-12-08
Publication date: 2023-06-09

Abstract

The invention discloses a compound with a structural formula I, which has good 3CL protease inhibition activity, obvious proliferation inhibition activity on virus infected cells and potential application value in treating diseases related to virus infection. The compound has good solubility and permeability, good in vivo metabolic stability, high in vivo exposure and high bioavailability, and is a potential patent medicine compound.

Description

Compound and composition for virus 3CL protease inhibitor and application thereof

Technical Field

The invention belongs to the technical field of drug molecule research and development, and particularly relates to a compound for a virus 3CL protease inhibitor and application thereof.

Background

The subfamily of orthocoronaviruses (school name: orthoronavirinae) is known as coronaviruses (Coronavir), a class of viruses that infect mammals and birds, belonging to the order of the Neuroviridae, and is a enveloped, positive single-stranded RNA virus. Coronaviruses have long been recognized as important pathogens causing respiratory and gastrointestinal diseases in birds and mammals. Six coronaviruses have been found to infect humans prior to SARS-CoV-2: HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, severe acute respiratory syndrome coronavirus (SARS-CoV) and middle east respiratory tract virus coronavirus (MERS-CoV). The first four are the limiting endemic strains responsible for the common cold, and the second two may cause severe respiratory epidemics. SARS is an atypical viral respiratory disease, leading to 8098 cases and 774 deaths in 17 countries (9.6% mortality) during a large-scale outbreak in 2003. MERS is a human and animal co-occurrence of respiratory tract infections, initially outbreak in the middle east 2012. According to the world health organization records MERS coronaviruses have hitherto led to 2494 infections and 858 deaths. Based on the high prevalence and broad distribution of coronaviruses, as well as their genetic diversity and frequent recombination of genomes, it constitutes a constant threat to humans.

Due to the current sudden public health event caused by covd-19, the united states Food and Drug Administration (FDA) has issued Emergency Use Authority (EUA) for the experimental drug adefovir. However, the effectiveness and safety of adefovir remains controversial. Day of the dayThe present Ministry of thick raw labor also approves Redexevir as a treatment for COVID-19. In addition, interferon-alpha and anti-Human Immunodeficiency Virus (HIV) drug lopinavir/ritonavir

Has been used to treat covd-19, but the evidence of its effectiveness is still limited, and the drug may have toxic side effects. Recently, the merck company reported that the first oral medicine Molnupiravir developed by the merck company treats stage III clinical mid-term data of patients with light and medium grade COVID-19 pneumonia, the hospitalization or mortality rate of treatment groups is 7.3 percent (28/385), the hospitalization or mortality rate of control groups is 7.3 percent (28/385), the mortality rate is 14.1 percent (53/377), and the latest reported medicine effective rate is only 30 percent and is far lower than 50 percent reported in mid-term. Therefore, there is an urgent need to develop highly specific and potent anti-coronavirus drugs against key viral targets, particularly SARS-CoV-2, which is also of great importance for the prevention and treatment of recurrence of future coronavirus epidemics.

The 3CL protease (also called as main protease) plays a vital role in the life cycle of coronaviruses and is highly conserved, and inhibition of the 3CL protease can effectively block viral RNA replication and transcription and further block viral proliferation, and is one of important targets for development of antiviral drugs. The oral 3CL protease inhibitor PF-07321332 (comparative compound 1) developed by the company pyroxene is in clinical trials for the combination therapy with ritonavir with the date of the phase II/III clinical trials currently published showing that it reduces the risk of hospitalization or death by 89%, but further investigation of its effectiveness is needed with reference to the experience of Molnupiravir and in order to slow down the metabolism and breakdown of the viral small molecule inhibitor PF-07321332 in vivo, the clinical treatment requires the combination of low doses of ritonavir to exert better efficacy. Therefore, there is an urgent need to develop a novel viral 3CL protease inhibitor which has a stronger efficacy and better pharmaceutical properties and can overcome drug resistance after long-term administration, as a novel effective therapeutic means for anti-coronavirus therapy.

Disclosure of Invention

The present invention relates to pharmaceutically active compounds and pharmaceutically acceptable salts thereof, which are useful in the treatment of respiratory diseases caused by viral infections.

The present invention provides a compound having structural formula I, an isomer thereof, or a pharmaceutically acceptable salt thereof.

Wherein:

ring A is selected from

L ₁ Selected from the group consisting of

L ₂ Selected from 5, 6 and 7 membered ring lactams;

R ₁ selected from alkynyl, C _1-3 Alkyl, C _3-7 Cycloalkyl, 4-to 7-membered heterocycloalkyl, 5-to 7-membered heteroaryl, which alkynyl, alkyl, cycloalkyl, heterocycloalkyl, heteroaryl may optionally be substituted with one or more C _1-3 Alkyl, C _1-3 Haloalkyl, halogen, amino, hydroxyl, cyano, nitro, benzene ring;

R ₂ selected from hydrogen, C _1-3 An alkyl group;

R ₃ and R is ₄ Each independently selected from hydrogen, C _1-5 Alkyl, C _3-7 Cycloalkyl;

R ₂ and R is ₃ May form, together with the atoms to which they are attached, a 4-or 5-membered heterocycloalkyl, which heterocycloalkyl may be optionally substituted with one or more halogen;

R ₃ and R is ₄ Can form C together with the atoms to which they are attached _3-5 Cycloalkyl;

R ₅ 、R ₆ and R is ₇ Each independently selected from hydrogen, C _1-5 Alkyl, C _1-5 Alkoxy group,

The alkyl group, alkyl groupOxy may be optionally substituted with one or more halogens;

R ₈ and R is ₉ Each independently selected from hydrogen, C _1-3 An alkyl group;

R ₅ and R is ₇ Can form a 5-or 6-membered ring together with the atoms to which they are attached;

R ₆ and R is ₈ Can form C together with the atoms to which they are attached _3-5 Cycloalkyl;

R ₇ and R is ₈ Can form C together with the atoms to which they are attached _3-5 Cycloalkyl groups, which may optionally be substituted with one or more C' s _1-3 Alkyl, halogen substituted;

R ₈ and R is ₉ Can form a 5-or 6-membered ring together with the atoms to which they are attached;

when ring A is

R ₃ And R is ₄ Any one of which is->

And the other is hydrogen, R ₁ Not CF ₃ 。

In some embodiments of the invention, the compound has a structure represented by formula ii:

wherein:

in some embodiments of the invention, the compound:

R ₅ 、R ₆ and R is ₇ Each independently ofAt the site selected from hydrogen, C _1-5 Alkyl, C _1-5 Alkoxy group,

The alkyl, alkoxy groups may be optionally substituted with one or more halogens;

R ₈ selected from hydrogen;

R ₉ selected from hydrogen, C _1-3 An alkyl group;

in some embodiments of the invention, the compound:

R ₅ and R is ₇ Together with the atoms to which they are attached, form a 5-or 6-membered ring;

R ₆ selected from hydrogen, C _1-5 Alkyl, C _1-5 Alkoxy group,

R ₈ selected from hydrogen;

R ₉ selected from hydrogen, C _1-3 An alkyl group;

in some embodiments of the invention, the compound:

R ₅ and R is ₇ Each independently selected from hydrogen, C _1-5 Alkyl, C _1-5 Alkoxy group,

R ₆ and R is ₈ Together with the atoms to which they are attached form C _3-5 Cycloalkyl;

R ₉ selected from hydrogen, C _1-3 An alkyl group;

in some embodiments of the invention, the compound is characterized by:

R ₅ selected from hydrogen, C _1-5 Alkyl, C _1-5 Alkoxy group,

The alkyl group, alkoxy groupOptionally substituted with one or more halogens;

R ₆ selected from hydrogen;

R ₇ and R is ₈ Together with the atoms to which they are attached form C _3-5 Cycloalkyl groups, which may optionally be substituted with one or more C' s _1-3 Alkyl, halogen substituted;

R ₉ selected from hydrogen, C _1-3 An alkyl group;

in some embodiments of the invention, the compound is characterized by:

R ₈ and R is ₉ Together with the atoms to which they are attached, may form a 5-or 6-membered ring.

In some embodiments of the invention, the compound is characterized in that ring a is selected from:

in some embodiments of the invention, L in the compound ₁ Selected from the group consisting of

In some embodiments of the invention, L in the compound ₂ Selected from the group consisting of

In some embodiments of the invention, R in the compound ₁ Selected from-CF ₃ 、

In some embodiments of the invention, the compound is of the structure:

The R is ₁ Preferred are fluorocyclopropyl (monofluoro-substituted, difluoro-substituted may occur on the same carbon atom or on different carbon atoms), trifluoromethyl.

In some embodiments of the invention, the compound is:

in some embodiments of the present invention, there is provided a pharmaceutical composition comprising a compound as described in any one of the preceding claims, an isomer thereof, a pharmaceutically acceptable salt, solvate or prodrug thereof.

In some embodiments of the invention, the use of a compound, isomer, pharmaceutically acceptable salt, solvate or prodrug thereof as described in any of the above is for the preparation of an inhibitor or a medicament for the inhibition of viral 3CL protease.

In some embodiments of the invention, the use of a compound, isomer, pharmaceutically acceptable salt, solvate or prodrug thereof as claimed in any of the above, wherein the medicament is for the treatment of a disease caused by a viral infection.

In some embodiments of the invention, the use of a compound, isomer, pharmaceutically acceptable salt, solvate or prodrug thereof as claimed in any of the above, wherein the disease is selected from pneumonia or asymptomatic infection by SARS-CoV-2 coronavirus infection.

Definition and description

The following terms and phrases used herein are intended to have the following meanings unless otherwise indicated. A particular term or phrase, unless otherwise specifically defined, should not be construed as being ambiguous or otherwise clear, but rather should be construed in a generic sense.

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

The term "isomer" means that a compound of the present invention may exist in a particular geometric or stereoisomeric form. The present invention contemplates all such compounds, including cis and trans isomers, (-) -and (+) -pairs of enantiomers, (R) -and (S) -enantiomers, diastereomers, (D) -isomers, (L) -isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are included within the scope of the present invention.

Unless otherwise indicated, the term "enantiomer" or "optical isomer" refers to stereoisomers that are mirror images of each other.

Unless otherwise indicated, the term "cis-trans isomer" or "geometric isomer" is caused by the inability of a double bond or a single bond of a ring-forming carbon atom to rotate freely.

Unless otherwise indicated, the term "diastereoisomer" refers to stereoisomers of a molecule having two or more chiral centers and having a non-mirror relationship between the molecules.

Unless otherwise indicated, "(D)" or "(+)" means dextrorotation, "(L)" or "(-)" means levorotatory, "(DL)" or "(strych)" means racemization.

Unless otherwise indicated, with solid wedge bonds

And wedge-shaped dotted bond->

Representing the absolute configuration of a solid centre, using straight solid keys +.>

And straight dotted bond->

Representing the relative configuration of the stereo centers, using wavy lines +.>

Representing a wedge solid key +.>

Or wedge-shaped dotted bond->

Or by wave lines->

Representing a straight solid line key->

And straight dotted bond->

The term "cis-trans isomer" refers to a configuration in which a double bond or a single bond of a ring-forming carbon atom in a molecule cannot rotate freely.

The term "enantiomer" refers to stereoisomers that are mirror images of each other.

The term "diastereoisomer" refers to a stereoisomer of a molecule having two or more chiral centers and having a non-mirror image relationship between the molecules.

The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention prepared from the compounds of the present invention which have the specified substituents found herein with relatively non-toxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts may be obtained by contacting neutral forms of such compounds with a sufficient amount of a base in pure solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic ammonia or magnesium salts or similar salts. When the compounds of the present invention contain relatively basic functional groups, the acid addition salts may be obtained by contacting the neutral form of such compounds with a sufficient amount of an acid in pure solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include mineral acid salts. The inorganic acid salt is selected from hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, nitrate, carbonate, bicarbonate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate and pyrophosphate; the organic acid salt is selected from formate, acetate, octanoate, isobutyrate, oxalate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, succinate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, salicylate, picrate, glutamate, ascorbate, camphordate, camphorsulfonate, and the like.

"optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

The term "substituted" means that any one or more hydrogen atoms on a particular atom is substituted with a substituent, and may include deuterium and variants of hydrogen, provided that the valence of the particular atom is normal and the substituted compound is stable. When the substituent is oxygen (i.e., =0), it means that two hydrogen atoms are substituted. Oxygen substitution does not occur on the aromatic group. The term "optionally substituted" means that the substituents may or may not be substituted, and the types and numbers of substituents may be arbitrary on the basis that they can be chemically achieved unless otherwise specified.

When any variable (e.g., R) occurs more than once in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if a group is substituted with 0 to 2R, the group may optionally be substituted with up to two R's, and R's in each case have independent options. Furthermore, combinations of substituents and/or variants thereof are only permissible if such combinations result in stable compounds.

Unless otherwise specified, the term "alkyl" is used to denote a straight or branched saturated hydrocarbon group, which may be monosubstituted (e.g. -CH ₂ F) Or polysubstituted (e.g. -CF) ₃ ) May be monovalent (e.g., methyl), divalent (e.g., methylene), or multivalent (e.g., methine). Unless otherwise specified, C _1-3 Alkyl groups include C1, C2 and C3 alkyl groups; c (C) _1-5 Alkyl groups include C1, C2, C3, C4 and C5 alkyl groups. Examples of alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, s-butyl, t-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like.

Unless otherwise specified, the term "cycloalkyl" includes any stable cyclic or polycyclic hydrocarbon group, any carbon atom being saturated, either mono-or polysubstituted, and either divalent or multivalent. Unless otherwise specified, C _3-7 Cycloalkyl includes C3, C4, C5, C6 and C7 cycloalkyl; c (C) _3-5 Cycloalkyl includes C3, C4 and C5 cycloalkyl. Examples of such cycloalkyl groups include, but are not limited to, cyclopropyl, norbornyl, [2.2.2 ]]Bicyclo octane, [4.4.0 ]]Bicyclodecane, etc.

Unless otherwise specified, the term "heterocycloalkyl" means cycloalkyl containing 1 to 4 heteroatoms selected from N, O and S.

Unless otherwise specified, the term "cyclic lactam" is expressed as

In the structure shown, n is a positive integer. Unless otherwise specified, 5-, 6-and 7-membered cyclic lactams are each denoted +.>

The structure shown.

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

Unless otherwise specified, the term "alkoxy" means an alkyl group attached to the remainder of the molecule through an oxygen atom, wherein the alkyl group has the meaning as described herein. Unless otherwise specified, C _1-5 Alkoxy includes C1, C2, C3, C4 and C5 alkoxy. Examples of alkoxy groups include, but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy and S-pentoxy. The alkoxy group may be optionally substituted with one or more substituents described herein.

The term "amine group" refers to-NH 2, -NH (alkyl) or-N (alkyl), unless otherwise specified.

The term "aryl" means, unless otherwise specified, a polyunsaturated aromatic alkane monocyclic ring, which may be mono-or polysubstituted.

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

Unless otherwise specified, the term "ring" includes any chemically stable cycloalkyl, heterocycloalkyl, aryl, heteroaryl group.

The compound has good 3CL protease inhibition activity, obvious proliferation inhibition activity on virus-infected cells, and potential application value in treating diseases related to virus infection. The compound has good solubility and permeability, good in vivo metabolic stability, high in vivo exposure and high bioavailability, and is a potential patent medicine compound.

Detailed Description

The present invention is described in detail below by way of examples, but is not meant to be limiting in any way. The present invention has been described in detail herein, and specific embodiments thereof are also disclosed, it will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the invention without departing from the spirit and scope of the invention.

EXAMPLE 1 (2S, 4R) -4- (tert-butoxy) -N- ((S) -1-cyano-2- ((S) -2-oxopyrrolidin-3-yl) ethyl) -1- ((S) -3, 3-dimethyl-2- (2, 2-trifluoroacetamido) butanoyl) pyrrolidine-2-carboxamide (A1)

Step 1: (2S, 4S) -2- ((t-Butoxycarbonyl) amino) -4- (cyanomethyl) glutarate dimethyl ester (Compound 1-3)

Dimethyl (t-Butoxycarbonyl) -L-glutamic acid (1-2) (6 g,21.8 mmol) was dissolved in 60mL anhydrous tetrahydrofuran, and Lithium Hexamethyldisilazide (LHDMS) was slowly added dropwise at-78deg.C under nitrogen protectionFuran solution (47 mL,1 mol/L) was stirred at-78deg.C for 1 hour after addition. 2-bromoacetonitrile (1.62 mL,23.3 mmol) was then slowly added dropwise, and the reaction was continued at-78deg.C for 1-2 hours. After the reaction was completed, the reaction was quenched by sequentially dropping pre-chilled methanol (3 mL) and pre-chilled tetrahydrofuran acetate solution (3 mL of acetic acid in 20mL of tetrahydrofuran), stirred for 30 minutes, and then transferred to room temperature and stirred. After the system was returned to room temperature, the solvent was distilled off under reduced pressure, and the residue was dissolved in ethyl acetate, washed with saturated brine to give an organic phase with anhydrous Na ₂ SO ₄ Drying, concentrating, purifying the residue by column chromatography to obtain 6g of light yellow oily substance with 88% yield, ¹ H NMR(400MHz,CDCl3)δ5.05-5.17(1H),4.31-4.44(1H),3.76-3.76(3H),3.75-3.75(3H),2.81-2.90(1H),2.76-2.81(1H),2.10-2.22(2H),1.43-1.45(9H)；ESI-MS:m/z＝315[M+1] ⁺ 。

step 2: (S) -2- (Boc-amino) -3- [ (S) -2-oxo-3-pyrrolidinyl ] propanoic acid methyl ester (intermediate 1-4)

Intermediate 1-3 (6 g,19 mmol) was dissolved in 90mL of methanol and CHCl was added ₃ (8 mL) and PtO ₂ (345 mg,1.52 mmol) was stirred under hydrogen atmosphere for 24 hours, the platinum dioxide solid was filtered off, acona (3.1 g,38 mmol) was added and the mixture was heated under reflux for 6 hours. After the completion of the reaction, the solvent was distilled off under reduced pressure, and the obtained residue was extracted with ethyl acetate and saturated brine to obtain anhydrous Na for an organic phase ₂ SO ₄ Drying, concentrating, and purifying the residue by column chromatography to give 2.5g of compound 1-4 in 46% yield, ESI-MS: m/z=287 [ M+1 ]] ⁺ 。

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

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

Step 4: (S) -1-cyano-2- ((S) -2-oxopyrrolidin-3-yl) ethyl) carbamic acid tert-butyl ester (intermediate 1-6)

Compound 1-5 (2.36 g,8.71 mmol) was dissolved in 20mL of dichloromethane and Burgess reagent was added(4.16 g,17.42 mmol) was stirred for 6 hours. Extraction with dichloromethane and saturated brine gave an organic phase which was then dried over anhydrous Na ₂ SO ₄ Drying and concentrating under reduced pressure. The residue was purified by column chromatography to give 2.5g of compounds 1-4 in 46% yield, ESI-MS: m/z=254 [ M+1 ]] ⁺ 。

Step 5: (S) -3, 3-dimethyl-2- (2, 2-trifluoroacetamido) butanoic acid (intermediate 1-9)

(S) -2-amino-3, 3-dimethylbutyric acid (1-8) (1.3 g,1 mmol) was dissolved in 20mL of methanol, cooled to 0℃and 3mL of triethylamine was added dropwise. After the reaction system was slowly warmed to room temperature, 1.7g of ethyl trifluoroacetate (1-7) was added thereto and stirred overnight. After the completion of the reaction, the reaction mixture was concentrated under reduced pressure, and the residue was purified by column chromatography to give 1.9g of Compound 1-9 in 86% yield. ¹ H NMR(400MHz,CDCl ₃ )δ7.32-7.42(1H),4.13-4.20(1H),0.99-1.01(9H)，ESI-MS:m/z＝228[M+1] ⁺ 。

Step 6: (S) -2-amino-3- ((S) -2-oxo-3-pyrrolidinyl) methylaminopropionitrile (intermediate 1-10)

Intermediate 1-6 (506 mg,2 mmol) was dissolved in 20mL dichloromethane, 5mL trifluoroacetic acid was slowly added dropwise under ice-bath conditions, and after addition, stirring at room temperature for 30 min, the crude compound 1-10 was obtained by concentrating under reduced pressure and used in the next step without further purification.

Step 7: tert-butyl (2S, 4R) -4- (tert-butoxy) -2- (((S) -1 cyano-2- ((S) -2-oxo-3-pyrrolidinyl) ethyl) carbamoyl) pyrrolidine-1 carboxylic acid ester (intermediate 1-12)

(2S, 3S) -4- (tert-Butoxy) -1- (tert-Butoxycarbonyl) pyrrolidine-2-carboxylic acid (1-11) (574 mg,2 mmol), 1-hydroxybenzotriazole (HOBt) (294 mg,2.2 mmol) and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCl) (420 mg,2.2 mmol) were dissolved in 30mL of dichloromethane, stirred for 10min under ice bath conditions, 0.98mL of diisopropylethylamine was added, stirring under ice bath was continued for 15min, and after slowly adding a dichloromethane solution in which intermediate 1-10 was dissolved, stirring overnight at room temperature. After completion of the reaction, the mixture was extracted with dichloromethane and saturated brine, and the organic phases were combined with anhydrous Na ₂ SO ₄ Drying, concentrating under reduced pressure, and separating the residue by column chromatography 350mg of intermediate 1-12 was obtained in 41% yield in ESI-MS: m/z=423 [ M+1 ]] ⁺ 。

Step 8: intermediates 1 to 13

Synthetic procedure referring to example 1, step 6, compounds 1-6 were replaced with compounds 1-12 to give compounds 1-13. Concentrating under reduced pressure, and taking into the next step without further purification.

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

Synthetic procedure referring to example 1, step 7, compounds 1-11 were replaced with 1-9, compounds 1-10 were replaced with 1-13 to give compound A1, ¹ h NMR (400 MHz, chloroform-d) delta 8.32 (d, J=6.6 Hz, 1H), 7.15 (d, J=8.8 Hz, 1H), 5.92 (s, 1H), 4.86-4.77 (m, 1H), 4.60-4.55 (m, 1H), 4.51-4.46 (m, 1H), 4.42-4.36 (m, 1H), 3.92-3.86 (m, 1H), 3.44-3.39 (m, 1H), 3.37-3.31 (m, 2H), 2.56-2.46 (m, 1H), 2.43-2.35 (m, 1H), 2.34-2.27 (m, 1H), 2.03-1.93 (m, 3H), 1.18 (s, 9H), 1.02 (s, 9H) [ yield ] 42%, ESI-3.39 (m/z=m+1 ]] ⁺ 。

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

Synthetic procedure referring to example 1, steps 7 to 9, (2 s,4 r) -1- (tert-butoxycarbonyl) -4- (4-fluorobenzyl) pyrrolidine-2-carboxylic acid 2-1 was substituted for 1-11 to give intermediate 2-2; replacing 1-12 with the intermediate 2-2 to obtain an intermediate 2-3; the intermediate 2-3 is substituted for 1-13 to obtain a compound A2, ¹ H NMR(400MHz,CDCl ₃ )δ9.29(d,J＝4.5Hz,1H),7.29–7.25(m,2H),7.24(d,J＝2.0Hz,1H),6.96(ddt,J＝8.5,7.4,1.1Hz,1H),6.72–6.68(m,2H),5.65(s,1H),4.85(tt,J＝4.6,1.2Hz,1H),4.80(dd,J＝9.3,1.1Hz,1H),4.60(d,J＝8.7Hz,1H),4.45(dt,J＝11.4,4.8Hz,1H),4.00–3.88(m,2H),3.18–3.10(m,1H),2.91–2.83(m,1H),2.82–2.77(m,1H),2.30–2.18(m,3H),2.16–2.07(m,1H),2.01–1.98(m,1H),1.04(s,9H).ESI-MS:m/z＝552[M+1] ⁺ 。

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

Synthesis procedure referring to example 1, steps 7 to 9, (S) -5- (t-butoxycarbonyl) -5-azaspiro [2.4]1-11 is replaced by heptane-6-carboxylic acid 3-1 to obtain an intermediate 3-2; replacing 1-12 with the intermediate 3-2 to obtain an intermediate 3-3; the intermediate 3-3 is substituted for 1-13 to obtain a compound A3, ¹ H NMR(400MHz,CDCl ₃ )δ8.33(d,J＝7.1Hz,1H),7.11(d,J＝9.3Hz,1H),6.10(s,1H),4.92–4.80(m,1H),4.56–4.48(m,2H),3.74(d,J＝9.6Hz,1H),3.54–3.49(m,1H),3.36–3.31(m,2H),2.67–2.58(m,1H),2.42–2.33(m,2H),2.24–2.16(m,2H),2.02–1.96(m,2H),1.03(s,9H),0.73–0.68(m,2H),0.63–0.57(m,2H).ESI-MS:m/z＝486[M+1] ⁺ 。

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

Synthetic procedure referring to example 1, steps 7 to 9, (3S) -2- (t-butoxycarbonyl) -2-azabicyclo [2.2.1]1-11 is replaced by heptane-3-carboxylic acid 4-1 to obtain intermediate 4-2; replacing 1-12 with the intermediate 4-2 to obtain an intermediate 4-3; the intermediate 4-3 is substituted for 1-13 to obtain a compound A4, ¹ H NMR(400MHz,CDCl ₃ )δ8.38(d,J＝6.8Hz,1H),7.87(d,J＝9.4Hz,1H),6.26(s,1H),4.75–4.67(m,1H),4.61(d,J＝9.4Hz,1H),4.50(s,1H),3.89(s,1H),3.36–3.31(m,2H),2.81–2.78(m,1H),2.47–2.31(m,3H),2.27–2.19(m,2H),2.19–2.15(m,3H),2.02–1.92(m,3H),1.03(s,9H).ESI-MS:m/z＝486[M+1] ⁺ 。

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

Synthetic procedure referring to example 1, steps 7 to 9, (2 s,4 r) -N-Boc-4-trifluoromethylproline 5-1 was substituted for 1-11 to afford intermediate 5-2; replacing 1-12 with the intermediate 5-2 to obtain an intermediate 5-3; intermediate 5-3 was substituted for 1-13 to give compound A5, ESI-MS: m/z=528 [ m+1 ]] ⁺ 。

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

Synthetic procedure referring to example 1, steps 7 to 9, (3S) -2- (t-butoxycarbonyl) -2-azabicyclo [2.1.1]Hexane-3-carboxylic acid 6-1 replaces 1-11 to obtain intermediate 6-2; replacing 1-12 with the intermediate 6-2 to obtain an intermediate 6-3; intermediate 6-3 was substituted for 1-13 to give compound A6, ESI-MS: m/z=472 [ m+1 ]] ⁺ 。

Example 7: (2S) -6, 6-dichloro-N- ((S) -1-cyano-2- ((S) -2-oxopyrrolidin-3-yl) ethyl) -3- ((S) -3, 3-dimethyl-2- (2, 2-trifluoroacetamido) butanoyl) -3-azabicyclo [3.1.0]Hexane-2-carboxamide

Synthesis procedure referring to example 1 Steps 7 to 9, (1S, 2S, 5R) -3- (tert-butoxycarbonyl) -6, 6-dichloro-3-azabicyclo [3.1.0 ]Hexane-2-carboxylic acid 7-1 replaces 1-11 to give intermediate 7-2; replacing 1-12 with the intermediate 7-2 to obtain an intermediate 7-3; intermediate 7-3 was substituted for 1-13 to give compound A7, ESI-MS: m/z=540 [ m+1 ]] ⁺ 。

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

Synthesis procedure referring to example 1, steps 7 to 9, (1S, 3AR,6 AS) -2- (tert-butoxycarbonyl) octahydrocyclopenta [ C]Pyrrole-1-carboxylic acid 8-1 replaces 1-11 to obtain intermediate 8-2; replacing 1-12 with the intermediate 8-2 to obtain an intermediate 8-3; intermediate 8-3 was substituted for 1-13 to give compound A8, ESI-MS: m/z=500 [ m+1 ]] ⁺ 。

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

Synthetic procedure referring to example 1, steps 7 to 9, (S) -1-tert-butoxycarbonyl-4, 4-dimethylpyrrolidine-2-carboxylic acid 9-1 was substituted for 1-11 to give intermediate 9-2; replacing 1-12 with the intermediate 9-2 to obtain an intermediate 9-3; intermediate 9-3 was substituted for 1-13 to give compound A9, ESI-MS: m/z=488 [ m+1 ]] ⁺ 。

Example 10: (2S) -4- (bicyclo [1.1.1] pentan-1-yl) -N- ((S) -1-cyano-2- ((S) -2-oxopyrrolidin-3-yl) ethyl) -1- ((S) -3, 3-dimethyl-2- (2, 2-trifluoroacetamido) butanoyl) pyrrole-2-carboxamide (A10)

Synthetic procedure (2S) -4- (bicyclo [ 1.1.1) with reference to example 1, steps 7 through 9]Pentane-1-yl) -1- (tert-butoxycarbonyl) pyrrole-2-carboxylic acid 10-1 replaces 1-11 to give intermediate 10-2; replacing 1-12 with the intermediate 10-2 to obtain an intermediate 10-3; intermediate 10-3 was substituted for 1-13 to give compound a10, ESI-MS: m/z=526 [ m ]+1] ⁺ 。

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

Step 1: tert-butyl ((S) -1- ((S) -2- (((S) -1-cyano-2- ((S) -2-oxopyrrolidin-3-yl) ethyl) pyrrol-1-yl) -3, 3-dimethyl-1-acetoacetyl-2-yl) carbamate (intermediate 11-5)

Synthetic procedure referring to example 1, steps 7 to 9, (t-butoxycarbonyl) -L-proline 11-1 was substituted for 1-11 to give intermediate 11-2; replacing 1-12 with the intermediate 11-2 to obtain an intermediate 11-3; intermediate 11-5, ESI-MS: m/z=464 [ M+1] +, was obtained by substituting intermediate 11-3 for 1-13 and N-Boc-L-tert-leucine for 1-9.

Step 2: tert-butyl ((S) -1- ((S) -2- (((S) -1-cyano-2- ((S) -2-oxopyrrolidin-3-yl) ethyl) pyrrol-1-yl) -3, 3-dimethyl-1-acetoacetyl-2-yl) carbamate (a 11)

Synthetic procedure referring to example 1, steps 8 to 9, 11-5 was substituted for 1-12 to afford intermediate 11-6; intermediate 11-6 was substituted for 1-13 and spiro [3.3] ]Heptane-2-carboxylic acid instead of 1-9 gave compound A11, ESI-MS: m/z=486 [ M+1 ]] ⁺ 。

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

Step 1: intermediate 12-2

Reference example 1 step 7, substituting intermediate 11-6 for 1-10, using (S) -5- (tert-butoxycarbonyl) -5-azaspiro [2.4]]Heptane-6-carboxylic acid (intermediate 12-1) instead of 1-11, intermediate 12-2, ESI-MS: m/z=587 [ M+1 ]] ⁺ 。

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

To the ethyl acetate solution of compound 12-2 was slowly added dropwise an HCl saturated ethyl acetate solution, and the mixture was stirred at room temperature for 30 minutes, followed by concentration under reduced pressure to obtain a white powder. To give the target compound A12, ESI-MS: m/z=486 [ M+1 ]] ⁺ 。

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

Step 1: (S) -2-amino-3- (1H-imidazol-4-yl) propionitrile (intermediate 13-2)

Synthetic procedure referring to example 1, step 7, compound 13-1 was substituted for compound 1-6 to afford intermediate 13-2. Step 2: (1R, 2S, 5S) -N- (1-cyano-2- (1H-imidazol-4-yl) ethyl) -3- ((S) -3, 3-dimethyl-2- (2, 2) -trifluoroacetylamino) butanoyl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide (A13)

Synthetic procedure referring to examples 1, steps 7 to 9, (t-butoxycarbonyl) -L-proline 11-1 was substituted for 1-11, intermediate 13-2 was substituted for intermediate 1-10 to give intermediate 13-3; replacing 1-12 with the intermediate 13-3 to obtain an intermediate 13-4; intermediate 13-4 was substituted for 1-13 to give compound a13, ESI-MS: m/z=443 [ m+1 ]] ⁺ 。

Example 14: (S) -N- ((S) -1-cyano-2- ((S) -2-oxopyrrolidin-3-yl) ethyl) -1- ((S) -3, 3-dimethyl-2- (spiro [3.3] heptane-2-carboxamide) butyryl) -5-azaspiro [2.4] heptane-6-carboxamide (A14)

Synthetic procedure referring to example 1, steps 7 to 9, intermediate 11-4 was substituted for 1-11 to afford intermediate 14-1; replacing 1-12 with the intermediate 14-1 to obtain an intermediate 14-2; intermediate 14-2 was substituted for 1-13 and spiro [3.3]]Heptane-2-carboxylic acid instead of 1-9 gave Compound A14, ESI-MS: m/z=512 [ M+1 ] ] ⁺ 。

Example 15: (S) -N- ((S) -1-cyano-2- ((S) -2-oxopyrrolidin-3-yl) ethyl) -5- (1- (2, 2-trifluoroacetamide) cyclopropyl-1-carbonyl) -5-azaspiro [2.4] heptane-6-carboxamide (A15)

Step 1:1- (2, 2-trifluoroacetamide) cyclopropyl-1-carboxylic acid (intermediate 15-2)

Synthesis procedure referring to example 1, step 5, compound 15-1 was substituted for Compound 1-6 to give intermediate 15-2, ESI-MS: m/z=198 [ M+1 ]] ⁺ 。

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

Synthetic procedure referring to example 1, steps 7-9, intermediate 15-3 was substituted for 1-11 to afford intermediate 15-4; replacing 1-12 with intermediate 15-4 to obtain intermediate 15-5; the intermediate 15-5 was substituted for 1-13 and 15-2 was substituted for 1-9 to give Compound A15, ESI-MS: m/z=470 [ M+1 ]] ⁺ 。

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

Step 1:1- (2, 2-trifluoroacetamide) cyclopropyl-1-carboxylic acid (intermediate 16-2)

Synthesis procedure referring to example 1, step 5, compound 16-1 was substituted for Compound 1-6 to give intermediate 16-2, ESI-MS: m/z=258 [ M+1 ] ] ⁺ 。

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

Synthesis procedure referring to example 1, step 7, substituting compound 15-5 for 1-11 and intermediate 16-2 for intermediate 1-10, gave compound A16, ESI-MS: m/z=502 [ M+1 ]] ⁺ 。

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

Synthesis procedure referring to example 1, step 7, substituting compound 3-3 for 1-11 and intermediate 16-2 for intermediate 1-10, gave compound A17, ESI-MS: m/z=488 [ M+1 ]] ⁺ 。

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

Step 1: synthesis of intermediate 18-2

Synthesis procedure referring to example 1, step 5, compound 18-1 was substituted for Compound 1-6 to give intermediate 18-2, ESI-MS: m/z=226 [ M+1 ]] ⁺ 。

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

Synthesis procedure referring to example 1, step 7, substituting compound 15-5 for 1-11 and intermediate 18-2 for intermediate 1-10, gave compound A18, ESI-MS: m/z=470 [ M ]1] ⁺ 。

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

Synthesis procedure referring to example 1, step 7, substituting compound 3-3 for 1-11 and intermediate 18-2 for intermediate 1-10, gave compound A19, ESI-MS: m/z=456 [ M+1 ]] ⁺ 。

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

Synthetic procedure referring to example 1, steps 7 to 9, intermediate 11-4 was substituted for 1-11 to afford intermediate 20-1; replacing 1-12 with the intermediate 20-1 to obtain an intermediate 20-2; intermediate 20-2 was substituted for 1-13 and compound 20-3 was substituted for 1-9 to give compound a20, ESI-MS: m/z=508 [ m+1 ]] ⁺ 。

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

Synthetic procedure referring to example 1, step 7, intermediate 20-3 was substituted for 1-11, intermediate 14-2 was substituted for 1-10 to give compound a21, ¹ H NMR(400MHz,Chloroform-d)δ8.18(d,J＝7.6Hz,1H),6.77(d,J＝9.2Hz,1H),6.36(s,1H),5.00–4.85(m,1H),4.60–4.55(m,2H),3.72–3.64(m,2H),3.38–3.31(m,2H),2.66–2.60(m,1H),2.43–2.33(m,4H),2.21–2.15(m,1H),2.09–1.99(m,2H),1.96–1.91(m,1H),1.65–1.59(m,1H),0.99(s,9H),0.71–0.66(m,2H),0.64–0.61(m,2H).ESI-MS:m/z＝494[M+1] ⁺ 。

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

Synthesis procedure reference example 16, substituting compound 22-2 for compound 16-1, gave compound A22, ESI-MS: m/z=500 [ M+1] ⁺ 。

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

Synthesis procedure referring to example 1, step 7, intermediate 22-3 was substituted for 1-11 and intermediate 3-3 was substituted for 1-10 to give Compound A23, ESI-MS: m/z=486 [ M+1 ]] ⁺ 。

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

Step 1. Intermediate 24-3

Synthesis procedure referring to example 1, steps 3-4, 24-1 was substituted for 1-4 to give intermediate 24-3, ESI-MS: m/z=196 [ M+1 ]] ⁺ 。

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

Synthetic procedure referring to example 1, steps 7 to 9, intermediate 24-3 was substituted for 1-11 to afford intermediate 24-4; replacing 1-12 with the intermediate 24-4 to obtain an intermediate 24-5; intermediate 24-5 was substituted for 1-13 and compound 22-2 was substituted for 1-9 to give compound a24, ESI-MS: m/z=514 [ m+1 ]] ⁺ 。

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

Synthetic procedure referring to example 1, steps 7 to 9, intermediate 24-3 was substituted for 1-11 to afford intermediate 25-1; replacing 1-12 with the intermediate 25-1 to obtain an intermediate 25-2; intermediate 25-2 was substituted for 1-13 and compound 22-2 was substituted for 1-9 to give compound a24, ESI-MS: m/z=500 [ m+1 ]] ⁺ 。

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

Synthesis procedure referring to example 1, step 7, intermediate 14-2 was substituted for 1-11, 1-fluorocyclopropane carboxylic acid and 1-10 was substituted to give Compound A26, ESI-MS: m/z=490 [ M+1 ]] ⁺ 。

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

Synthetic procedure referring to example 1, step 7, intermediate 20-2 was substituted for 1-11, 1-fluorocyclopropane carboxylic acid was substituted for 1-10 to give compound a27, ¹ H NMR(400MHz,Chloroform-d)δ8.24(d,J＝6.9Hz,1H),7.03(dd,J＝9.3,3.7Hz,1H),5.92(s,1H),4.94–4.85(m,1H),4.62–4.51(m,2H),3.70–3.61(m,2H),3.38–3.34(m,2H),2.70–2.58(m,1H),2.48–2.30(m,2H),2.20(dd,J＝12.6,6.2Hz,1H),2.02–1.82(m,3H),1.35–1.29(m,4H),1.04(s,9H),0.69–0.54(m,4H).ESI-MS:m/z＝476[M+1] ⁺ 。

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

Synthesis procedure referring to example 1, step 7, intermediate 14-2 was substituted for 1-11, 1-fluorocyclopropane carboxylic acid and 1-10 was substituted to give compound A28, ESI-MS: m/z=512 [ M+1 ]] ⁺ 。

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

Synthetic procedure referring to example 1, step 7, intermediate 20-2 was substituted for 1-11, 1-fluorocyclopropane carboxylic acid was substituted for 1-10 to give compound a29, ¹ H NMR(400MHz,Chloroform-d)δ8.22(d,J＝7.3Hz,1H),7.79(d,J＝17.1Hz,2H),6.52(d,J＝9.6Hz,1H),5.97(s,1H),4.91(ddd,J＝10.7,7.2,5.4Hz,1H),4.72(d,J＝9.5Hz,1H),4.51(dd,J＝7.9,6.4Hz,1H),3.89(s,3H),3.70(s,2H),3.37–3.27(m,2H),2.68–2.55(m,1H),2.49–2.38(m,1H),2.37–2.28(m,1H),2.21–2.14(m,1H),1.99–1.83(m,3H),1.04(s,9H),0.89–0.80(m,2H),0.68–0.62(m,2H).ESI-MS:m/z＝498[M+1] ⁺ 。

example 30: (1R, 2S, 5S) -N- ((S) -1-cyano-2- ((S) -2-oxopyrrolidin-3-yl) ethyl) -3- ((S) -3, 3-dimethyl-2- (6-methylpyrazine-2-amide) butyryl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide (A30)

Synthesis procedure referring to example 1, step 7, intermediate 14-2 was substituted for 1-11, 1-fluorocyclopropane carboxylic acid and 1-10 was substituted to give Compound A30, ESI-MS: m/z=524 [ M+1 ] ] ⁺ 。

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

Synthesis procedure referring to example 1, step 7, intermediate 20-2 was substituted for 1-11, 6-methylpyrazine-2-carboxylic acid 32-1 and 1-10 to give Compound A31, ESI-MS: m/z=510 [ M+1 ]] ⁺ 。

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

Step 1:4- ((Boc) amino) -3-chlorobenzoic acid (32-2)

4-amino-3-chlorobenzoic acid 32-1 (348 mg,2 mmol) was dissolved in methylene chloride, and sodium hydrogen carbonate (504 mg,6 mmol) and di-tert-butyl dicarbonate (504 mg,3 mmol) were added under ice-bath and stirred overnight. Washing with dilute hydrochloric acid and saturated salt water after the reaction, concentrating the organic layer under reduced pressureThus, compound 32-2, ESI-MS: m/z=272 [ M+1 ]] ⁺ 。

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

Synthesis procedure referring to example 1, step 7, compound 32-2 obtained in the previous step was substituted for 1-11, intermediate 13-2 was substituted for intermediate 1-10 to give compound A32, ESI-MS: m/z=657 [ M+1 ]] ⁺ 。

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

Synthesis procedure referring to example 12, step 2, compound 32-3 obtained in the previous step was substituted for 12-2 to give Compound A32, ESI-MS: m/z=557 [ M+1 ]] ⁺ 。

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

Step 1:4- ((Boc) amino) -3-chlorobenzoic acid (32-2)

Synthesis procedure reference example 32, step 1

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

Synthesis procedure referring to example 1, step 7, compound 32-2 obtained in the previous step was substituted for 1-11, intermediate 20-2 was substituted for intermediate 1-10, and Compound A, ESI-MS: m/z=643 [ M+1 ] ] ⁺ 。

Synthesis procedure referring to example 12, step 2, compound 33-1 obtained in the previous step was substituted for 12-2 to give compound A33, ESI-MS: m/z=543 [ M+1 ]] ⁺ 。

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

Synthesis procedure referring to example 1, step 7, intermediate 14-2 was substituted for 1-11,2,5-dichlorobenzoic acid 34-1 and 1-10 to give Compound A34, ESI-MS: m/z=576 [ M+1 ]] ⁺ 。

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

Synthesis procedure referring to example 1, step 7, 2, 5-dichlorobenzoic acid 35-1 was substituted for 1-11 and intermediate 20-1 was substituted for intermediate 1-10 to give Compound A35, ESI-MS: m/z=562 [ M+1 ]] ⁺ 。

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

Synthesis procedure with reference to example 1, step 7, 2-morpholinoacetic acid 36-1 was substituted for 1-11, intermediate 14-2 was substituted for intermediate 1-10 to give Compound A36，ESI-MS:m/z＝531[M+1] ⁺ 。

Example 37: ((S) -N- ((S) -1-cyano-2- ((S) -2-oxopyrrolidin-3-yl) ethyl) -5- ((S) -3, 3-dimethyl-2- (2-morpholinoacetamido) butanoyl) -5-azaspiro [2.4] heptane-6-carboxamide (A37)

Synthesis procedure referring to example 1, step 7, 2-morpholinoacetic acid 36-1 was substituted for 1-11, intermediate 20-1 was substituted for intermediate 1-10 to give Compound A37, ESI-MS: m/z=517 [ M+1 ]] ⁺ 。

Example 38: n- ((S) -1- ((1R, 2S, 5S) -2- ((S) -1-cyano-2- ((S) -2-oxopyrrolidin-3-yl) ethyl) carbamoyl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-3-yl) -3, 3-dimethyl-1-oxobutan-2-yl) -2-methylthiazole-5-carboxamide (A38)

Synthetic procedure referring to example 1, step 7, substituting 2-methylthiazole-5-carboxylic acid 38-1 for 1-11 and intermediate 14-2 for intermediate 1-10, gave compound a38, ESI-MS: m/z=529 [ m+1 ]] ⁺ 。

Example 39: n- ((S) -1- ((S) -6- ((S) -1-cyano-2- ((S) -2-oxopyrrolidin-3-yl) ethyl) carbamoyl) -5-azaspiro [2.4] heptane-5-yl) -3, 3-dimethyl-1-oxobutan-2-yl) -2-methylthiazole-5-carboxamide (A39)

Synthesis procedure referring to example 1, step 7, 2-methylthiazole-5-carboxylic acid 38-1 was substituted for 1-11 and intermediate 20-1 was substituted for intermediate 1-10 to give Compound A39, ESI-MS: m/z=515 [ M+1 ] ] ⁺ 。

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

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

Compound 14-2 (403 mg,1 mmol) was dissolved in 20mL of dry dichloromethane, 0.43mL of triethylamine was added dropwise at 0℃and after 20min of reaction, trifluoromethanesulfonyl chloride (169 mg,1 mmol) was added and stirred overnight. After completion of the reaction, the mixture was extracted with dichloromethane and saturated brine, and the organic phases were combined with anhydrous Na ₂ SO ₄ Drying, concentrating under reduced pressure, and separating the residue by column chromatography to give 250mg of compound A40 in 47% yield, ESI-MS: m/z=536 [ M+1 ]] ⁺ 。

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

Step 1: (S) -N- ((S) -1-cyano-2- ((S) -2-oxopyrrolidin-3-yl) ethyl) -5- ((S) -3, 3-dimethyl-2- ((trifluoromethyl) sulfonamide) butanoyl) -5-azaspiro [2.4 ]Step for the synthesis of heptane-6-carboxamide (A41) referring to example 40, step 1, intermediate 20-1 was substituted for intermediate 14-2 to give compound A41, ¹ HNMR(400MHz,Chloroform-d)δ8.25(d,J＝8.0Hz,1H),7.48(d,J＝13.9Hz,1H),6.42(s,1H),4.97(ddd,J＝10.7,8.0,5.2Hz,1H),4.51(t,J＝7.8Hz,1H),3.94(d,J＝9.8Hz,1H),3.74(d,J＝9.6Hz,1H),3.30(dd,J＝9.9,5.7Hz,3H),2.72–2.61(m,1H),2.47–2.31(m,2H),2.26–2.17(m,1H),1.98–1.88(m,3H),1.04(s,9H),0.74–0.66(m,2H),0.64–0.53(m,2H).ESI-MS:m/z＝522[M+1] ⁺ 。

example 42: (1R, 2S, 5S) -N- ((S) -1-cyano-2- ((S) -2-oxopyrrolidin-3-yl) ethyl) -3- ((S) -2- (cyclopropanesulphonamide) -3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide (A42)

Synthesis procedure referring to example 40, step 1, cyclopropanesulfonyl chloride 42-1 was substituted for 40-1 to give Compound A42, ESI-MS: m/z=508 [ M+1]] ⁺ 。

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

Synthesis procedure referring to example 40, step 1, cyclopropanesulfonyl chloride 42-1 was substituted for 40-1 and intermediate 20-1 was substituted for intermediate 14-2 to give Compound A43, ESI-MS: m/z=494 [ M+1]] ⁺ 。

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

Synthetic procedure referring to example 40, step 1, p-toluenesulfonyl chloride 44-1 was substituted for 40-1 to give compound a44, ESI-MS: m/z=558 [ m+1] +.

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

Synthesis procedure referring to example 40, step 1, p-toluenesulfonyl chloride 44-1 was substituted for 40-1, intermediate 20-1 was substituted for intermediate 14-2 to give Compound A45, ESI-MS: m/z=544 [ M+1 ]] ⁺

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

Synthesis procedure referring to example 1, step 7, 6-phenylpicolinic acid 46-1 was substituted for 1-11 and intermediate 14-2 was substituted for intermediate 1-10 to give Compound A46, ESI-MS: m/z=585 [ M+1 ]] ⁺ 。

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

Synthesis procedure referring to example 1, step 7, 6-phenylpicolinic acid 46-1 was substituted for 1-11 and intermediate 20-1 was substituted for intermediate 1-10 to give Compound A47, ESI-MS: m/z=571 [ M+1 ]] ⁺ 。

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

Synthesis procedure referring to example 1, step 7, 2-cyanoacetic acid 48-1 was substituted for 1-11, intermediate 14-2 was substituted for intermediate 1-10 to give Compound A48, ESI-MS: m/z=471 [ M+1]] ⁺ 。

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

Synthesis procedure referring to example 1, step 7, 2-cyanoacetic acid 48-1 was substituted for 1-11, intermediate 20-1 was substituted for intermediate 1-10 to give Compound A49, ESI-MS: m/z=457 [ M+1]] ⁺ 。

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

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

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

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

Synthesis procedure referring to example 40, step 1, 50-1 was substituted for 14-2 and cyclopropanesulfonyl chloride 42-1 was substituted for 40-1 to give Compound A50, ESI-MS: m/z=522 [ M+1 ]] ⁺ 。

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

Synthesis procedure referring to example 50, steps 1-3, compound 20-1 was substituted for 14-2 to give Compound A51, ESI-MS: m/z=508 [ M+1 ]] ⁺ 。

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

Synthetic procedure referring to example 40 step 1, 5- (trifluoromethyl) pyridine-2-sulfonyl chloride 52-1 was substituted for 40-1 to give compound a52, ESI-MS: m/z=613 [ m+1 ]] ⁺ 。

Example 53 (S) -N- ((S) -1-cyano-2- ((S) -2-oxopyrrolidin-3-yl) ethyl) -5- ((S) -3, 3-dimethyl-2- ((5- (trifluoromethyl) pyridine) -2-sulfanyl) butanoyl) -5-azaspiro [2.4] heptane-6-carboxamide (A53)

Synthesis procedure referring to example 40, step 1, substituting compound 20-1 for 14-2 and 5- (trifluoromethyl) pyridine-2-sulfonyl chloride 52-1 for 40-1 gave compound A53, ESI-MS: m/z=599 [ M+1 ]] ⁺ 。

Example 54 (1R, 2S, 5S) -3- ((S) -2- (but-2-alkynylamido) -3, 3-dimethylbutyryl) -N- ((S) -1-cyano-2- ((S) -2-oxopyrrolidin-3-yl) ethyl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide (A54)

Synthesis procedure referring to example 40, step 1, 2-butynoic acid 54-1 was substituted for 40-1 to give Compound A54, ESI-MS: m/z=470 [ M+1 ]] ⁺ 。

Example 55 (S) -5- ((S) -2- (but-2-alkynylamido) -3, 3-dimethylbutyryl) -N- ((S) -1-cyano-2- ((S) -2-oxopyrrolidin-3-yl) ethyl) -5-azaspiro [2.4] heptane-6-carboxamide (A55)

Synthesis procedure referring to example 1, step 7, intermediate 20-1 was substituted for 14-2 and 2-butynoic acid 54-1 was substituted for 1-11 to give Compound A55, ESI-MS: m/z=456 [ M+1 ]] ⁺ 。

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

Synthesis procedure referring to example 1, step 7, 4- (trifluoromethyl) picolinic acid 56-1 was substituted for 1-11, intermediate 14-2 was substituted for intermediate 1-10 to give Compound A56, ESI-MS: m/z=577 [ M+1 ] ] ⁺ 。

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

Synthetic procedure referring to example 1, step 7, 4- (trifluoromethyl) picolinic acid 56-1 was substituted for 1-11, intermediate 20-1 was substituted for intermediate 1-10 to give compound A57, ¹ H NMR(400MHz,Chloroform-d)δ8.83(dq,J＝5.0,0.7Hz,1H),8.65(d,J＝9.8Hz,1H),8.42(ddt,J＝21.2,1.6,0.8Hz,1H),8.23(d,J＝7.2Hz,1H),7.72(ddd,J＝5.0,1.8,0.7Hz,1H),6.10(s,1H),4.99(ddd,J＝10.3,7.2,5.9Hz,1H),4.81(d,J＝9.8Hz,1H),4.61(dd,J＝8.1,5.9Hz,1H),3.85–3.67(m,2H),3.44–3.37(m,2H),2.75–2.60(m,1H),2.55–2.33(m,2H),2.09–1.96(m,2H),1.93–1.82(m,2H),1.16(s,9H),0.82–0.73(m,2H),0.71–0.57(m,2H).ESI-MS:m/z＝563[M+1] ⁺ 。

EXAMPLE 58 2019 evaluation of novel coronavirus 3CL protease inhibitory Activity

Assay of Compound pair 2019 novel coronavirus 3CL protease (2019-nCoV 3CL ^pro ) The enzyme level inhibitory activity of inhibitors against 3CL protease was determined using fluorescence resonance energy transfer (fluorescence resonance energy transfer FRET) technique. 92. Mu.L according to Assay Buffer, 2019-nCoV 3CL ^pro 1 uL of Assay Reagent was prepared and 93 uL of buffer Assay Reagent was added to each well in a 96 well blackboard, along with 5 uL of compound (final concentration 0.5 uM) and 2 uL of Substrate. Fluorescent parameters are measured by using a Tecan multifunctional enzyme-labeled instrument, excitation wavelength and emission wavelength are 325nm and 393nm respectively, and data are read after 10min at 37 ℃. Negative and positive controls were used, wherein no compound was added to the negative control, PF-07321332 was used as the positive control, and the remainder were the same. The data obtained were processed using the software GraphPad Prism 7 and the experimental results are shown in table 1.

TABLE 1 2019 novel coronavirus 3CL protease inhibitory Activity

The experimental results show that the compound has certain 3CL protease inhibition activity, wherein the activity of the compounds A5, A10, A11, A23, A40, A41 and A43 is equal to or better than that of the comparative compounds.

EXAMPLE 59 evaluation of Compound 2019 novel coronavirus replication inhibitory Activity

Vero cells were inoculated in batches with SARS-CoV-2 (delta strain) at a fold infection (MOI) of 0.01 in BSL-3 laboratory. Virus-inoculated cells were then added to ready-made compound plates at 4000 cells/well in DMEM (medium with various amino acids and glucose, dulbecco's modified eagle medium) containing 2% heat-inactivated fetal bovine serum. Cells at 37℃and 5% CO ₂ The conditions were incubated for 3 days at which time the virus-induced CPE (cytopathic effect (cytopathic effect, CPE)) was 90% under untreated infection control conditions. Cell viability was assessed using CCK 8. The test compounds were tested alone or in the presence of specified concentrations of the P-glycoprotein (P-gp) inhibitor CP-100356.

TABLE 2Vero cell 2019 novel coronavirus infection CPE after application of compounds

Compounds of formula (I)	CPE at 1. Mu.M
		Comparative Compound (+1. Mu.M CP-100356)	<10％
A1(+1μM CP-100356)	40-50％
		A3(+1μM CP-100356)	<10％
A4(+1μM CP-100356)	80％
		A21(+1μM CP-100356)	<10％
A27(+1μM CP-100356)	<10％
		A41(+1μM CP-100356)	<10％
Comparative Compound 1	>80％
		A1	>80％
A3	>80％
		A4	>80％

The experimental results show that the compound (added with the P-gp inhibitor) has a certain antiviral effect, wherein the in-vitro drug effect of the compounds A3, A21, A27 and A41 is equivalent to that of the comparative compound 1 at the drug concentration of 1 mu M.

Example 60 cytotoxicity test:

in the BSL-2 laboratory, cytotoxicity of compounds was assessed in parallel with uninfected cellular vero using off-the-shelf compound plates. Drug concentrations were set at 1 μm and 10 μm. The non-toxic index is that the cell number is 90% -110% before the administration.

Toxicity of the compounds of Table 3 against Normal cells

From experiments, it appears that for normal VERO cells, compounds A3, A4, A21, A27, A41 are less toxic than the control compounds and the safety window is greater than the control compounds.

Example 61 plasma stability test:

1. with 5mM MgCl ₂ (K/Mg-buffer) 0.1M potassium phosphate buffer, pH 7.4.+ -. 0.1.

2. Plasma preparation: frozen human plasma was rapidly thawed at 37 ℃.

3. Test compound and internal standard compound solution:

0.5mM solution A: mu.L of 10mM stock solution was added to 95. Mu.L of ACN. 0.01mM solution B: to 980. Mu.L of 0.1. 0.1M K/Mg-buffer was added 20. Mu.L of solution A.

4. Plasma was preheated with solution B at 37 ℃ for 5min.

5. 90. Mu.L of pre-warmed plasma was added at each time point of 0, 5, 15, 30, 60, 120min, respectively.

6. To the wells of the 0min plate, 10. Mu. L B solution, 400. Mu.L of ACN containing Internal Standard (IS) were added, respectively.

7. For the other time points, 10 μl of preheated solution B was added to wells designated at time points 5, 15, 30, 60, 120min, respectively.

8. 400. Mu.L of IS-containing ACN was added to the corresponding wells at 5, 15, 30, 60, 120min, respectively, and the reaction stopped.

9. After quenching, shaking-up is carried out for 5 minutes (600 rpm) and stored at-20℃if necessary.

Samples were thawed at room temperature and centrifuged at 6000rpm for 20 minutes prior to lc/MS analysis.

11. 100. Mu.L of supernatant per well was transferred to 96-well sample plates containing 100. Mu.L of ultrapure water for LC/MS analysis.

Table 4 compound plasma stability

The results show that the compound of the invention has good plasma stability and half-life (T _1/2 ) Is larger than 289.1min.

Example 62 liver microsome stability test:

1. with 5mM MgCl at pH 7.41 ₂ 100mM K-buffer was pre-heated.

2. Test solution preparation

0.5mM solution A: 10mM stock solution of compound was added to 5. Mu.L, and reference was made to 95. Mu.L of ACN.

Microsomal 1.5 μm solution (0.75 mg/mL): mu.L of 500. Mu.M solution and 18.75. Mu.L of 20Mg/mL liver microsomes were added to 479.75. Mu.L of K/Mg-buffer.

3. A K/Mg-buffer NADPH solution (6 mM,5 Mg/mL) was used.

4. 30. Mu.M human liver microsomes at a concentration of 0.75mg/mL were added to the plates at various time points (0, 15,30,45,60 min).

5. Preincubation was carried out for 5 minutes at 37 ℃.

At 6.0min, 150. Mu.L of IS-containing ACN was added before 15. Mu.L of NADPH stock (6 mM) was added.

NADPH was dissolved in K/mg buffer to prepare 6mm,5mg/mL NADPH stock.

7. At other time points, 15. Mu.L of NADPH stock (6 mM) was added to the wells, the reaction was started and timed.

At 8.15min, 30min, 45min, and 60min, 150 μl of ACN containing IS was added to the corresponding wells, and the reaction was stopped.

9. After quenching, shaking up for 10 minutes (600 rpm) and then 6000 rpm/separating the cores for 15 minutes.

10. 80. Mu.L of supernatant per well was transferred to a 96-well sample plate (containing 140. Mu.L of pure water) for LC/MS analysis.

Table 5 compound liver microsomal stability

The results show that the compounds of the invention have moderate liver microsomal stability, half-life (T _1/2 ) For 46.4min, clearance (Cl) _int ) 29.9mL/min/kg.

EXAMPLE 63 pharmacokinetic testing

The samples used a vehicle of 2% (v/v) Tween80/98% 0.5% (w/v) methylcellulose. Blood samples were collected at various time points following oral dosing of a single dose of 10mg/kg in SD rats.

Standard curve and quality control sample preparation treatment: and diluting the mixed stock solution of the compounds with 50% methanol water to obtain standard working solutions with the concentrations of 20, 40, 100, 200, 400, 1000, 2000, 4000 and 10000ng/mL and quality control working solutions with the concentrations of 60, 600 and 8000 ng/mL. A standard curve working solution and a quality control working solution with the concentration of 1.00, 2.00, 5.00, 10.00, 20.00, 50.00, 100.00, 200.00, 500.00ng/mL and quality control samples with the concentration of 3.00, 30.00 and 400.00ng/mL are respectively taken, 2.50 mu L of acetonitrile (containing internal standard verapamil 2 ng/mL) is respectively added, vortex oscillation is carried out for 3min, centrifugation is carried out for 10min at 20000rcf and 4 ℃, and the supernatant is taken for LC-MS/MS analysis.

Unknown sample preparation: plasma samples were taken at 50. Mu.L, 200. Mu.L of acetonitrile (containing internal standard verapamil 2 ng/mL) was added, and after vortexing for 3min, centrifuged at 20000rcf at 4℃for 10min, and the supernatant was taken for LC-MS/MS analysis.

Unknown samples were diluted 10-fold for preparation: 45. Mu.L of blank plasma is taken, 5. Mu.L of plasma sample is added, 200. Mu.L of acetonitrile (containing internal standard verapamil 2 ng/mL) is added, vortex oscillation is carried out for 3min, centrifugation is carried out for 10min at 4 ℃ for 20000rcf, and the supernatant is taken for LC-MS/MS analysis.

TABLE 6 pharmacokinetic Properties of the Compounds of the invention in rats

Conclusion: the compound of the invention has good drug absorption in rats and pharmacokinetic advantage, and Cmax and AUC of the compound A3 are superior to those of the comparative compound 1.

Example 64 tissue distribution test

Drug lung tissue distribution: SD rats were dosed orally with the compound of the example at a dose of 30mg/kg, with lysozyme: 0.5% (w/v) MC solution of 2% (v/v) Tween 80 was taken 0.25, 1, 2, 4, 6 hours after administration, SD rat lung tissue was homogenized, 200. Mu.L acetonitrile (containing internal standard verapamil 2 ng/mL) was added with blood sample, protein was precipitated with methanol solution, vortexed for 3min, centrifuged at 20000rcf at 4℃for 10min, and the supernatant was taken for LC-MS/MS analysis.

TABLE 7 tissue distribution of the compounds of the invention in rats

Compounds of formula (I)	Lung/plasma (ng)	Proportion of
			A3	4886/1357	3.6
A21	4924/1201	4.1
			A41	6973/1516	4.6
Comparative Compound 1	1007/1157	0.87

Conclusion: the compound of the invention has good absorption in rats, higher exposure and higher lung targeting.

Claims

1. A compound having the structure of formula I:

or an isomer of the structure shown in formula I or a pharmaceutically acceptable salt thereof;

wherein:

ring A is selected from

L ₁ Selected from the group consisting of

L ₂ Selected from 5, 6 and 7 membered ring lactams;

R ₂ selected from hydrogen, C _1-3 An alkyl group;

R ₃ and R is ₄ Each independently selected from hydrogen, C _1-5 Alkyl, C _3-7 Cycloalkyl; or R is ₂ And R is ₃ May form, together with the atoms to which they are attached, a 4-or 5-membered heterocycloalkyl, which heterocycloalkyl may be optionally substituted with one or more halogen;

or R is ₃ And R is ₄ Can form C together with the atoms to which they are attached _3-5 Cycloalkyl; r is R ₅ 、R ₆ And R is ₇ Each independently selected from hydrogen, C _1-5 Alkyl, C _1-5 Alkoxy group,

When ring A is

R ₃ And R is ₄ Any one of which is->

And the other is hydrogen, R ₁ Not CF ₃ 。

2. The compound of claim 1, having the structure of formula ii:

or an isomer of the structure shown in formula II or a pharmaceutically acceptable salt thereof;

wherein:

L ₁ selected from the group consisting of

3. A compound according to claim 1 or 2, characterized in that:

R ₈ selected from hydrogen;

R ₉ selected from hydrogen, C _1-3 An alkyl group;

or (b)

R ₆ selected from hydrogen, C _1-5 Alkyl, C _1-5 Alkoxy group,

R ₈ selected from hydrogen;

R ₉ selected from hydrogen, C _1-3 An alkyl group;

or (b)

R ₉ selected from hydrogen, C _1-3 An alkyl group;

or (b)

R ₅ Selected from hydrogen, C _1-5 Alkyl, C _1-5 Alkoxy radicalA base group,

R ₆ selected from hydrogen;

R ₉ selected from hydrogen, C _1-3 An alkyl group;

or (b)

4. A compound according to any one of claims 1 to 3, wherein ring a is selected from:

5. a compound according to any one of claims 1 to 4, wherein L ₁ Selected from the group consisting of

6. A compound according to any one of claims 1 to 5, wherein L ₂ Selected from the group consisting of

7. A compound according to any one of claims 1 to 6, wherein R ₁ Selected from-CF ₃ 、

8. The compound of claim 1, having a structure represented by the following formula III or III

9. The compound of claim 8, wherein R ₁ Preferred are fluorocyclopropyl and trifluoromethyl.

10. A compound according to claim 1, wherein the compound is selected from the group consisting of:

11. a pharmaceutical composition comprising a compound according to any one of claims 1 to 10, an isomer, a pharmaceutically acceptable salt, solvate or prodrug thereof.

12. Use of a compound, isomer, pharmaceutically acceptable salt, solvate or prodrug or composition thereof according to any of claims 1-11 for the preparation of an inhibitor or a medicament for the inhibition of viral 3CL protease.

13. The use according to claim 12, wherein the medicament is for the treatment of a disease caused by a viral infection.

14. The use according to claim 13, wherein the disease is selected from the group consisting of pneumonia or asymptomatic infections caused by SARS-CoV-2 coronavirus infection.