CN117858867A

CN117858867A - SARS-COV-2 MPRO inhibitor compounds

Info

Publication number: CN117858867A
Application number: CN202280032654.5A
Authority: CN
Inventors: 迈尔斯·斯图尔特·康格里夫; 约翰·安德鲁·克里斯托弗; 马克·皮克沃斯; 克里斯·德格拉夫; 艾丽西娅·佩雷斯·伊格韦洛; 乔纳森·斯蒂芬·梅森
Original assignee: Heptares Therapeutics Ltd
Current assignee: Nxera Pharma UK Ltd
Priority date: 2021-04-01
Filing date: 2022-04-01
Publication date: 2024-04-09
Also published as: GB202107026D0

Abstract

The invention described herein relates to a compound of formula (1 b) or a salt thereof, wherein R ¹ 、R ^1a 、R ² 、R ³ And R is ⁵ As described hereinDefinition, and their use in the treatment of novel coronaviruses and related viruses and conditions associated with novel coronaviruses.

Description

SARS-COV-2 MPRO inhibitor compounds

Technical Field

The present application relates to novel compounds and their use as inhibitors of SARS-CoV-2 main protease (Mpro). The compounds described herein are useful for treating SARS-CoV-2 and related viruses and SARS-CoV-2:Mpro-associated disorders (dis). The application also relates to pharmaceutical compositions comprising these compounds, as well as the preparation and use of these compounds and compositions in the treatment of SARS-CoV-2 and related viruses and SARS-CoV-2:Mpro-related disorders. The compounds and compositions are useful for preventing death or complications from chronic underlying diseases or co-diseases in patients infected with SARS-CoV-2 and related viruses.

Background

Coronaviruses are long-standing in nature and are transmitted to humans by infectious diseases in animals, which often cause mild respiratory diseases such as the common cold. However, over the last two decades, the outbreak of new human coronavirus infections leading to severe respiratory diseases has become a major global health problem. This includes severe acute respiratory syndrome coronavirus (SARS-CoV) outbreak in 2002-2004, middle east respiratory syndrome coronavirus (MERS-CoV) in 2012-2015, and severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) in which a coronavirus strain was recently developed, which was found in 2019 and was causative of viral pneumonia in 2019-2020 coronavirus disease (COVID-19). Although these incidents have had a tragedy and widespread impact and the periodic appearance of new human coronaviruses has increased the likelihood of future outbreaks, we have not yet been effective antiviral treatments for coronavirus infections.

SARS-CoV-2 comprises a large RNA genome of about 30kb, two thirds of which are encoded by two multimeric proteins pp1a and pp1b (Hegyi et al Journal of General Virology (3): 595-99). These polyproteins are processed into 16 nonstructural proteins (nsp) which are released from the long polypeptide chain by two viral cysteine proteases, papain-like protease (nsp 3) and 3C-like protease (nsp 5). The latter, also called the main protease (Mpro), cleaves the viral polyprotein at 11 sites, yielding 12 nonstructural proteins (nsp 5-16). These include those involved in replication and transcription mechanisms, such as RNA-dependent RNA polymerase (nsp 12) and helicase (nsp 13). The important role of Mpro in viral replication has been demonstrated in mutagenesis experiments (Kim et al, virology 208 (1): 1-8; stobart et al, journal of Virology 86 (9): 4801-10), which makes it an attractive target for designing inhibitors for the treatment of coronavirus infection. Furthermore, there is no human protease with similar cleavage specificity, so selective inhibitors of Mpro are likely to be non-toxic (Anand et al, 2003 Science 300 (5626): 1763-67).

Protease inhibitors have been promising for the treatment of viral diseases (Bacon et al, the New England Journal of Medicine (13): 1207-17), and the similarity of the SARS-CoV-2 Mpro active site to other viral proteases has driven efforts to identify clinically approved drugs that can be reused for the treatment of COVID-19 (Riva et al, nature, 586:113-119). Screening for 18 viral protease inhibitors designed for the treatment of Human Immunodeficiency Virus (HIV) and Hepatitis C Virus (HCV) established the preclinical inhibitor GC376 against the HCV drug Boceprevir (boceprevir) and against the Feline Infectious Peritonitis Virus (FIPV) as an inhibitor of SARS-CoV-2 Mpro (Fu et al, nature Communications (1): 4417). Although GC376 shows a ratio of boscalid (IC ₅₀ =8μΜ), more potent inhibition of recombinant protease activity (IC ₅₀ ＝0.15μM) However, GC376 has shown side effects in tests performed on cats, causing potential safety problems (Pedersen et al, journal of Feline Medicine and Surgery 20 (4): 378-92). In a different study, bossep Wei Ye was identified with Telaprevir (telaprevir) as an inhibitor of SARS-CoV-2 Mpro, although both agents inhibited the IC of SARS-CoV-2 Mpro ₅₀ Value of>1. Mu.M (Anson et al, 2020, doi:10.21203/rs.3.Rs-26344/v 1). In addition to SARS-CoV-2 Mpro, the inhibitory effect of Bossep Wei Hete Lapidevir on Mpro proteases from other eight coronaviruses, including SARS, MERS, HKU1, HKU4, HKU5, NL63, FIPV and IBV, was also evaluated. In this selection, boscalid was able to inhibit all tested coronavirus proteases except NL63, and telaprevir showed a similar broad spectrum of activity, which showed inhibitory activity against SARS, HKU4, HKU5, NL63 and IBV. Although these drugs have insufficient antiviral activity against SARS-CoV-2 Mpro for clinical development, their ability to inhibit a wide range of polyproteinase highlights the potential for broad-spectrum antiviral drug design, which is capable of treating not only SARS-CoV-2 infection but also other human coronaviruses and potentially novel coronaviruses that may occur in the future.

Similar sequences between the active sites of SARS-CoV and SARS-CoV-2 Mpro were also used to identify the SARS-CoV-2 Mpro inhibitor PF-07304114, PF-07304814 being a phosphate prodrug of PF-00835231, originally designed for the treatment of SARS-CoV (Bora et al, bioRxiv, 2020.09.12.293498). PF-00835231 inhibited SARS-CoV-2 Mpro K _i Has a value of 0.27nM and shows broad inhibitory activity against another 10 coronavirus strains, K _i The value is 0.03-4nM. This translates to an activity of about 1. Mu.M in a cell-based live virus assay. The activity of PF-00835231 in combination with Remdesivir (a nucleoside RNA-dependent RNA polymerase inhibitor) was also evaluated, as antiviral agents targeting different aspects of the viral replication process may produce a synergistic effect when used in combination. Indeed, PF-00835231 and Rede-sivir show a synergistic or additive effect in cell-based antiviral assays, indicating that the Mpro inhibitor acts in combination with antiviral drugs and other modes of actionCombinations of formulas may exhibit clinical benefit.

In 2020, a crystal structure of a complex of SARS-CoV-2 Mpro and N3 (a Michael acceptor inhibitor) was disclosed (Jin et al, nature 582 (7811): 289-93), thereby enabling virtual screening of SARS-CoV-2 Mpro inhibitors and structure-based drug design (SBDD). Efforts to these SBDDs include the design of peptidomimetic alpha-keto amides as broad spectrum inhibitors of coronaviruses and enteroviruses, two of the most promising inhibitors being IC to enteroviruses EV-A71 and CVB3 and coronaviruses SARS-CoV and NL63 in recombinant inhibition assays against proteases ₅₀ The value is 0.71-12.27. Mu.M (Zhang et al, 2020, journal of Medicinal Chemistry 63 (9): 4562-4578). The observed activity in the recombinant protease assay approximately matches the antiviral activity in the cell-based live virus assay, IC in both systems ₅₀ Values within 10-fold indicate good activity in protease inhibition assays as a good indicator of antiviral activity.

Currently, there are no targeted therapeutic agents to treat covd-19 and the effective treatment options remain very limited. Despite many ongoing research activities and many ongoing clinical trials, only adefovir and fampirvir (favipiravir) have been approved for limited use in the treatment of SARS-CoV-2 infection in selected countries, but have only shown limited efficacy (Zhou et al, ACS Pharmacology & Translational Science (5): 813-834). There is a need for targeted therapeutic agents for the treatment of SARS-CoV-2 infection, and for the reasons stated above, SARS-CoV-2 Mpro represents an attractive drug target for SARS-CoV-2. The compounds disclosed herein have been shown to be inhibitors of SARS-CoV-2 Mpro and thus represent potential candidates for the treatment of coronavirus infections and related disorders, including but not limited to COVID-19.

Disclosure of Invention

The present invention provides compounds having SARS-CoV-2:Mpro inhibitor activity.

The present invention provides a compound of formula (1 b), or a salt thereof:

wherein:

R ¹ and R is ^1a Independently H, C optionally substituted with 1-6 fluorine or chlorine atoms _1-6 Saturated hydrocarbon radicals or benzyl radicals optionally substituted by 1 to 6 fluorine or chlorine atoms, or R ¹ And R is ^1a Are linked together to form a saturated ring optionally containing additional heteroatoms;

R ² is C containing cycloalkyl and optionally substituted with one or more substituents selected from fluoro or hydroxy _3-5 Saturated hydrocarbon groups;

R ³ is a saturated radical containing 3 to 5 carbon atoms and optionally containing cycloalkyl groups or optionally containing saturated rings containing oxygen heteroatoms and optionally substituted by one or more substituents selected from fluorine or hydroxyl groups, or R ³ Is CH ₂ Aryl, CH (CH) ₃ ) Aryl or C (CH) ₃ ) ₂ An aryl group; and is also provided with

R ⁵ Is C _2-8 Hydrocarbyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, C _2-8 The hydrocarbyl group optionally contains one or more rings or double bonds and is optionally substituted with one or more groups selected from the group consisting of: fluorine, chlorine, bromine, cyano, hydroxy, methoxy, amino.

The compounds of the invention are useful as SARS-CoV-2:Mpro inhibitors. The compounds of the invention are useful for treating SARS-CoV-2 and related viruses or SARS-CoV-2 related diseases or conditions. The compounds of the invention are useful for preventing death or complications from chronic underlying disease or comorbidity in patients infected with SARS-CoV-2 and related viruses. Such chronic underlying diseases or co-diseases may include, for example, hypertension, obesity, chronic lung disease (tuberculosis (TB), asthma and cystic fibrosis), diabetes and cardiovascular diseases (coronary heart disease, congenital heart disease and heart failure). The compounds of the invention are useful in the manufacture of medicaments. The compounds or medicaments are useful for treating, preventing, ameliorating, controlling or reducing the risk of SARS-CoV-2 and related viruses and diseases or conditions associated with SARS-CoV-2:Mpro. The compounds or medicaments are useful for treating, preventing, ameliorating, controlling or reducing the risk of chronic underlying diseases or co-diseases in patients infected with SARS-CoV-2 and related viruses.

The compounds of the present invention may be used as a single agent or in combination with one or more other agents. The compounds of the invention are useful for treating SARS-CoV-2 and related viruses or diseases or conditions associated therewith.

Detailed Description

The present invention relates to novel compounds. The invention also relates to the use of the novel compounds as SARS-CoV-2:Mpro inhibitors. The invention also relates to the use of the novel compounds in the preparation of a medicament for SARS-CoV-2:Mpro inhibitor. The invention further relates to compounds, compositions and medicaments useful for treating SARS-CoV-2 and related viruses or diseases or conditions associated therewith.

The present invention provides a compound of formula (1 b), or a salt thereof:

wherein:

R ⁵ Is C _2-8 Hydrocarbyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, C _2-8 The hydrocarbyl group optionally contains one or more rings or double bonds,and optionally substituted with one or more groups selected from: fluorine, chlorine, bromine, cyano, hydroxy, methoxy, amino.

Also provided are compounds of formula (1 a) or salts thereof:

wherein:

q is CN or a group of the formula:

R ² is C containing cycloalkyl and optionally substituted with one or more substituents selected from fluoro or hydroxy _3-5 Saturated hydrocarbon radicals, or R ² Is a saturated ring containing an oxygen heteroatom and optionally substituted with one or more substituents selected from fluorine, methyl or hydroxy;

R ³ is a saturated group containing 3 to 5 carbon atoms and optionally containing cycloalkyl or optionally containing a saturated ring containing an oxygen heteroatom and optionally substituted with one or more substituents selected from fluorine or hydroxy, or R ³ Is CH ₂ Aryl, CH (CH) ₃ ) Aryl or C (CH) ₃ ) ₂ An aryl group; and is also provided with

R ⁴ Is H or CO-R ⁵ Wherein R is ⁵ Is C _2-8 Hydrocarbyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, C _2-8 The hydrocarbyl group optionally contains one or more rings or double bonds and is optionally substituted with one or more groups selected from the group consisting of: fluorine, chlorine, bromine, cyano, hydroxy, methoxy, amino.

Also provided are compounds of formula (1) or salts thereof:

wherein:

Provided are compounds of formula (2) or salts thereof:

wherein:

R ¹ is H, C optionally substituted by 1 to 6 fluorine or chlorine atoms _1-6 Saturated hydrocarbon groups or benzyl groups optionally substituted with 1 to 6 fluorine or chlorine atoms;

Provided are compounds of formula (2) or salts thereof:

wherein:

R ² c is cycloalkyl-containing _3-5 Saturated hydrocarbon groups;

R ³ a saturated group containing 3 to 5 carbon atoms and optionally containing cycloalkyl groups or optionally containing a saturated ring containing an oxygen heteroatom; and is also provided with

R ⁴ Is C _2-8 Hydrocarbyl groups optionally substituted with 1 to 6 fluorine or chlorine atoms and optionally containing one or more rings or double bonds.

Provided are compounds of formula (3) or salts thereof:

wherein:

Provided are compounds of formula (2 b) or salts thereof:

wherein:

R ⁵ Is C _2-8 Hydrocarbyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, C _2-8 The hydrocarbyl group is optionally substituted with 1 to 6 fluorine or chlorine atoms and optionally contains one or more rings or double bonds, and is optionally substituted with one or more groups selected from the group consisting of: fluorine, chlorine, bromine, cyano, hydroxy, methoxy, amino.

Provided are compounds of formula (2 b) or salts thereof:

wherein:

R ² c is cycloalkyl-containing _3-5 Saturated hydrocarbon groups;

R ⁵ Is C _2-8 Hydrocarbyl groups optionally substituted with 1 to 6 fluorine or chlorine atoms and optionally containing one or more rings or double bonds.

In the compounds herein, Q may be CN. Q may be a group of the formula:

q may be selected from the following groups:

CN，

in the compounds herein, R ^1a May be H. R is R ^1a May be C optionally substituted with 1 to 6 fluorine or chlorine atoms _1-6 Saturated hydrocarbon groups. R is R ^1a May be methyl. R is R ^1a Can be cyclopropyl. R is R ^1a May be benzyl optionally substituted with 1 to 6 fluorine or chlorine atoms. R is R ^1a Can be selected from-CH ₂ -cyclopropyl, -CH ₂ -chlorophenyl, -CH ₂ -phenyl, methylcyclopropyl, 1-dimethylcyclopropyl, 1, 2-dimethylcyclopropyl, difluorocyclopropyl.

In the compounds herein, R ¹ May be H. R is R ¹ May be C optionally substituted with 1 to 6 fluorine or chlorine atoms _1-6 Saturated hydrocarbon groups. R is R ¹ May be methyl. R is R ¹ Can be cyclopropyl. R is R ¹ May be benzyl optionally substituted with 1 to 6 fluorine or chlorine atoms. R is R ¹ Can be selected from-CH ₂ -cyclopropyl, -CH ₂ -chlorophenyl, -CH ₂ -phenyl, methylcyclopropyl, 1-dimethylcyclopropyl, 1, 2-dimethylcyclopropyl, difluorocyclopropyl.

R ¹ May be selected from the following groups:

H，CH ₃ ，

R ¹ can be H, CH ₃ Benzyl, cyclopropyl or

R ^1a May be selected from H and methyl. R is R ^1a May be H. R is R ¹ 、R ^1a May be H. R is R ¹ 、R ^1a Can be all-CH ₃ 。

R ¹ And R is ^1a May be linked together to form a saturated ring optionally containing additional heteroatoms. The ring may contain 3 to 6 atoms. The heteroatom may be O or N. The heteroatom may be O. The ring may be an alkyl chain (CH ₂ ) _n Wherein n is 2 to 5.n may be 2, 3, 4 or 5.R is R ¹ And R is ^1a May be linked to form a 3 to 6 membered ring. R is R ¹ And R is ^1a May be linked to form an aziridine ring, an azetidine ring, a pyrrolidine ring, a piperidine ring or a morpholine ring. R is R ¹ And R is ^1a May be linked to form an azetidine ring. R is R ¹ And R is ^1a May be linked together to form an azetidine or aziridine ring.

In the compounds herein, R ² May be C containing cycloalkyl and optionally substituted with one or more substituents selected from fluoro or hydroxy _3-5 Saturated hydrocarbon radicals, or R ² May be a saturated ring containing an oxygen heteroatom and optionally substituted with one or more substituents selected from fluoro, methyl or hydroxy. R is R ² May be C containing cycloalkyl and optionally substituted with one or more substituents selected from fluoro or hydroxy _3-5 Saturated hydrocarbon groups. R is R ² May be C containing cycloalkyl _3-5 Saturated hydrocarbon groups. R is R ² May be a saturated ring containing oxygen heteroatoms. The ring may contain 3 to 6 atoms, one of which is O. The ring may be optionally substituted with one or more substituents selected from fluorine, methyl or hydroxy. R is R ² Can be selected from cyclobutyl, cyclopropyl, methylcyclopropyl.

R ² May be selected from the following groups:

R ² may be selected from the following groups:

R ² may be

In the compounds herein, R ³ Can be a saturated radical containing 3 to 5 carbon atoms and optionally containing cycloalkyl groups or optionally containing saturated rings containing oxygen heteroatoms and optionally substituted by one or more substituents selected from fluorine or hydroxyl groups, or R ³ Can be CH ₂ Aryl, CH (CH) ₃ ) Aryl or C (CH) ₃ ) ₂ Aryl groups. R is R ³ May be a saturated group containing 3 to 5 carbon atoms and optionally containing cycloalkyl groups or optionally containing a saturated ring containing an oxygen heteroatom and optionally substituted with one or more substituents selected from fluorine or hydroxy groups. R is R ³ May be a saturated group containing 3 to 5 carbon atoms and containing cycloalkyl groups. R is R ³ May be a saturated group containing a saturated ring containing an oxygen heteroatom. The ring may contain 3 to 6 atoms, one of which is O. The ring may contain one or more substituents selected from fluorine, methyl or hydroxy. R is R ³ Can be CH ₂ Aryl, CH (CH) ₃ ) Aryl or C (CH) ₃ ) ₂ Aryl groups. The aryl group may be phenyl. R is R ³ Can be selected from-CH ₂ -cyclopropyl, -CH (CH) ₃ ) ₂ 、-C(CH ₃ ) ₃ Cyclopropyl, oxacyclopentane, oxetane, -CH (CH) ₃ )CH ₂ CH ₃ Cyclobutyl, C (CH) ₃ ) ₂ Ph、CH ₂ Ph。

R ³ May be selected from the following groups:

in the compounds herein, R ⁴ Can be H or CO-R ⁵ Wherein R is ⁵ Is C _2-8 Hydrocarbyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, C _2-8 The hydrocarbyl group optionally contains one or more rings or double bonds and is optionally substituted with one or more groups selected from the group consisting of: fluorine, chlorine, bromine, cyano, hydroxy, methoxy, amino.

In the compounds herein, R ⁵ May be C _2-8 Hydrocarbyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, C _2-8 The hydrocarbyl group is optionally substituted with 1 to 6 fluorine or chlorine atoms and optionally contains one or more rings or double bonds, and is optionally substituted with one or more groups selected from the group consisting of: fluorine, chlorine, bromine, cyano, hydroxy, methoxy, amino.

R ⁵ May be C _2-8 Hydrocarbyl groups optionally containing one or more rings or double bonds. R is R ⁵ Can be selected from cyclopropyl, cyclobutyl, cyclopentyl, ethyl, -CH (CH) ₃ ) ₂ 、-CH ₂ (CH ₃ ) ₂ 、-CH(CH ₃ )CH ₂ CH ₃ 、-CH ₂ CF ₃ 、CH(CF ₃ ) ₂ Methyl cyclopropyl, vinyl (-ch=ch) ₂ ) Bicyclo (1, 1) pentanes and cubanes.

R ⁵ May be selected from the following groups:

/>

R ⁵ may be selected from the following groups:

provided are compounds of formula (1):

wherein:

R ^1a is H or methyl;

R ¹ is H, methyl, benzyl or cyclopropyl;

or R is ¹ And R is ^1a Linked together to form a saturated ring of 3 to 6 atoms;

R ² selected from the following groups:

R ³ selected from the following groups:

and

R ⁴ is CO-R ⁵ Wherein R is ⁵ Selected from the following groups:

/>

these compounds may be compounds of formula (1 a), (1 b), (1) or (1 i) or salts thereof:

therein Q, R ¹ 、R ^1a 、R ² 、R ³ 、R ⁴ And R is ⁵ As defined herein.

These compounds may be compounds of formula (2), (2 a), (2 i), (2 b), (2 ba) or (2 bi) or salts thereof:

therein Q, R ¹ 、R ² 、R ³ And R is ⁵ As defined herein.

These compounds may be compounds of formula (3), (3 a), (3 b) or (3 i) or salts thereof:

/>

The compound may be selected from any one of examples 1 to 35 as shown in table 1 or an isomer or salt thereof.

The compound may be selected from the following substances or salts thereof:

(1 r,2S, 5S) -N- (4-amino-1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- ((S) -2-isobutyramide-3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2S, 5S) -N- [ 3-amino-1- (cyclopropylmethyl) -2, 3-dioxo-propyl ] -3- [ (2S) -2- (cyclopropanecarbonylamino) -3-methylbutanoyl ] -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2s,5 s) -N- (4-amino-1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- (isobutyryl-L-allo-isoleucine) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2s,5 s) -3- (acryl-L-valyl) -N- (4-amino-1-cyclopropyl-3, 4-dioxobutan-2-yl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2s,5 s) -N- (4-amino-1- ((1 r,2 s) -2-methylcyclopropyl) -3, 4-dioxobutan-2-yl) -3- (isobutyryl-L-valyl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2S, 5S) -N- (4-amino-1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- ((S) -3-cyclopropyl-2-isobutyrylaminopropionyl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2s,5 s) -N- (4-amino-1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- (isobutyryl-L-valyl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2S, 5S) -N- (4-amino-1-cyclopropyl-3, 4-dioxobutan-2-yl) -6, 6-dimethyl-3- (((S) -2-methylbutanoyl) -L-valyl) -3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2S, 5S) -N- (4-amino-1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- ((S) -2- (cyclopropanecarboxamide) -2-cyclopropylacetyl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2S, 5S) -N- (4-amino-1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- ((S) -2-cyclopropyl-2-isobutyramide acetyl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2s,5 s) -N- (4- (benzylamino) -1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- (isobutyryl-L-valyl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2s,5 s) -N- (1-cyclopropyl-4- (cyclopropylamino) -3, 4-dioxobutan-2-yl) -3- (isobutyryl-L-valyl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2s,5 s) -N- (4-amino-1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- (2-isobutyramide-2- (oxetan-3-yl) acetyl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2S, 5S) -N- (4-amino-1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- ((S) -2- (cyclopropanecarboxamide) -3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2S, 5S) -3- ((S) -2-acrylamido-3, 3-dimethylbutyryl) -N- (4-amino-1-cyclopropyl-3, 4-dioxobutan-2-yl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2S, 5S) -3- ((S) -2-acrylamido-3, 3-dimethylbutyryl) -N- (4- (benzylamino) -1-cyclopropyl-3, 4-dioxobutan-2-yl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2S, 5S) -N- (4- (benzylamino) -1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- ((S) -2-isobutyramide-3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2S, 5S) -N- (4- (benzylamino) -1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- ((S) -2- (cyclopropanecarboxamide) -3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2S, 5S) -3- ((S) -2- (cyclopropanecarboxamide) -3, 3-dimethylbutyryl) -N- (1-cyclopropyl-4- (cyclopropylamino) -3, 4-dioxobutan-2-yl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2S, 5S) -3- ((S) -2-acrylamido-3, 3-dimethylbutyryl) -N- (1-cyclopropyl-4- (cyclopropylamino) -3, 4-dioxobutan-2-yl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2S, 5S) -N- (1-cyclopropyl-4- (cyclopropylamino) -3, 4-dioxobutan-2-yl) -3- ((S) -2-isobutyramide-3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2S, 5S) -N- (1-cyclopropyl-4- (methylamino) -3, 4-dioxobutan-2-yl) -3- ((S) -2-isobutyramide-3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2S, 5S) -N- (1-cyclopropyl-4- (dimethylamino) -3, 4-dioxobutan-2-yl) -3- ((S) -2-isobutyramide-3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2S, 5S) -N- (4- (aziridin-1-yl) -1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- ((S) -2-isobutyramide-3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2S, 5S) -N- (4-amino-1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- ((S) -2-cyclobutyl-2-isobutyramide acetyl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2S, 5S) -N- (4-amino-1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- ((S) -2-isobutyramidoyl-3-methyl-3-phenylbutyryl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2S, 5S) -N- (1-cyano-2-cyclopropylethyl) -3- ((S) -2-isobutyramide-3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2S, 5S) -N- (4- (azetidin-1-yl) -1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- ((S) -2-isobutyramide-3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2s,5 s) -3- ((cyclopropanecarbonyl) -L-valyl) -N- (1-cyclopropyl-4- (cyclopropylamino) -3, 4-dioxobutan-2-yl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2s,5 s) -N- (4-amino-1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- (2-isobutyramide-2- (tetrahydrofuran-3-yl) acetyl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2S, 5S) -N- (1-cyclopropyl-4- (((S) -2, 2-dimethylcyclopropyl) amino) -3, 4-dioxobutan-2-yl) -3- ((S) -2-isobutyramidol-3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2s,5 s) -N- (4-amino-1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- (isobutyryl-L-phenylpropionyl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2S, 5S) -N- (4-amino-1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- ((S) -3, 3-dimethyl-2- (3, 3-trifluoro-2- (trifluoromethyl) propanamido) butanoyl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2S, 5S) -N- (4-amino-1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- ((S) -3, 3-dimethyl-2- (2, 2-trifluoroacetamido) butanoyl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide;

(1 r,2S, 5S) -N- (4- (azetidin-1-yl) -1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- ((S) -3, 3-dimethyl-2- (2, 2-trifluoroacetamido) butanoyl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide.

Further embodiments include the use of a compound of the invention or a salt thereof or a pharmaceutical composition comprising a compound of the invention as an inhibitor of SARS-CoV-2:Mpro. The compounds of the invention are useful as SARS-CoV-2:Mpro inhibitors. The compounds of the invention are useful for treating SARS-CoV-2 or a disease or condition associated with SARS-CoV-2. The compounds of the present invention are useful for preventing death or complications from chronic underlying diseases or co-diseases in patients infected with SARS-CoV-2. Such chronic underlying diseases or co-diseases may include, for example, hypertension, obesity, chronic lung disease (tuberculosis, asthma, and cystic fibrosis), diabetes, and cardiovascular diseases (coronary heart disease, congenital heart disease, and heart failure). The compounds of the invention are useful in the manufacture of medicaments. The compounds or medicaments are useful for treating, preventing, ameliorating, controlling or reducing the risk of SARS-CoV-2 and diseases or conditions associated with SARS-CoV-2:Mpro. The compounds or medicaments are useful for treating, preventing, ameliorating, controlling or reducing the risk of chronic underlying diseases or co-complications in patients suffering from SARS-CoV-2 infection.

The compounds of the present invention may be used as a single agent or in combination with one or more other drugs. The compounds of the invention are useful for treating SARS-CoV-2 or diseases or conditions associated therewith.

As provided herein, the compounds described herein or salts thereof, and the compositions described herein may be administered with agents that treat any of the diseases and conditions disclosed herein.

The compounds of the invention may exist in the form of prodrugs (prodrugs). "prodrug" refers to any compound that is converted, for example, in vivo, to a biologically active compound of the invention. For example, some prodrugs are esters or phosphates (e.g., physiologically acceptable metabolically labile esters) of the active compound. In the metabolic process, an ester group (-C (=o) OR a phosphate group (P (=o) (OH) ₂ -OR) is cleaved to yield the active agent. Such esters may be formed by esterification, for example of the hydroxyl groups present in the parent compound, where appropriate, if desired, by first protecting and then deprotecting any other reactive groups present in the parent compound. Other functional groups present in the active compound, such as amide groups or amino groups, may be used to form the prodrug. In addition, some prodrugs are enzymatically activated to produce the active compound, or are produced in a further step Compounds that produce the active compound upon chemical reaction (e.g., ADEPT, GDEPT, LIDEPT, etc.). For example, the prodrug may be a sugar derivative or other glycoside conjugate, or may be an amino acid ester derivative.

Thus, there is provided a prodrug of a compound as defined herein, wherein the compound contains a functional group convertible to a hydroxyl group, an amide group or an amino group under physiological conditions.

Definition of the definition

In this application, the following definitions apply unless otherwise indicated.

The term "SARS-CoV-2:Mpro inhibitor" as used herein refers to any compound that binds to and modulates SARS-CoV-2:Mpro function.

The term "treatment" in connection with the use of any of the compounds described herein, including compounds of formula (1 b), is used to describe any form of intervention in which the compound is administered to a subject suffering from or at risk of suffering from the disease or disorder or a subject at risk of suffering from the disease or disorder. Thus, the term "treatment" includes prophylactic (prophoric) treatment and treatment that reveals measurable or detectable symptoms of a disease or disorder.

The term "therapeutically effective amount" (e.g., a method of treatment with respect to a disease or disorder) refers to an amount of a compound that is effective to produce a desired therapeutic effect. For example, if the condition is pain, the therapeutically effective amount is an amount sufficient to provide the desired level of pain relief. The desired level of pain relief may be, for example, complete elimination of pain or reduction of pain severity.

Unless otherwise indicated, terms such as "benzyl", "bicyclo", "hydrocarbon", "heterocycle", "carbocycle", "alkyl", "aryl", "amino", "heteroaryl", "cycloalkyl" and "halogen" are used in their conventional sense (e.g., as defined in IUPAC Gold Book). "optionally substituted" as applied to any group means that the group may be substituted with one or more substituents which may be the same or different, if desired.

“C _1-4 Saturated hydrocarbon groupThe term "saturated hydrocarbon group" in "means a hydrocarbon group having no carbon-carbon double bond or triple bond. Thus, the saturated hydrocarbon group may be alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl or alkylcycloalkylalkyl. C (C) _1-4 Examples of saturated hydrocarbon groups include C _1-4 Alkyl, cyclopropyl, cyclobutyl, and cyclopropylmethyl.

The term "cycloalkyl" as used herein includes monocyclic cycloalkyl groups (such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl, for example), as well as bicyclic and tricyclic groups, where the particular number of carbon atoms permits. Bicyclic cycloalkyl groups include bridged ring systems such as bicycloheptane, bicyclooctane and adamantane.

For any of the compounds described having chiral centers, the invention extends to all optical isomers of those compounds, whether in racemic form or as resolved enantiomers. The invention described herein relates to all crystalline forms, solvates and hydrates of any of the disclosed compounds, regardless of how prepared. For any compound disclosed herein having an acid or base center (e.g., carboxylate or amino), all salt forms of the compound are included herein. In the case of pharmaceutical use, the salt should be considered as a pharmaceutically acceptable salt.

Salts or pharmaceutically acceptable salts that may be mentioned include acid addition salts and base addition salts. Such salts may be formed by conventional methods, for example, optionally in a solvent, or in a salt-insoluble medium, by reacting the free acid or free base form of the compound with one or more equivalents of the appropriate acid or base, and then removing the solvent or medium using standard techniques (e.g., in vacuo, by freeze drying or filtration). Salts may also be prepared by, for example, exchanging the counter ion of a compound in salt form with another counter ion using a suitable ion exchange resin.

Examples of pharmaceutically acceptable salts include acid addition salts derived from inorganic and organic acids, and salts derived from metals such as sodium, magnesium, potassium and calcium.

Examples of acid addition salts include acid addition salts formed with: acetic acid, 2-dichloroacetic acid, adipic acid, alginic acid, arylsulfonic acid (e.g., benzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1, 5-disulfonic acid, and p-toluenesulfonic acid), ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzoic acid, 4-acetamidobenzoic acid, butyric acid, (+) camphoric acid, camphorsulfonic acid, (+) - (1S) -camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclohexylsulfamic acid, dodecylsulfuric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, mucic acid (gallic acid), gentisic acid, glucoheptylic acid gluconic acid (e.g., D-gluconic acid), glucuronic acid (e.g., D-glucuronic acid), glutamic acid (e.g., L-glutamic acid), alpha-ketoglutaric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxyethanesulfonic acid, lactic acid (e.g., (+) -L-lactic acid and (+ -) -DL-lactic acid), lactobionic acid, maleic acid, malic acid (e.g., (-) -L-malic acid), malonic acid, (+ -) -DL-mandelic acid, metaphosphoric acid, methanesulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, L-pyroglutamic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, tartaric acid (e.g. (+) -L-tartaric acid), thiocyanic acid, undecylenic acid and valeric acid.

Any solvate of these compounds and salts thereof is also included. Preferred solvates are those formed by incorporating into the solid state structure (e.g., crystalline structure) of the compounds of the present invention a non-toxic pharmaceutically acceptable solvent molecule (hereinafter referred to as solvating solvent). Examples of such solvents include water, alcohols (such as ethanol, isopropanol, and butanol), and dimethylsulfoxide. Solvates may be prepared by recrystallising the compounds of the invention from a solvent or solvent mixture containing a solvating solvent. Whether a solvate has formed in any given case can be determined by analysis of the crystals of the compound using well known standard techniques such as thermogravimetric analysis (TGA), differential Scanning Calorimetry (DSC) and X-ray crystallography.

The solvate may be a stoichiometric or non-stoichiometric solvate. Specific solvates may be hydrates, examples of which include hemihydrate, monohydrate and dihydrate. For a more detailed discussion of solvates and methods for preparing and characterizing them, see Bryn et al, solid-State Chemistry of Drugs, second edition, published by SSCI corporation of Silafeet, indiana, USA, 1999, ISBN 0-967-06710-3.

The term "pharmaceutical composition" in the context of the present invention refers to a composition comprising an active agent and additionally comprising one or more pharmaceutically acceptable carriers. Depending on the mode of administration and the nature of the dosage form, the composition may further comprise ingredients selected from, for example, the following: diluents, adjuvants, excipients, carriers, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, antibacterial agents, antifungal agents, lubricants, and dispersing agents. The compositions may take the form of, for example, tablets, dragees, powders, elixirs, syrups, liquid preparations (including suspensions), sprays, inhalants, tablets, lozenges, emulsions, solutions, cachets, granules, capsules and suppositories, as well as liquid preparations for injection (including liposomal preparations).

The compounds of the invention may contain one or more isotopic substitutes and the scope of reference to a particular element includes all isotopes of that element. For example, reference to hydrogen includes ¹ H、 ² H (D) and ³ h (T). Similarly, the scope of references to carbon and oxygen includes, respectively ¹² C、 ¹³ C and C ¹⁴ C, C and C ¹⁶ O and ¹⁸ o. In a similar manner, the scope of reference to a particular functional group also includes isotopic variations unless the context indicates otherwise. For example, reference to an alkyl (e.g., ethyl) or alkoxy (e.g., methoxy) group also includes variants in which one or more hydrogen atoms in the group are in the deuterium or tritium isotope form, e.g., all five hydrogen atoms in the ethyl group are in the deuterium isotope form (perdeuteroethyl) or all three hydrogen atoms in the methoxy group are in the deuterium isotope form (tridecylmethoxy). Isotopes may be radioactive or non-radioactive.

The therapeutic dosage may vary depending on the requirements of the patient, the severity of the disease being treated and the compound being used. Determination of the appropriate dosage for a particular situation is within the skill of the art. Typically, treatment is initiated with a smaller dose than the optimal dose of the compound. Thereafter, the dose is increased in small increments until the optimal effect is reached in this case. For convenience, the total daily dose may be administered in several portions of the day, if desired.

Of course, the size of the effective dose of a compound will vary with the severity of the condition being treated and the particular compound and its route of administration. The selection of an appropriate dosage is within the ability of one of ordinary skill in the art without undue burden. In general, the daily dosage may range from about 10 μg to about 30mg per kilogram of human and non-human animal body weight, preferably from about 50 μg to about 30mg per kilogram of human and non-human animal body weight, such as from about 50 μg to about 10mg per kilogram of human and non-human animal body weight, such as from about 100 μg to about 30mg per kilogram of human and non-human animal body weight, such as from about 100 μg to about 10mg per kilogram of human and non-human animal body weight, and most preferably from about 100 μg to about 1mg per kilogram of human and non-human animal body weight.

Pharmaceutical preparation

Although the active compounds may be administered alone, it is preferred that they be administered in the form of a pharmaceutical composition (e.g., formulation).

Thus, in some embodiments of the present invention, there is provided a pharmaceutical composition comprising at least one compound of the present invention and at least one pharmaceutically acceptable excipient.

Pharmaceutically acceptable excipients may be selected from, for example, carriers (e.g., solid, liquid or semi-solid carriers), adjuvants, diluents (e.g., solid diluents such as fillers or bulking agents), and liquid diluents such as solvents and co-solvents), granulating agents, binders, glidants, coating agents, release controlling agents (e.g., release-delaying or release-delaying polymers or waxes), binders, disintegrants, buffers, lubricants, preservatives, antifungal and antibacterial agents, antioxidants, buffers, tonicity adjusting agents, thickening agents, flavoring agents, sweeteners, colors, plasticizers, taste masking agents, stabilizers or any other excipient conventionally used in pharmaceutical compositions.

The term "pharmaceutically acceptable" as used herein refers to 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 subjects (e.g., human subjects) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each excipient must also be "acceptable", i.e., compatible with the other ingredients of the formulation.

Pharmaceutical compositions containing the compounds of the invention may be formulated according to known techniques, see for example Remington's Pharmaceutical Sciences, mack Publishing Company, easton, PA, USA. The pharmaceutical composition may be in any form suitable for oral, parenteral, intravenous, intramuscular, intrathecal, subcutaneous, topical, intranasal, intrabronchial, sublingual, buccal, ocular, aural, rectal, intravaginal or transdermal administration.

Pharmaceutical dosage forms suitable for oral administration include tablets (coated or uncoated), capsules (hard or soft shell), caplets, pills, troches, syrups, solutions, powders, granules, elixirs and suspensions, sublingual tablets, wafers or patches (such as oral patches).

The composition may be a tablet composition or a capsule composition. Tablet compositions may contain a unit dose of the active compound in combination with an inert diluent or carrier, such as a sugar or sugar alcohol, e.g. lactose, sucrose, sorbitol or mannitol; and/or non-sugar derived diluents such as sodium carbonate, calcium phosphate, calcium carbonate or cellulose or derivatives thereof (e.g. microcrystalline cellulose (MCC), methylcellulose, ethylcellulose, hydroxypropyl methylcellulose) and starches (e.g. corn starch). Tablets may also contain standard ingredients such as binders and granulating agents (e.g. polyvinylpyrrolidone), disintegrating agents (e.g. swellable crosslinked polymers such as crosslinked carboxymethylcellulose), lubricating agents (e.g. stearates), preserving agents (e.g. parabens), antioxidants (e.g. BHT), buffering agents (e.g. phosphate or citrate buffers) and effervescent agents (e.g. citrate/bicarbonate mixtures). Such excipients are well known and need not be discussed in detail herein.

Tablets may be designed to release the drug upon contact with gastric fluid (immediate release tablets), or to release the drug in a controlled manner over an extended period of time (controlled release tablets), or to release the drug in specific areas of the gastrointestinal tract.

The pharmaceutical composition generally comprises from about 1% (w/w) to about 95%, preferably% (w/w) of the active ingredient and from 99% (w/w) to 5% (w/w) of a pharmaceutically acceptable excipient (e.g. as defined above) or a combination of these excipients. Preferably, the composition comprises about 20% (w/w) to about 90% (w/w) active ingredient and 80% (w/w) to 10% pharmaceutical excipient or combination of excipients. The pharmaceutical composition comprises from about 1% to about 95%, preferably from about 20% to about 90%, of the active ingredient. The pharmaceutical compositions according to the invention may be in the form of, for example, unit dosage forms, for example, ampoules, vials, suppositories, pre-filled syringes, dragees, powders, tablets or capsules.

Tablets and capsules may contain, for example, 0-20% disintegrant, 0-5% lubricant, 0-5% glidant and/or 0-99% (w/w) filler and/or bulking agent (depending on the drug dosage). They may also contain 0-10% (w/w) of a polymeric binder, 0-5% (w/w) of an antioxidant, 0-5% (w/w) of a pigment. In addition, sustained release tablets typically contain 0-99% (w/w) of a controlled release (e.g., delayed) polymer (depending on the dosage). Film coatings for tablets or capsules typically contain 0-10% (w/w) polymer, 0-3% (w/w) pigment and/or 0-2% (w/w) plasticizer.

The composition may be a parenteral composition. Parenteral formulations typically contain 0-20% (w/w) buffer, 0-50% (w/w) co-solvent, and/or 0-99% (w/w) water for injection (WFI) (depending on the dosage and whether lyophilized). The intramuscular depot (formulation of intramuscular depots) may also contain 0-99% (w/w) of an oil.

The pharmaceutical formulation may be provided to the patient in a "patient pack" which contains the entire course of treatment in a single package, typically a blister pack.

The compounds of the present invention are typically present in unit dosage form and thus typically contain sufficient compound to provide the desired level of biological activity. For example, the formulation may contain 1ng to 2g of active ingredient, for example 1ng to 2mg of active ingredient. Within these ranges, the specific subrange of the compound is 0.1mg to 2g of active ingredient (more typically 10mg to 1g, e.g., 50mg to 500 mg), or 1 μg to 20mg (e.g., 1 μg to 10mg, e.g., 0.1mg to 2mg of active ingredient).

For oral compositions, unit dosage forms may contain from 1mg to 2g, more typically from 10mg to 1g, for example from 50mg to 1g, for example from 100mg to 1g of active compound.

The active compound will be administered to a patient (e.g., a human or animal patient) in need thereof in an amount (effective amount) sufficient to achieve the desired therapeutic effect. The precise amount of compound administered can be determined by the physician of skill in the art according to standard procedures.

These compounds may be administered with other agents, for example, other agents for treating SARS-CoV-2 subjects. These compounds may be co-administered with HIV drugs known to block cypP 450-mediated metabolism, such as ritonavir (ritonavir) or lopinavir (lopinavir)/ritonavir combination.

Examples

The present invention will now be described by referring to the following examples shown in table 1, but the present invention is not limited thereto.

TABLE 1

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Preparation of the Compounds of the invention

The compounds of the present invention can be prepared by routes including, but not limited to, those detailed in scheme 1. Details of many standard transformations, such as those in the following schemes and other methods that can be used to perform the same, can be found in standard reference textbooks, for example, "Organic Synthesis", M.B.Smith, mcGraw-Hill (1994), "Advanced Organic Chemistry", 4 th edition, J.March, john Wiley & Sons (1992) or "Protective Groups in Organic Synthesis", 3 rd edition, T.W.Greene, john Wiley & Sons (1999).

Nitrogen protected derivatives of alpha-amino acids (e.g., boc or Fmoc derivatives) are commercially available or may be prepared by standard transformations known to those skilled in the art, including those detailed in the synthesis of the intermediates and the synthesis section of the examples below. Similarly, ester derivatives of alpha-amino acids (e.g., methyl or ethyl esters) are commercially available or may be prepared by standard transformations known to those skilled in the art, including those detailed in the synthesis of the intermediates and the synthesis section of the examples below.

The acid functionality in the nitrogen-protected derivative of the alpha-amino acid may be coupled with an amine to give the corresponding amide derivative (e.g. scheme 1, step vi; schemes 2 and 3, step ix; scheme 4, step iv), as may other carboxylic acids (e.g. scheme 3, step xi; scheme 4, step viii). The amide functionality may also be introduced by other means, for example by reaction in a base (e.g., et ₃ N) in a solvent such as MeOH, typically at room temperature, with ethyl 2, 2-trifluoroacetate (e.g., scheme 4, step vii). The derivative of the alpha-amino acid may be coupled with a carboxylic acid to give the corresponding amide derivative (e.g., scheme 1, step viii, step x; scheme 2, step v, step vii; scheme 3, step v, step vii; scheme 4, step ii). The ester functions present in the alpha-amino acid derivative, or for example in the amide coupling product with the alpha-amino acid derivative, may be hydrolysed under acidic or basic conditions, for example typically at 0 ℃ or room temperature, using lithium hydroxide monohydrate in a solvent (such as THF, methanol or H ₂ O or a mixture of these solvents) (e.g., scheme 2, step viii; route 4, step iii, step vi). Hydrolysis to produce a carboxylic acid which can then be reacted with an amine or derivative of an alpha-amino acid under amide coupling conditions (e.g., those described in detail above) (e.g., scheme 1, step vi; scheme 2 or 3, step ix; scheme Step xi; route 4, step viii).

The nitrogen protection present in the alpha-amino acid derivative or in the coupled product of the amide and the alpha-amino acid derivative may be removed under conditions known to those skilled in the art. The Boc group may be removed under acidic conditions, for example using a solution of HCl in EtOAc at room temperature, TFA in solvent (e.g. DCM) at room temperature, HCl in a solvent mixture (e.g. 1, 4-dioxane/DCM) at room temperature, or with another transformation, for example hydrolysis of the nitrile group to the methyl ester in the presence of TMSCl in methanol at elevated temperature (e.g. 60 ℃). The Fmoc group can be removed under basic conditions, e.g. in 20% piperidine in DMF at room temperature.

The amide coupling reaction conditions will typically be in a solvent (e.g., DCM or DMF), typically at room temperature, using one or more coupling agents, such as T3P, HATU, or a combination of these agents (e.g., EDCI (typically hydrochloride) and HOBt), with or without a suitable base (e.g., DIPEA, NMM or Et ₃ N). Alternatively, derivatives of alpha-amino acids may be coupled with an acid chloride (e.g., cyclopropanecarbonyl chloride) in a solvent (e.g., DCM) under conditions of a suitable base (e.g., DIPEA), typically at room temperature, to form an amide derivative.

The acid functionality present in the alpha-amino acid derivative may be converted to an aldehyde by a process comprising the steps of: the formation of an N-methoxy-N-methylamide derivative (commonly known as Weinreb amide) is followed by reduction to an aldehyde using the following conditions: for example using lithium aluminum hydride in a solvent such as THF, typically at 0 ℃ (e.g. schemes 1, 2 and 3, steps i and ii). The aldehyde can then be used in a series of steps to form a substituted or unsubstituted ketoamide functionality. For example, in one such reaction, a reaction is carried out in the presence of a base (e.g., et ₃ N) in a solvent (e.g., DCM), typically at 0 ℃ or room temperature, with acetone cyanohydrin (e.g., schemes 1 and 3, step iii) to give 1-cyano, 1-hydroxy derivatives. The cyano (also referred to as nitrile) functionality in the 1-cyano, 1-hydroxy derivative can be hydrolyzed to the primary amide, e.g., scheme 1, step iv, in a solvent, e.g., DMSO, typically at 0 ℃ or room temperature, e.g., using hydrogen peroxide in the presence of a base, e.g., potassium carbonate. Or alternativelyThe cyano functionality can be converted to the methyl ester by reaction with methanol under acidic conditions, e.g., in the presence of tmcl, at elevated temperature (e.g., 60 ℃) (e.g., scheme 3, step iv). Similar conversion to ethyl ester can be achieved by reacting the primary amide group with ethanol and thionyl chloride at elevated temperature (e.g., 65 ℃) (e.g., scheme 4, step i). In a second conversion of the aldehyde, reaction with an isonitrile (e.g., an alkyl or benzyl isonitrile) in the presence of AcOH in a solvent (e.g., DCM), typically at room temperature, will yield an alkylamino or benzylamino substituted 1-oxo-2-ylacetate derivative (e.g., scheme 2, step iii). Et at room temperature, for example ₃ Removal of the acetate substituent under conditions in a solution of N in MeOH affords substituted hydroxyketoamide derivatives (e.g., scheme 2, step iv). The hydroxyl groups derived from aldehydes by the above method, at this stage or after further conversion, can be oxidized to ketones (e.g. schemes 1, 2 and 3, steps xi, x, xii, respectively) using an oxidizing agent (e.g. IBX or DMP) in a suitable solvent (e.g. DMSO), typically at 0 ℃ or room temperature.

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Scheme 1

General procedure

When the preparation route is not included, the relevant intermediates are commercially available. Commercial reagents were used without further purification. Room temperature refers to about 20-27 ℃. ¹ H Nuclear Magnetic Resonance (NMR) spectra were recorded at 300 or 400MHz using a Bruker instrument. Chemical shift values are expressed in parts per million (ppm), i.e., (delta) values. The following abbreviations are used for multiplicity of NMR signals: s=singlet, br=broad, d=doublet, t=triplet, q=quartet, quin=quintet, h=heptad, dd=doublet, dt=doublet, m=multiplet. Coupling constants are listed as J values in units ofHz. NMR and mass spectral results were corrected to account for background peaks. TLC for monitoring the reaction refers to TLC operation using silica gel as the stationary phase.

LCMS experiments were performed under the following conditions, LCMS data shown in the following format: mass ion, retention time.

Method AInstrument: shimadzu LCMS-2020; chromatographic column: chromolith@flash RP-18E 25-2MM or Kinetex EVO C18.1x30mm, 5 μm; gradient [ time (min)/solvent B in A (%)]:0.0/5, 0.8/95, 1.2/95, 1.21/5, 1.55/5 (solvent a=0.0375% tfa in water (v/v), b=0.01875% tfa in MeCN (v/v)); ultraviolet detection is carried out at 220nm and 254nm; column temperature 50 ℃; the flow rate was 1.5mL/min.

Method A2.Instrument: agilent 1200 LC with Agilent 6110 MSD; chromatographic column: agilent ZORBAX SB-Aq,2.1 x 50mm,5 μm; gradient [ time (min)/solvent B in A (%)]:0.0/1, 0.4/1, 3.4/90, 3.91/1, 4.5/1 (solvent a=0.0375% tfa in water (v/v), b=0.01875% tfa in MeCN (v/v)); ultraviolet detection is carried out at 220nm and 254nm; column temperature 50 ℃; the flow rate was 0.8mL/min.

Method B.Instrument: shimadzu LCMS-2020; chromatographic column: kineex EVO C18.1 x30mm,5 μm; method B gradient [ time (min)/solvent B in A (%)]:0.0/0, 0.8/60, 1.2/60, 1.21/0, 1.55/0 (solvent a=0.025% nh) ₃ ·H ₂ O (25%, w/w) in aqueous solution (v/v), b=mecn; ultraviolet detection is carried out at 210-254nm; column temperature 40 ℃ or 50 ℃; the flow rate is 0.8 or 1.5mL/min.

Method CInstrument: shimadzu LCMS-2020; chromatographic column: chromolith Flash RP-18e 25x2.0mm or Kinetex EVO C18.1 x 30mm,5 μm; gradient [ time (min)/solvent B in A (%)]:0.0/0, 0.8/60, 1.2/60, 1.21/0, 1.55/0 (solvent a=0.0375% tfa in water (v/v), b=0.01875% tfa in MeCN (v/v)); ultraviolet detection is carried out at 210-254nm; column temperature 50 ℃; the flow rate was 0.8mL/min.

Methods D and D2Instrument: agilent G1956A; chromatographic column: XBridge C18,2.1 x 50mm,5 μm; method D gradient [ time (min)/solvent B in A (%)]：0.0/5、0.4/5、3.4/90、3.90/100、3.91/5 (solvent a=0.05% nh ₃ ·H ₂ O (25%, w/w) in water (v/v), b=mecn; method D2 gradient [ time (min)/solvent B in A (%)]:0.0/5, 0.4/5, 3.4/90, 4.00/100, 4.01/5 (solvent A=0.05% NH) ₃ ·H ₂ O (25%, w/w) in water (v/v), b=mecn; ultraviolet detection is carried out at 220nm and 254nm; column temperature 40 ℃; the flow rate was 0.8mL/min.

Method EInstrument: agilent G1956A; chromatographic column: XBridge C18,2.1 x 50mm,5 μm; gradient [ time (min)/solvent B in A (%)]:0.0/1, 0.4/1, 3.4/90, 4.00/100, 4.01/1 (solvent a=0.05% nh) ₃ ·H ₂ O (25%, w/w) in water (v/v), b=mecn; ultraviolet detection is carried out at 220nm and 254nm; column temperature 40 ℃; the flow rate was 0.8mL/min.

Methods F and F2Instrument: method F: an Acquity UPLC equipped with a PDA and a QDA detector; method F2: an acquisition H-Class equipped with PDA and QDA detectors; chromatographic column: c18 50 x 2.1mm,1.6 μm; gradient [ time (min)/solvent B in A (%)]:0.0/3, 0.2/3, 2.7/98, 3.00/100, 3.50/100, 3.51/3 (solvent a=0.1% hco) ₂ Aqueous H, b=0.1% hco ₂ Aqueous solution of H, acetonitrile (10:90)); column temperature is 35 ℃; the flow rate was 0.8mL/min.

Method F3Instrument: an AcquityUPLC equipped with PDA and QDA detector; chromatographic column: c18 50 x 2.1mm,2.5 μm; gradient [ time (min)/solvent B in A (%)]:0.0/3, 0.2/3, 2.7/98, 3.00/100, 3.50/100, 3.51/3 (solvent a=0.1% hco) ₂ Aqueous H, b=0.1% hco ₂ Aqueous solution of H, acetonitrile (10:90)); column temperature is 30 ℃; the flow rate was 0.8mL/min.

Method GInstrument: an AcquityUPLC equipped with PDA and QDA detector; chromatographic column: c18 50 x 2.1mm,2.5 μm; gradient [ time (min)/solvent B in A (%)]:0.0/3, 0.2/3, 2.7/98, 3.0/100, 3.5/100, 3.51/3 (solvent a=5 mM (NH) ₄ )HCO ₃ B=mecn); column temperature is 35 ℃; the flow rate was 1.0mL/min.

Method HInstrument: waterselliance 2690 and 996PDA detectors equipped with micromass ZQ; chromatographic column: c18 150 x 4.6mm,3.5 μm; Gradient [ time (min)/solvent B in A (%)]:0.0/10, 7.0/90, 9.0/100, 14.0/100, 14.01/10 (solvent a=0.1% tfa in water, b=mecn); column temperature: room temperature; the flow rate was 1.0mL/min.

Method IInstrument: an Acquity UPLC equipped with a PDA and a QDA detector; chromatographic column: c18 50 x 2.1mm,2.5 μm; gradient [ time (min)/solvent B in A (%)]:0.0/3, 0.2/3, 2.7/98, 3/100, 3.5/100, 3.51/3 (solvent a=0.1% hco) ₂ Aqueous H, b=0.1% hco ₂ H aqueous solution, acetonitrile (10:90)); column temperature is 30 ℃; the flow rate was 0.8mL/min.

Method JInstrument: agilent 1100 LC with Agilent G1956A; chromatographic column: waters XBLridge C18.1.50 mm,5 μm; gradient [ time (min)/solvent B in A (%)]:0.0/1, 0.4/1, 3.4/90, 4.0/100, 4.01/1, 4.5/1 (solvent a=0.05% nh) ₃ ·H ₂ O (25%, w/w) in water (v/v), b=mecn; ultraviolet detection is carried out at 220nm and 254nm; column temperature 40 ℃; the flow rate was 0.8mL/min.

Method KInstrument: agilent Infinity II G6125C; chromatographic column: c18 150 x 4.6mm,3.5 μm; gradient [ time (min)/solvent B in A (%)]:0.0/10, 7.0/90, 9.0/100, 14.0/100, 14.01/10, 17.0/10 (solvent a=0.1% nh) ₃ B=mecn); column temperature 25 ℃; the flow rate was 1.0mL/min.

Method IInstrument: agilent 1290 info II series LC/6125 quadrupole MSD SL; chromatographic column: waters XB ridge C8X 4.6mm,3.5 μm; gradient [ time (min)/solvent B in A (%)]:0.0/5,2.5/95,4.0/95,4.5/5,6.0/5 (solvent A=0.1% TFA H) ₂ O solution: meCN (95:5), b=0.1% tfa in MeCN); ultraviolet detection is carried out at 210nm to 400nm; column temperature 25 ℃; the flow rate was 1.5mL/min.

Abbreviations (abbreviations)

AcOH = acetic acid

Boc=tert-butoxycarbonyl group

Cdi=n, N-carbonyldiimidazole

Dcm=dichloromethane

Dmf=n, N-dimethylformamide

Dmp=dess-martin oxidizer (Dess-Martin Periodinane)

Dipea=n, N-diisopropylethylamine

DMSO = dimethyl sulfoxide

Edci=1-ethyl-3- (3-dimethylaminopropyl) carbodiimide

EtOAc = ethyl acetate

Fmoc=fluorenylmethoxycarbonyl or (((9H-fluoren-9-yl) methoxy) carbonyl)

h=h

HATU = 1- [ bis (dimethylamino) methylene ] -1H-1,2, 3-triazolo [4,5-b ] pyridinium 3-oxide hexafluorophosphate

Hobt=1-hydroxybenzotriazole

HPLC = high performance liquid chromatography

Ibx=2-iodoxybenzoic acid

L=l

Lc=liquid chromatography

Mecn=acetonitrile

min = min

Ms=mass spectrum

Nmm=n-methylmorpholine

Nmr=nuclear magnetic resonance

Mtbe=methyl tert-butyl ether

rt=room temperature

T3p=propylphosphonic anhydride

TFA = trifluoroacetic acid

TFAA = trifluoroacetic anhydride

THF = tetrahydrofuran

TLC = thin layer chromatography

Tmscl=trimethylchlorosilane

Uplc=ultra high performance liquid chromatography

The prefixes n-, s-, i-, t-and tert-are their usual meanings: positive, secondary, iso and tertiary.

Synthesis of intermediates

Intermediate 1: (1R, 2S, 5S) -N- [ 3-amino-1- (cyclopropylmethyl) -2-hydroxy-3-oxo-propyl]-6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxamide

Step 1: to a mixture of methyl 2- (tert-butoxycarbonylamino) -2-dimethoxyphosphorylacetate (551 g,1.85 mol) in DCM (2.5L) was slowly added 1, 3-tetramethylguanidine (214 g,1.85mol,233 mL) at 0deg.C. After stirring at 0deg.C for 30min, cyclopropanecarbaldehyde (100 g,1.43mol,107 mL) was added to the reaction mixture at 0deg.C, and the mixture was stirred at N ₂ The resulting mixture was stirred at 25℃for 12h. Adding H ₂ O (2L), the resulting mixture was extracted with DCM (800 mL. Times.3), and the combined organic layers were washed with brine (1L) and with anhydrous Na ₂ SO ₄ Dried, filtered and concentrated in vacuo. Purification by gradient silica gel column chromatography (petroleum ether: etOAc 1:0 to 10:1) afforded (E) -2- (tert-butoxycarbonylamino) -3-cyclopropyl-prop-2-enoic acid methyl ester (374 g,1.55 mol) as a white solid.

LCMS (method a): m/z 142.2 (M-100+H) at 0.79 min.

¹ H NMR:(400MHz,DMSO-d ₆ )δ0.59-0.64(2H,m),0.87-0.93(2H,m),1.38(9H,br s),1.59-1.71(1H,m),3.62(3H,s),5.61-5.98(1H,m),8.37(1H,br s)。

Step 2: at 0 ℃ and N ₂ Next, BH was slowly added dropwise to a solution of (E) -2- (tert-butoxycarbonylamino) -3-cyclopropyl-prop-2-enoic acid methyl ester (374 g,1.55 mol) in THF (2.5L) ₃ -Me ₂ S (Me of 10M) ₂ S solution, 620 mL). The reaction mixture was taken up in N ₂ Stirring was carried out at 25℃for 24h. The reaction mixture was quenched by dropwise addition of MeOH (2L) at 0deg.C, and quenched under N ₂ The resulting mixture was stirred at 25 ℃ for 1h and then concentrated in vacuo. Purification by gradient silica gel column chromatography (petroleum ether: etOAc1:0 to 10:1) afforded methyl 2- (tert-butoxycarbonylamino) -3-cyclopropyl-propanoate (213 g, crude product) as a colorless oil.

LCMS (method a): m/z 144.2 (M-100+H) at 0.83 min.

¹ H NMR:(400MHz,DMSO-d ₆ )δ-0.05-0.01(1H,m),0.07-0.13(1H,m),0.35-0.41(2H,m),0.73-0.79(1H,m),1.33(1H,br s),1.38(9H,s),1.57-1.64(1H,m),3.61(3H,s),3.96-4.05(1H,m),7.26(1H,br d,J＝7.6Hz)。

Step 3: to methyl 2- (tert-butoxycarbonylamino) -3-cyclopropyl-propanoate (213 g,875 mmol) in methanol (600 mL), THF (600 mL) and H at 25 ℃ ₂ To the mixture in O (600 mL) was added LiOH H ₂ O (73.5 g,1.75 mol). Before vacuum concentration, at N ₂ The resulting mixture was stirred at 25℃for 2h. Aqueous HCl (1N) was added to adjust the pH to about 7, and the resulting mixture was extracted with ethyl acetate (600 ml×3). The combined organic layers were washed with brine (800 mL), dried over anhydrous Na ₂ SO ₄ Dried, filtered and concentrated in vacuo to give 2- (tert-butoxycarbonylamino) -3-cyclopropyl-propionic acid (197 g, crude product) as a colourless oil.

LCMS (method a): m/z 130.2 (M-100+H) at 0.73 min.

¹ H NMR:(400MHz,DMSO-d ₆ )δ-0.01(1H,br s),0.07(1H,br s),0.35(2H,br s),0.73(1H,br s),1.37(9H,br s),1.42-1.46(1H,m),1.54(1H,br s),3.81(1H,br s),6.64(1H,br s)。

Step 4: to a mixture of 2- (tert-butoxycarbonylamino) -3-cyclopropyl-propionic acid (197g, 859 mmol) in DMF (800 mL) was slowly added HOBt (139 g,1.03 mol) and EDCI. HCl (198g, 1.03 mol) at 25 ℃. After stirring at 25℃for about 12min, N-methoxymethylamine hydrochloride (101 g,1.03 mol) and Et were added ₃ N (104 g,1.03 mol), and at N ₂ The resulting mixture was stirred at 25℃for 2h. Adding H ₂ O (1.5L) and the resulting mixture was extracted with EtOAc (800 mL. Times.3). The combined organic layers were washed with brine (500 mL. Times.3), dried over anhydrous Na ₂ SO ₄ Dried, filtered and concentrated in vacuo. Purification by gradient silica gel column chromatography (petroleum ether: etOAc 1:0 to 10:1) afforded N- [1- (cyclopropylmethyl) -2- [ methoxy (methyl) amino as a white solid]-2-oxo-ethyl]Tert-butyl carbamate (209 g,0.77 mol).

LCMS (method a): m/z 173.2 (M-100+H) at 0.81 min.

¹ H NMR:(400MHz,DMSO-d ₆ )δ-0.06-0.01(1H,m),0.07-0.13(1H,m),0.35-0.41(2H,m),0.74-0.78(1H,m),1.14-1.22(1H,m),1.37(9H,s),1.58-1.66(1H,m),3.09(3H,s),3.73(3H,s),4.44(1H,br d,J＝3.2Hz),7.01(1H,br d,J＝8.4Hz)。

Step 5: at 0℃to N- [1- (cyclopropylmethyl) -2- [ methoxy (methyl) amino group]-2-oxo-ethyl]To a mixture of tert-butyl carbamate (189 g,659 mmol) in THF (1.5L) was slowly added LiAlH ₄ (25.0 g,659 mmol) and under N ₂ The reaction mixture was stirred at 25℃for 1h. Dropping H at 0 DEG C ₂ O (25 mL), the resulting mixture was filtered and the residue was washed with THF (1L). The filtrate was treated with anhydrous Na ₂ SO ₄ Dried, filtered and concentrated in vacuo. The residue was purified by gradient silica gel column chromatography (petroleum ether: etOAc 1:0 to 5:1) to give N- [1- (cyclopropylmethyl) -2-oxo-ethyl as a pale yellow oil]Tert-butyl carbamate (105 g, crude product).

LCMS (method A) M/z 114.2 (M-100+H) at 0.75 min.

¹ H NMR:(400MHz,DMSO-d ₆ )δ0.06-0.08(2H,m),0.38-0.40(2H,m),0.76-0.78(1H,m),1.40(9H,s),1.44-1.48(2H,m),3.86-3.92(1H,m),7.31(1H,br d,J＝7.2Hz),9.45(1H,s)。

Step 6: at 0℃to N- [1- (cyclopropylmethyl) -2-oxo-ethyl ]]Tert-butyl carbamate (121 g,567 mmol) in DCM (1L) was slowly added Et ₃ N (86.1 g,851mmol,118 mL) and 2-hydroxy-2-methylpropanenitrile (74.7 g,878mmol,80.2 mL). At N ₂ The reaction mixture was stirred at 25℃for 12h. By addition of saturated NaHCO at 0deg.C ₃ The reaction mixture was quenched with aqueous solution (500 mL) and the resulting mixture was extracted with DCM (300 ml×3). The combined organic layers were washed with brine (500 mL), dried over anhydrous Na ₂ SO ₄ Dried, filtered and concentrated in vacuo. Purification by gradient silica gel column chromatography (petroleum ether: etOAc 1:0 to 5:1) afforded N- [ 2-cyano-1- (cyclopropylmethyl) -2-hydroxy-ethyl as a pale yellow oil ]Tert-butyl carbamate (69.2 g, crude product).

¹ H NMR:(400MHz,DMSO-d ₆ )δ-0.06-0.01(1H,m),0.09-0.15(1H,m),0.35-0.41(2H,m),0.66-0.75(1H,m),1.33-1.35(1H,m),1.39(9H,s),1.48-1.56(1H,m),3.60-3.71(1H,m),6.49-6.60(1H,m),6.93-7.07(1H,m)。

Step 7: to N- [ 2-cyano-1- (cyclopropylmethyl) -2-hydroxy-ethyl at 0deg.C]Tert-butyl carbamate (71.0 g,295 mmol) and K ₂ CO ₃ (81.7 g,591 mmol) in DMSO (500 mL) in a slow drop of H ₂ O ₂ (449 g,3.96mol,380mL,30% purity, w/w). At N ₂ The reaction mixture was stirred at 0-25℃for 12h. By H ₂ The reaction mixture was diluted with O (1L) and quenched at 0deg.C by the addition of saturated Na ₂ S ₂ O ₃ (1L) aqueous solution was slowly quenched. The resulting mixture was acidified to about pH 8 with 1N aqueous HCl and then extracted with DCM (500 ml×3). The combined organic layers were washed with brine (500 mL) and saturated Na ₂ S ₂ O ₃ Aqueous (500 mL) wash with anhydrous Na ₂ SO ₄ Dried, filtered and concentrated in vacuo. Purification by gradient silica gel column chromatography (petroleum ether: etOAc 10:1 to 0:1) afforded N- [ 3-amino-1- (cyclopropylmethyl) -2-hydroxy-3-oxo-propyl]Tert-butyl carbamate (35.6 g,0.14 mol).

LCMS (method a): m/z 159.2 (M-100+H) at 0.70 min.

¹ H NMR:(400MHz,DMSO-d ₆ )δ0.01-0.05(2H,m),0.37(2H,br d,J＝7.6Hz),0.63-0.67(1H,m),1.36(9H,s),1.39(2H,s),3.77-3.82(1H,m),3.88-3.91(1H,m),5.44(1H,d,J＝6.4Hz),5.95(1H,br d,J＝9.2Hz),7.20(2H,br d,J＝10.8Hz)。

Step 8: HCl/EtOAc (4N, 34 mL) was added to N- [ 3-amino-1- (cyclopropylmethyl) -2-hydroxy-3-oxo-propyl ] at 25 ℃]A mixture of tert-butyl carbamate (5.60 g,21.7 mmol) in EtOAc (30 mL). At N ₂ The resulting mixture was stirred at 25℃for 1h. After concentration in vacuo, purification by preparative HPLC (HCl as additive) afforded 3-amino-4-cyclopropyl-2-hydroxy-butanamide hydrochloride (3.30 g,20.9 mmol) as a white solid.

LCMS (method B): m/z 158.9 (M+H) at 0.38 min.

¹ H NMR:(400MHz,DMSO-d ₆ )δ-0.06-0.13(2H,m),0.37-0.46(2H,m),0.74-0.85(1H,m),1.21-1.51(2H,m),3.45-3.47(1H,m),4.11-4.28(1H,m),6.18-6.57(1H,m),7.42-7.55(2H,m),7.95(1H,br s),8.25(1H,br s)。

Preparative HPLC (HCl) method for purification in step 8. Instrument: shimadzu LC-20AP; chromatographic column: phenomenex luna C18 250×80mm×10 μm; mobile phase: a=0.05% aqueous hcl (v/v), b=mecn; gradient: 0-10% B in A within 15 min; the flow rate is 150mL/min; column temperature: 40 ℃; wavelength: 220nm,254nm.

Step 9: to 3-amino-4-cyclopropyl-2-hydroxybutyramide (2.00 g,12.6 mmol) and (1R, 2S, 5S) -3-tert-butoxycarbonyl-6, 6-dimethyl-3-azabicyclo [3.1.0 ] at 25 ℃]To a mixture of hexane-2-carboxylic acid (2.97 g,11.6 mmol) in DMF (30 mL) was slowly added Et ₃ N (2.56 g,25.3mmol,3.52 mL) and T3P (12.1 g,19.0mmol,11.3mL,50% purity in EtOAc). At N ₂ The resulting mixture was stirred at 25℃for 2H, then H was added ₂ O (50 mL) and extracted with EtOAc (50 mL. Times.3). The combined organic layers were washed with brine (50 mL), dried over anhydrous Na ₂ SO ₄ Dried, filtered and concentrated in vacuo. By gradient silica gel column chromatography (petroleum ether: etOAc 10:1 to 1:1) followed by preparative HPLC (NH ₄ HCO ₃ As additive) to give (1 r,2s,5 s) -2- [ [ 3-amino-1- (cyclopropylmethyl) -2-hydroxy-3-oxo-propyl ] as a white solid]Carbamoyl radicals]-6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-3-carboxylic acid tert-butyl ester (1.43 g,3.62 mmol).

LCMS (method C): m/z 396.2 (M+H) at 0.99 min.

¹ H NMR:(400MHz,DMSO-d ₆ )δ-0.15-0.11(2H,m),0.23-0.41(2H,m),0.62-0.76(1H,m),0.83-0.87(1H,m),0.87-1.03(6H,m),1.27-1.36(9H,m),1.36-1.40(2H,m),1.42-1.62(1H,m),3.23-3.31(1H,m),3.43-3.58(1H,m),3.83-4.02(2H,m),4.03-4.23(1H,m),5.43-5.88(1H,m),7.12-7.29(2H,m),7.39-8.06(1H,m)。

Preparative HPLC (NH ₄ HCO ₃ ) The method. Instrument: shimadzu LC-20AP; chromatographic column: kromasil Eternity XT 250X 80mm X10 μm; mobile phase: a=10 mM NH ₄ HCO ₃ Aqueous solution (v/v), b=mecn; gradient: 37-67% B in A within 20 min; flow rate: 140mL/min; column temperature: 40 ℃; wavelength: 220nm,254nm.

Step 10: at 25 ℃, to (1R, 2S, 5S) -2- [ [ 3-amino-1- (cyclopropylmethyl) -2-hydroxy-3-oxo-propyl group]Carbamoyl radicals]-6, 6-dimethyl-3-azabicyclo [3.1.0]To a mixture of tert-butyl hexane-3-carboxylate (1.40 g,3.24 mmol) in EtOAc (5 mL) was added HCl/EtOAc (4N, 15.3 mL) and followed by N ₂ The resulting mixture was stirred at 25℃for 1h. Vacuum concentrating to obtain pale yellow solid (1R, 2S, 5S) -N- [ 3-amino-1- (cyclopropylmethyl) -2-hydroxy-3-oxo-propyl]-6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxamide (intermediate 1,1.15g,3.89 mmol).

LCMS (method C): m/z 296.1 (M+H) at 0.66 min.

¹ H NMR:(400MHz,DMSO-d ₆ )δ-0.16-0.17(2H,m),0.27-0.44(2H,m),0.60-0.72(1H,m),1.02-1.09(6H,m),1.29-1.64(2H,m),1.65-1.89(2H,m),3.02-3.07(1H,m),3.60(2H,br s),3.89-3.98(1H,m),4.18-4.29(1H,m),7.08-7.38(2H,m),8.35-8.72(1H,m),8.72-8.94(1H,m),9.87-10.23(1H,m)。

Intermediate 2: (1R, 2S, 5S) -N- [ 3-amino-1- (cyclopropylmethyl) -2-hydroxy-3-oxopropyl]-3- [ (2S) -2-amino-3-methyl-butyryl]-6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxamide

Step 1: to (1R, 2S, 5S) -N- [ 3-amino-1- (cyclopropylmethyl) -2-hydroxy-3-oxo-propyl group at 25 ℃]-6, 6-dimethyl-3-azabicyclo [3.1.0]Et is slowly added to a mixture of hexane-2-carboxamide intermediate 1 (280 mg,0.95 mmol) and (tert-butoxycarbonyl) -L-valine (309 mg,1.42 mmol) in DMF (3 mL) ₃ N (192 mg,1.90mmol,0.26 mL) and T3P (255 mg,1.42mmol,0.85mL,50% purity, w/w). At N ₂ Next, the obtained mixture was mixedThe mixture was stirred at 25℃for 12h. Adding H ₂ O (50 mL) and the resulting mixture was extracted with EtOAc (20 mL. Times.3). The combined organic layers were washed with brine (30 mL), dried over anhydrous Na ₂ SO ₄ Dried, filtered and concentrated in vacuo. The residue was purified by preparative HPLC (formic acid as additive) to give N- [ (1S) -1- [ (1 r,2S, 5S) -2- [ [ 3-amino-1- (cyclopropylmethyl) -2-hydroxy-3-oxo-propyl ] as a white solid]Carbamoyl radicals]-6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-3-carbonyl]-2-methyl-propyl ]Tert-butyl carbamate (183 mg,0.37 mmol).

LCMS (method C): m/z 495.2 (M+H), at 1.04 min.

¹ H NMR:(400MHz,DMSO-d ₆ )δ-0.15-0.08(2H,m),0.26-0.41(2H,m),0.63-0.72(1H,m),0.76-0.99(12H,m),1.00(2H,br s),1.14-1.30(1H,m),1.30-1.39(9H,m),1.40-1.45(1H,m),1.77-1.95(1H,m),3.70-3.84(2H,m),3.85-3.97(2H,m),4.04-4.29(2H,m),4.98-5.96(1H,m),6.88-7.00(1H,m),7.12-7.28(2H,m),7.37-7.81(1H,m)。

The preparative HPLC (formic acid) method for purification in step 1. Instrument: shimadzu LC-20AP; chromatographic column: phenomenex luna C18 150×40mm×15 μm; mobile phase: a=0.225% formic acid in water (v/v), b=mecn; gradient: 33-63% B in A within 10 min; flow rate: 60mL/min; column temperature: 40 ℃; wavelength: 220nm,254nm.

Step 2: HCl/EtOAc (4N, 3 mL) was added to N- [ (1S) -1- [ (1R, 2S, 5S) -2- [ [ 3-amino-1- (cyclopropylmethyl) -2-hydroxy-3-oxo-propyl ] at 25 ℃]Carbamoyl radicals]-6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-3-carbonyl]-2-methyl-propyl]Tert-butyl carbamate (150 mg,0.30 mmol) in a mixture of EtOAc (3 mL) and in N ₂ The resulting mixture was stirred at 25℃for 1h. The reaction mixture was concentrated in vacuo to give (1 r,2s,5 s) -N- [ 3-amino-1- (cyclopropylmethyl) -2-hydroxy-3-oxo-propyl ] as a pale yellow solid]-3- [ (2S) -2-amino-3-methyl-butyryl]-6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxamide (125 mg, crude product).

LCMS (method C): m/z 395.2 (M+H) at 0.73 min.

Intermediate 3 : (1R, 2S, 5S) -N- (4-amino-3-hydroxy-1- ((1R, 2S) -2-methylcyclopropyl) -4-oxobutan-2-yl) -6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxamide

Step 1: at N ₂ n-BuLi (2.5M in n-hexane, 344 mL) was added dropwise to a solution of ethyl 2-diethoxyphosphorylacetate (193 g,861mmol,171 mL) in 2-methyltetrahydrofuran (1L) at 25 ℃. After stirring the resulting mixture at 25℃for 30min, (2R) -2-methyl oxirane (50.0 g,861mmol,60.3 mL) was added at 25 ℃. The resulting mixture was stirred in a 5L autoclave at 150℃for 12h under a pressure of 15 Psi. The reaction mixture was cooled to 25℃and distilled at 100℃under reduced pressure (ca. 0.03 bar) to give the product of step 1 as a colourless oil, (2S) -2-methylcyclopropane carboxylate (91.0 g,710 mmol).

¹ H NMR:(400MHz,CDCl ₃ )δ0.63-0.69(1H,m),1.11(3H,d,J＝6.0Hz),1.13-1.18(1H,m),1.24-1.28(3H,m),1.34-1.44(2H,m),4.07-4.15(2H,m)。

Step 2: at N ₂ And at 0 ℃, alMe ₃ (2M in PhMe, 78.0mL,156.0 mmol) was added dropwise to a mixture of N-methoxymethylamine hydrochloride (15.2 g,156 mmol) in PhMe (100 mL). The resulting mixture was stirred at 25℃for 30min, then a solution of ethyl (2S) -2-methylcyclopropane carboxylate (10.0 g,78.0 mmol) in PhMe (150 mL) was added at 0 ℃. The resulting mixture was stirred at 25℃for 12H, then H was added ₂ O (100 mL). The aqueous phase was extracted with ethyl acetate (100 ml×3) and the combined organic phases were washed with brine (100 ml×3), dried (Na ₂ SO ₄ ) Filtered and concentrated in vacuo. The residue was purified by gradient silica gel column chromatography eluting with petroleum ether in ethyl acetate 20:1 to 5:1 to give (1 s,2 s) -N-methoxy-N, 2-dimethyl-cyclopropanecarboxamide as a yellow oil (3.00 g,21.0 mmol).

LCMS (method a): m/z 144.2 (M+H) at 0.60 min.

¹ H NMR:(400MHz,CDCl ₃ )δ0.61-0.68(1H,m),1.13(3H,d,J＝6.0Hz),1.16-1.21(1H,m),1.31-1.41(1H,m),1.78-1.90(1H,m),3.20(3H,s),3.75(3H,s)。

Step 3: at N ₂ And DIBAL-H (1M, 23.1mL,23.1mmol in PhMe) was added dropwise to a mixture of (1S, 2S) -N-methoxy-N, 2-dimethyl-cyclopropanecarboxamide (3.00 g,21.0 mmol) in THF (30 mL) at-70 ℃. The resulting mixture was stirred at-70℃for 30min. In addition, at N ₂ And n-BuLi (2.5M in n-hexane, 17.6mL,35.2 mmol) was added dropwise to a solution of methyl 2- (tert-butoxycarbonylamino) -2-dimethoxyphosphorylacetate (6.85 g,23.1 mmol) in THF (70 mL) at-70℃and the resulting mixture was stirred at-70℃for 30min and then added to the first reaction mixture at-70 ℃. After stirring at 25℃for 12h, water (100 mL) was added at 0℃and the resulting mixture was extracted with EtOAc (100 mL. Times.3). The combined organic layers were treated with anhydrous Na ₂ SO ₄ Dried, filtered and concentrated in vacuo. Purification by gradient silica gel column chromatography eluting with petroleum ether/ethyl acetate 10:1 to 5:1 afforded (E) -2- (tert-butoxycarbonylamino) -3- [ (1S, 2S) -2-methylcyclopropyl) as a yellow oil]Methyl prop-2-enoate (3.40 g,13.3 mmol).

LCMS (method a): m/z 200.1 (M-56+H) at 0.85 min.

¹ H NMR:(400MHz,CDCl ₃ )δ0.77-0.85(2H,m),1.04-1.11(1H,m),1.12-1.17(3H,m),1.35-1.42(1H,m),1.48(9H,s),3.75(3H,s),5.74-5.96(1H,m),6.06(1H,d,J＝10.8Hz)。

Step 4-12: (1R, 2S, 5S) -N- (4-amino-3-hydroxy-1- ((1R, 2S) -2-methylcyclopropyl) -4-oxobutan-2-yl) -6, 6-dimethyl-3-azabicyclo [3.1.0 ] using procedures similar to those detailed for the preparation of intermediate 1 (steps 2-10) from methyl (E) -2- (tert-butoxycarbonylamino) -3- [ (1S, 2S) -2-methylcyclopropyl) prop-2-enoate]Hexane-2-carboxamide hydrochloride (intermediate 3,0.39g,1.26mmol, pale yellow solid).

LCMS (method B): m/z 310.2 (M+H) at 1.00 min.

¹ H NMR:(400MHz,CD ₃ OD)δ0.10-0.61(4H,m),0.94-1.05(3H,m),1.11-1.19(6H,m),1.21-1.49(2H,m),1.52-1.70(1H,m),1.78-1.85(1H,m),3.21-3.30(1H,m),3.71-3.79(1H,m),4.08-4.20(2H,m),4.29-4.48(1H,m)。

Intermediate 4: isobutyryl-L-valine

Step 1: to a mixture of methyl (2S) -2-amino-3-butanoate hydrochloride (5.00 g,29.8 mL) in DCM (50 mL) was added DIPEA (11.6 g,89.5mmol,15.6 mL) and 2-methylpropanoyl chloride (3.81 g,35.8 mmol) at 0deg.C. The resulting mixture was stirred at 25℃for 12h, then DCM (100 mL) and water (80 mL) were added. The organic phase was washed with water (2X 80 mL) and dried over anhydrous Na ₂ SO ₄ Dried, filtered and concentrated in vacuo to give methyl (2S) -3-methyl-2- (2-methylpropanamido) butyrate (6.00 g,29.8 mmol) as a yellow oil.

LCMS (method a): m/z 202.2 (M+H) at 0.35 min.

Step 2: at 0 ℃, liOH.H ₂ O (2.50 g,59.6 mmol) was added to methyl (2S) -3-methyl-2- (2-methylpropanamido) butyrate (4.00 g,19.9 mmol) in THF (20 mL), meOH (6 mL) and H ₂ O (6 mL) and the resulting mixture was stirred at 25℃for 1h. Aqueous 2N HCl was added to adjust to pH about 7, followed by EtOAc (100 mL). The phases were separated and the organic phase was washed with water (80 mL. Times.3) and dried over anhydrous Na ₂ SO ₄ Dried, filtered and concentrated in vacuo. Purification by preparative HPLC (formic acid as additive) afforded (2S) -3-methyl-2- (2-methylpropanamino) butanoic acid as a white solid (intermediate 4,1.60g,8.55 mmol).

LCMS (method a): m/z 188.2 (M+H) at 0.37 min.

¹ H NMR:(400MHz,DMSO-d ₆ )δ0.96-0.99(6H,m),1.11-1.14(6H,m),2.12-2.20(1H,m),2.56-2.64(1H,m),4.00(1H,d,J＝6.0Hz)。

A preparative HPLC (formic acid) method for purifying intermediate 4. Instrument: shimadzu LC-20AP; chromatographic column: phenomenex Synergi Max-RP C18 is 250 multiplied by 50mm multiplied by 10 mu m; mobile phase: a=0.225% formic acid in water (v/v), b=mecn; gradient: 1-30% B in A within 20 min; flow rate: 150mL/min; column temperature: 40 ℃; wavelength: 220nm,254nm.

Intermediate 5: (1R, 2S, 5S) -3- (isobutyryl-L-valyl) -6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxylic acid

Step 1: HATU (10.5 g,27.7 mmol) was added to a solution of (tert-butoxycarbonyl) -L-valine (5.00 g,23.0 mmol) in DMF (30 mL) and the mixture was stirred at room temperature for 10min. Adding (1R, 2S, 5S) -6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxylic acid methyl ester hydrochloride (5.66 g,27.7 mmol) and DIPEA (11.9 ml,69.1 mmol) and the reaction mixture was stirred at room temperature for 2h. The reaction mixture was partitioned between cold water (250 mL) and EtOAc (100 mL), the aqueous phase was extracted with EtOAc (2×70 mL), and the combined organic phases were dried (Na ₂ SO ₄ ). After concentration in vacuo, purification by reverse phase gradient flash column chromatography on C18 silica eluting with 0-50% MeCN in water afforded (1R, 2S, 5S) -3- ((tert-butoxycarbonyl) -L-valyl) -6, 6-dimethyl-3-azabicyclo [ 3.1.0) as a yellow viscous solid]Hexane-2-carboxylic acid methyl ester (5.90 g,16.0 mmol).

LCMS (method F): m/z 269.3 (M-100), at 2.24 min.

Step 2: TFA (6 mL) was added dropwise to (1R, 2S, 5S) -3- ((tert-butoxycarbonyl) -L-valyl) -6, 6-dimethyl-3-azabicyclo [ 3.1.0) at 0deg.C]A solution of hexane-2-carboxylic acid methyl ester (5.90 g,16.0 mmol) in DCM (50 mL). After stirring at room temperature for 2h, concentration in vacuo afforded (1R, 2S, 5S) -3- (L-valyl) -6, 6-dimethyl-3-azabicyclo [ 3.1.0) as a yellow viscous solid ]Hexane-2-carboxylic acid methyl ester triacetate.

LCMS (method F): m/z 269.3 (M+H) at 0.93 min.

Step 3: et at 0 ℃ ₃ N (7.17 mL,51.0 mmol) was added dropwise to (1R, 2S, 5S) -3- (L-valyl) -6, 6-dimethyl-3-azabicyclo [ 3.1.0)]Hexane-2-carboxylic acid methyl ester trifluoroacetate (6.50 g,17.0 mmol) in THF (100 mL) and the reaction mixture was stirred at 0deg.C for 10min. Isobutyryl chloride (1.80 mL,17.0 mmol) was added, after stirring at room temperature for 1h, water (250 mL) and ethyl acetate (100 mL) were added, and the phases were separated. The aqueous layer was extracted with EtOAc (2×100 mL) and the combined organic layers were dried (Na ₂ SO ₄ ) And concentrated in vacuo. Purification by reverse phase gradient flash column chromatography on C18 silica gel eluting with 0-40% MeCN in water afforded (1R, 2S, 5S) -3- (isobutyryl-L-valyl) -6, 6-dimethyl-3-azabicyclo [ 3.1.0) as a white solid]Hexane-2-carboxylic acid methyl ester (4.10 g,12.1 mmol).

LCMS (method F): m/z 339.3 (M+H), at 1.79 min.

Step 4: liOH.H ₂ O (2.48 g,60.6 mmol) was added to (1R, 2S, 5S) -3- (isobutyryl-L-valyl) -6, 6-dimethyl-3-azabicyclo [ 3.1.0)]Hexane-2-carboxylic acid methyl ester (4.10 g,12.1 mmol) in THF (20 mL) and water (10 mL) and the mixture was stirred at room temperature for 2h. Water was added and the mixture was acidified to pH about 5 with ice AcOH (20 mL). The aqueous layer was extracted with 10% MeOH in DCM (3X 200 mL). The combined organic layers were dried (Na ₂ SO ₄ ) And concentrated in vacuo to give (1R, 2S, 5S) -3- (isobutyryl-L-valyl) -6, 6-dimethyl-3-azabicyclo [3.1.0 ] as a white solid]Hexane-2-carboxylic acid (intermediate 5,3.30g,10.2 mmol) was used without further purification.

LCMS (method F): m/z 325.3 (M+H), at 1.49 min.

Intermediate 6: 3-amino-N-benzyl-4-cyclopropyl-2-hydroxybutyramide

Step 1: (S) -2- (((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -3-cyclopropylpropionic acid (12.0 g,34.2 mmol), EDCI. HCl (7.20 g,37.5 mmol) and HOBt (5.06 g,37.5 mmol) were held in the chamberDissolve in DMF (20 mL) at temperature and stir for 20min. N, O-dimethylhydroxylamine hydrochloride (3.65 g,37.5 mmol) and Et were added ₃ N (5.26 mL,37.5 mmol) and the reaction mixture was stirred at room temperature for 2h. After partitioning between water (200 mL) and EtOAc (100 mL), the aqueous layer was extracted with EtOAc (2X 70 mL). The combined organic layers were dried (Na ₂ SO ₄ ) And concentrated in vacuo. Trituration with n-hexane afforded (9H-fluoren-9-yl) methyl (S) - (3-cyclopropyl-1- (methoxy (methyl) amino) -1-oxopropan-2-yl) carbamate (12.0 g,30.4 mmol) as a pale brown oil.

LCMS (method F): m/z 417.1 (M+Na), at 2.28 min.

Step 2: at N ₂ Lithium aluminum hydride (2M in THF, 15.0mL,30.0 mmol) was added dropwise to a solution of (9H-fluoren-9-yl) methyl (S) - (3-cyclopropyl-1- (methoxy (methyl) amino) -1-oxopropan-2-yl) carbamate (12.0 g,30.5 mmol) in dry THF (50 mL) at-78deg.C. At N ₂ And stirring at-78deg.C for 2 hr, adding saturated NH ₄ Aqueous Cl (70 mL). The mixture was filtered through celite and the residue was washed with EtOAc (100 mL). The filtrate was concentrated in vacuo to give the crude (9H-fluoren-9-yl) methyl (S) - (1-cyclopropyl-3-oxopropan-2-yl) carbamate (9.20 g) as a pale yellow solid, which was used in the next step without purification.

LCMS (method G): m/z 336.6 (M+H), at 2.33 and 2.70 min.

Step 3: benzyl isonitrile (3.56 g,30.2 mmol) and glacial AcOH (4.71 mL,82.4 mmol) were added to a solution of (9H-fluoren-9-yl) methyl (S) - (1-cyclopropyl-3-oxopropan-2-yl) carbamate (9.20 g,27.5 mmol) in DCM (30 mL) at 0deg.C. After stirring at room temperature for 2h, 1N aqueous HCl (20 mL), water (70 mL) and DCM (100 mL) were added. The phases were separated and the aqueous phase was extracted with DCM (2X 100 mL). The combined organic layers were washed with saturated NaHCO ₃ Aqueous (100 mL) was washed and dried (Na ₂ SO ₄ ). After filtration and concentration in vacuo, trituration with n-hexane afforded (3S) -3- (((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -1- (benzylamino) -4-cyclopropyl-1-oxobutan-2-yl acetate (14.0 g,2.73 mmol) as a white solid.

LCMS (method F): m/z 513.0 (M+H), at 2.50 and 2.53 min.

Step 4 : et is added to ₃ N (8.00 mL,54.6 mmol) was added to a solution of (3S) -3- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -1- (benzylamino) -4-cyclopropyl-1-oxobutan-2-yl acetate (14.0 g,27.3 mmol) in MeOH (200 mL) and the reaction mixture stirred at room temperature for 2H. After concentration in vacuo, the resulting solid was suspended in water (100 mL) and then filtered. After drying, trituration with n-hexane afforded (9H-fluoren-9-yl) methyl ((2S) -4- (benzylamino) -1-cyclopropyl-3-hydroxy-4-oxobutan-2-yl) carbamate (10.0 g,21.3 mmol) as a white solid.

LCMS (method F): m/z 471.3 (M+H), at 2.43 and 2.46 min.

Step 5: (9H-fluoren-9-yl) methyl ((2S) -4- (benzylamino) -1-cyclopropyl-3-hydroxy-4-oxobutan-2-yl) carbamate (10.0 g,21.3 mmol) was dissolved in 20% piperidine in DMF (100 mL) and stirred at room temperature for 40min. After dilution with cold water (500 mL), the resulting suspension was filtered through celite. The filtrate was concentrated in vacuo and triturated with MeCN (70 mL) to give (3S) -3-amino-N-benzyl-4-cyclopropyl-2-hydroxybutyramide (intermediate 6,3.00g,7.01 mmol) as a white solid.

LCMS (method F): m/z 249.3 (M+H), at 0.83 and 0.91 min.

¹ H NMR:(400MHz,DMSO-d ₆ )δ-0.14--0.19(m,1H),-0.03--0.06(m,1H),0.28-0.33(m,1H),0.35-0.39(m,1H),0.77-0.81(q,1H,J＝5.2Hz),0.91-0.97(m,1H),1.24-1.31(m,1H),1.53(br s,2H),2.93-2.95(t,1H,J＝4.4Hz),3.83(s,1H),4.21-4.32(m,2H),5.56(s,1H),7.22-7.319(m,5H),8.29-8.32(t,1H,J＝6.0Hz).

Intermediate 7: 3-amino-4-cyclopropyl-2-hydroxybutyric acid methyl ester hydrochloride

Step 1: at N ₂ And (S) -2- ((tert-butoxycarbonyl) amino) -3-cyclopropylpropionic acid (2.42 g,10.6 mmol) was dissolved at 0 ℃CSolution in PhMe (25 mL). CDI (1.75 g,10.8 mmol) was added at 0deg.C and the reaction mixture was stirred at 0deg.C for 2h. N, O-dimethylhydroxylamine hydrochloride (1.34 g,13.7 mmol) and DIPEA (1.86 mL,10.8 mmol) were added at 0deg.C and the reaction mixture was stirred at room temperature for 16h. After partitioning between water (50 mL) and EtOAc (50 mL), the aqueous phase was extracted with EtOAc (2X 50 mL). The combined organic phases were dried (Na ₂ SO ₄ ) Filtration and concentration in vacuo gave (S) - (3-cyclopropyl-1- (methoxy (methyl) amino) -1-oxopropan-2-yl) carbamic acid tert-butyl ester (2.00 g,7.35 mmol) as an off-white solid.

LCMS (method F2): m/z 273.1 (M+H) at 2.04 min.

Step 2: at N ₂ And at 0 ℃, liAlH ₄ (2.5M in THF, 2.95mL,7.35 mmol) was added to a solution of tert-butyl (S) - (3-cyclopropyl-1- (methoxy (methyl) amino) -1-oxopropan-2-yl) carbamate (2.00 g,7.35 mmol) in THF (20 mL) and the reaction mixture stirred at room temperature for 1h. At saturation NH ₄ After partitioning between aqueous Cl (50 mL) and EtOAc (60 mL), the aqueous phase was extracted with EtOAc (2X 60 mL). The combined organic phases were dried (Na ₂ SO ₄ ) And concentrated in vacuo to give tert-butyl (1-cyclopropyl-3-oxopropan-2-yl) carbamate (1.30 g,6.10 mmol) as a yellow viscous material, which was used without further purification.

TLC: rf0.6 (EtOAc/hexane, 3:7).

Step 3: sodium carbonate (1.94 g,18.3 mmol) was added to a solution of tert-butyl (1-cyclopropyl-3-oxopropan-2-yl) carbamate (1.30 g,6.09 mmol) in PhMe (5 mL) and water (2 mL) and the reaction mixture was stirred at room temperature for 15min. Acetone cyanohydrin (0.84 mL,9.14 mmol) was added and the reaction mixture was stirred at room temperature for 1h. After dilution with water (20 mL) and acidification to pH of about 5 with 2N HCl (8 mL), the mixture was extracted with EtOAc (3X 60 mL). The combined organic phases were dried (Na ₂ SO ₄ ) And concentrated in vacuo. Purification by flash column chromatography on a normal phase gradient of silica eluting with 0-20% EtOAc in n-hexane afforded tert-butyl (1-cyano-3-cyclopropyl-1-hydroxypropan-2-yl) carbamate as a brown viscous material (1.00 g,4.16 mmol).

LCMS (method F2): m/z 185.2 (M-56) at 1.68 min.

Step 4: trimethylchlorosilane (2 mL,15.7 mmol) was added to a solution of tert-butyl (1-cyano-3-cyclopropyl-1-hydroxypropyl-2-yl) carbamate (1.00 g,4.16 mmol) in MeOH (15 mL) and the mixture stirred at 60℃for 3h. After concentration in vacuo, purification by reverse phase gradient flash column chromatography (silica gel C18) eluting with 0-12% MeCN in water afforded methyl 3-amino-4-cyclopropyl-2-hydroxybutyrate hydrochloride as a yellow viscous material (0.80 g,3.81 mmol).

LCMS (method F2): m/z 174.0 (M+H), at 0.36min, the purity was about 69%.

Intermediate 8: 4-cyclopropyl-2-hydroxy-3- ((1R, 2S, 5S) -3- (isobutyryl-L-valyl) -6, 6-dimethyl-3-azabicyclo [ 3.1.0)]Hexane-2-carboxamido) butyric acid

Step 1: EDCl & HCl (0.35 g,1.85 mmol) and HOBt (0.21 g,1.54 mmol) were added to (1R, 2S, 5S) -3- (isobutyryl-L-valyl) -6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxylic acid (intermediate 5,0.50g,1.54 mmol) and 3-amino-4-cyclopropyl-2-hydroxybutyric acid methyl ester hydrochloride (intermediate 7,0.35g,1.70 mmol) in EtOAc (5 mL) and water (0.5 mL). After stirring at room temperature for 16h, the reaction mixture was partitioned between water (30 mL) and EtOAc (50 mL). The phases were separated and the aqueous phase was extracted with EtOAc (2X 50 mL). The combined organic phases were dried (Na ₂ SO ₄ ) Filtered and concentrated in vacuo. Purification by reverse phase gradient flash column chromatography (silica gel C18), eluting with 0-40% MeCN in water, afforded 4-cyclopropyl-2-hydroxy-3- ((1R, 2S, 5S) -3- (isobutyryl-L-valyl) -6, 6-dimethyl-3-azabicyclo [ 3.1.0) as an off-white solid]Hexane-2-carboxamide) methyl butyrate (0.40 g,0.83 mmol).

LCMS (method F): m/z 480.3 (M+H) at 1.56 min.

Step 2: liOH monohydrate [ ]89mg,2.18 mmol) was added to 4-cyclopropyl-2-hydroxy-3- ((1R, 2S, 5S) -3- (isobutyryl-L-valyl) -6, 6-dimethyl-3-azabicyclo [ 3.1.0)]A solution of methyl hexane-2-carboxamido) butyrate (0.21 g,0.43 mmol) in 1, 4-dioxane (1 mL) and water (1 mL). After stirring at room temperature for 3h, water (50 mL) was added and the reaction mixture was extracted with EtOAc (2X 30 mL). The aqueous phase was acidified to pH of about 2 with 1N HCl (1.5 mL) and extracted with EtOAc (3X 30 mL). The combined organic phases were dried (Na ₂ SO ₄ ) And concentrated in vacuo to give 4-cyclopropyl-2-hydroxy-3- ((1R, 2S, 5S) -3- (isobutyryl-L-valyl) -6, 6-dimethyl-3-azabicyclo [ 3.1.0) as a brown viscous material]Hexane-2-carboxamide) butyric acid (0.18 g,0.39 mmol).

LCMS (method F2): m/z 466.1 (M+H), at 1.69, 1.77 and 1.83 min.

Intermediate 9: n- ((1S) -1- ((1R, 2S, 5S) -2- ((3-amino-1- (cyclopropylmethyl) -2-hydroxy-3-oxo-propyl) carbamoyl) -6, 6-dimethyl-3-azabicyclo [ 3.1.0)]Hexane-3-carbonyl) -2-methyl-2-phenyl-propyl-carbamic acid tert-butyl ester

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Step 1: at N ₂ and-78deg.C, et is added dropwise to a mixture of 3-methyl-3-phenyl-butyric acid (4.50 g,25.3 mmol) in THF (60 mL) ₃ N (3.83 g,37.9mmol,5.27 mL) and 2, 2-dimethylpropionyl chloride (3.35 g,27.8mmol,3.42 mL). The reaction mixture was stirred at-78 ℃ until a white solid formed. The reaction mixture was warmed to 0 ℃ and stirred for 1h, then cooled to-78 ℃ (mixture a). At N ₂ And n-BuLi (2.5M in n-hexane, 20.2 mL) (mixture B) was added dropwise to a solution of (4S) -4-isopropyloxazolidin-2-one (6.52 g,50.5 mmol) in THF (80 mL) at-78deg.C. Mixture B was added to mixture a at-78 ℃ and the reaction mixture was stirred at-78 ℃ for 2h and then at 25 ℃ for 12h. The reaction mixture was treated with saturated NH at 0deg.C ₄ Aqueous Cl (100 mL) was slowly quenched and then stirred at 25℃for 1h. The mixture was washed with ethyl acetate (50 mL. Times.3)) And (5) extracting. The combined organic phases were washed with brine (100 mL), dried over anhydrous Na ₂ SO ₄ Dried, filtered and concentrated in vacuo. The residue was purified by gradient flash column chromatography on silica gel eluting with petroleum ether to petroleum ether/ethyl acetate 5/1 followed by preparative reverse phase HPLC (formic acid as additive) to give (4S) -4-isopropyl-3- (3-methyl-3-phenyl-butyryl) oxazolidin-2-one (6.15 g,21.3 mmol) as a yellow oil.

LCMS (method a): m/z 290.2 (M+H) at 0.91 min.

¹ H NMR:(400MHz,CDCl ₃ )δ0.74(3H,d,J＝6.8Hz),0.81(3H,d,J＝6.8Hz),1.50(6H,d,J＝1.6Hz),2.12-2.20(1H,m),3.36(2H,d,J＝4.0Hz),4.01-4.09(2H,m),4.20-4.24(1H,m),7.15-7.21(1H,m),7.27-7.33(2H,m),7.37-7.42(2H,m).

A preparative reverse phase HPLC (formic acid) method for purifying intermediate 9, step 1. Instrument: shimadzu LC-20AP; chromatographic column: phenomenex Luna C18 250×80mm×15 μm; mobile phase: a=0.225% formic acid in water (v/v), b=mecn; gradient: 55-85% B in A within 35 min; flow rate: 140mL/min; column temperature: 40 ℃; wavelength: 220nm,254nm.

Step 2: at N ₂ And KHMDS (1M in THF, 22.8 mL) was added dropwise to a mixture of (4S) -4-isopropyl-3- (3-methyl-3-phenyl-butyryl) oxazolidin-2-one (6.00 g,20.7 mmol) in THF (50 mL) at-78deg.C. The reaction mixture was stirred at-78 ℃ for 1h. A solution of N-diazo-2, 4, 6-triisopropyl-benzenesulfonamide (8.02 g,25.9 mmol) in THF (30 mL) was then added dropwise at-78deg.C. The reaction mixture was stirred at-78℃for 0.5h, then HOAc (5.73 g,95.4mmol,5.46 mL) was added at-78 ℃. The reaction mixture was stirred at 40℃for 2h, then at 0℃with NH ₄ Aqueous Cl (100 mL) was quenched and extracted with ethyl acetate (50 mL. Times.3). The combined organic phases were washed with brine (50 mL), saturated aqueous sodium bicarbonate (50 mL), and with anhydrous Na ₂ SO ₄ Dried, filtered and concentrated in vacuo. Purification by gradient flash column chromatography on silica gel eluting with petroleum ether to petroleum ether/ethyl acetate 20/1 afforded (4S) -3- [ (2S) -2-azido-3-methyl-3-phenyl-butanoyl as a pale yellow oil ]-4-isopropyl-oxazolidin-2-one (5.92 g,17.9 mmol).

LCMS (method a): m/z 303.2 (M+H-28) +, at 0.94 min.

¹ H NMR:(400MHz,CDCl ₃ )δ0.83(6H,dd,J＝15.6,7.2Hz),1.54-1.56(6H,m),2.28-2.34(1H,m),3.57(1H,t,J＝8.4Hz),3.87-3.91(1H,m),3.97(1H,dd,J＝8.8,2.0Hz),5.66(1H,s),7.25-7.28(1H,m),7.30-7.36(2H,m),7.38-7.43(2H,m)。

Step 3: at H ₂ (15 PSI) under reduced pressure, (4S) -3- [ (2S) -2-azido-3-methyl-3-phenyl-butyryl]-4-isopropyl-oxazolidin-2-one (5.70 g,17.3 mmol), boc ₂ A mixture of O (8.28 g,38.0mmol,8.72 mL) and Pd/C (500 mg,10% purity, w/w) in EtOAc (50 mL) was stirred at 25℃for 12h. The reaction mixture was filtered and the filtrate was concentrated in vacuo. The residue obtained is purified by gradient flash column chromatography on silica gel eluting with petroleum ether to petroleum ether/ethyl acetate 10/1, followed by purification by preparative HPLC (formic acid as additive) and then by normal phase preparative HPLC (NH ₃ ·H ₂ O as additive) to give N- [ (1S) -1- [ (4S) -4-isopropyl-2-oxo-oxazolidine-3-carbonyl as a pale yellow oil]-2-methyl-2-phenyl-propyl]Tert-butyl carbamate (6.50 g,16.1 mmol).

LCMS (method a): m/z 405.2 (M+H) at 0.95 min.

¹ H NMR:(400MHz,CDCl ₃ )δ0.76-0.83(6H,m),1.41(3H,s),1.43(9H,s),1.48(3H,s),2.21-2.29(1H,m),3.45(1H,t,J＝8.4Hz),3.79-3.83(1H,m),3.90(1H,dd,J＝8.8,2.0Hz),4.86-5.32(1H,m),6.13(1H,d,J＝10.0Hz),7.20-7.26(1H,m),7.28-7.34(2H,m),7.41(2H,d,J＝7.6Hz)。

A preparative reverse phase HPLC (formic acid) method for purifying intermediate 9, step 3. Instrument: shimadzu LC-20AP; chromatographic column:Phenomenex Luna C18 250×80mm×10μmthe method comprises the steps of carrying out a first treatment on the surface of the Mobile phase: a=0.225% formic acid in water (v/v), b=mecn; gradient: 48-78% B in A within 20 min; flow rate: 100mL/min; column temperature: 40 ℃; wavelength: 220nm,254nm.

Preparative normal phase HPLC (NH ₃ ·H ₂ O) method, step 3. Instrument: shimadzu LC-20AP; chromatographic column: welch Ultimate XB-SiOH 250X 50mm X10 μm; mobile phase: a=hexane, b=0.1% nh ₃ ·H ₂ O in EtOH (v/v), 2% B in A over 15 min; flow rate: 100mL/min; column temperature: 40 ℃; wavelength: 220nm,254nm.

Step 4: at N ₂ And at 0deg.C, to N- [ (1S) -1- [ (4S) -4-isopropyl-2-oxo-oxazolidine-3-carbonyl]-2-methyl-2-phenyl-propyl]Tert-butyl carbamate (3.00 g,7.42 mmol) and aqueous 1NLiOH (22.3 mL) in THF (80 mL) and H ₂ H was added dropwise to the mixture in O (20 mL) ₂ O ₂ Aqueous (10.1 g,89.0mmol,8.60 mL). The reaction mixture was stirred at 25℃for 12H, then H was added at 0 ℃ ₂ O (100 mL) and saturated Na ₂ SO ₃ Aqueous solution (300 mL). The mixture was stirred at 25 ℃ for 1h, then the pH was adjusted to about 7 by adding 1N aqueous HCl and the mixture was taken up in CH ₂ Cl ₂ (50 mL. Times.3) extraction. The combined organic phases were washed with brine (100 mL) and saturated Na ₂ SO ₃ Aqueous (100 mL) wash with anhydrous Na ₂ SO ₄ Dried, filtered and concentrated in vacuo. Purification on silica gel by gradient flash column chromatography eluting with petroleum ether/ethyl acetate (10/1 to 1/1) followed by preparative HPLC (formic acid as additive) and preparative HPLC (NH) ₄ HCO ₃ As an additive), to give (2S) -2- (tert-butoxycarbonylamino) -3-methyl-3-phenyl-butyric acid (1.53 g,5.22 mmol) as a pale yellow solid.

LCMS (method A2): m/z 194.1 (M+H-100) ⁺ At 2.87 min.

¹ H NMR:(400MHz,DMSO-d ₆ )δ1.28(9H,s),1.30(6H,d,J＝5.6Hz),4.01(1H,d,J＝8.8Hz),5.86(1H,d,J＝9.2Hz),7.07-7.13(1H,m),7.22(2H,t,J＝7.6Hz),7.32(2H,d,J＝7.6Hz)。

A preparative reverse phase HPLC (formic acid) method for purifying intermediate 9, step 4. Instrument: shimadzu LC-20AP; chromatographic column: phenomenex Luna C18 250X 70mm X10 μm; mobile phase: a=0.225% formic acid in water (v/v), b=mecn; gradient: 30-60% B in A within 20 min; flow rate: 140mL/min; column temperature: 40 ℃; wavelength: 220nm,254nm.

Preparative reverse phase HPLC (NH ₄ HCO ₃ ) The method comprises a step 4. Instrument: shimadzu LC-20AP; chromatographic column: waters Xbridge 150X 25mm X5 μm; mobile phase: a=10 mM NH ₄ HCO ₃ Aqueous solution (v/v), b=mecn; gradient: 14-44% B in A within 10 min; flow rate: 25mL/min; column temperature: 40 ℃; wavelength: 220nm,254nm.

Step 5: a mixture of (2S) -2- (tert-butoxycarbonylamino) -3-methyl-3-phenylbutyric acid (intermediate 9, step 4 product, 669mg,2.28 mmol), HOBt (308 mg,2.28 mmol) and EDCI (437 mg,2.28 mmol) in DMF (5 mL) was stirred at 25℃for 0.2h. Adding (1R, 2S, 5S) -N- [ 3-amino-1- (cyclopropylmethyl) -2-hydroxy-3-oxopropyl ]-6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxamide (intermediate 1, 40mg,1.52 mmol) and Et ₃ N (308 mg,3.04mmol,0.42 mL) and the reaction mixture was stirred at 25℃for 2h. The reaction mixture was treated with H ₂ O (50 mL) was diluted and the mixture was extracted with EtOAc (30 mL. Times.3). The combined organic phases were washed with brine (30 mL), dried over anhydrous Na ₂ SO ₄ Dried, filtered and concentrated in vacuo. The residue was purified by preparative HPLC (formic acid as additive) and then by preparative HPLC (NH ₄ HCO ₃ As additive) to yield N- ((1S) -1- ((1 r,2S, 5S) -2- ((3-amino-1- (cyclopropylmethyl) -2-hydroxy-3-oxo-propyl) carbamoyl) -6, 6-dimethyl-3-azabicyclo [ 3.1.0) as a pale yellow solid]Hexane-3-carbonyl) -2-methyl-2-phenyl-propyl) carbamic acid tert-butyl ester (intermediate 9, 42.0mg,0.07 mmol).

LCMS (method a): m/z 571.2 (M+H) ⁺ At 0.90 min.

¹ H NMR:(400MHz,CDCl ₃ )δ0.06-0.22(2H,m),0.40-0.58(2H,m),0.66-0.76(1H,m),0.76-0.89(3H,m),0.96-0.99(3H,m),1.22-1.37(2H,m),1.38-1.43(15H,m),1.44-1.48(2H,m),3.69-3.86(1H,m),4.20-4.33(2H,m),4.61-4.76(1H,m),4.92-5.28(1H,m),5.46-5.71(1H,m),7.29-7.43(5H,m)。

A preparative reverse phase HPLC (formic acid) method for purifying intermediate 9, step 5. Instrument: shimadzu LC-20AP; chromatographic column: phenomenex Luna C18 75X 30mm X3 μm; mobile phase: a=0.225% formic acid in water (v/v), b=mecn; gradient: 45-75% B in A within 7 min; flow rate: 25mL/min; column temperature: 40 ℃; wavelength: 220nm,254nm.

Preparative reverse phase HPLC (NH ₄ HCO ₃ ) The method comprises a step 5. Instrument: shimadzu LC-20AP; chromatographic column: waters Xbridge 150X 25mm X5 μm; mobile phase: a=10 mM NH ₄ HCO ₃ Aqueous solution (v/v), b=mecn; gradient: 32-62% B in A within 10 min; flow rate: 25mL/min; column temperature: 40 ℃; wavelength: 220nm,254nm.

Intermediate 10: 4-cyclopropyl-2-hydroxy-3- ((1 r,2S, 5S) -3- ((S) -2-isobutyramide-3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [ 3.1.0)]Hexane-2-carboxamido) butyric acid

Step 1: (S) -2- ((tert-Butoxycarbonyl) amino) -3, 3-dimethylbutyric acid (5.00 g,21.6 mmol) and (1R, 2S, 5S) -6, 6-dimethyl-3-azabicyclo [ 3.1.0)]Hexane-2-carboxylic acid methyl ester hydrochloride (5.34 g,26.0 mmol) was dissolved in DMF (30 mL), HATU (9.89 g,26.0 mmol) was added and the reaction mixture was stirred at room temperature for 30min, then DIPEA (11.3 mL,64.9 mmol) was added and the reaction mixture was stirred at room temperature for 3h. Cold water (100 mL) was added and the resulting precipitate was collected by filtration to give the crude product (1R, 2S, 5S) -3- ((S) -2- ((tert-butoxycarbonyl) amino) -3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [3.1.0 ] as a brown viscous solid ]Hexane-2-carboxylic acid methyl ester (9.30 g).

LCMS (method F3): m/z 327.4 (M-56), at 2.35 and 2.37 min.

Step 2: at N ₂ Next, a solution of 4N HCl in 1, 4-dioxane (90 mL) was added to (1R, 2S, 5S) -3- ((S) -2- ((tert-butoxycarbonyl) amino) -3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxylic acid methyl ester (9.30 g) in DCM (30mL). After stirring at room temperature for 2h, the reaction mixture was concentrated in vacuo and purified by trituration with ether (3X 10 mL) to give (1R, 2S, 5S) -3- ((S) -2-amino-3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [ 3.1.0) as a white solid]Hexane-2-carboxylic acid methyl ester hydrochloride (6.70 g,21.0 mmol).

LCMS (method F3): m/z 283.5 (M+H), at 1.14 and 1.28 min.

Step 3: et at 0 ℃ ₃ N (8.80 mL,63.2 mmol) was added dropwise to (1R, 2S, 5S) -3- ((S) -2-amino-3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [ 3.1.0)]Hexane-2-carboxylic acid methyl ester hydrochloride (6.70 g,21.0 mmol) in THF (40 mL) and the reaction mixture was stirred for 15min. Isobutyryl chloride (2.23 g,21.1 mmol) was added and the reaction mixture was stirred at room temperature for 30min, then water (200 mL) was added and extracted with EtOAc (3X 100 mL). The combined organic phases were dried (Na ₂ SO ₄ ) Concentrated in vacuo and purified by reverse phase gradient flash column chromatography (silica gel C18), eluting with 0% to 42% MeCN in water, to give (1R, 2S, 5S) -3- ((S) -2-isobutyramide-3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [3.1.0 ] as a brown viscous solid]Hexane-2-carboxylic acid methyl ester (4.90 g,13.9 mmol).

LCMS (method F3): m/z 353.5 (M+H), at 1.88 and 1.96 min.

Step 4: liOH monohydrate (2.92 g,69.6 mmol) was added to (1R, 2S, 5S) -3- ((S) -2-isobutyramide-3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [ 3.1.0)]Hexane-2-carboxylic acid methyl ester (4.90 g,13.9 mmol) in 1, 4-dioxane (20 mL) and water (15 mL) and the reaction mixture was stirred at room temperature for 4h, then added (200 mL) and EtOAc (100 mL). The phases were separated, the aqueous phase was acidified to pH of about 2 with 1N HCl (15 mL) and extracted with EtOAc (2X 100 mL). The combined organic phases were dried (Na ₂ SO ₄ ) And concentrated in vacuo to give the crude product (1R, 2S, 5S) -3- ((S) -2-isobutyramide-3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [3.1.0 ] as a white solid]Hexane-2-carboxylic acid (4.80 g).

LCMS (method F3): m/z 339.5 (M+H), at 1.59 and 1.74 min.

Step 5: HATU (3.37 g,8.86 mmol) was added to (1R, 2S, 5S) -3- ((S) -2-isobutyramide-3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [ 3.1.0) ]Hexane-2-carboxylic acid (1.50 g,4.43 mmol) and 3-amino-4-cyclopropyl-2-hydroxybutyric acid methyl ester hydrochloride (1.11 g,5.32 mmol) in DMF and the reaction mixture was stirred at room temperature for 15min. N-methylmorpholine (1.95 mL,17.7 mmol) was added and the reaction mixture was stirred at room temperature for 30min and then concentrated in vacuo. Purification by reverse phase gradient flash column chromatography (silica gel C18), eluting with 0% to 46% MeCN in water, afforded 4-cyclopropyl-2-hydroxy-3- ((1R, 2S, 5S) -3- ((S) -2-isobutyramide-3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [3.1.0 ] as a white solid]Hexane-2-carboxamide) methyl butyrate (1.70 g,3.44 mmol).

LCMS (method F3): m/z 494.5 (M+H), at 1.69 and 1.75 min.

Step 6: liOH monohydrate (0.11 g,2.53 mmol) was added to 4-cyclopropyl-2-hydroxy-3- ((1R, 2S, 5S) -3- ((S) -2-isobutyramide-3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [ 3.1.0)]Hexane-2-carboxamido) butanoic acid methyl ester (0.25 g,0.51 mmol) in 1, 4-dioxane (2 mL) and water (2 mL) and the reaction mixture was stirred at room temperature for 2h. Water (70 mL) and EtOAc (50 mL) were added and the aqueous layer was acidified to pH of about 2 with 1N HCl (3 mL) and extracted with EtOAc (2X 50 mL). The combined organic phases were dried (Na ₂ SO ₄ ) And concentrated in vacuo to give 4-cyclopropyl-2-hydroxy-3- ((1R, 2S, 5S) -3- ((S) -2-isobutyramide-3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [3.1.0 ] as a white solid]Hexane-2-carboxamide) butyric acid (intermediate 10,0.20g,0.39 mmol).

LCMS (method F3): m/z 480.5 (M+H), at 1.55 and 1.58 min.

Synthesis example

Example 1: (1R, 2S, 5S) -N- (4-amino-1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- ((S) -2-isobutyramide-3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxamide

Step 1: to a mixture of (2S) -2- (tert-butoxycarbonylamino) -3, 3-dimethylbutyric acid (305 mg,1.32 mmol) in DMF (5 mL) was slowly added HOBt (178 mg,1.32 mmol) and EDCI. HCl (255 mg,1.32 mmol), and the mixture was stirred for about 12min, followed by the addition of (1R, 2S, 5S) -N- [ 3-amino-1- (cyclopropylmethyl) -2-hydroxy-3-oxo-propyl group]-6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxamide (intermediate 1, 300mg,1.02 mmol) and Et ₃ N (206 mg,2.03mmol,0.28 mL). At N ₂ The reaction mixture was stirred at 25℃for 2h. Adding H ₂ O (50 mL), and the mixture was extracted with EtOAc (20 mL. Times.3). The combined organic phases were washed with brine (30 mL), dried over anhydrous Na ₂ SO ₄ Dried, filtered and concentrated in vacuo. The residue was purified by preparative HPLC (formic acid as additive) to give N- [ (1S) -1- [ (1 r,2S, 5S) -2- [ [ 3-amino-1- (cyclopropylmethyl) -2-hydroxy-3-oxo-propyl ] as a white solid]Carbamoyl radicals]-6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-3-carbonyl]-2, 2-dimethyl-propyl]Tert-butyl carbamate (251 mg,0.49 mmol) was a white solid.

LCMS (method a): m/z 509.2 (M+H) at 1.08 min.

¹ H NMR:(400MHz,CDCl ₃ )δ0.05-0.27(2H,m),0.41-0.53(2H,m),0.68-0.81(1H,m),0.86-0.88(3H,m),0.97(3H,d,J＝3.6Hz),0.98-1.06(9H,m),1.39(9H,s),1.43(2H,br d,J＝6.8Hz),1.52-1.57(1H,m),1.66-1.89(1H,m),3.78-4.19(3H,m),4.19-4.34(2H,m),4.34-4.48(1H,m),6.78-7.20(1H,m)。

The preparative HPLC (formic acid) method for purification in step 1. Instrument: shimadzu LC-20AP; chromatographic column: phenomenex luna C18 150×40mm×15 μm; mobile phase: a=0.225% formic acid in water (v/v), b=mecn; gradient: 40-70% B in A within 10 min; flow rate: 60mL/min; column temperature: 40 ℃; wavelength: 220nm,254nm.

Step 2: a solution of HCl in EtOAc (4N, 5 mL) was added to N- [ (1S) -1- [ (1R, 2S, 5S) -2- [ [ 3-amino-1- (cyclopropylmethyl) -2-hydroxy-3-oxo-propyl ] at 25 ℃]Carbamoyl radicals]-6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-3-carbonyl]-2, 2-dimethyl-propyl]Tert-butyl carbamate (220 mg,0.43 mmol) in EtOAc (5 mL) and in N ₂ The resulting mixture was stirred at 25℃for 1h. The reaction mixture was concentrated in vacuo to give (1 r,2s,5 s) -N- [ 3-amino-1- (cyclopropylmethyl) -2-hydroxy-3-oxo-propyl ] as a white solid ]-3- [ (2S) -2-amino-3, 3-dimethyl-butyryl]-6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxamide (185 mg), which was used without purification.

LCMS (method a): m/z 409.2 (M+H) at 0.68 min.

Step 3: to (1R, 2S, 5S) -N- [ 3-amino-1- (cyclopropylmethyl) -2-hydroxy-3-oxo-propyl group at 0 ℃]-3- [ (2S) -2-amino-3, 3-dimethylbutyryl]-6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxamide (180 mg, crude product) and Et ₃ N (0.12 mL,0.88 mmol) to a mixture of 2-methylpropanoyl chloride (0.06 mL,0.57 mmol) in DCM (5 mL) and under N ₂ The resulting mixture was stirred at 25℃for 1h. The reaction mixture was concentrated in vacuo and purified by preparative HPLC (formic acid as additive) to give the desired step 3 product (1 r,2s,5 s) -N- [ 3-amino-1- (cyclopropylmethyl) -2-hydroxy-3-oxo-propyl]-3- [ (2S) -3, 3-dimethyl-2- (2-methylpropionylamino) butanoyl]-6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxamide (121 mg,0.25 mmol) as a white solid.

LCMS (method a): m/z 479.2 (M+H) at 0.77 min.

¹ H NMR:(400MHz,CDCl ₃ )δ0.05-0.26(2H,m),0.39-0.53(2H,m),0.68-0.81(1H,m),0.81-0.88(3H,m),0.92-0.99(6H,m),0.99-1.07(9H,m),1.13(3H,d,J＝7.2Hz),1.17-1.39(1H,m),1.41-1.60(2H,m),1.70-1.82(1H,m),2.32-2.44(1H,m),3.81-4.21(3H,m),4.25-4.32(1H,m),4.38-4.52(1H,m),4.54-4.66(1H,m),6.10-6.27(1H,m),6.76-7.00(1H,m)。

The preparative HPLC (formic acid) method for purification of step 3. Instrument: shimadzu LC-20AP; chromatographic column: unisil 3-100 C18 Ultra 150 ×25mm×3μm; mobile phase: a=0.225% formic acid in water (v/v), b=mecn; gradient: 30-60% B in A within 10 min; flow rate: 25mL/min; column temperature: 40 ℃; wavelength: 220nm,254nm.

Step 4: to (1R, 2S, 5S) -N- [ 3-amino-1- (cyclopropylmethyl) -2-hydroxy-3-oxo-propyl group at 0 ℃]-3- [ (2S) -3, 3-dimethyl-2- (2-methylpropionylamino) butanoyl]-6, 6-dimethyl-3-azabicyclo [3.1.0]To a mixture of hexane-2-carboxamide (110 mg,0.23 mmol) in DMSO (3 mL) was added 2-iodobenzoic acid (161 mg,0.57 mmol) and under N ₂ The resulting mixture was stirred at 25℃for 12h. Adding H ₂ O (0.5 mL), and the resulting mixture was filtered and concentrated in vacuo. The residue was purified by prep HPLC (NH ₄ HCO ₃ As additive) to give (1 r,2s,5 s) -N- [ 3-amino-1- (cyclopropylmethyl) -2, 3-dioxopropyl) as a white solid of example 1]-3- [ (2S) -3, 3-dimethyl-2- (2-methylpropionylamino) butanoyl]-6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxamide (55.5 mg,0.12 mmol).

LCMS (method D): m/z 477.3 (M+H) at 2.69 min.

¹ H NMR:(400MHz,CDCl ₃ )δ0.00-0.16(2H,m),0.39-0.50(2H,m),0.69-0.76(1H,m),0.81(2H,d,J＝2.4Hz),0.95-1.01(9H,m),1.03(2H,s),1.08(3H,d,J＝6.8Hz),1.14(3H,d,J＝6.8Hz),1.46-1.81(6H,m),2.33-2.40(1H,m),3.76-3.86(1H,m),4.01(1H,br d,J＝10.8Hz),4.46(1H,d,J＝13.6Hz),4.60-4.69(1H,m),5.35-5.40(1H,m),5.74(1H,br s),6.08-6.16(1H,m),6.79(1H,br d,J＝10.8Hz),7.27-7.53(1H,m)。

Preparative HPLC (NH for purification of example 1 ₄ HCO ₃ ) The method. Instrument: shimadzu LC-20AP; chromatographic column: waters Xbridge 150X 25mm X5 μm; mobile phase: a=10 mM NH ₄ HCO ₃ Aqueous solution (v/v), b=mecn; gradient: 25-55% B in A within 10 min; flow rate: 25mL/min; column temperature: 40 ℃; wavelength: 220nm,254nm.

Example 2: (1R, 2S, 5S) -N- [ 3-amino-1- (cyclopropylmethyl) -2, 3-dioxopropyl ]-3- [ (2S) -2- (cyclopropanecarbonylamino) -3-methylbutanoyl]-6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxamide

Step 1 and 2: the title compound (34.3 mg,0.07mmol, white solid) was prepared from intermediate 2 (125 mg,0.32 mmol) and cyclopropanecarbonyl chloride (39.8 mg,0.38mmol,0.03 ml) using procedures similar to those detailed in example 1 (steps 3 and 4). Example 2 by preparative HPLC (NH ₄ HCO ₃ As an additive).

LCMS (method D): m/z 461.3 (M+H) at 2.41 min.

¹ H NMR:(400MHz,CDCl ₃ )δ0.03-0.13(2H,m),0.45-0.49(2H,m),0.67-0.77(4H,m),0.86(3H,s),0.93-0.96(6H,m),1.03(3H,s),1.05(1H,br d,J＝3.6Hz),1.37-1.41(1H,m),1.48-1.51(1H,m),1.62-1.68(1H,m),1.75-1.83(2H,m),2.00-2.06(1H,m),3.77-3.84(1H,m),3.97(1H,br d,J＝10.4Hz),4.44-4.55(2H,m),5.26-5.43(1H,m),5.69-5.91(1H,m),6.33-6.44(1H,m),6.75-6.85(1H,m),7.26-7.49(1H,m)。

Preparative HPLC (NH for purification of example 2 ₄ HCO ₃ ) The method. Instrument: shimadzu LC-20AP; chromatographic column: waters Xbridge 150X 25mm X5 μm; mobile phase: a=10 mM NH ₄ HCO ₃ Aqueous solution (v/v), b=mecn; gradient: 15-45% B in A within 10 min; flow rate: 25mL/min; column temperature: 40 ℃; wavelength: 220nm,254nm.

Example 3: (1R, 2S, 5S) -N- (4-amino-1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- (isobutyryl-L-allo-isoleucine) -6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxamide

The title compound (50.0 mg,0.10mmol, white solid) was prepared from intermediate 1 (380 mg,1.29 mmol) and (2 s,3 r) -2- (tert-butoxycarbonylamino) -3-methyl-pentanoic acid (372 mg,1.61 mmol) using procedures similar to those detailed for intermediate 1 (step 90) and example 1 (steps 2-4). Example 3 by preparative HPLC (NH ₄ HCO ₃ As a means ofAdditives) purification.

LCMS (method D): m/z 477.3 (M+H), at 2.59 min.

¹ H NMR:(400MHz,CDCl ₃ )δ0.00-0.16(2H,m),0.41-0.52(2H,m),0.66-0.77(1H,m),0.84(3H,s),0.85-0.88(3H,m),0.89-0.96(3H,m),1.03(3H,s),1.06-1.10(3H,m),1.13(3H,m),1.45-1.56(2H,m),1.63-1.70(1H,m),1.71-1.90(4H,m),2.30-2.42(1H,m),3.77-3.84(1H,m),4.04(1H,d,J＝10.8Hz),4.44(1H,d,J＝9.6Hz),4.51-4.58(1H,m),5.35-5.40(1H,m),5.96(1H,br s),6.15(1H,brt,J＝9.8Hz),6.80(1H,br d,J＝11.2Hz),7.27-7.47(1H,m)。

Preparative HPLC (NH for purification of example 3 ₄ HCO ₃ ) The method. Instrument: shimadzu LC-20AP; chromatographic column: waters Xbridge 150X 25mm X5 μm; mobile phase: a=10 mM NH ₄ HCO ₃ Aqueous solution (v/v), b=mecn; gradient: 22-52% B in A within 10 min; flow rate: 25mL/min; column temperature: 40 ℃; wavelength: 220nm,254nm.

Example 4: (1R, 2S, 5S) -3- (acryl-L-valyl) -N- (4-amino-1-cyclopropyl-3, 4-dioxobutan-2-yl) -6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxamide

The title compound (32.4 mg,0.07mmol, white solid) was prepared from intermediate 2 (130 mg,0.33 mmol) and prop-2-enoyl chloride (38.8 mg,0.43mmol,0.03 ml) using procedures similar to those detailed in example 1 (steps 3 and 4). By preparative HPLC (NH ₄ HCO ₃ As additive, followed by formic acid as additive) example 4 was purified twice.

LCMS (method D): m/z 447.0 (M+H), at 2.21 min.

¹ H NMR:(400MHz,CDCl ₃ )δ0.00-0.14(2H,m),0.40-0.51(2H,m),0.68-0.74(1H,m),0.84-0.89(3H,m),0.89-0.93(3H,m),0.95-1.00(3H,m),1.02-1.07(3H,m),1.48-1.56(1H,m),1.61-1.67(1H,m),1.73-1.81(1H,m),1.86-2.09(2H,m),3.80-3.88(1H,m),3.96(1H,br d,J＝10.4Hz),4.40-4.49(1H,m),4.56-4.66(1H,m),5.25-5.43(1H,m),5.64(1H,br d,J＝10.4Hz),5.81-6.01(1H,m),6.08-6.17(1H,m),6.24-6.31(1H,m),6.36-6.62(1H,m),6.77-6.88(1H,m),7.26-7.45(1H,m)。

Preparative HPLC (NH for purification of example 4 ₄ HCO ₃ ) The method. Instrument: shimadzu LC-20AP; chromatographic column: waters Xbridge 150X 25mm X5 μm; mobile phase: a=10 mM NH ₄ HCO ₃ Aqueous solution (v/v), b=mecn; gradient: 15-45% B in A within 10 min; flow rate: 25mL/min; column temperature: 40 ℃; wavelength: 220nm,254nm.

The preparative HPLC (formic acid) method for purification of example 4. Instrument: shimadzu LC-20AP; chromatographic column: shim-pack C18×25mm×10μm; mobile phase: a=0.225% formic acid in water (v/v), b=mecn; gradient: 12-42% B in A within 10 min; flow rate: 25mL/min; column temperature: 40 ℃; wavelength: 220nm,254nm.

Example 5: (1R, 2S, 5S) -N- (4-amino-1- ((1R, 2S) -2-methylcyclopropyl) -3, 4-dioxobutan-2-yl) -3- (isobutyryl-L-valyl) -6, 6-dimethyl-3-azabicyclo [ 3.1.0)]Hexane-2-carboxamide

The title compound (16.0 mg,0.03mmol, yellow solid) was prepared from (1 r,2S, 5S) -N- (4-amino-3-hydroxy-1- ((1 r, 2S) -2-methylcyclopropyl) -4-oxobutan-2-yl) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide hydrochloride (intermediate 3,0.37g,1.07 mmol) and (2S) -2- (tert-butoxycarbonylamino) -3-methyl-butyric acid using procedures similar to those detailed in example 1 (steps 1-4). Example 5 purification by preparative HPLC (formic acid as additive).

LCMS (method D2): m/z 477.3 (M+H), at 2.59 min.

¹ H NMR:(400MHz,CD ₃ OD)δ0.10-0.30(2H,m),0.34-0.62(2H,m),0.88-1.14(23H,m),1.40-1.59(2H,m),1.94-2.17(1H,m),2.43-2.57(1H,m),3.89-4.09(2H,m),4.10-4.53(3H,m)。

The preparative HPLC (formic acid) method for purification of example 5. Instrument: shimadzu LC-20AP; chromatographic column: shim-pack C18×25mm×10μm; mobile phase: a=0.225% formic acid in water (v/v), b=mecn; gradient: 25-55% B in A within 10 min; flow rate: 25mL/min; column temperature: 25 ℃; wavelength: 220nm,254nm.

Example 6: (1R, 2S, 5S) -N- (4-amino-1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- ((S) -3-cyclopropyl-2-isobutyrylaminopropionyl) -6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxamide

From (1R, 2S, 5S) -N- [ 3-amino-1- (cyclopropylmethyl) -2-hydroxy-3-oxo-propyl, using procedures similar to those detailed for intermediate 1 (step 9) and example 1 (steps 2-4)]-6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxamide (intermediate 1, 380mg,1.29 mmol) and (2S) -2- (tert-butoxycarbonylamino) -3-cyclopropylpropionic acid (383 mg,1.67 mmol) the title compound (76.3 mg,0.16mmol, white solid) was prepared. Example 6 preparation by HPLC (NH) ₄ HCO ₃ As an additive).

LCMS (method D): m/z 475.6 (M+H), at 2.51 min.

¹ H NMR:(400MHz,CDCl ₃ )δ-0.02-0.14(4H,m),0.39-0.50(4H,m),0.62-0.77(2H,m),0.85-0.95(3H,m),1.03(3H,s),1.06-1.10(3H,m),1.11-1.13(3H,m),1.39-1.53(2H,m),1.56-1.64(1H,m),1.64-1.77(2H,m),1.77-1.89(1H,m),2.31-2.41(1H,m),3.80-3.86(1H,m),3.91-4.00(1H,m),4.43(1H,d,J＝11.2Hz),4.70-4.80(1H,m),5.29-5.43(1H,m),6.27-6.42(1H,m),6.48(1H,br dd,J＝18.8,8.0Hz),6.85(1H,br d,J＝9.6Hz),7.19-7.38(1H,m)。

Preparative HPLC (NH for purification of example 6 ₄ HCO ₃ ) The method. Instrument: shimadzu LC-20AP; chromatographic column: waters Xbridge 150X 25mm X5 μm; mobile phase: a=10 mM NH ₄ HCO ₃ Aqueous solution (v/v), b=mecn; gradient: 18-48% B in A within 10 min; flow rate: 25mL/min; column temperature: 40 ℃; wavelength: 220nm,254nm.

Example 7: (1R, 2S, 5S) -N- (4-amino-1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- (isobutyryl-L-valyl) -6, 6-dimethyl-3-azabicyclo [3.1.0 ]Hexane-2-carboxamide

From (1R, 2S, 5S) -N- [ 3-amino-1- (cyclopropylmethyl) -2-hydroxy-3-oxo-propyl, using procedures similar to those detailed for intermediate 1 (step 1) and example 1 (step 4)]-6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxamide (intermediate 1, 180mg,0.61 mmol) and (2S) -3-methyl-2- (2-methylpropanamido) butanoic acid (intermediate 4, 114mg,0.61 mmol) the title compound (16.1 mg,0.03mmol, white solid) was prepared. Example 7 preparation by preparative HPLC (formic acid as additive followed by NH ₄ HCO ₃ As an additive) was purified twice.

LCMS (method E): m/z 463.3 (M+H) at 2.73 min.

¹ H NMR:(400MHz,CDCl ₃ )δ0.05-0.08(1H,m),0.11-0.13(1H,m),0.47-0.50(2H,m),0.70-0.75(1H,m),0.87(3H,s),0.92-0.96(6H,m),1.05(3H,s),1.12-1.16(6H,m),1.52(1H,br s),1.68-1.72(1H,m),1.76-1.88(2H,m),2.01-2.08(1H,m),2.36-2.41(1H,m),3.80-3.85(1H,m),3.98(1H,br d,J＝10.8Hz),4.47(1H,d,J＝12.0Hz),4.50-4.58(1H,m),5.35-5.43(1H,m),5.45-5.53(1H,m),5.98-6.06(1H,m),6.75-6.78(1H,m),7.21-7.44(1H,m)。

The preparative HPLC (formic acid) method for purification of example 7. Instrument: shimadzu LC-20AP; chromatographic column: unisil 3-100 C18 Ultra 150 ×50mm×3μm; mobile phase: a=0.225% formic acid in water (v/v), b=mecn; gradient: 25-55% B in A within 10 min; flow rate: 25mL/min; column temperature: 40 ℃; wavelength: 220nm,254nm.

Preparative HPLC (NH ₄ HCO ₃ ) The method. Instrument: shimadzu LC-20AP; chromatographic column: waters Xbridge 150X 25mm X5 μm; mobile phase: a=10 mM NH ₄ HCO ₃ Aqueous solution (v/v), b=mecn; gradient: 21-51% B in A within 9 min; flow rate: 25mL/min; column temperature: 40 ℃; wavelength: 220nm,254nm.

Example 8: (1R, 2S, 5S) -N- (4-amino-1-cyclopropyl-3, 4-dioxobutan-2-yl) -6, 6-dimethyl-3- (((S) -2-methylbutanoyl) -L-valyl) -3-azabicyclo [ 3.1.0)]Hexane-2-carboxamide

The title compound (41.5 mg,0.06mmol, white solid) was prepared from (1 r,2S, 5S) -N- [ 3-amino-1- (cyclopropylmethyl) -2-hydroxy-3-oxo-propyl ] -3- [ (2S) -2-amino-3-methyl-butyryl ] -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide (intermediate 2, 120mg,0.28 mmol) and (2S) -2-methylbutanoic acid (28.4 mg,0.28 mol) using procedures similar to those detailed in example 1 (steps 1 and 4). Example 8 was purified by preparative HPLC (formic acid as additive).

LCMS (method D2): m/z 477.0 (M+H) at 2.60 min.

¹ H NMR:(400MHz,CDCl ₃ )δ0.00-0.20(2H,m),0.42-0.55(2H,m),0.68-0.76(1H,m),0.82-0.88(6H,m),0.91-0.99(6H,m),1.05(3H,s),1.14(3H,d,J＝6.8Hz),1.36-1.42(1H,m),1.48-1.54(1H,m),1.56-1.72(2H,m),1.75-1.89(2H,m),1.95-2.09(1H,m),2.11-2.22(1H,m),3.75-3.90(1H,m),4.01-4.04(1H,m),4.41-4.65(2H,m),5.39(1H,br s),5.60-5.83(1H,m),6.04-6.25(1H,m),6.78(1H,br d,J＝11.6Hz),7.42(1H,br d,J＝7.2Hz)。

The preparative HPLC (formic acid) method for purification of example 8. Instrument: shimadzu LC-20AP; chromatographic column: phenomenex luna C18 150X 25mm,10 μm; mobile phase: a=0.225% formic acid in water (v/v), b=mecn; gradient: 32-62% B in A within 10 min; flow rate: 25mL/min; column temperature: 40 ℃; wavelength: 220nm,254nm.

Example 9: (1R, 2S, 5S) -N- (4-amino-1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- ((S) -2- (cyclopropanecarboxamide) -2-cyclopropylacetyl) -6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxamide

The title compound (10.9 mg,0.02mmol, white solid) was prepared from intermediate 1 (560 mg,1.90 mmol) and (2S) -2- (tert-butoxycarbonylamino) -2-cyclopropylacetic acid (612 mg,2.84 mmol) using procedures similar to those detailed for intermediate 1 (step 1) and example 1 (steps 2-4). Example 9 by preparative HPLC (NH ₄ HCO ₃ As an additive).

LCMS (method D2): m/z 459.3 (M+H) at 2.44 min.

¹ H NMR:(400MHz,CDCl ₃ )δ0.01-0.19(2H,m),0.40-0.61(6H,m),0.67-0.81(3H,m),0.86-0.94(3H,m),0.94-1.03(2H,m),1.03-1.10(3H,m),1.10-1.23(1H,m),1.36-1.42(1H,m),1.46-1.53(1H,m),1.66-1.71(1H,m),1.72-1.89(2H,m),3.76-3.99(2H,m),4.22-4.45(1H,m),4.45-4.66(1H,m),5.36-5.64(1H,m),6.34-6.59(1H,m),6.66-6.83(1H,m)。

Preparative HPLC (NH for purification of example 9 ₄ HCO ₃ ) The method. Instrument: shimadzu LC-20AP; chromatographic column: waters Xbridge 150X 25mm X5 μm; mobile phase: a=10 mM NH ₄ HCO ₃ Aqueous solution (v/v), b=mecn; gradient: 15-45% B in A within 10 min; flow rate: 25mL/min; column temperature: 40 ℃; wavelength: 220nm,254nm.

Example 10: (1R, 2S, 5S) -N- (4-amino-1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- ((S) -2-cyclopropyl-2-isobutyramide acetyl) -6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxamide

The title compound was prepared from intermediate 1 (560 mg,1.90 mmol) and (2S) -2- (tert-butoxycarbonylamino) -2-cyclopropylacetic acid (612 mg,2.84 mmol) using procedures similar to those detailed for intermediate 1 (step 1) and example 1 (steps 2-4) (20.2 mg,0.04mmol, white solid). Example 10 by preparative HPLC (NH ₄ HCO ₃ As an additive).

LCMS (method D2): m/z 461.5 (M+H) at 2.48 min.

¹ H NMR:(400MHz,CDCl ₃ )δ0.01-0.06(1H,m),0.09-0.14(1H,m),0.40-0.52(6H,m),0.68-0.73(1H,m),0.89(3H,s),1.04(3H,s),1.13(6H,dd,J＝15.6,6.8Hz),1.47-1.53(1H,m),1.68(1H,dd,J＝7.6,2.0Hz),1.78-1.83(2H,m),1.84-1.90(1H,m),2.34-2.42(1H,m),3.82-3.90(2H,m),4.36(1H,br t,J＝7.8Hz),4.46(1H,d,J＝16.8Hz),5.36-5.45(1H,m),5.77-5.91(1H,m),6.32-6.34(1H,m),6.75-6.87(1H,m),7.22-7.34(1H,m)。

Preparative HPLC (NH for purification of example 10 ₄ HCO ₃ ) The method. Instrument: shimadzu LC-20AP; chromatographic column: waters Xbridge 150X 25mm X5 μm; mobile phase: a=10 mM NH ₄ HCO ₃ Aqueous solution (v/v), b=mecn; gradient: 15-45% B in A within 10 min; flow rate: 25mL/min; column temperature: 40 ℃; wavelength: 220nm,254nm.

Example 11: (1R, 2S, 5S) -N- (4- (benzylamino) -1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- (isobutyryl-L-valyl) -6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxamide

Step 1: HATU (1.23 g,3.23 mmol) was added to (1R, 2S, 5S) -3- (isobutyryl-L-valyl) -6, 6-dimethyl-3-azabicyclo [ 3.1.0)]A solution of hexane-2-carboxylic acid (intermediate 5,0.70g,2.16 mmol) in DMF (10 mL) was added and the reaction mixture was stirred at room temperature for 10min. 3-amino-N-benzyl-4-cyclopropyl-2-hydroxybutyramide (intermediate 6,0.53g,2.16 mmol) and DIPEA (1.11 mL,6.47 mmol) were added and after stirring at room temperature for 18h the reaction mixture was partitioned between cold water (150 mL) and EtOAc (70 mL). The phases were separated and the aqueous layer was extracted with EtOAc (3X 70 mL). The combined organic phases were dried (Na ₂ SO ₄ ) Filtered and concentrated in vacuo. By reversed phase gradient columnChromatography (C18 silica gel) eluting with 0-60% MeCN in water gave (1R, 2S, 5S) -N- ((2S) -4- (benzylamino) -1-cyclopropyl-3-hydroxy-4-oxobutan-2-yl) -3- (isobutyryl-L-valyl) -6, 6-dimethyl-3-azabicyclo [3.1.0 ] as a white solid]Hexane-2-carboxamide (0.59 g,1.06 mmol).

LCMS (method G): m/z 555.9 (M+H), at 2.20,2.25,2.38 min.

Step 2: IBX (0.40 g,1.44 mmol) was added to (1R, 2S, 5S) -N- ((2S) -4- (benzylamino) -1-cyclopropyl-3-hydroxy-4-oxobutan-2-yl) -3- (isobutyryl-L-valyl) -6, 6-dimethyl-3-azabicyclo [ 3.1.0)]A solution of hexane-2-carboxamide (0.40 g,0.72 mmol) in DMSO (5 mL) was added and the reaction mixture was stirred at 60℃for 2h. Water (50 mL) and EtOAc (30 mL) were added, the phases were separated, and the aqueous phase was extracted with EtOAc (2X 50 mL). The combined organic phases were dried (Na ₂ SO ₄ ) Filtered and concentrated in vacuo. Purification by reverse phase gradient column chromatography (C18 silica gel) eluting with 0-60% MeCN in water afforded example 11 (0.22 g,0.40 mmol) as a white solid.

LCMS (method H): m/z 553.3 (M+H), at 7.70, 7.95 and 8.29 min.

¹ H NMR:(400MHz,DMSO-d ₆ )δ0.08-0.07(m,2H),0.41-0.35(m,2H),0.77-0.69(m,9H),1.01-0.83(m,10H),1.32(d,1H,J＝7.6Hz),1.49-1.40(m,2H),1.79-1.69(m,1H),1.91-1.90(m,1H),2.45-2.41(m,1H),3.75-3.72(m,1H),3.89(d,1H,J＝10.4Hz),4.11(t,1H,J＝9.2Hz),4.43-4.25(m,3H),5.10-5.05(m,1H),7.33-7.24(m,5H),7.97(d,1H,J＝8.4Hz),8.50(d,1H,J＝6.4Hz),9.26(t,1H,J＝6.0Hz)。

Example 12: (1R, 2S, 5S) -N- (1-cyclopropyl-4- (cyclopropylamino) -3, 4-dioxobutan-2-yl) -3- (isobutyryl-L-valyl) -6, 6-dimethyl-3-azabicyclo [3.1.0 ]Hexane-2-carboxamide

Step 1: EDCI. HCl (0.19 g,1.03 mmol) and HOBt (0.11 g,0.86 mmol) were added at 0deg.CInto 4-cyclopropyl-2-hydroxy-3- ((1R, 2S, 5S) -3- (isobutyryl-L-valyl) -6, 6-dimethyl-3-azabicyclo [ 3.1.0)]Hexane-2-carboxamide) butyric acid (intermediate 8,0.40g,0.86 mmol) and cyclopropylamine (59 mg,1.03 mmol) in EtOAc (4 mL) and H ₂ O (1 mL) and the reaction mixture was stirred at room temperature for 3h and then concentrated in vacuo. Purification by reverse phase gradient flash column chromatography (C18 silica gel) eluting with 0-46% MeCN in water gave (1R, 2S, 5S) -N- (1-cyclopropyl-4- (cyclopropylamino) -3-hydroxy-4-oxobutan-2-yl) -3- (isobutyryl-L-valyl) -6, 6-dimethyl-3-azabicyclo [3.1.0 ] as an off-white solid]Hexane-2-carboxamide (0.25 g,0.50 mmol).

LCMS (method I): m/z 505.6 (M+H), at 1.51, 1.57, 1.66 min.

Step 2: DMP (0.31 g,0.74 mmol) was added to (1R, 2S, 5S) -N- (1-cyclopropyl-4- (cyclopropylamino) -3-hydroxy-4-oxobutan-2-yl) -3- (isobutyryl-L-valyl) -6, 6-dimethyl-3-azabicyclo [3.1.0]A solution of hexane-2-carboxamide (0.25 g,0.49 mmol) in EtOAc (3 mL) and the reaction mixture was stirred at room temperature for 48h. After filtration through celite (celite) and washing with EtOAc (50 mL), the filtrate was concentrated in vacuo. Purification by reverse phase gradient flash column chromatography (C18 silica gel) eluting with 0-65% MeCN in water and 0.1% formic acid as modifier afforded example 12 (31 mg,0.06 mmol) as an off-white solid.

LCMS (method I): m/z 503.5 (M+H), at 1.48,1.75 min.

¹ H NMR:(400MHz,DMSO-d ₆ )δ0.08-0.03(m,2H),0.42-0.37(m,2H),0.56(s,2H),0.65-0.63(d,2H,J＝7.2Hz),0.75-0.74(m,9H),1.11-0.82(m,10H),1.48-1.23(m,3H),1.89-1.69(m,1H),1.92-1.90(m,1H),2.50-2.33(m,1H),2.74-2.60(m,1H),3.74-3.70(m,1H),3.91-3.88(m,1H),4.12-4.07(m,1H),4.29(s,1H),5.05-5.01(m,1H),7.98-7.96(d,1H,J＝8.4Hz),8.47-8.46(d,1H,J＝6.4Hz),8.75-8.74(d,1H,J＝4.8Hz)。

EXAMPLE 26: (1R, 2S, 5S) -N- (4-amino-1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- ((S) -2-isobutyramide-3-methyl-3-phenylbutyryl) -6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxamide

Using procedures similar to those detailed in example 1 (Steps 2-4), the title compound (18.2 mg,0.03mmol, white solid) was prepared from intermediate 9 (31.0 mg,0.05 mmol). Example 26 by preparative HPLC (NH ₄ HCO ₃ As an additive).

LCMS (method J): m/z 539.3 (M+H) at 2.80 min.

¹ H NMR:(400MHz,CDCl ₃ )δ0.03-0.22(2H,m),0.43-0.57(2H,m),0.71-0.78(3H,m),0.78-0.89(1H,m),0.94-0.98(3H,m),1.02-1.08(6H,m),1.27-1.32(1H,m),1.40-1.48(6H,m),1.52-1.62(1H,m),1.80-1.91(2H,m),2.27-2.35(1H,m),2.83-2.89(1H,m),3.69-3.72(1H,m),4.32-4.36(1H,m),4.97-5.01(1H,m),5.27-5.43(1H,m),5.72-5.88(1H,m),6.09-6.20(1H,m),6.81(1H,brs),7.17-7.23(1H,m),7.26-7.34(2H,m),7.38(2H,d,J＝7.6Hz),7.41-7.52(1H,m)。

Preparative HPLC (NH) for purification of example 26 ₄ HCO ₃ ) The method. Instrument: shimadzu LC-20AP; chromatographic column: waters Xbridge 150X 25mm X5 μm; mobile phase: a=10 mM NH ₄ HCO ₃ Aqueous solution (v/v), b=mecn; gradient: 30-60% B in A within 10 min; flow rate: 25mL/min; column temperature: 40 ℃; wavelength: 220nm,254nm.

EXAMPLE 28: (1R, 2S, 5S) -N- (4- (azetidin-1-yl) -1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- ((S) -2-isobutyramide-3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxamide

Step 1: n-methylmorpholine (0.17 mL,1.59 mmol) was added to 4-cyclopropyl-2-hydroxy-3- ((1R, 2S, 5S) -3- ((S) -2-isobutyramide-3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [ 3.1.0) ]Hexane-2-carboxamido) butyric acidIntermediate 10,0.19g,0.40 mmol) and azetidine (32 mg,0.48 mmol) in DMF (2.8 mL) and then stirring the reaction mixture at room temperature for 4h. Water (80 mL) was added and the mixture was extracted with EtOAc (3X 25 mL). The combined organic layers were dried (Na ₂ SO ₄ ) And concentrated in vacuo. Purification by reverse phase gradient flash column chromatography (silica gel C18), eluting with 0% to 60% MeCN in water, afforded (1R, 2S, 5S) -N- (4- (azetidin-1-yl) -1-cyclopropyl-3-hydroxy-4-oxobutan-2-yl) -3- ((S) -2-isobutyramide-3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [ 3.1.0) as a brown solid]Hexane-2-carboxamide (0.15 g,0.29 mmol).

LCMS (method F3): m/z 519.4 (M+H), at 1.58, 1.65 and 1.70 min.

Step 2: dess-Martin oxidizer (0.17 g,0.41 mmol) was added to (1R, 2S, 5S) -N- (4- (azetidin-1-yl) -1-cyclopropyl-3-hydroxy-4-oxobutan-2-yl) -3- ((S) -2-isobutyramide-3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxamide (0.14 g,0.27 mmol) in EtOAc (3 mL) and the reaction mixture was stirred at room temperature for 16h then filtered through a bed of celite and washed with EtOAc (100 mL). The filtrate was concentrated in vacuo and purified by reverse phase gradient flash column chromatography (silica gel C18), eluting with a (water+0.1% formic acid) solution of 0% to 90% mecn, to give (1 r,2S, 5S) -N- (4- (azetidin-1-yl) -1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- ((S) -2-isobutyramide-3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [ 3.1.0) as a white solid of example 28 ]Hexane-2-carboxamide (57 mg,0.11 mmol).

LCMS (method K): m/z 517.0 (M+H), at 7.49 min.

¹ H NMR:(400MHz,DMSO-d ₆ )δ0.03-0.11(m,2H),0.35-0.43(m,2H),0.80-1.03(m,22H),1.29-1.31(m,1H),1.43-1.50(m,2H),1.69-1.71(m,1H),2.23-2.27(t,2H,J＝7.8Hz),2.54-2.59(m,1H),3.77-3.85(m,2H),3.88-4.00(m,2H),4.20-4.33(m,3H),4.39-4.41(d,1H,J＝9.6Hz),4.72-4.77(m,1H),7.74-7.76(d,1H,J＝9.2Hz),8.52-8.53(d,1H,J＝6.4Hz)。

Example 34: (1R, 2S, 5S) -N- (4-amino-1-cyclopropyl)-3, 4-dioxobutan-2-yl) -3- ((S) -3, 3-dimethyl-2- (2, 2-trifluoroacetamido) butanoyl) -6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxamide

Step 1: to (1R, 2S, 5S) -N- [ 3-amino-1- (cyclopropylmethyl) -2-hydroxy-3-oxo-propyl group at 0 ℃]-3- [ (2S) -2-amino-3, 3-dimethylbutyryl]-6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxamide (example 1, step 2 product, 400mg,0.98 mmol) in CH ₂ Cl ₂ Et is added dropwise to the mixture in (5 mL) ₃ N (0.20 mL,1.47 mmol) and TFAA (0.20 mL,1.47mmol,0.20 mL) in CH ₂ Cl ₂ (2 mL) of the solution. The reaction mixture was stirred at 25℃for 2H, then H was added ₂ O (40 mL). After extraction with ethyl acetate (20 mL. Times.3), the combined organic phases were washed with brine (30 mL. Times.3) and with anhydrous Na ₂ SO ₄ Dried, filtered and concentrated in vacuo. Purification by preparative HPLC (formic acid as additive) afforded (1 r,2S, 5S) -N- (4-amino-1-cyclopropyl-3-hydroxy-4-oxobutan-2-yl) -3- ((S) -3, 3-dimethyl-2- (2, 2-trifluoroacetamido) butanoyl) -6, 6-dimethyl-3-azabicyclo [3.1.0 ] as a white solid ]Hexane-2-carboxamide (170 mg,0.34 mmol).

LCMS (method a): m/z 505.2 (M+H) at 0.81 min.

¹ H NMR:(400MHz,MeOD-d ₄ )δ-0.08-0.21(2H,m),0.26-0.53(2H,m),0.64-1.14(16H,m),1.16-2.32(4H,m),3.60-4.73(6H,m)。

The preparative HPLC (formic acid) method for purification in step 1. Instrument: shimadzu LC-20AP; chromatographic column: unisil 3-100 C18 Ultra 150 ×50mm×3μm; mobile phase: a=0.225% formic acid in water (v/v), b=mecn; gradient: 30-60% B in A within 10 min; flow rate: 25mL/min; column temperature: 40 ℃; wavelength: 220nm,254nm.

Step 2: IBX (305 mg,1.09 mmol) was added to (1R, 2S, 5S) -N- (4-amino-1-cyclopropyl-3-hydroxy-4-oxobutan-2-yl) -3- ((S) -3, 3-dimethyl-2- (2, 2-trifluoroacetamido) butanoyl) -6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxamide (220 mg,0.44 mmol) in DMSO (3 mL) and stirring the reaction mixture at 25℃for 2H, then adding H ₂ O (20 mL) and extracted with ethyl acetate (20 mL. Times.3). The combined organic phases were washed with brine (30 mL. Times.3), dried over anhydrous Na ₂ SO ₄ Dried, filtered and concentrated in vacuo. Purification by preparative HPLC (formic acid as additive) afforded (1 r,2S, 5S) -N- (4-amino-1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- ((S) -3, 3-dimethyl-2- (2, 2-trifluoroacetamido) butanoyl) -6, 6-dimethyl-3-azabicyclo [3.1.0 ] as a white solid ]Hexane-2-carboxamide (195 mg,0.39 mmol).

LCMS (method J): m/z 503.3 (M+H) at 3.04 min.

¹ H NMR:(400MHz,DMSO-d ₆ )δ-0.14-0.16(2H,m),0.24-0.49(2H,m),0.56-1.20(16H,m),1.34-1.77(4H,m),3.47-3.99(2H,m),4.17-4.64(2H,m),4.74-5.38(1H,m),7.66-7.85(1H,m),7.86-8.11(1H,m),8.26-8.74(1H,m),9.15-9.59(1H,m)。

The preparative HPLC (formic acid) method for purification in step 1. Instrument: shimadzu LC-20AP; chromatographic column: unisil 3-100 C18 Ultra 150 ×50mm×3μm; mobile phase: a=0.225% formic acid in water (v/v), b=mecn; gradient: 33-63% B in A within 10 min; flow rate: 25mL/min; column temperature: 40 ℃; wavelength: 220nm,254nm.

Example 35: (1R, 2S, 5S) -N- (4- (azetidin-1-yl) -1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- ((S) -3, 3-dimethyl-2- (2, 2-trifluoroacetamido) butanoyl) -6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxamide

Step 1: liOH (154 mg,6.37 mmol) was added to (1R, 2S, 5S) -3- ((S) -2- ((tert-butoxycarbonyl) amino) -3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxylic acid methyl ester (intermediate 10, step 1 product, 2.50g,6.37 mmol) in THF/MeOH/H ₂ In suspension in O (32 mL, 10:3:3) and the mixture was stirred at room temperature for 3h. Water (10 mL) and MTBE (10 mL) were added and the phases separated. The aqueous layer was acidified to pH 2-3 with 1.5N HCl and extracted with DCM (2X 30 mL). The combined organic phases were treated with anhydrous Na ₂ SO ₄ Dried, and concentrated in vacuo to give (1R, 2S, 5S) -3- ((S) -2- ((tert-butoxycarbonyl) amino) -3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [3.1.0 as a white solid]Hexane-2-carboxylic acid (2.12 g,5.60 mmol), which was used in the next step without further purification.

LCMS (method L): m/z 369.3 (M+H) at 2.26 min.

¹ H NMR:(400MHz,DMSO-d ₆ )δ12.53(br s,1H),6.68(d,J＝9.6Hz,1H),4.13(s,1H),4.05(d,J＝9.6Hz,1H),3.91(d,J＝10.4Hz,1H),3.79-3.74(m,1H),1.51-1.47(m,1H),1.41-1.37(m,1H),1.35(s,9H),1.01(s,3H),0.94(s,9H),0.84(s,3H)。

Step 2: SOCl was added at 0deg.C ₂ (1.15 mL,15.6 mmol) was added to a stirred solution of 3-amino-4-cyclopropyl-2-hydroxybutyramide (2.50 g,15.6 mmol) in EtOH (20 mL) and the reaction mixture was then stirred at 65℃for 15h. Concentrated in vacuo and azeotroped with toluene (2X 20 mL) to give crude ethyl 3-amino-4-cyclopropyl-2-hydroxybutyrate (2.10 g) as a brown semi-solid, which was used in the next step without further purification.

LCMS (method L): m/z 188.3 (M+H) at 1.03 min.

Step 3: to (1R, 2S, 5S) -3- ((S) -2- ((tert-butoxycarbonyl) amino) -3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [3.1.0 ] at room temperature]To a suspension of hexane-2-carboxylic acid (2.45 g,6.25 mmol) and ethyl 3-amino-4-cyclopropyl-2-hydroxybutyrate (1.65 g,7.50 mmol) in DCM (20 mL) was added DIPEA (3.37 mL,18.8 mmol) followed by T3P (50% in EtOAc) at 0deg.C (5.58 mL,9.38 mmol). The resulting reaction mixture was stirred at room temperature for 3h, and 10% NaHCO was added at room temperature ₃ Aqueous solution, and the mixture was extracted with DCM (2×30 mL). The combined organic phases were treated with anhydrous Na ₂ SO ₄ Dried and concentrated in vacuo. Purification by gradient flash column chromatography on silica gel with 0-50%Petroleum ether solution elution of EtOAc afforded a pale yellow gum 3- ((1 r,2S, 5S) -3- ((S) -2- ((tert-butoxycarbonyl) amino) -3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxamido) -4-cyclopropyl-2-hydroxybutyric acid ethyl ester (2.30 g,4.19 mmol).

LCMS (method L): m/z 538.4 (M+H), at 2.43-2.47 min.

¹ H NMR:(400MHz,DMSO-d ₆ )δ7.90(d,J＝8.8Hz,1H),6.57(d,J＝9.2Hz,1H),5.57-5.50(m,1H),4.27(d,J＝2.8Hz,1H),4.11-4.09(m,1H),4.04-4.00(m,4H),3.95-3.85(m,1H),3.82-3.71(m,1H),1.40(s,9H),1.18-1.13(m,6H),1.05-0.95(m,4H),0.93-0.83(m,10H),0.72-0.61(m,1H),0.35-0.09(m,2H),0.15--0.15(m,2H)。

Step 4: to 3- ((1R, 2S, 5S) -3- ((S) -2- ((tert-butoxycarbonyl) amino) -3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [ 3.1.0)]To a solution of hexane-2-carboxamide) -4-cyclopropyl-2-hydroxybutyric acid ethyl ester (2.20 g,4.01 mmol) in DCM (10 mL) was added a solution of 4M HCl in 1, 4-dioxane (5 mL,20 mmol) and the reaction mixture was stirred at room temperature for 3h. The supernatant layer was decanted from the reaction mixture and the solid gum was triturated with MTBE (10 mL). The MTBE layer was decanted and the residue was dried in vacuo to give 3- ((1 r,2S, 5S) -3- ((S) -2-amino-3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [ 3.1.0) as an off-white solid ]Hexane-2-carboxamide) -4-cyclopropyl-2-hydroxybutyric acid ethyl ester hydrochloride (1.80 g), which was used in the next step without further purification.

LCMS (method L): m/z 438.4 (M+H), at 1.42-1.79 min.

Step 5: liOH (292 mg,12.2 mmol) was added to 3- ((1R, 2S, 5S) -3- ((S) -2-amino-3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [ 3.1.0) at room temperature]Hexane-2-carboxamido) -4-cyclopropyl-2-hydroxybutyric acid ethyl ester hydrochloride (1.45 g,2.45 mmol) in THF/MeOH/H ₂ In suspension in O (16 mL, 10:3:3), the mixture was acidified to pH about 2-3 with 1.5N aqueous HCl and the reaction mixture was concentrated in vacuo and azeotroped with toluene (3X 20 mL) to give the crude product 3- ((1R, 2S, 5S) -3- ((S) -2-amino-3, 3-Dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [3.1.0]Hexane-2-carboxamido) -4-cyclopropyl-2-hydroxybutyric acid hydrochloride (2.2 g), which is used in the next step without further purification.

LCMS (method L): m/z 410.3 (M+H), at 1.05-1.53 min.

Step 6: trimethylamine (1.04 mL,7.38 mmol) and ethyl 2, 2-trifluoroacetate (0.48 mL,4.92 mmol) were added to 3- ((1R, 2S, 5S) -3- ((S) -2-amino-3, 3-dimethylbutyryl) -6, 6-dimethyl-3-azabicyclo [ 3.1.0) ]Hexane-2-carboxamide) -4-cyclopropyl-2-hydroxybutyric acid hydrochloride (2.4 g,2.46 mmol) in MeOH (20 mL) and the reaction mixture was stirred at room temperature for 16h. The reaction mixture was then acidified with a solution of 4M HCl in 1, 4-dioxane (3 mL,12 mmol) and concentrated in vacuo, followed by addition of water (10 mL) and extraction with EtOAc (3X 25 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated in vacuo, and azeotroped with toluene (3×20 mL) to give an off-white solid crude 4-cyclopropyl-3- ((1 r,2S, 5S) -3- ((S) -3, 3-dimethyl-2- (2, 2-trifluoroacetamido) butanoyl) -6, 6-dimethyl-3-azabicyclo [ 3.1.0)]Hexane-2-carboxamide) -2-hydroxybutyric acid (1.20 g), which was used in the next step without further purification.

LCMS (method L): m/z 506.4 (M+H), at 2.03-2.09 min.

Step 7: n-methylmorpholine (NMM, 1.37mL,12.4 mmol) was added to stirred azetidine hydrochloride (467 mg,4.94 mmol) and 4-cyclopropyl-3- ((1R, 2S, 5S) -3- ((S) -3, 3-dimethyl-2- (2, 2-trifluoroacetamido) butanoyl) -6, 6-dimethyl-3-azabicyclo [ 3.1.0)]A solution of hexane-2-carboxamido) -2-hydroxybutyric acid (1.42 g,2.47 mmol) in DMF (5 mL) was stirred at room temperature for 5min. HATU (1.42 g,3.71 mmol) was added at 0 ℃ and the reaction mixture was stirred at room temperature for 2h, then water (2 mL) was added and concentrated in vacuo. Purification by gradient reverse phase flash column chromatography on silica gel C18 eluting with 0% to 70% MeCN in water gave (1R, 2S, 5S) -N- (4- (azetidin-1-yl) -1-cyclopropyl-3-hydroxy-4-oxobutan-2-yl) -3- ((S) -3, 3-dimethyl-2- (2, 2-trifluoroacetamido) butanoyl) -6, 6-dimethyl-3- Azabicyclo [3.1.0]Hexane-2-carboxamide (0.75 g,1.18 mmol).

LCMS (method L): m/z 545.3 (M+H) at 2.06-2.14 min.

¹ H NMR:(400MHz,DMSO-d ₆ )δ9.43-9.31(m,1H),7.94-7.71(m,1H),5.22-5.01(m,1H),4.43-4.31(m,1H),4.30-4.17(m,3H),4.15-3.70(m,4H),2.28-2.18(m,2H),1.57-1.48(m,2H),1.31-1.22(m,2H),1.21-0.82(m,17H),0.76-0.65(m,1H),0.39-0.17(m,2H),0.15--0.19(m,2H).

Step 8: dess-Martin oxidizer (1.17 g,2.75 mmol) was added to (1R, 2S, 5S) -N- (4- (azetidin-1-yl) -1-cyclopropyl-3-hydroxy-4-oxobutan-2-yl) -3- ((S) -3, 3-dimethyl-2- (2, 2-trifluoroacetamido) butanoyl) -6, 6-dimethyl-3-azabicyclo [ 3.1.0) at room temperature]Hexane-2-carboxamide (0.50 g,0.92 mmol) in EtOAc (10 mL) the reaction mixture was stirred at room temperature for 2h then EtOAc (10 mL) was added. The mixture was filtered through celite, and the residue was rinsed with EtOAc (50 mL). The filtrate was concentrated in vacuo and combined with another batch of (1R, 2S, 5S) -N- (4- (azetidin-1-yl) -1-cyclopropyl-3-hydroxy-4-oxobutan-2-yl) -3- ((S) -3, 3-dimethyl-2- (2, 2-trifluoroacetamido) butanoyl) -6, 6-dimethyl-3-azabicyclo [3.1.0]The crude filtrates from hexane-2-carboxamide preparation were combined. Purification by gradient reverse phase flash column chromatography on silica gel C18 with 0% to 80% MeCN (H ₂ O+0.1% TFA) to give (1R, 2S, 5S) -N- (4- (azetidin-1-yl) -1-cyclopropyl-3, 4-dioxobutan-2-yl) -3- ((S) -3, 3-dimethyl-2- (2, 2-trifluoroacetamido) -6, 6-dimethyl-3-azabicyclo [ 3.1.0) as a white solid ]Hexane-2-carboxamide (example 35,185mg,0.33 mmol).

LCMS (method L): m/z 543.3 (M+H) at 2.34 min.

¹ H NMR:(400MHz,DMSO-d ₆ )δ9.40-9.35(m,1H),8.73-8.58(m,1H),4.81-4.51(m,1H),4.44-4.18(m,2H),3.97-3.69(m,4H),2.34-2.22(m,2H),1.73-1.34(m,3H),1,34-1.28(m,1H),1.06-0.76(m,18H),0.42-0.36(m,2H),0.18--0.05(m,2H)。

Examples 13-25, 27, 29-33 were synthesized using procedures similar to those detailed above.

Biological activity

Structural design of SARA-CoV-2 Mpro

SARS CoV-2-Mpro (major protease/3C-like protease, uniProt ID: P0DTD 1) protein sequence up to and including its autocleavage border, and the preceding N-terminal 5 amino acid residues, including the P1 glutamine residue, are codon optimized for E.coli expression and cloned into pET26b (Merck, # US 169862-3) or pGEX6P1 (Fisher Scientific, # 10350355) vectors using BamHI and XhoI sites. Thus, the expression construct is characterized by the native viral N-terminal sequence, and a C-terminal modified 3C protease cleavage site (LEVLFQGK), with an optional lysine residue at the P2' position, followed by a polyhistidine (His-8) tag.

Protein expression and protein purification

Chemically competent BL21 (DE 3) -RIL E.coli (Agilent, # 230240) cells were transformed with the relevant coronavirus Mpro construct and grown overnight at 37℃on LB agar plates supplemented with the appropriate antibiotics. All incubation steps were performed at 37 ℃ unless otherwise indicated. One colony was grown in 15mL of antibiotic-supplemented LB medium for about 2 hours, taking care not to exceed the Optical Density (OD) density of 2.0 measured by a spectrophotometer at 600 nm. The preculture was used to inoculate 500mL of expression culture: LB medium for IPTG-induced expression or auto-induction super broth (Formedium, # AIMSB 0210). Expression was induced in LB medium at an OD of 0.7-1.0 by addition of IPTG at a final concentration of 0.5 mM. The culture was then grown overnight at 18 ℃. In auto-induction expression, once an OD of 0.7-1.0 was observed, the temperature was reduced to 18 ℃ and then grown overnight. Cells were harvested by centrifugation and frozen until use.

Thawed cells were resuspended in resuspension buffer: 20mM Tris-HCl pH 8.0, 150mM NaCl and DNase I (Merck# 4716728001) and cleaved by ultrasound. The lysate was clarified by centrifugation at 23,000rcf for 15min at 4 ℃. The supernatant was loaded onto 5mLNiNTA resin (Cytiva, # 17-5248-02) at a flow rate of 0.5 mL/min. The resin was washed with the same buffer containing 20mM imidazole as described above. The Mpro protein was eluted using the same buffer containing 250mM imidazole. The target protein was further purified in a resuspension buffer using a Superdex S7516/60pg (GE, # GE 28-9893-33) column. Protein purity was assessed by SDS-PAGE and identity was confirmed by mass spectrometry. The purified protein was concentrated and frozen for later use.

SARS-Cov-2 Mpro enzyme assay

The activity of SARS-Cov-2 Mpro was determined in a Fluorescence Resonance Energy Transfer (FRET) based enzyme assay using the FRET substrate Dabcyl-KTSAVLQSGFRKM-E (Edans) -amide. Briefly, 100nL of test compound (ranging in concentration from 10. Mu.M to 0.00051. Mu.M) was pre-incubated with 5. Mu.L of 5nM (final concentration) Mpro enzyme for 20min at room temperature in assay buffer containing 20mM Tris (pH 7.5), 100mM NaCl and 1mM EDTA. The reaction was initiated by adding 5. Mu.L of 25. Mu.M (final concentration) FRET substrate (Dabcyl-KTSAVLQSGFRKM-E (Edans) -amide). Fluorescence intensities generated at ex=360 nm/em=490 nm were measured every 90 seconds over 60min using a PHERAstar reader (BMG Labtech) at room temperature. Using MARS software (BMG Labtech), the linear portion of the reaction was selected and the rate of RFU per minute was calculated. Besseprevir was used as a reference standard compound. Determination of pIC using 4PL GraphPad Prism ₅₀ And pKi, data expressed as mean n=2±sd. The pKi values of the compounds of the present invention are shown in Table 2.

TABLE 2

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Claims

1. A compound of formula (1 b), or a salt thereof:

wherein:

R ⁵ Is C _2-8 Hydrocarbyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl radicals, the said C _2-8 The hydrocarbyl group optionally contains one or more rings or double bonds and is optionally substituted with one or more groups selected from the group consisting of: fluorine, chlorine, bromine, cyano, hydroxy, methoxy, amino.

2. The compound of claim 1, wherein R ¹ H, CH of a shape of H, CH ₃ Benzyl, cyclopropyl or

3. The compound according to claim 1 or 2, wherein R ^1a H.

4. The compound of claim 1, wherein R ¹ And R is ^1a Are all H.

5. According to claim1, wherein R is ¹ And R is ^1a Are all-CH ₃ 。

6. The compound of claim 1, wherein R ¹ And R is ^1a And are linked together to form a saturated ring of 3 to 6 atoms.

7. The compound of claim 1, wherein R ¹ And R is ^1a And joined together to form an azetidine ring or an aziridine ring.

8. The compound according to any one of claims 1 to 7, wherein R ² Selected from:

9. the compound according to any one of claims 1 to 8, wherein R ³ Selected from:

10. the compound according to any one of claims 1 to 9, wherein R ⁵ Selected from:

11. the compound of claim 1, which is a compound of formula (3 b) or a salt thereof:

12. the compound of claim 1, selected from the following compounds or salts thereof:

13. The compound according to any one of claims 1 to 12, having SARS-CoV-2Mpro inhibitor activity.

14. A pharmaceutical composition comprising a compound of any one of claims 1 to 13 and a pharmaceutically acceptable excipient.

15. A compound or composition according to any one of claims 1 to 14 for use in the treatment of SARS-CoV-2 or for use in the treatment of a condition associated with SARS-CoV-2.