EA047028B1 - COMPOUND, REPRESENTING A SHORT PEPTIDE BASED ON PROLINE WITH A MODIFIED RING, AND ITS APPLICATION - Google Patents

Description

This application claims priority to the following applications:

CN 202110413867X, filed April 16, 2021;

CN 2021105177436, filed May 12, 2021;

CN 2021106375805, filed June 8, 2021;

CN 2021106592421, filed June 11, 2021;

CN 2021108795702, filed July 30, 2021;

CN 2021110408784, filed September 6, 2021;

CN 2021110888122, filed September 16, 2021;

CN 2021113070430, filed November 5, 2021;

CN 2021113430120, filed November 12, 2021;

CN 2021114339622, filed November 29, 2021;

CN 2021115671634, filed December 20, 2021;

CN 2022100298871, filed January 12, 2022;

CN 2022101700462, filed February 23, 2022.

Field of technology to which the invention relates

The present invention relates to the field of medicinal chemistry, in particular to a short ring-modified proline-based peptide compound and its use.

BACKGROUND OF THE INVENTION

There are currently seven varieties of coronaviruses that can infect humans, namely HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV (SARS), MERS-CoV (MERS) and COVID-19, which first discovered in December 2019, and COVID-19 changes from alpha, beta to delta... and then to omicron and is still spreading rapidly and constantly changing. 2022 marks the third year of the COVID-19 pandemic, with total confirmed cases exceeding 500 million and total deaths exceeding 6 million, with confirmed COVID-19 cases exceeding 1 million in approximately 50 countries. The epidemiological mode of transmission, general direction of spread and pandemic level are much higher than those of influenza A, H1N1, in 2009. SARS-CoV-2 is a single-stranded positive-strand RNA virus and is characterized by high homology to SARS-CoV and MERS-CoV. After the virus infects and enters a host cell, with the help of the host cell, the RNA genetic material first mediates the translation and expression of two polyprotein precursors (pp1a and pp1ab), and the polyprotein precursors undergo intramolecular cleavage by 3CL protease and PL protease to produce multiple unstructured proteins. Because the 3CL protease is responsible for cleavage at at least 11 sites, the 3CL protease is also called the major protease (Mpro). Unstructured proteins are involved in the production of subgenic RNA and four structural proteins (E protein, M protein, S protein and N protein) of the virus, thus completing the multiplication and release of viral progeny; 3CL protease is a cysteine protease, and the active form of 3CL protease is a homodimer. Coronavirus 3CL protease is relatively conserved, and 3CL protease substrates from different coronaviruses share common characteristics. Since there is no protease homologous to 3CL protease in the human body, 3CL protease has become one of the ideal targets against coronavirus.

Contents of the present invention

The present invention provides a compound represented by formula (X) or a pharmaceutically acceptable salt thereof

Where

G selected from ring A selected from C _3-10 cycloalkyl,

3-10 membered heterocycloalkyl, ^-^ aryl and 5-10 membered heteroaryl;

each R ₁ is independently selected from halogen, ORn, CN, CH ₃ S((O) _m , -NH(R ₁₂ ), C _1-3 αlkyl and C _1-3 haloalkyl;

- 1 047028

Rn is selected from H, C _1-3 alkyl, C _1-3 haloalkyl, CH ₃ (OCH ₂ CH ₂ ) _P - and H(OCH ₂ CH ₂ )q-;

R ₁₂ is selected from C _1-3 alkyl, C _1-3 haloalkyl, CH3CO- and CH3SO2-;

m is selected from 0, 1 and 2;

p and q are selected from 1, 2, 3, 4, 5 and 6;

n is selected from 0, 1, 2, 3 and 4;

X is selected from -CH(R ₃ )-, -CH2CH2-, O, S, Se, SO2 and -N(R ₃ )-, and -CH2CH2- is optionally substituted with 1,2,3 or 4 R;

each R is independently selected from halogen, OH, NH2, CN, C _1-3 alkyl and C _1-3 haloalkyl;

each R ₃ is independently selected from H, C _1-3 alkyl, C ^ alkoxy, C _1-3 haloalkyl, C _1-3 haloalkoxy, ^-sharal and 5-10 membered heteroaryl, and C _6-10 aryl and 5- The 10-membered heteroaryl is optionally substituted with 1, 2 or 3 R ₃₁ ;

each of R2 and R4 is independently selected from H, C _1-3 alkyl, C _1-3 alkoxy, C _1-3 haloalkyl, C _1-3 haloalkoxy, C _6-10 aryl and 5-10 membered heteroaryl and

C _6-10 aryl and 5-10 membered heteroaryl are optionally substituted with 1, 2 or 3 R ₂₁ or

R2 and R4 together with the atoms to which they are attached form C _5-8 cycloalkyl, 5-6 membered heterocycloalkyl and 5-6 membered heterocycloalkenyl, and C _5-8 cycloalkyl, 5-6 membered heterocycloalkyl and 5-6 -membered heterocycloalkenyl is optionally and independently substituted with 1 or 2 Ra;

each Ra is independently selected from H, halogen, C _1-3 alkyl, C _1-3 alkoxy, C _1-3 haloalkoxy, C _3-6 cycloalkyl, C _6-10 aryl and 5-10 membered heteroaryl, and C _3-6 cycloalkyl, C _6-10 aryl and 5-10 membered heteroaryl are optionally substituted with 1, 2 or 3 R ₄₁ ;

R ₂₁ , R ₃₁ and R 41 are each independently selected from halogen, OH, NH2, CN, C _1-3 alkyl, C _1-3 alkoxy, C _1-3 haloalkyl and C _1-3 haloalkoxy;

R ₅ is selected from C _1-3 alkyl, C _1-3 haloalkyl, C _1-3 alkoxy, C _1-3 haloalkoxy, -CH2-R6 and -CH2-O-R6;

R6 is selected from phenyl, and phenyl is optionally substituted with 1, 2 or 3 _R61 ;

R ₆₁ is selected from halogen, C _1-3 alkyl, C _1-3 haloalkyl, C _1-3 alkoxy and C _1-3 haloalkoxy.

The present invention also provides a compound represented by formulas (X-1) and (X-2), or a pharmaceutically acceptable salt thereof wherein each Rb is independently selected from H, halogen, C _1-3 alkyl, C _1-3 alkoxy, C _{1 -3} haloalkoxy and C _3-6 cycloalkyl;

or two Rb at adjacent carbon atoms or at the same carbon atom together with the atoms to which they are attached form cyclopropyl or cyclobutyl;

t selected from 1 and 2;

R1, _R5 , n and ring A are defined herein.

The present invention also provides the compound represented by formula (IV), or a pharmaceutically acceptable salt thereof, wherein ring A is selected from C _3-10 cycloalkyl, 3-10 membered heterocycloalkyl, C _6-10 aryl and 5-10 membered heteroaryl;

each R1 is independently selected from halogen, OR11, CN, CH ₃ S((O) _m , -NH(R ₁₂ ), C _1-3 alkyl and C _1-3 haloalkyl;

R11 is selected from H, C _1-3 alkyl, C _1-3 haloalkyl, CH ₃ (OCH ₂ CH ₂ ) _p - and H(OCH ₂ CH ₂ ) _q -;

R ₁₂ is selected from ^^alkyl, C _1-3 haloalkyl, CH3CO- and CH3SO2-;

m is selected from 0, 1 and 2;

p and q are selected from 1, 2, 3, 4, 5 and 6;

n is selected from 0, 1, 2, 3 and 4;

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X is selected from -CH(R ₃ )-, -CH2CH2-, O, S, Se, SO2 and -N(R ₃ )- and

-CH ₂ CH ₂ -optionally substituted with 1, 2, 3 or 4 R;

each R is independently selected from halogen, OH, NH2, CN, C ₁ . ₃ αalkyl and C _1-3 haloalkyl;

each R3 is independently selected from H, C ₁ . ₃ alkyl, C _1-3 alkoxy, C ₁ . ₃ haloalkyl, C ₁ . ₃ haloalkoxy, C _6-10 aryl and 5-10 membered heteroaryl and C _6-10 aryl and 5-10 membered heteroaryl optionally substituted with 1, 2 or 3 R ₃ i;

each of R2 and R is independently selected from H, C _1-3 alkyl, C _1-3 alkoxy, C _1-3 haloalkyl, C _1-3 haloalkoxy, C _6-10 aryl and 5-10 membered heteroaryl and C _{6- 10} aryl and 5-10 membered heteroaryl are optionally substituted with 1, 2 or 3 R21;

or

R2 and R4 together with the atoms to which they are attached form C _5-8 cycloalkyl, 5-6 membered heterocycloalkyl and 5-6 membered heterocycloalkenyl, and C _5-8 cycloalkyl, 5-6 membered heterocycloalkyl and 5-6 -membered heterocycloalkenyl is optionally substituted with 1 or 2 Ra;

each Ra is independently selected from H, halogen, C _1-3 alkyl, C _1-3 alkoxy, C _1-3 haloalkoxy, C ₆₁₀ aryl and 5-10 membered heteroaryl, and C _6-10 aryl and 5-10 member heteroaryl is optionally substituted with 1, 2 or 3 R41;

R ₂₁ , R ₃₁ and R 41 are each independently selected from halogen, OH, NH2, CN, C _1-3 alkyl, C _1-3 alkoxy, C _1-3 haloalkyl and C _1-3 haloalkoxy;

heterocycloalkyl, heterocycloalkenyl and heteroaryl contain 1, 2 or 3 heteroatoms or groups of heteroatoms independently selected from O, S, SO2, N, P and Se.

In some embodiments of the present invention, R1 is selected from F, Cl, Br, I, methyl, OH, CN, CH3O-, CH3S-, CH3S(O)-, CH3SO2-, CH3NH-, CH3CONH-, CH3SO2NH-, CH3OCH2CH2O-, CH3(OCH2CH2)2O-, CH3(OCH2CH2)4O-, HOCH2CH2O-, H(OCH2CH2)2O- and H(OCH2CH2)4O-, and other variables are defined herein.

In some embodiments of the present invention, ring A is selected from C _5-9 cycloalkyl, 5-8 membered heterocycloalkyl, adamantyl and phenyl, and other variables are defined herein.

In some embodiments of the present invention, ring A is selected from

and other variables are defined in this document.

youIn some embodiments of the present invention, a structural fragment of branes from

- 3 047028

and other variables are defined in this document.

In some embodiments of the present invention, R _a is selected from H and methyl, and other variables are defined herein.

In some embodiments of the present invention, the structural fragment Y/Y/

selected from

R _a ^R =

Ra ’ and other variables are defined in this document.

^R2 you In some embodiments of the present invention, a structural fragment of branes from

and other variables are defined in this document.

The present invention also provides a compound or a pharmaceutically acceptable salt thereof, and the compound is selected from

where R1, R _a , n, X and ring A are defined herein.

The present invention also provides a compound or a pharmaceutically acceptable salt thereof, and the compound is selected from

where R1, R _a , n, X and ring A are defined herein.

The present invention provides a compound represented by formula (VIII) or a pharmaceutically acceptable salt thereof

Where

- 4 047028

X is selected from -CH(R ₃ )-, -CH2CH2-, O, S, Se, SO2 and -N(R ₃ )-, and -CH2CH2- is optionally substituted with 1, 2, 3 or 4 R;

each R is independently selected from halogen, OH, NH2, CN, C _1-3 alkyl and C _1-3 haloalkyl;

each R ₃ is independently selected from H, C _1-3 alkyl, C _1-3 alkoxy, C _1-3 haloalkyl, C _1-3 haloalkoxy, C _6-10 aryl and 5-10 membered heteroaryl, and C ₆ . ₁₀ aryl and 5-10 membered heteroaryl are optionally substituted with 1, 2 or 3 R ₃₁ ;

each of R2 and R4 is independently selected from H, C _1-3 alkyl, C _1-3 alkoxy, C _1-3 haloalkyl, C _1-3 haloalkoxy, C _6-10 aryl and 5-10 membered heteroaryl and C _{6- 10} aryl and 5-10 membered heteroaryl are optionally substituted with 1, 2 or 3 R ₂₁ or

R2 and R4 together with the atoms to which they are attached form C _5-8 cycloalkyl, 5-6 membered heterocycloalkyl and 5-6 membered heterocycloalkenyl and C _5-8 cycloalkyl, 5-6 membered heterocycloalkyl and 5-6 -membered heterocycloalkenyl is optionally and independently substituted with 1 or 2 R _a ;

each R _a is independently selected from H, halogen, C _1-3 alkyl, C _1-3 alkoxy, C _1-3 haloalkoxy, C _6-10 aryl and 5-10 membered heteroaryl, and C _6-10 aryl and 5-10 -membered heteroaryl is optionally substituted with 1, 2 or 3 R ₄₁ ;

R ₂₁ , R ₃₁ and R 41 are each independently selected from halogen, OH, NH2, CN, C _1-3 alkyl, C _1-3 alkoxy, C _1-3 haloalkyl and C _1-3 haloalkoxy;

heterocycloalkyl, heterocycloalkenyl and heteroaryl contain 1, 2 or 3 heteroatoms or groups of heteroatoms independently selected from O, S, SO ₂ , N, P and Se.

In some embodiments of the present invention, the compound is selected from the structure represented by formula (VIII-1)

Where

R2 and R4 together with the atoms to which they are attached form C _5-8 cycloalkyl and C _5-8 cycloalkyl is optionally substituted with 1 or 2 Ra;

R _a is defined herein.

In some embodiments of the present invention, each R _a is independently selected from H, F and CH ₃ and other variables are defined herein.

In some embodiments of the present invention, the structural fragment Y i 1 _

Г\ _а is selected from

<a _and

d other variables are defined in this document.

In some embodiments of the present invention, the structural fragment

selected from

other variables are defined in this document.

In some embodiments of the present invention, each Rb is independently selected from H, F, methyl, ethyl, isopropyl, cyclopropyl and cyclobutyl, and other variables are defined herein.

In some embodiments of the present invention, a brane structural fragment of

You-

- 5 047028

and other variables are defined in this document.

In some embodiments of the present invention, the structural moiety is selected from

In some embodiments of the present invention, each R ₁ is independently selected from halogen, C _1-3 alkyl, C| _-3 apsoxy, C _1-3 haloalkyl and C _1-3 haloalkoxy and other variables are defined herein.

In some embodiments of the present invention, each R ₁ is independently selected from F, C 1 and methyl, and other variables are defined herein.

In some embodiments of the present invention, ring A is selected from C _5-10 cycloalkyl and phenyl, and other variables are defined herein.

In some embodiments of the present invention, ring A is selected from cyclohexyl, spiro[3.3]heptyl, bicyclo[2.2.2]octyl, adamantyl and phenyl, and other variables are defined herein.

In some embodiments of the present invention, ring A is selected from

and other variables are defined in this document.

In some embodiments of the present invention, the structural moiety ^(Rl)n is selected from

and other variables are defined in this document.

In some embodiments of the present invention, R5 is selected from -CF ₃ , -OCH ₃ , and other variables as defined herein.

The present invention provides a compound represented by the following formula, or a pharmaceutically acceptable salt thereof, and the compound is selected from

(Ri) _n (IV) where ring A is selected from C _3-10 cycloalkyl, 3-10 membered heterocycloalkyl, C _6-10 aryl and 5-10 membered heteroaryl;

each R1 is independently selected from halogen, OR11, CN, CH ₃ S((Q)m, -NH(R ₁₂ ), C _1-3 alkyl and C _1-3 haloalkyl;

Rii is selected from H, C _1-3 alkyl, C _1-3 haloalkyl, CH ₃ (OCH ₂ CH ₂ )p- and H(OCH ₂ CH ₂ )q-;

R ₁₂ is selected from C _1-3 alkyl, C _1-3 haloalkyl, CH3CO- and CH ₃ SO ₂ -;

- 6 047028 m selected from 0, 1 and 2;

p and q are selected from 1, 2, 3, 4, 5 and 6;

n selected from 0, 1,2, 3 and 4;

X is selected from -CH(R3)-, -CH2CH2-, O, S, Se, SO2 and -N(R3)-, and -CH2CH2—optionally substituted with 1, 2, 3 or 4 R;

each R is independently selected from halogen, OH, NH ₂ , CN, u-alkyl and u-alkyl;

each R ₃ is independently selected from H, C _1-3 alkyl, C _1-; ^kokcu, C _1-3 haloalkyl, i-haloalkoxy, i-^aryl and 5-10 membered heteroaryl, and Q-yuaryl and 5-10 membered heteroaryl are optionally substituted with 1, 2 or 3 R ₃₁ ;

each of R ₂ and R 4 is independently selected from H, C ₁ - ₃ alkyl, u-alkoxy, C ₁ - ₃ haloalkyl, C ₁ - ₃ haloalkoxy, u-aryl and 5-10 membered heteroaryl and Q-yuaryl and 5 -10-membered heteroaryl is optionally substituted with 1, 2 or 3 R ₂₁ ;

or

_R2 and R4, together with the atoms to which they are attached, form u-alkloalkyl, 5-6-membered heterocycloalkyl and 5-6-membered heterocycloalkenyl, and u-alkloalkyl, 5-6-membered heterocycloalkyl and 5-6-membered heterocycloalkenyl optionally substituted with 1 or 2 Ra;

each Ra is independently selected from H, halogen, C ₁ - ₃ alkyl, C _1-; ^kokcu, C ₁ - ₃ haloalkoxy, u-^aryl and 5-10 membered heteroaryl and u-^aryl and 5-10 membered heteroaryl optionally substituted with 1, 2 or 3 R ₄₁ ;

R ₂₁ , R ₃₁ and R ₄₁ are each independently selected from halogen, OH, NH ₂ , CN, C ₁ - ₃ alkyl, u-alkoxy, C ₁ - ₃ haloalkyl and C ₁ - ₃ haloalkoxy;

heterocycloalkyl, heterocycloalkenyl and heteroaryl contain 1, 2 or 3 heteroatoms or groups of heteroatoms independently selected from O, S, SO ₂ , N, P and Se.

In some embodiments of the present invention, R1 is selected from F, Cl, Br, I, methyl, OH, CN, CH3O-, CH3S-, CH3S(O)-, CH3SO2-, CH3NH-, CH3CONH-, CH3SO2NH-, CH3OCH2CH2O-, CH ₃ (OCH ₂ CH ₂ ) ₂ O-, CH ₃ (OCH ₂ CH ₂ ) ₄ O-, HOCH ₂ CH ₂ O-, H(OCH ₂ CH ₂ ) ₂ O- and H(OCH ₂ CH ₂ ) ₄ O- and other variables are defined in this document.

In some embodiments of the present invention, Ring A is selected from n-cycloalkyl, 5-8 membered heterocycloalkyl, adamantyl and phenyl, and other variables as defined herein.

In some embodiments of the present invention, ring A is selected from and other variables are defined herein.

In some embodiments of the present invention, the structural moiety ^(Rl)n is selected from

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and other variables are defined in this document.

In some embodiments of the present invention, Ra is selected from H and methyl, and other variables are defined herein.

In some embodiments of the present invention, the structural fragment \ / \ /

N·

selected from ^: a ^a and

^R i 'a ' and other variables are defined herein.

In some embodiments of the present invention, the structural moiety is selected from

and other variables are defined in this document.

The present invention provides a compound represented by a pharmaceutically acceptable salt thereof, and the compound is selected from the following formula, or

wherein ring A is selected from C _3-10 cycloalkyl, 3-10 membered heterocycloalkyl and phenyl;

each R1 is independently selected from halogen and C _1-3 alkyl and C _1-3 alkyl is optionally substituted with 1, 2 or 3 F;

n is selected from 0, 1, 2, 3 and 4;

R ₂ represents H, R ₃ and R ₄ together with the carbon atoms to which they are attached form C\ _-6 iiukjocLikuj, and C _3-b cycloalkyl is optionally substituted with 1 or 2 Ra; or

R3 represents H, R2 and R4 together with the carbon atoms to which they are attached form C _5-8 cycloalkyl, and C _5-8 cycloalkyl is optionally substituted with 1 or 2Ra;

each Ra is independently selected from H and C _1-3 alkyl;

heterocycloalkyl contains 1, 2 or 3 heteroatoms independently selected from O, S, N, P and Se;

carbon atom co* represents a chiral carbon atom that defines the being

- 8 047028 form of the single (R) or (S) enantiomer or a form rich in the single enantiomer.

The present invention provides a compound represented by the following formula, or a pharmaceutically acceptable salt thereof, and the compound is selected from wherein ring A is selected from C _3-10 cycloalkyl, 3-10 membered heterocycloalkyl and phenyl;

each R1 is independently selected from halogen, OR11, CN, CH ₃ S((Q)m, NHR ₁₂ and C _1-3 alkyl and C _1-3 alkyl is optionally substituted with 1, 2 or 3 F;

Rii is selected from H, C _1-3 alkyl, CH ₃ (OCH ₂ CH ₂ )p- and H(OCH ₂ CH ₂ )q- and C _1-3 alkyl is optionally substituted with 1, 2 or 3 halogen atoms;

R ₁₂ is selected from C _1-3 alkyl, CH3CO- and CH ₃ SO ₂ - and C _1-3 alkyl is optionally substituted with 1, 2 or 3 halogen atoms;

m is selected from 0, 1 and 2;

p and q are selected from 1, 2, 3, 4, 5 and 6;

n is selected from 0, 1, 2, 3 and 4;

R2 represents H, _R3 and R4 together with the carbon atoms to which they are attached form _C3-6 cycloalkyl and _C3-6 cycloalkyl is optionally substituted with 1 or 2R _a ;

or

R ₃ represents H, R ₂ and R ₄ together with the carbon atoms to which they are attached form C _5-8 cycloalkyl, and C _5-8 cycloalkyl is optionally substituted with 1 or 2R _a ;

each Ra is independently selected from H and C _1-3 alkyl;

heterocycloalkyl contains 1, 2 or 3 heteroatoms or groups of heteroatoms independently selected from Q, S, SO ₂ , N, P and Se;

carbon atom co* is a chiral carbon atom that determines the existence of a form of a single (R) or (S) enantiomer or a form rich in a single enantiomer.

In some embodiments of the present invention, R1 is selected from F, Cl, Br, I, methyl, OH, CN, CH3O-, CH3S-, _CH3S (O)-, CH3SO2-, CH3NH-, CH3CONH-, CH3SO2NH-, CH3OCH2CH2O -, CH3(OCH2CH2)2O-, CH3(OCH2CH2)4O-, HOCH2CH2O-, R(OCH2CH2)2O- and ^OC^CH^Q- and other variables are defined herein.

In some embodiments of the present invention, R ₁ is selected from F and methyl, and other variables are defined herein.

In some embodiments of the present invention, ring A is selected from C _5-9 cycloalkyl, 5-8 membered heterocycloalkyl, adamantyl and phenyl, and other variables are defined herein.

In some embodiments of the present invention, ring A is selected from and other variables are defined herein.

In some embodiments of the present invention, ring A is selected from C _5-9 cycloalkyl, 5-8 membered heterocycloalkyl and phenyl, and other variables are defined herein.

In some embodiments of the present invention, the ^(Ri)n structural moiety is selected from

- 9 047028

and other variables are defined in this document.

In some embodiments of the present invention, ring A is selected from

О''d θ 0 ό> <d ό О Ρ ? ??

//I/

and other variables are defined in this document.

In some embodiments of the present invention, the structural moiety ^(Rl)n is selected from

and other variables are defined in this document.

In some embodiments of the present invention, Ra is selected from H and methyl, and other variables are defined herein.

In some embodiments of the present invention, the structural fragment

selected from

and other variables are defined in this document.

In some embodiments of the present invention, the structural fragment

- 10 047028 p' ..

selected from ^and ' and other variables defined in this document.

The present invention also provides a compound or a pharmaceutically acceptable salt thereof, and the compound is selected from

where R ₁ , n and ring A are defined herein.

The present invention provides a compound represented by the following formula, or a pharmaceutically acceptable salt thereof, and the compound is selected from

Where

optionally and independently substituted with 1, 2 or 3 Ra';

ring A' is selected from C _3-6 cycloalkyl and C _3-6 cycloalkyl is optionally substituted with 1 or 2 Ra';

ring B is selected from C _4-8 cycloalkyl, C _5-8 cycloalkenyl, 3-8 membered heterocycloalkyl and 5-8 membered heterocycloalkenyl and C _4-8 cycloalkyl, C _5-8 cycloalkenyl, _3-8 membered heterocycloalkyl and 58 member heterocycloalkenyl is optionally substituted with 1 or 2 R _a' ;

ring C selected from

ring D is selected from C _4-8 cycloalkyl, C _5-8 cycloalkenyl and 5-8 membered heterocycloalkyl and C _3-6 cycloalkyl is optionally substituted with 1 or 2 Ra';

each Ra‘ is independently selected from F and methyl;

heterocycloalkyl contains 1, 2 or 3 heteroatoms or groups of heteroatoms independently selected from O, S, N and NH.

In some embodiments of the present invention, the compound is selected from formulas (I'11), (I'-12), (I'-13), (I'-14), (I'-15), (I'-16) and (I'-17)

- 11 047028

wherein rings A', B, C and D are defined herein.

In some embodiments of the present invention, the structural fragment

selected from ⁿ

And

’ and other variables are defined in this document.

youIn some embodiments of the present invention, a structural fragment of branes from

where W is selected from CH2, NH, N(CH ₃ ), O, S and SO2 and other variables are defined herein.

youIn some embodiments of the present invention, a structural fragment of branes from

where W is selected from CH2, NH, O and S and other variables are defined herein.

In some embodiments of the present invention, a brane structural fragment of

You-

- 12 047028

and other variables are defined in this document.

youIn some embodiments of the present invention, a structural fragment of branes from

and other variables are defined in this document.

In some embodiments of the present invention, a brane structural fragment of

You-

other variables are defined in this document.

In some embodiments of the present invention, the structural fragment

you ⁿ ' bran out and

’ and other variables are defined in this document.

Other embodiments of the present invention are derived from any combination of the above variables.

The present invention provides a compound represented by the following formula, or a pharmaceutically acceptable salt thereof, and the compound is selected from

- 13 047028

- 14 047028

- 15 047028

The present invention also provides a pharmaceutical composition containing the compound or a pharmaceutically acceptable salt thereof. Additionally, the pharmaceutical composition may also contain a pharmaceutically acceptable excipient.

The present invention also provides the use of a compound or a pharmaceutically acceptable salt or pharmaceutical composition thereof in the manufacture of a medicament for the treatment of a disease associated with 3CL protease.

In some embodiments of the present invention, the disease associated with the 3CL protease is a coronavirus infection.

In some embodiments of the present invention, the coronavirus infection is a COVID-19 infection.

The present invention also provides a method of treating coronavirus infection, comprising administering a therapeutically effective amount of a compound or a pharmaceutically acceptable salt or pharmaceutical composition thereof to an individual in need thereof.

The present invention also provides the following synthesis routes.

- 16 047028

Way 1:

Way 2:

Reference 1: Aza-Diels-Alder cycloaddition reaction refers to the method described in Tetrahedron, 2009, vol. 65, no. 14, p. 2806-2817.

Way 3:

backfill 2 o

Cl OH

Reference 2: Bioorganic and Medicinal Chemistry Letters, 2001, vol. 11, no. 22, p. 2911-2915;

Link 3: Organic Letters, 2020, vol. 22, No. 17, p. 6863-6867.

Way 4:

The present invention also provides the following research method.

1. Evaluation of antiviral activity and cytotoxicity of compounds in an in vitro study using a new coronavirus replicon system

The compound is diluted twofold and 0.3 μl per well is added to a 384-well microplate.

- 17 047028 holes. SARS-CoV-2 replicon RNA was electroporated into Huh7 cells and then inoculated with 60 μl of the mixture in a microwell plate containing a two-fold dilution of the compound at a density of 4000/well. At the same time, a ZPE control (cells electroporated with the SARSCoV-2 replicon without compound treatment) and an HPE control (a control that is a culture medium) are obtained, and the final concentration of DMSO in the culture medium is 0.5%, and the cells are cultured in an incubator at 5% CO ₂ and 37°C for 1 day. The number of GFP-expressing cells in each well was determined using an Acumen instrument, and the data was used to analyze antiviral activity. The experimental conditions for cytotoxicity are the same as for the experiment for antiviral activity. After incubating the compound with cells for 1 day, CellTiter Glo reagent is added in the dark to detect cell viability, and cell viability in each well is determined using a BioTek microplate reader and the data is used to analyze the cytotoxicity of the sample. The antiviral activity and cell viability of the samples were analyzed using non-linear approximation using GraphPad Prism software and the half-maximal effective concentration (EC ₅₀ ) and half-maximal cytotoxic concentration (CC ₅₀ ) of the samples were calculated.

2. In vitro study of activity and toxicity against the new coronavirus

Core viruses are obtained from African green monkey kidney cells (Vero) from the American Type Culture Collection (ATCC), cat. No. CCL-81. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM, WelGene) supplemented with 10% fetal bovine serum (Gibco) and 1% double antibody solution (Gibco). DMEM supplemented with 2% fetal bovine serum (Gibco) and 1% double antibody solution (Gibco) was used as the experimental culture medium.

The new coronavirus strain eCoV/KOR/KCDC03/2020 is provided by the Korea Center for Disease Control and Prevention (KCDC), serial number NCCP43326.

Cell seeding

After trypsin digestion of Vero cells, Vero cells are diluted to 480,000 cells per ml using experimental culture medium. The diluted cells are added to a 384-well assay plate at 25 µl and 12,000 cells per well using an automatic dispenser. Cells are cultured overnight in an incubator at 5% CO ₂ and 37°C.

Compound Treatment and Viral Infection On day two, compound and CP-100356 (CP-100356, as used herein, refers to compound

) diluted with DMSO and added the diluted compound to the assay wells using an automatic liquid dispensing station. Then, 25 μl of SARS-CoV-2 virus solution diluted with experimental culture medium is added to each well at an MOI of 0.0125. Cell control (cells not treated with compound or viral infection), and control without compound treatment (cells infected with virus without treatment with compound with 0.5% DMSO), and control CP-100356 (cells infected with virus treated with 2 μM CP-100356) were administered. 100356). The final volume of cell culture medium in each well is 50 μl. Cells are cultured in an incubator at 5% CO ₂ and 37°C for 24 hours.

Immunofluorescence staining (1) 24 hours after virus infection, 17 μl of 16% paraformaldehyde was added to each well. The samples containing the virus are then left at room temperature for 30 minutes.

(2) The supernatant is aspirated and the plate is washed twice with DPBS.

(3) Add 25 µL of 0.25% Tritonx-100 to each well and leave at room temperature for 20 min.

(4) Aspirate 0.25% TritonX-100 and wash the plate twice with DPBS.

(5) Add 25 μL of diluted primary antibody (1:3000 dilution) to each well and incubate at 37°C for 1 h.

(6) The primary antibody solution is aspirated and the plate is washed twice with DPBS.

(7) Add 25 μl of diluted sheep anti-rabbit IgG secondary antibody labeled with Alexa Fluor 488 (1:2000 dilution) and 2.5 μg/ml Hoechst 33342 (1:4000 dilution) to each well and incubate for 1 h at 37°C.

(8) The secondary antibody and Hoechst solution are aspirated and the plate is washed twice with DPBS.

(9) Apply Operetta multi-parameter image analyzer to read the plate

- 18 047028 tov and install the device with excitation at 488/405, objective 20/, 5 fields of view per well.

3. Data analysis

Columbus software is used to quantify the total number of cells (Hoechst-stained cell number) and the number of cells infected with the novel coronavirus (Alexa Fluor 488-labeled cell number) in images obtained by reading a multiparameter image analyzer. The proportion of infected cells and the total number of cells are used to analyze the antiviral activity and cytotoxicity of the compounds. The calculation formula is as follows:

Inhibition rate (%) = 100 - (proportion of infected cells in test wells - average proportion of infected cells in cell control wells)/(average proportion of infected cells in cell control wells without compound treatment - average proportion of infected cells in cell control wells ) /100

Cell viability (%)=total number of cells in test wells/average total number of cells in control wells without compound treatment/100

XLfit 4 software is used to analyze the effect of compounds on inhibitory activity and cell viability using non-linear approximation and calculate the IC ₅₀ and CC ₅₀ values of the compounds. The approximation method is a sigmoidal dose-response relationship. The formula for calculating IC ₅₀ and CC ₅₀ is as follows: Y=lower point + (upper point)/(1+(IC ₅₀ /X)Hill slope).

3. In vitro pharmacokinetic study

3.1 Hepatocyte Metabolic Stability Study (HMS)

Mix 1 µM of the compound with hepatocytes of CD-1 mice, SD rats, Beagle dogs, Cynomolgus macaques and humans at 37°C and incubate at various points in time (usually up to 90 minutes), and 7- ethoxycoumarin (7-EC, 30 μM) to evaluate phase I and phase II metabolic activity in the hepatocyte incubation system. The incubation solution is removed at each time point and the reaction is stopped by precipitation of the protein with the organic phase. The supernatant is collected and LC/MS-MS is used to determine the amount of compound remaining at each time point.

3.2 Stability study against liver microsomes (MMS)

Test compounds are incubated with liver microsomes from CD-1 mice, SD rats, beagle dogs, cynomolgus monkeys and humans to assess the stability of the compounds. The test compounds are diluted to obtain 10 μM samples, pre-incubated with five types of liver microsomes for 10 min, and then the NADPH regeneration system working solution is added to the incubation plate at each time point to initiate the reaction. Finally, after 0, 5, 10, 20, 30, and 60 min, stopping solution is added to the reaction plate to stop the reaction. Test compounds and control compounds are detected using the LCMS/MS method.

3.3 Study of the level of binding to blood plasma proteins (PPB)

Protein binding levels of test compounds in the blood plasma of CD-1 mice, Sprague-Dawley rats, Beagle dogs, cynomolgus monkeys and humans were determined by the equilibrium dialysis method.

Way

Blood plasma samples with concentrations of 0.2, 2 and 25 μM are obtained from the above five types of blood plasma and placed in a 96-well equilibrium dialysis device, and dialyzed using phosphate buffered saline at a temperature of 37 ± 1 ° C in for 4 hours. Warfarin was used as a control compound in this experiment. Concentrations of test and control compounds in samples were determined using liquid chromatography coupled to mass spectrometry (LC/MS/MS). Analyte and internal standard retention times, chromatogram collection, and chromatogram integration were processed using Analyst software (ABSCIEX, Framingham, MA, USA).

3.4. CYP enzyme inhibition study (drug interactions)

Pooled human liver microsomes (HLM) are used as a source of CYP enzymes, and the marker substrates of the five CYP isoenzymes are incubated with different concentrations of test compounds in the presence of the cofactor NADPH, and the concentrations of metabolites of the marker substrates in the incubation system are determined. From the resulting dose-response curve, the IC50 values of the test compounds are calculated depending on the specific reaction of the marker substrates catalyzed by CYP isoenzymes.

4. Research regarding physico-chemical properties

4.1 Membrane permeability study

Membrane permeability to test compounds is assessed in MDR ₁ -MDCK II cells. The test compounds are diluted in transport buffer to obtain a sample from the final concentration

- 19 047028 tion 2 µM and injected in both directions (A-B and B-A). After injection, the plates with cells are incubated at 37°C in an incubator containing 5% CO ₂ and at saturating humidity for 150 minutes. After 150 min of incubation, samples are collected and the LC/MS/MS method is used to semi-quantitatively determine the concentrations of test compounds and control compounds in transport samples.

4.2 Solubility study

Kinetic solubility (KS) is measured by shaking the flask method.

Kinetic solubility is the maximum concentration achieved by a compound after equilibration in buffer under DMSO solubilization conditions. Kinetic solubility using the shake flask method was determined by high performance liquid chromatography-ultraviolet spectrophotometry. The concentration of the stock solution of the compound whose kinetic solubility is to be measured is 10 mM dimethyl sulfoxide solution, and the sample solution with 2% dimethyl sulfoxide is prepared by dilution with buffer, the theoretical concentration being 200 μM. The mixture is shaken at room temperature for 24 hours to achieve equilibrium and the equilibrium solution is filtered through an aspiration filter plate. The filtrate is analyzed by high performance liquid chromatography to obtain the peak area of absorption in the ultraviolet region, and the results are calculated using the external standard method in combination with a dilution factor.

5. In vivo pharmacokinetics (PK) study

Pharmacokinetic studies were carried out using mice, rats, beagle dogs or cynomolgus macaques. For the RK study of a single administration of a compound, the test compound is administered through intravenous injection or through intragastric administration once according to a certain dose. For the PK cassette assay, the test compound is administered by intravenous injection or intragastric administration at a specific dose per compound. Blood plasma is collected before administration (0) and at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 hours after administration (sampling time point can be adjusted according to the properties of each compound ). Plasma concentration values are processed using a nonparametric pharmacokinetic software model and pharmacokinetic parameters are calculated using the linear log trapezoidal method.

6. Efficacy of OS43 in vivo

A mouse model of coronavirus (COV) infection (10 days old) is used to evaluate the antiviral efficacy of test compounds in vivo by observing survival rates. Description of a specific method. Mice are inoculated with the virus via a nasal drip and are continuously administered the test compound for 7 days from day 0 to day 6 by intraperitoneal injection once daily, with the first timed administration occurring 2 hours before virus inoculation. Animals are monitored continuously from day 0 to day 14, and their weight, health status, and survival are recorded.

Technical effect

The compounds of the present invention have adequate in vitro activity against the novel coronavirus Mpro protease, adequate in vitro activity against the coronavirus at the cellular level, and are free of cytotoxicity and have adequate pharmacokinetic properties. Following administration of a compound of the present invention in combination with ritonavir, exposure is approximately 20 times greater than that of the drug alone and higher exposure is observed in the lungs of rats.

Brief description of graphic materials

Fig. 1 - method of binding compound 15 to the 6WTT protein;

fig. 2 - method of binding of compound 16 to the 6WTT protein;

fig. 3 - method of binding of compound 17 to the 6WTT protein;

fig. 4 - method of binding of compound 18 to the 6WTT protein;

fig. 5 - method of binding of compound 19 to the 6WTT protein;

fig. 6 - method of binding compound 20 to the 6WTT protein;

fig. 7 - method of binding of compound 21 to the 6WTT protein;

fig. 8 - method of binding of compound 22 to the 6WTT protein;

fig. 9 - method of binding of compound 23 to the 6WTT protein;

fig. 10 - method of binding compound 24 to the 6WTT protein;

fig. 11 - method of binding compound 25 to the 6WTT protein;

fig. 12 - method of binding compound 26 to the 6WTT protein;

fig. 13 - method of binding compound 27 to the 6WTT protein.

Definition and Description

Unless otherwise specified, the following terms and expressions used in this document are intended to have the following meanings. A specific term or expression in the absence of an exact one

- 20 047028 definitions should not be considered vague or unclear, but should be understood in accordance with their ordinary meaning. If a trade name appears in this document, it is assumed that it refers to the corresponding product or its active ingredient.

The term pharmaceutically acceptable is used herein to refer to those compounds, materials, compositions and/or dosage forms that, within the bounds of sound medical judgment, are suitable for use in human and animal tissues without excessive toxicity, irritation, allergic reaction or other problems or complications. in accordance with a reasonable benefit/risk ratio.

The term pharmaceutically acceptable salt refers to a salt of a compound of the present invention that is prepared by reacting a compound containing a particular substituent of the present invention with a relatively non-toxic acid or base. If a compound of the present invention contains a relatively acidic functional group, a base addition salt can be prepared by bringing the compound in neutral form into contact with a sufficient amount of base in a pure solution or suitable inert solvent. Pharmaceutically acceptable base addition salt includes sodium, potassium, calcium, ammonium, organic amine or magnesium salts or the like. If the compound of the present invention contains a relatively basic functional group, an acid addition salt can be prepared by bringing the compound in neutral form into contact with a sufficient amount of acid in a pure solution or suitable inert solvent. Examples of the pharmaceutically acceptable acid addition salt include an inorganic acid salt, wherein the inorganic acid includes, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid and etc.; and an organic acid salt, where the organic acid includes, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid , p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, etc.; and a salt of an amino acid (such as arginine and the like), and a salt of an organic acid such as glucuronic acid and the like. Certain specific compounds of the present invention contain both basic and acidic functional groups, thus they can be converted into any base addition salt or acid addition salt.

The pharmaceutically acceptable salt of the present invention can be synthesized from a starting compound that contains an acidic or basic moiety by a conventional chemical method. Typically, such a salt can be prepared by reacting the compound in free acid or base form with a stoichiometric amount of the corresponding base or acid in water or an organic solvent or a mixture thereof.

The term pharmaceutically acceptable excipient refers to inert substances that are administered along with the active ingredient and provide a benefit to the administration of the active ingredient, including, without limitation, any glidant, sweetener, diluent, preservative, colorant/colorant, flavor enhancer, surfactant a wetting agent, dispersant, disintegrating agent, suspending agent and stabilizer that are acceptable to humans or animals (such as livestock) and licensed by the State Drug and Food Administration.

As used herein and are familiar in the art, the terms treatment or delivery of treatment represent a method of obtaining useful or desired results, including clinical results. Beneficial or desired clinical results may include, but are not limited to, reduction in tumor progression, reduction in tumor size, reduction in tumor growth rate, reduction in tumor invasion and metastasis potential, alleviation or reduction in intensity of one or more symptoms or conditions, reduction in disease severity, stability (i.e., absence of worsening) of the disease state, prevention of the spread of the disease, delay or slowing of the progression of the disease, alleviation or temporary relief of the disease state and remission (partial or total), detectable or undetectable. Treatment or implementation of treatment may also mean prolonged survival compared to the expected survival if no treatment was received.

The term pharmaceutical composition refers to a mixture of one or more compounds of the present invention or salts thereof and a pharmaceutically acceptable excipient. The purpose of the pharmaceutical composition is to facilitate the administration of the compound of the present invention into the body.

The therapeutic dose of the compound of the present invention can be determined according to, for example, the specific treatment application, the route of administration of the compound, the health status and condition of the patient, and the judgment of the attending physician. The ratio or concentration of the compounds present

- 21 047028 of the invention in a pharmaceutical composition may not be fixed, depending on various factors including dose, chemical properties (eg, hydrophobicity) and route of administration.

The term treatment means the administration of a compound or composition described herein for the purpose of alleviating or eliminating a disease or one or more symptoms associated with a disease, and includes:

(i) suppression of a disease or disease state, in other words, stopping its development;

(ii) alleviation of a disease or painful condition, even if the disease or painful condition has abated.

The term "therapeutically effective amount" means an amount of a compound of the present invention that (i) provides treatment for a particular disease, condition or disorder, (ii) alleviates, ameliorates or eliminates one or more symptoms of a particular disease, condition or disorder, or (iii) prevents or delays the onset of one or more symptoms of a specific disease, condition or disorder described herein. The amount of a compound of the present invention that constitutes a therapeutically effective amount varies depending on the compound, the disease state and its severity, the route of administration and the age of the mammal being treated, but can be routinely determined by one skilled in the art according to his or her own knowledge. and the present invention.

Unless otherwise required by the present invention, throughout the specification and following claims, the word include and its variants, such as contains and containing, are to be construed in an open and inclusive sense, in other words, include without limitation.

References herein to the phrase in some embodiments, or in one embodiment, or in another embodiment, or in certain embodiments mean that the specific elements, structures, or features associated with the embodiment are included in at least one embodiment. Thus, occurrences of a phrase in some embodiments, or in one embodiment, or in another embodiment, or in certain embodiments at different places in the description are not always necessarily referring to the same embodiment. Additionally, specific elements, structures, or features may be combined into one or more embodiments in any suitable manner.

Unless otherwise specified, the term isomer is intended to include geometric isomers, cis-trans isomers, stereoisomers, enantiomers, optical isomers, diastereomers and tautomers.

The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis and trans isomers, (-) and (+) enantiomers, and (R) and (b) enantiomers. diastereoisomers, (D)isomers, (b)-isomers, as well as racemic and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present invention. Substituents such as alkyl may contain additional asymmetric carbon atoms. All of these isomers and mixtures thereof are included within the scope of the present invention.

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

Unless otherwise noted, the term cis-trans isomer or geometric isomer refers to the inability of the double bonds or single bonds at the ring-forming carbon atoms to rotate freely.

Unless otherwise specified, the term diastereomer refers to a stereoisomer in which the molecule contains two or more chiral centers and the molecules are not related to each other as mirror images.

Unless otherwise noted, (+) refers to right-hand rotation, (-) refers to left-hand rotation, and/or (±) refers to racemization.

Unless otherwise stated, the absolute configuration of a stereogenic center is represented by a wedge-shaped solid bond (Ύ) and a wedge-shaped dotted bond ( ), and the relative configuration of a stereogenic center is represented by a straight solid bond (^) and a straight dashed bond (''), a wavy line (^) is used for representations of a wedge-shaped solid connection (<*) or a wedge-shaped dotted connection (·'') or a wavy line (<) are used to represent a straight solid connection () or a straight dotted connection ( ).

The term optional or optional means that a subsequent event or condition may be realized, but not necessarily, so that the term includes the case in which the event or condition is realized and the case in which the event or condition is not realized.

The term substituted means that one or more hydrogen atoms at a particular atom are replaced by a substituent, including deuterium and hydrogen variants, provided that the valence con

- 22 047028 crete atom is normal, and the substituted compound is stable. If the substituent is an oxygen atom (ie =O), then this means that two hydrogen atoms are substituted. Positions on the aromatic ring cannot be replaced by a ketone. The term optionally substituted means that the atom may or may not be replaced by a substituent unless otherwise indicated, and the type and number of substituents may be arbitrary as long as it is chemically achievable.

If any variable (such as R) appears more than once in the structure or structure of a compound, then the definition of the variable in each occurrence is independent. Thus, for example, if a group is substituted with 0-2 R's, then that group may optionally be substituted with up to two R's, the definition of R being independent in each case. Moreover, the combination of a substituent and/or its variable is valid only if the combination results in the formation of a stable compound.

If the number of the linker group is 0, such as -(CRR) ₀ -, then this means that the linker group is a single bond.

If one of the variables is selected from a single link, this means that the two groups connected by the single link are directly related. For example, if the L in A-L-Z represents a single bond, then the A-L-Z structure actually represents A-Z.

When a substituent is vacant, it means that the substituent does not exist, for example, when X is vacant in A-X, the structure A-X is actually A. When a listed substituent does not indicate by which atom it is bonded to the group to be replaced, such a substituent can be connected through any of its atoms. For example, if pyridyl functions as a substituent, it can be attached to the substituent group via any carbon atom on the pyridine ring.

If the listed linker group does not indicate the direction of binding, then the direction of binding is arbitrary; for example, if the linker group L contained in ’ is -M-W-, then -M-W- can link ring A and ring B to form in the direction corresponding to the left-to-right reading order and to form in the opposite direction to the left-to-right reading order. A combination of linker groups, substituents and/or variables thereof is permissible only if such combination can lead to the formation of a stable compound.

Unless otherwise stated, when a group contains one or more linkable sites, any one or more sites in the group may be linked to other groups through chemical bonds. If the type of compound of a chemical bond is not established, and an H atom is present in the added site, then the number of H atoms in the specified site will correspondingly decrease by the number of chemical bonds added, and in the case of the addition of a chemical bond, the group becomes of the corresponding valency. The chemical bond between a site and other groups can be represented by a straight solid bond (^), a straight dotted bond (''''), or a wavy line (^^'). For example, a straight continuous bond in -OCH ₃ means that the group is attached to other groups via an oxygen atom in the group; straight dotted bonds in ⁿ mean that the group is attached to other groups at either end of the nitrogen atom in the group; The wavy lines in mean that the phenyl group is attached to other groups through the carbon atoms at position 1 and position 2.

Unless otherwise specified, the terms single-isomer-enriched, isomer-enriched, single-enantiomer-enriched, or enantiomer-enriched refer to a content of one of the isomers or enantiomers of less than 100% and a content of an isomer or enantiomer greater than or equal to 60%, or greater than or equal to 70 %, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99% , or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.

Unless otherwise specified, the term isomer excess or enantiomeric excess refers to the difference in the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90%, and the content of another isomer or enantiomer is 10%, then the excess of the isomer or enantiomer (ee value) is 80%.

Unless otherwise specified, the term C _1-3 alkyl refers to a linear or branched saturated carbon

- 23 047028 lehydrogen group containing from 1 to 3 carbon atoms. _C1-3 alkyl includes _C1-2- and _C2-3 alkyl, etc.; it can be monovalent (such as methyl), divalent (such as methylene), or polyvalent (such as methine). Examples of C _1-3 alkyl include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.

Unless otherwise specified, the term '^-daloalkyl' refers to monohaloalkyl and polyhaloalkyl containing from 1 to 3 carbon atoms. C _1-3 haloalkyl includes C _1-2- , C _2-3- , C _3- , C _2- and C 1 haloalkyl and the like. Examples of C _1-3 haloalkyl include, but are not limited to, trifluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, pentafluoroethyl, pentachloroethyl, 3-bromopropyl and the like.

Unless otherwise specified, the term C _1-3 alkoxy refers to an alkyl group containing from 1 to 3 carbon atoms that are attached to the rest of the molecule by an oxygen atom. _C1-3 alkoxy includes _C1-2- , _C2-3- , _C3- , _C2 alkoxy and the like. Examples of C _1-3 alkoxy include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), and the like.

Unless otherwise specified, the term C _1-3 haloalkoxy refers to monohaloalkoxy and polyhaloalkoxy containing from 1 to 3 carbon atoms. C _1-3 haloalkyl includes C _1-2- , C _2-3- , C _3- , C _2- and C 1 haloalkoxy and the like. Examples of C _1-3 haloalkyl include, but are not limited to, trifluoromethoxy, trichloromethoxy, 2,2,2-trifluoroethoxy, pentafluoroethoxy, pentachloroethoxy, 3-bromopropoxy and the like.

Unless otherwise stated, the number of atoms in a ring is usually defined as the number of members of the ring, for example, a 5-7 membered ring refers to a ring that has 5-7 atoms arranged in a circle.

Unless otherwise stated, _Cn-n+m or _Cn - _Cn+m includes any particular occurrence of n to n+m carbon atoms, for example, _C1-12 includes C1, _C2 , _C3 , _C4 . _C5 , _C6 , _C7 , _C8 , _C9 , Xiu, Si and _C12 , and any range from n to n+m is also included, for example Cm2 includes _C1-3 , _C1-6 , _{C1 -9} , C _3-6 , C _3-9 , C _3-12 , C _6-9 , C ₆ -i2 and C _9-12 , etc.; similarly, n-membered to n+m-membered means that the number of atoms in the ring is from n to n+m, for example, a 3-12 membered ring includes a 3-membered ring, a 4-membered ring, a 5-membered ring , 6-membered ring, 7-membered ring, 8-membered ring, 9-membered ring, 10-membered ring, 11-membered ring and 12-membered ring, and any range from n to n+m is also included, e.g. 3- A 12 membered ring includes a 3-6 membered ring, a 3-9 membered ring, a 5-6 membered ring, a 5-7 membered ring, a 6-7 membered ring, a 6-8 membered ring and a 6-10 membered ring. ring, etc.

Unless otherwise specified, C _3-10 cycloalkyl refers to a saturated cyclic hydrocarbon group of 3-10 carbon atoms, including monocyclic, bicyclic and tricyclic systems, where the bicyclic and tricyclic systems include a spiro ring, a fused ring and a bridged ring. ^-yucycloalkyl includes C _3-8- , C _3-6- , C _3-5- , C _4-10- , C _4-8- , C _4-6- , C _4-5- , ^^-or Ocycloalkyl, etc.; it can be monovalent, divalent or polyvalent. Examples of C _3-10 cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl, [2.2.2]dicyclooctyl and the like.

Unless otherwise specified, C _5-8 cycloalkyl refers to a saturated cyclic hydrocarbon group of 5-8 carbon atoms, including monocyclic and bicyclic systems, where bicyclic systems include a spiro ring, a fused ring, and a bridged ring. _C5-8 cycloalkyl includes _C5-6- , _C5-7- , _C5-8- , C-^cycloalkyl and the like. It can be monovalent, divalent or polyvalent. Examples of C _5-8 cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl, [2.2.2]dicyclooctyl and the like.

Unless otherwise specified, C _4-8 cycloalkyl refers to a saturated cyclic hydrocarbon group of 4-8 carbon atoms, including monocyclic and bicyclic systems, where bicyclic systems include a spiro ring, a fused ring, and a bridged ring. ^^cycloali^ includes C _4-7- , C _4-6- , C _4-5- , C _5-8- or Ocycloalkyl, etc. It can be monovalent, divalent or polyvalent. Examples of C _4-8 cycloalkyl include, but are not limited to, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl, [2.2.2]dicyclooctyl and the like.

Unless otherwise specified, _C3-6 cycloalkyl refers to a saturated cyclic hydrocarbon group of 3-6 carbon atoms, which is a monocyclic and bicyclic system, and ^(Cycloalkyl includes _C3-5- , _C4-5- and ^-cycloalkyl and the like; it may be monovalent, divalent or polyvalent. Examples of C _3-6 cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.

Unless otherwise specified, C _5-8 cycloalkenyl refers to a partially unsaturated cyclic hydrocarbon group of 5-8 carbon atoms containing at least one carbon-carbon double bond, including monocyclic and bicyclic systems, where bicyclic systems include a spiro ring, a fused ring and a bridged ring, and any ring in this system is non-aromatic. Ocycloalkenyl includes _C5-6- , _C5-7- , _C6-8 or _C7-8 cycloalkenyl and the like. It can be monovalent, divalent or polyvalent. Examples of Oscikloalkenyl include, but are not limited to, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl and the like.

Unless otherwise specified, the term 3-10 membered heterocycloalkyl, alone or in combination with other terms, refers to a saturated cyclic group consisting of 3-10 ring atoms, where 1, 2, 3 or 4 of the ring atoms are heteroatoms, independently selected from O, S and N, and os

- 24 047028 tal represent carbon atoms, where the nitrogen atoms are optionally quaternized, and the nitrogen and sulfur heteroatoms may optionally be oxidized (ie NO and S(O)p, p equals 1 or 2). It includes monocyclic, bicyclic and tricyclic systems, where the bicyclic and tricyclic systems include a spiro ring, a fused ring and a bridged ring. Additionally, for a 3- to 10-membered heterocycloalkyl, the heteroatom may occupy a position linking the heterocycloalkyl to the rest of the molecule. 3-10 membered heterocycloalkyl includes 3-8 membered, 3-6 membered, 3-5 membered, 4-6 membered, 5-6 membered, 4 membered, 5 membered and 6 membered heterocycloalkyl, etc. P. Examples of 3 to 10 membered heterocycloalkyl include, but are not limited to, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothienyl (including tetrahydrothiophen-2-yl and tetrahydrothiophen-3-yl, etc.), tetrahydrofuranyl (including tetrahydrofuran-2- yl, etc.), tetrahydropyranyl, piperidinyl (including 1-piperidinyl, 2-piperidinyl and 3-piperidinyl, etc.), piperazinyl (including 1-piperazinyl and 2-piperazinyl, etc.), morpholinyl (including 3 -morpholinyl and 4-morpholinyl, etc.), dioxynyl, dithianyl, isoxazolidinyl, isothiazolidinyl, 1,2-oxazinyl, 1,2-thiazinyl, hexahydropyridazinyl, homopiperazinyl, homopiperidinyl or dioxacycloheptyl, etc.

Unless otherwise specified, the term 3-8 membered heterocycloalkyl, alone or in combination with other terms, refers to a saturated cyclic group consisting of 3-8 ring atoms, where 1, 2, 3 or 4 ring atoms are heteroatoms, regardless selected from O, S and N, and the remainder are carbon atoms, wherein the nitrogen atoms are optionally quaternized, and the nitrogen and sulfur heteroatoms may optionally be oxidized (ie NO and S(O) _p , p is 1 or 2). It includes monocyclic and bicyclic systems, where bicyclic systems include a spiro ring, a fused ring, and a bridged ring. In addition, with respect to a 3-8 membered heterocycloalkyl, the heteroatom may occupy a position linking the heterocycloalkyl to the rest of the molecule. 3-8 membered heterocycloalkyl includes 3-6 membered, 3-5 membered, 4-6 membered, 5-6 membered, 4 membered, 5 membered and 6 membered heterocycloalkyl and the like. Examples of 3-8 membered heterocycloalkyl include, but are not limited to, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothienyl (including tetrahydrothiophen-2-yl and tetrahydrothiophen-3-yl, etc.), tetrahydrofuranyl (including tetrahydrofuran-2-yl and the like), tetrahydropyranyl, piperidinyl (including 1-piperidinyl, 2-piperidinyl and 3-piperidinyl, etc.), piperazinyl (including 1-piperazinyl and 2-piperazinyl, etc.), morpholinyl (including 3-morpholinyl and 4-morpholinyl, etc.), dioxynyl, dithianyl, isoxazolidinyl, isothiazolidinyl, 1,2-oxazinyl, 1,2-thiazinyl, hexahydropyridazinyl, homopiperazinyl, homopiperidinyl or dioxacycloheptyl and the like.

Unless otherwise specified, the term 5-8 membered heterocycloalkyl, alone or in combination with other terms, refers to a saturated cyclic group consisting of 5-8 ring atoms, where 1, 2, 3 or 4 ring atoms are heteroatoms, regardless selected from O, S and N, and the remainder are carbon atoms, wherein the nitrogen atoms are optionally quaternized, and the nitrogen and sulfur heteroatoms may optionally be oxidized (ie NO and S(O)p, p is 1 or 2). It includes monocyclic and bicyclic systems, where bicyclic systems include a spiro ring, a fused ring, and a bridged ring. In addition, in relation to a 5-8 membered heterocycloalkyl, the heteroatom may occupy a position linking the heterocycloalkyl to the rest of the molecule. 5-8 membered heterocycloalkyl includes 5-6 membered, 5-7 membered, 6-8 membered and 7-8 membered heterocycloalkyl and the like. Examples of 5 to 8 membered heterocycloalkyl include, but are not limited to, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothienyl (including tetrahydrothiophen-2-yl and tetrahydrothiophen-3-yl and the like), tetrahydrofuranyl (including tetrahydrofuran-2-yl and the like). ), tetrahydropyranyl, piperidinyl (including 1-piperidinyl, 2-piperidinyl and 3-piperidinyl, etc.), piperazinyl (including 1-piperazinyl and 2-piperazinyl, etc.), morpholinyl (including 3-morpholinyl and 4 -morpholinyl, etc.), dioxynyl, dithianyl, isoxazolidinyl, isothiazolidinyl, 1,2-oxazinyl, 1,2thiazinyl, hexahydropyridazinyl, homopiperazinyl, homopiperidinyl or dioxacycloheptyl, etc.

Unless otherwise specified, the term 5-6 membered heterocycloalkyl, alone or in combination with other terms, refers to a saturated cyclic group of 5-6 ring atoms, where 1, 2, 3 or 4 ring atoms are heteroatoms, regardless selected from O, S, N, P and Se, and the remainder are carbon atoms, wherein the nitrogen atoms are optionally quaternized, and the heteroatoms of nitrogen, sulfur and phosphorus may be optionally oxidized (i.e. NO, S(O) _p and P (O) _p , p equals 1 or 2). It includes monocyclic and bicyclic systems, where bicyclic systems include a spiro ring, a fused ring, and a bridged ring. In addition, in relation to a 5-6 membered heterocycloalkyl, the heteroatom may occupy a position linking the heterocycloalkyl to the rest of the molecule. 5-6 membered heterocycloalkyl includes 5-membered and 6-membered heterocycloalkyl and the like. Examples of 5-6 membered heterocycloalkyl include, but are not limited to, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothienyl (including tetrahydrothiophen-2yl and tetrahydrothiophen-3-yl and the like), tetrahydrofuranyl (including tetrahydrofuran-2-yl and the like), tetrahydropyranyl, piperidinyl (including 1-piperidinyl, 2-piperidinyl and 3-piperidinyl, etc.), piperazinyl (including 1-piperazinyl and 2-piperazinyl, etc.), morpholinyl (including 3-morpholinyl and 4-morpholinyl And

- 25 047028

the like), dioxynyl, dithianyl, isoxazolidinyl, isothiazolidinyl, 1,2-oxazinyl, 1,2-thiazinyl or hexahydropyridazinyl, etc.

Unless otherwise specified, the term 5-8 membered heterocycloalkenyl, alone or in combination with other terms, appropriately means a partially unsaturated cyclic group of 5-8 ring atoms containing at least one carbon-carbon double bond, where 1, 2, 3 or 4 of the ring atoms are heteroatoms independently selected from O, S and N, and the remainder are carbon atoms, where the nitrogen atoms are optionally quaternized, and the nitrogen and sulfur heteroatoms may be optionally oxidized (i.e. NO and S (O)p, p equals 1 or 2). It includes monocyclic, bicyclic and tricyclic systems, where the bicyclic and tricyclic systems include a spiro ring, a fused ring and a bridged ring, and any ring in the systems is non-aromatic. In addition, with respect to a 5-8 membered heterocycloalkenyl, the heteroatom may occupy a position linking the heterocycloalkenyl to the rest of the molecule. 5-8 membered heterocycloalkenyl includes 5-7 membered, 5-6 membered, 4-5 membered, 4 membered, 5 membered and 6 membered heterocycloalkenyl and the like. Examples of 5-8 membered heterocycloalkenyl include, but are not limited to

Unless otherwise specified, the term 5-6 membered heterocycloalkenyl, alone or in combination with other terms, refers to a partially unsaturated cyclic group consisting of 5-6 ring atoms, where 1, 2, 3 or 4 of the ring atoms are heteroatoms, independently selected from O, S, N, P or Se, and the remainder are carbon atoms, wherein the nitrogen atoms are optionally quaternized, and the nitrogen, sulfur and phosphorus heteroatoms may be optionally oxidized (i.e. NO, S(O) _p and Р(О) _р , р equals 1 or 2). It includes monocyclic and bicyclic systems, where bicyclic systems include a spiro ring, a fused ring, and a bridged ring, and any of the rings in the system is non-aromatic. In addition, in relation to a 5-6 membered heterocycloalkenyl, the heteroatom may occupy a position linking the heterocycloalkenyl to the rest of the molecule. 5-6 membered heterocycloalkenyl includes 5-membered and 6-membered heterocycloalkenyl and the like.

Examples of 5-6 membered heterocycloalkenyl include, but are not limited to, or C/ ^N and the like.

Unless otherwise indicated, the terms aromatic _C6-10 ring and Oz-yuaryl may be used interchangeably in the present invention, and the term aromatic _C6-10 ring or Oz-yuaryl means a cyclic hydrocarbon group with a conjugated π-electron system consisting of 6- 10 carbon atoms, which can be a monocyclic, fused bicyclic or fused tricyclic system, where each ring is aromatic. It may be monovalent, divalent or polyvalent, and C^-aryl includes _C6-9- , _C9- , _C10- and _C6 aryl and the like. Examples of ^-^aryl include, but are not limited to, phenyl, naphthyl (including 1-naphthyl and 2-naphthyl, and the like).

Unless otherwise indicated, the terms 5-10 membered heteroaromatic ring and 5-10 membered heteroaryl may be used interchangeably in the present invention, and the term 5-10 membered heteroaryl means a cyclic group with a conjugated π electron system consisting of 5-10 ring atoms, of which 1, 2, 3 or 4 ring atoms are heteroatoms independently selected from O, S, N, P or Se, and the remainder are carbon atoms. It may be a monocyclic, fused bicyclic or fused tricyclic system, where each of the rings is aromatic, where the nitrogen atoms are optionally quaternized, and the nitrogen and sulfur heteroatoms may be optionally oxidized (i.e. NO, S(O) _p and P( O) _p , p equals 1 or 2). The 5-10 membered heteroaryl may be attached to the rest of the molecule via a heteroatom or carbon atom. 5-10 membered heteroaryl includes 5-8 membered, 5-7 membered, 5-6 membered, 5 membered and 6 membered heteroaryl and the like. Examples of 5 to 10 membered heteroaryl include, but are not limited to, pyrrolyl (including N-pyrrolyl, 2-pyrrolyl and 3-pyrrolyl, etc.), pyrazolyl (including 2-pyrazolyl and 3-pyrazolyl, etc.), imidazolyl (including N -imidazolyl, 2-imidazolyl, 4-imidazolyl and 5-imidazolyl, etc.), oxazolyl (including 2-oxazolyl, 4-oxazolyl and 5-oxazolyl, etc.), triazolyl (1H1,2,3-triazolyl, 2H-1,2,3-triazolyl, III-1,2,4-triazolyl and 4H-1,2,4-triazolyl, etc.), tetrazolyl, isoxazolyl (3-isoxazolyl, 4-isoxazolyl and 5- isoxazolyl, etc.), thiazolyl (including 2-thiazolyl, 4-thiazolyl and 5-thiazolyl, etc.), furyl (including 2-furyl and 3-furyl, etc.), thienyl (including 2 -thienyl and 3-thienyl, etc.), pyridyl (including 2-pyridyl, 3-pyridyl and 4-pyridyl, etc.), pyrazinyl, pyrimidinyl (including 2-pyrimidinyl and 4-pyrimidinyl, etc.) , benzothiazolyl (including 5-benzothiazolyl and the like), purinyl, benzimidazolyl (including 2-benzimidazolyl and the like), benzoxazolyl, indolyl (including 5-indolyl and the like), isoquinolyl (including 1-isoquinolyl and 5-isoquinolinyl and the like), quinoxalinyl (including 2-quinoxalinyl and 5-quinoxalinyl and the like) or quinolyl (including 3-quinolyl and 6-quinolyl and the like).

- 26 047028

Unless otherwise specified, the term halogen or halogen, by itself or as part of another substituent, refers to a fluorine, chlorine, bromine or iodine atom.

Optically active (R)- and (b)-isomers or D- and L-isomers can be prepared using chiral synthesis, or chiral reagents, or other conventional techniques. If one kind of enantiomer of a particular compound of the present invention is to be prepared, the net required enantiomer can be obtained by asymmetric synthesis or by derivatization with chiral auxiliaries, followed by separation of the resulting mixture of diastereoisomers and cleavage of the auxiliary group. Alternatively, if the molecule contains a basic functional group (such as amino) or an acidic functional group (such as carboxyl), the compound is reacted with the appropriate optically active acid or base to form a diastereoisomer salt, which is then subjected to diastereomeric resolution by a conventional method, known in the art to obtain the pure enantiomer. In addition, the enantiomer and diastereoisomer are usually isolated by chromatography, which uses a chiral stationary phase and is optionally combined with a chemical derivatization method (eg, carbamate derived from an amine).

The compound of the present invention may contain a non-naturally occurring fraction of an atomic isotope of one or more atoms that make up the compound. For example, the compound may be radiolabeled using a radioactive isotope such as tritium ( ^3H ), iodine-125 ( ^125I ), or C-14 ( ^14C ). As another example, deuterated drugs can be formed by replacing hydrogen with deuterium, wherein the bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon, and compared with non-deuterated drugs, deuterated drugs have the advantage of reducing toxicity and side effects, increasing the stability of the drug, enhancing the effectiveness and prolonging the half-life of the drug, etc. All isotopic variants of the compound of the present invention, whether radioactive or not, are included within the scope of the present invention.

The compounds of the present invention can be prepared by various synthetic routes known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis routes, and equivalent alternatives known to those skilled in the art. fields, with preferred embodiments including, without limitation, embodiments of the present invention.

The structure of the compounds of the present invention can be confirmed by conventional methods known to those skilled in the art, and if the present invention includes an absolute configuration of a compound, then the absolute configuration can be confirmed by means of conventional techniques in the art. For example, in the case of single crystal X-ray diffraction (SXRD), the absolute configuration can be confirmed by collecting diffraction intensity data of the grown single crystal using a Bruker D8 Venture diffractometer with a CuKa light source and the following scan mode: φ/ω scan, and after collection With corresponding data, the crystal structure can be further analyzed in a direct manner (Shelxs97).

The solvents used in the present invention are commercially available.

In the present invention the following abbreviations are used:

ACN means acetonitrile; Boc means tert-butoxycarbonyl; PE means petroleum ether; EA or EtOAc means ethyl acetate; Pre-HPLC stands for Preparative High Performance Liquid Chromatography; °C means degrees Celsius; DCM means dichloromethane; TEBBE reagent means hydrocarbyl titanocene, bis(cyclopentadienyl)a-chloro(dimethylaluminum)a-methylene titanium, CAS: 67719-69-1.

The compounds of the present invention are named according to traditional principles of nomenclature in the art or using ChemDraw® software, and for commercially available compounds, names are used according to the supplier's catalogue.

Detailed Description of the Preferred Embodiment

The present invention is described in detail using the embodiments presented below, but this does not mean that there are any contrary limitations with respect to the present invention. This document describes the present invention in detail and also discloses specific embodiments thereof; It will be apparent to one skilled in the art that modifications and improvements to the embodiments of the present invention are made without departing from the spirit and scope of the present invention.

- 27 047028

Calculation option 1. Simulation of the binding method of compounds 15-27 and protein:

The molecular docking method was carried out using GlideSP ^[1] in Maestro (Schrodinger version 2017-2) and with default settings. The cocrystal structure with PDB ID code 6 WTT was chosen as the template for docking. To obtain the protein, hydrogen atoms were added using the Protein Preparation Wizard module from Maestro ^[2] and the OPLS3 force field was applied. To obtain ligands, 3D structures were generated, and energy minimization calculations were performed using LigPrep ^[3] . A 30 A docking grid was prepared using ligand centroids from the 7BV2 crystal structure. Ligands were then removed and embodiment compounds were placed into molecular docking. The type of interaction between the protein receptor and the ligand was analyzed and then a suitable conformation for docking was selected and saved according to the calculated docking parameters and Global Strain values. The results of modeling the binding of compounds 15-27 and the 6WTT protein are shown in FIG. 1-13.

[1] Glide, Schrodinger, LLC, New York, NY, 2017.

[2] Maestro, Schrodinger, LLC, New York, NY, 2017.

[3] LigPrep, Schrodinger, LLC, New York, NY, 2017.

Conclusion: The compounds of the present invention exhibit proper compatibility with the 6WTT protein.

Option 1.

Synthesis route:

- 28 047028

Stage 1. Synthesis of compound BB-1-2

Compound BB-1-1 (11 g, 38.42 mmol) was added to a solution of ammonia in methanol (7 M, 54.88 ml) and the tube was sealed, and then the reaction mixture was gradually heated to 50°C and stirred continuously for 16 hours. The reaction mixture was concentrated under reduced pressure and dissolved in an appropriate amount of DCM, then concentrated again. The residue was not purified to obtain compound BB-1-2. ^1H NMR (400 MHz, CDCl ₃ ) δ=7.23-7.07 (m, 1H), 6.57-6.36 (m, 1H), 6.125.96 (m, 1H), 5.95 -5.82 (m, 1H), 4.44-4.28 (m, 1H), 3.46-3.23 (m, 2H), 2.60-2.49 (m, 1H), 2 .46-2.29 (m, 1H), 2.15-2.00 (m, 1H), 1.96-1.78 (m, 2H), 1.55-1.37 (m, 9H) .

Stage 2. Synthesis of the hydrochloride salt of compound BB-1

Compound BB-1-2 (2 g, 7.37 mmol) was added to ethyl acetate (10 ml) and a 4 M solution of hydrogen chloride in ethyl acetate (20 ml) was added, and then the reaction mixture was continuously stirred at 20°C for 3 h. The reaction mixture was filtered to obtain a white solid, which was quickly transferred to a flask (high sensitivity to moisture absorption) and concentrated under reduced pressure. The hydrochloride salt of compound BB-1 was obtained. 1H NMR (400 MHz, CD3OD) δ=4.06-3.99 (m, 1H), 3.69-3.60 (m, 2H), 3.44-3.37 (m, 2H), 2 .83-2.70 (m, 1H), 2.48-2.37 (m, 1H), 2.11-2.02 (m, 2H), 1.94-1.81 (m, 1H) , 1.65-1.55 (m, 2H).

Stage 3. Synthesis of compound 1-2

At 0°C, to a solution of compound 1-1 (400.00 mg, 1.66 mmol) in methanol (2 ml) was added toluene (4 ml), (trimethylsilyl)diazomethane (2 M, 1.66 ml) and allowed to proceed reaction in the reaction mixture at 20°C for 16 hours. The reaction mixture was concentrated under reduced pressure to obtain the crude product. The crude product was separated by silica gel column chromatography (petroleum ether/ethyl acetate=10:1) to give compound 1-2. 1H NMR (400 MHz, CDCl ₃ ) δ=4.39-4.20 (m, 1H), 3.87-3.68 (m, 4H), 2.74-2.62 (m, 1H), 1.98-1.87 (m, 1H), 1.83-1.61 (m, 3H), 1.561.49 (m, 1H), 1.48-1.36 (m, 9H), 1. 30-1.21 (m, 1H).

Step 4. Synthesis of the hydrochloride salt of compound 1-3

Ethyl acetate hydrochloride (4 M, 5 ml) was added to the reaction flask with compound 1-2 (0.28 g, 1.10 mmol) and the reaction mixture was allowed to proceed at 20°C for 2 hours. The reaction mixture was concentrated at reduced pressure to obtain a crude product. The hydrochloride salt of compound 1-3 was obtained. ^1H NMR (400 MHz, CD3OD) δ=4.21-4.07 (m, 2H), 3.94-3.79 (m, 3H), 3.03-2.91 (m, 1H), 2.04-2.03 (m, 1H), 2.05-2.00 (m, 1H), 1.93-1.80 (m, 3H), 1.73 (s, 2H).

Step 5. Synthesis of compound 1-4

At 0°C, ^)-2-((tert-butoxycarbonyl)amino)-2-(4 -fluorophenyl)acetic acid (0.5 g, 1.86 mmol), NN-diisopropylethylamine (719.95 mg, 5.57 mmol) and 2-(7-azabenzotriazol-1yl)-KKK^tetramethyluronium hexafluorophosphate (1.06 g, 2.79 mmol) and allowed the reaction to proceed at 20°C for 16 hours. The reaction mixture was poured into 5% citric acid solution to separate the phases, then the aqueous phase was extracted with ethyl acetate (50 mlx2) and the organic phase dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain the crude product. The crude product was separated by silica gel column chromatography (petroleum ether/ethyl acetate=3:1) to give compound 1-4. 1H NMR (400 MHz, CDCl ₃ ) δ=7.60-7.33 (m, 2H), 7.16-6.93 (m, 2H), 5.77 (br d, J=7.9 Hz , 1H), 5.50-5.36 (m, 1H), 3.87-3.68 (m, 3H), 2.81-2.59 (m, 1H), 1.88-1.68 (m, 3H), 1.60 (s, 1H), 1.48-1.39 (m, 9H), 1.34-1.19 (m, 4H).

Step 6. Synthesis of compound 1-5

Lithium hydroxide monohydrate (148.66 mg, 3.54 mmol) was added to a solution of compound 1-4 (0.72 g, 1.77 mmol) in tetrahydrofuran (10 ml) and water (5 ml) and allowed to react in the reaction mixture at 20°C for 16 hours. 50 ml of 5% aqueous citric acid solution was added to the reaction mixture and 50 ml of ethyl acetate was added to separate the phases, then the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain the crude product . Compound 1-5 was obtained. 1H NMR (400 MHz, CDCl ₃ ) δ=7.52-7.34 (m, 2H), 7.137.01 (m, 2H), 5.99 (br d, J=7.3 Hz, 1H), 5.55-5.36 (m, 1H), 4.45-4.18 (m, 1H), 4.08-3.98 (m, 1H), 3.06-2.87 (m, 1H ), 2.01-1.72 (m, 3H), 1.69-1.52 (m, 1H), 1.47-1.37 (m, 10H), 1.27 (brt, J=7 ,1 Hz, 1H).

Step 7. Synthesis of compound 1-6

At 0°C, to a solution of compound 1-5 (0.65 g, 1.66 mmol) in butanone (10 ml) was added the hydrochloride salt of compound BB-1 (412.75 mg, 1.99 mmol), N,N- diisopropylethylamine (642.23 mg, 4.97 mmol, 865.54 µl), 1-hydroxybenzotriazole (268.58 mg, 1.99 mmol) and 1-(3-dimethylaminopropyl)-3ethylcarbodiimide hydrochloride (381.04 mg, 1 .99 mmol) and allowed the reaction to proceed in the reaction mixture at 20°C for 16 hours. The reaction mixture was poured into 20 ml of water, a stirred solution of dichloromethane and methanol (at a volume ratio of 5:1) (50 mlx2) was added for extraction and washed organic phase with 5% citric acid solution (50 mlx1), and washed with

- 29 047028 water (50 mlx1), then the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain the crude product. The crude product was separated by silica gel column chromatography (dichloromethane/methanol=15:1) to give compound 1-6. 1H NMR (400 MHz, DMSO-d ₆ ) δ=8.52-7.98 (m, 1H), 7.71-7.58 (m, 1H), 7.547.39 (m, 2H), 7, 29-7.08 (m, 3H), 7.03 (br s, 1H), 5.46 (br d, J=8.5 Hz, 1H), 4.50-4.18 (m, 1H) , 4.13 (br s, 1H), 3.51 (br s, 1H), 3.15-3.05 (m, 1H), 2.61-2.46 (m, 5H), 2.37 -1.84 (m, 3H), 1.82-1.49 (m, 5H), 1.45-1.28 (m, 9H), 1.25-1.16 (m, 1H).

Step 8. Synthesis of trifluoroacetate salt of compound 1-7

At 0°C, trifluoroacetic acid (1 ml) was added to a solution of compound 1-6 (0.3 g, 515.41 μmol) in dichloromethane (3 ml) and the reaction mixture was allowed to proceed at 20°C for 2 hours. The reaction mixture was concentrated under reduced pressure to obtain the crude product. The trifluoroacetate salt of compound 1-7 was obtained.

Step 9. Synthesis of compound 1

At 0°C, pyridine (492.38 mg, 6.22 mmol, 502.43 μl) and trifluoroacetic anhydride were added to a solution of the trifluoroacetate salt of compound 1-7 (0.25 g, 518.73 μmol) in tetrahydrofuran (2 ml). (272.37 mg, 1.30 mmol, 180.38 µl) and allowed the reaction mixture to proceed at 20°C for 2 The reaction mixture was quenched with 20 ml water and added ethyl acetate (20 mlx2) for extraction, then organic the phase was washed with 5% citric acid (20 mlx1), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain the crude product. The crude product was separated by preparative HPLC (column type: Phenomenex C18 75*30 mm*3 μm; mobile phase: [H ₂ O (NH ₄ HCO ₃ )-ACN]; ACN%: 20-60%, 8 min) to obtain compound 1. 1H NMR (400 MHz, DMSO-d ₆ ) δ=9.42-9.07 (m, 1H), 8.33-7.97 (m, 1H), 6.97-6.83 ( m, 1H), 6.78-6.61 (m, 2H), 6.46-6.34 (m, 2H), 4.95-4.82 (m, 1H), 4.28-4, 02 (m, 1H), 3.28-3.15 (m, 1H), 3.06-2.98 (m, 1H), 2.40-2.16 (m, 2H), 1.58- 1.50 (m, 1H), 1.41-1.16 (m, 2H), 1.04-0.81 (m, 6H), 0.64-0.47 (m, 1H), 0. 41 (br d, J=9.3 Hz, 1H).

Embodiment 2.

Synthesis route:

Step 1. Synthesis of compound 2-1

At 0°C, to a solution of the hydrochloride salt of compound 1-3 (671.62 mg, 2.61 mmol) in N,Ndimethylformamide (5 ml) was added Boc-L-cyclohexylglycine (0.5 g, 2.61 mmol), N ,Ndiisopropylethylamine (1.01 g, 7.83 mmol), 2-(7-azabenzotriazol-1-yl)-HHH^tetramethyluronium hexa fluorophosphate (1.49 g, 3.92 mmol) and ensured the reaction in the reaction mixture at 20°C for 16 hours. The reaction mixture was poured into 5% citric acid solution to separate the phases, then the aqueous phase was extracted with ethyl acetate (50 mlx2) and the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain the crude product. The crude product was separated by silica gel column chromatography (petroleum ether/ethyl acetate=3:1) to give compound 2-1. ¹ H NMR (400 MHz, CDCl ₃ ) δ=5.16 (br d, J=9.4 Hz, 1H), 4.48-4.34 (m, 1H), 4.32-4.23 ( m, 1H), 4.08-4.00 (m, 1H), 3.71 (s, 3H), 2.77-2.69 (m, 1H), 2.05-1.99 (m, 1H), 1.91-1.62 (m, 11H), 1.45-1.41 (m, 9H), 1.23-1.03 (m, 4H),

Step 2. Synthesis of compound 2-2

Lithium hydroxide monohydrate (212.72 mg, 5.07 mmol) was added to a solution of compound 2-1 (1 g, 2.53 mmol) in tetrahydrofuran (10 ml) and water (5 ml) and allowed the reaction mixture to proceed at 20°C for 16 hours. 50 ml of 5% aqueous citric acid solution was added to the reaction mixture and 50 ml of ethyl acetate was added to separate the phases, and the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to obtain the crude product. By

- 30 047028 received connection 2-2. 1H NMR (400 MHz, CDCI3) δ=5.17 (br d, J=9.7 Hz, 1H), 4.39 (br s, 1H), 4.35-4.24 (m, 1H), 4.16-4.11 (m, 1H), 3.04-2.93 (m, 1H), 1.96 (br d, J=10.3 Hz, 1H), 1.87-1.53 (m, 11H), 1.44 (s, 9H), 1.190.95 (m, 4H).

Stage 3. Synthesis of compound 2-3

At 0°C, the hydrochloride salt of compound BB-1 (438.81 mg, 2.11 mmol), K,K- diisopropylethylamine (682.77 mg, 5.28 mmol, 920.18 µl), 1-hydroxybenzotriazole (285.53 mg, 2.11 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (405.09 mg , 2.11 mmol) and allowed the reaction to proceed at 20°C for 16 hours. The reaction mixture was poured into 20 ml of water and extracted with a stirred solution of dichloromethane and methanol (dichloromethane: methanol = 10:1, 50 ml x 2), and the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to obtain the crude product. The crude product was separated by silica gel column chromatography (dichloromethane:methanol=20:1) to give compound 2-3. 1H NMR (400 MHz, DMSO-d ₆ ) δ=8.08 (br d, J=8.4 Hz, 1H), 7.75-7.55 (m, 1H), 7.32-7.18 (m, 1H), 7.08-6.94 (m, 1H), 6.87-6.64 (m, 1H), 4.424.31 (m, 1H), 4.20 (ddd, J=3 ,8, 8.2, 11.6 Hz, 1H), 4.11-4.00 (m, 3H), 3.14-2.96 (m, 2H), 2.55 (br s, 1H) , 2.422.32 (m, 1H), 2.21-2.09 (m, 1H), 2.04-1.95 (m, 1H), 1.93-1.81 (m, 1H), 1 .78-1.49 (m, 11H), 1.40-1.24 (m, 11H), 1.14-0.86 (m, 5H).

Step 4. Synthesis of trifluoroacetate salt of compound 2-4

At 0°C, trifluoroacetic acid (5 ml) was added to a solution of compound 2-3 (0.86 g, 1.61 mmol) in dichloromethane (15 ml) and the reaction mixture was allowed to proceed at 20°C for 2 hours. The reaction mixture was concentrated under reduced pressure to obtain the crude product. The trifluoroacetate salt of compound 2-4 was obtained.

Step 5. Synthesis of compound 2

At 0°C, pyridine (1.41 g, 17.87 mmol) and trifluoroacetic anhydride (782.03 mg) were added to a solution of the trifluoroacetate salt of compound 2-4 (0.7 g, 1.49 mmol) in tetrahydrofuran (10 ml). , 3.72 mmol) and allowed the reaction to proceed in the reaction mixture at 20°C for 2 hours. 20 ml of water was added to the reaction mixture to quench and ethyl acetate (20 mlx2) was added for extraction, then the organic phase was washed with 5% citric acid acid (20 mlx1), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain the crude product. The crude product was separated by preparative HPLC (column type: Phenomenex C18 75*30 mm*3 μm; mobile phase: [H ₂ O (NH ₄ HCO ₃ )-ACN]; ACN%: 25-65%, 8 min.) with obtaining compound 2. Ή NMR (400 MHz, DMSO-d6) δ=9.67 (br d, J=7.2 Hz, 1H), 8.90-8.74 (m, 1H), 7.767.60 ( m, 1H), 5.05-4.88 (m, 1H), 4.56-4.46 (m, 1H), 4.45-4.29 (m, 1H), 3.75 (s, 1H), 3.18-3.02 (m, 2H), 2.462.38 (m, 1H), 2.16-2.04 (m, 3H), 1.87-1.59 (m, 11H) , 1.39-1.26 (m, 2H), 1.16-0.95 (m, 5H).

Embodiment 3.

Synthesis route:

Step 1. Synthesis of compound 3-2

Compound 3-1 (1 g, 3.23 mmol) was dissolved in N-dimethylformamide (10 ml) and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (1 .84 g, 4.85 mmol). The reaction mixture was stirred at 20°C for 0.5 h, then diisopropylethylamine (2.09 g, 16.16 mmol) and the hydrochloride salt of compound 1-3 (601.92 mg, 3.88 mmol) were added and allowed to proceed in the reaction mixture at 20°C for 16 hours. Ethyl was added to the reaction system

- 31 047028 acetate (50 ml) and added 3% citric acid solution (25 ml) and saturated saline solution (25 ml) for extraction to separate the organic phase, and the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:0 to 10:1) to give compound 3-2. ^1H NMR (400 MHz, CD3OD) δ=6.27 (br d, J=9.6 Hz, 1H), 4.62 (br s, 1H), 4.19 (d, J=9.6 Hz , 1H), 3.99 (s, 1H), 3.74-3.68 (m, 3H), 2.71 (br s, 1H), 1.92-1.49 (m, 21H), 1 .44 (s.9H).

Step 2. Synthesis of compound 3-3

Compound 3-2 (1.2 g, 2.69 mmol) was dissolved in tetrahydrofuran (9 ml) and water (4 ml) and lithium hydroxide monohydrate (225.52 mg, 5.37 mmol) was added and the reaction mixture stirred at 20°C for 16 hours. Ethyl acetate (60 ml) and 3% citric acid (30 ml) were added to the reaction system for extraction to separate the organic phase, and the organic phase was washed with saturated saline (30 mlx2) until neutral, dried over anhydrous sodium sulfate, filtered and concentrated. Compound 3-3 was obtained. ^1H NMR (400 MHz, CD3OD) δ=4.60 (br s, 1H), 4.22-4.15 (m, 1H), 3.95 (s, 1H), 2.74 (br s, 1H), 2.05-1.94 (m, 6H), 1.87-1.61 (m, 15H), 1.46-1.40 (m, 9H).

Stage 3. Synthesis of compound 3-4

Compound 3-3 (1.1 g, 2.54 mmol) was dissolved in 2-butanone (12 ml), then 1-hydroxybenzotriazole (343.62 mg, 2.54 mmol) and 1-(3 hydrochloride salt) were added -dimethylaminopropyl)-3ethylcarbodiimide (585.01 mg, 3.05 mmol) and the reaction mixture was stirred at 20°C for 0.5 h. Then diisopropylethylamine (1.64 g, 12.72 mmol) and the hydrochloride salt were added compound BB-1 (580.89 mg, 2.80 mmol) and the reaction mixture was continuously stirred at 20°C for 16 hours. Dichloromethane (60 ml) and 3% citric acid (30 ml) were added to the reaction system for extraction with separating the organic phase and the organic phase was washed with saturated saline (30 mlx2) until neutral with separation of the organic phase, and the organic phase was dried over anhydrous sodium sulfate, filtered and evaporated to dryness by rotary evaporation. The residue was purified by silica gel column chromatography (dichloromethane:methanol=1:0 to 10:1) to give compound 3-4. 1H NMR (400 MHz, CD3OD) δ=4.58 (br s, 1H), 4.51-4.41 (m, 1H), 4.20 (s, 1H), 3.95 (s, 1H) , 3.36-3.30 (m, 2H), 2.72 (br s, 1H), 2.69-2.59 (m, 1H), 2.43-2.30 (m, 1H), 2.22 (br d, J=9.8 Hz, 1H), 2.15-2.07 (m, 1H), 1.97 (br s, 4H), 1.77-1.61 (m, 18H), 1.44 (s.9H).

Step 4. Synthesis of trifluoroacetate salt of compound 3-5

Compound 3-4 (1.1 g, 1.88 mmol) was dissolved in dichloromethane (20 ml), and trifluoroacetic acid (5 ml) was added, the reaction mixture was stirred at 20°C for 1 hour, and the reaction system was directly evaporated to dryness using an oil pump, a small amount of dichloromethane was added and evaporated by rotary evaporation, and the above steps were repeated until the product took the form of a white foam. The trifluoroacetate salt of compound 3-5 was obtained. 1H NMR (400 MHz, CD3OD) δ=4.55 (s, 1H), 4.49 (dd, J=3.6, 11.9 Hz, 1H), 4.02 (s, 1H), 3, 85 (s, 1H), 3.30-3.19 (m, 2H), 2.77 (br s, 1H), 2.72-2.63 (m, 1H), 2.45-2.31 (m, 1H), 2.23 (br d, J=10.0 Hz, 1H), 2.17-2.10 (m, 1H), 2.04 (br s, 4H), 1.94- 1.67 (m, 21H). [M+1]+=486.3.

Step 5. Synthesis of compound 3

The trifluoroacetate salt of compound 3-5 (900 mg, 1.85 mmol) was dissolved in tetrahydrofuran (10 ml), then pyridine (1.47 g, 18.53 mmol, 1.50 ml) and trifluoroacetic acid were added to the resulting solution at 0°C. anhydride (973.13 mg, 4.63 mmol, 644.46 μl) and the temperature was slowly raised to 20°C, then the reaction mixture was stirred for 16 hours. Dichloromethane (50 ml) and 3% citric acid ( 25 ml) for extraction to separate the organic phase, and the organic phase was extracted with saturated brine (25 ml) to separate the organic phase, and the organic phase was dried over anhydrous sodium sulfate, filtered, and the crude product was separated by preparative HPLC to obtain compound 3. 1H NMR (400 MHz, CD3OD) δ=5.06 (dd, J=4.7, 11.3 Hz, 1H), 4.66 (s, 1H), 4.61 (s, 1H), 3, 87 (s, 1H), 3.32-3.18 (m, 2H), 2.73-2.63 (m, 2H), 2.39-2.27 (m, 3H), 2.01 ( br s, 4H), 1.84 (br s, 2H), 1.81 (br s, 2H), 1.77-1.65 (m, 11H), 1.63-1.41 (m, 3H ).

Embodiment 4.

- 32 047028

Synthesis route:

Step 1. Synthesis of compound 4-2

Compound 4-1 (2.74 g, 17.76 mmol) was dissolved in tetrahydrofuran (27.4 ml) and the reaction system was purged with nitrogen three times, cooled to 0°C, then a solution of borane in tetrahydrofuran was slowly added dropwise. 35.52 ml, 1 M). The reaction mixture was stirred at 20°C for 16 hours, sodium hydroxide solution (80 ml, 1 M) was added at 0°C, extracted twice with methyl tert-butyl ether (100 ml), the organic phases were combined and washed with 10% citric acid (80 mlx2) and saturated saline (80 ml), dried over anhydrous sodium sulfate, filtered and concentrated. Compound 4-2 was obtained. 1H NMR (400 MHz, CDCl ₃ ) δ ppm 3.19 (s, 2H) 1.50-1.59 (m, 7H) 1.29-1.38 (m, 6H).

Stage 2. Synthesis of compound 4-3

Compound 4-2 (2.26 g, 16.09 mmol) was dissolved in dichloromethane (67.8 mL) and Dess-Martin periodinane (10.24 g, 24.14 mmol) was added to the reaction system. The reaction mixture was stirred at 20°C for 16 hours. Sodium thiosulfate (50 ml) and saturated sodium bicarbonate solution (70 ml) were added to the reaction mixture and extracted twice with dichloromethane (100 ml). The combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated. Compound 4-3 was obtained. 1H NMR (400 MHz, CDCl ₃ ) δ ppm 9.41 (s, 1H) 1.60 (s, 13H),

Step 3. Synthesis of compound 4-4

Compound 4-3 (1.97 g, 14.25 mmol) was dissolved in methanol (137.9 mL), and R-phenylglycinol compound (2.35 g, 17.10 mmol) was added to the reaction system, and the reaction mixture was stirred at 20°C for 2 hours. The reaction mixture was cooled to 0°C, trimethylsilyl cyanide (9.90 g, 99.78 mmol) was added and stirred at 50°C for 16 hours. The reaction mixture was directly evaporated to dryness by rotary evaporation. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate=10:1) to give compound 4-4. 1H NMR (400 MHz, CDCl ₃ ) δ ppm 7.28-7.39 (m, 5 H) 4.06 (dd, J=9.10, 4.06 Hz, 1H) 3.80 (dd, J =10.96, 3.95 Hz, 1H) 3.58 (t, J=9.98 Hz, 1H) 2.89 (s, 1H) 1.56-1.65 (m, 13H).

Step 4. Synthesis of the hydrochloride salt of compound 4-5

Compound 4-4 (1.42 g, 4.99 mmol) was dissolved in hydrochloric acid (28.4 ml) and glacial acetic acid (7.1 ml) and the reaction mixture was stirred at 80°C for 16 hours. The reaction mixture cooled to 0°C to precipitate solids and filtered. The hydrochloride salt of compound 4-5 was obtained. [M+1]+=303.2.

Step 5. Synthesis of the hydrochloride salt of compound 4-6

The hydrochloride salt of compound 4-5 (2.24 g, 7.38 mmol) was dissolved in methanol (112 ml) and glacial acetic acid (22.4 ml), then 20% wet palladium hydroxide (0.448 g, 638, 02 µmol). 50 psi was introduced into the reaction system. inch of hydrogen, and the reaction mixture was heated to 50°C and stirred for 18 hours. The reaction mixture was filtered and directly evaporated to dryness by rotary evaporation and slurried with methyl tert-butyl ether (40 ml) and filtered. The hydrochloride salt of compound 4-6 was obtained. 1H NMR (400 MHz, CD ₃ OD) δ ppm 3.53 (s, 1H) 1.47-1.71 (m, 13H) 3.53 (s, 1H),

Step 6. Synthesis of compound 4-7

The hydrochloride salt of compound 4-6 (50 mg, 272.86 µmol) was dissolved in 1,4-dioxane (0.375 ml) and water (1 ml), then anhydrous sodium carbonate (115.68 mg, 1.09 mmol) was added ) and di-tert-butyl dicarbonate (119.10 mg, 545.71 µmol) and the reaction mixture was stirred at 20°C for 16 hours. Water (5 ml) and 5% citric acid (10 ml) were added to the reaction mixture, extracted

- 33 047028 was washed twice with ethyl acetate (20 ml) and the organic phases were combined, washed with brine (20 ml), dried over anhydrous sodium sulfate, filtered and evaporated to dryness by rotary evaporation. Compound 4-7 was obtained. 1H NMR (400 MHz, CDCl ₃ ) δ ppm 4.98 (br d, J=8.63 Hz, 1H) 3.97-4.05 (m, 1H) 1.43-1.62 (m, 22H ).

Step 7. Synthesis of compound 4-8

Compound 4-7 (250 mg, 882.26 μmol) was dissolved in NN-dimethylformamide (2.5 ml), then 1-hydroxybenzotriazole (357.64 mg, 2.65 mmol) and 1-(3dimethylaminopropyl) hydrochloride salt were added )-3-ethylcarbodiimide (338.26 mg, 1.76 mmol) and the reaction mixture was stirred at 20°C for 0.5 h, then N,N-diisopropylethylamine (342.08 mg, 2.65 mmol ) and the hydrochloride salt of compound 1-3 (136.92 mg, 882.26 µmol) and the reaction mixture was stirred at 20°C for 16 hours. Water (10 ml) was added to the reaction mixture, extracted twice with ethyl acetate (20 ml ), then the organic phases were combined, washed twice with 5% citric acid (15 ml) and washed four times with brine (10 ml), then dried over anhydrous sodium sulfate, filtered and evaporated to dryness by rotary evaporation. The residue was purified by column chromatography (petroleum ether:ethyl acetate=8:1) to obtain compound 4-8. 1H NMR (400 MHz, CDCl ₃ ) δ ppm 5.20 (br d, J=9.63 Hz, 1H) 4.48-4.54 (m, 1H) 4.22-4.30 (m, 1H ) 4.02-4.11 (m, 1H) 3.76 (s, 3H) 2.76 (br s, 1H) 2.05 (br d, J=10.38 Hz, 1H) 1.68- 1.84 (m, 4H) 1.60 (s, 13H) 1.46(s, 9H).

Step 8. Synthesis of compound 4-9

Compound 4-8 (300 mg, 713.37 µmol) was dissolved in tetrahydrofuran (3 ml) and water (1 ml), then lithium hydroxide monohydrate (59.87 mg, 1.43 mmol) was added and the reaction mixture was stirred at 20°C for 16 hours. Water (10 ml) and 5% citric acid (15 ml) were added to the reaction mixture and extracted twice with ethyl acetate (15 ml). The organic phases were combined, washed with brine (15 ml), dried over anhydrous sodium sulfate, filtered and evaporated to dryness by rotary evaporation. Compound 4-9 was obtained without purification. 1H NMR (400 MHz, CDC13) δ ppm 5.07-5.17 (m, 1H) 4.42-4.47 (m, 1H) 4.26 (br d, J=9.88 Hz, 1H) 4.17 (s, 1H) 3.07 (br s, 1H) 1.70-1.98 (m, 5H) 1.46-1.67 (m, 13H).

Step 9. Synthesis of compound 4-10

Compound 4-9 (266 mg, 654.36 µmol) was dissolved in 2-butanone, then 1-hydroxybenzotriazole (88.42 mg, 654.34 µmol) and 1-(3-dimethylaminopropyl)-3ethylcarbodiimide hydrochloride salt ( 150.53 mg, 785.21 μmol), the reaction mixture was stirred at 20°C for 0.5 h and N,N-diisopropylethylamine (338.28 mg, 2.62 mmol) and the hydrochloride salt of compound BB- 1 (134.42 mg, 785.21 µmol), and the reaction mixture was stirred at 20°C for 16 hours. Water (10 ml) was added to the reaction mixture, extracted twice with dichloromethane (20 ml), the organic phases were combined and washed twice with 5% citric acid (15 ml), washed twice with brine (10 ml), and then dried over anhydrous sodium sulfate, filtered and evaporated to dryness by rotary evaporation. The residue was purified by column chromatography (dichloromethane:methanol = 20:1) to give compound 4-10. [M+1]+=560.4.

Step 10. Synthesis of trifluoroacetate salt of compound 4-11

Compound 4-10 (166 mg, 296.59 µmol) was dissolved in dichloromethane (1.8 ml) and trifluoroacetic acid (0.6 ml) and the reaction mixture was stirred at 20°C for 2 hours. The reaction system was directly evaporated to dryness condition using an oil pump, a small amount of dichloromethane was added and evaporated by rotary evaporation, and the above steps were repeated until the product took the form of a light yellow foam. The trifluoroacetate salt of compound 4-11 was obtained. [M+1]+=460.4.

Step 11. Synthesis of compound 4

The trifluoroacetate salt of compound 4-11 (136 mg, 295.92 µmol) was dissolved in tetrahydrofuran (1.4 ml), cooled to 0°C, then pyridine (79.10 mg, 2.07 mmol) and trifluoroacetic anhydride were added (210.03 mg, 1.18 mmol). The temperature of the reaction mixture was raised to room temperature of 20° C. and stirred for 16 hours. The reaction mixture was extracted twice with water (10 ml), extracted twice with dichloromethane (10 ml) and the organic phases were combined, washed twice with 3% citric acid (10 ml), and washed twice with brine (10 ml), dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was separated by preparative HPLC (column type: C18-2 100*30 mm*5 μm; mobile phase: [H ₂ O (NH ₄ HCO ₃ )-ACN]; ACN%: 30-50%, 20 min) with obtaining compound 4. 1H NMR (400 MHz, CDC13) δ=9.39-8.06 (m, 1H), 7.14-6.89 (m, 1H), 6.10-5.81 (m, 1H), 4.99-4.69 (m, 1H), 4.63-4.39 (m, 1H), 4.03-3.86 (m, 1H), 3.48-3.26 ( m, 2H), 2.98-2.79 (m, 1H), 2.61-1.17 (m, 25H).

- 34 047028

Embodiment 5.

Synthesis route:

Step 1. Synthesis of compound 5-2

Compound 5-1 (5 g, 54.32 mmol) was dissolved in methanol (50 mL) and refluxed at 70° C. for 48 hours. The reaction system was concentrated under reduced pressure to give the crude title product. The high purity crude product was directly used in the next reaction to obtain compound 5-2. 1H NMR (400 MHz, CDCl ₃ ) δ=4.81 (s, 1H), 3.77 (s, 3H), 3.43 (s, 3H).

Step 2. Synthesis of compound 5-3

Compound 5-2 was dissolved in toluene (3 ml), cooled to 0°C, and compound ^)-(+)-phenethylamine (1.5 g, 12.38 mmol, 1.60 ml) was slowly added dropwise. , and the reaction mixture was stirred at 20°C for 1 hour. Ethyl acetate (60 ml) and saturated brine (30 ml) were added to the reaction system to extract and separate the organic phase, the organic phase was dried over anhydrous sodium sulfate and evaporated to dryness by rotary evaporation to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:0 to 5:1) to give the target compound 5-3. 1H NMR (400 MHz, CDCl ₃ ) δ=7.95-7.56 (m, 1H), 7.31-7.17 (m, 5H), 4.71-4.40 (m, 1H), 3.95-3.71 (m, 3H), 1.67-1.51 (m, 3H).

Stage 3. Synthesis of compound 5-4

Compound 5-3 (0.5 g, 2.61 mmol) was dissolved in 2,2,2-trifluoroethanol (5 ml) and trifluoroacetic acid (313.04 mg, 2.75 mmol, 203.28 μl) was added. , cooled to -10°C, stirred for 1 hour, while the temperature was controlled at -10°C and cyclopentadiene (207.40 mg, 3.14 mmol) was slowly added dropwise to the resulting mixture, and the reaction mixture was continuously stirred for 0.5 h. The reaction system was concentrated under reduced pressure, then methyl tert-butyl ether (60 ml) and a saturated sodium bicarbonate solution (30 mlx2) were added to the resulting solution, stirred for 10 min, extracted with separation of the organic phase, the organic phase dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate=1:0 to 5:1) to give compound 5-4. 1H NMR (400 MHz, CDCl ₃ ) δ=7.34-7.18 (m, 5H), 6.59-6.41 (m, 1H), 6.31 (dd, J=1.6, 5 .6 Hz, 1H), 4.35 (br d, J=1.3 Hz, 1H), 3.39 (s, 3H), 3.18-3.03 (m, 1H), 2.95 ( br s, 1H), 2.332.22 (m, 1H), 2.14 (brd, J=8.4 Hz, 1H), 1.54-1.41 (m, 4H). [M+1] ⁺ =258.2.

Step 4. Synthesis of compound 5-5

Compound 5-4 (100.00 mg, 388.61 µmol) was dissolved in tetrahydrofuran (1.25 ml), cooled to -70°C, and the borane-tetrahydrofuran complex (1 M, 427.47) was slowly added dropwise. µl), and the temperature of the reaction mixture was slowly raised to 20°C and stirred for 1 hour. The reaction mixture was cooled to 0°C and a 10% aqueous solution of sodium hydroxide (0.55 ml) and a 30% solution of hydrogen peroxide were added to the resulting mixture (220.28 mg, 1.94 mmol, 186.68 µl), then the temperature was slowly raised to 20°C and the reaction mixture was stirred for 1 hour.

- 35 047028 system was added saturated sodium thiosulfate solution (10 ml), stirred for 10 minutes to quench the reaction and added saturated saline solution (20 ml) and ethyl acetate (60 mlx2) for extraction to separate the organic phase. A small amount of sample solution was taken and the pH was adjusted with 3% citric acid until it was less than 8, and after the starch iodide paper test showed negative results, the mixture was dried over anhydrous sodium sulfate and concentrated at 30°C under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate=1:0 to 5:1) to give compound 5-5. ^1H NMR (400 MHz, CDC13) δ=7.30-7.13 (m, 5H), 3.93 (br d, J=6.5 Hz, 1H), 3.78 (br s, 1H) , 3.70-3.54 (m, 1H), 3.39-3.32 (m, 1H), 3.31-3.24 (m, 3H), 2.49-2.40 (m, 1H), 2.26 (s, 1H), 2.09-2.00 (m, 1H), 1.72 (br d, J=10.1 Hz, 1H), 1.46 (br d, J =6.5 Hz, 1H), 1.41-1.33 (m, 3H). [M+1]+ =276.1.

Stage 5. Synthesis of compound 5-6

Compound 5-5 (800 mg, 2.91 mmol) was dissolved in acetonitrile (10 ml), and 2-iodoxybenzoic acid (976.31 mg, 3.49 mmol) was added and the reaction mixture was allowed to proceed at 75 °C for 1 hour. The reaction system was dried over anhydrous sodium sulfate, filtered, the filter cake was washed with acetonitrile (10 ml) and the filtrates were combined and concentrated to obtain compound 5-6. [M+1]+=274.0.

Step 6. Synthesis of compound 5-7

Compound 5-6 was dissolved in tetrahydrofuran (20 ml), cooled to 0°C, and under a nitrogen atmosphere, a 0.5 M solution of bis(cyclopentadienyl)^chloro(dimethylaluminum)^-methylene titanium in toluene (0.5 M, 4.02 ml), the temperature was slowly raised to 20°C and the reaction mixture was stirred for 2.5 hours. The temperature was lowered to 0°C and saturated sodium bicarbonate solution (1 ml) was added to the reaction system to quench the reaction, then methyl tert-butyl ether (10 ml) was added to the resulting mixture, and anhydrous sodium sulfate was added to dry it, and the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:0 to 10:1) to give compound 5-7. ¹ H NMR (400 MHz, CDCl ₃ ) δ=7.28-7.09 (m, 5H), 4.92 (br s, 1H), 4.71 (s, 1H), 3.81 (s, 1H), 3.48 (q, J=6.5 Hz, 1H), 3.28-3.15 (m, 3H), 2.76-2.69 (m, 2H), 2.65 (br d, J=16.8 Hz, 1H), 2.21 (br d, J=9.8 Hz, 1H), 2.08-1.95 (m, 1H), 1.43 (d, J= 9.8 Hz, 1H), 1.31 (d, J=6.5 Hz, 3H). [M+1]+=272.0.

Step 7. Synthesis of the hydrochloride salt of compound 5-8

Compound 5-7 (310 mg, 1.14 mmol) was dissolved in ethanol (5 ml), 12 M hydrochloric acid (285.61 μl) and wet palladium on carbon (1 g, palladium content 10%) were added, and allowed the reaction to proceed in the reaction mixture under a hydrogen atmosphere using a balloon at 15 psi. inch and at 20°C for 16 hours. The reaction system was filtered through diatomaceous earth and the filtrate was evaporated to dryness by rotary evaporation to obtain the hydrochloride salt of compound 5-8. 1H NMR (400 MHz, CD3OD) δ=4.17-4.00 (m, 1H), 3.88-3.81 (m, 3H), 3.74-3.41 (m, 1H), 2 .55-2.20 (m, 1H), 2.17-1.99 (m, 1H), 1.96-1.71 (m, 3H), 1.36-1.23 (m, 2H) , 1.18-1.01 (m, 3H). [M+1]+=170.0.

Step 8. Synthesis of compound 5-9

Compound 1-b (311.63 mg, 1.35 mmol) was dissolved in N.N-dimethylformamide (3 ml), then O-(7-azabenzotriazol-1-yl)-H,HHH-tetramethyluronium hexafluorophosphate (640, 38 mg, 1.68 mmol) and diisopropylethylamine (725.55 mg, 5.61 mmol, 977.83 μl) and the reaction mixture was stirred for 30 min, then the hydrochloride salt of compound 5-8 (190 mg, 1 .12 mmol) and the reaction mixture was continuously stirred at 20°C for 2.5 hours. Ethyl acetate (60 ml) and 3% citric acid solution (30 ml) were added to the reaction system for extraction to separate the organic phase, and the organic phase was extracted with using saturated saline (30 ml) to separate the organic phase, and dried the organic phase over anhydrous sodium sulfate, concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EA=1:0 to 10:1) to give compound 5-9. 1H NMR (400 MHz, CDC13) δ=6.62-6.36 (m, 1H), 5.26-5.12 (m, 1H), 4.68-4.63 (m, 1H), 4 .34-4.26 (m, 1H), 3.74-3.63 (m, 3H), 2.30-2.18 (m, 1H), 2.11-2.02 (m, 1H) , 1.62-1.52 (m, 4H), 1.50 (br s, 3H), 1.13-1.07 (m, 9H), 1.05 (br s, 9H). [M+1]+=383.3.

Step 9. Synthesis of compound 5-10

Compound 5-9 (0.2 g, 522.89 µmol) was dissolved in tetrahydrofuran (1.5 ml) and water (0.5 ml), and lithium hydroxide monohydrate (43.88 mg, 1.05 mmol) was added. and allowed the reaction to proceed in the reaction mixture at 20°C for 16 hours. Dichloromethane (60 ml) and 3% citric acid (30 ml) were added to the reaction system for extraction to separate the organic phase, saturated saline solution (30 ml) for extraction to separate the organic phase, and the organic phase was dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain compound 5-10. [M+1]+=369.3.

Step 10. Synthesis of compound 5-11

Compound 5-10 (190 mg, 515.65 µmol) was dissolved in N,N-dimethylformamide (2 ml), then

- 36 047028 1-hydroxybenzotriazole (69.67 mg, 515.65 µmol) and 1-(3dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride salt (118.62 mg, 618.78 µmol) were added and the reaction mixture was stirred for 30 minutes , then diisopropylethylamine (333.21 mg, 2.58 mmol, 449.07 μl) and the hydrochloride salt of compound BB-1 (128.49 mg, 618.78 μmol) were added to the resulting mixture and the reaction was allowed to proceed in the reaction mixture at 20° C for 2 hours. Ethyl acetate (50 ml) and 3% citric acid (25 ml) were added to the reaction system to extract and separate the organic phase, extract the organic phase with brine (25 ml) and separate the organic phase, and dry the organic phase. phase over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:methanol=1:0 to 30:1) to give compound 5-11. [M+1]+=522.4.

Step 11. Synthesis of trifluoroacetate salt of compound 5-12

Compound 5-11 was dissolved in dichloromethane (1 ml), then trifluoroacetic acid (0.3 ml) was added and the reaction mixture was allowed to proceed at 20°C for 1 hour. The reaction system was concentrated under reduced pressure to obtain trifluoroacetate salt connections 5-12. [M+1] ⁺ =422.3.

Step 12. Synthesis of compound 5

The trifluoroacetate salt of compound 5-12 (80 mg) was dissolved in dichloromethane (1 ml), then pyridine (150.12 mg, 1.90 mmol, 153.18 μl) and trifluoroacetic anhydride (99.65 mg, 474.46 µmol, 65.99 µl), the temperature of the reaction mixture was slowly increased to 20°C and the reaction was allowed to proceed for 2 hours. A 3% solution of citric acid (30 ml) and dichloromethane (60 mlx2) were added to the reaction system for organic phase extraction, wash the organic phase with brine (60 ml) until neutral and concentrate under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:methanol=10:1) and then purified by preparative high performance liquid chromatography (column type: Phenomenex Luna 80*30mm*3μm; mobile phase: [water (hydrochloric acid)acetonitrile] ; acetonitrile%: 20-40%, 8 min) to obtain compound 5. 1H NMR (400 MHz, CDCl ₃ ) δ=9.55-8.15 (m, 1H), 7.21-7.02 (m , 1H), 6.63-5.95 (m, 1H), 4.66 (br d, J=6.0 Hz, 5H), 3.72-3.31 (m, 2H), 2.33 -1.33 (m, 9H), 1.19-1.02 (m, 12H). [M+1] ⁺ =500.3.

Embodiment 6.

Synthesis route:

or 6A

Step 1. Synthesis of compound 6-2

Compound 6-1 (30 g, 198.46 mmol) and glyoxylic acid (18.27 g, 198.46 mmol, 3.68 ml) were dissolved in anhydrous toluene (300 ml) and the reaction mixture was stirred at 40°C for 3 hours. The reaction mixture was filtered and concentrated to give compound 6-2. [M+1] ⁺ =226.2.

Step 2. Synthesis of compound 6-3

- 37 047028

Compound 6-2 was dissolved in anhydrous methanol (420 ml), sulfuric acid (18.73 g, 190.94 mmol, 10.18 ml) was added and the reaction mixture was stirred at 20°C for 48 hours. methyl tert-butyl ether (300 ml) and water (150 ml) were added for extraction and the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to give the crude product of the desired product. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate=2:1) to give compound 6-3. 1H NMR (400 MHz, DMSO-de) δ=8.52-8.46 (m, 1H), 7.40-7.30 (m, 6H), 5.08 (s, 2H), 3.683.64 (m, 3H), 3.26 (s, 3H).

Stage 3. Synthesis of compound 6-4

Compound 6-3 (26 g, 102.67 mmol) was dissolved in sulfuric acid (10.07 g, 102.67 mmol, 5.47 ml) and anhydrous toluene (260 ml) was added to the mixture and heated to 70°C. Phosphorus trichloride (49.35 mg, 359.33 µmol) was slowly added dropwise to the resulting mixture, and the reaction mixture was continuously stirred at 75°C for 16 hours. The reaction mixture was then concentrated under reduced pressure, and anhydrous toluene (200 ml) was added to dilute and then evaporated by rotary evaporation under reduced pressure, which was repeated three times. Finally, anhydrous toluene (200 ml) was added, trimethylphosphite (15.29 g, 123.20 mmol, 14.56 ml) was slowly added dropwise to the concentrated solution at 75°C, then heated to 90°C and allowed to react at for 1.5 hours. The reaction mixture was poured into saturated aqueous sodium bicarbonate (360 ml) to quench the reaction, ethyl acetate (360 ml) was added with phase separation and the organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate=2:1) to give compound 6-4. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ=7.41-7.30 (m, 5H), 5.09-5.07 (m, 2H), 4.89-4.83 (m, 1H), 3.733.63 (m, 9H).

Step 4. Synthesis of compound 6-6

At 20°C, compound 6-4 (20 g, 60.38 mmol) was dissolved in acetonitrile (50 ml), and 1,8-diazabicyclo[5.4.0]undec-7-ene (11.03 g, 72 .45 mmol, 10.92 ml), the reaction mixture was stirred for 0.5 h, then a solution of compound 6-5 (6.65 g, 60.38 mmol) in acetonitrile (20 ml) was added and the reaction mixture was stirred for 16 hours. Ethyl acetate (40 ml) and water (40 ml) were added to the reaction mixture with phase separation, the organic phases were combined and evaporated by rotary evaporation under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate=2:1) to give compound 6-6. 1H NMR (400 MHz, CDCl ₃ ) δ=7.41-7.30 (m, 5H), 6.12 (br s, 1H), 5.13 (s, 2H), 3.77-3.71 (m, 3H), 3.11 (s, 2H), 2.87-2.81 (m, 2H), 2.10 (br s, 4H), 1.89-1.79 (m, 2H) .

Step 5. Synthesis of the hydrochloride salt of compound 6-7

Compound 6-6 (11 g, 34.88 mmol) was dissolved in anhydrous methanol (5 ml) and 10% wet palladium/carbon (2.20 g, 7.33 mol) was added, followed by hydrogen at 15 lbs. sq. inch and the reaction mixture was stirred at 30°C for 18 hours. The reaction mixture was filtered through diatomite and then concentrated under reduced pressure to obtain the crude product. The crude product was not further purified. The hydrochloride salt of compound 6-7 was obtained. 1H NMR (400 MHz, DMSO-d ₆ ) δ=3.60-3.57 (m, 3H), 3.32 (s, 2H), 3.17-3.12 (m, 1H), 1, 98-1.73 (m, 11H).

Stage 6. Synthesis of compound 6-8

Compound 6-7 (6 g, 32.74 mmol) was added to a solution of water (30 ml) and anhydrous tetrahydrofuran (30 ml), then potassium carbonate (13.58 g, 98.23 mmol) and Boc anhydride were added. (21.44 g, 98.23 mmol, 22.57 ml) and the reaction mixture was stirred at 20°C for 16 hours. 5% citric acid (15 ml) was added to the reaction mixture and then water (40 ml) was added. extracted with ethyl acetate (40 ml) with phase separation. The aqueous phase was extracted with ethyl acetate (30 ml), then the organic phases were combined, washed with brine (30 ml), dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether:ethyl acetate=5:1) to obtain compound 6-8. 1H NMR (400 MHz, DMSO-d ₆ ) δ=3.84-3.83 (m, 1H), 3.57-3.52 (m, 1H), 1.99-1.91 (m, 12H ), 1.87-1.76 (m, 11H).

Step 7. Synthesis of compound 6-9

Compound 6-8 (0.8 g, 2.82 mmol) was dissolved in a solution of anhydrous tetrahydrofuran (6 ml) and water (2 ml), and lithium hydroxide monohydrate (236.95 mg, 5.65 mmol) was added to the reaction mixture. then the reaction mixture was stirred at 20°C for 16 hours. Ethyl acetate (5 ml) was added to the reaction mixture, washed with water (5 ml), then 5% citric acid (15 ml) was added to the aqueous phase, then water was added ( 5 ml) and extracted with ethyl acetate (5 mlx2). The organic phases were combined, washed with brine (15 ml), dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was not further purified to provide compound 6-9. [M+1]+=270.34.

Step 8. Synthesis of compound 6-10

- 38 047028

Compound 6-9 (3 g, 11.14 mmol) was added to Ν,N-dimethylformamide (30 ml), then 1-hydroxybenzotriazole (6.02 g, 44.55 mmol) and 1-(3dimethylaminopropyl) hydrochloride salt were added )-3-ethylcarbodiimide (5.80 g, 44.90 mmol, 7.82 ml) and the reaction mixture was stirred at 20°C for 0.5 h, then N.N-diisopropylethylamine (6.41 g, 33 .42 mmol) and the hydrochloride salt of compound 1-3 (2.13 g, 13.76 mmol) and the reaction mixture was stirred at 20°C for 20 hours. 5% citric acid (15 ml) was added to the reaction mixture, then added water (40 ml) and extracted with ethyl acetate (40 ml). The aqueous phase was then extracted with ethyl acetate (30 ml) and the organic phases were combined, washed with brine (30 ml), dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether:ethyl acetate=1:1) to obtain compound 6-10. [M+1]+=406.25.

Step 9. Synthesis of compound 6-11

Compound 6-10 (300.00 mg, 737.98 µmol) was added to a mixture of anhydrous tetrahydrofuran (2.5 ml) and water (1 ml), then lithium hydroxide monohydrate (61.94 mg, 1.48 mmol) and the reaction mixture was stirred at 20°C for 16 hours. Ethyl acetate (10 ml) was added to the reaction mixture, washed with water (10 ml), then 5% citric acid (2 ml) was added to the aqueous phase, extracted with using ethyl acetate (10 mlx2). The organic phases were combined, washed with brine (10 ml), dried over anhydrous sodium sulfate and concentrated under reduced pressure. Compound 6-11 was obtained from the crude product without further purification. [M+1]+=392.23.

Step 10. Synthesis of compound 6-12

Compound 6-11 (300.00 mg, 764.35 µmol) was added to 2-butanone (6 ml), then 1-hydroxybenzotriazole (103.28 mg, 764.35 µmol) and 1-(3dimethylaminopropyl) hydrochloride salt were added )-3-ethylcarbodiimide (175.83 mg, 917.23 μmol) and the reaction mixture was stirred at 20°C for 0.5 h. N,N-diisopropylethylamine (263.43 mg, 2.04 mmol) was added to the reaction system , 355.03 µl) and the hydrochloride salt of compound BB-1 (158.72 mg, 764.35 µmol) and the reaction mixture was stirred at 20°C for 16 hours. 5% citric acid (5 ml) was added to the reaction mixture, then water (10 ml) was added and extracted with ethyl acetate (10 mlx2). The aqueous phase was extracted with ethyl acetate (10 ml) and the organic phases were combined, washed with brine (5 ml), dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (dichloromethane:anhydrous methanol =10:1) to obtain compound 6-12. [M+1]+=545.32.

Step 11. Synthesis of compound 6-13

Compound 6-12 (0.1 g, 183.26 µmol) was dissolved in dichloromethane (1 ml), then trifluoroacetic acid (41.79 mg, 366.52 µmol, 27.14 µl) was added and the reaction mixture was stirred at 20°C for 1 hour. The reaction mixture was then evaporated to dryness by rotary evaporation, dichloromethane was added and concentrated under reduced pressure, and the above steps were repeated three times, then the concentrated solid was dissolved in anhydrous methanol (5 ml). Methyl trifluoroacetate (1.44 g, 11.22 mmol, 1.13 mL) was added to the reaction system, and triethylamine (681.33 mg, 6.73 mmol, 937.17 μL) was added thereto. The reaction mixture was stirred at 38°C for 12 hours. The reaction mixture was directly concentrated under reduced pressure and purified by column chromatography (petroleum ether:ethyl acetate=1:1) to obtain compound 6-13. [M+1]+=541.25.

Step 12. Synthesis of compound 6

Compound 6-13 (0.1 g, 184.65 µmol) was dissolved in dichloromethane (1 ml), then methyl N(triethylammoniumsulfonyl)carbamate (110.01 mg, 461.63 µmol) was added and the reaction mixture was stirred at 25°C for 16 hours, then dichloromethane (10 ml) was added to the reaction mixture, saturated sodium bicarbonate solution (5 ml) was added to the resulting extraction with phase separation, saturated saline solution (5 ml) was added to the organic phase with phase separation , then the organic phase was dried over sodium sulfate and concentrated under reduced pressure to obtain the crude product, and the crude product was separated by preparative HPLC (column type: C18 100 * 30 mm * 10 μm; mobile phase: [H ₂ O(NH ₄ HCO ₃ )-ACN]; ACN%: 40-60%, 8 min) to obtain the crude product. The crude product was separated using the SFC analytical method: column type: Chiralpak AD-3, ext. dia. 150x4.6 mm, 3 µm, mobile phase: A: CO ₂ , B: EtOH (0.1% IPAm, v/v), gradient: time A% B%, 0.0-0.5 min. , V% from 10 to 50%, retention for 4.5 min, 4.5-5.0 min, V% from 50 to 10%, flow rate: 2.5 ml/min., column temperature: 35° C, ABPR: 2000 psi. inch. The retention time of compound 6A was 1.97 minutes and the retention time of compound 6 was 2.28 minutes. The crude product was separated by SFC (column type: DAICEL CHIRALPAK AD (250 mm*30 mm, 10 μm); mobile phase: [0.1% NH ₃ H ₂ O-EtOH]%: 16%-16%, 10 min) to give compound 6. 1H NMR (400 MHz, CDCl ₃ ) δ=7.22-7.17 (m, 1H), 4.88-4.79 (m, 1H), 4.75-4.67 ( m, 1H), 4.38-4.33 (m, 1H), 3.53-3.32 (m, 2H), 2.93-2.84 (m, 1H), 2.71-2, 58 (m, 1H), 2.33- 39 047028

2.05 (m, 3H), 2.04-1.74 (m, 16H), 1.72-1.60 (m, 2H), 1.57-1.41 (m, 3H).

Embodiment 7.

Step 1. Synthesis of compound 7-2

Compound 7-1 (5 g, 35.16 mmol) was dissolved in tetrahydrofuran (50 ml) and the reaction system was purged with nitrogen three times, cooled to 0°C, and a solution of borane in tetrahydrofuran (70.33 ml) was slowly added dropwise. , 1M). The reaction mixture was stirred for 16 hours at 20°C. Sodium hydroxide solution (80 ml, 1 M) was added to the reaction mixture at 0°C, extracted twice with methyl tert-butyl ether (100 ml), the organic phases were combined and washed with 10% citric acid (80 ml x 2) and brine (80 ml), dried over anhydrous sodium sulfate, filtered and concentrated. Compound 7-2 was obtained. 1H NMR (400 MHz, CDCl ₃ ) δ ppm 3.28 (s, 2H) 1.22-1.46 (m, 10H) 0.85-0.87 (m, 3H).

Step 2. Synthesis of compound 7-3

Compound 7-2 (4 g, 31.20 mmol) was dissolved in acetonitrile (40 ml) and 2-iodobenzoic acid (13.10 g, 46.80 mmol) was added to the reaction system. The reaction mixture was stirred at 50°C for 16 hours. The reaction system was filtered and the filtrate was used directly in the next reaction without purification to obtain a solution of compound 7-3 in acetonitrile.

Step 3. Synthesis of compound 7-4

A solution of compound 7-3 (4 g, 31.37 mmol) in acetonitrile was dissolved in methanol (60 ml) and the compound R-phenylglycinol (5.22 g, 38.04 mmol) was added to the reaction system and the reaction mixture stirred at 20°C for 2 hours. The reaction system was cooled to 0°C, trimethylsilyl cyanide (22.01 g, 221.88 mmol) was added and the reaction mixture was stirred at 50°C for 16 hours. The reaction mixture was directly evaporated to dryness by rotary evaporation. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate=3:1) to give compound 7-4. [M+1]+=273.3. 1H NMR (400 MHz, CDCl ₃ ) δ=7.42-7.29 (m, 5H), 4.17-4.01 (m, 1H), 3.99-3.68 (m, 2H), 3.58-3.39 (m, 1H), 1.60-1.16 (m, 10H), 1.10-1.04 (m, 3H).

Step 4. Synthesis of compound 7-5

Compound 7-4 (5 g, 18.36 mmol) was dissolved in methanol (50 ml) and dichloromethane (50 ml), cooled to 0°C and lead tetraacetate (13.56 g, 27.53 mmol) was added. and the reaction mixture was stirred at 0°C for 15 minutes. The reaction mixture was poured into saturated sodium bicarbonate solution (45 ml), extracted three times with dichloromethane (45 ml). The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain the crude product. Compound 7-5 was obtained. [M+1]+=241.2.

Step 5. Synthesis of the hydrochloride salt of compound 7-6

Compound 7-5 (3 g, 12.48 mmol) was dissolved in 6 M hydrochloric acid (300 ml), heated to 100°C and the reaction mixture was stirred for 24 hours. The reaction system was extracted three times with chloroform (300 ml) , the aqueous phase was collected and concentrated under reduced pressure. The hydrochloride salt of compound 7-6 was obtained. 1H NMR (400 MHz, CD ₃ OD) δ=4.47 (s, 1H), 1.66-1.42 (m, 10H), 1.22-1.14 (m, 3H).

- 40 047028

Step 6. Synthesis of compound 7-7

The hydrochloride salt of compound 7-6 (1 g, 5.84 mmol) was dissolved in tetrahydrofuran (10 ml) and water (10 ml), then anhydrous potassium carbonate (2.42 g, 17.52 mmol) and di- tert-butyl dicarbonate (2.55 g, 11.68 mmol) and the reaction mixture were stirred at 20°C for 16 hours. The reaction mixture was adjusted to pH 3 using 1 M KHSO₄ and the mixture was extracted four times with dichloromethane (40 ml), then the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. Compound 7-7 was obtained. [M-1] =270.3.

Step 7. Synthesis of compound 7-8

Compound 7-7 (350 mg, 1.29 mmol) was dissolved in MH-dimethylformamide (4 ml) and 2-(7-azabenzotriazol-1-yl)-CMI.I-tetramethyluronium hexafluorophosphate (735.65 mg, 1.93 mmol), and the reaction mixture was stirred at 20°C for 0.5 h. N,Ndiisopropylethylamine (833.51 mg, 6.45 mmol) and the hydrochloride salt of compound 1-3 (260.23 mg , 1.68 mmol), then the reaction mixture was stirred at 20°C for 16 hours. Water (10 ml) was added to the reaction mixture, extracted twice with ethyl acetate (20 ml) and the organic phases were combined, washed twice with 5% citric acid (15 ml) and washed four times with brine (10 ml), dried over anhydrous sodium sulfate, filtered and evaporated to dryness by rotary evaporation. The residue was purified by column chromatography (petroleum ether:ethyl acetate=5:1) to obtain compound 7-8. [M+1]+=409.4.

Step 8. Synthesis of compound 7-9

Compound 7-8 (120 mg, 293.74 µmol) was dissolved in tetrahydrofuran (6 ml), methanol (2 ml) and water (2 ml), then lithium hydroxide monohydrate (36.98 mg, 881.21 µmol) was added ) and stirred the reaction mixture at 20°C for 16 hours. 3% citric acid (20 ml) was added to the reaction mixture, extracted twice with ethyl acetate (20 ml) and the organic phases were combined, washed with brine (20 ml ), dried over anhydrous sodium sulfate, filtered and evaporated to dryness by rotary evaporation to give compound 7-9. [M+1]+=391.3.

Step 9. Synthesis of compound 7-10

Compound 7-9 (30 mg, 76.04 µmol) was dissolved in N,N-dimethylformamide (2 ml), then 1-hydroxybenzotriazole (10.28 mg, 76.04 µmol) and 1-(3dimethylaminopropyl) hydrochloride salt were added )-3-ethylcarbodiimide (17.49 mg, 91.25 µmol) and the reaction mixture was stirred at 20°C for 0.5 h. N,N-diisopropylethylamine (39.31 mg, 304.18 µmol) was added to the resulting mixture. and the hydrochloride salt of compound BB-1 (19.53 mg, 114.07 µmol), then the reaction mixture was stirred at 20°C for 16 hours. Water (10 ml) was added to the reaction mixture, extracted twice with dichloromethane (20 ml ), the organic phases were combined, washed twice with 5% citric acid (15 ml) and washed twice with brine (10 ml), then dried over anhydrous sodium sulfate, filtered and evaporated to dryness by rotary evaporation. The residue was purified by column chromatography (dichloromethane:methanol =20:1) to obtain compound 7-10. [M+1]+=548.4.

Step 10. Synthesis of trifluoroacetate salt of compound 7-11

Compound 7-10 (150 mg, 273.88 µmol) was dissolved in dichloromethane (3 ml) and trifluoroacetic acid (1 ml) and the reaction mixture was stirred at 20°C for 2 hours. The reaction mixture was directly evaporated to dryness by rotary evaporation using an oil pump, dichloromethane was added and evaporated by rotary evaporation and the above steps were repeated. The trifluoroacetate salt of compound 7-11 was obtained.

Step 11. Synthesis of compound 7-12

The trifluoroacetate salt of compound 7-11 (120 mg, 268.11 µmol) was dissolved in methanol (2 ml), then triethylamine (162.78 mg, 1.61 mmol) and methyl trifluoroacetate (343.32 mg, 2.68 mmol). The reaction mixture was heated to 38°C and stirred for 16 hours. The reaction system was directly concentrated under reduced pressure, then dissolved in water (10 ml) and ethyl acetate (20 ml) and adjusted to acidic pH with 3% citric acid (10 ml), then the mixture was extracted three times with ethyl acetate (20 ml) and then washed twice with brine (10 ml), dried over anhydrous sodium sulfate, filtered and concentrated. Compound 7-12 was obtained. [M+1]+=544.3.

Step 12. Synthesis of compound 7

Compound 7-12 (130 mg, 239.16 µmol) was dissolved in dichloromethane (4 ml), then tetrahydrofuran (0.4 ml) and Burgess' reagent (142.48 mg, 597.89 µmol) were added and the reaction mixture was stirred at 25°C for 2 hours. Dichloromethane (10 ml) was added to the reaction system, washed with saturated sodium bicarbonate (5 ml), then washed with brine (5 ml) to obtain an organic phase and the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. Crude

- 41 047028 the product was separated by preparative HPLC (column type: C18 100*30 mm*10 μm; mobile phase: [water (NH ₄ HCO ₃ )-ACN]; ACN %: 35-55%, 8 min) to obtain the compound 7. 1H NMR (400 MHz,

CDCl3) δ=4.92-4.81 (m, 1H), 4.78-4.63 (m, 1H), 4.60 (br s, 1H), 3.50-3.34 (m, 2H), 2.93-2.79 (m, 1H), 2.30 (br s, 2H), 2.10 (br s, 2H), 1.94-1.86 (m, 1H), 1 .75 (br s, 2H), 1.71-1.60 (m, 4H), 1.33 (br d, J=16.5 Hz, 10H), 1.301.17 (m, 1H), 1, 16-0.96 (m, 3H).

Embodiment 8.

Synthesis route:

Step 1. Synthesis of compound 8-1

Compound 5-6 (2 g, 7.32 mmol) was dissolved in tetrahydrofuran (20 ml) and the reaction system was purged with nitrogen three times, cooled to -70°C, then lithium diisopropylamide (2 M, 7. 32 ml) and allowed the reaction mixture to proceed for 1 hour. Subsequently, methyl iodide (2.60 g, 73.17 mmol, 4.56 ml) was slowly added dropwise at -70°C and the temperature was slowly raised to 20°C. and continued to allow the reaction to proceed for 1 hour. Under a nitrogen atmosphere, saturated ammonium chloride solution (100 ml) was slowly added dropwise to the reaction system to quench the reaction, then extracted twice with ethyl acetate (200 ml) and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate=1:0 to 10:1) to give compound 8-1. ¹ H NMR (400 MHz, CDCl ₃ ) δ=7.34-7.27 (m, 3H), 7.26-7.18 (m, 2H), 3.87-3.77 (m, 1H) , 3.67-3.59 (m, 1H), 3.25 (s, 3H), 2.96 (s, 1H), 2.67-2.64 (m, 1H), 2.64-2 .55 (m, 2H), 1.94 (d, J=10.7 Hz, 1H), 1.43 (d, J=6.5 Hz, 3H), 1.14-1.09 (m, 3H).

Step 2. Synthesis of compound 8-2

Compound 8-1 (600 mg, 2.09 mmol) was dissolved in H^dimethylformamide (6 ml), then p-toluenesulfonylhydrazide (466.62 mg, 2.51 mmol) and trifluoromethanesulfonic acid (36.69 mg, 239.58 µmol, 21.58 µl), heated to 100°C, then sodium cyanoborohydride (393.65 mg, 6.26 mmol) was added and the reaction mixture was allowed to proceed for 2 hours. Ethyl acetate ( 60 ml), washed with saturated sodium bicarbonate solution (30 ml) and saturated brine (30 ml) to separate the organic phase, and dried the organic phase over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:0 to 10:1) to give compound 8-2. 1H NMR (400 MHz, CDCl ₃ ) δ=7.37-7.29 (m, 2H), 7.25 (br d, J=7.1 Hz, 3H), 3.67-3.50 (m , 1H), 3.48-3.34 (m, 1H), 3.32-3.15 (m, 3H), 2.51 (s, 1H), 2.35-2.23 (m, 2H ), 2.02-1.94 (m, 1H), 1.70-1.61 (m, 1H), 1.46-1.41 (m, 1H), 1.36 (d, J=6 .5 Hz, 3H), 1.18-1.10 (m, 1H), 0.96 (d, J=7.1 Hz, 3H).

Step 3. Synthesis of the hydrochloride salt of compound 8-3

Compound 8-2 (150 mg, 548.71 µmol) was dissolved in ethanol (4 ml), then 12 M hydrochloric acid (137.18 µl) and wet palladium on carbon (0.5 g, palladium content 10%) were added. ) and allowed the reaction to proceed in the reaction mixture under a hydrogen atmosphere using a balloon at 15 psi. inch and at 20°C for 16 hours. The reaction system was filtered through diatomaceous earth and evaporated to dryness by rotary evaporation to obtain hyd

- 42 047028 hydrochloride salt of compound 8-3. [M+1]+=170.1.

Step 4. Synthesis of compound 8-4

Compound 1-b (114.81 mg, 496.39 µmol) was dissolved in N.N-dimethylformamide (2 ml), then O-(7-azabenzotriazol-1-yl)-KKKN-tetramethyluronium hexafluorophosphate (235.93 mg) was added , 620.49 µmol) and diisopropylethylamine (267.31 mg, 2.07 mmol, 360.26 µl) and stirred the reaction mixture for 30 min, then the hydrochloride salt of compound 8-3 (70 mg) and the reaction mixture were added stirred continuously at 20°C for 2.5 hours. Ethyl acetate (40 ml) was added to the reaction system, then extracted with 3% citric acid solution (30 ml) and saturated sodium bicarbonate solution (20 ml) to separate the organic phase and extracted with saturated brine (20 ml) to separate the organic phase, and dried the organic phase over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain compound 8-4. [M+1]+=383.3.

Step 5. Synthesis of compound 8-5

Compound 8-4 (125 mg, 326.80 µmol) was dissolved in a mixed solvent of tetrahydrofuran (3 ml), water (1 ml) and methanol (1 ml), and lithium hydroxide monohydrate (41.14 mg, 980. 41 µmol) and allowed the reaction to proceed at 30°C for 16 hours. Dichloromethane (40 ml) and 3% citric acid (20 ml) were added to the reaction system for extraction to separate the organic phase. Then, saturated brine (20 ml) was added for extraction to separate the organic phase, and the organic phase was dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain compound 8-5. [M+1]+=369.3.

Step 6. Synthesis of compound 8-6

Compound 8-5 (115 mg, 312.10 µmol) was dissolved in N,N-dimethylformamide (2 ml), then 1-hydroxybenzotriazole (50.61 mg, 374.53 µmol) and 1-(3dimethylaminopropyl hydrochloride salt) were added )-3-ethylcarbodiimide (89.75 mg, 468.16 µmol), stirred for 30 min, then diisopropylethylamine (161.35 mg, 1.25 mmol, 217.45 µl) and BB-1 hydrochloride salt were added (64.12 mg, 374.53 μmol) and allowed the reaction to proceed in the reaction mixture at 20°C for 2 hours. Ethyl acetate (50 ml) and 3% citric acid (25 ml) were added to the reaction system for extraction to separate organic phase, extracted the organic phase with brine (25 ml) to separate the organic phase, and dried the organic phase over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (dichloromethane:methanol=1:0 to 30:1) to give compound 8-6. [M+1]+=522.4.

Step 7. Synthesis of trifluoroacetate salt of compound 8-7

Compound 8-6 (240 mg, 460.08 µmol, 1 eq.) was dissolved in dichloromethane (1 ml), then trifluoroacetic acid (0.3 ml) was added and the reaction mixture was allowed to proceed at 20°C for 1 hour. The reaction system was directly concentrated under reduced pressure to obtain the trifluoroacetate salt of compound 8-7. [M+1]+=422.3.

Step 8. Synthesis of compound 8-8

The trifluoroacetate salt of compound 8-7 (180 mg, 427.01 μmol) was dissolved in methanol (2 ml), then triethylamine (172.84 mg, 1.71 mmol, 237.74 μl) and methyl trifluoroacetate (546.79 mg, 4.27 mmol, 430.54 μl) and allowed the reaction to proceed in the reaction mixture for 3 hours at 38°C. The reaction system was concentrated under reduced pressure, then dissolved in water (10 ml) and ethyl acetate (30 mlx2). The pH was adjusted to acidic with 3% citric acid (5 ml) and the mixture was extracted to separate the organic phase, and the organic phase was washed with brine (15 ml), dried and concentrated to give compound 8-8. [M+1]+=518.3.

Step 9. Synthesis of compound 8

Compound 8-8 (200 mg, 386.44 µmol) was dissolved in dichloromethane (2 ml) and Burgess' reagent (230.23 mg, 966.11 µmol) was added and the reaction mixture was stirred at 25°C for 16 h .1H NMR (400 MHz, CD3OD) δ=5.07-4.99 (m, 2H), 4.79-4.71 (m, 1H), 4.29-4.18 (m, 1H), 3.83-3.74 (m, 1H), 3.28-3.23 (m, 1H), 2.76-2.65 (m, 1H), 2.63-2.57 (m, 1H ), 2.38-2.26 (m, 2H), 2.19-2.10 (m, 1H), 2.021.95 (m, 1H), 1.90-1.75 (m, 3H), 1.67-1.60(m, 1H), 1.33-1.26 (m, 1H), 1.15-1.04 (m, 9H), 0.99 (d,J=7.1 Hz, 3H). [M+1]+=500.3.

- 43 047028

Embodiment 9.

Synthesis route:

Step 1. Synthesis of compound 9-2

Compound 9-1 (2 g, 6.15 mmol) was dissolved in methanol (8 ml) and toluene (24 ml) and cooled to 0°C, and a solution of (trimethylsilyl)diazomethane (6.15 ml) was slowly added dropwise. , 2 M) in n-hexane. The reaction mixture was stirred for 16 hours at 20°C. The reaction mixture was directly evaporated to dryness by rotary evaporation and then purified by column chromatography (petroleum ether:ethyl acetate=3:1) to obtain compound 9-2. 1H NMR (400 MHz, CDCl ₃ ) δ=5.15-5.07 (m, 1H), 3.74 (s, 3H), 2.25 (br s, 1H), 1.72-1.41 (m, 23H). [M-99] ⁺ =240.2.

Step 2. Synthesis of compound 9-3

Compound 9-2 (100 mg, 294.62 µmol) was dissolved in dichloromethane (1 ml), cooled to 0°C and diethylaminosulfur trifluoride (94.98 mg, 589.23 µmol) was added to the reaction system. The reaction mixture was stirred at 20°C for 16 hours. The reaction mixture at 0°C was slowly added to saturated sodium bicarbonate solution (20 ml) and extracted twice with dichloromethane (20 ml). The combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated to give compound 9-3. 1H NMR (400 MHz, CDCl ₃ ) δ=5.20-4.99 (m, 1H), 3.75 (s, 3H), 2.32 (br s, 1H), 1.88-1.43 (m, 23H). [M-55] ⁺ =286.1.

Step 3. Synthesis of compound 9-4

Compound 9-3 (700 mg, 2.05 mmol) was dissolved in tetrahydrofuran (7 ml), water (2.3 ml) and methanol (2.3 ml), then lithium hydroxide monohydrate (430.19 mg, 10.25 mmol) and stirred the reaction mixture at 20°C for 16 hours. Water (10 ml) and 5% citric acid (15 ml) were added to the reaction mixture, extracted twice with ethyl acetate (15 ml) and the organic phases were combined , washed with brine (15 ml), dried over anhydrous sodium sulfate, filtered and evaporated to dryness by rotary evaporation. Compound 9-4 was obtained without purification. ^1H NMR (400 MHz, CDCl ₃ ) δ=5.31 (s, 1H), 5.10 (br d, J=9.5 Hz, 1H), 4.20-4.09 (m, 1H) , 2.34 (br s, 1H), 1.90-1.75 (m, 7H), 1.58 (br s, 5H), 1.46 (s, 9H). [M55] ⁺ =272.2.

Step 4. Synthesis of compound 9-5

Compound 9-4 (776 mg, 2.37 mmol) was dissolved in H^dimethylformamide (8 ml), then 1-hydroxybenzotriazole (960.83 mg, 7.11 mmol) and 1-(3dimethylaminopropyl)-hydroxychloride salt were added. 3-ethylcarbodiimide (908.77 mg, 4.74 mmol) and stirred the reaction mixture at 20°C for 0.5 hours. To this was added ^^diisopropylethylamine (919.02 mg, 7.11 mmol) and the hydrochloride salt of the compound 1-3 (367.85 mg, 2.37 mmol) and stirred the reaction mixture at 20°C for 16 hours. Water (15 ml) was added to the reaction mixture, extracted twice with ethyl acetate (30 ml) and the organic phases were combined , washed twice with 5% citric acid (30 ml) and washed four times with brine (20 ml), dried over anhydrous sodium sulfate, filtered and evaporated to dryness by rotary evaporation. The residue was purified by column chromatography (petroleum ether:ethyl acetate=8:1) to obtain compound 9-5. 1H NMR (400 MHz, CDCl3) δ=5.32-5.21 (m, 1H), 4.49 (br s, 1H), 4.32 (br d,

J=9.8 Hz, 1H), 4.07 (s, 1H), 3.73 (s, 3H), 2.75 (br s, 1H), 2.33 (br s, 1H), 1, 89-1.53 (m, 18H), 1.44 (s, 9H).

[M+1]+=465.3.

Step 5. Synthesis of compound 9-6

Compound 9-5 (740 mg, 1.59 mmol) was dissolved in tetrahydrofuran (7.4 ml), water (2.47 ml) and methanol (2.47 ml), then lithium hydroxide monohydrate (200.51 mg, 4.78 mmol) and stirred the reaction mixture at 20°C for 16 hours. Water (20 ml) and 5% citric acid (25 ml) were added to the reaction mixture, extracted twice with ethyl acetate (40 ml) and combined organic phases, washed with brine (40 ml), dried over anhydrous sodium sulfate, filtered and evaporated to dryness by rotary evaporation. Compound 9-6 was obtained without purification. ¹ H NMR (400 MHz, CDCl ₃ ) δ=5.30 (br d,J= 9.8 Hz, 1H), 4.48 (br s, 1H), 4.36 (br d, J=9, 9 Hz, 1H), 4.14 (s, 1H), 2.97 (br s, 1H), 2.31 (br s, 1H), 2.01-1.50 (m, 19H), 1, 44 (s, 9H). [M-55] ⁺ =395.2.

Step 6. Synthesis of compound 9-7

Compound 9-6 (710 mg, 1.58 mmol) was dissolved in 2-butanone (7 ml), then 1-hydroxybenzotriazole (255.52 mg, 1.89 mmol) and 1-(3-dimethylaminopropyl) hydrochloride salt were added )3-ethylcarbodiimide (453.14 mg, 2.36 mmol) and stirred the reaction mixture at 20°C for 0.5 h. N.N-diisopropylethylamine (814.67 mg, 6.30 mmol) and the hydrochloride salt were added to the resulting compound BB-1 (323.74 mg, 1.89 mmol) and stirred the reaction mixture at 20°C for 16 hours. Water (15 ml) was added to the reaction mixture, extracted twice with ethyl acetate (30 ml) and combined organic phase, washed twice with 5% citric acid (30 ml) and washed four times with brine (20 ml), dried over anhydrous sodium sulfate, filtered and evaporated to dryness by rotary evaporation. The residue was purified by column chromatography (dichloromethane:methanol =20:1) to obtain compound 9-7. 1H NMR (400 MHz, CDC13) δ=7.96 (br d, J=6.3 Hz, 1H), 5.83-5.74 (m, 1H), 5.57-5.44 (m, 1H), 5.25 (br d, J=9.9 Hz, 1H), 4.54-4.48 (m, 1H), 4.37 (br d, J=9.9 Hz, 1H), 4.05-3.97 (m, 1H), 3.43-3.27 (m, 2H), 2.95-2.76 (m, 1H), 2.60-1.30 (m, 34H ). [M+1]+=604.4.

Step 7. Synthesis of trifluoroacetate salt of compound 9-8

Compound 9-7 (760 mg, 1.26 mmol) was dissolved in dichloromethane (7.6 ml) and trifluoroacetic acid (2.7 ml) and the reaction mixture was stirred at 20°C for 2 hours. The reaction mixture was directly evaporated to dryness condition by rotary evaporation using an oil pump, dichloromethane was added and evaporated by rotary evaporation, and the above steps were repeated.

The trifluoroacetate salt of compound 9-8 was obtained. [M+1]+=504.4.

Step 8. Synthesis of compound 9-9

The trifluoroacetate salt of compound 9-8 (300 mg, 595.70 μmol) was dissolved in methanol (6 ml), then triethylamine (241.12 mg, 2.38 mmol) and methyl trifluoroacetate (762.79 mg, 5.96 mmol). The reaction mixture was heated to 38°C and stirred for 16 hours. The reaction mixture was directly dried and dissolved by adding water (10 ml) and ethyl acetate (10 ml) and adjusted the pH of the solution to acidic by adding 5% citric acid (10 ml ). The phases were separated, extracted twice with ethyl acetate (10 ml) and the organic phases were combined and washed twice with brine (10 ml), dried over anhydrous sodium sulfate, filtered and concentrated to give compound 9-9 without purification. [M+1]+=600.3.

Step 9. Synthesis of compound 9

Compound 9-9 was dissolved in dichloromethane (2.8 ml) and then Burgess' reagent (278.20 mg, 1.17 mmol) was added thereto. The reaction mixture was heated to 25°C and stirred for 2 hours. Sodium bicarbonate solution (10 ml) and saturated brine (5 ml) were added to the reaction mixture, extracted twice with dichloromethane (15 ml) and the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was separated by preparative HPLC (column type: C18 100*30 mm*10 μm; mobile phase: [water (NH ₄ HCO ₃ )-ACN]; ACN%: 35% - 55%, 8 min.) to obtain the compound 9. 1H NMR (400 MHz, CDC13) δ=9.50-8.17 (m, 1H), 7.12-6.93 (m, 1H), 5.92-5.73 (m, 1H) , 4.95-4.77 (m, 1H), 4.72-4.57 (m, 1H), 4.46 (s, 1H), 3.97-3.86 (m, 1H), 3 .38 (br dd, J=4.0, 8.9 Hz, 1H), 2.87-2.78 (m, 1H), 2.63-1.22 (m, 26H). [M+1]+=582.3.

- 45 047028

Embodiment 10.

Synthesis route:

Step 1. Synthesis of the hydrochloride salt of compound 10-1

Compound 5-5 (3 g, 10.90 mmol) was dissolved in ethanol (80 ml), then hydrochloric acid (1.19 g, 32.69 mmol) and wet palladium on carbon (15 g, 10.68 mmol) were added. mmol). The reaction mixture was stirred at 20°C for 16 hours. The reaction mixture was filtered through diatomaceous earth and directly evaporated to dryness by rotary evaporation to obtain the crude hydrochloride salt product of compound 10-1. [M+1]+=172.0.

Step 2. Synthesis of compound 10-2

Compound 1-b (1.87 g, 10.90 mmol) was dissolved in N-dimethylformamide (20 ml), then O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium was added hexafluorophosphate (4.78 g, 12.58 mmol) and diisopropylethylamine (4.34 g, 33.55 mmol), after stirring for 30 min, the hydrochloride salt of compound 10-1 (190 mg, 1.12 mmol) was added. . The reaction mixture was stirred at 20°C for 16 hours. Water (15 ml) was added to the reaction mixture, extracted twice with ethyl acetate (60 ml) and the organic phases were combined, washed twice with 5% citric acid (30 ml) and washed four times with saline (20 ml), dried over anhydrous sodium sulfate, filtered and evaporated to dryness by rotary evaporation. The residue was purified by column chromatography (petroleum ether:ethyl acetate=3:1) to obtain compound 10-2. ^1H NMR (400 MHz, CDCl ₃ ) δ=5.28-5.16 (m, 1H), 4.50 (br s, 1H), 4.28 (d, J=9.8 Hz, 1H) , 3.92 (s, 1H), 3.74 (s, 3H), 2.81 (s, lH), 2.67 (s, 1H), 2.17 (brdd, J=6.1, 12 .7 Hz, 1H), 1.99-1.93 (m, 1H), 1.90-1.84 (m, 1H), 1.59 (br d, J=13.3 Hz, 2H), 1.43 (s, 9H), 1.04 (s, 9H). [M+1] ⁺ =385.2.

Step 3. Synthesis of compound 10-3

Compound 10-2 (500 mg, 1.30 mmol) was dissolved in acetonitrile (7.5 ml), then 2-iodobenzoic acid (976, 31 mg, 3.49 mmol) was added and the reaction mixture was stirred at 60°C for 16 hours. The reaction mixture was filtered through diatomaceous earth and evaporated to dryness by rotary evaporation. Compound 10-3 was obtained without purification. [M-55] ⁺ =327.1.

Step 4. Synthesis of compound 10-4

Compound 10-3 (480 mg, 1.26 mmol) was dissolved in dichloromethane (4.8 ml), cooled to 0°C, diethylaminosulfur trifluoride (1.01 g, 6.28 mmol) was added and the reaction mixture was stirred at 20°C for 16 hours. Saturated sodium bicarbonate solution (20 ml) was added slowly to the reaction mixture at 0°C, extracted twice with dichloromethane (20 ml) and the combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated. Compound 10-4 was obtained without purification. [M-100] ⁺ =304.0.

Step 5. Synthesis of compound 10-5

Compound 10-4 (475 mg, 1.17 mmol) was dissolved in tetrahydrofuran (5.5 ml), water (1.84 ml) and methanol (1.84 ml), then lithium hydroxide monohydrate (147.84 mg, 3.52 mmol) and stirred the reaction mixture at 20°C for 16 hours. Water (20

- 46 047028 ml) and 5% citric acid (25 ml), extracted twice with ethyl acetate (40 ml), combined the organic phases and washed with brine (40 ml), dried over anhydrous sodium sulfate, filtered and evaporated to dry state by rotary evaporation. Compound 10-5 was obtained without purification. 1H NMR (400 MHz, CDCl ₃ ) δ=5.18 (br d, J=9.8 Hz, 1H), 4.65-4.53 (m, 2H), 4.31 (d, J=9 .9 Hz, 1H), 3.33 (br d, J=7.3 Hz, 1H), 2.55-2.41 (m, 1H), 2.33-2.07 (m, 3H), 1.45 (s, 9H), 1.03 (s, 9H). [M-55] ⁺ =335.1.

Step 6. Synthesis of compound 10-6

Compound 10-5 (200 mg, 512.27 µmol) was dissolved in 2-butanone (2 ml), then 1-hydroxybenzotriazole (83.06 mg, 614.72 µmol) and 1-(3dimethylaminopropyl)- hydrochloride salt were added. 3-ethylcarbodiimide (264.83 mg, 2.05 mmol) and the reaction mixture was stirred at 20°C for 0.5 h. To this was added ^^diisopropylethylamine (264.83 mg, 2.05 mmol) and the hydrochloride salt of the compound BB-1 (105.24 mg), then the reaction mixture was stirred at 20°C for 16 hours. Water (15 ml) was added to the reaction mixture, extracted twice with ethyl acetate (30 ml) and the organic phases were combined, washed twice with with 5% citric acid (30 ml) and washed four times with brine (20 ml), dried over anhydrous sodium sulfate, filtered and evaporated to dryness by rotary evaporation. The residue was purified by column chromatography (dichloromethane:methanol=20:1) to obtain compound 10-6. 1H NMR (400 MHz, CDCl ₃ ) δ=8.19 (br d, J=6.8 Hz, 1H), 5.78 (br s, 1H), 5.49 (br s, 1H), 5, 19 (br d, J=10.0 Hz, 1H), 4.61 (br s, 1H), 4.44 (s, 1H), 4.34 (d, J=10.3 Hz, 1H), 3.38 (br d, J=6.5 Hz, 2H), 3.10 (br d, J=6.5 Hz, 1H), 2.32-2.20 (m, 3H), 2.02 -1.82 (m, 6H), 1.44 (s, 9H), 1.05 (s, 9H). [M+1] ⁺ =544.3.

Step 7. Synthesis of trifluoroacetate salt of compound 10-7

Compound 10-6 (180 mg, 331.12 μmol) was dissolved in dichloromethane (2 ml) and trifluoroacetic acid (0.7 ml) and the reaction mixture was stirred at 20°C for 2 hours. The reaction mixture was directly dried using an oil pump, a small amount of dichloromethane was added and dried, and the above steps were repeated until the product took the form of a light yellow foam. The trifluoroacetate salt of compound 10-7 was obtained. [M+1] ⁺ =444.3.

Step 8. Synthesis of compound 10-8

The trifluoroacetate salt of compound 10-7 (145 mg, 326.95 μmol) was dissolved in methanol (3.2 ml), and then triethylamine (132.34 mg, 1.31 mmol) and methyl trifluoroacetate (418.66 mg, 3 .27 mmol). The reaction mixture was heated to 38°C and stirred for 16 hours. The reaction mixture was directly evaporated to dryness by rotary evaporation, dissolved in water (10 ml) and ethyl acetate (10 ml) and adjusted to acidic pH by adding 5% citric acid (10 ml). The phases were separated, extracted twice with ethyl acetate (10 ml), combined the organic phases and washed twice with brine (10 ml) x 3, dried over anhydrous sodium sulfate, filtered, concentrated to give compound 10-8 without purification. [M+1] ⁺ =540.3.

Step 9. Synthesis of compound 10

Compound 10-8 (170 mg, 315.11 µmol) was dissolved in dichloromethane (2.8 ml) and then Burgess' reagent (187.73 mg, 787.77 µmol) was added thereto. The reaction mixture was heated to 25°C and stirred for 2 hours. Sodium bicarbonate solution (10 ml) and saturated saline solution (5 ml) were added to the reaction mixture, extracted twice with dichloromethane (15 ml) and the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was separated by preparative HPLC (column type: C18 100*30 mm* 10 μm; mobile phase: [H ₂ O (NH ₄ HCO ₃ )-ACN]; ACN%: 35-55%, 8 min) to obtain the compound 10. ¹ H NMR (400 MHz, CDCl ₃ ) δ=9.79-8.65 (m, 1H), 7.18-7.01 (m, 1H), 6.34-6.20 (m, 1H), 4.64-4.58 (m, 2H), 4.454.37 (m, 1H), 3.44-3.30 (m, 3H), 3.12-3.05 (m, 1H) , 2.63-2.47 (m, 3H), 2.40-2.12 (m, 6H), 1.06 (s, 9H). [M+1] ⁺ =522.3.

Embodiment 11.

Synthesis route:

- 47 047028

Step 1. Synthesis of compound 11-1

Compound 10-3 (0.7 g, 1.83 mmol) was dissolved in tetrahydrofuran (14 ml) and TEBBE reagent (0.5 M, 14.64 ml) was added at 0°C. The reaction mixture was stirred at 0°C for 1 hour, then heated to 15°C and stirred continuously for 3 hours. The reaction mixture was slowly poured into saturated sodium bicarbonate solution (50 ml), filtered through diatomite, extracted with ethyl acetate (30 mlx3) and washed with saturated saline (30 mlx2). The residue was purified by column chromatography (petroleum ether:ethyl acetate=5:1) to obtain compound 11-1. [M+1]+=381.1.

Step 2. Synthesis of compound 11-2

Under a nitrogen atmosphere, diethylzinc (1 M, 13.14 mL) was slowly added to 1,2-dichloroethane (80 mL) at 0° C. After stirring for 0.25 h, diiodomethane (7.04 g, 26.28 mmol, 2.12 mL) was slowly added to the reaction mixture at 0°C and stirred for 0.25 h. Trifluoroacetic acid was slowly added to the reaction system (149.84 mg, 1.31 mmol, 97.30 μl), stirred continuously for 0.5 h. A solution of compound 11-1 (0.5 g, 1.31 mmol) in 1,2dichloroethane was added to the reaction system (5 ml), heated to 20°C and stirred continuously for 12 hours. The reaction mixture was quenched with saturated sodium bicarbonate solution (200 ml), extracted with dichloromethane (100 ml), dried over anhydrous sodium sulfate, filtered and concentrated . The crude product was purified by preparative HPLC (column type: Phenomenex luna C18 80*40 mm*3 μm; mobile phase: [H2O (HCl)-acetonitrile]; acetonitrile%: 1-30%, 7 min) to obtain compound 11-2 . [M+1]+=295.2.

Step 3. Synthesis of compound 11-3

Compound 11-2 (0.1 g, 339.69 µmol) was dissolved in 1,4-dioxane (3 ml), then a solution of potassium carbonate (187.79 mg, 1.36 mmol) and di-tert- butyl dicarbonate (111.20 mg, 509.53 µmol, 117.06 µl) in water (1 ml) and stirred the reaction mixture at 15°C for 12 hours. The reaction mixture was poured into water (30 ml), extracted with ethyl acetate (20 mlx3), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (petroleum ether:ethyl acetate=5:1) to obtain compound 11-3. [M+1]+=395.2.

Step 4. Synthesis of compound 11-4

Compound 11-3 (88.13 mg, 223.40 µmol) was dissolved in tetrahydrofuran (2 ml) and methanol (0.6 ml) and lithium hydroxide monohydrate (28.12 mg, 670.21 µmol) was added, dissolved in water (0.6 ml). The reaction mixture was stirred at 15°C for 2 hours. The pH was adjusted to 5 with 3% citric acid and the mixture was extracted with ethyl acetate (20 mlx3), dried over anhydrous sodium sulfate, filtered and concentrated to give compound 11-4. [M+1]+=381.3.

Step 5. Synthesis of compound 11-5

Compound 11-4 (0.056 g, 148.79 µmol) was dissolved in N-dimethylformamide (2 ml), then O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate was added to the reaction system (84.86 mg, 223.18 µmol) and the reaction mixture was stirred at 15°C for 0.5 h. Diisopropylethylamine (76.92 mg, 595.16 µmol, 103.67 µl) was then added to the reaction mixture, to the reaction system, a solution of the hydrochloride salt of compound BB-1 (43.26 mg, 208.31 µmol) dissolved in N-dimethylformamide (0.5 ml) was added and the reaction mixture was stirred at 15°C for 12 hours. The reaction mixture was diluted with water (20 ml), extracted with ethyl acetate (20 mlx3) and washed the organic phase with 3% citric acid (20 ml), washed with saturated sodium chloride (20 ml), dried over anhydrous sodium sulfate, filtered and concentrated to give connections 11-5. [M+1]+=534.4.

Step 6. Synthesis of compound 11-6

Compound 11-5 (0.02 g, 37.48 µmol) was dissolved in dichloromethane (2 ml) and trifluoroacetic acid (141.02 mg, 1.24 mmol, 91.57 µl) was added to the reaction system. The reaction mixture was stirred at 15°C for 1 hour. The reaction mixture was directly quenched with sodium bicarbonate solution (10 ml), extracted with dichloromethane (5 mlx5) and the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to give compound 11-6. [M+1]+=434.2.

Step 7. Synthesis of compound 11

Compound 11-6 (0.03 g, 69.20 µmol) was dissolved in dichloromethane (1 ml) and trifluoroacetic anhydride (58.13 mg, 276.79 µmol, 38.50 µl) was added to the reaction system. The reaction mixture was stirred at 15°C for 1 hour. The reaction mixture was directly quenched with sodium bicarbonate solution (10 ml), extracted with dichloromethane (5 mlx5) and the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was separated by preparative HPLC (column type: Waters Xbridge BEH C18, 100 * 30 mm * 10 μm; mobile phase: [H ₂ O (NH ₄ HCO ₂ )acetonitrile]; acetonitrile%: 10-50%, 8 min) to obtain compound 11. [M+1] ⁺ =512.2.

In the version about the existence of 12.

Synthesis route:

Step 1. Synthesis of compound 12-2

Compound 12-1 (2.36 g, 7.64 mmol) was dissolved in H^dimethylformamide (17 ml), then O-(7-azabenzotriαzol-1-yl)-N,N,N,N-tetramethyluronium was added hexafluorophosphate (4.36 g, 11.46 mmol) and diisopropylethylamine (3.95 g, 30.55 mmol), after stirring for 30 min, the hydrochloride salt of compound 10-1 (1.7 g, 8.19 mmol) and the reaction mixture was stirred at 15 °C for 1 hour. Water (15 ml) was added to the reaction mixture, extracted twice with ethyl acetate (60 ml) and the organic phases were combined, washed twice with 5% citric acid (30 ml ) and washed four times with brine (20 ml), dried over anhydrous sodium sulfate, filtered and evaporated to dryness by rotary evaporation. The residue was purified by column chromatography (petroleum ether:ethyl acetate=1:1) to obtain compound 12-2. 1H NMR (400 MHz, CDCl ₃ ) δ=5.22 (br d, J=9.76 Hz, 1H), 4.50-4.62 (m, 1H), 4.09-4.18 (m , 2H), 3.74 (br s, 3H), 1.93-2.04 (m, 4H), 1.87 (br d, J=9.88 Hz, 1H), 1.53-1, 77 (m, 15H), 1.39-1.46 (m, 9H), 1.27 (t, J=7.13 Hz, 2H). [M+1] ⁺ =463.58.

Step 2. Synthesis of compound 12-3

Compound 12-2 (3 g, 6.49 mmol) was dissolved in acetonitrile (45 ml), 2-iodoxybenzoic acid (3.63 g, 12.97 mmol) was added and the reaction mixture was allowed to proceed at 60°C for 16 hours. The reaction mixture was filtered through diatomaceous earth and evaporated to dryness by rotary evaporation under reduced pressure. Compound 12-3 was obtained. [M+1] ⁺ =461.56.

Step 3. Synthesis of compound 12-4

Compound 12-3 (4 g, 8.69 mmol) was dissolved in tetrahydrofuran (80 ml), cooled to 0°C, and the resulting reagent TEBBE (bis(cyclopentadienyl)^-chloro(dimethylaluminum)^- 49 047028 methylene titanium) was added ( 10.24 g, 34.74 mmol) and allowed the reaction mixture to proceed at 15°C for 3 hours. Saturated sodium bicarbonate solution (100 ml) was slowly added to the reaction mixture, extracted three times with ethyl acetate (50 ml) , the combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. Compound 12-4 was obtained. ¹ H NMR (400 MHz, CDC13) δ=5.227-5.251 (br d, J=9.6 Hz, 2 H), 4.862 (br s, 1H), 4.636 (br s, 1H), 4.191-4.215 (m , 2H), 3.768 (br s, 3H), 3.117 (br s, 1H), 2.369 (br s, 2H), 2.115 (t, J=14.4 Hz, 1H), 2.010-2.046 (br s, 3H), 1.618-1.701 (m, 13H), 1.432 (br s, 9H). [M+1]+=459.59.

Step 4. Synthesis of compound 12-5

Compound 12-4 (1.90 g, 4.14 mmol) was dissolved in methanol (191 ml), added to another one-neck flask containing a solution of wet palladium on carbon (9.55 g, palladium content 5%) in methanol, reaction the system was purged with a hydrogen cylinder three times and the reaction mixture was allowed to proceed at 15° C. and 15 psi. inch for 2 hours. The reaction mixture was filtered through diatomite and evaporated to dryness by rotary evaporation under reduced pressure. Compound 12-5 was obtained. [M+1]+=461.60.

Step 5. Synthesis of compound 12-6

Compound 12-5 (1.8 g, 3.91 mmol) was dissolved in tetrahydrofuran (40 ml) and methanol (13 ml), then cooled to 0°C, and lithium hydroxide monohydrate (983.94 mg, 23. 46 mmol) and allowed the reaction to proceed in the reaction mixture at 15°C for 40 hours. Water (30 ml) was added to the reaction mixture and the pH was adjusted to 4-5 with 5% citric acid (20 ml), then the mixture was extracted twice with ethyl acetate (50 ml) and the organic phases were combined, washed twice with saturated sodium chloride (30 ml), dried over anhydrous sodium sulfate, filtered and evaporated to dryness by rotary evaporation. Compound 12-6 was obtained without purification. 1H NMR (400 MHz, DMSO-d ₆ ) δ=1.424 (br s, 9H) 1.594-1.655 (m, 14 H) 1.846 (br s, 8 H) 2.030 (br s, 1H) 2.540-2.548 (m, 1H) 3.240-3.367 (br d, J=50.8 Hz, 1H) 3.633-3.668 (m, 1H) 3.777-3.823 (m, 1H) 4.446 (br s, 1H) 6.309-6.332 (br d, J= 9.2, 1H). [M+1]+=447.58.

Step 6. Synthesis of compound 12-7

Compound 12-6 (1.3 g, 2.91 mmol) was dissolved in NN-dimethylformamide (12 ml), then 1-hydroxybenzotriazole (472.01 mg, 3.49 mmol), 1-(3-dimethylaminopropyl) )-3ethylcarbodiimide (837.07 mg, 4.37 mmol) and the reaction mixture was stirred at 15°C for 30 minutes. The hydrochloride salt of compound BB-1 (747.27 mg, 4.37 mmol) and diisopropylethylamine (1.50 g, 11.64 mmol) were added to the resulting mixture, and the reaction mixture was allowed to proceed at 15°C for 2 hours. the reaction mixture was added with water (50 ml), extracted twice with ethyl acetate (50 ml) and the organic phases were combined, washed twice with 5% citric acid (30 ml) and washed twice with brine (20 ml), dried over anhydrous sodium sulfate, filtered and evaporated to dryness by rotary evaporation. The residue was purified by column chromatography (dichloromethane:methanol=20:1) to obtain compound 12-7. 1H NMR (400 MHz, CDCl ₃ ) δ=7.730-7.748 (br d, J=7.2 Hz, 1H), 6.162 (br s, 1H) 7.270 (br s, 1H), 5.569 (br d, 1H) , 5.288 (br s, 1H), 4.510-4.567 (m, 1H), 4.430 (br d, 1H), 4.331 (br d, 1H), 4.1834.208 (br d, J=10 Hz, 1H) , 3.322-3.360 (t, J=7.6 Hz, 2H), 2.658-2.666 (m, 1H), 2.459 (m, 2 H), 2.017-2.076 (m, 1H), 1.990 (m, 8H), 1.602-1.780 (m, 13H), 1.424 (m, 9H), 1.046 (br s, 1H), 0.976-1.029 (br d, J=21.2 Hz, 3H). [M+1]+=600.76.

Step 7. Synthesis of compound 12-8

Compound 12-7 (920.00 mg, 1.53 mmol) was dissolved in dichloromethane (18.4 ml), then trifluoroacetic acid (7.89 g, 69.22 mmol) was added and the reaction mixture was allowed to proceed at 15°C for 1 hour. The reaction mixture was directly evaporated to dryness by rotary evaporation. Compound 12-8 was obtained. [M+1]+=500.64.

Step 8. Synthesis of compound 12-9

Compound 12-8 (420.00 mg, 840.60 μmol) was dissolved in methanol (8.4 ml), then triethylamine (510.36 mg, 5.04 mmol) was added, and methyl trifluoroacetate (1.29 g, 10.09 mmol), heated the reaction mixture to 38°C and stirred for 16 hours. The reaction mixture was concentrated under reduced pressure, and ethyl acetate (50 ml) and water (20 ml) were added. The pH of the reaction mixture was adjusted to acidic by adding 3% citric acid, the mixture was extracted three times by adding ethyl acetate (30 ml) and the organic phases were combined, washed with saturated sodium chloride solution (30 ml), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (dichloromethane:methanol =10:1) to obtain compound 12-9. 1H NMR (400 MHz, CDCl ₃ ) δ=7.819-7.832 (br d, J=5.2 Hz, 1H), 7.006-7.176 (m, 2H), 5.636-5.947 (m, 3H), 4.343 (br s , 3H), 3.339-3.477 (m, 3H), 2.666 (br s, 1H), 2.487-2.503 (m, 3 H), 2.161-2.186 (m, 3H), 2.000-2.020 (br d, J=8 Hz, 4H), 1.856-1.878 (m, 2H), 1.567-1.652 (m, 10H), 1.003-1.061 (m, 5H). [M+1]+=596.65.

Step 9. Synthesis of compound 12

Compound 12-9 (390.00 mg, 654.74 µmol) was dissolved in dichloromethane (7.8 ml) and tetrahydrofur

- 50 047028 not (0.78 ml), cooled to 0°C, then the Burgess reagent (390.07 mg, 1.64 mmol) was added to the resulting mixture and the reaction was allowed to proceed in the reaction mixture at 15°C for 1 hour. The reaction mixture was added with water (30 ml), extracted twice with ethyl acetate (30 ml) and the organic phases were combined, sodium bicarbonate solution (30 ml) was added and stirred for 20 min, washed with brine (30 ml), dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was separated by preparative HPLC (column type: C18 100*30 mm* 10 μm; mobile phase: [H2O (NH ₄ HCO ₃ )acetonitrile]; acetonitrile%: 35-55%, 8 min.) to obtain compound 12. 1H NMR (400 MHz, CDCl ₃ ) δ=8.23 (br d, J=7.00 Hz, 1H), 6.89-7.04 (m, 1H), 5.77-5.86 (m , 1H), 4.94 (br d, J=9.44, 6.91 Hz, 1H), 4.51 (br d, J=9.26 Hz, 1H), 4.40 (br s, 1H ), 4.22-4.35 (m, 1H), 3.05-3.81 (m, 3H), 2.71 (br d, J=3.63 Hz, 1H), 2.39-2 .66 (m, 2H), 2.12-2.34 (m, 3H), 1.79-2.09 (m, 6H), 1.61-1.73 (m, 11H), 1.43 (m, 1H), 1.17 (br d, J=6.88 Hz, 1H), 0.98-1.14 (m, 3H). [M+1]+ =578.64.

Embodiment 13.

Synthesis route:

3-5 13-2 13

Step 1. Synthesis of compound 13-2

Compound 13-1 (100.50 mg, 669.25 μmol) was dissolved in H^dimethylformamide (25 ml), then O-(7-azabenzotriazol-1-yl)-H^H^tetramethyluronium hexafluorophosphate (293, 62 mg, 772.21 µmol) and H^diisopropylethylamine (266.14 mg, 2.06 mmol, 358.67 µl) and stirred the reaction mixture for 0.5 hours. The trifluoroacetate salt of compound 3-5 (0 .25 g) and the reaction mixture was stirred at 20°C for 16 hours. The reaction mixture was extracted by adding water (15 ml) and ethyl acetate (30 ml x 2) and the organic phases were combined, washed with 5% citric acid (10 ml ) and saline solution (20 mlx4), dried over anhydrous sodium sulfate, filtered and evaporated to dryness by rotary evaporation. The residue was purified by column chromatography (dichloromethane:methanol=5:1) to obtain compound 13-2. [M+1] ⁺ =618.7.

Stage 2. Synthesis of compound 13

Compound 13-2 (0.2 g, 323.74 µmol) was dissolved in dichloromethane (6 ml) and tetrahydrofuran (0.6 ml), Burgess' reagent (115.73 mg, 485.61 µmol) was added and allowed to proceed reaction in the reaction mixture at 15°C for 1 hour. The reaction mixture was washed with saturated sodium bicarbonate (5 ml), extracted with dichloromethane (10 ml), washed with saturated saline (10 ml), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was separated by preparative HPLC (column type: Waters Xbridge Prep OBD C18 150*40 mm*10 μm; mobile phase: [water (KI4HCO3)-acetonitrile]; acetonitrile%: 35-65%, 8 min) and the fraction was concentrated at reduced pressure to obtain compound 13. [M+1] ⁺ =600.7. 1H NMR (400 MHz, CDCl ₃ ) δ=8.16 (d, J=7.5 Hz, 1H), 7.18 (s, 4H), 4.98 (q, J=7.9 Hz, 1H ), 4.62-4.43 (m, 2H), 3.98 (s, 1H), 3.69-3.48 (m, 3H), 3.43-3.27 (m, 2H), 2.372.34 (m, 3H), 1.88-1.36 (m, 27H).

Embodiment 14.

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Synthesis route:

Step 1. Synthesis of compound 14-2

Compound 14-1 (36.44 mg, 214.16 µmol) was dissolved in H^dimethylformamide (10 ml), then C-(7-azabenzotriazol-1-yl)-HHH^tetramethyluronium hexafluorophosphate (93.96 mg) was added , 247.11 µmol) and ^^diisopropylethylamine (85.17 mg, 658.95 µmol, 114.78 µl) and stirred the reaction mixture for 0.5 h, the trifluoroacetate salt of compound 3-5 (0.08 d) and stirred the reaction mixture at 20°C for 16 hours. Water (15 ml) was added to the reaction mixture, extracted with ethyl acetate (30 ml x 2) and the organic phases were combined, washed with 5% citric acid (10 ml) and saline solution (20 mlx4), dried over anhydrous sodium sulfate, filtered and evaporated to dryness by rotary evaporation. The residue was purified by column chromatography (dichloromethane:methanol=5:1) to obtain compound 14-2. [M+1]+=638.7.

Step 3. Synthesis of compound 14

Compound 14-2 (0.1 g, 156.80 µmol) was dissolved in dichloromethane (3 ml) and tetrahydrofuran (0.3 ml), Burgess' reagent (56.05 mg, 235.21 µmol) was added and allowed to proceed reaction in the reaction mixture at 15°C for 1 hour. The reaction mixture was washed with saturated sodium bicarbonate (5 ml), extracted with dichloromethane (10 ml), washed with saturated saline (10 ml), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was separated by preparative HPLC (column type: Waters Xbridge Prep OBD C18 150*40 mm*10 µm; mobile phase: [water (NH^HCOs^^tohutp^]; acetonitrile%: 20-70%, 8 min) and concentrated the fraction under reduced pressure to obtain compound 14. [M+1] ⁺ =620.7. 1H NMR (400 MHz, CDCl ₃ ) δ=8.23 (br d, J=7.5 Hz, 1H), 7 .03-7.03 (m, 1H), 7.21-6.85 (m, 3H), 5.00 (q, J=7.8 Hz, 1H), 4.69-4.40 (m , 3H), 4.04-3.94 (m, 1H), 3.453.26 (m, 2H), 2.95-2.79 (m, 1H), 2.66-1.24 (m, 23H ).

Embodiment 15.

Synthesis route:

Step 1. Synthesis of compound 15-1

At 0°C, N-Boc-L-tert-leucine (0.21 g, 1.10 mmol), N,N-diisopropylethylamine (426.49 mg, 3.30 mmol, 574.79 μl), 2- (7-azobenzotriazol-1-yl)-HHH^tetramethyluronium hexafluorophosphate (627.38 mg, 1.65 mmol) to a solution of the hydrochloride salt of compound 1-3 (305.30 mg, 1.32 mmol) in N,Ndimethylformamide (2 ml) and allowed the reaction mixture to proceed at 20°C for 16 hours. The reaction mixture was poured into 5% citric acid solution with phase separation, the aqueous phase was extracted with ethyl acetate (20 ml*2) and the organic phase was dried over anhydrous sulfate sodium, filtered and concentrated under reduced pressure to obtain the crude product. The crude product was separated by silica gel column chromatography (petroleum ether/ethyl acetate=3:1) to give compound 15-1. ¹ H NMR (400 MHz, CDCl ₃ ) δ=5.21 (br d, J=9.6 Hz, 1H), 4.48-4.37 (m, 1H), 4.30-4.21 ( m, 1H), 4.01-3.95 (m, 1H), 3.68-3.60 (m, 3H), 2.73-2.61 (m, 1H), 1.97-1, 87 (m, 1H), 1.79-1.56 (m, 4H), 1.39-1.33 (m, 10H), 0.97 (s, 9H).

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Step 2. Synthesis of compound 15-2

Lithium hydroxide monohydrate (51.25 mg, 1.22 mmol) was added to a solution of compound 15-1 (0.3 g, 814.19 μmol) in tetrahydrofuran (2 ml) and water (1 ml) and allowed to react in the reaction mixture at 20°C for 16 hours. 20 ml of 5% aqueous citric acid solution was added to the reaction mixture, 20 ml of ethyl acetate was added with phase separation, and the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to obtain the crude product. Compound 15-2 was obtained. ¹ H NMR (400 MHz, CDCl ₃ ) δ=5.24 (d, J=9.9 Hz, 1H), 4.51-4.45 (m, 1H), 4.41-4.33 (m , 1H), 4.18-4.15 (m, 1H), 3.06-2.99 (m, 1H), 1.99-1.89 (m, 1H), 1.85-1.75 (m, 3H), 1.59-1.49 (m, 2H), 1.461.42 (m, 9H), 1.05-1.01 (m, 9H).

Step 3. Synthesis of compound 15-3

At 0°C, the reaction of compound 15-2 (0.28 g, -methylimidazole (291.87 mg, 3.55 mmol), H^H^tetramethylchloroformamidinium hexafluorophosphate (265.98 mg, 947.97 μmol) at 20 °C for 16 hours. The hydrochloride salt of compound BB-1 (196.85 mg, 947.97 µmol) was added to a solution of H^dimethylformamide (3 ml, 789.98 µmol) and the reaction mixture was poured into 20 ml of water N. Added mixed solution of dichloromethane and methanol (volume ratio 10:1) for extraction (20 ml*2) and the organic phase was washed with 5% citric acid solution (20 ml*1), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the crude product. The crude product was separated by silica gel column chromatography (dichloromethane/methanol=20:1) to give compound 15-3 (400 MHz, CDCl ₃ ) δ=4.71-4.23 (m, 3H), 3.97 (br s, 1H), 3.35 (br d, J=7.1 Hz, 2H), 2.83-2.74 (m, 1H), 2.61-2.31 (m, 2H), 2.09 (br s, 1H), 2.04-1.92 (m, 2H), 1.88-1.64 (m, 4H), 1.57-1.34 ( m, 11H), 1.11-0.89 (m, 9H).

Step 4. Synthesis of trifluoroacetate salt of compound 15-4

At 0°C, trifluoroacetic acid (2 ml) was added to a solution of compound 15-3 (0.2 g, 393.99 μmol) in dichloromethane (6 ml) and the reaction mixture was allowed to proceed at 20°C for 2 hours. The reaction mixture was concentrated under reduced pressure to obtain the trifluoroacetate salt of compound 15-4.

Step 5. Synthesis of compound 15

At 0°C, pyridine (187.07 mg, 2.37 mmol, 190.89 μl), trifluoroacetic anhydride (206.97 mg, 985.43 μmol, 137.07 μl) was added to a solution of trifluoroacetate salt of compound 15-4 ( 0.175 g, 394.17 µmol) in tetrahydrofuran (2 ml) and allowed the reaction mixture to proceed at 20°C for 4 hours. The reaction mixture was quenched with 20 ml of water, extracted with ethyl acetate (20 ml * 2) and the organic phase was washed with 5% citric acid (20 ml*1), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain the crude product. The crude product was separated by preparative HPLC (column type: Waters Xbridge BEH C18, 100*25 mm*5 µm; mobile phase: [H ₂ O (NH ₄ HCO ₃ )-ACN]; ACN%: 20-50%, 10 min ) to give compound 15. 1H NMR (400 MHz, CDCl ₃ ) δ=9.43-8.31 (m, 1H), 7.11-6.95 (m, 1H), 5.93-5.71 (m, 1H), 4.92-4.76 (m, 1H), 4.72-4.62 (m, 1H), 4.55-4.44 (m, 1H), 4.01-3 .89 (m, 1H), 3.483.30 (m, 2H), 2.88-2.78 (m, 1H), 2.62-2.38 (m, 2H), 2.34-2.15 (m, 2H), 2.04-1.75 (m, 4H), 1.71-1.58 (m, 3H), 1.57-1.41 (m, 2H), 1.07-0 .92 (m, 9H).

Embodiment 16.

Synthesis route:

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Step 1. Synthesis of compound 16-2

The hydrochloride salt of compound 16-1 (949.93 mg, 4.32 mmol) was added to N,Ndimethylformamide (10 ml), then 2-(7-azabenzotriazole-Gyl)-CCKNtetramethyluronium hexafluorophosphate (1.97 g, 5 .19 mmol) and stirred the reaction mixture for 0.5 h, then diisopropylethylamine (1.40 g, 10.81 mmol) and N-Boc-L-tert-leucine (1 g, 4.32 mmol) were added. and stirred the reaction mixture at 20°C for 16 hours. The reaction mixture was washed with methyl tert-butyl ether (50 ml), water (20 ml), 3% citric acid (20 ml*2) and saturated sodium chloride solution (20 ml) and dried the organic phase over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate=3:1) to give compound 16-2. ¹ H NMR (400 MHz, CDCl ₃ ) δ=5.27-5.18 (m, 1H), 4.36 (d, J=4.1 Hz, 1H), 4.33-4.28 (m , 1H), 4.234.13 (m, 2H), 3.89-3.80 (m, 1H), 3.79-3.70 (m, 1H), 2.77-2.61 (m, 2H ), 1.98-1.82 (m, 2H), 1.80-1.70 (m, 1H), 1.69-1.60 (m, 2H), 1.54-1.47 (m , 1H), 1.46-1.41 (m, 9H), 1.29-1.25 (m, 3H), 1.06-1.00 (m, 9H).

Step 2. Synthesis of compound 16-3

Compound 16-2 (0.2 g, 504.39 µmol) was added to tetrahydrofuran (3 ml), to which was added a solution of lithium hydroxide monohydrate (63.50 mg, 1.51 mmol) in water (1.5 ml) and stirred the reaction mixture at 20°C for 16 hours. The crude product was neutralized with 3% citric acid solution (20 ml), extracted with ethyl acetate (30 ml) and washed the organic phase with saturated sodium chloride solution (10 ml), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. Compound 16-3 was obtained without purification. 1H NMR (400 MHz, CD3OD) δ=4.32-4.25 (m, 2H), 3.90-3.81 (m, 2H), 2.85-2.67 (m, 2H), 2 .01-1.86 (m, 2H), 1.79-1.50 (m, 5H), 1.44 (s, 9H), 1.06-1.00 (m, 9H).

Step 3. Synthesis of compound 16-4

Compound 16-3 (0.35 g, 949.88 µmol), the hydrochloride salt of compound BB-1 (197.25 mg, 949.88 µmol) was added to NN-dimethylformamide (4 ml), cooled to 0°C, then 1-methylimidazole (272.95 mg, 3.32 mmol) and KMCN-tetramethylchloroformamidinium hexafluorophosphate (399.78 mg, 1.42 mmol) were added to the resulting mixture, and the reaction mixture was gradually heated to 20°C and stirred for 16 hours. the mixture was washed with ethyl acetate (50 ml), water (20 ml), 3% citric acid (20 ml*2) and saturated sodium chloride solution (20 ml) and dried the organic phase over anhydrous sodium sulfate, filtered and concentrated under reduced pressure . The residue was purified by silica gel column chromatography (dichloromethane:methanol=20:1) to give compound 16-4. 1H NMR (400 MHz, CDCl ₃ ) δ=8.15-7.87 (m, 1H), 7.24-7.14 (m, 1H), 6.19-5.95 (m, 1H), 5.81-5.55 (m, 1H), 5.44-5.24 (m, 1H), 4.51-3.72 (m, 5H), 3.49-3.27 (m, 2H ), 2.86-2.64 (m, 2H), 2.55-2.26 (m, 2H), 1.97-1.75 (m, 5H), 1.73-1.55 (m , 3H), 1.51-1.36 (m, 10H), 1.06-0.90 (m, 9H).

Step 4. Synthesis of trifluoroacetate salt of compound 16-5

Compound 16-4 (0.31 g, 594.27 µmol) was added to dichloromethane (3 ml), trifluoroacetic acid (1 ml) was added and the reaction mixture was stirred at 20°C for 2 hours. The reaction mixture was concentrated under reduced pressure. The trifluoroacetate salt of compound 16-5 was obtained. [M+1]+=422.30

Step 5. Synthesis of compound 9

The trifluoroacetate salt of compound 16-5 (240 mg, 448.13 μmol) was added to tetrahydrofuran (3 ml), cooled to 0°C, then pyridine (212.68 mg, 2.69 mmol) was added to the resulting drops of trifluoroacetic anhydride (235.30 mg, 1.12 mmol), the reaction mixture was gradually heated to 20°C and stirred for 1 hour. The reaction mixture was washed with ethyl acetate (50 ml), water (10 ml), 3% citric acid acid (20 ml*2) and saturated sodium chloride solution (10 ml) and dried the organic phase over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:methanol=20:1) to give compound 9. 1H NMR (400 MHz, DMSO- _d6 ) δ=8.748.25 (m, 2H), 7.30-7.12 (m, 1H), 4.99-4.83 (m, 1H), 4.60-4.47 (m, 1H), 4.21-4.14 (m, 1H), 3.92-3 .82 (m, 1H), 3.65-3.57 (m, 1H), 3.25-3.09 (m, 2H), 2.80-2.70 (m, 1H), 2.62 -2.54 (m, 1H), 2.46-2.38 (m, 1H), 2.28-2.14 (m, 2H), 1.94-1.55 (m, 7H), 1 .47-1.36 (m, 1H), 1.02 (s, 9H).

Biological research

Experimental Embodiment 1. In vitro assessment of the activity of the test compound against the Mpro protease of the novel coronavirus

1. Materials for experiments.

1.1 Reagents and consumables.

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Table 1. Names and brands of reagents and consumables

Name of reagent and consumables Trademark 1 Tris Sigma 2 EDTA Sigma 3 NaCl Sigma 4 384 well plate Perkin Elmer 5 Dimethyl sulfoxide (DMSO) Sigma 6 Substrate (Dabcyl- KTSAVLQSGFRKM-(Edans)) GenScript 7 Mpro SARS-CoV-2 WuXi AppTec 8 GC376 TargetMol

1.2 Devices

Table 2. Devices and brands

Device Trademark 1 Microplate reader SpectraMax M2e Molecular Devices 2 Dosing station Labcyte liquids Echo 655 3 High Speed Benchtop Centrifuge Eppendorf

2. Experimental method.

The compound was dissolved in DMSO and diluted using Echo655 with a 3-fold gradient according to the concentration requirements to obtain 10 concentration values and for each concentration value, the assay was duplicated and the diluted solution was added to a 384-well plate. Mpro protein and substrate were diluted with test buffer (100 mM NaCl, 20 mM Tris-HCl, 1 mM EDTA) and Mpro protein was added to a 384-well test plate, incubated with the compound for 30 min at room temperature, then substrate was added and wherein the Mpro protein concentration tested was 25 nM, and the substrate concentration tested was 25 μM. After incubation for 60 minutes in a constant temperature incubator of 30°C, the fluorescence signal value at Ex/Em=340 nm/490 nm was read using a microplate reader. At the same time, the background value of a well containing substrate and compound, but not containing Mpro protein, was set as a control.

3. Data analysis

1) The degree of inhibition was calculated using the following formula:

Inhibition rate % = [(compound- _BO compound)-(/PE-BO7PE)]/[(HPE-BGhpe)-(ZPE-BGzpe)] * 100% #HPE: 100% inhibition control containing 25 nM Mpro protein + 25 µM substrate + 1 µM GC376

ZPE: no inhibition control containing 25 nM Mpro protein + 25 µM substrate, no compound

Compound: test compound well containing 25 nM Mpro protein + 25 µM substrate + compound

BG: background value of control well containing 25 µM substrate + compound, not containing Mpro protein

2) Log (agonist) versus response - nonlinear slope fitting analysis was performed on the rate of inhibition data (rate of inhibition) of a compound using GraphPad Prism software and the IC ₅₀ value of the compound was obtained.

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Table 3. In vitro activity of test compounds against the Mpro protease of the new coronavirus

Conclusion: The compounds of the present invention have adequate in vitro activity against the novel coronavirus Mpro protease.

Experimental embodiment 2: Evaluation of in vitro activity of compounds against coronavirus in a cytopathic model

1. Materials for experiments.

1.1 Reagents and consumables.

Table 4. Names and brands of reagents and consumables

Name of reagent and consumables Trademark 1 MEM Wednesday Sigma 2 L-glutamine Gibco 3 Non-essential amino acid Gibco 4 Double antibody (penicillin-streptomycin solution) Well Cl one 5 Fetal bovine serum (FBS) ExCell 6 Phosphate Buffered Saline (DPBS) Corning 7 0.25% trypsin Gibco 8 CellTiter Gio Cell Activity Assay Kit Promega 9 Remdesivir M.C.E. 10 96-well plate Grenier

1.2 Devices

Table 5. Devices and brands

Device Trademark 1 Microplate reader BioTek 2 Cell counting device Beckman 3 COG incubator Thermo

1.3 Cells and viruses

MRC5 cells and coronavirus OC43 HCoV were purchased from ATCC.

MRC5 cells were cultured in MEM (Sigma) supplemented with 10% fetal bovine serum (Excell), 1% double antibody solution (Hyclone), 1% L-glutamine (Gibco), and 1% non-essential amino acid solution (Gibco). MEM medium (Sigma) supplemented with 5% fetal bovine serum (Excell), 1% double antibody solution (Hyclone), 1% L-glutamine (Gibco), and 1% non-essential amino acid solution (Gibco) was used as an experimental culture medium. environment.

2. Experimental method

Table 6. Methods for studying viruses used in this study

Virus (strain) Cell Connection processing time (days)/endpoint method Control connection Detection reagent OS43 HCoV, YuotsShzo/hole 20,000 MNC5 cells/well 5/CPE Remdesivir Cell Titer Gio.

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Cells were inoculated into a 96-microwell plate at a defined density (Table 6) and cultured overnight in an incubator at 5% CO2 and 37°C. On day two, compound was added after double dilution (8 concentrations, duplicate wells) at 50 μl per well. The diluted virus was then added to the cells at 100 TCID ₅₀ per well, 50 μl per well. Cellular control (cells without compound treatment or viral infection), viral control (virus-infected cells without compound treatment), and culture medium control (culture medium only) were added. The final volume of culture medium in this experiment was 200 μl, and the final concentration of DMSO in the culture medium was 0.5%. Cells were cultured in a 5% CO2 incubator at 33°C for 5 days. Cell viability was determined using the CellTiter Glo Cell Viability Assay Kit (Promega). Cytotoxicity experiments were performed under the same conditions as the antiviral activity experiments, but without viral infection.

3. Data analysis

The antiviral activity and cytotoxicity of the compound are presented as the degree of inhibition (%) and cell viability (%) of the compound against the cytopathic effect caused by the virus at different concentrations, respectively. The calculation formula is as follows:

Inhibition rate (%) = (test well reading - viral control average) / (cell control average - viral control average) x 100

Cell viability (%) = (reading value of the test well - average value of the culture medium control) / (average value of the cell control - average value of the culture medium control) x 100

To perform a nonlinear fit analysis regarding the degree of inhibition and cell viability of a compound, GraphPad Prism was used and the half-maximal effective concentration (EC50) and half-maximal cytotoxic concentration ( _CC50 ) of the compound were calculated.

Table 7. Assessment of in vitro activity of compounds against coronavirus in a cytoplastic model

Connection number EC ₅ o (nM) CC ₅ o (nM) 2 697 >10000 3 62 >10000 4 141 >10000 5 205 >10000 AND 3.5 >10000 15 191 >10000 16 83 >10000

Conclusion: The compounds of the present invention have adequate in vitro activity against coronavirus at the cellular level and are not cytotoxic.

Embodiment 3: Novel Coronavirus Activity and Toxicity Study

3.1. Source of cells and type of new coronavirus

Core viruses were obtained from African green monkey kidney cells (Vero) from the American Type Culture Collection (ATCC), cat. No. CCL-81. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM, WelGene) supplemented with 10% fetal bovine serum (Gibco) and 1% double antibody solution (Gibco). DMEM supplemented with 2% fetal bovine serum (Gibco) and 1% double antibody solution (Gibco) was used as the experimental culture medium.

The new coronavirus strain eCoV/KOR/KCDC03/2020 was provided by the Korea Center for Disease Control and Prevention (KCDC), serial number NCCP43326.

3.2. Experimental method

Cell seeding

After trypsin digestion of Vero cells, Vero cells were diluted to 480,000 cells per ml using experimental culture medium. The diluted cells were added to a 384-well assay plate at 25 μL and 12,000 cells per well using an automatic dispenser. Cells were cultured overnight in an incubator at 5% CO2 and 37°C.

Compound Treatment and Viral Infection

On day two, compound and CP-100356 were diluted in DMSO and the diluted compound was added to the assay wells using an automatic liquid dispensing station. Then, 25 μL of SARS-CoV-2 virus solution diluted with experimental culture medium was added to each well at an MOI of 0.0125. A cell control (cells without compound treatment or viral infection) and a control without compound treatment (cells infected with

- 57 047028 virus without treatment with a compound with 0.5% DMSO), and control CP-100356 (cells infected with virus, treated with 2 μM CP-100356). The final volume of cell culture medium in each well was 50 μl. Cells were cultured in an incubator at 5% _CO2 and 37°C for 24 hours.

Immunofluorescence staining (1) 24 h after virus infection, 17 μl of 16% paraformaldehyde was added to each well. The samples containing the virus were then left at room temperature for 30 minutes.

(2) The supernatant was aspirated and the plate was washed twice with DPB S.

(3) 25 μL of 0.25% Tritonx-100 was added to each well and left at room temperature for 20 min.

(4) Aspirate 0.25% TritonX-100 and wash the plate twice with DPBS.

(5) 25 μL of diluted primary antibody (1:3000 dilution) was added to each well and incubated at 37 °C for 1 h.

(6) The primary antibody solution was aspirated and the plate was washed twice with DPBS.

(7) 25 μl of diluted sheep anti-rabbit IgG secondary antibody labeled with Alexa Fluor 488 (1:2000 dilution) and 2.5 μg/ml Hoechst 33342 (1:4000 dilution) were added to each well and incubated for 1 h at 37°C.

(8) The secondary antibody and Hoechst solution were aspirated and the plate was washed twice with DPBS.

(9) An Operetta multiparameter image analyzer was used to read the plates and the instrument was set at: excitation at 488/405, 20x objective, 5 fields of view per well.

Data analysis

Columbus software was used to quantify the total cell number (Hoechst-stained cell number) and the number of cells infected with the novel coronavirus (Alexa Fluor 488-labeled cell number) in images obtained by multiparameter image analyzer readout. The proportion of infected cells and the total number of cells were used to analyze the antiviral activity and cytotoxicity of the compounds. The calculation formula is as follows:

Inhibition rate (%) = 100 - (proportion of infected cells in test wells - average proportion of infected cells in cell control wells)/(average proportion of infected cells in cell control wells without compound treatment - average proportion of infected cells in cell control wells ) x 100

Cell viability (%)=total number of cells in test wells/average total number of cells in control wells without compound treatment x 100

XLfit 4 software was used to analyze the effect of compounds on inhibitory activity and cell viability using non-linear approximation, and the IC ₅₀ and CC ₅₀ values of the compounds were calculated. The approximation method was a sigmoidal dose-response relationship. The formula for calculating IC ₅₀ and CC ₅₀ is as follows: Y = lower point + (upper point) / (1 + (1C ₅₀ /X)Hill slope).

Table 8. Assessment of in vitro activity of compounds against wild-type coronavirus in a cytoplastic model

Connection number EU so (nM) CC ₅₀ (nM) 3 35 >5000 4 thirty >5000 5 20 >5000 9 19 >5000 eleven 3.5 >5000

Conclusion: The compounds of the present invention exhibit adequate in vitro activity against the novel coronavirus.

Experimental Embodiment 4: Pharmacokinetic Study in Mice

In this study, male C57BL/6J mice were selected as test animals and the LC/MS/MS method was used to quantitatively measure the plasma concentration of test compound 11 at various time points after oral administration and injection into mice, in order to evaluate the pharmacokinetic characteristics of the test subject. drugs in mice.

The test compound dissolved in 30% PEG400+70% normal saline was administered to mice (fasted after an overnight fast, 6-8 weeks old) via intragastric administration. 25 μl of blood was collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 hours after administration, respectively, and placed in commercially available anticoagulant tubes with pre-added EDTA-K2, centrifuged at 4°C , 3200 g for 10 min to obtain blood plasma and after the blood plasma sample was processed, the concentration in blood plasma was determined by LC-MS/MS method.

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Table 9. Pharmacokinetic parameters of compound 11 in mice

RK parameter intravenous® 3 mg/kg orally® 10 mg/kg Stakh (NM) n. O. 1855 Ttah (Ch.) n. O. 0.25 T1/2 (H.) 0.2 n. O. Vdss (l/kg) 0.9 n. O. SCml/min./kg) 68.5 n. O. AUCo-last(HM-4.) 1447 1519

n. O. means absence.

Conclusion: The compounds of the present invention are rapidly eliminated in mice, resulting in low levels of compound exposure and approximately 30% oral bioavailability.

Experimental Embodiment 5: Pharmacokinetic Study of the Compound of the Present Invention in Combination with Ritonavir

In this study, male C57BL/6J mice were selected as test animals and LC/MS/MS was used to quantify plasma concentrations of mice in combination with ritonavir at various time points in order to evaluate the pharmacokinetic characteristics of the test drug in mice. .

Initially, mice were administered ritonavir at a dose of 10 mg/kg via intragastric administration at 12 noon. And 0h, and then the test compound was dissolved in 30% PEG400+70% normal saline and administered to mice via intragastric administration (fasting after an overnight fast, 6 to 8 weeks of age). 25 μl of blood was collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 hours after administration to animals, placed in a commercially available anticoagulant tube with pre-added EDTA-K2 and centrifuged at 4°C , 3200 g for 10 minutes to obtain blood plasma. After the blood plasma samples were processed, the plasma concentration was determined by the LC-MS/MS method.

Table 10. Pharmacokinetics of compound 11 in combination with ritonavir in mice

RK parameter orally® 10 mg/kg Stakh (NM) 10090 Ttah (h.) 0.5 AUCo-last(HM-4.) 28683

Conclusion: When the compound of the present invention is used in combination with ritonavir, the effect is approximately 20 times greater than when the drug is used alone.

Experimental Embodiment 6: Tissue Distribution Study in Rats

In this study, male SD rats were selected as test animals, and drug concentrations corresponding to test compounds in the blood plasma and lungs of rats were quantified by LC/MS/MS method at different time points to evaluate the pharmacokinetic characteristics of the test drugs in rats.

Initially, rats were administered ritonavir at a dose of 10 mg/kg via intragastric administration at -12 and 0 hours, and the test compound at a dose of 30 mg/kg was dissolved in a solution of 10% solutol + 30% PEG 400 + 2% Tween 80+H ₂ O 58 % and administered to rats via intragastric administration (on an empty stomach after an overnight fast). 40 μl of blood was collected from the saphenous vein of rats at 0.25, 1 and 6 hours after administration, placed in an anticoagulant tube with added EDTA-K2, centrifuged at 4°C, 3200 g for 10 min to obtain blood plasma, and some were killed. animals after 0.25, 1 and 6 hours, respectively, to collect lung tissue. After the blood plasma samples were processed, the plasma concentration was determined by the LC-MS/MS method.

Table 11. Pharmacokinetic parameters of compound 11 in rats

Concentration and ratio in tissue at each time point Lung/blood plasma (nM) Ratio 0.25 hours 15800/2860 5.5/1 1 hour 18450/5490 3.2/1 6 hours 7850/4875 1.6/1 AUC(0-6 h) (h.nmol/l) 76840/29371 2.6/1

Conclusion: After administration of the compound of the present invention in combination with ritonavir, higher exposure was observed in the lungs of rats.

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Claims (18)

CLAIM 1. The compound represented by formula (X) or a pharmaceutically acceptable salt thereof (X) where G is selected from ring A is selected from C _3-10 cycloalkyl and C _6-10 aryl; each R1 is independently selected from halogen, OR11, CN, C _1-3 αlkyl and C _1-3 haloαlkyl; R11 is selected from H and C _1-3 alkyl; n is selected from 0, 1, 2, 3 and 4; X represents -CH2; R2 and _R4 together with the atoms to which they are attached form ^^cycloalkyl, and said ^^cycloalkyl is optionally and independently substituted with 1 or 2R _a ; each R _a is independently selected from H, halogen, C _1-3 alkyl, C _1-3 alkoxy and C _1-3 haloalkoxy; R5 is selected from ^^alkyl, C _1-3 haloalkyl, -CH2-R6 and -CH2-O-R6; R6 is selected from phenyl, and said phenyl is optionally substituted with 1, 2 or 3 _R61 ; R61 is selected from halogen, ^^alkyl, ^^haloalkyl, ^^alkoxy and ^^halogenal^^^ 2. The compound or a pharmaceutically acceptable salt thereof according to claim 1, where the compound is selected from the structures represented by formulas (X-1) and (X-2) (X-1) (X-2) where each Rb is independently selected from H, halogen, C _1-3 αlkyl, C _1- ^kokcu and C _1-3 halogenα-alkoxy; or two Rb at adjacent carbon atoms or at the same carbon atom together with the atoms to which they are attached form cyclopropyl; t selected from 1 and 2; R1, R5, n and ring A are defined in paragraph 1. 3. The compound or pharmaceutically acceptable salt thereof according to claim 2, wherein each Rb is independently selected from H, F, methyl, ethyl and isopropyl. 4. A compound or a pharmaceutically acceptable salt thereof according to claim 2, wherein the structural fragment is selected from 5. The compound or its pharmaceutically acceptable salt according to claim 4, where the structural fragment selected from 6. The compound or pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein each R1 is independently selected from halogen, C _1-3 alkyl, C _1-3 alkoxy and C _1-3 haloalkyl. - ⁷ AND X— ⁷ . 7. The compound or pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein each R1 is independently selected from F, Cl and methyl. 8. A compound or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein ring A is selected from - 60 047028 With _5-10 cycloalkyl and phenyl. 9. The compound or pharmaceutically acceptable salt thereof according to claim 8, wherein ring A is selected from cyclohexyl, spiro[3.3]heptyl, bicyclo[2.2.2]octyl, adamantyl and phenyl. 10. The compound or a pharmaceutically acceptable salt thereof according to claim 9, wherein ring A is selected from 11. A compound or a pharmaceutically acceptable salt thereof according to claim 1 or 2, where structural fragment 1 ^(Rl)n is selected from 12. The compound or its pharmaceutically acceptable salt according to claim 1 or 2, where R5 is selected from -CF ₃ , 13. A compound represented by the following formula, or a pharmaceutically acceptable salt thereof 14. A pharmaceutical composition containing a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13. 15. The pharmaceutical composition according to claim 14, wherein said pharmaceutical composition contains a pharmaceutically acceptable excipient. 16. Use of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13 or a pharmaceutical composition according to claim 14 or 15 in the manufacture of a medicinal product intended for the treatment of a disease associated with 3CL protease. 17. Use according to claim 16, wherein the disease associated with the 3CL protease is a coronavirus infection. 18. Use according to claim 17, wherein the coronavirus infection is a COVID-19 infection. -