RU2839468C1 - Sulphonylbenzamide derivative and conjugates thereof, process for preparation and use thereof - Google Patents

Abstract

FIELD: chemistry; pharmaceutics.

SUBSTANCE: invention relates to a compound of formula (D), wherein R¹-R¹⁴ are defined in the claims, or a pharmaceutically acceptable salt thereof, a method for preparation thereof, a pharmaceutical composition containing said compound for the treatment of cancer by receptor regulation.

EFFECT: disclosed is a compound for treating cancer by regulating receptors.

24 cl, 2 dwg, 6 tbl, 24 ex

Description

AREA OF TECHNOLOGY

The present invention relates to a sulfonyl benzamide derivative and a ligand-drug conjugate (ADC) thereof. In particular, the present invention relates to a sulfonyl benzamide derivative with a completely new structure and an antibody-drug conjugate thereof, a method for producing the same, a pharmaceutical composition containing the conjugate, and a use of the conjugate or the pharmaceutical composition.

PRIOR ART

An important feature that distinguishes tumor cells from normal cells is that apoptosis is inhibited in tumor cells, giving them a greater survival advantage. BCL-2 family proteins are important factors in regulating apoptosis inhibition.

The BCL-2 family proteins are mainly present on the mitochondrial membranes and can be classified into two main groups according to their functions: antiapoptotic proteins and proapoptotic proteins. Antiapoptotic protein includes BCL-2, BCL-xL, BCL-w, MCL-1, etc. Proapoptotic protein includes Bax, Bak, and BH3-only protein. Bax and Bak, when activated, are converted from harmless monomers to deadly oligomers that form pores in the outer mitochondrial membrane, increase the permeability of the cell's mitochondrial membrane, and promote the release of cytochrome C and the like into the cytoplasm, resulting in cell death. BH3-only protein contains only the BH3 domain. In the surviving state of the cell, the BH3-only protein (e.g., Bim) binds to the antiapoptotic protein. When the cell is exposed to external stress, the binding balance is disrupted and the BH3-only protein is released to bind to BAX on the mitochondria, thereby promoting the formation of BAX/BAK polymers, promoting the release of cytochrome c and SMAC into the cytoplasm and activating the subsequent apoptotic pathway.

Available preclinical data show that the dual-targeted BCL2/BCL-xL inhibitor ABT263 is not only effective against hematological tumors, but also has good inhibitory effects on solid tumors, especially small cell lung cancer. However, the clinical toxicity of ABT263 to platelets limited its efficacy and ultimately led to the termination of clinical trials.

There is still a need to explore dual-targeted BCL2/BCL-xL inhibitor so that it can be used as monotherapy or in combination therapy or ADC to meet clinical requirements.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides a compound of general formula (D) or a pharmaceutically acceptable salt thereof:

Where:

R ¹ is selected from the group consisting of deuterium, hydrogen, alkyl and deuterated alkyl;

R ² is selected from the group consisting of alkyl and hydroxyalkyl;

or R ¹ and R ² together with the atom to which they are attached form a heterocyclyl optionally further substituted with a substituent selected from the group consisting of halogen, hydroxy, alkyl, hydroxyalkyl, alkoxy and cycloalkyl;

R ³ is selected from the group consisting of hydrogen, hydroxyalkyl, alkyl, deuterated alkyl, cycloalkyl and cycloalkylalkyl;

R ⁴ is selected from the group consisting of hydrogen, halogen, deuterated alkyl and alkyl;

R ⁵ is selected from the group consisting of halogen and haloalkyl;

R ⁶ is selected from the group consisting of alkyl, amino, hydroxy and alkoxy;

R ⁷ is selected from the group consisting of hydrogen, halogen, alkyl, deuterated alkyl, hydroxy and alkoxy;

each of R ⁸ and R ⁹ is independently selected from the group consisting of hydrogen, alkyl, deuterated alkyl, and cycloalkyl; or R ⁸ and R ⁹ together with the atom to which they are attached form a cycloalkyl;

each of R ¹⁰ and R ¹¹ is independently selected from the group consisting of hydrogen, alkyl, deuterated alkyl, and cycloalkyl; or R ¹⁰ and R ¹¹ together with the atom to which they are attached form a cycloalkyl;

R ¹² is selected from the group consisting of hydrogen, deuterium, deuterated alkyl, halogen, hydroxy, alkyl, hydroxyalkyl, alkoxy and cycloalkyl;

R ¹³ is selected from the group consisting of hydrogen, deuterium, deuterated alkyl, halogen, hydroxy, alkyl, hydroxyalkyl, alkoxy and cycloalkyl;

R ¹⁴ is selected from the group consisting of hydrogen, deuterium, deuterated alkyl, halogen, hydroxy, alkyl, hydroxyalkyl, alkoxy and cycloalkyl; and

r is selected from the group consisting of 0, 1, 2, and 3;

provided that if R ¹ and R ² together with the atom to which they are attached do not form a heterocyclyl, R ⁵ is haloalkyl.

In some other embodiments, the present invention provides a compound of general formula (D) or a pharmaceutically acceptable salt thereof, wherein R ¹ is hydrogen and R ² is alkyl.

In some other embodiments, the present invention provides a compound of general formula (D) or a pharmaceutically acceptable salt thereof, wherein R ¹ and R ² together with the atom to which they are attached form a heterocyclyl; preferably, the heterocyclyl optionally further comprises 1-3 heteroatoms selected from the group consisting of nitrogen and oxygen; more preferably, R ¹ and R ² together with the atom to which they are attached form a 3-6-membered heterocyclyl; preferably, the 3-6-membered heterocyclyl optionally further comprises 1-3 heteroatoms selected from the group consisting of nitrogen and oxygen; and most preferably, R ¹ and R ² together with the atom to which they are attached form pyrrolidinyl.

In some other embodiments, the present invention provides a compound of general formula (D) or a pharmaceutically acceptable salt thereof, which is a compound of general formula (D-I) or a pharmaceutically acceptable salt thereof:

where m is selected from the group consisting of 0, 1, 2, and 3, and

r and R ³ -R ¹⁴ are as defined in general formula (D).

In some other embodiments of the present invention, there is provided a compound of general formula (D) or a pharmaceutically acceptable salt thereof, which is a compound of general formula (D-II) or a pharmaceutically acceptable salt thereof:

where m is selected from the group consisting of 0, 1, 2, and 3, and

r and R ³ -R ¹¹ are as defined in general formula (D).

In some other embodiments of the present invention, there is provided a compound of general formula (D) or a pharmaceutically acceptable salt thereof, which is a compound of general formula (D-III) or a pharmaceutically acceptable salt thereof:

where m is selected from the group consisting of 0, 1, 2, and 3, and

r and R ³ -R ¹¹ are as defined in general formula (D). In some other embodiments, the present invention provides a compound of general formula (D) or a pharmaceutically acceptable salt thereof, which is a compound of general formula (D-IV) or a pharmaceutically acceptable salt thereof:

where m is selected from the group consisting of 0, 1, 2, and 3, and

R ³ -R ⁹ are as defined in general formula (D).

In some other embodiments, the present invention provides a compound of general formula (D) or a pharmaceutically acceptable salt thereof, wherein m is 2.

In some other embodiments of the present invention, there is provided a compound of general formula (D) or a pharmaceutically acceptable salt thereof, wherein R⁵ is chlorine.

In some other embodiments, the present invention provides a compound of general formula (D) or a pharmaceutically acceptable salt thereof, wherein R ⁵ is trifluoromethyl.

In some other embodiments, the present invention provides a compound of general formula (D) or a pharmaceutically acceptable salt thereof, wherein R ³ is selected from the group consisting of hydrogen, hydroxyalkyl, and alkyl; preferably hydroxyalkyl; and more preferably C _1-6 hydroxyalkyl.

In some other embodiments, the present invention provides a compound of general formula (D) or a pharmaceutically acceptable salt thereof, which is a compound of general formula (D-V) or a pharmaceutically acceptable salt thereof:

where t is selected from the group consisting of 0, 1, 2 and 3, preferably 1, and

r, R ¹ , R ² , R ⁴ and R ⁶ -R ¹⁴ are as defined in general formula (D).

In some other embodiments, the present invention provides a compound of general formula (D) or a pharmaceutically acceptable salt thereof, which is a compound of general formula (D-VI) or a pharmaceutically acceptable salt thereof:

where t is selected from the group consisting of 0, 1, 2 and 3, preferably 1, and

r, R ¹ , R ² , R ⁴ and R ⁶ -R ¹¹ are as defined in general formula (D).

In some other embodiments of the present invention, there is provided a compound of general formula (D) or a pharmaceutically acceptable salt thereof, which is a compound of general formula (D-VII) or a pharmaceutically acceptable salt thereof:

where t is selected from the group consisting of 0, 1, 2 and 3, preferably 1, and

r and R ¹ -R ¹¹ are as defined in general formula (D).

In some other embodiments, the present invention provides a compound of general formula (D) or a pharmaceutically acceptable salt thereof, wherein R ⁴ is selected from the group consisting of hydrogen and alkyl; preferably hydrogen and C _1-6 alkyl; and more preferably hydrogen.

In some other embodiments, the present invention provides a compound of general formula (D) or a pharmaceutically acceptable salt thereof, wherein R ⁶ is selected from the group consisting of hydroxy and alkoxy; preferably hydroxy and C _1-6 alkoxy; and more preferably hydroxy.

In some other embodiments, the present invention provides a compound of general formula (D) or a pharmaceutically acceptable salt thereof, wherein R ⁷ , R ⁸ and R ⁹ are hydrogen.

In some other embodiments, the present invention provides a compound of general formula (D) or a pharmaceutically acceptable salt thereof, wherein each of R ¹⁰ and R ¹¹ is independently selected from the group consisting of hydrogen and alkyl; preferably hydrogen and C _1-6 alkyl; and more preferably hydrogen.

In some other embodiments, the present invention provides a compound of general formula (D) or a pharmaceutically acceptable salt thereof, wherein r is 0 or 1, preferably 0.

In some other embodiments of the present invention, a compound of general formula (D) is provided, including, but not limited to:

or any pharmaceutically acceptable salt thereof.

The present invention further provides a compound of the general formula (DA1) or (DB1) or a pharmaceutically acceptable salt thereof,

Where:

G ₁ represents an amino protecting group, preferably Boc (tert-butoxycarbonyl group);

m is selected from the group consisting of 0, 1, 2, and 3, and

r and R ⁴ -R ¹¹ are as defined in general formula (D).

In some other embodiments, the present invention provides a compound of general formula (DA1) or (DB1), or a pharmaceutically acceptable salt thereof, which is selected from any of the following compounds:

And

The present invention further provides a method for producing a compound of general formula (D-III) or a pharmaceutically acceptable salt thereof, which comprises:

subjecting a compound of general formula (DA1) or a pharmaceutically acceptable salt thereof to a deprotection reaction in an acidic medium to obtain a compound of general formula (D-III) or a pharmaceutically acceptable salt thereof; preferably, the reagent used in the acidic medium is HCl;

Where:

G ₁ represents an amino protecting group, preferably Boc;

m is selected from the group consisting of 0, 1, 2, and 3;

R ³ is hydrogen; and

r and R ⁴ -R ¹¹ are as defined in general formula (D).

The present invention further provides a compound of the general formula (DA2) or (DB2) or a pharmaceutically acceptable salt thereof,

Where:

m is selected from the group consisting of 0, 1, 2, and 3, and

r and R ⁴ -R ¹¹ are as defined in general formula (D).

In some other embodiments, the present invention provides a compound of general formula (DA2) or (DB2), or a pharmaceutically acceptable salt thereof, which is selected from any of the following compounds:

The present invention further provides a method for producing a compound of general formula (D-III) or a pharmaceutically acceptable salt thereof, which comprises:

subjecting a compound of general formula (DA2) or a pharmaceutically acceptable salt thereof to a substitution reaction in an alkaline medium to obtain a compound of general formula (D-III) or a pharmaceutically acceptable salt thereof; preferably, the reagent used in the alkaline medium is triethylamine;

Where:

m is selected from the group consisting of 0, 1, 2, and 3;

R ³ is hydroxyalkyl; and

r and R ⁴ -R ¹¹ are as defined in general formula (D).

The present invention further provides a compound of the general formula (DA3), (DB3) or (DC3) or a pharmaceutically acceptable salt thereof,

Where:

G ₂ represents a hydroxy protecting group, preferably TBS;

t is selected from the group consisting of 0, 1, 2, and 3, and

r, R ¹ , R ² , R ⁴ -R ¹¹ are as defined in the general formula (D).

In some other embodiments, the present invention provides a compound of general formula (DA3), (DB3) or (DC3), or a pharmaceutically acceptable salt thereof, which is selected from any of the following compounds:

The present invention further provides a method for producing a compound of general formula (D-VI) or a pharmaceutically acceptable salt thereof, which comprises:

subjecting a compound of general formula (DA3) or a pharmaceutically acceptable salt thereof to a deprotection reaction in an alkaline medium to obtain a compound of general formula (D-VI) or a pharmaceutically acceptable salt thereof; and the reagent used in the alkaline medium is tetrabutylammonium fluoride;

Where:

G ₂ represents a hydroxy protecting group, preferably TBS;

t is selected from the group consisting of 0, 1, 2, and 3, and

r, R ¹ , R ² , R ⁴ -R ¹¹ are as defined in the general formula (D).

The present invention further provides a ligand-drug conjugate having a structure of formula (-D), or a pharmaceutically acceptable salt thereof:

Where:

R ¹ is selected from the group consisting of deuterium, hydrogen, alkyl and deuterated alkyl;

R ² is selected from the group consisting of alkyl and hydroxyalkyl;

or R ¹ and R ² together with the atom to which they are attached form a heterocyclyl optionally further substituted with a substituent selected from the group consisting of halogen, hydroxy, alkyl, hydroxyalkyl, alkoxy and cycloalkyl;

R ^3a is selected from the group consisting of a bond and -(CH ₂ ) _t -CH ₂ -O-; the alkyl end is bonded to the N atom of the drug moiety, and the -O- end is bonded to the linker;

R ⁴ is selected from the group consisting of hydrogen, halogen, deuterated alkyl and alkyl;

R ⁵ is selected from the group consisting of halogen and haloalkyl;

R ⁶ is selected from the group consisting of alkyl, amino, hydroxy and alkoxy;

R ⁷ is selected from the group consisting of hydrogen, halogen, alkyl, deuterated alkyl, hydroxy and alkoxy;

each of R ⁸ and R ⁹ is independently selected from the group consisting of hydrogen, alkyl, deuterated alkyl, and cycloalkyl; or R ⁸ and R ⁹ together with the atom to which they are attached form a cycloalkyl;

each of R ¹⁰ and R ¹¹ is independently selected from the group consisting of hydrogen, alkyl, deuterated alkyl, and cycloalkyl; or R ¹⁰ and R ¹¹ together with the atom to which they are attached form a cycloalkyl;

R ¹² is selected from the group consisting of hydrogen, deuterium, deuterated alkyl, halogen, hydroxy, alkyl, hydroxyalkyl, alkoxy and cycloalkyl;

R ¹³ is selected from the group consisting of hydrogen, deuterium, deuterated alkyl, halogen, hydroxy, alkyl, hydroxyalkyl, alkoxy and cycloalkyl;

R ¹⁴ is selected from the group consisting of hydrogen, deuterium, deuterated alkyl, halogen, hydroxy, alkyl, hydroxyalkyl, alkoxy and cycloalkyl;

r is selected from the group consisting of 0, 1, 2, and 3;

t is selected from the group consisting of 0, 1, 2, and 3, and

The wavy line indicates covalent binding to a linker moiety or to a ligand.

In some other embodiments of the present invention, a ligand-drug conjugate having a structure of formula (-D), or a pharmaceutically acceptable salt thereof, wherein formula (-D) is selected from the group consisting of structures of formulas (-di) and (-DII):

t is selected from the group consisting of 0, 1, 2, and 3, and

wavy line r, R ¹ , R ² and R ⁴ -R ¹⁴ are as defined in the general formula (-D).

In some other embodiments, the present invention provides a ligand-drug conjugate having a structure of formula (-D), or a pharmaceutically acceptable salt thereof, wherein formula (-D) is selected from the group consisting of structures of formulas (-DIII), (-DIV), and (-DV):

Where:

t is selected from the group consisting of 0, 1, 2 and 3;

m is selected from the group consisting of 0, 1, 2, and 3, and

wavy line r, R ¹ , R ² and R ⁴ -R ¹¹ are as defined in the general formula (-D);

preferably in formula (-DV), R ¹ and R ² do not form heterocyclyl.

In some other embodiments, the present invention provides a ligand-drug conjugate having a structure of formula (-D), or a pharmaceutically acceptable salt thereof, wherein formula (-D) is selected from the group consisting of structures of formulas (-DVI) and (-DVII):

Where:

t is selected from the group consisting of 0, 1, 2 and 3;

m is selected from the group consisting of 0, 1, 2, and 3, and

wavy line, r and R ⁵ -R ⁹ are as defined in the general formula (-D).

In some other embodiments, the present invention provides a ligand-drug conjugate having a structure of formula (-D), or a pharmaceutically acceptable salt thereof, which is a ligand-drug conjugate of the general formula (Pc-L-D), or a pharmaceutically acceptable salt thereof,

Where:

R ¹ is selected from the group consisting of deuterium, hydrogen, alkyl and deuterated alkyl;

R ² is selected from the group consisting of alkyl and hydroxyalkyl;

or R ¹ and R ² together with the atom to which they are attached form a heterocyclyl optionally further substituted with a substituent selected from the group consisting of halogen, hydroxy, alkyl, hydroxyalkyl, alkoxy and cycloalkyl;

R _3a is selected from the group consisting of the bond and -(CH ₂ ) _t -CH ₂ -O-;

R ₄ is selected from the group consisting of hydrogen, halogen, deuterated alkyl and alkyl;

R ₅ is selected from the group consisting of halogen and haloalkyl;

R ₆ is selected from the group consisting of alkyl, amino, hydroxy and alkoxy;

R ₇ is selected from the group consisting of hydrogen, halogen, alkyl, deuterated alkyl, hydroxy and alkoxy;

each of R ₈ and R ₉ is independently selected from the group consisting of hydrogen, alkyl, deuterated alkyl, and cycloalkyl; or R ₈ and R ₉ together with the atom to which they are attached form a cycloalkyl;

each of R ₁₀ and R ₁₁ is independently selected from the group consisting of hydrogen, alkyl, deuterated alkyl, and cycloalkyl; or R ₁₀ and R ₁₁ together with the atom to which they are attached form a cycloalkyl;

R ₁₂ is selected from the group consisting of hydrogen, deuterium, deuterated alkyl, halogen, hydroxy, alkyl, hydroxyalkyl, alkoxy and cycloalkyl;

R ₁₃ is selected from the group consisting of hydrogen, deuterium, deuterated alkyl, halogen, hydroxy, alkyl, hydroxyalkyl, alkoxy and cycloalkyl;

R ₁₄ is selected from the group consisting of hydrogen, deuterium, deuterated alkyl, halogen, hydroxy, alkyl, hydroxyalkyl, alkoxy and cycloalkyl;

r is selected from the group consisting of 0, 1, 2, and 3;

t is selected from the group consisting of 0, 1, 2 and 3;

n is a decimal number between 1 and 10; and

Rc is a ligand; and L is a linker moiety.

In some other embodiments, the present invention provides a ligand-drug conjugate having the structure of formula (-D), or a pharmaceutically acceptable salt thereof, which is a ligand-drug conjugate of the general formula (Pc-L-DIII), (Pc-L-DIV) or (Pc-L-DV), or a pharmaceutically acceptable salt thereof:

Where:

t is selected from the group consisting of 0, 1, 2, and 3; m is selected from the group consisting of 0, 1, 2, and 3, and

r, R ₁ , R ₂ , R ₄ -R ₁₁ , Pc, L and n are as defined in the general formula (Pc-LD).

In some other embodiments, the present invention provides a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, wherein n is an integer or decimal number from 1 to 8; preferably an integer or decimal number from 2 to 8.

In some other embodiments, the present invention provides a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, wherein Pc is an antibody.

In some other embodiments, the present invention provides a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody is selected from the group consisting of an anti-HER2 (ErbB2) antibody, an anti-Trop-2 antibody, an anti-CD79b antibody, an anti-EGFR antibody, an anti-B7-H3 antibody, an anti-c-Met antibody, an anti-HER3 (ErbB3) antibody, an anti-HER4 (ErbB4) antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD30 antibody, an anti-CD33 antibody, an anti-CD44 antibody, an anti-CD56 antibody, an anti-CD70 antibody, an anti-CD73 antibody, an anti-CD105 antibody, an anti-CEA antibody, an anti-A33 antibody, an anti-Cripto antibody, an anti-EphA2 antibody, an anti-G250 antibody, an anti-MUC1 antibody, an anti-Lewis Y antibody, an anti-VEGFR antibody, an anti-GPNMB antibody, anti-integrin antibodies, anti-PSMA antibodies, anti-tenascin-C antibodies, anti-SLC44A4 antibodies and anti-mesothelin antibodies; preferably trastuzumab, pertuzumab, nimotuzumab, enoblituzumb, emibetuzumab, inotuzumab, pinatuzumab, brentuximab, gemtuzumab, bivatuzumab, lorvotuzumab, cBR96 and glematumamab.

In some other embodiments, the present invention provides a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the linker moiety -L- is -L ^a -L ^b -L ^c -, where

L ^a is selected from the group consisting of wherein W is selected from the group consisting of -C _1-6 alkyl-, -C _1-6 alkyl-cycloalkyl-, and linear -heteroalkyl- of 1-6 atoms, wherein heteroalkyl contains from 1 to 3 heteroatoms selected from the group consisting of N, O, and S, wherein -C _1-6 alkyl-, -C _1-6 alkyl-cycloalkyl-, and linear -heteroalkyl- of 1-6 atoms are each independently and optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy, and cycloalkyl;

L ^b is a peptide residue or chemical bond consisting of 2-7 amino acids that are optionally substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl; and

L ^c is selected from the group consisting of -NR ¹⁷ (CR ¹⁸ R ¹⁹ )t-, -NH-C(R ¹⁸ R ¹⁹ )-OC(R ²⁰ R ²¹ )-C(O)-, -NH-R ²² -(CH ₂ ) _q -OC(O)-, -C(O)NR ¹⁷ , -C(O)NR ¹⁷ (CH ₂ ) _q - and a chemical bond, where q is an integer from 1 to 6;

R ¹⁷ is selected from the group consisting of hydrogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl;

R ¹⁸ and R ¹⁹ are the same or different and are each independently selected from the group consisting of hydrogen, halogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl;

R ²⁰ is selected from the group consisting of alkyl, cycloalkylalkyl and cycloalkyl;

R ²¹ is selected from the group consisting of hydrogen, alkyl and haloalkyl; or R ²⁰ and R ²¹ together with the carbon atom to which they are attached form C _3-6 cycloalkyl;

R ²² is selected from the group consisting of aryl and heteroaryl.

In some other embodiments, the present invention provides a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, wherein W is selected from the group consisting of -( _CH2 ) _2- and -( _CH2 ) _5- .

In some other embodiments, the present invention provides a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the peptide residue L ^b is an amino acid residue formed from one or more amino acids selected from the group consisting of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid; preferably a tetrapeptide residue, a dipeptide residue and a chemical bond; and more preferably a glycine-glycine-phenylalanine-glycine tetrapeptide residue or a valine-citrulline dipeptide residue.

In some other embodiments, the present invention provides a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the linker unit L ^c is selected from the group consisting of -NH-C(R ¹⁸ R ¹⁹ )-OC(R ²⁰ R ²¹ )-C(O)-, -NH-R ²² -(CH ₂ ) _q -OC(O)- and a chemical bond, q is an integer from 1 to 6; and

R ²² is selected from the group consisting of aryl and heteroaryl;

preferably L ^c is selected from the group consisting of the following structural formulas:

R ¹⁸ and R ¹⁹ are the same or different and are each independently selected from the group consisting of hydrogen, halogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl;

R ²⁰ is selected from the group consisting of alkyl, cycloalkylalkyl and cycloalkyl;

R ²¹ is selected from the group consisting of hydrogen, alkyl and haloalkyl; or R ²⁰ and R ²¹ together with the carbon atom to which they are attached form C _3-6 cycloalkyl.

In some other embodiments, the present invention provides a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the linker moiety -L- is -L ^a -, wherein -L ^a - is as defined above.

In some other embodiments, the present invention provides a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the linker moiety -L- is -L ^a -L ^b -L ^c -, where

L ^a is selected from the group consisting of where W is selected from the group consisting of -( _CH2 ) _2- and -( _CH2 ) _5- ;

L ^b is selected from the group consisting of a tetrapeptide residue and a dipeptide residue; preferably a glycine-glycine-phenylalanine-glycine tetrapeptide residue or a valine-citrulline dipeptide residue; and

L ^c is selected from the group consisting of the following structural formulas:

R ¹⁸ and R ¹⁹ are the same or different and are each independently selected from the group consisting of hydrogen, halogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl;

R ²⁰ is selected from the group consisting of alkyl, cycloalkylalkyl and cycloalkyl;

R ²¹ is selected from the group consisting of hydrogen, alkyl and haloalkyl; or R ²⁰ and R ²¹ together with the carbon atom to which they are attached form C _3-6 cycloalkyl.

In some other embodiments, the present invention provides a ligand-drug conjugate or a pharmaceutically acceptable salt thereof that comprises a linking moiety -L-, wherein -L- is selected from the group consisting of:

where the a-end is linked to the Pc ligand and the b-end is linked to the R ^3a end of the drug.

In some other embodiments, the present invention provides a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the linker moiety -L- is -L ^a -L ^b -L ^c -, where

L ^a is selected from the group consisting of where W is selected from the group consisting of -( _CH2 ) _2- and -( _CH2 ) _5- ;

L ^b is selected from the group consisting of a tetrapeptide residue and a dipeptide residue; preferably a glycine-glycine-phenylalanine-glycine tetrapeptide residue or a valine-citrulline dipeptide residue; and

L ^c has the following structural formula:

The present invention further provides a compound of the general formula (Lu-D) or a pharmaceutically acceptable salt thereof:

Where:

L ^a ' is selected from the group consisting of, where W is selected from the group consisting of -(CH ₂ ) ₂ - and -(CH ₂ ) ₅ -;

L ^b is a peptide residue or chemical bond consisting of 2-7 amino acids that are optionally substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl; and

L ^c has the following structural formula:

R ¹ is selected from the group consisting of deuterium, hydrogen, alkyl and deuterated alkyl;

R ² is selected from the group consisting of alkyl and hydroxyalkyl;

or R ¹ and R ² together with the atom to which they are attached form a heterocyclyl optionally further substituted with a substituent selected from the group consisting of halogen, hydroxy, alkyl, hydroxyalkyl, alkoxy and cycloalkyl;

R ^3a is selected from the group consisting of the bond and -(CH ₂ ) _t -CH ₂ -O-;

R ⁴ is selected from the group consisting of hydrogen, halogen, deuterated alkyl and alkyl;

R ⁵ is selected from the group consisting of halogen and haloalkyl;

R ⁶ is selected from the group consisting of alkyl, amino, hydroxy and alkoxy;

R ⁷ is selected from the group consisting of hydrogen, halogen, alkyl, deuterated alkyl, hydroxy and alkoxy;

each of R ⁸ and R ⁹ is independently selected from the group consisting of hydrogen, alkyl, deuterated alkyl, and cycloalkyl; or R ⁸ and R ⁹ together with the atom to which they are attached form a cycloalkyl;

each of R ¹⁰ and R ¹¹ is independently selected from the group consisting of hydrogen, alkyl,

deuterated alkyl and cycloalkyl; or R ¹⁰ and R ¹¹ together with the atom to which they are attached form cycloalkyl;

R ¹² is selected from the group consisting of hydrogen, deuterium, deuterated alkyl, halogen, hydroxy, alkyl, hydroxyalkyl, alkoxy and cycloalkyl;

R ¹³ is selected from the group consisting of hydrogen, deuterium, deuterated alkyl, halogen, hydroxy, alkyl, hydroxyalkyl, alkoxy and cycloalkyl;

R ¹⁴ is selected from the group consisting of hydrogen, deuterium, deuterated alkyl, halogen, hydroxy, alkyl, hydroxyalkyl, alkoxy and cycloalkyl;

r is selected from the group consisting of 0, 1, 2, and 3; and

t is selected from the group consisting of 0, 1, 2, and 3.

In some other embodiments of the present invention, there is provided a compound of the general formula (Lu-D) or a pharmaceutically acceptable salt thereof, which is a compound as shown below:

The present invention further provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of general formula (D) or a pharmaceutically acceptable salt thereof, or a ligand-drug conjugate having a structure of formula (-D) or a pharmaceutically acceptable salt thereof, or a ligand-drug conjugate of general formula (Pc-L-D) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

The present invention further provides the use of a compound of general formula (D) or a pharmaceutically acceptable salt thereof, or a ligand-drug conjugate having a structure of formula (-D), or a pharmaceutically acceptable salt thereof, or a ligand-drug conjugate of general formula (Pc-L-D), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described above, in the preparation of a Bcl-2 and/or Bcl-XL inhibitor.

The present invention further provides the use of a compound of general formula (D) or a pharmaceutically acceptable salt thereof, or a ligand-drug conjugate having a structure of formula (-D), or a pharmaceutically acceptable salt thereof, or a ligand-drug conjugate of general formula (Pc-L-D), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described above, in the preparation of a medicament for the treatment or prevention of a tumor.

The present invention provides the use of a compound of general formula (D) or a pharmaceutically acceptable salt thereof, or a ligand-drug conjugate having a structure of formula (-D), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a ligand-drug conjugate of general formula (Pc-L-D) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described above, in the preparation of a medicament for the treatment and/or prevention of cancer, wherein the cancer is preferably melanoma, liver cancer, kidney cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, nasopharyngeal cancer, colorectal cancer, colon cancer, rectal cancer, pancreatic cancer, cervical cancer, ovarian cancer, breast cancer, bladder cancer, prostate cancer, leukemia, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia, acute myelogenous leukemia, mantle cell lymphoma, diffuse large B-cell lymphoma, follicular central lymphoma, non-Hodgkin's lymphoma, T-cell lymphoma, B-cell lymphoma, squamous cell carcinoma of the head and neck, cervical cancer, thyroid cancer, lymphoma, sarcoma, neuroblastoma, brain tumor, myeloma, astrocytoma and glioma.

Another aspect of the present invention further relates to a method for treating or preventing cancer, which comprises administering a therapeutically effective dose of a compound of general formula (D) or a pharmaceutically acceptable salt thereof, or a ligand-drug conjugate having a structure of formula (-D) or a pharmaceutically acceptable salt thereof, or a ligand-drug conjugate of general formula (Pc-L-D) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, as described above; wherein the cancer is preferably melanoma, liver cancer, kidney cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, nasopharyngeal cancer, colorectal cancer, colon cancer, rectal cancer, pancreatic cancer, cervical cancer, ovarian cancer, breast cancer, bladder cancer, prostate cancer, leukemia, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia, acute myelogenous leukemia, mantle cell lymphoma, diffuse large B-cell lymphoma, follicular central lymphoma, non-Hodgkin's lymphoma, T-cell lymphoma, B-cell lymphoma, squamous cell carcinoma of the head and neck, cervical cancer, thyroid cancer, lymphoma, sarcoma, neuroblastoma, brain tumor, myeloma, astrocytoma and glioma.

The active compound (e.g., a ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to the present invention) can be formulated in a form suitable for administration by any suitable route, preferably in a unit dose form or in a single dose form that can be self-administered by a subject. A unit dose of the active compound or composition of the present invention can be in a tablet, capsule, cachet, vial, powder, granule, lozenge, suppository, powder for reconstitution, or liquid preparation.

The dosage of the active compound or composition used in the treatment method of the present invention generally varies depending on the severity of the disease, the weight of the subject and the potency of the active compound. Typically, a suitable single dose may be from 0.1 to 1000 mg.

The pharmaceutical composition of the present invention may contain, in addition to the active compound, one or more auxiliary substances selected from the group consisting of a filler, a diluent, a binder, a wetting agent, a disintegrant, an excipient, and the like. Depending on the route of administration, the composition may contain from 0.1 to 99% by weight of the active compound.

The pharmaceutical composition containing the active ingredient may be in a form suitable for oral administration, for example, in the form of a tablet, dragee, lozenge, aqueous or oily suspension, dispersible powder or granule, emulsion, hard or soft capsule or syrup or tincture. Oral compositions may be prepared according to any method known in the art for preparing pharmaceutical compositions. Such oral compositions may contain a binder, filler, lubricant, disintegrant or pharmaceutically acceptable wetting agent, and may also contain one or more ingredients selected from the group consisting of a sweetener, a flavoring agent, a coloring agent and a preservative, in order to provide a pharmaceutical composition that is pleasing in appearance and taste.

The aqueous suspension contains the active substance and an excipient which is used for mixing and is suitable for obtaining the aqueous suspension. The aqueous suspension may also contain one or more preservatives, one or more colorants, one or more corrective substances and one or more sweeteners.

An oil suspension may be formulated by suspending the active ingredient in a vegetable oil. The oil suspension may contain a thickening agent. Sweetening agents and flavoring agents as described above may be added to provide an attractive formulation.

Pharmaceutical compositions can also be prepared as follows: dispersible powders or granules for the preparation of aqueous suspensions provide the active ingredient, and water is added to mix the active ingredient with one or more dispersing agents, wetting agents, suspending agents or preservatives. Other excipients such as sweetening agents, flavoring agents and coloring agents can also be added. Antioxidants such as ascorbic acid are added to preserve these compositions.

The pharmaceutical composition of the present invention may also be in the form of an oil-in-water emulsion.

The pharmaceutical composition may be in the form of a sterile aqueous injection solution. Available and acceptable carriers or solvents include water, Ringer's solution and isotonic sodium chloride solution. The sterile injection preparation may be a sterile oil-in-water microemulsion for injection, in which the active ingredient is dissolved in the oil phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. The oil solution is then added to a mixture of water and glycerol and processed to form a microemulsion. The injection or microemulsion can be administered locally into the bloodstream of a subject in large quantities. Alternatively, it may be advisable to administer solutions and microemulsions in such a way as to maintain a constant circulating concentration of the compound of the present invention. To maintain such a constant concentration, a continuous intravenous delivery device can be used. An example of such a device is the Deltec CADD-PLUS.TM.5400 intravenous injection pump.

The pharmaceutical composition may be in the form of a sterile aqueous or oily suspension for injection for intramuscular and subcutaneous administration.

The suspension can be prepared according to the prior art using those suitable dispersing agents or wetting agents and suspending agents as disclosed above. The sterile injection composition can also be a sterile injection or suspension prepared in a parenterally acceptable non-toxic diluent or solvent. In addition, a sterile fixed oil can be commonly used as a solvent or suspending medium.

The compound of the present invention can be administered in the form of a suppository for rectal administration. Such a pharmaceutical composition can be prepared by mixing the drug with a suitable non-irritating excipient that is a solid at ambient temperature but is liquid in the rectum and will therefore dissolve in the rectum to release the drug. Such materials include cocoa butter, glycerogelatin, hydrogenated vegetable oils, polyethylene glycols of various molecular weights, and mixtures of fatty acid esters of polyethylene glycols.

As is well known to those skilled in the art, the dosage of the drug administered depends on many factors, including, but not limited to, the activity of the particular compound used, the age of the subject, the body weight of the subject, the health of the subject, the behavior of the subject, the diet of the subject, the time of administration, the route of administration, the rate of excretion, the combination of drugs, and the like. In addition, the optimal treatment regimen, such as the route of administration, the daily dose of the compound of the general formula, or the type of pharmaceutically acceptable salts, can be tested in accordance with conventional treatment regimens.

The present invention relates to conjugates comprising a targeting (binding) fragment and a therapeutic fragment, which is a drug, with improved ability to target various cancer cells, infectious pathogens and/or for use in the treatment of autoimmune diseases. The targeting fragment of the antibody is linked to the therapeutic fragment of the compound via an intracellularly cleavable bond, which increases therapeutic efficacy.

For many years, the goal of scientists in the field of specific drug therapy has been to use monoclonal antibodies (MAbs) to specifically deliver toxic agents to human cancers. Conjugates of tumor-associated MAbs with appropriate toxic agents have been developed, but they have had mixed success in cancer treatment and have been of little use in other diseases such as infectious diseases and autoimmune diseases. Toxic agents are the most commonly used chemotherapeutic drugs. There is a further need to develop a more effective conjugate of an antibody with an intracellularly cleavable linker for the treatment of cancer, pathogens and other diseases.

Detailed description of the invention

Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Although any methods and materials similar or equivalent to those described herein can also be used to practice or test the present invention, the preferred methods and materials are described herein. In the description and claims of the present invention, the following terms are used in accordance with the definitions given below.

When a trade name is used in this description, it is intended to include the composition of the commercial product under the said trade name and the medicinal product and the active component of the medicinal product of the commercial product under the said trade name.

The term "ligand" refers to a macromolecular compound capable of recognizing and binding to an antigen or receptor associated with a target cell. The role of the ligand is to present a drug to the target cell population to which the ligand binds, and ligands include, but are not limited to, protein hormones, lectins, growth factors, antibodies, or other molecules capable of binding to cells. In embodiments of the present invention, the ligand is designated as Pc, and the ligand may form a bond with the binding moiety through a heteroatom on the ligand, and is preferably an antibody or an antigen-binding fragment thereof, wherein the antibody is selected from the group consisting of a chimeric antibody, a humanized antibody, a fully human antibody, and a murine antibody; preferably a monoclonal antibody.

The term "drug" refers to a cytotoxic drug or immunomodulator. A cytotoxic drug may have a chemical molecule on a tumor cell that is potent enough to disrupt its normal growth. A cytotoxic drug can kill tumor cells in general at a high enough concentration; however, due to the lack of specificity, a cytotoxic drug may induce apoptosis of normal cells while killing tumor cells, resulting in serious side effects. The term includes toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, radioisotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, and radioactive isotopes of Lu), chemotherapeutic drugs, antibiotics, and nucleolytic enzymes. In such an embodiment of the present invention, the drug is designated as D and may be a BCL2 inhibitor, a BCL-XL inhibitor, or a dual-target BCL2/BCL-XL inhibitor.

The term "linker unit", "linker" or "linker moiety" refers to a chemical structural fragment or linkage that is linked to a ligand at one end and to a drug at the other end, and may also be linked to other linkers, and then linked to a drug.

The linker comprises an extender, a spacer and an amino acid knot and can be synthesized by methods known in the art, such as those described in US 2005-0238649 A1. The linker can be a "cleavable linker" that facilitates the release of drugs in cells. For example, acid-labile linkers (e.g., hydrazones), protease-sensitive linkers (e.g., peptidase-sensitive), photolabile linkers, dimethyl linkers or disulfide-containing linkers can be used (Chari et al., Cancer Research 52: 127-131 (1992); US Patent No. 5,208,020).

The term "ligand-drug conjugate" means that the ligand is linked to a biologically active drug via a linker moiety. In the present specification, the "ligand-drug conjugate" is preferably an antibody-drug conjugate (ADC), which means that a monoclonal antibody or antibody fragment is linked to a biologically active toxic drug via a stable linker moiety.

The three-letter and one-letter codes for amino acids used in this specification are disclosed in J. biol. chem, 243, p3558 (1968).

"Antibody" as described herein is used in the broadest sense and includes various antibody structures, including, but not limited to, a monoclonal antibody, a polyclonal antibody, a murine antibody, a chimeric antibody, a humanized antibody, a multispecific antibody (e.g., a bispecific antibody), and antigen-binding fragments, so long as they exhibit the desired antigen-binding activity and specificity.

Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art, for example, as described in Chapters 5-8 and 15 of Antibodies: A Laboratory Manual, Cold Spring Harbor Press. Likewise, antigen-binding fragments can also be produced by conventional methods. The antibody or antigen-binding fragment described in the present invention is genetically engineered to contain one or more additional human FRs in a non-human CDR. Human germline FR sequences can be obtained from the ImMunoGeneTics (IMGT) website at http://imgt.cines.fr or from the Journal of Immunoglobulins, 2001ISBN012441351, by comparison with the human antibody germline variable region gene database using MOE software.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group which is a linear or branched group containing from 1 to 20 carbon atoms, preferably to alkyl containing from 1 to 12 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) carbon atoms, more preferably to alkyl containing from 1 to 10 carbon atoms, and most preferably to alkyl containing from 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl and their various isomers with side chains, etc. More preferable is a lower alkyl having 1 to 6 carbon atoms, and non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. The alkyl may be substituted or unsubstituted, and when substituted, the substitution with a substituent may be at any available site on the compound, wherein the substituent is preferably one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo.

The term "heteroalkyl" refers to an alkyl containing one or more heteroatoms selected from the group consisting of N, O and S, wherein alkyl is as defined above.

The term "alkylene" refers to a saturated linear or branched aliphatic hydrocarbon group having 2 residues derived from a parent alkane by the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms, which is a linear or branched group containing from 1 to 20 carbon atoms, preferably an alkylene group containing from 1 to 12 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) carbon atoms, and more preferably an alkylene group containing from 1 to 6 carbon atoms. Non-limiting examples of alkylene include, but are not limited to, methylene ( _-CH2- ), 1,1-ethylidene (-CH( _{CH3 )} -), 1,2-ethylidene ( _-CH2CH2 )-, 1,1-propylidene (-CH( _CH2CH3 )-), _1,2 -propylidene ( _-CH2CH ( _CH3 )-), 1,3-propylidene ( _{-CH2CH2CH2- )} , _1,4 - _butylidene ( _{-CH2CH2CH2CH2- )} , _{1,5 - butylidene (-CH2CH2CH2CH2CH2- ) ,} and the like _. The alkyl may be substituted or unsubstituted, and when substituted, the substitution with a substituent may be at any available site on the compound, wherein the substituent is preferably independently and optionally selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

The term "alkoxy" refers to the group -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl or cycloalkyl is as defined above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy and cyclohexyloxy. Alkoxy may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio and heterocycloalkylthio.

The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent. The cycloalkyl ring contains from 3 to 20 carbon atoms, preferably from 3 to 12 carbon atoms, more preferably from 3 to 10 carbon atoms, and most preferably from 3 to 8 carbon atoms. Non-limiting examples of monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like. Polycyclic cycloalkyl includes spirocycloalkyl, fused cycloalkyl, and bridged cycloalkyl.

The term "cycloalkylene" refers to a cycloalkyl group having two moieties derived by the removal of two hydrogen atoms from a parent cycloalkyl. The cycloalkyl group is as defined above.

The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing from 3 to 20 ring atoms, wherein one or more ring atoms are heteroatoms selected from the group consisting of nitrogen, oxygen and S(O) _m (where m is an integer from 0 to 2), excluding the cyclic moiety -O-O-, -O-S- or -SS-, and the remaining ring atoms are carbon atoms. Heterocyclyl preferably contains from 3 to 12 ring atoms, of which from 1 to 4 (e.g. 1, 2, 3 and 4) are heteroatoms; more preferably from 3 to 10 ring atoms; more preferably 3 to 8 (e.g. 3, 4, 5, 6, 7 and 8) ring atoms of which 1 to 3 (e.g. 1, 2 and 3) are heteroatoms; more preferably 3 to 6 ring atoms of which 1 to 3 are heteroatoms; and most preferably 5 to 6 ring atoms of which 1 to 3 are heteroatoms. Non-limiting examples of monocyclic heterocyclyl include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocyclyl includes spiroheterocyclyl, fused heterocyclyl and bridged heterocyclyl.

The term "spiroheterocyclyl" refers to a 5- to 20-membered polycyclic heterocyclyl group in which the monocyclic rings share one atom (called a spiro atom), wherein one or more ring atoms are heteroatoms selected from the group consisting of nitrogen, oxygen, and S(O) _m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon atoms. These rings may contain one or more double bonds, but none of them has a completely conjugated π-electron system. Preferably, spiroheterocyclyl is 6- to 14-membered, and more preferably 7- to 10-membered (e.g., 7-membered, 8-membered, 9-membered, or 10-membered). According to the number of spiroatoms shared between the rings, spiroheterocyclyl may be monospiroheterocyclyl, bispiroheterocyclyl or polyspiroheterocyclyl, preferably monospiroheterocyclyl and bispiroheterocyclyl, and more preferably 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered monospiroheterocyclyl. Non-limiting examples of spiroheterocyclyl include:

The term "fused heterocyclyl" refers to a 5- to 20-membered polycyclic heterocyclyl group in which each ring shares a pair of adjacent atoms with other rings in a system wherein one or more rings may contain one or more double bonds but none has a completely conjugated π-electron system, wherein one or more ring atoms are heteroatoms selected from the group consisting of nitrogen, oxygen, and S(O) _m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon atoms. Preferably, the fused heterocyclyl is 6- to 14-membered, and more preferably 7- to 10-membered (e.g., 7-membered, 8-membered, 9-membered, or 10-membered). According to the number of rings formed, the fused heterocyclyl may be a bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclyl, preferably a bicyclic or tricyclic fused heterocyclyl, and more preferably a 5-membered/5-membered or 5-membered/6-membered bicyclic fused heterocyclyl. Non-limiting examples of the fused heterocyclyl include:

The term "bridged heterocyclyl" refers to a 5- to 14-membered polycyclic heterocyclyl group in which any two rings share two carbon atoms that are not directly attached to each other, which rings may contain one or more double bonds but none of which has a completely conjugated π-electron system, wherein one or more ring atoms are heteroatoms selected from the group consisting of nitrogen, oxygen, and S(O) _m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon atoms. Preferably, the bridged heterocyclyl is 6- to 14-membered, and more preferably 7- to 10-membered (e.g., 7-membered, 8-membered, 9-membered, or 10-membered). According to the number of rings formed, the bridged heterocyclyl may be bicyclic, tricyclic, tetracyclic or polycyclic, preferably bicyclic, tricyclic or tetracyclic, and more preferably bicyclic or tricyclic. Non-limiting examples of bridged heterocyclyl include:

A heterocyclyl ring may be fused to an aryl, heteroaryl, or cycloalkyl ring, wherein the ring attached to the parent structure is a heterocyclyl; non-limiting examples include, but are not limited to:

Heterocyclyl may be optionally substituted or unsubstituted, and when substituted, the substituent preferably represents one or more of the following groups independently selected from the group consisting of: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo.

The term "heterocyclylene" refers to a heterocyclyl group having two moieties derived by the removal of two hydrogen atoms from identical or different ring atoms of a parent heterocyclyl. Heterocyclyl is as defined above.

The term "aryl" refers to a 6- to 14-membered, preferably 6- to 10-membered carbon monocyclic or fused polycyclic (i.e., rings sharing a pair of adjacent carbon atoms) group having a conjugated π-electron system, such as phenyl and naphthyl, preferably phenyl. The aryl ring may be fused to a heteroaryl, heterocyclyl, or cycloalkyl ring, wherein the ring attached to the parent structure is an aryl ring; non-limiting examples include, but are not limited to:

Aryl may be substituted or unsubstituted, and when substituted, the substituent preferably represents one or more of the following groups independently selected from the group consisting of: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio and heterocycloalkylthio.

The term "heteroaryl" refers to a heteroaromatic system containing from 1 to 4 heteroatoms and from 5 to 14 ring atoms, wherein the heteroatoms are selected from the group consisting of oxygen, sulfur and nitrogen. Heteroaryl is preferably 5-10 membered, more preferably 5- or 6-membered, such as furanyl, thienyl, pyridinyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl and tetrazolyl. The heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring attached to the parent structure is heteroaryl; non-limiting examples include, but are not limited to:

Heteroaryl may be optionally substituted or unsubstituted, and when substituted, the substituent preferably represents one or more of the following groups independently selected from the group consisting of: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio and heterocycloalkylthio.

The term "amino protecting group" refers to a group that can be easily removed and is intended to protect the amino group from being altered when the reaction is carried out elsewhere in the molecule. Non-limiting examples of amino protecting groups include 9-fluorenylmethoxycarbonyl, tert-butoxycarbonyl, acetyl, benzyl, allyl, p-methoxybenzyl, etc. These groups may be optionally substituted with 1-3 substituents selected from the group consisting of halogen, alkoxy, and nitro. The amino protecting group is preferably 9-fluorenylmethoxycarbonyl.

The term "alkenyl" refers to an alkyl compound containing carbon-carbon double bonds in the molecule, wherein alkyl is as defined above. Alkenyl may be substituted or unsubstituted, and when substituted, the substituent is preferably one or more groups independently selected from one or more hydrogen, alkyl, alkoxy, halogen, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl substituents.

The term "alkynyl" refers to an alkyl compound containing a carbon-carbon triple bond in the molecule, wherein alkyl is as defined above. Alkynyl may be substituted or unsubstituted, and when substituted, the substituent is preferably one or more groups independently selected from one or more hydrogen, alkyl, alkoxy, halogen, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl substituents.

The term "cycloalkylalkyl" refers to an alkyl group substituted by one or more cycloalkyl groups, preferably one cycloalkyl group, wherein alkyl is as defined above and cycloalkyl is as defined above.

The term "haloalkyl" refers to an alkyl group substituted by one or more halogens, wherein alkyl is as defined above.

The term "deuterated alkyl" refers to an alkyl group substituted with one or more deuterium atoms, wherein alkyl is as defined above.

The term "hydroxy" refers to the -OH group.

The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

The term "amino" refers to -NH ₂ .

The term "nitro" refers to -NO ₂ .

The term "acylamino" refers to -C(O)N(alkyl) or -C(O)N(cycloalkyl), where alkyl and cycloalkyl are as defined above.

The term "carboxylate group" refers to -C(O)O(alkyl) or C(O)O(cycloalkyl), where alkyl and cycloalkyl are as defined above.

In the chemical formula, the abbreviation "Me" refers to methyl.

In a chemical formula, the abbreviation "Ph" refers to phenyl.

The present invention also comprises various deuterated forms of the compound of formula (D). Each available hydrogen atom attached to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize deuterated forms of the compound of general formula (D) according to the relevant literature. Commercially available deuterated starting materials can be used to prepare deuterated forms of the compound of formula (D), or they can be synthesized using conventional methods with deuterated reagents, including, but not limited to, deuterated borane, trideuterated borane in tetrahydrofuran, deuterated lithium aluminum hydride, deuterated iodoethane, deuterated iodomethane, and the like.

The term “optional” or “optionally” means that the event or circumstance described subsequently may, but need not, occur, and that such description includes instances where the event or circumstance does or does not occur. For example, “a heterocyclyl group optionally substituted with alkyl” means that alkyl may, but need not, be present, and that the description includes instances where the heterocyclyl group is or is not substituted with alkyl.

The term "substituted" means that one or more, preferably up to 5, and more preferably from 1 to 3 hydrogen atoms in the group are independently replaced by the appropriate number of substituents. It goes without saying that the substituent is only in its possible chemical position, and those skilled in the art will be able to determine (experimentally or theoretically) possible or impossible substitution without much effort. For example, it may be unstable when an amino or hydroxy having a free hydrogen is bonded to a carbon atom having an unsaturated (e.g. olefinic) bond.

The term "pharmaceutical composition" refers to a mixture containing one or more compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The pharmaceutical composition is intended to facilitate administration to the body, which facilitates absorption of the active ingredient, thereby exerting biological activity.

The term "pharmaceutically acceptable salt" refers to a salt of a ligand-drug conjugate of the present invention or a salt of a compound described herein. Such salts are safe and effective when used in mammals and have the desired biological activity. The ligand-drug conjugate of the present invention contains at least one amino group and, thus, can form a salt with an acid. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodate, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, mesylate, ethanesulfonate, benzenesulfonate and p-toluenesulfonate.

The term "drug content" refers to the average amount of cytotoxic drugs contained in each ligand in the conjugate molecules and can also be referred to as the drug to antibody ratio (DAR). The drug content may be from 0 to 12, preferably from 1 to 10, cytotoxic drugs attached to each ligand (Pc). In embodiments of the present invention, the drug content is denoted as n, and illustratively, can be on average 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, in the range of 0 to 12, preferably 1 to 10, and more preferably 1 to 8, or 2 to 8, or 2 to 7, or 3 to 8, or 3 to 7, or 3 to 6, or 4 to 7, or 4 to 6, or 4 to 5. The drug content per ADC molecule after the coupling reaction can be characterized by conventional methods such as UV/Visible spectroscopy (UV-UV), mass spectrometry, ELISA (enzyme-linked immunosorbent assay), CE-SDS (sodium dodecyl sulfate capillary electrophoresis) and HPLC (high performance liquid chromatography).

The ligand-cytotoxic drug conjugate content may be controlled by the following non-limiting methods, including:

(1) control of the molar ratio of coupling reagent to ligand,

(2) control of reaction time and temperature, and

(3) selection of different reaction reagents.

For common pharmaceutical formulations, see Chinese Pharmacopoeia.

The term "carrier" for a drug according to the present invention refers to a system that can change the way a drug enters the human body and the distribution of the drug in the human body, control the release rate of the drug, and deliver the drug to the target organ. The release and targeting system based on the drug carrier can reduce the degradation and loss of the drug, reduce side effects, and improve bioavailability. For example, polymeric surfactants that can be used as carriers can self-assemble due to their unique amphiphilic structures to form various forms of aggregates, such as micelles, microemulsions, gels, liquid crystals, and vesicles, as preferred examples. The aggregates have the ability to encapsulate drug molecules and have good membrane permeability, and therefore can be used as excellent drug carriers.

The term "excipient" is an addition, other than the main drug, to a pharmaceutical composition. It can also be called an auxiliary substance. For example, binders, fillers, disintegrating agents and lubricants in tablets; a main part in semi-solid ointment and cream preparations; preservatives, antioxidants, flavoring agents, co-solvents, emulsifiers, solubilizers, tonicity adjusting agents, coloring agents and the like in liquid formulations can be called excipients.

The term "diluent", also called filler, is primarily used to increase the weight and volume of a tablet. The addition of a diluent not only ensures a certain volume, but also reduces the dose deviation of the main ingredients, and improves the compressibility of the drug, etc. When a drug in the form of a tablet contains oily components, it is necessary to add an absorbent to absorb the oily components so as to maintain a "dry" state and thus facilitate the preparation of a tablet. Examples include starch, lactose, inorganic calcium salts, microcrystalline cellulose, etc.

Alkaline providing reagents include organic and inorganic bases, wherein organic bases include, but are not limited to, triethylamine, diethylamine, N-methylmorpholine, pyridine, piperidine, N,N-diisopropylethylamine, n-butyl lithium, lithium diisopropylamide, potassium acetate, sodium tert-butoxide, tetrabutylammonium fluoride or potassium tert-butoxide, and inorganic bases include, but are not limited to, sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide and lithium hydroxide, preferably N,N-diisopropylethylamine.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Fig. 1: Inhibitory effect of the compounds of the present invention on RS4;11 human acute lymphoblastic leukemia on murine subcutaneous xenograft tumor.

Fig. 2: Body weight change induced by RS4;11 inhibition of human acute lymphoblastic leukemia on murine subcutaneous xenograft tumor.

DETAILED DESCRIPTION OF THE INVENTION

The following examples further illustrate the present invention, but the present invention is not limited thereto.

The experimental procedures without conditions indicated in the examples of the present invention were generally carried out according to conventional conditions or according to the conditions recommended by the manufacturer of the starting materials or commercial products. Reagents without indication of a specific origin are commercially available conventional reagents.

I. Synthesis of compounds

Examples

The structure of the compound was determined using nuclear magnetic resonance (NMR) spectroscopy and/or mass spectrometry (MS). The NMR shift (δ) is given in units of 10-6 (ppm). NMR spectra were determined using a Bruker AVANCE NEO 500M nuclear magnetic resonance instrument, with deuterated dimethyl sulfoxide (DMSO-d ₆ ), deuterated chloroform (CDCl3) and deuterated methanol (CD ₃ OD) as solvents and tetramethylsilane (TMS) as an internal standard.

Mass spectra were determined using an Agilent 1200/1290 DAD-6110/6120 Quadrupole MS liquid chromatography-mass spectrometry system (manufacturer: Agilent; MS model: 6110/6120 Quadrupole MS), Waters ACQuity UPLC-QD/SQD (manufacturer: Waters, MS model: Waters ACQuity Qda Detector/Waters SQ Detector) and THERMO Ultimate 3000-Q Exactive (manufacturer: THERMO, MS model: THERMO Q Exactive).

High performance liquid chromatography (HPLC) analysis was performed using Agilent HPLC 1200DAD, Agilent HPLC 1200VWD and Waters HPLC e2695-2489 high performance liquid chromatographs.

Chiral HPLC analysis was performed using an Agilent 1260 DAD high-performance liquid chromatograph.

Preparative high-performance liquid chromatography was performed using Waters 2545-2767, Waters 2767-SQ Detecor2, Shimadzu LC-20AP and Gilson GX-281 preparative chromatographs.

Chiral preparative HPLC was performed using a Shimadzu LC-20AP preparative chromatograph.

The rapid preparation instrument used is CombiFlash (flash chromatography) - Combiflash Rf200 (TELEDYNE ISCO).

Huanghai HSGF254 or Qingdao GF254 silica gel plates with specifications from 0.15 mm to 0.2 mm were used for thin layer chromatography (TLC) and 0.4 mm to 0.5 mm for TLC separation and purification.

Yantai Huanghai 200-300 mesh silica gel was commonly used as a carrier in column chromatography.

The mean kinase inhibition and IC ₅₀ value were determined using a NovoStar microplate reader (BMG, Germany).

The known starting materials described herein can be synthesized using or according to methods known in the art or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc., Chembee Chemicals and others.

In the examples, all reactions can be carried out under argon or nitrogen atmosphere unless otherwise stated.

Argon atmosphere or nitrogen atmosphere means that the reaction flask is connected to a cylinder containing about 1 liter of argon or nitrogen.

Hydrogen atmosphere means that the reaction flask is connected to a cylinder containing about 1 liter of hydrogen.

Parr 3916EKX hydrogenator, Qinglan QL-500 hydrogenator or HC2-SS hydrogenator were used in pressure hydrogenation reactions.

Hydrogenation reactions typically involve 3 cycles of vacuum and hydrogen purge.

A CEM Discover-S 908860 microwave reactor was used for microwave-assisted reactions.

In the examples, solution refers to an aqueous solution unless otherwise stated.

In the examples, the reaction temperature refers to room temperature, i.e. from 20 to 30°C, unless otherwise stated.

The progress of the reaction in the examples was monitored by thin layer chromatography (TLC). The developing solvent for the reactions, the elution system for column chromatography purification, and the developing solvent system for thin layer chromatography were: A: dichloromethane/methanol system, and B: n-hexane/ethyl acetate system. The volume ratio of the solvents was adjusted according to the polarity of the compound or by adding a small amount of basic or acidic reagents such as triethylamine and acetic acid.

Example 2-1

4-(4-((R)-(4'-chloro-[1,1'-biphenyl]-2-yl)(hydroxy)methyl)piperidin-1-yl)-N-((4-(((R)-1-(phenylthio)-3-((R)-pyrrolidin-2-yl)propan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide 1

Stage 1

Tert-butyl(R,Z)-2-(2-(((benzyloxy)carbonyl)amino)-3-methoxy-3-oxoprop-1-en-1-yl)pyrrolidin-1-carboxylate 1c

Methyl 2-(((benzyloxy)carbonyl)amino)-2-(dimethoxyphosphoryl)acetate 1b (1 g, 3.02 mmol, Accela ChemBio) was dissolved in dichloromethane (10 mL) and the solution was cooled to 0 °C under an ice bath. 1,8-diazabicyclo[5.4.0]undec-7-ene (356 mg, 2.34 mmol, Accela ChemBio) was added dropwise and the mixture was stirred at 0 °C for 20 min, followed by the addition of a solution of tert-butyl (R)-2-formylpyrrolidine-1-carboxylate 1a (500 mg, 2.51 mmol, PharmaBlock) in dichloromethane (5 mL). After dropwise addition, the ice bath was removed and the mixture was warmed to room temperature and reacted for 48 h. The reaction solution was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using eluent system B to give the title product 1c (830 g, yield: 81.8%).

MS m/z (mass/charge) (ESI (electrospray ionization)): 305.0 [M+H-100].

Stage 2

tert-Butyl(R)-2-((R)-2-amino-3-methoxy-3-oxopropyl)pyrrolidine-1-carboxylate 1d

1c (520 mg, 1.28 mmol) was dissolved in isopropanol (10 ml) and palladium on carbon (100 mg, 10% content, dry basis) was added. The mixture was purged with hydrogen 3 times and stirred at room temperature for 16 h. The reaction solution was filtered through celite and the filtrate was concentrated to give the title product 1d (300 mg, yield: 85.7%), which was directly used in the next step without purification.

MS m/z(ESI): 273.1 [M+1].

Stage 3

tert-Butyl(R)-2-((R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methoxy-3-oxopropyl)pyrrolidine-1-carboxylate 1e

The crude product 1d (300 mg, 1.10 mmol) was dissolved in 1,4-dioxane (4 mL), and water (1 mL), sodium bicarbonate (278 mg, 3.31 mmol), and fluorenyl methoxycarbonyl chloride (285 mg, 1.10 mmol, Accela ChemBio) were added. The mixture was stirred at room temperature for 1.5 h. Water (10 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, and filtered to remove the drying agent, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using eluent system B to give the title product 1e (396 mg, yield: 72.7%).

MS m/z (ESI): 395.1 [M+H-100].

Stage 4

tert-Butyl(R)-2-((R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-hydroxypropyl)-(pyrrolidine-1-carboxylate 1f

1e (200 mg, 0.40 mmol) was dissolved in tetrahydrofuran (10 ml) and ethanol (10 ml) was added followed by the successive addition of sodium borohydride (92 mg, 2.43 mmol) and lithium chloride (112 mg, 2.64 mmol). The mixture was stirred at 28 °C for 2.5 h. The reaction solution was cooled in an ice bath and saturated ammonium chloride solution (10 ml) was added dropwise to quench the reaction. Water (10 ml) was added and the mixture was extracted with ethyl acetate (10 ml 3 times). The organic phases were combined, washed with saturated sodium chloride solution (10 ml), dried over anhydrous sodium sulfate and filtered to remove the drying agent, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography with eluent system B to give the title product 1f (144 mg, yield: 76.3%).

MS m/z (ESI): 367.1 [M+H-100].

Stage 5

Tert-butyl(R)-2-((R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(phenylthio)propyl)pyrrolidine-1-carboxylate 1g

1f (144 mg, 0.31 mmol) was dissolved in toluene (5 mL), and diphenyl disulfide (225 mg, 1.03 mmol, adamas) and tributylphosphine (208 mg, 1.03 mmol) were added. The reaction system was purged with nitrogen 3 times and heated to 80 °C and stirred for 12 h. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with eluent system B to give the title product 1g (168 mg, 97.4%).

MS m/z (ESI): 459.1 [M+H-100].

Stage 6

Tert-butyl(R)-2-((R)-2-amino-3-(phenylthio)propyl)pyrrolidine-1-carboxylate 1h

1g (168 mg, 0.30 mmol) was dissolved in dichloromethane (2 ml) and diethylamine (2 ml) was added. The mixture was stirred at room temperature for 3 h. The reaction solution was concentrated under reduced pressure to remove the organic solvent and concentrated with toluene (5 ml) under reduced pressure to give the crude title product 1h (101 mg), which was directly used in the next step without purification.

MS m/z(ESI): 337.1 [M+1].

Stage 7

tert-Butyl(R)-2-((R)-3-(phenylthio)-2-((4-sulfamoyl-2-((trifluoromethyl)sulfonyl)phenyl)amino)propyl)pyrrolidine-1-carboxylate 1j

Crude 1h (101 mg, 0.30 mmol) was dissolved in N,N-dimethylformamide (5 mL), and 4-fluoro-3-((trifluoromethyl)sulfonyl)benzenesulfonamide 1i (102 mg, 0.33 mmol, Bide) and N,N-diisopropylethylamine (388 mg, 3.00 mmol) were added. The reaction system was purged with nitrogen 3 times and heated to 50 °C and stirred for 8 h. Water (10 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (10 mL 3 times). The organic phases were combined, washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, and filtered to remove the drying agent, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography on silica gel with eluent system B to give the title product 1j (129 mg, 68.9%).

MS m/z (ESI): 524.0 [M+H-100].

Stage 8

tert-Butyl (R)-2-((R)-2-((4-(N-(4-(4-((R)-(4'-chloro-[1,1'-biphenyl]-2-yl)(hydroxy)methyl)piperidin-1-yl)benzyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-3-(phenylthio)propyl)pyrrolidine-1-carboxylate 1l

1j (22 mg, 0.035 mmol) was dissolved in dichloromethane (3 ml) and 1k (16 mg, 0.038 mmol, synthesized by the method presented for intermediate 40 on page 79 of the description in patent "CN 103153954 B"), 4-dimethylaminopyridine (8.6 mg, 0.070 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (20.3 mg, 0.106 mmol) were added. After the addition, the mixture was stirred at room temperature for 4 hours under nitrogen atmosphere, 1k (11 mg, 0.026 mmol) was added and stirred at room temperature overnight after the addition. The reaction solution was washed successively with water (5 ml) and saturated sodium chloride solution (5 ml), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by thin-layer chromatography with a developing solvent system A to give the title product 1 L (28 mg, yield: 77.2%).

MS m/z (ESI): 1027.0 [M+1].

Stage 9

4-(4-((R)-(4'-chloro-[1,1'-biphenyl]-2-yl)(hydroxy)methyl)piperidin-1-yl)-N-((4-(((R)-1-(phenylthio)-3-((R)-pyrrolidin-2-yl)propan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide 1

1 L (14 mg, 0.014 mmol) was dissolved in dichloromethane (0.2 mL), and a solution of 4 M hydrogen chloride in dioxane (0.5 mL) was added. The mixture was stirred at room temperature for 30 min. The reaction solution was concentrated under reduced pressure to remove the organic solvent. The residue was suspended with diethyl ether and filtered. The resulting solid was purified by high performance liquid chromatography (separation conditions: column: Sharpsil-T Prep C18; mobile phase: ammonium bicarbonate, water, acetonitrile), and the appropriate fraction was collected and concentrated under reduced pressure to give the title product 1 (5 mg, yield: 39.6%).

MS m/z (ESI): 927.2 [M+1].

^1H NMR (500 MHz, CD ₃ OD) δ 8.25 (s, 1H), 8.06 (d, 1H), 7.82 (d, 2H), 7.62 (d, 1H), 7.48-7.31 (m, 8H), 7.26 (t, 2H), 7.20 (t, 2H), 6.82 (d, 2H), 6.74 (d, 1H), 4.44 (d, 1H), 4.00-3.95 (m, 1H), 3.78 (d, 1H), 3.61 (d, 1H), 3.52-3.45 (m, 2H), 3.26-3.17 (m, 2H), 2.68 (t, 2H), 2.53 (t, 2H), 2.27-1.92 (m, 6H), 1.81-1.59 (m, 4H), 1.27-1.21 (m, 1H), 1.18-1.12 (m, 1H), 1.02-0.90 (m, 2H).

Example 2-2

4-(4-(((R)-(4'-chloro-[1,1'-biphenyl]-2-yl)(hydroxy)methyl)piperidin-1-yl)-N-((4-(((R)-1-((R)-1-(2-hydroxyethyl)pyrrolidin-2-yl)-3-(phenylthio)propan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide 2

1 (13 mg, 0.014 mmol) was placed in a reaction flask, and acetonitrile (5 mL) was added, followed by 2-bromoethanol (26 mg, 0.210 mmol, adamas) and triethylamine (11 mg, 0.112 mmol). The reaction system was purged with nitrogen 3 times and heated to 70 °C and stirred for 7 h. The reaction solution was concentrated under reduced pressure. The resulting residue was purified by high performance liquid chromatography (separation conditions: column: Sharpsil-T Prep C18; mobile phase: ammonium bicarbonate, water, acetonitrile), and the appropriate fraction was collected and concentrated under reduced pressure to give the title product 2 (3 mg, yield: 22.0%).

MS m/z(ESI): 971.2 [M+1].

^1H NMR (500 MHz, CD ₃ OD) δ 8.26 (s, 1H), 8.03 (d, 1H), 7.80 (d, 2H), 7.62 (d, 1H), 7.46-7.36 (m, 5H), 7.35-7.27 (m, 5H), 7.24 (d, 1H), 7.18 (d, 1H), 6.81 (d, 2H), 6.63 (d, 1H), 4.42 (d, 1H), 3.91-3.77 (m, 4H), 3.63 (d, 2H), 3.25-3.16 (m, 2H), 2.67 (t, 2H), 2.54 (t, 2H), 2.36-2.29 (m, 2H), 2.23-2.13 (m, 3H), 2.09-1.97 (m, 4H), 1.81-1.71 (m, 2H), 1.65-1.59 (m, 2H), 1.27-1.21 (m, 1H), 1.17-1.11 (m, 1H), 1.02-0.90 (m, 2H).

Example 2-3

4-(4-((R)-(4'-chloro-[1,1'-biphenyl]-2-yl)(methoxy)methyl)piperidin-1-yl)-N-((4-(((R)-1-(phenylthio)-3-((R)-pyrrolidin-2-yl)propan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide 3

Stage 1

Ethyl (R)-4-(4-((4'-chloro-[1,1'-biphenyl]-2-yl)(methoxy)methyl)piperidin-1-yl)benzoate 3b

Ethyl (R)-4-(4-((4'-chloro-[1,1'-benphenyl]-2-yl)(hydroxy)methyl)piperidin-1-yl)benzoate 3a (60 mg, 0.133 mmol, synthesized based on the method described for intermediate 11 on page 63 of the description in patent "CN 103153954 B") was dissolved in 2 ml of a mixed solvent of N,N-dimethylformamide and tetrahydrofuran (V:V=1:1). The solution was purged with nitrogen three times and iodomethane (190 mg, 1.34 mmol) and 60% sodium hydride (11 mg, 0.275 mmol) were added successively. The mixture was stirred at room temperature for 3 h. The reaction solution was cooled in an ice bath and saturated ammonium chloride solution (5 ml) was added to quench the reaction. Water (5 ml) was added and the mixture was extracted with ethyl acetate (10 ml 3 times). The organic phases were combined, washed with saturated sodium chloride solution (10 ml), dried over anhydrous sodium sulfate and filtered to remove the drying agent, and the filtrate was concentrated under reduced pressure.

The resulting residue was purified by silica gel column chromatography using eluent system B to give the title product 3b (57 mg, yield: 92.1%).

MS m/z (ESI): 464.1 [M+1].

Stage 2

(R)-4-(4-((4'-chloro-[1,1'-biphenyl]-2-yl)(methoxy)methyl)piperidin-1-yl)benzoic acid 3c

3b (57 mg, 0.122 mmol) was dissolved in 3 ml of a mixed solvent of tetrahydrofuran, methanol and water (V:V:V=4:1:1), and lithium hydroxide monohydrate (26 mg, 0.620 mmol) was added thereto. The mixture was heated to 50 °C and stirred for 16 h. The reaction solution was concentrated under reduced pressure, adjusted to pH 2-3 with 1 M hydrochloric acid and filtered. The filter cake was dissolved in 10 ml of dichloromethane, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to give the title product 3c (53 mg, yield: 98.9%).

MS m/z (ESI): 436.1 [M+1].

Stage 3

tert-Butyl (R)-2-((R)-2-((4-(N-(4-(4-((R)-(4'-chloro-[1,1'-biphenyl]-2-yl)(methoxy)methyl)piperidin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-3-(phenylthio)propyl)pyrrolidine-1-carboxylate 3d

1j (32 mg, 0.051 mmol) was dissolved in dichloromethane (5 ml), and 3c (24 mg, 0.055 mmol) was added, followed by 4-dimethylaminopyridine (13 mg, 0.106 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (30 mg, 0.156 mmol). After the addition, the mixture was stirred at room temperature for 4 h under nitrogen atmosphere, 3c (22 mg, 0.051 mmol) was added and stirred at room temperature overnight after the addition. The reaction solution was washed successively with water (10 ml) and saturated sodium chloride solution (10 ml), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with evolving solvent system A to give the title product 3d (53 mg, yield: 99.6%).

MS m/z (ESI): 1041.1 [M+1].

Stage 4

4-(4-((R)-(4'-chloro-[1,1'-biphenyl]-2-yl)(methoxy)methyl)piperidin-1-yl)-N-((4-(((R)-1-(phenylthio)-3-((R)-pyrrolidin-2-yl)propan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide 3

3d (30 mg, 0.028 mmol) was dissolved in dichloromethane (0.4 ml), and 4 M hydrogen chloride in dioxane (1.2 ml) was added. The mixture was stirred at room temperature for 40 min. The reaction solution was concentrated under reduced pressure to remove the organic solvent. The residue was suspended with diethyl ether and filtered. The resulting solid was purified by high performance liquid chromatography (separation conditions: column: Sharpsil-T Prep C18; mobile phase: ammonium bicarbonate, water, acetonitrile), and the appropriate fraction was collected and concentrated under reduced pressure to give the title product 3 (10 mg, yield: 36.9%).

MS m/z (ESI): 941.1 [M+1].

¹ H NMR (500 MHz, CD ₃ OD) δ 8.26 (s, 1H), 8.04 (dd, 1H), 7.81 (d, 2H), 7.50 (d, 1H), 7.47-7.41 (m, 3H), 7.40-7.32 (m, 3H), 7.30-7.23 (m, 4H), 7.22-7.16 (m, 2H), 6.81 (d, 2H), 6.72 (d, 1H), 4.06 (d, 1H), 3.99-3.93 (m, 1H), 3.77 (d, 1H), 3.66 (d, 1H), 3.55-3.49 (m, 1H), 3.23 (d, 2H), 3.17 (s, 3H), 2.61 (t, 2H), 2.51 (t, 2H), 2.25-1.93 (m, 6H), 1.72-1.58 (m, 4H), 1.27-1.23 (m, 1H), 1.20-1.11 (m, 2H), 0.91 (t, 1H).

Example 2-4

4-(4-(((R)-(4'-chloro-[1,1'-biphenyl]-2-yl)(methoxy)methyl)piperidin-1-yl)-N-((4-(((R)-1-((R)-1-(2-hydroxyethyl)pyrrolidin-2-yl)-3-(phenylthio)propan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide 4

3 (15 mg, 0.015 mmol) was placed in a reaction flask, and acetonitrile (5 mL) was added, followed by 2-bromoethanol (29 mg, 0.232 mmol) and triethylamine (16, 0.158 mmol). The reaction system was purged with nitrogen 3 times and heated to 70 °C and stirred for 16 h. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: Sharpsil-T Prep C18; mobile phase: ammonium bicarbonate, water, acetonitrile), and the appropriate fraction was collected and concentrated under reduced pressure to give the title product 4 (2.5 mg, yield: 16.5%).

MS m/z (ESI): 985.1 [M+1].

¹ H NMR (500 MHz, CD ₃ OD) δ 8.27 (s, 1H), 8.03 (dd, 1H), 7.81 (d, 2H), 7.51 (d, 1H), 7.47-7.39 (m, 5H), 7.35 (t, 1H), 7.32-7.21 (m, 5H), 7.18 (d, 1H), 6.82 (d, 2H), 6.63 (d, 1H), 4.06 (d, 1H), 3.90-3.75 (m, 4H), 3.69-3.63 (m, 1H), 3.27-3.20 (m, 2H), 3.17 (s, 3H), 2.62 (t, 2H), 2.52 (t, 2H), 2.36-2.28 (m, 2H), 2.23-2.13 (m, 3H), 2.09-1.94 (m, 4H), 1.78-1.58 (m, 4H), 1.28-1.24 (m, 1H), 1.21-1.13 (m, 2H), 0.91 (t, 1H).

Example 2-5

4-(4-(((R)-(4'-chloro-[1,1'-biphenyl]-2-yl)(hydroxy)methyl)piperidin-1-yl)-N-((4-(((R)-1-((R)-1-(3-hydroxypropyl)pyrrolidin-2-yl)-3-(phenylthio)propan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide 5

1 (10 mg, 0.011 mmol) was placed in a reaction flask, and acetonitrile (3 mL) was added, followed by 3-bromopropanol (15 mg, 0.108 mmol, Adamas) and triethylamine (11 mg, 0.108 mmol). The reaction system was purged with nitrogen 3 times and heated to 70 °C and stirred for 16 h. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: Sharpsil-T Prep C18; mobile phase: ammonium bicarbonate, water, acetonitrile), and the appropriate fraction was collected and concentrated under reduced pressure to give the title product 5 (2.5 mg, yield: 23.5%).

MS m/z (ESI): 985.1 [M+1].

¹ H NMR (500 MHz, CD ₃ OD) δ 8.26 (s, 1H), 8.03 (d, 1H), 7.81 (d, 2H), 7.61 (d, 1H), 7.46-7.39 (m, 5H), 7.35-7.26 (m, 5H), 7.22 (t, 1H), 7.18 (d, 1H), 6.81 (d, 2H), 6.62 (d, 1H), 4.43 (d, 1H), 3.89-3.84 (m, 1H), 3.79 (d, 1H), 3.67 (t, 2H), 3.63 (d, 2H), 3.25-3.15 (m, 2H), 2.67 (t, 2H), 2.53 (t, 2H), 2.32-2.26 (m, 2H), 2.19 (t, 2H), 2.15-2.11 (m, 1H), 2.08-1.95 (m, 4H), 1.92-1.84 (m, 2H), 1.79-1.67 (m, 2H), 1.65-1.58 (m, 2H), 1.26-1.22 (m, 1H), 1.16-1.12 (m, 1H), 1.02-0.96 (m, 1H), 0.91 (t, 1H).

Example 2-6

4-(4-((S)-hydroxy(4'-(trifluoromethyl)-[1,1'-biphenyl]-2-yl)methyl)piperidin-1-yl)-N-((4-(((R)-4-((2-hydroxyethyl)(methyl)amino)-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide 6

Stage 1

tert-Butyl 4-(hydroxy(4'-(trifluoromethyl)-[1,1'-diphenyl]-2-yl)methyl)piperidine-1-carboxylate 6b

2-bromo-4'-(trifluoromethyl)-1,1'-biphenyl 6a (3.3 g, 10.95 mmol, synthesized according to the method described on page 53 of Example 53 in the patent "US 2004/171833, 2004, A1") was placed in a flask, and tetrahydrofuran (50 ml) was added. After cooling the mixture to -78 °C under a nitrogen atmosphere, n-butyl lithium (1.40 g, 21.92 mmol) was added dropwise into the reaction flask at an internal temperature below -65 °C, and after the addition, the mixture was stirred at -78 °C for 40 min, followed by the addition of a solution of tert-butylformyl piperidine-1-carboxylate (4.67 g, 21.92 mmol) in tetrahydrofuran (20 ml). After the addition, the resulting mixture was heated to 0°C and reacted for three hours, and then a saturated aqueous ammonium chloride solution was added dropwise to quench the reaction. The reaction solution was concentrated under reduced pressure and extracted with ethyl acetate (40 ml 3 times). The organic phase was combined, washed successively with water (40 ml) and saturated brine (30 ml), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using eluent system B to give the title product 6b (5.5 g, yield: more than 100%).

MS m/z (ESI): 434.1 [M-1].

Stage 2

Piperidin-4-yl(4'-(trifluoromethyl)-[1,1'-biphenyl]-2-yl)methanol 6c

6b (5.5 g, 12.63 mmol) was placed in a reaction flask, and a solution of 4 M hydrogen chloride in dioxane (20 ml) was added under an ice bath. After the addition, the mixture was naturally warmed to room temperature and stirred for 2 h. The reaction solution was concentrated under reduced pressure, and toluene (10 ml) was added to the residue. The resulting mixture was concentrated under reduced pressure and dried with an oil pump to give the crude title product 6c (4.23 g, yield: 100%), which was directly used in the next step without purification.

MS m/z (ESI): 336.0 [M+1].

Stage 3

Ethyl (R)-4-(4-(hydroxy(4'-(trifluoromethyl)-[1,1'-biphenyl]-2-yl)methyl)piperidin-1-yl)benzoate 6d

Ethyl (S)-4-(4-(hydroxy(4'-(trifluoromethyl)-[1,1'-biphenyl]-2-yl)methyl)piperidin-1-yl)benzoate 6e

Crude 6c (270 mg, 0.805 mmol) was placed in a reaction flask and dimethyl sulfoxide (3 mL) was added followed by sequential addition of ethyl p-fluorobenzoate (270 mg, 1.61 mmol, Accela ChemBio) and N,N-diisopropylethylamine (1.04 g, 8.05 mmol). After addition, the mixture was heated to 120 °C and reacted for 24 h under nitrogen atmosphere. To the reaction solution, 5 mL of water was added and the mixture was extracted with ethyl acetate (4 mL 5 times). The organic phases were combined, washed successively with water (4 mL) and saturated brine (4 mL), dried over anhydrous sodium sulfate, filtered and concentrated. The obtained residue was purified by column chromatography on silica gel with eluent system B to give a mixture (360 mg), which was submitted to chiral preparative HPLC (preparation conditions: column: CHIRALPAK IG, 0.50 cm i.d. *25 cm L, 0.1 μm; mobile phase: MeOH=100%; detection wavelength: UV 214 nm; temperature: 38°C) to give two fractions, Peak 1 with a lower retention time (which was 6d (73 mg)) and Peak 2 with a higher retention time (which was 6e (72 mg)).

MS m/z (ESI): 484.1 [M+1].

Peak 1: chiral HPLC analysis: retention time: 4.008 min, chiral purity: 100% (column: CHIRALPAK IG 150*4.6 mm, 5 μm (with guard column); mobile phase: n-hexane/ethanol=70/30 (v/v)).

Peak 2: chiral HPLC analysis: retention time: 6.289 min, chiral purity: 100% (CHIRALPAK IG 150*4.6 mm, 5 μm column (with guard column); mobile phase: n-hexane/ethanol=70/30 (v/v)).

Stage 4

(R)-4-(4-(hydroxy(4'-(trifluoromethyl)-[1,1'-biphenyl]-2-yl)methyl)piperidin-1-yl)benzoic acid 6f

6e (70 mg, 0.145 mmol) was placed in a flask, and tetrahydrofuran (2 ml), water (0.5 ml), and methanol (0.5 ml) were added, followed by lithium hydroxide monohydrate (30.3 mg, 0.723 mmol). The mixture was heated to 50 °C and reacted for 16 h under nitrogen atmosphere. The reaction solution was concentrated under reduced pressure, and water (5 ml) was added to the residue. The resulting mixture was cooled in an ice bath, adjusted to pH 4 with 1 N hydrochloric acid, and filtered. The filter cake was washed with n-hexane (5 ml) and oil pump dried to give the title product 6f (63 mg, yield: 95.54%).

MS m/z (ESI): 456.0 [M+1].

Stage 5

N-((4-(((R)-4-((2-((tert-butyldimethylsilyl)oxy)ethyl)(methyl)amino)-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)-4-(4-((S)-hydroxy(4'-(trifluoromethyl)-[1, 1'-biphenyl]-2-yl)methyl)piperidin-1-yl)benzamide 6h

6g (30 mg, 0.040 mmol, synthesized with reference to the method presented for intermediate 67 on page 90 of the description in patent "CN 103153954 B") was dissolved in dichloromethane (3 ml) and 6f (25 mg, 0.055 mmol) was added, followed by the sequential addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (17.5 mg, 0.091 mmol) and 4-dimethylaminopyridine (11.2 mg, 0.091 mmol). After the addition, the mixture was stirred at room temperature for 4 hours under nitrogen atmosphere, 6f (16.6 mg, 0.037 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (10.5 mg, 0.055 mmol) and 4-dimethylaminopyridine (6.7 mg, 0.055 mmol) were added thereto, and stirred at room temperature overnight after the addition. The reaction solution was washed with water (5 mL) and saturated brine (5 mL) successively, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography (elution system A) to give the title product 6h (40 mg, yield: 79.9%).

MS m/z (ESI): 1093.4 [M+1].

Stage 6

4-(4-((S)-hydroxy(4'-(trifluoromethyl)-[1,1'-biphenyl]-2-yl)methyl)piperidin-1-yl)-N-((4-(((R)-4-((2-hydroxyethyl)(methyl)amino)-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide 6

To tetrahydrofuran (2 mL) was added 6h. (40 mg, 0.036 mmol) and tetrabutylammonium fluoride (16.4 mg, 0.073 mmol). The mixture was stirred at room temperature for 4 h. The reaction solution was concentrated under reduced pressure, and the residue was dissolved in dichloromethane (8 mL) and washed with saturated ammonium chloride solution (10 mL 2 times). The organic phase was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate (8 mL), washed successively with saturated ammonium chloride solution (10 mL 6 times), water (10 mL) and saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The resulting solid was purified by high performance liquid chromatography (separation conditions: column: Sharpsil-T Prep C18; mobile phase: ammonium bicarbonate, water, acetonitrile), and the appropriate fraction was collected and concentrated under reduced pressure to give the title product 6 (17 mg, yield: 47.5%).

MS m/z (ESI): 979.2 [M+1].

^1H NMR (500 MHz, CD ₃ OD) δ 8.28 (d, 1H), 8.09-8.03 (m, 1H), 7.80 (d, 2H), 7.74 (d, 2H), 7.66 (d, 1H), 7.55 (d, 2H), 7.48 (t, 1H), 7.42-7.34 (m, 2H), 7.29-7.16 (m, 3H), 6.81 (dd, 2H), 4.41 (d, 1H), 4.02-3.97 (m, 1H), 3.84-3.75 (m, 2H), 3.64 (d, 1H), 3.37 (s, 2H), 3.30-3.18 (m, 3H), 3.12-3.06 (m, 2H), 2.75 (s, 2H), 2.72-2.63 (m, 1H), 2.59-2.50 (m, 1H), 2.29-2.19 (m, 2H), 2.08-2.02 (m, 2H), 1.82-1.72 (m, 1H), 1.66-1.58 (m, 1H), 1.39-1.31 (m, 4H), 1.27-1.12 (m, 2H), 1.03-0.89 (m, 2H).

Example 2-7

4-(4-((R)-hydroxy(4'-(trifluoromethyl)-[1,1'-biphenyl]-2-yl)methyl)piperidin-1-yl)-N-((4-(((3R)-1-((2-hydroxyethyl)(methyl)amino)-4-(phenylthio)pentan-3-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)sulfonyl)benzamide 7

Stage 1

Methyl (R)-3-(((benzyloxy)carbonyl)amino)-4-oxobutanoate 7b

Oxalyl chloride (772 mg, 6.08 mmol) was dissolved in dichloromethane (5 ml) and the solution was purged with nitrogen three times, cooled to -78 °C, and then a solution of dimethyl sulfoxide (950 mg, 12.16 mmol) in dichloromethane (5 ml) was added dropwise. After the dropwise addition, the mixture was stirred at -78 °C for 30 min and a solution of methyl (R)-3-(((benzyloxy)carbonyl)amino)-4-hydroxybutyrate 7a (650 mg, 2.43 mmol, synthesized by the method described for intermediate 4 on page 59 of the description of patent "CN 103153954 B") in dichloromethane (5 ml) was added dropwise. After the dropwise addition, the mixture was stirred at -78°C for 1 hour, and N,N-diisopropylethylamine (3.14 g, 24.29 mmol) was added dropwise. After the dropwise addition, the resulting mixture was heated to -60°C and stirred for 30 min, and then warmed to 0°C and stirred for 30 min. 1 M cold hydrochloric acid (20 ml) was added at 0°C to quench the reaction, and the mixture was subjected to liquid separation. The organic phase was washed with a phosphate buffer solution (30 ml) of pH 7 and a saturated sodium chloride solution (30 ml), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography with eluent system B to give the title product 7b (540 mg, yield: 83.7%).

MS m/z (ESI): 266.2 [M+1].

Stage 2

Methyl (3R)-3-(((benzyloxy)carbonyl)amino)-4-hydroxypentanoate 7c

7b (490 mg, 1.95 mmol) was dissolved in diethyl ether (15 mL), and the solution was purged with nitrogen three times and cooled to -78 °C, after which a solution of 1 M methylmagnesium bromide in tetrahydrofuran (2 mL, 2.03 mmol) was added dropwise. After the dropwise addition, the mixture was naturally warmed to 0 °C and stirred for 3 h. At 0 °C, a saturated ammonium chloride solution (20 mL) was added to quench the reaction. Water (10 mL) was added and the mixture was extracted with ethyl acetate (10 mL 3 times). The organic phases were combined, washed with a saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate and filtered to remove the drying agent, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography with eluent system B to give the title product 7c (188 mg, yield: 36.2%).

MS m/z (ESI): 282.1 [M+1].

Stage 3

Methyl (3R-3-(((benzyloxy)carbonyl)amino)-4-((methylsulfonyl)oxy)pentanoate 7d

7c (220 mg, 0.782 mmol) was dissolved in dichloromethane (8 ml), and pyridine (93 mg, 1.176 mmol) and 4-dimethylaminopyridine (9.5 mg, 0.078 mmol) were added. The mixture was purged with nitrogen three times and cooled to 0 °C, and methanesulfonyl anhydride (164 mg, 0.941 mmol) was slowly added. After the addition, the mixture was warmed to room temperature and stirred for 1.5 h. Water (20 ml) was added to the reaction solution, and the mixture was extracted with dichloromethane (8 ml 3 times). The organic phases were combined, washed with saturated sodium chloride solution (10 ml), dried over anhydrous sodium sulfate and filtered to remove the drying agent, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using eluent system B to give the title product 7d (216 mg, yield: 76.8%).

MS m/z (ESI): 360.0 [M+1].

Stage 4

Methyl (3R)-3-(((benzyloxy)carbonyl)amino)-4-(phenylthio)pentanoate 7e

7d (216 mg, 0.601 mmol) was dissolved in toluene (8 ml) and sodium thiophenolate (397 mg, 3.004 mmol) was added. The mixture was purged with nitrogen three times and heated to 105 °C and stirred for 16 h. After cooling the reaction solution to room temperature, water (20 ml) was added and the mixture was extracted with ethyl acetate (10 ml 3 times). The organic phases were combined, washed with saturated sodium chloride solution (30 ml), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with eluent system B to give the title product 7e (120 mg, yield: 53.5%).

MS m/z (ESI): 374.1 [M+1].

Stage 5

Methyl (3R)-4-(phenylthio)-3-((4-sulfamoyl-2-((trifluoromethyl)sulfonyl)phenyl)amino)pentanoate 7f

7e (112 mg, 0.300 mmol) was dissolved in tetrahydrofuran (1.5 mL), and trifluoroacetic acid (5 mL) was added. The mixture was heated to 72 °C and stirred for 1.5 h. The reaction solution was concentrated under reduced pressure with dichloromethane (20 mL) and dried in vacuo for 2 h. The residue was dissolved in N,N-dimethylformamide (4 mL), and 1i (93 mg, 0.302 mmol) and N,N-diisopropylethylamine (388 mg, 3.00 mmol) were added. The reaction system was purged with nitrogen 3 times and heated to 50 °C and stirred for 24 h. Water (20 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (10 mL 3 times). The organic phases were combined, washed with saturated sodium chloride solution (20 ml), dried over anhydrous sodium sulfate and filtered to remove the drying agent, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with eluent system B to give the title product 7f (119 mg, 75.3%).

MS m/z (ESI): 524.9 [M-1].

Stage 6

(3R)-4-(phenylthio)-3-((4-sulfamoyl-2-((trifluoromethyl)sulfonyl)phenyl)amino)pentanoic acid 7g

7f (119 mg, 0.226 mmol) was dissolved in 5 ml of a mixed solvent of tetrahydrofuran, methanol and water (V:V:V=3:1:1), and lithium hydroxide monohydrate (29 mg, 0.691 mmol) was added thereto. The mixture was stirred at room temperature for 3 h. The reaction solution was concentrated under reduced pressure, adjusted to pH 3-4 with 1 M hydrochloric acid, and extracted with dichloromethane (8 ml 3 times). The organic phases were combined, washed with saturated sodium chloride solution (20 ml), dried over anhydrous sodium sulfate and filtered to remove the drying agent, and the filtrate was concentrated under reduced pressure to obtain the title product 7 g (115 mg, yield: 99.3%).

MS m/z (ESI): 511.0 [M-1].

Stage 7

(3R)-N-(2-((tert-butyldimethylsilyl)oxy)ethyl)-N-methyl-4-(phenylthio)-3-((4-sulfamoyl-2-((trifluoromethyl)sulfonyl)phenyl)amino)pentanamide 7h

7g (115 mg, 0.224 mmol) was dissolved in tetrahydrofuran (3 ml), and 2-((tert-butyldimethylsilyl)oxy)-N-methylethyl-1-amine (43 mg, 0.227 mmol, Accela ChemBio), 3-(diethoxy-o-acyloxy)-1,2,3-benzotriazin-4-one (134 mg, 0.448 mmol), and N,N-diisopropylethylamine (58 mg, 0.448 mmol) were added successively. After the addition, the mixture was stirred at room temperature for 3 hours under nitrogen atmosphere. The reaction solution was concentrated, and the residue was dissolved in ethyl acetate (10 ml), washed successively with a saturated aqueous solution of ammonium chloride (20 ml), a saturated solution of sodium bicarbonate (20 ml) and a saturated solution of sodium chloride (20 ml). The organic phase was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using eluent system B to give the title product 7h (107 mg, yield: 69.7%).

MS m/z (ESI): 684.0 [M+1].

Stage 8

4-(((3R)-1-((2-((tert-butyldimethylsilyl)oxy)ethyl)(methyl)amino)-4-(phenylthio)pentan-3-yl)amino)-3-((trifluoromethyl)sulfonyl)benzenesulfonamide 7i

7 h (107 mg, 0.156 mmol) was placed in a reaction flask, and 1 M borane-tetrahydrofuran complex solution (5 ml) was added thereto. After the addition, the mixture was heated to 60 °C and stirred for 24 h. The reaction system was cooled to 0 °C, and 4 ml of methanol was added dropwise to quench the reaction. The mixture was concentrated under reduced pressure, and a solution of 7 M ammonia in methanol (10 ml) was added to the residue. The mixture was stirred at room temperature for 48 h after sealing, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using eluent system B to give the title product 7i (10 mg, yield: 9.5%).

MS m/z (ESI): 670.1 [M+1].

Stage 9

(R)-4-(4-(hydroxy(4'-(trifluoromethyl)-[1,1'-biphenyl]-2-yl)methyl)piperidin-1-yl)benzoic acid 7j

6d (70 mg, 0.145 mmol) was placed in a flask, and tetrahydrofuran (2 ml), water (0.5 ml), and methanol (0.5 ml) were added, followed by lithium hydroxide monohydrate (30 mg, 0.72 mmol). The mixture was heated to 50 °C and reacted for 16 h under nitrogen atmosphere. The reaction solution was concentrated under reduced pressure, and water (5 ml) was added to the residue. The resulting mixture was cooled in an ice bath, adjusted to pH 4 with 1 M hydrochloric acid, and filtered. The filter cake was washed with n-hexane (5 ml) and oil pump dried to give the title product 7j (63 mg, yield: 95.5%).

MS m/z (ESI): 456.0 [M+1].

Stage 10

N-((4-(((3R)-1-((2-((tert-butyldimethylsilyl)oxy)ethyl)(methyl)amino)-4-(phenylthio)pentan-3-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)-4-(4-((R)-hydroxy(4'-(trifluoromethyl)-[1,1'-biphenyl]-2-yl)methyl)piperidin-1-yl)benzamide 7k

7i (16 mg, 0.024 mmol) was dissolved in dichloromethane (4 ml), and 7j (13 mg, 0.028 mmol) was added, followed by 4-dimethylaminopyridine (6 mg, 0.049 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (14 mg, 0.073 mmol). After the addition, the mixture was purged with nitrogen three times, stirred at room temperature for 4 h, 7j (13 mg, 0.028 mmol) was added and stirred at room temperature for 16 h after the addition. The reaction solution was washed successively with water (8 ml) and saturated sodium chloride solution (8 ml), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with evolving solvent system A to give the title product 7k (14 mg, yield: 52.9%).

MS m/z (ESI): 1107.2 [M+1].

Stage 11

4-(4-((R)-hydroxy(4'-(trifluoromethyl)-[1,1'-biphenyl]-2-yl)methyl)piperidin-1-yl)-N-((4-(((3R)-1-((2-hydroxyethyl)(methyl)amino)-4-(phenylthio)pentan-3-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)sulfonyl)benzamide 7

7k (14 mg, 0.013 mmol) was dissolved in tetrahydrofuran (3 ml), and tetrabutylammonium fluoride (6 mg, 0.027 mmol) was added. The mixture was stirred at room temperature for 1 h. The reaction solution was concentrated under reduced pressure, and the residue was dissolved in dichloromethane (5 ml) and washed with saturated ammonium chloride solution (5 ml 2 times). The organic phase was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate (8 ml), washed successively with saturated ammonium chloride solution (5 ml 6 times), water (10 ml), and saturated sodium chloride solution (10 ml), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting solid was purified by high performance liquid chromatography (separation conditions: column: Sharpsil-T Prep C18; mobile phase: ammonium bicarbonate, water, acetonitrile), and the appropriate fraction was collected and concentrated under reduced pressure to give the title product 7 (4.5 mg, yield: 35.8%).

MS m/z (ESI): 993.2 [M+1].

^1H NMR (500 MHz, CD ₃ OD) δ 8.33 (s, 1H), 8.02 (d, 1H), 7.76 (dd, 4H), 7.65 (d, 1H), 7.56 (dd, 4H), 7.48 (t, 1H), 7.44-7.33 (m, 4H), 7.21 (d, 1H), 6.81 (d, 2H), 6.60 (d, 1H), 4.41 (d, 1H), 3.97-3.92 (m, 1H), 3.84-3.72 (m, 3H), 3.69-3.56 (m, 3H), 2.76-2.62 (m, 4H), 2.55 (t, 2H), 2.32-2.17 (m, 3H), 2.10-1.87 (m, 4H), 1.82-1.72 (m, 2H), 1.64-1.58 (m, 2H), 1.27-1.22 (m, 1H), 1.21-1.12 (m, 2H), 1.03-0.96 (m, 1H), 0.92 (t, 1H).

Example 2-8

4-(4-((R)-(4'-chloro-[1,1'-biphenyl]-2-yl)(hydroxy)methyl)piperidin-1-yl)-N-((4-(((R)-1-(phenylthio)-3-((S)-pyrrolidin-2-yl)propan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide 8

Stage 1

Tert-butyl(S,)-2-(2-(((benzyloxy)carbonyl)amino)-3-methoxy-3-oxoprop-1-en-1-yl)pyrrolidin-1-carboxylate 8b

1b (2.49 g, 7.53 mmol, Accela ChemBio) was dissolved in tetrahydrofuran (20 mL) and the solution was cooled to 0 °C in an ice bath, followed by dropwise addition of N,N-lithium diisopropylamide (661 mg, 6.17 mmol). The mixture was stirred at 0 °C for 1 h. The reaction system was cooled to -78 °C and a solution of tert-butyl (S)-2-formylpyrrolidine-1-carboxylate 8a (1.00 g, 5.02 mmol, PharmaBlock) in tetrahydrofuran (20 mL) was added dropwise. After dropwise addition, the dry ice-acetone bath was removed, and the mixture was warmed to room temperature and reacted for 12 h. Saturated ammonium chloride solution (20 ml) was added to quench the reaction, and the mixture was extracted with ethyl acetate (20 ml 3 times). The organic phase was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with eluent system B to give the title product 8b (1.90 g, yield: 94.1%).

MS m/z (ESI): 305.0 [M+H-100].

Stage 2

Tert-butyl(2S)-2-(2-amino-3-methoxy-3-oxopropyl)pyrrolidine-1-carboxylate 8c

8b (1.90 g, 4.7 mmol) was dissolved in isopropanol (50 mL) and palladium on carbon (380 mg, 10% content, dry basis) was added. The mixture was purged with hydrogen 3 times and stirred at room temperature for 8 h. The reaction solution was filtered through celite and the filter cake was washed with ethyl acetate (20 mL) and methanol (20 mL). The filtrate was concentrated to give the crude title product 8c (1.28 g), which was used directly in the next step without purification.

Stage 3

Tert-butyl(S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methoxy-3-oxopropyl)pyrrolidine-1-carboxylate 8d

Tert-butyl(S)-2-((R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methoxy-3-oxopropyl)pyrrolidine-1-carboxylate 8e

Crude 8c (1.28 g, 4.7 mmol) was dissolved in 1,4-dioxane (40 mL), and water (10 mL), sodium bicarbonate (1.17 g, 14.1 mmol), and fluorenyl methoxycarbonyl chloride (1.21 g, 4.7 mmol) were added. The mixture was stirred at room temperature for 3 h. Water (30 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (20 mL x 3). The organic fraction was dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography with eluent system A to afford the title product 8d (1.03 g, 44.2%, peak 1) and 8e (513 mg, 22.1%, peak 2).

MS m/z (ESI): 495.2 [M+1].

Peak 1: UPLC analysis: retention time: 2.77 min (column: ACQUITY UPLC BENC18 50*2.1 mm, 1.7 μm; mobile phase: acetonitrile/water/formic acid=10/90/0.1 (v/v/v) to 95/5/0.1 (v/v/v) gradient elution).

Peak 2: UPLC analysis: retention time: 2.69 min (column: ACQUITY UPLC BENC18 50*2.1 mm, 1.7 μm; mobile phase: acetonitrile/water/formic acid=50/50/0.1 (v/v/v) to 95/5/0.1 (v/v/v) gradient elution).

Stage 4

tert-Butyl(S)-2-((R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-hydroxypropyl)-(pyrrolidine-1-carboxylate 8f

8e (169 mg, 0.34 mmol) was dissolved in tetrahydrofuran (8 mL), and ethanol (8 mL), sodium borohydride (77 mg, 2.05 mmol), and lithium chloride (94 mg, 2.22 mmol) were added. The mixture was stirred at 28 °C for 2.5 h, and saturated ammonium chloride (5 mL) was added to quench the reaction. The solvent was concentrated to dryness, and the residue was dissolved in ethyl acetate (10 mL), and washed successively with water (10 mL) and saturated sodium chloride (10 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography with eluent system B to give the title product 8f (125 mg, 78.4%).

MS m/z (ESI): 467.2 [M+1].

Stage 5

Tert-butyl(S)-2-((R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(phenylthio)propyl)pyrrolidine-1-carboxylate 8g

8f (120 mg, 0.26 mmol) was dissolved in toluene (5 mL) and diphenyl disulfide (168 mg, 0.77 mmol) and tributylphosphine (156 mg, 0.77 mmol) were added. The mixture was heated to 80 °C and reacted for 12 h under nitrogen atmosphere. The solvent was concentrated to dryness. The resulting residue was purified by silica gel column chromatography with eluent system B to give the title product 8g (135 mg, 93.9%).

MS m/z (ESI): 559.2 [M+1].

Stage 6

Tert-butyl(S)-2-((R)-2-amino-3-(phenylthio)propyl)pyrrolidine-1-carboxylate 8h

8g (135 mg, 0.24 mmol) was dissolved in dichloromethane (4 ml) and diethylamine (4 ml) was added. The mixture was stirred at room temperature for 4 h. The reaction solution was concentrated under reduced pressure to remove the organic solvent to give the title product 8h (81.6 mg), which was directly used in the next step without purification.

Stage 7

tert-Butyl(S)-2-((R)-4-(phenylthio)-3-((4-sulfamoyl-2-((trifluoromethyl)sulfonyl)phenyl)amino)propyl)pyrrolidine-1-carboxylate 8i

Crude 8h (81.6 mg, 0.24 mmol) was dissolved in N,N-dimethylformamide (5 mL), and 1i (82 mg, 0.27 mmol) and N,N-diisopropylethylamine (314 mg, 2.4 mmol) were added. The reaction system was heated to 50 °C and stirred for 12 h under nitrogen atmosphere. Water (10 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (5 mL 3 times). The organic phase was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with eluent system B to give the title product 8i (105 mg, 69.3%).

MS m/z (ESI): 622.1 [M-1].

Stage 8

tert-Butyl (S)-2-((R)-2-((4-(N-(4-(4-((R)-(4'-chloro-[1,1'-biphenyl]-2-yl)(hydroxy)methyl)piperidin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-3-(phenylthio)propyl)pyrrolidine-1-carboxylate 8j

8i (30 mg, 0.048 mmol) was dissolved in dichloromethane (3 mL) and 1k (24 mg, 0.058 mmol) was added followed by sequential addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (19 mg, 0.096 mmol) and 4-dimethylaminopyridine (12 mg, 0.096 mmol). After addition, the mixture was stirred at room temperature for 4 h under nitrogen atmosphere, 1k (12 mg, 0.038 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (19 mg, 0.096 mmol) and 4-dimethylaminopyridine (12 mg, 0.096 mmol) were added and stirred at room temperature overnight after addition. The reaction solution was washed successively with water (5 ml) and saturated brine (5 ml), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with a developing solvent system A to give the title product 8j (30 mg, yield: 60.7%).

MS m/z (ESI): 1027.2 [M+1].

Stage 9

4-(4-((R)-(4'-chloro-[1,1'-biphenyl]-2-yl)(hydroxy)methyl)piperidin-1-yl)-N-((4-(((R)-1-(phenylthio)-3-((S)-pyrrolidin-2-yl)propan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide 8

8j (30 mg, 0.029 mmol) was dissolved in dichloromethane (2 ml), and 4 M hydrogen chloride in 1,4-dioxane (2 ml) was added. The resulting mixture was stirred at room temperature for 20 min. The reaction solution was concentrated under reduced pressure to remove the organic solvent. The residue was dissolved in dichloromethane and concentrated under reduced pressure to dryness, which was repeated three times. The resulting solid was purified by high performance liquid chromatography (separation conditions: column: Sharpsil-T Prep C18; mobile phase: ammonium bicarbonate, water, acetonitrile), and the appropriate fraction was collected and concentrated under reduced pressure to give the title product 8 (25 mg, yield: 92.3%).

MS m/z (ESI): 927.1 [M+1].

¹ H NMR (500 MHz, CD ₃ OD) δ 8.24 (d, 1H), 8.05-8.01 (m, 1H), 7.82 (d, 2H), 7.60 (d, 1H), 7.45-7.39 (m, 3H), 7.37 (d, 2H), 7.34-7.28 (m, 3H), 7.23 (t, 2H), 7.20-7.14 (m, 2H), 6.80 (d, 2H), 6.72 (d, 1H), 4.42 (d, 1H), 3.97 (br, 1H), 3.84-3.74 (m, 1H), 3.27-3.21 (m, 2H), 3.19-3.07 (m, 3H), 3.06-2.97 (m, 1H), 2.69-2.61 (m, 1H), 2.55-2.48 (m, 1H), 2.16-2.00 (m, 5H), 1.98-1.89 (m, 2H), 1.88-1.80 (m, 1H), 1.78-1.69 (m, 1H), 1.63-1.57 (m, 2H), 1.54-1.49 (m, 1H), 1.26-1.20 (m, 1H), 1.15-1.04 (m, 1H), 1.01-0.94 (m, 1H).

Example 2-9

4-(4-(((R)-(4'-chloro-[1,1'-biphenyl]-2-yl)(hydroxy)methyl)piperidin-1-yl)-N-((4-(((R)-1-((S)-1-(2-hydroxyethyl)pyrrolidin-2-yl)-3-(phenylthio)propan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide 9

8 (15 mg, 0.016 mmol) was dissolved in acetonitrile (2 ml), and triethylamine (16 mg, 0.16 mmol) and bromoethanol (20 mg, 0.16 mmol) were added successively. The reaction system was heated to 70 °C and reacted for 12 h under nitrogen atmosphere. The obtained solid was purified by high performance liquid chromatography (separation conditions: column: Sharpsil-T Prep C18; mobile phase: ammonium bicarbonate, water, acetonitrile), and the appropriate fraction was collected and concentrated under reduced pressure to give the title product 9 (10 mg, yield: 63.4%).

MS m/z (ESI): 971.2 [M+1].

¹ H NMR (500 MHz, CD ₃ OD) δ 8.25 (d, 1H), 8.06 (dd, 1H), 7.80 (d, 2H), 7.61 (dd, 1H), 7.49-7.35 (m, 5H), 7.35-7.28 (m, 3H), 7.24 (dd, 2H), 7.20-7.14 (m, 2H), 6.79 (t, 3H), 4.42 (d, 1H), 4.02 (t, 1H), 3.84-3.70 (m, 3H), 3.61 (d, 2H), 3.28-3.14 (m, 2H), 3.06 (br, 1H), 2.96 (br, 1H), 2.71-2.59 (m, 1H), 2.56-2.45 (m, 1H), 2.45-2.35 (m, 1H), 2.32-2.22 (m, 1H), 2.10-1.97 (m, 4H), 1.96-1.86 (m, 1H), 1.85-1.69 (m, 2H), 1.60 (t, 2H), 1.26-1.18 (m, 1H), 1.06-1.08 (m, 1H), 1.01-0.92 (m, 1H), 0.92-1.86 (m, 1H).

Example 2-10

4-(4-((R)-hydroxy(4'-(trifluoromethyl)-[1,1'-biphenyl]-2-yl)methyl)piperidin-1-yl)-N-((4-(((R)-4-((2-hydroxyethyl)(methyl)amino)-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide 10

Stage 1

N-((4-(((R)-4-((2-((tert-butyldimethylsilyl)oxy)ethyl)(methyl)amino)-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)-4-(4-((R)-hydroxy(4'-(trifluoromethyl)-[1,1'-biphenyl]-2-yl)methyl)piperidin-1-yl)benzamide 10a

6g (30 mg, 0.040 mmol) was dissolved in dichloromethane (3 ml) and 7j (25 mg, 0.055 mmol) was added, followed by the sequential addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (17.5 mg, 0.091 mmol) and 4-dimethylaminopyridine (11.2 mg, 0.091 mmol). After the addition, the mixture was stirred at room temperature for 4 hours under nitrogen atmosphere, 7j (16.6 mg, 0.037 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (10.5 mg, 0.055 mmol) and 4-dimethylaminopyridine (6.7 mg, 0.055 mmol) were added and stirred at room temperature overnight after the addition. The reaction solution was washed successively with water (5 mL) and saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with evolving solvent system A to give the title product 10a (40 mg, yield: 79.9%).

MS m/z (ESI): 1093.4 [M+1].

Stage 2

4-(4-((R)-hydroxy(4'-(trifluoromethyl)-[1,1'-biphenyl]-2-yl)methyl)piperidin-1-yl)-N-((4-(((R)-4-((2-hydroxyethyl)(methyl)amino)-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide 10

To tetrahydrofuran (2 mL) were added 10a (40 mg, 0.036 mmol) and tetrabutylammonium fluoride (16 mg, 0.073 mmol). The mixture was stirred at room temperature for 4 h. The reaction solution was concentrated under reduced pressure, and the residue was dissolved in dichloromethane (8 mL) and washed with saturated ammonium chloride solution (10 mL 2 times). The organic phase was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate (8 mL), washed successively with saturated ammonium chloride solution (10 mL 6 times), water (10 mL), and saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by high performance liquid chromatography (separation conditions: column: Sharpsil-T Prep C18; mobile phase: ammonium bicarbonate, water, acetonitrile), and the appropriate fraction was collected and concentrated under reduced pressure to give the title product 10 (17 mg, yield: 47.5%).

MS m/z (ESI): 979.2 [M+1].

^1H NMR (500 MHz, CD ₃ OD) δ 8.26 (d, 1H), 8.04 (dd, 1H), 7.79 (d, 2H), 7.72 (d, 2H), 7.64 (d, 1H), 7.53 (d, 2H), 7.46 (t, 1H), 7.40-7.31 (m, 3H), 7.29-7.12 (m, 4H), 6.86-6.71 (m, 3H), 4.39 (d, 1H), 3.98 (br, 1H), 3.84-3.71 (m, 3H), 3.62 (d, 1H), 3.26-3.16 (m, 3H), 3.14-2.96 (m, 2H), 2.80-2.60 (m, 4H), 2.56-2.49 (m, 1H), 2.19 (t, 1H), 2.11-1.93 (m, 3H), 1.76 (d, 1H), 1.27-1.18 (m, 1H), 1.17-1.10 (m, 1H), 1.03-0.93 (m, 1H), 0.94-0.83 (m, 1H).

Example 2-11

4-(4-(((R)-(4'-chloro-[1,1'-biphenyl]-2-yl)(hydroxy)methyl)piperidin-1-yl)-N-((4-(((R)-1-((S)-1-(3-hydroxypropyl)pyrrolidin-2-yl)-3-(phenylthio)propan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide 11

8 (20 mg, 0.021 mmol) was dissolved in acetonitrile (2 ml), and triethylamine (22 mg, 0.22 mmol) and bromoethanol (30 mg, 0.22 mmol) were added successively. The reaction system was heated to 70 °C and reacted for 12 h under nitrogen atmosphere. The obtained solid was purified by high performance liquid chromatography (separation conditions: column: Sharpsil-T Prep C18; mobile phase: ammonium bicarbonate, water, acetonitrile), and the appropriate fraction was collected and concentrated under reduced pressure to give the title product 11 (13 mg, yield: 61.1%).

MS m/z (ESI): 985.1 [M+1].

^1H NMR (500 MHz, CD ₃ OD) δ 8.28 (d, 1H), 8.09 (dd, 1H), 7.86-7.78 (m, 2H), 7.63 (dd, 1H), 7.48-7.38 (m, 5H), 7.37-7.31 (m, 3H), 7.27 (dd, 2H), 7.23-7.17 (m, 2H), 6.81 (t, 3H), 4.44 (d, 1H), 4.08-4.00 (m, 1H), 3.81 (d, 1H), 3.72-3.55 (m, 4H), 3.25 (dd, 2H), 3.05 (s, 1H), 2.94 (s, 1H), 2.73-2.63 (m, 1H), 2.59-2.50 (m, 1H), 2.48-2.39 (m, 1H), 2.29 (s, 1H), 2.21 (t, 1H), 2.10-2.01 (m, 4H), 1.97-1.70 (m, 5H), 1.62 (s, 1H), 1.31-1.20 (m, 3H), 1.15 (d, 1H), 1.04-0.95 (m, 1H), 0.93 (d, 1H).

Example 2-12

4-(4-((R)-hydroxy(4'-(trifluoromethyl)-[1,1'-biphenyl]-2-yl)methyl)piperidin-1-yl)-N-((4-(((R)-1-(phenylthio)-3-((S)-pyrrolidin-2-yl)propan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide 12

Stage 1

tert-Butyl(S)-2-((R)-2-((4-(N-(4-(4-((R)-hydroxy(4'-(trifluoromethyl)-[1,1'-biphenyl]-2-yl)methyl)piperidin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-3-(phenylthio)propyl)pyrrolidine-1-carboxylate 12a

8i (80 mg, 0.13 mmol) was dissolved in dichloromethane (3 ml) and 7j (70 mg, 0.15 mmol) was added followed by sequential addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (49 mg, 0.26 mmol) and 4-dimethylaminopyridine (32 mg, 0.26 mmol). After addition, the mixture was stirred at room temperature for 4 h under nitrogen atmosphere, 7j (47 mg, 0.10 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (49 mg, 0.26 mmol) and 4-dimethylaminopyridine (32 mg, 0.26 mmol) were added and stirred at room temperature overnight after addition. The reaction solution was washed successively with water (5 ml) and saturated brine (5 ml), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with a developing solvent system A to give the title product 12a (80 mg, yield: 58.8%).

MS m/z (ESI): 1059.0 [M-1].

Stage 2

4-(4-((R)-hydroxy(4'-(trifluoromethyl)-[1,1'-biphenyl]-2-yl)methyl)piperidin-1-yl)-N-((4-(((R)-1-(phenylthio)-3-((S)-pyrrolidin-2-yl)propan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide 12

12a (50 mg, 0.047 mmol) was dissolved in dichloromethane (2 ml) and 4 M hydrogen chloride solution in 1,4-dioxane (2 ml) was added. The resulting mixture was stirred at room temperature for 20 min. The reaction solution was concentrated under reduced pressure to remove the organic solvent. The residue was dissolved in dichloromethane and concentrated under reduced pressure to dryness, which was repeated three times. The obtained residue was purified by high performance liquid chromatography (separation conditions: column: Sharpsil-T Prep C18; mobile phase: ammonium bicarbonate, water, acetonitrile), and the appropriate fraction was collected and concentrated under reduced pressure to give the title product 12 (6 mg, yield: 66.2%).

MS m/z (ESI): 961.2 [M+1].

¹ H NMR (500 MHz, CD ₃ OD) δ 8.23 (s, 1H), 8.03 (d, 1H), 7.80 (d, 2H), 7.72 (d, 2H), 7.64 (d, 1H), 7.53 (d, 2H), 7.47 (t, 1H), 7.35 (t, 3H), 7.27-7.14 (m, 4H), 6.79 (d, 2H), 6.73 (d, 1H), 4.39 (d, 1H), 3.98 (s, 1H), 3.81-3.73 (m, 1H), 3.63-3.58 (m, 1H), 3.56-3.50 (m, 1H), 3.21-3.13 (m, 2H), 2.64 (t, 1H), 2.50 (t, 1H), 2.33-2.25 (m, 1H), 2.25-2.20 (m, 1H), 2.11-1.91 (m, 6H), 1.80-1.65 (m, 2H), 1.64-1.54 (m, 2H), 1.25-1.17 (m, 2H), 1.16-1.10 (m, 1H), 1.00-0.93 (m, 1H).

Example 2-13

4-(4-((R)-hydroxy(4'-(trifluoromethyl)-[1,1'-biphenyl]-2-yl)methyl)piperidin-1-yl)-N-((4-(((R)-1-((S)-1-(2-hydroxyethyl)pyrrolidin-2-yl)-3-(phenylthio)propan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide 13

12 (30 mg, 0.031 mmol) was dissolved in acetonitrile (4 mL), and triethylamine (32 mg, 0.31 mmol) and bromoethanol (39 mg, 0.31 mmol) were added successively. The reaction system was heated to 70 °C and reacted for 12 h under nitrogen atmosphere. The reaction solution was concentrated under reduced pressure. The residue was purified by high performance liquid chromatography (separation conditions: column: Sharpsil-T Prep C18; mobile phase: ammonium bicarbonate, water, acetonitrile), and the appropriate fraction was collected and concentrated under reduced pressure to give the title product 13 (25 mg, yield: 56.9%).

MS m/z (ESI): 1005.1 [M+1].

¹ H NMR (500 MHz, CD ₃ OD) δ 8.27 (d, 1H), 8.08 (dd, 1H), 7.85-7.79 (m, 2H), 7.74 (d, 2H), 7.68-7.63 (m, 1H), 7.55 (d, 2H), 7.50-7.46 (m, 1H), 7.42-7.35 (m, 3H), 7.29-7.18 (m, 4H), 6.87-6.69 (m, 3H), 4.41 (d, 1H), 4.03 (br, 1H), 3.83-3.73 (m, 3H), 3.67-3.56 (m, 2H), 3.25-3.18(m, 2H), 3.14-2.86 (m, 2H), 2.72-2.59 (m, 1H), 2.58-2.47 (m, 1H), 2.46-2.36 (m, 1H), 2.33-2.23 (m, 1H), 2.10-1.97 (m, 4H), 1.96-1.87 (m, 1H), 1.84-1.72 (m, 2H), 1.66-1.55 (m, 1H), 1.28-1.19 (m, 1H), 1.16 (d, 1H), 1.03-0.95 (m, 1H), 0.95-0.89 (m, 1H).

Example 2-14

4-(4-((R)-hydroxy(4'-(trifluoromethyl)-[1,1'-biphenyl]-2-yl)methyl)piperidin-1-yl)-N-((4-(((2R)-4-((2-hydroxyethyl)(methyl)amino)-3-methyl-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide 14

Example 2-15

4-(4-((R)-hydroxy(4'-(trifluoromethyl)-[1,1'-biphenyl]-2-yl)methyl)piperidin-1-yl)-N-((4-(((R)-5-((2-hydroxyethyl)(methyl)amino)-1-(phenylthio)pentan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide 15

Example 2-16

4-(4-((R)-hydroxy(4'-(trifluoromethyl)-[1,1'-biphenyl]-2-yl)methyl)piperidin-1-yl)-N-((4-(((R)-1-((R)-1-(2-hydroxyethyl)pyrrolidin-2-yl)-3-(phenylthio)propan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide 16

Example 2-17

4-(4-(((R)-(4'-chloro-[1,1'-biphenyl]-2-yl)(hydroxy)methyl)piperidin-1-yl)-N-((4-(((R)-1-((R)-1-methylpyrrolidin-2-yl)-3-(phenylthio)propan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide 17

Example 2-18

4-(4-(((R)-(4'-chloro-[1,1'-biphenyl]-2-yl)(hydroxy)methyl)piperidin-1-yl)-N-((4-(((R)-1-((S)-1-methylpyrrolidin-2-yl)-3-(phenylthio)propan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide 18

Example 2-19

4-(4-((R)-hydroxy(4'-(trifluoromethyl)-[1,1'-biphenyl]-2-yl)methyl)piperidin-1-yl)-N-((4-(((R)-1-((S)-1-methylpyrrolidin-2-yl)-3-(phenylthio)propan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide 19

Example 2-20

4-(4-(((R)-(4'-chloro-[1,1'-biphenyl]-2-yl)(hydroxy)methyl)piperidin-1-yl)-N-((4-(((R)-1-((S)-1-(2-hydroxyethyl)-2-methylpyrrolidin-2-yl)-3-(phenylthio)propan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide 20

Example 2-21

4-(4-((R)-hydroxy(4'-(trifluoromethyl)-[1,1'-biphenyl]-2-yl)methyl)piperidin-1-yl)-N-((4-(((R)-1-((S)-1-(2-hydroxyethyl)-2-methylpyrrolidin-2-yl)-3-(phenylthio)propan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide 21

Example 2-22

4-(4-(((R)-(4'-chloro-[1,1'-biphenyl]-2-yl)(hydroxy)methyl)piperidin-1-yl)-N-((4-(((2R)-1-(1-(2-hydroxyethyl)azetidin-2-yl)-3-(phenylthio)propan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide 22

Example 2-23

Example 2-24 AZD4320

The compound was prepared according to the method described in Example 3 (positive control compound AZD4320) on pages 160-161 of patent WO 2017251.

Example 3-1 LD-1

4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)enzyl (R)-2-((R)-2-((4-(N-(4-((R)-(4'-chloro-[1,1'-biphenyl]-2-yl)(hydroxy)methyl)piperidin-1-yl)benzoyl)sulfamoyl)-2-((trifluoromethyl)sulfonyl)phenyl)amino)-3-(phenylthio)propyl)pyrrolidine-1-carboxylate LD-1

To 0.5 mL of N,N-dimethylformamide was added 1 (3 mg, 0.003 mmol), and 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate LD-1a (3.1 mg, 0.004 mmol, Haoyuan) was added, followed by 0.1 mL of pyridine. The mixture was purged with argon three times, and 1-hydroxybenzotriazole (2 mg, 0.014 mmol) and N,N-diisopropylethylamine (2 mg, 0.015 mmol) were added. The resulting mixture was stirred at room temperature for 16 h. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18; mobile phase: ammonium acetate, water, acetonitrile), and the appropriate fractions were collected and concentrated under reduced pressure to give the title product LD-1 (2 mg, yield: 40.5%).

MS m/z (ESI): 1525.0 [M+1].

¹ H NMR (500 MHz, CD ₃ OD) δ 8.17 (s, 1H), 7.92-7.80 (m, 3H), 7.64-7.51 (m, 3H), 7.46-7.09 (m, 12H), 6.84-6.74 (m, 3H), 6.48-6.40 (m, 2H), 5.35 (t, 4H), 4.41 (d, 1H), 4.15 (d, 1H), 4.00-3.71 (m, 6H), 3.66-3.54 (m, 2H), 3.44 (t, 2H), 3.22-3.05 (m, 6H), 2.77 (t, 1H), 2.72-2.62(m, 2H), 2.53 (t, 2H), 2.23 (t, 2H), 2.18 (t, 4H), 2.11-2.06 (m, 2H), 1.96-1.82 (m, 4H), 1.80-1.71 (m, 2H), 1.65-1.51 (m, 8H), 1.17-1.09 (m, 2H), 1.00-0.89 (m, 9H).

Biological assessment

Test Example 1: Analysis of the binding activity of compounds according to this description for BCL-xL

The in vitro BCL-xLin binding activity was tested as follows.

Reagents used in this experiment: His-tagged BCL-xL protein (R&D, Cat#894-BX-050); biotin-tagged BIM protein (R&D, Cat#3526/1); binding buffer (cisbio, Cat#62DLBDDF); assay buffer (cisbio, Cat#62DB1FDG); MAb cryptate to 6His-Eu (cisbio, Cat#61HI2KLA); and affinity streptomycin coupled to XL665 (cisbio, Cat#611SAXLA).

The in vitro binding assay described below can determine the binding activity of test compounds to BCL-xL.

The test compounds were dissolved in dimethyl sulfoxide and diluted to the gradient concentrations required for the experiment, and the compounds at different concentrations formulated in dimethyl sulfoxide were diluted 100-fold with binding buffer. BCL-xL was diluted to the appropriate concentration with binding buffer and added to the white 384-well plate at 4 μl/well. Then, the diluted test compound was added to the white 384-well plate at 2 μl/well. The plate was incubated at 25°C for 1 hour after uniform mixing of the mixture. Biotin-labeled BIM protein was diluted to the appropriate concentration with binding buffer and added to the white 384-well plate at 4 μl/well. The plate was incubated at 25°C for 2 hours after uniform mixing of the mixture. The MAb cryptate to 6His-Eu and affinity streptomycin coupled to XL665 were each diluted to the appropriate concentration with assay buffer and added to a white 384-well plate at 5 μl/well. The plate was incubated at 25°C for 2-4 hours after uniformly mixing the mixture. The signal values were read using HTRF software in a microplate reader (BMG Labtech, model: PHERAstar FS).

The test results are shown in Table 1.

Test Example 2: Analysis of the binding activity of compounds according to this description for BCL-2

In vitro BCL-2 binding activity was tested as follows. The reagents used in this experiment were: His-tagged BCL-2 protein (R&D systems, Cat. No. 827-BC-050); biotin-tagged BIM protein (R&D, Cat. No. 3526/1); binding buffer (cisbio, Cat. No. 62DLBDDF); assay buffer (cisbio, Cat. No. 62DB1FDG); MAb cryptate to 6His-Eu (cisbio, Cat. No. 61HI2KLA); and affinity streptomycin coupled to XL665 (cisbio, Cat. No. 611SAXLA).

The in vitro binding assay described below can determine the binding activity of test compounds to BCL-2.

The test compounds were dissolved in dimethyl sulfoxide and diluted to the gradient concentrations required for the experiment, and the compounds at different concentrations formulated in dimethyl sulfoxide were diluted 100-fold with binding buffer. BCL-2 was diluted to the appropriate concentration with binding buffer and added to the white 384-well plate at 4 μl/well. Then, the diluted test compound was added to the white 384-well plate at 2 μl/well. The plate was incubated at 25°C for 1 hour after uniformly mixing the mixture. Biotin-labeled BIM protein was diluted to the appropriate concentration with binding buffer and added to the white 384-well plate at 4 μl/well. The plate was incubated at 25°C for 2 hours after uniformly mixing the mixture. The MAb cryptate to 6His-Eu and affinity streptomycin coupled to XL665 were each diluted to the appropriate concentration with assay buffer and added to a white 384-well plate at 5 μl/well. The plate was incubated at 25°C for 2-4 hours after uniformly mixing the mixture. The signal values were read using the HTRF program in a microplate reader (BMG Labtech, model: PHERAstar FS). The test results are shown in Table 2.

Test Example 3. Determination of the inhibitory activity of the compounds of the present invention on the proliferation of RS4;11 cells and NCI-H146 cells

The in vitro inhibitory activity against the proliferation of RS4;11 cells and NCI-H146 cells was tested as follows.

Reagents used in this experiment: RPMI-1640 medium (HyCLone, Cat.#SH30243018); fetal bovine serum (Gibco, Cat.#10099-141); pancreatin (Gibco, Cat.#25200-072); cell titer-Glo (Promega, Cat.#G7571).

Materials used: RS4;11 cells (Nanjing Kebai Biotechnology Co., Ltd, Cat. No. CBP60641); and NCI-H 146 cells (ATCC, Cat. No. HTB-173).

The in vitro cell proliferation assay described below can determine the inhibitory activity of test compounds on the proliferation of RS4;11 cells and NCI-H146 cells.

RS4;11 cells (or NCI-H146 cells) were cultured in RPMI-1640 culture medium containing 10% FBS and passaged 2-3 times per week at a passage ratio of 1:3 or 1:5. During passage, the cells were transferred to a centrifuge tube and centrifuged at 1200 rpm for 3 min. The supernatant was discarded and fresh culture medium was added to resuspend the cells. 90 μl of the cell suspension was added to a 96-well cell culture plate, where RS4;11 cells and NCI-H146 cells were added at a density of 2.22× ¹⁰⁵ cells/ml. Only 100 μl of complete medium (RPMI-1640 medium containing 10% FBS) was added to the periphery of the 96-well plate. The plate was incubated in an incubator for 24 h (37°C, 5% CO ₂ ).

The test sample was diluted to the proper concentration gradient with dimethyl sulfoxide as required for the experiment. 5 μL of the test compound solution prepared in the gradient concentration was added to 95 μL of fresh culture medium and mixed uniformly, and then 10 μL of the culture medium containing the said compound was added to the cell culture plate. The cell culture plate was incubated in an incubator for 3 days (37°C, 5% _CO2 ). 50 μL of CellTiter-Glo reagent was added to each well of the 96-well cell culture plate. The plate was left to stand for 5-10 minutes away from light at room temperature. The chemiluminescence signals were read using a microplate reader (BMG Labtech, model: PHERAstar FS), and the data were processed using GraphPad software. The results are shown in Table 3.

Test Example 4. Toxicity Test on SD (Sprague-Dawley) Rats by Repeated Intravenous Administration for 7 Days

1. Purpose

This experiment was conducted to evaluate the toxicity of compound 9 and AZD4320 in SD rats by repeated intravenous administration for 7 days.

2. Test connections

Storage conditions: Stored in a dehydrator at room temperature away from light. Compound 9, 100% pure, molecular weight: 976.1. Compound AZD4320, 100% pure, molecular weight: 945.5.

3. Experimental plan and procedures

3.1 Experimental animals and conditions of maintenance

30 SD rats, weighing about 180-200g and aged 6-7 weeks, half male and half female, purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd., SPF grade (specific pathogen free). Animal Certification No.: 200917Aazz0619000298 and 20200917Aazz0619000104. Production License No.: SCXK (Zhejiang) 2019-0001.

Housing conditions: SPF class, animals were placed in plastic and transparent rat cages, 2-3 per cage. Room temperature was 20-26°C, and relative humidity was 40-70%. Lighting was controlled in a 12/12 light/dark cycle. Mice ate pelleted food freely in unlimited quantities.

3.2 Grouping of animals

Rats were randomly divided into the following 3 groups: vehicle control group, 9-1 mg/kg compound receiving group and AZD4320-1 mg/kg compound receiving group, with 6 rats in each group, half male and half female.

3.3 Experimental procedures

The dosage of the test sample for the 7-day toxicity test in rats: 1 mg/kg (1 time/day) for 7 consecutive days. Administration route: intravenous, dosing volume: 10 ml/kg, administration frequency and dosing period: daily in the morning for 7 days.

Carrier: 1.5% DMA+98.5% (10% HS-15), where DMA is N,N-dimethylacetamide (Shanghai Titan Scientific Co., Ltd., Cat. No. 01013630); and HS-15 is polyethyleneglycol stearate 15 (Solutol HS15) (Shanghai Xietai Chemical Co., Ltd., Cat. No. 50253404).

3.4 Experimental equipment Automatic biochemical analyzer Hitachi 7100 Automatic blood cell analyzer Siemens Advia 2120i Automatic coagulation analyzer Sysmex ca1500

Toxicokinetic data collection: AB 4000 liquid chromatograph/mass spectrometer

3.5 Data expression and statistical processing

The results of the detection were entered and statistically processed in Excel computer.

Clinical symptoms, food intake and general visual observation results during dissection were not statistically calculated, and parameters such as body weight, organ weight and clinical examination were calculated using the T-test.

4. Results

SD rats were intravenously administered 1 mg/kg compound 9 or 1 mg/kg AZD4320 at 5 ml/kg for 7 repeated days.

No significant abnormalities were observed in the clinical observation of animals in the compound 9 group during treatment. Compared with the vehicle control group, there was no significant difference in body weight and body weight growth rate of animals (p>0.05) among the dosing groups, and no significant changes in food intake were observed.

Platelets of rats in the compound 9 group were slightly reduced but within the background normal range, while platelets of AZD 4320 rats were more significantly reduced (p<0.01). The results are shown in Table 4.

5. Conclusion

Under the conditions of this experiment, compared with the vehicle control group and the AZD 4320 positive control group, compound 9 reduced platelet count less than the positive control group and had the advantage of higher safety.

Test Example 5. Pharmacokinetic evaluation of the compounds of the present invention in mice

1. Abstract

The drug concentration in the plasma of test animals (mice) at different time points after intravenous (iv) administration of compound 10 and AZD4320 was determined by LC/MS/MS. The pharmacokinetic behavior of the compounds of the present invention in mice was studied and their pharmacokinetic profile was assessed.

2. Methodology

2.1 Test connections

Compound 10

2.2 Test animals

18 female C57 mice, randomly divided into 2 groups, were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd, Animal Production License No.: SCXK (Shanghai) 2017-0005.

2.3. Preparation of the medicinal product

A certain amount of compound 10 was weighed, dissolved by adding 5% by volume DMSO and 5% Tween 80 (Shanghai Titan Scientific Co., Ltd.), and then prepared into 0.1 mg/mL clear solution by adding 90% normal saline. Compound AZD4320 was formulated in the same way as compound 10.

2.4. Introduction

Intravenous administration: Compounds 10 and AZD4320 were administered to mice separately intravenously at a dose of 2.0 mg/kg and a volume of 20.0 ml/kg.

3. Procedures

Mice were intravenously injected with compound 10 and 0.1 ml of blood was collected before administration and 5 min, 0.25 h, 0.5 h, 1.0 h, 2.0 h, 4.0 h, 8.0 h, 11.0 h and 24.0 h after administration, placed in an EDTA-K2 anticoagulation tube, centrifuged at 10,000 rpm for 1 min (4°C). Plasma was separated within 1 hour and stored at -20°C for testing. The process from blood collection to centrifugation was carried out in an ice bath.

Mice were intravenously injected with AZD4320 compound and 0.1 ml of blood was collected before administration and 5 min, 0.25 h, 0.5 h, 1.0 h, 2.0 h, 4.0 h, 8.0 h, 11.0 h and 24.0 h after administration, placed in an EDTA-K2 anticoagulation tube, centrifuged at 10,000 rpm for 1 min (4°C). Plasma was separated within 1 hour and stored at -20°C for testing. The process from blood collection to centrifugation was carried out in an ice bath.

The test compounds were determined at different concentrations in mouse plasma after administration: 10 μl of mouse plasma at each time point after administration was mixed with 200 μl of methanol (containing a solution of internal standard camptothecin (100 ng/ml)); the mixture was vortexed for 1 min and centrifuged for 10 min (18,000 rpm), and 3 μl of the supernatant was taken for LC/MS/MS analysis.

4. Pharmacokinetic parameters

Test Case 6. Therapeutic Effect on RS4;11 Human Acute Lymphoblastic Leukemia in Mouse Subcutaneous Xenograft Tumor

This experiment was conducted to evaluate and compare the therapeutic effect of the compounds of the present invention on RS4;11 acute lymphoblastic leukemia mouse subcutaneous xenograft tumor.

1. Experimental animals and conditions of maintenance

NOD-Scid mice (non-obese diabetic mice with severe combined immunodeficiency), 5 weeks old, purchased from Beijing Huafukang Biotechnology Co., Ltd. Production License No.: SCXK (Beijing) 2019-0008, Animal Certificate No.: 110322211100992176. Housing environment: SPF grade. 6 mice in the vehicle group and 8 mice in the compound group.

2. Test connections

Groups receiving compound 9, compound 10 and carrier.

5 mg of the compound was added to 250 μL of DMSO and vortexed to obtain a 20 mg/mL solution, which was diluted to the desired concentration with 30% hydroxypropyl-β-cyclodextrin (HP-β-CD, InnoStar Biotechnology Nantong Co., Ltd, Nantong).

3. Experimental plan and procedures

Human acute lymphoblastic leukemia RS4;11 was purchased from the American Type Culture Collection. RS4;11 cells were cultured in a 10 cm Petri dish with RPMI 1640 culture medium (Gibco) containing 10% fetal bovine serum (Gibco) and penicillin and streptomycin in an incubator at 37°C containing 5% CO ₂ . Cells were passaged twice a week and then harvested, counted, and inoculated when they were in the exponential growth phase.

Each mouse was subcutaneously inoculated with 1× ¹⁰⁷ RS4;11 cells. When the tumors grew to 100-200 ^mm3 , the mice were grouped according to tumor volume and were injected intravenously (i.v.) once a week (1 time/week) for a total of 4 times, with an injection volume of 0.1 ml/10 g body weight and a dose of 10 mg/kg.~

4. Results

The experimental endpoint was to examine the effect of the drug on tumor growth, and the specific endpoint was T/C% or tumor growth inhibition (TGI%).

Tumor diameters were measured twice weekly using calipers, and tumor volume (V) was calculated using the following formula:

V=1/2×a×b ² , where a and b denote the length and width, respectively.

T/C(%)=(T- _T0 )/(C- _C0 )×100, where T and C were the tumor volumes of animals at the end of the experiment in the treatment group and the control group, respectively; _T0 and _C0 were the tumor volumes of animals at the beginning of the experiment in the treatment group and the control group, respectively.

% tumor growth inhibition (TGI%) = 100 - T/C (%);

When the tumor began to regress, tumor growth inhibition % (TGI%) = 100 - (T - T ₀ ) / T ₀ × 100;

If the tumor had a reduced volume compared to the initial volume, i.e. T less than _T0 or C less than _C0 , this was defined as partial regression (PR) of the tumor; if the tumor completely disappeared, this was defined as complete regression (CR) of the tumor.

Conclusion: Compound 9 (10 mg/kg, IV, 1 time/week, 3 times) and compound 10 (10 mg/kg, IV, 1 time/week, 3 times) exerted significant inhibitory effects on the growth of RS4;11 human acute lymphoblastic leukemia on murine subcutaneous xenograft tumor (Fig. 1), with tumor inhibition rates of 72% and 82%, respectively; all tumor-bearing mice tolerated the above drugs well, and no symptoms such as significant weight loss were observed (Fig. 2).

Claims (72)

1. A compound of general formula (D) or a pharmaceutically acceptable salt thereof: Where: R ¹ represents hydrogen; R ² is selected from the group consisting of C _1-6 alkyl and C _1-6 hydroxyalkyl; or R ¹ and R ² together with the atom to which they are attached form a 3- to 6-membered heterocyclyl; R ³ is selected from the group consisting of hydrogen, C _1-6 hydroxyalkyl and C _1-6 alkyl; R ⁴ is selected from the group consisting of hydrogen and C _1-6 alkyl; R ⁵ is selected from the group consisting of halogen and C _1-6 haloalkyl; R ⁶ is selected from the group consisting of hydroxy and C _1-6 alkoxy; R ⁷ is hydrogen; each of R ⁸ and R ⁹ is independently selected from the group consisting of hydrogen and C _1-6 alkyl; each of R ¹⁰ and R ¹¹ is independently selected from the group consisting of hydrogen and C _1-6 alkyl; R ¹² is hydrogen; R ¹³ is hydrogen; R ¹⁴ is hydrogen; and r is selected from the group consisting of 0, 1, 2, and 3; provided that if R ¹ and R ² together with the atom to which they are attached do not form a 3- to 6-membered heterocyclyl, R ⁵ is haloalkyl. 2. A compound of general formula (D) or a pharmaceutically acceptable salt thereof according to claim 1, wherein R ¹ and R ² together with the atom to which they are attached form pyrrolidinyl. 3. A compound of general formula (D) or a pharmaceutically acceptable salt thereof according to claim 1, which is a compound of general formula (D-I) or a pharmaceutically acceptable salt thereof: where m is selected from the group consisting of 0, 1, 2, and 3, and r and R ³ -R ¹⁴ are as defined in paragraph 1. 4. A compound of general formula (D) or a pharmaceutically acceptable salt thereof according to claim 1, which is a compound of general formula (D-II) or a pharmaceutically acceptable salt thereof: where m is selected from the group consisting of 0, 1, 2, and 3, and r and R ³ -R ¹¹ are as defined in paragraph 1. 5. A compound of general formula (D) or a pharmaceutically acceptable salt thereof according to claim 1, which is a compound of general formula (D-III) or a pharmaceutically acceptable salt thereof: where m is selected from the group consisting of 0, 1, 2, and 3, and r and R ³ -R ¹¹ are as defined in paragraph 1. 6. A compound of general formula (D) or a pharmaceutically acceptable salt thereof according to claim 1, which is a compound of general formula (D-IV) or a pharmaceutically acceptable salt thereof: Where m is selected from the group consisting of 0, 1, 2, and 3, and R ³ -R ⁹ are as defined in paragraph 1. 7. A compound of general formula (D) or a pharmaceutically acceptable salt thereof according to claim 3, where m is 2. 8. A compound of general formula (D) or a pharmaceutically acceptable salt thereof according to claim 1, wherein R ⁵ is chlorine. 9. A compound of general formula (D) or a pharmaceutically acceptable salt thereof according to claim 1, wherein R ⁵ is trifluoromethyl. 10. A compound of general formula (D) or a pharmaceutically acceptable salt thereof according to claim 1, wherein R ³ is C _1-6 hydroxyalkyl. 11. A compound of general formula (D) or a pharmaceutically acceptable salt thereof according to claim 1, which is a compound of general formula (D-V) or a pharmaceutically acceptable salt thereof: where t is selected from the group consisting of 0, 1, 2, and 3, and r, R ¹ , R ² , R ⁴ and R ⁶ -R ¹⁴ are as defined in paragraph 1. 12. A compound of general formula (D) or a pharmaceutically acceptable salt thereof according to claim 1, which is a compound of general formula (D-VI) or a pharmaceutically acceptable salt thereof: where t is selected from the group consisting of 0, 1, 2, and 3, and r, R ¹ , R ² , R ⁴ and R ⁶ -R ¹¹ are as defined in paragraph 1. 13. A compound of general formula (D) or a pharmaceutically acceptable salt thereof according to claim 12, which is a compound of general formula (D-VI) or a pharmaceutically acceptable salt thereof, where t is equal to 1. 14. A compound of general formula (D) or a pharmaceutically acceptable salt thereof according to claim 1, which is a compound of general formula (D-VII) or a pharmaceutically acceptable salt thereof: where r and R ¹ -R ¹¹ are as defined in paragraph 1. 15. A compound of general formula (D) or a pharmaceutically acceptable salt thereof according to claim 1, wherein R ⁴ is hydrogen. 16. A compound of general formula (D) or a pharmaceutically acceptable salt thereof according to claim 1, wherein R ⁶ is hydroxy. 17. A compound of general formula (D) or a pharmaceutically acceptable salt thereof according to claim 1, wherein R ⁷ , R ⁸ , R ⁹ , R ¹⁰ and R ¹¹ represent hydrogen. 18. A compound of general formula (D) or a pharmaceutically acceptable salt thereof according to claim 1, wherein r is 0 or 1. 19. A compound of general formula (D) or a pharmaceutically acceptable salt thereof according to claim 1, where r is 0. 20. A compound of general formula (D) or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is selected from the group consisting of: or a pharmaceutically acceptable salt thereof. 21. A method for producing a compound of general formula (D-III) or a pharmaceutically acceptable salt thereof, comprising: subjecting a compound of general formula (DA2) or a pharmaceutically acceptable salt thereof to a substitution reaction in an alkaline medium to obtain a compound of general formula (D-III) or a pharmaceutically acceptable salt thereof; wherein the reagent used in the alkaline medium is triethylamine, Where: m is selected from the group consisting of 0, 1, 2, and 3, and R ³ is C _1-6 hydroxyalkyl; and r and R ⁴ -R ¹¹ are as defined in paragraph 1. 22. A pharmaceutical composition for treating cancer or a tumor, containing a therapeutically effective amount of a compound of general formula (D) or a pharmaceutically acceptable salt thereof according to any one of claims 1-20 and a pharmaceutically acceptable carrier, diluent or excipient. 23. A compound of general formula (D) or a pharmaceutically acceptable salt thereof according to any one of claims 1-20 or a pharmaceutical composition according to claim 22 for use in inhibiting Bcl-2 and/or Bcl-XL. 24. A compound of general formula (D) or a pharmaceutically acceptable salt thereof according to any one of claims 1-20 or a pharmaceutical composition according to claim 22 for use in the treatment of cancer or a tumor, wherein the cancer is selected from the group consisting of: melanoma, liver cancer, kidney cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, nasopharyngeal cancer, colorectal cancer, colon cancer, rectal cancer, pancreatic cancer, cervical cancer, ovarian cancer, breast cancer, bladder cancer, prostate cancer, leukemia, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia, acute myelogenous leukemia, mantle cell lymphoma, diffuse large B-cell lymphoma, follicular central lymphoma, non-Hodgkin's lymphoma, T-cell lymphoma, B-cell lymphoma, squamous cell carcinoma of the head and neck, cervical cancer uterine, thyroid cancer, lymphoma, sarcoma, neuroblastoma, brain tumors, myeloma, astrocytomas and gliomas.