WO2018223909A1 - Chimeric molecule and preparation therefor and use thereof - Google Patents

Abstract

Provided is a chimeric molecule consisting of a small molecule compound unit of a target protein, an E3 ubiquitin ligase binding unit and a linkage unit. The chimeric molecule can bind to a BRD protein, enable the BRD protein to be more easily degraded by proteases, and thus play a role in inhibiting cell proliferation. The chimeric molecule can be used as a drug for the degradation of the BRD protein to treat cancers or coronary artery diseases.

Description

Chimeric molecule and preparation and application thereof Technical field

The invention belongs to the field of compound medicines, and in particular to a chimeric molecule.

Background technique

Modern molecular biology regulates the expression level of proteins from three basic levels: first, at the DNA level, by gene knockout, thereby inactivating the DNA of the target protein; secondly, at the mRNA level, through small RNA, and the target protein The mRNA binds, thereby inhibiting the translation and expression of mRNA; again, at the protein level, the amount and activity of the target protein are adjusted by modification of the translated target protein, such as methylation, phosphorylation, glycosylation and the like. The chimeric molecule (PROTAC) referred to in this patent is based on protein levels, regulates the expression of a target protein, and is used to treat diseases. The BRD4 chimeric molecule of the present patent has a heterologous bifunctional molecule composed of a target protein (BRD4), an E3 ubiquitin ligase recognition group, and a linking group; the purpose of the coupling target is to interact with the ligand BRD4 in the molecule. Binding; the purpose of the E3 ubiquitin ligase recognition group is to bind to the target protein, resulting in ubiquitination of the target protein, and finally the target protein is degraded by the proteasome.

From the knockout of functional genes at the gene level to the interference of small RNA at the mRNA level, more biochemical techniques have been used to study protein expression regulation; chimeric molecules do not affect the expression of DNA and mRNA. Direct regulation of protein expression at the protein level is therefore more ethically acceptable. At the same time, chimeric molecules, as a new protein regulation method, have advantages that other methods do not have, and may become an alternative to gene therapy. Chimeric molecules target the degradation of functional proteins, and it is possible to develop drugs into new therapeutic methods.

Summary of the invention

It is an object of the present invention to provide a chimeric molecule and its preparation and use.

The present invention first provides a compound represented by the formula (I), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof:

M-L-N (I)

Wherein L is a linking unit, and one atom on M and N is substituted by one end of the linking group L, respectively;

Unit M is as shown in equation (a) or (c):

Unit N is as shown in equation (b):

Wherein one of R ₁ and R ₂ is selected from the group consisting of halogen, -NH-SO ₂ -R ₇ , hydrogen, cyano, nitro, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl a heterocyclic aryl group, wherein said alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heterocyclic aryl group is independently independently optionally further selected from one or more selected from the group consisting of ruthenium atoms Substituted by a halogen, cyano, nitro, oxo, alkyl, haloalkyl, hydroxyalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heterocyclic aryl substituent ;

Wherein R _{7 is} selected from the group consisting of hydrogen, cyano, nitro, alkyl, cycloalkyl, heterocycloalkyl, aryl, heterocyclic aryl;

Another structure of R ₁ and R ₂ is selected from the group consisting of -A-, wherein A is selected from -O-, -COX- or -XCO-, -NH-, -HNCONH-, -NH-SO ₂ -R ₇ , - SO ₂ - or -CH ₂ -, X is selected from -CH ₂ -, -NH-, -SO ₂ -, -O-, acylamino, ester or carbonyl, and A is bonded to linking unit L;

R ₃ , R ₄ and R ₆ are each independently selected from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heterocyclic aryl, wherein Said alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heterocyclic aryl group, respectively, optionally further optionally one or more selected from the group consisting of a halogen atom, a halogen, a cyano group, a nitrate Substituted with a substituent of an oxo group, an oxo group, an alkyl group, a haloalkyl group, a hydroxyalkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heterocyclic aryl group;

R _{5 is} selected from the group consisting of alkyl, alkoxy, amino, amido, ester, carbonyl, and any combination of the above.

Further, one of R ₁ and R ₂ is selected from the group consisting of halogen, -NH-SO ₂ -R _{7 ,} hydrogen, and another of R ₁ and R ₂ is selected from -A-, wherein A is selected from -O-, -COX- or -XCO-, X is selected from -CH ₂ -, -NH-, -SO ₂ -, -O-, acylamino, ester or carbonyl, and R _{7 is} selected from hydrogen, cyano, nitro, alkane Base, cycloalkyl, heterocycloalkyl, aryl, heterocyclic aryl.

Further, the X is selected from -CH ₂ -, -NH-, -O-, an ester group or a carbonyl group.

Further, the M structure is represented by the formula (a1) or the formula (a2):

Wherein A _{1 is} selected from -COX- or -XCO-, and X is selected from -CH ₂ -, -NH-, -O-, ester or carbonyl;

R ₃ and R ₆ are each independently selected from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heterocyclic aryl, wherein The alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heterocyclic aryl groups are each independently optionally further selected from one or more selected from the group consisting of a halogen atom, a halogen, a cyano group, a nitro group, and an oxygen group. Substituted with a substituent of an alkyl group, an alkyl group, a halogenated alkyl group, a hydroxyalkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heterocyclic aryl group;

R _{7 is} selected from the group consisting of hydrogen, cyano, nitro, alkyl, cycloalkyl, heterocycloalkyl, aryl, heterocyclic aryl.

Further, the R _{6 is} selected from a C ₁ -C ₄ alkyl group; preferably, the R _{6 is} selected from a methyl group.

Further, the R _{7 is} selected from a C ₁ -C ₄ alkyl group; preferably, the R ₇ is an ethyl group.

Further, the M structure is represented by the formula (a3) or the formula (a4):

Wherein A _{1 is} selected from -COX- or -XCO-, and X is selected from -CH ₂ -, -NH-, -O-, ester or carbonyl;

R _{3 is} selected from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heterocyclic aryl, wherein said alkyl, alkenyl, The alkynyl, cycloalkyl, heterocycloalkyl, aryl or heterocyclic aryl group, respectively, is optionally further optionally further selected from one or more selected from the group consisting of a halogen atom, a halogen, a cyano group, a nitro group, an oxo group, an alkyl group, Substituted by a substituent of a haloalkyl group, a hydroxyalkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heterocyclic aryl group.

Further, R _{3 is} selected from the group consisting of hydrogen, halogen, and alkyl.

Further, the M structure is represented by (a5) or (a6):

Wherein A _{1 is} selected from -COX- or -XCO-; and X is selected from -CH ₂ -, -NH-, -O-, ester or carbonyl.

Further, the X is selected from -NH- or -O-.

Further, the M structure is:

Further, the L is selected from the group consisting of -(CH ₂ CH ₂ OCH ₂ )n-CO-, -(CH ₂ CH ₂ O)n-CH ₂ CO-, -(CH ₂ OCH ₂ CH ₂ CH ₂ O)n -CH ₂ CO-, -(CH ₂ OCH ₂ )n-CO-, -(CH ₂ OCH ₂ CH ₂ )n-CH ₂ CO- or -(CH ₂ CH ₂ OCH ₂ )n-CH ₂ CO-, Where n is an integer from 0 to 20.

Further, n is an integer of 1 to 3.

Further, R _{4 is} selected from H or CH ₃ (S or R).

Further, the R _{5 is} selected from an alkyl group or an amino group.

Further, the N structure is

Wherein R _{4 is} selected from H or CH ₃ (S or R).

Further, the N structure is represented by (b1), (b2) or (b3);

Further, the structure of the compound is as shown in formula (II):

L is as defined in claim 12 or 13.

Further, the structure of the compound is as shown in formula (III):

L is as defined in claim 12 or 13.

Further, the structure of the compound is as shown in formula (IV):

L is as defined in claim 12 or 13.

Further, the structure of the compound is as shown in the formula (V):

L is as defined in claim 12 or 13.

Further, the structure of the compound is as shown in formula (VI):

L is as defined in claim 12 or 13.

Further, the structure of the compound is as shown in formula (VII):

L is as defined in claim 12 or 13.

Further, the structure of the compound is as shown in formula (VIII):

L is as defined in claim 12 or 13.

Further, the structure of the compound is as shown in formula (IX):

L is as defined in claim 12 or 13.

Further, the structure of the compound is as shown in the formula (X):

L is as defined in claim 12 or 13.

Further, the structure of the compound is as shown in the formula (XI):

L is as defined in claim 12 or 13.

Further, the structure of the compound is as shown in the formula (XII):

L is as defined in claim 12 or 13.

Further, the structure of the compound is as shown in the formula (XIII):

L is as defined in claim 12 or 13.

Further, the compound is one of the following compounds:

The present invention also provides the use of the compound, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof, for the preparation of a BRD4 inhibitor or a protein degradation agent.

Further, the medicament is a medicament for treating cancer or coronary artery disease.

Further, the cancer is prostate cancer, non-small cell lung cancer, breast cancer, melanoma, leukemia or rectal cancer.

The present invention also provides a pharmaceutical composition comprising the aforementioned compound or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient, together with a pharmaceutically acceptable adjuvant Prepared preparation.

Definitions of terms of use in connection with the present invention: Unless otherwise stated, the initial definitions provided herein by the group or term apply to the group or term of the entire specification; for terms not specifically defined herein, it should be based on the disclosure and context. Given the meanings that those skilled in the art can give to them.

"Substitution" means that a hydrogen atom in a molecule is replaced by a different atom or molecule.

The minimum and maximum values of the carbon atom content in the hydrocarbon group are represented by a prefix, for example, the prefix (C _a - C _b ) alkyl group indicates any alkyl group having "a" to "b" carbon atoms. Thus, for example, (C ₁ -C ₆ )alkyl means an alkyl group containing from 1 to 6 carbon atoms.

The C ₁ -C ₄ alkyl group means an alkyl group of C ₁ , C ₂ , C ₃ , C ₄ , that is, a linear or branched alkyl group having 1 to 4 carbon atoms, such as a methyl group or an ethyl group. , propyl, isopropyl, butyl, isobutyl, tert-butyl, sec-butyl and the like.

The cycloalkyl group means a cyclic alkyl group such as a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group or the like.

The halogen means a fluorine atom, a bromine atom, a chlorine atom, or an iodine atom.

The term "pharmaceutically acceptable" means that a carrier, carrier, diluent, adjuvant, and/or salt formed is generally chemically or physically compatible with the other ingredients that constitute a pharmaceutical dosage form, and is physiologically Compatible with the receptor.

The terms "salt" and "pharmaceutically acceptable salt" refer to the above-mentioned compounds or stereoisomers thereof, acid and/or basic salts formed with inorganic and/or organic acids and bases, and also includes zwitterionic salts (within Salts) also include quaternary ammonium salts such as alkylammonium salts. These salts can be obtained directly in the final isolation and purification of the compounds. It can also be obtained by mixing the above compound, or a stereoisomer thereof, with a certain amount of an acid or a base as appropriate (for example, an equivalent amount). These salts may be precipitated in a solution and collected by filtration, or recovered after evaporation of the solvent, or may be obtained by lyophilization after reaction in an aqueous medium.

It is apparent that various other modifications, substitutions and changes can be made in the form of the above-described embodiments of the present invention.

The above content of the present invention will be further described in detail below by way of specific embodiments in the form of embodiments. However, the scope of the above-mentioned subject matter of the present invention should not be construed as being limited to the following examples. Any technique implemented based on the above description of the present invention is within the scope of the present invention.

DRAWINGS

Figure 1 is an electropherogram of the BRD4 protein of the compound of the present invention.

detailed description

Example 1. Compound 1 (2S, 4R)-1-((S)-13-tert-butyl-1-(4-(2,4-difluorophenoxy)-3-(6-methyl-7) -oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridin-4-yl)phenyl)-1,11-dioxo-5,9-dioxo-2,12 Synthesis of -diazatetradecyl-14-carbonyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)phenyl)tetrahydropyrrole-2-amide (1)

(1) Synthesis of intermediate 1(E)-2-(5-bromo-2-methoxy-3-nitropyridin-4-yl)-N,N-dimethylethylamine

To a 10 L reaction flask containing N,N-dimethylformamide (4 L) was added 5-bromo-2-methoxy-4-methyl-3-nitropyridine (200 g, 0.8 mol), N N-dimethylformamide dimethyl acetal (571.2 g, 4.8 mol), lithium methoxide (0.9 g, 0.024 mol), and the mixture was stirred at 110 ° C for 3 hours. After the reaction solution was cooled to room temperature, it was poured into ice water (12 L), and the solid was analyzed, filtered, washed with water (1 L), and dried. A brownish red solid powder intermediate 1 (240 g) was obtained in a yield of 98%.

(2) Synthesis of intermediate 2 4-bromo-7-methoxy-1H-pyrrolo[2,3-c]pyridine

Ethyl acetate (5 L) was added to a 10 L reaction flask to reduce iron powder (223 g, 3.97 mol), acetic acid (2.3 L, 39.7 mol), and after heating to 80 ° C, intermediate compound 1 (240 g, 0.79 mol) was added. ). After the addition was completed, the reaction was continued at this temperature for 30 minutes. Cool, filter by suction, spin dry, and beat with a mixed solvent of ethanol (1 L) and water (1 L). After suction filtration and drying, Intermediate Compound 2 (140 g) was obtained in a yield of 78%.

(3) Synthesis of intermediate 3 4-bromo-7-methoxy-1-p-toluenesulfonyl-1H-pyrrolo[2,3-c]pyridine

N,N-dimethylformamide (2 L), Intermediate Compound 2 (140 g, 0.62 mol) was added to a 5 L reaction flask. After the solution was dissolved, it was cooled to 0 ° C in an ice water bath, and NaH (40 g, 60%, 0.99 mol) was added to the reaction mixture, and the temperature of the reaction was controlled to not exceed 10 °C. After the addition of NaH to stop bubbling, p-toluenesulfonyl chloride (177 g, 0.93 mol) was added and stirred at room temperature overnight. After the reaction was completed, the reaction mixture was poured into 6 L of water to precipitate a solid, which was filtered, and the solid was dissolved in ethyl acetate (200 mL), and 600 mL of n-hexane was added to precipitate a solid, which was filtered to give intermediate compound 3 (188 g). .

(4) Synthesis of intermediate 4 4-bromo-1-toluenesulfonyl-1H-pyrrolo[2,3-c]pyridine-7(6H)-one

To a 5 L reaction flask was added 1,4-dioxane 2L, methanol (78 g, 2.45 mol). Acetyl chloride (154 g, 1.96 mol) was added to the reaction flask at room temperature. After the addition was completed, stirring was continued for 1 hour. Intermediate compound 3 (188 g, 0.49 mol) was added, and the mixture was warmed to 80 ° C and stirred overnight. After completion of the reaction, the mixture was dried, and then pulverized with 300 mL of methyl tert-butyl ether and filtered to give Intermediate Compound 4 (139 g).

(5) Synthesis of intermediate 5 4-bromo-6-methyl-1-toluenesulfonyl-1H-pyrrolo[2,3-c]pyridine-7(6H)-one

To a 5 L reaction flask was added 1,4-dioxane 2L, intermediate compound 4 (79 g, 0.21 mol), cesium carbonate (118 g, 0.32 mol), methyl iodide (92 g, 0.64 mol), and stirred at room temperature overnight. . After completion of the reaction, the mixture was filtered and dried to give Intermediate Compound 5 (75 g).

(6) Intermediate 6 6-Methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-toluenesulfonyl Synthesis of -1H-pyrrolo[2,3-c]pyridine-7(6H)-one

To a 2 L reaction flask was added 800 mL of 1,4-dioxane, intermediate compound 5 (40 g, 0.1 mol), diboronic acid pinacol ester (105 g, 0.4 mol), potassium acetate (20.4 g, 0.2 mol). After three times of nitrogen substitution, tetrakistriphenylphosphine palladium (12 g, 0.01 mol) was added, and the mixture was replaced with nitrogen three times, heated to 110 ° C, and stirred overnight. After completion of the reaction, filtration and column chromatography gave Intermediate Compound 6 (40 g).

(7) Synthesis of intermediate 8 methyl 3-bromo-4-(2,4-difluorophenoxy)benzoate

Intermediate compound 7 (5 g, 21.5 mmol), 2,4-difluorophenol (4.2 g, 32.3 mmol), cesium carbonate (12.5 g, 38.3 mmol) and 150 mL DMSO were added to a 250 mL reaction flask, and the temperature was raised to 110 °C. Stir overnight. After the reaction was completed, 200 mL of water was added, and ethyl acetate was evaporated, and dried over anhydrous sodium sulfate.

(8) Intermediate 9 (2,4-difluorophenoxy)-3-(6-methyl-7-oxo-1-toluenesulfonyl-6,7-dihydro-1H-pyrrolo[2 Synthesis of 3-(3-pyridin-4-yl)benzoic acid methyl ester

Intermediate compound 8 (500 mg, 1.46 mmol), intermediate compound 6 (750 mg, 1.75 mmol), tetratriphenylphosphine palladium (168 mg, 0.146 mmol), potassium carbonate (400 mg, 2.9 mmol) was added to a 100 mL reaction flask. And 50 mL of N,N-dimethylformamide and 1-2 drops of water, three times with nitrogen, warmed to 90 ° C, and stirred overnight. After the reaction was completed, 50 mL of water was added, and ethyl acetate was evaporated.

(9) Intermediate 10 (2,4-difluorophenoxy)-3-(6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridine Synthesis of -4-yl)benzoic acid:

Intermediate compound 9 (350 mg, 0.62 mmol), 5NKOH, 5 mL of tetrahydrofuran was added to a 50 mL reaction flask, and the mixture was warmed to 80 ° C and stirred for 4 hours. After completion of the reaction, the tetrahydrofuran was concentrated to remove the residue, and the mixture was filtered, washed with water, and then evaporated to dryness to afford Intermediate Compound 10 (185 mg, 75%).

(10) Synthesis of intermediate 12 ((2-(allyloxy)ethoxy)methyl)benzene

Intermediate compound 11 (10 g, 66 mmol), 100 ml of tetrahydrofuran was added to a 250 mL reaction flask, and sodium hydride (1.89 g, 79 mmol) was added thereto, and the mixture was stirred for half an hour, and bromopropene (8 g, 66 mmol) was added thereto, and the temperature was raised to 40. After stirring for 30 minutes at 0<0>C, EtOAc (EtOAc)EtOAc.

(11) Synthesis of intermediate 13 3-(2-(benzyloxy)ethoxy)propan-1-ol

The intermediate compound 12 (2.5 g, 13 mmol), 25 mL of tetrahydrofuran was added to a 250 ml reaction flask, and a solution of borane in tetrahydrofuran (20 mL, 1 mol/L) was added thereto, and the mixture was heated to 25 °C, stirred for 2 hours, and saturated. 7 mL of an aqueous solution of sodium acetate and 6 mL of 30% hydrogen peroxide were stirred for 1 hour. After completion of the reaction, the mixture was extracted with ethyl acetate and dried over anhydrous sodium sulfate.

(12) Synthesis of intermediate 14 2-(3-(2-(benzyloxy)ethoxy)propoxy)acetate

Intermediate compound 13 (1.6 g, 7.7 mmol) was added to a 250 mL reaction flask, and 15 mL of dichloromethane was added, and ethyl diazoacetate (0.9 g, 7.7 mmol) was added thereto, and 0.1 mL of boron trifluoride etherate solution was added thereto. After stirring for 30 minutes, after completion of the reaction, 10 mL of water was added, and the organic layer was dried over anhydrous sodium sulfate.

(13) Synthesis of intermediate 15-ethyl 2-(3-(2-hydroxyethoxy)propoxy)acetate

Intermediate compound 14 (1.3 g), 0.25 mL of acetic acid, 0.2 g of palladium on carbon, 10 mL of methanol, and three times of hydrogen were added to a 100 mL reaction flask, and stirred at room temperature for 5 hours. After completion of the reaction, filtration, concentration and column chromatography gave Intermediate Compound 15 (0.77 g, yield 82%).

(14) Synthesis of intermediate 16 2-(3-(2-((methylsulfonyl)oxy)ethoxy)propoxy)acetate

Intermediate compound 15 (0.77 g, 3.7 mmol), triethylamine (560 mg, 5.55 mmol), 10 mL dichloromethane was added to a 100 mL reaction flask, and methanesulfonyl chloride (500 mg, 4.44 mol) was added in an ice bath and stirred at room temperature. After 2 hours, after completion of the reaction, 1N HCl was added to adjust pH <5, extracted with dichloromethane, dried over anhydrous sodium sulfate, and concentrated to give intermediate compound 16 directly.

(15) Synthesis of intermediate 17 2-(3-(2-(1,3-dioxoisoindolin-2-yl)ethoxy)propoxy)acetate

To a 100 mL reaction flask was added intermediate compound 16, o-phenylsuccinimide (0.4 g), potassium carbonate (0.5 g), and 10 mL of N,N-dimethylformamide, warmed to 95 degrees and stirred for 3 hours. . After completion of the reaction, 10 mL of water was added, and ethyl acetate was evaporated and dried over anhydrous sodium sulfate.

(16) Synthesis of intermediate 18 2-(3-(2-aminoethoxy)propoxy)acetate

Intermediate compound 17 (0.7 g, 2 mmol), 0.3 g of hydrazine hydrate, 10 mL of methanol were added to a 100 mL reaction flask, and the mixture was heated to 70 ° C, and stirred for 1 hour. After the reaction was completed, concentrated, water was added, and ethyl acetate was evaporated. Drying over sodium sulfate and column chromatography gave intermediate compound 18 (0.36 g, yield 84%).

(17) Intermediate 19 2-(3-(2-(4-(2,4-difluorophenoxy)-3-(6-methyl-7-oxo-6,7-dihydro-1H) Synthesis of pyridyl[2,3-c]pyridin-4-yl)benzoylamino)ethoxy)propoxy)acetate

Intermediate compound 10 (200 mg, 0.5 mmol), intermediate compound 18 (104 mg, 0.5 mmol), 2-(7-azobenzotriazole)-N,N,N', was added to a 100 mL reaction flask. N'-Tetramethylurea hexafluorophosphate HATU (300 mg, 0.8 mmol), N,N-diisopropylethylamine DIPEA (208 mg, 1.6 mmol), 20 mL DMF, stirred at room temperature overnight, 10 mL water, acetic acid The ethyl ester was extracted, dried over anhydrous sodium sulfate, and evaporated

(18) Intermediate 20 2-(3-(2-(4-(2,4-Difluorophenoxy)-3-(6-methyl-7-oxo-6,7-dihydro-1H) Synthesis of pyrrolo[2,3-c]pyridine-4-benzoylamino)ethoxy)propoxy)acetic acid

Add intermediate compound 19 (180 mg, 0.3 mmol), 2N lithium hydroxide aqueous solution (1 mL), methanol 2 mL, and warm to 60 degrees. After the reaction is completed, add 2NHCl to adjust pH<4, and filter to obtain the middle. Compound 20 (150 mg, yield 88%).

(19) Synthesis of Compound 2: Compound 2 (2S, 4R)-1-((S)-13-tert-butyl-1-(4-(2,4-difluorophenoxy)-3-(6- Methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridin-4-yl)phenyl)-1,11-dioxo-5,9-dioxo -2,12-diazatetradecyl-14-carbonyl)-4-hydroxy-N-((S)-1-4-(4-methylthiazol-5-yl)phenyl)ethyl)tetra Preparation of hydropyrrole-2-amide

Intermediate compound 20 (100 mg, 0.18 mmol), intermediate compound 21 (synthesized according to US20170327469) (84 mg, 0.18 mmol), 2-(7-azobenzotriazole)-N, was added to a 50 mL reaction flask. N,N',N'-tetramethyluronium hexafluorophosphate HATU (76 mg, 0.2 mmol), N,N-diisopropylethylamine DIPEA (52 mg, 0.4 mmol), 10 mL DMF, 10mL of water was added, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated and column chromatography, to obtain 98mg of compound 2 as a pale yellow solid, in 56% yield, MS: mass spectrum 982 (M ⁺ H +). ^{1 H NMR (400MHz, DMSO)} δ11.98 (s, 1H), 8.95 (s, 1H), 8.45-8.44 (m, 1H), 8.34-8.33 (m, 1H), 7.85-7.84 (m, 1H) , 7.63-7.62 (m, 1H), 7.47-7.36 (m, 8H), 7.07–6.95 (m, 2H), 6.9-6.89 (m, 1H), 6.25 (s, 1H), 5.16 (s, 1H) , 4.97–4.85 (m, 1H), 4.53-4.51 (m, 1H), 4.46-4.42 (m, 1H), 4.24 (s, 1H), 4.01–3.83 (m, 2H), 3.66-3.60 (m) , 10H), 3.55 (s, 3H), 2.43 (s, 3H), 2.07 - 1.97 (m, 1H), 1.92 - 1.85 (m, 2H), 1.82 - 1.75 (m, 1H), 1.44-1.38 (m , 3H), 0.91 (s, 9H).

Example 2, Compound 5 (2S,4R)-1-((S)-12-tert-butyl-1-(4-(2,4-difluorophenoxy)-3-(6-methyl-7) -oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridin-4-yl)phenyl)-1,10-dioxo-5,8-dioxo-2,11 -diazotridecyl-13-carbonyl)-4-hydroxy-N-((S)-1-4-(4-methylthiazol-5-yl)phenyl)ethyl)tetrahydropyrrole-2 - Preparation of amide

In the same manner as in Example 1, an intermediate compound 22 was added to a 50 mL reaction flask (according to the synthesis method of the intermediate compound 20, wherein the step (12) was synthesized by substituting ethylene glycol for the intermediate compound 13) (100 mg, 0.18 mmol), Intermediate compound 21 (synthesized according to US20170327469) (84 mg, 0.18 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate HATU ( 76 mg, 0.2 mmol), N,N-diisopropylethylamine DIPEA (52 mg, 0.4 mmol), 10 mL DMF, EtOAc. Chromatography gave 95 mg of compound 5 as a pale yellow solid, yield 55%, mass 968 (M+H ⁺ ). ¹ H NMR (400 MHz, DMSO) δ 12.03 (s, 1H), 8.97 (s, 1H), 8.45-8.41 (m, 1H), 8.34 - 8.32 (m, 1H), 7.86-7.84 (m, 1H) , 7.65-7.63 (m, 1H), 7.48-7.35 (m, 8H), 7.06–6.93 (m, 2H), 6.91-6.89 (m, 1H), 6.27 (s, 1H), 5.17 (s, 1H) , 4.98–4.87 (m, 1H), 4.55-4.52 (m, 1H), 4.47-4.45 (m, 1H), 4.25 (s, 1H), 4.02–3.87 (m, 2H), 3.68-3.63 (m) , 10H), 3.53 (s, 3H), 2.41 (s, 3H), 2.06 - 1.99 (m, 1H), 1.85 - 1.77 (m, 1H), 1.45-1.39 (m, 3H), 0.93 (s, 9H) ).

Example 3, Compound 8 (2S,4R)-1-((S)-15-tert-butyl-1-(4-(2,4-difluorophenoxy)-3-(6-methyl-7) -oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridin-4-yl)phenyl)-1,13-dioxo-5,8,11-trioxo-2 ,14-diazahexadecyl-16-carbonyl)-4-hydroxy-N-((S)-1-4-(4-methylthiazol-5-yl)phenyl)ethyl)tetrahydropyrrole Preparation of -2-amide

In the same manner as in Example 1, an intermediate compound 23 was added to a 50 mL reaction flask (according to the synthesis method of the intermediate compound 20, wherein the step (12) was synthesized from diethylene glycol instead of the intermediate compound 13) (100 mg, 0.18) Ment), intermediate compound 21 (synthesized according to US20170327469) (84 mg, 0.18 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate The ester HATU (76 mg, 0.2 mmol), N,N-diisopropylethylamine DIPEA (52 mg, 0.4 mmol), 10 mL DMF, EtOAc. concentrated and column chromatography to give 100mg product 8 as a pale yellow solid, in 56% yield, mass spectrum 1012 (M ⁺ H +). ¹ H NMR (400 MHz, DMSO) δ 12.01 (s, 1H), 8.99 (s, 1H), 8.43-8.40 (m, 1H), 8.36-8.33 (m, 1H), 7.88-7.85 (m, 1H) , 7.66-7.64 (m, 1H), 7.46-7.37 (m, 8H), 7.05–6.98 (m, 2H), 6.93-6.88 (m, 1H), 6.25 (s, 1H), 5.15 (s, 1H) , 4.97–4.89 (m, 1H), 4.57-4.53 (m, 1H), 4.48-4.46 (m, 1H), 4.23 (s, 1H), 4.01–3.89 (m, 2H), 3.69-3.65 (m, 14H), 3.51(s,3H), 2.47(s,3H), 2.08–1.97(m,1H),1.88–1.79(m,1H),1.47-1.37(m,3H),0.96(s,9H) .

Example 4, Compound 11 (2S, 4R)-1-((S)-2-(2-(3-(2-((4-(2)4-difluorophenoxy)-3-(6-) Methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridin-4-yl)phenyl)amino)-2-oxoethoxy)propan)acetamide Preparation of bis(tert-butyl)-4-hydroxy-N-((S)-1-4-(4-methylthiazol-5-yl)phenyl)ethyl)tetrahydropyrrole-2-amide

(1) Synthesis of intermediate 24 2-bromo-1-(2,4-difluorophenoxy)-4-nitrobenzene

To a 250 mL reaction flask was added 3-bromo-4-fluoronitrobenzene (5.5 g, 25 mmol), 2,4-difluorophenol (5 g, 38 mmol), cesium carbonate (12.5 g, 50 mmol) and 100 mL DMSO. Degree, stir for 4 hours. After completion of the reaction, 200 mL of water was added, filtered, and dried to give Intermediate Compound 24 (5.1 g).

(2) Synthesis of intermediate 25 3-bromo-4-(2,4-difluorophenoxy)aniline

Intermediate compound 24 (2 g, 6 mmol), iron powder (1.8 g, 32 mmol), 20 mL of tetrahydrofuran, 20 mL of ethanol and 10 mL of water were added to a 250 mL reaction flask, and the mixture was warmed to 100 ° and stirred for 4 hours. After completion of the reaction, ethyl acetate was extracted and dried over anhydrous sodium sulfate.

(3) Intermediate 26 4-(5-Amino-2-(2,4-difluorophenoxy)phenyl)-6-methyl-1-p-toluenesulfonyl-1,6-dihydro-7H Synthesis of pyrrolo[2,3-c]pyridine-7-one

Intermediate compound 25 (500 mg, 1.6 mmol), intermediate compound 6 (750 mg, 1.75 mmol), tetratriphenylphosphine palladium (168 mg, 0.146 mmol), potassium carbonate (400 mg, 2.9 mmol) was added to a 100 mL reaction flask. And 50 mL of N,N-dimethylformamide and 1-2 drops of water, three times with nitrogen, warmed to 90 ° C, and stirred overnight. After the reaction was completed, 50 mL of water was added, and ethyl acetate was evaporated.

(4) Intermediate 27 4-(5-Amino-2-(2,4-difluorophenoxy)phenyl)-6-methyl-1,6-dihydro-7H-pyrrolo[2,3 Synthesis of -c]pyridine-7-one

Intermediate compound 26 (450 mg, 0.86 mmol), 5N KOH, 5 mL of tetrahydrofuran was added to a 50 mL reaction flask, and the mixture was warmed to 80 ° C and stirred for 4 hours. After completion of the reaction, the tetrahydrofuran was concentrated to remove the residue, and then the mixture was filtered.

(5) Synthesis of intermediate 28 2-(3-hydroxypropoxy)acetic acid ethyl acetate

Add propylene glycol (10g, 131mmol) to a 250mL reaction flask, 100mL dichloromethane, add diazoacetic acid ethyl acetate (3g, 26mmol), add three drops of boron trifluoride etherate solution, stir for 30 minutes, the reaction is completed Thereafter, 100 mL of water was added, and the organic layer was dried over anhydrous sodium sulfate.

(6) Synthesis of intermediate 29 2-(3-(2-ethoxy-2-oxoethoxy)propoxy)acetic acid tert-butyl ester

Intermediate compound 28 (1.2 g, 7.4 mmol) was added to a 250 mL reaction flask, 50 mL of dichloromethane, and ethyl diazoacetate (1.2 g, 8.4 mmol) was added, and three drops of boron trifluoride etherate solution were added. After stirring for 30 minutes, after completion of the reaction, 50 mL of water was added, and the organic layer was dried over anhydrous sodium sulfate.

(7) Synthesis of intermediate 30 2-(3-(2-(tert-butoxy)-2-oxoethoxy)propoxy)acetic acid

The intermediate compound 29 (1.1 g, 4 mmol), 4N potassium hydroxide, 10 mL of water and 10 mL of tetrahydrofuran was added to a 100 mL reaction flask, and stirred at room temperature for 30 minutes. After completion of the reaction, the pH was adjusted to 4 with 2N HCl and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and evaporated

(8) Intermediate 31 (3-(2-((4-(2,4-difluorophenoxy)-3-(6-methyl-7-oxo-6,7-dihydro-1H-) Synthesis of pyrrolo[2,3-c]pyridin-4-yl)phenyl)amino)-2-oxoethoxy)propoxy)acetate

Intermediate compound 27 (240 mg, 0.8 mmol), intermediate compound 28 (240 mg, 0.96 mmol), 2-(7-azobenzotriazole)-N,N,N', was added to a 50 mL reaction flask. N'-Tetramethylurea hexafluorophosphate HATU (480 mg, 1.26 mmol), N,N-diisopropylethylamine DIPEA (164 mg, 1.26 mmol), 10 mL DMF, stirred at room temperature overnight, 10 mL water, acetic acid The ethyl ester was extracted, dried over anhydrous sodium sulfate, and evaporated

(9) Intermediate 32 2-(3-(2-((4-(2,4-difluorophenoxy)-3-(6-methyl-7-oxo-6,7-dihydro-) Synthesis of 1H-pyrrolo[2,3-c]pyridin-4-yl)phenyl)amino)-2-oxoethoxy)propoxy)acetic acid

Intermediate compound 31 (170 mg, 0.28 mmol), 10 mL of formic acid was added to a 50 mL reaction flask, warmed to 40 ° and stirred overnight. After completion of the reaction, concentration was carried out, and the title compound was obtained to afford Intermediate Compound 32 (130 mg, yield: 85%).

(10) Synthesis of Compound 11

Intermediate compound 32 (100 mg, 0.18 mmol), intermediate compound 21 (synthesized according to US20170327469) (84 mg, 0.18 mmol), 2-(7-azobenzotriazole)-N, was added to a 50 mL reaction flask. N,N',N'-tetramethyluronium hexafluorophosphate HATU (76 mg, 0.2 mmol), N,N-diisopropylethylamine DIPEA (52 mg, 0.4 mmol), 10 mL DMF, After adding 10 mL of water, ethyl acetate was extracted, dried over anhydrous sodium sulfate. ¹ H NMR (400 MHz, DMSO) δ 12.02 (s, 1H), 9.79 (s, 1H), 8.98 (s, 1H), 8.44 - 8.42 (m, 1H), 7.84-7.83 (m, 1H), 7.62 -7.61 (m, 1H), 7.49-7.34 (m, 8H), 7.08–6.93 (m, 2H), 6.91-6.9 (m, 1H), 6.28 (s, 1H), 5.14 (s, 1H), 4.95 – 4.80 (m, 1H), 4.55-4.53 (m, 1H), 4.44-4.43 (m, 1H), 4.28 (s, 1H), 4.06 (s, 2H), 4.01 – 3.86 (m, 2H), 3.65 -3.61 (m, 6H), 3.53 (s, 3H), 2.45 (s, 3H), 2.09 - 1.99 (m, 1H), 1.94 - 1.82 (m, 2H), 1.81 - 1.70 (m, 1H), 1.4 -1.36 (m, 3H), 0.89 (s, 9H).

Example 5, Compound 14 (2S, 4R)-1-((S)-2-(2-(2-(2-((4-(2)4-difluorophenoxy)-3-(6-) Methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridin-4-yl)phenyl)amino)-2-oxoethoxy)propan)acetamide Preparation of bis(tert-butyl)-4-hydroxy-N-((S)-1-4-(4-methylthiazol-5-yl)phenyl)ethyl)tetrahydropyrrole-2-amide

Intermediate compound 33 was added to a 50 mL reaction flask (according to the synthesis method of intermediate compound 32, ethylene glycol was used instead of propylene glycol as a raw material (100 mg, 0.18 mmol), and intermediate compound 21 (synthesized according to US20170327469) (84 mg, 0.18 mmol) , 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate HATU (76 mg, 0.2 mmol), N,N-diisopropyl Ethylamine DIPEA (52 mg, 0.4 mmol), 10 mL DMF, EtOAc EtOAc. 54%, mass 955 (M+H+). ¹ H NMR (400 MHz, DMSO) δ 12.03 (s, 1H), 9.77 (s, 1H), 8.96 (s, 1H), 8.45-8.41 (m, 1H) ), 7.86-7.85 (m, 1H), 7.63-7.62 (m, 1H), 7.48-7.33 (m, 8H), 7.07–6.95 (m, 2H), 6.93-6.91 (m, 1H), 6.25 (s) , 1H), 5.11 (s, 1H), 4.91 - 4.85 (m, 1H), 4.57 - 4.51 (m, 1H), 4.46 - 4.42 (m, 1H), 4.27 (s, 1H), 4.05 (s, 2H) ), 4.01–3.88 (m, 2H), 3.69-3.62 (m, 6H), 3.51 (s, 3H), 2.47 (s, 3H), 2.05–1.97 (m, 1H), 1.80–1.75 (m, 1H) ), 1.42-1.38 (m, 3H), 0.92 (s, 9H).

Example 6. Compound 17 (2S,4R)-1-((S)-2-tert-butyl-14-((4-(2,4-difluorophenoxy)-3-(6-methyl-) 7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridin-4-yl)phenyl)amino)-4,14-dioxo-6,9,12-three Oxy-3-nitrotridecyl-1-carbonyl)-4-hydroxy-N-((S)-1-4-(4-methylthiazol-5-yl)phenyl)ethyl)tetrahydropyrrole Preparation of -2-amide

Intermediate compound 34 was added to a 50 mL reaction flask (according to the synthesis method of intermediate compound 32, diethylene glycol was used instead of propylene glycol as raw material (100 mg, 0.18 mmol), and intermediate compound 21 (synthesized according to US20170327469) (84 mg, 0.18) Ment), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate HATU (76 mg, 0.2 mmol), N,N-diisopropyl Ethylethylamine DIPEA (52 mg, 0.4 mmol), 10 mL DMF, EtOAc EtOAc EtOAc. The rate is 55%, mass spectrum 999 (M+H+). ¹ H NMR (400 MHz, DMSO) δ 11.98 (s, 1H), 9.79 (s, 1H), 8.91 (s, 1H), 8.47-8.43 (m, 1H), 7.87-7.83 (m, 1H), 7.65-7.61 (m, 1H), 7.49-7.37 (m, 8H), 7.09–6.99 (m, 2H), 6.95-6.92 (m, 1H), 6.26 ( s, 1H), 5.13 (s, 1H), 4.93 - 4.87 (m, 1H), 4.59 - 4.53 (m, 1H), 4.47 - 4.45 (m, 1H), 4.29 (s, 1H), 4.07 (s, 2H), 4.03–3.89 (m, 2H), 3.67-3.61 (m, 10H), 3.53 (s, 3H), 2.48 (s, 3H), 2.07–1.99 (m, 1H), 1.83–1.79 (m, 1H), 1.44-1.37 (m, 3H), 0.9 (s, 9H).

Example 7, Compound 29 (2S,4R)-1-((S)-2-(2-(3-(2-(4-(4-(ethanesulfonamide))-2-(6-methyl-) 7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridin-4-yl)phenoxy)-3-fluorophenoxy)ethoxy)propan)acetamide -3,3-dimethylbutoxy)-4-hydroxy-N-((S)-1-4-(4-methylthiazol-5-yl)phenyl)ethyl)tetrahydropyrrole- Preparation of 2-amide

(1) Intermediate 36 N-(4-(2-Fluoro-4-methoxyphenoxy)-3-(6-methyl-7-oxo-1-p-toluenesulfonyl-6,7- Synthesis of Dihydro-1H-pyrrolo[2,3-c]pyridin-4-yl)phenyl)ethanesulfonamide

Intermediate compound 35 was added to a 50 mL reaction flask (according to the synthesis method of intermediate compound 26, 2-fluoro-4-methoxyphenol was used instead of 2,4-difluorophenol as a raw material (1 g, 1.87 mmol), three Ethylamine (283 mg, 2.8 mmol) and 20 mL of dichloromethane were added to ethyl sulfonyl chloride (360 mg, 2.8 mmol). After the reaction was completed, 20 mL of water was added, dichloromethane was evaporated, dried over anhydrous sodium sulfate After chromatography, intermediate compound 36 (0.95 g, yield 84%) was obtained.

(2) Intermediate 37 N-(4-(2-Fluoro-4-methoxyphenoxy)-3-(6-methyl-7-oxo-6,7-dihydro-1H-pyrrole Synthesis of [2,3-c]pyridin-4-yl)phenyl)ethanesulfonamide

Intermediate compound 36 (0.9 g, 1.43 mmol), 5NKOH, 5 mL of tetrahydrofuran was added to a 50 mL reaction flask, and the mixture was warmed to 80 ° C and stirred for 4 hours. After completion of the reaction, the tetrahydrofuran was concentrated to remove the residue, and then the mixture was evaporated to ethyl ether.

(3) Intermediate 38 N-(4-(2-Fluoro-4-hydroxyphenoxy)-3-(6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2 Synthesis of 3-(3-pyridin-4-yl)phenyl)ethanesulfonamide

Intermediate compound 37 (0.6 g, 1.27 mmol), 10 mL of HBr aqueous solution, 10 mL of tetrahydrofuran was added to a 50 mL reaction flask, and the mixture was warmed to 80 ° C and stirred for 4 hours. After completion of the reaction, ethyl acetate was extracted, dried over anhydrous sodium sulfate

(4) Intermediate 39 2-(3-(2-(4-(4-(ethylsulfonylamino))-2-(6-methyl-7-oxo-6,7-dihydro-1H- Synthesis of pyrrolo[2,3-c]phenoxy)-3-fluorophenoxy)ethoxy)propoxy)acetate

Intermediate compound 38 (400 mg, 0.87 mmol), intermediate compound 15 (180 mg, 0.87 mmol), triphenylphosphine (262 mg, 1 mmol) was added to a 50 mL reaction flask, and diisopropyl azodicarboxylate was added to the ice bath. The ester (220 mg, 1.1 mmol) was stirred for 4 h. After the reaction was completed, ethyl acetate was evaporated.

(5) Intermediate 40 2-(3-(2-(4-(4-(ethylsulfonamido))-2-(6-methyl-7-oxo-6,7-dihydro-1H- Synthesis of pyrrolo[2,3-c]pyridin-4-yl)phenoxy)-3-fluorophenoxy)ethoxy)propoxy)acetic acid

Intermediate compound 39 (220 mg, 0.34 mmol), 5NKOH, 5 mL of tetrahydrofuran was added to a 50 mL reaction flask, and the mixture was warmed to 80 ° C and stirred for 4 hours. After the reaction was completed, the tetrahydrofuran was concentrated to remove the residue, and then the mixture was evaporated to ethyl ether.

(6) Synthesis of Compound 29

Intermediate compound 40 (100 mg, 0.18 mmol), intermediate compound 21 (synthesized according to US20170327469) (84 mg, 0.18 mmol), 2-(7-azobenzotriazole)-N, was added to a 50 mL reaction flask. N,N',N'-tetramethyluronium hexafluorophosphate HATU (76 mg, 0.2 mmol), N,N-diisopropylethylamine DIPEA (52 mg, 0.4 mmol), 10 mL DMF, After adding 10 mL of water, ethyl acetate was evaporated, dried over anhydrous sodium sulfate. ¹ H NMR (400 MHz, DMSO) δ 12.02 (s, 1 H), 9.58 (s, 1H), 8.98 (s, 1H), 8.44 - 8.42 (m, 1H), 7.84-7.83 (m, 1H), 7.62 -7.61 (m, 1H), 7.49-7.34 (m, 8H), 7.08–6.93 (m, 2H), 6.91-6.9 (m, 1H), 6.28 (s, 1H), 5.14 (s, 1H), 4.95 – 4.80 (m, 1H), 4.55-4.53 (m, 1H), 4.44-4.43 (m, 1H), 4.28 (s, 1H), 4.06 (s, 2H), 4.01–3.86 (m, 2H), 3.65 -3.61(m,8H),3.53(s,3H),3.07-3.04(m,2H),2.45(s,3H),2.09–1.99(m,1H),1.94–1.82(m,2H),1.81 –1.70 (m, 1H), 1.4-1.36 (m, 3H), 1.25-1.23 (m, 3H), 0.89 (s, 9H).

Example 8, Compound 37 (2S,4R)-1-S)-13-(tert-butyl)-1-4-(2,4-difluorophenoxy)-3-(6-methyl-7 -oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridin-4-yl)phenyl)amino)-1,11 dioxo-5,9-dioxa-2 ,12-diazatetradecane-14 yl)-4-hydroxy N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2- Preparation of formamide

(1) Intermediate 41 2-(3-(2-(3-(4-(2,4-difluorophenoxy)-3-(6-methyl-7-oxo-1-toluenesulfonyl) Synthesis of ethyl -6,7-dihydro-1H-pyrrolo[2,3-3-c]pyridin-4-yl)phenyl)ureido)ethoxy)propoxy)acetate

To a 50 mL reaction flask was added triphosgene (88 mg, 0.296 mmol), 5 mL of tetrahydrofuran, and an intermediate compound 18 (220 mg, 1.07 mmol) and triethylamine (60 mg, 0.6 mmol) in 5 mL of THF. In a minute, intermediate compound 26 (100 mg, 0.19 mmol) was added and stirred for 1 hour. After the reaction was completed, ethyl acetate was evaporated and dried over anhydrous sodium sulfate.

(2) Intermediate 42 2-(3-(2-(3-(4-(2,4-difluorophenoxy)-3-(6-methyl-7-oxo-6,7-di) Synthesis of hydrogen-1H-pyrrolo[2,3-c]pyridin-4-yl)phenyl)ureido)ethoxy)propoxy)acetic acid

Intermediate compound 41 (56 mg, 0.074 mmol), 5NKOH, 5 mL of tetrahydrofuran was added to a 50 mL reaction flask, and the mixture was warmed to 80 ° C and stirred for 4 hours. After completion of the reaction, the tetrahydrofuran was concentrated to remove the residue, and the mixture was evaporated to ethyl ether.

(3) Synthesis of Compound 37

Intermediate compound 42 (42 mg, 0.074 mmol), intermediate compound 21 (synthesized according to US20170327469) (36 mg, 0.074 mmol), 2-(7-azobenzotriazole)-N, was added to a 50 mL reaction flask. N,N',N'-tetramethyluronium hexafluorophosphate HATU (38 mg, 0.1 mmol), N,N-diisopropylethylamine DIPEA (26 mg, 0.2 mmol), 10 mL DMF, After adding 10 mL of water, ethyl acetate was evaporated, dried over anhydrous sodium sulfate. ¹ H NMR (400 MHz, DMSO) δ 12.00 (s, 1 H), 9.45 (s, 1H), 8.98 (s, 1H), 8.49-8.48 (m, 1H), 7.65-7.64 (m, 1H), 7.46 –7.16(m,8H),7.02-7.01(m,1H),6.93-6.92(m,3H), 6.86-6.84(m,2H),6.26(s,1H),4.96–4.83(m,1H) , 4.54-4.53 (m, 1H), 4.46-4.45 (m, 2H), 4.28 (s, 2H), 4.00–3.84 (m, 2H), 3.66–3.47 (m, 13H), 2.45 (s, 3H) , 2.1-2.08 (m, 1H), 1.84–1.71 (m, 1H), 1.36-1.33 (m, 3H), 0.90 (s, 9H).

Example 9. Compound 38 (2S,4R)-1-((S)-2-(2-(3-(2-(N-(4-(2)4-difluorophenoxy)-3-) (6-Methyl-7-oxo-6-1,7-dihydro-1H-pyrrolo[2,3-c]pyridin-4-yl)phenyl)ethylsulfonylamino)ethoxy) Propoxy)acetamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl) Preparation of pyrrolidine-2-carboxamide

(1) Intermediate 43 N-(4-(2,4-difluorophenoxy)-3-(6-methyl-7-oxo-1-p-toluenesulfonyl-6,7-dihydro- Synthesis of 1H-pyrrolo[2,3-c]pyridin-4-yl)phenyl)ethanesulfonamide

Intermediate compound 26 (1 g, 1.92 mmol), triethylamine (283 mg, 2.8 mmol) and 20 mL dichloromethane were added to a 50 mL reaction flask, and ethyl sulfonyl chloride (360 mg, 2.8 mmol) was added. After completion of the reaction, 20 mL of water was added, and the mixture was extracted with methylene chloride and dried over anhydrous sodium sulfate.

(2) Intermediate 44 2-(3-(2-(N-(4-(2,4-difluorophenoxy)-3-(6-methyl-7-oxo-1-toluenesulfonyl) Synthesis of ethyl -6,7-dihydro-1H-pyrrolo[2,3-3-c]pyridin-4-yl)phenyl)ethylsulfonylamino)ethoxy)propoxy)acetate

Add intermediate compound 43 (150 mg, 0.244 mmol), intermediate compound 16 (70 mg, 0.244 mmol), cesium carbonate (163 mg, 0.5 mmo), 10 mg potassium iodide and 20 mL DMSO, 10 mL water, and warm to 100 deg. Stir overnight. After completion of the reaction, 20 mL of water was added, and ethyl acetate was evaporated.

(3) Intermediate 45 2-(3-(2-(N-(4-(2,4-difluorophenoxy)-3-(6-methyl-7-oxo-6,7-di) Synthesis of hydrogen-1H-pyrrolo[2,3-c]pyridin-4-yl)phenyl)ethylsulfonylamino)ethoxy)propoxy)acetic acid

Intermediate compound 44 (110 mg, 0.137 mmol), 5NKOH, 5 mL of tetrahydrofuran was added to a 50 mL reaction flask, and the mixture was warmed to 80 ° C and stirred for 4 hours. After the reaction was completed, the THF was evaporated to dryness.

(4) Synthesis of Compound 38

Intermediate compound 45 (46 mg, 0.074 mmol), intermediate compound 21 (synthesized according to US20170327469) (36 mg, 0.074 mmol), 2-(7-azobenzotriazole)-N, was added to a 50 mL reaction flask. N,N',N'-tetramethyluronium hexafluorophosphate HATU (38 mg, 0.1 mmol), N,N-diisopropylethylamine DIPEA (26 mg, 0.2 mmol), 10 mL DMF, After adding 10 mL of water, ethyl acetate was evaporated, dried over anhydrous sodium sulfate. ^{1 H NMR (400MHz, DMSO)} δ11.98 (s, 1H), 8.91 (s, 1H), 8.42-8.4 (m, 1H), 7.81-7.8 (m, 1H), 7.65-7.63 (m, 1H) , 7.45-7.31 (m, 8H), 7.02–6.91 (m, 2H), 6.93-6.91 (m, 1H), 6.24 (s, 1H), 5.19 (s, 1H), 4.91–4.81 (m, 1H) , 4.59-4.55 (m, 1H), 4.47-4.45 (m, 1H), 4.24 (s, 1H), 4.01 (s, 2H), 4.0–3.89 (m, 2H), 3.67-3.62 (m, 8H) , 3.55 (s, 3H), 3.09 - 3.01 (m, 2H), 2.47 (s, 3H), 2.05 - 1.93 (m, 1H), 1.97 - 1.89 (m, 2H), 1.85 - 1.73 (m, 1H) , 1.45-1.39 (m, 3H), 1.29-1.25 (m, 3H), 0.93 (s, 9H).

Example 10, Compound 39 (2S,4R)-1-((S)-13-(tert-butyl)-1-(4-fluoro-3-(6-methyl-7-oxo-6,7) -dihydro-1H-pyrrolo[2,1,3-c]pyridin-4-yl)phenyl)-1,11 dioxo-5,9-dioxa-2,12-diazatetradecane Synthesis of alkyl-14 acyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

Intermediate compound 46 was added to a 50 mL reaction flask (synthesized from 3-bromo-4-fluorobenzoic acid methyl ester in place of intermediate compound 8 according to the synthesis method of intermediate compound 20) (78 mg, 0.18 mmol), intermediate Compound 21 (synthesized according to US20170327469) (84 mg, 0.18 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate HATU (76 mg, 0.2 mmol), N,N-diisopropylethylamine DIPEA (52 mg, 0.4 mmol), 10 mL DMF, EtOAc EtOAc. Yield 58 mg of product 39 as a pale yellow solid (yield: 37.5%), mass spectrum 858 (M+H ⁺ ). ¹ H NMR (400 MHz, DMSO) δ 12.14 (s, 1H), 8.98 (s, 1H), 8.60-8.58 (m, 1H), 8.44-8.43 (m, 1H), 8.02-8.01 (m, 1H) , 7.95–7.85 (m, 1H), 7.49–7.26 (m, 10H), 6.16 (s, 1H), 5.14–5.13 (m, 1H), 4.95–4.80 (m, 1H), 4.53-4.52 (m, 1H), 4.44-4.42 (m, 1H), 4.28 (s, 1H), 3.89-3.88 (m, 2H), 3.59 (s, 3H), 3.55–3.43 (m, 9H), 2.45 (s, 4H) , 2.04-2.03 (m, 1H), 1.85–1.69 (m, 1H), 1.36-1.34 (m, 3H), 0.92 (s, 9H).

The beneficial effects of the present invention will be described below by way of test examples.

Test Example 1. Biological determination of the inhibitory effect of the compound of the present invention on proliferation of CWR22RV1 cells

(1) Experimental materials:

CWR22RV1 cells (Chinese Academy of Sciences Cell Bank, TCHu100)

FBS (Gibco, Cat. No. 10099-141)

0.01M PBS (Biosharp, Cat. No. 162262)

RIPM1640 (Hyclone, Cat. No. 308090.01)

Penicillin-streptomycin solution (Hyclone, Cat. No. SV30010)

Cell counting kit-8 (Signalway Antibody, Cat. No.CP002)

DMSO (Sigma, Cat. No. D5879)

Centrifuge tube, 15ml (Excell Bio, Cat.No.CS015-0001)

Petri dish, (Excell Bio, Cat. No. CS016-0128)

96-well cell culture cluster (Corning, Cat. No. 3599)

(2) Experimental method

A. Buffer preparation

B. Experimental steps

1CWR22RV1 cells were subcultured with cell culture medium, and cells grown in good condition were inoculated into 96-well plates at 80 μL per well, and the number of cells per well was 1500, and cultured overnight at 37 ° C in a 5% CO ₂ cell incubator.

2 The drug was formulated with dimethyl sulfoxide (DMSO) as a 30 mM stock solution. Dilute 3 times with DMSO before use, then dilute by 3 times to obtain 9 concentration gradients, and then dilute the compound at each concentration 200 times with the culture solution (to ensure the DMSO concentration in the culture system is 0.1%). The concentration is made to repeat 2 holes. 20 μL of the diluted compound was added to the cell culture well (final concentration of 10 μM, 3.3 μM, 1.1 μM...), and gently mixed by shaking. In addition, three cell-only negative control wells and three blank control wells (only 20 μL of culture medium diluted with 200 μL of DMSO) were placed.

(3) Result detection

1 After 6 days of culture, 10 μL of CCK-8 was added to each well, and incubation was continued for 2.5 hours at 37 ° C in a 5% CO ₂ cell incubator.

2 The absorbance (OD value) was measured at 450 nm using a multi-function microplate reader.

3 Data were analyzed by the Dose-response-inhibition equation in the software GraphPad Prism6, and the IC50 value was obtained.

The IC50 (nM) of the inhibition of the activity of the compound of the present invention on CWR22RV1 is shown in Table 1.

IC50 of inhibition of CWR22RV1 activity by the compounds of Table 1

Compound	IC50(nM)
Compound 2	6
Compound 5	8
Compound 8	8
Compound 11	2
Compound 14	5
Compound 17	8
Compound 29	250

Compound 37	15000
Compound 38	1.6
Compound 39	3

Test Example 2: Biological determination of protein expression of BRD4 (Bromodomain containing protein 4) of the present invention

(1) Experimental materials

CWR22RV1 cells (Chinese Academy of Sciences Cell Bank, TCHu100)

FBS (Gibco, Cat. No. 10099-141)

0.01M PBS (Biosharp, Cat. No. 162262)

RIPM1640 (Hyclone, Cat. No. 308090.01)

Penicillin-streptomycin solution (Hyclone, Cat. No. SV30010)

DMSO (Sigma, Cat. No. D5879)

Centrifuge tube, 15ml (Excell Bio, Cat.No.CS015-0001)

Petri dish, (Excell Bio, Cat. No. CS016-0128)

6-well cell culture cluster (Corning, Cat. No. 3516)

RIPA Lysis Buffer (Beyotime, Cat. No. P0013B)

Protein Buffer Buffer (Beyotime, Cat.No.P0015L)

BCA Protein Quantification Kit (Beyotime, Cat.No.P0012)

SDS-PAGE Gel Preparation Kit (Chengdu Yihe Technology Co., Ltd., Cat.No.PG112)

Anti-β-tubulin mouse monoclonal antibody (Zen Bioscience, Cat. No.200608)

Anti-BRD4 (E2A7X) rabbit monoclonal antibody (CST, Cat. No.13440)

Peroxidase Affinipure (HRP) goat anti-mouse IgG (Zen Bioscience, Cat. No. 511103)

Peroxidase Affinipure (HRP) goat anti-mouse IgG (Zen Bioscience, Cat. No. 511203)

TBST (Biosharp, Cat. No. BL601A)

ECL Chemiluminescence Kit (Beyotime, Cat.No.P0018)

(2) Experimental method

A. Buffer preparation

B. Experimental steps

1CWR22RV1 cells were subcultured with cell culture medium, and cells grown in good condition were inoculated into 6-well plates, 2 ml per well, and the number of cells per well was 1 million, and cultured overnight at 37 ° C in a 5% CO ₂ cell incubator.

2 The drug was formulated with dimethyl sulfoxide (DMSO) as a 30 mM stock solution. Dilute 3 times with DMSO before use, and add 2 μl of the diluted compound to the cell culture well (to ensure that the DMSO concentration in the culture system is 0.1%). Repeat 2 wells for each concentration and mix gently by shaking. A negative control well (plus equal amount of DMSO) and a positive control well were also set.

3 After 24 hours of culture, cells were lysed with RIPA cell lysate, proteins were extracted, and protein concentration was measured using a BCA kit. Add 5 times concentrated protein loading buffer, heat at 100 ° C for 5 minutes and store the sample at -20 ° C.

4 The amount of protein per well of 30 μg was applied to a polyacrylamide gel and electrophoresed.

5 Protein was transferred from polyacrylamide gel to PVDF membrane, blocked with 5% skim milk for 1 hour at room temperature, primary antibody (Anti-BRD4 (E2A7X) rabbit mAb and Anti-β-Tubulin Mouse mAb) incubated overnight at 4 °C, TBST The solution was washed three times for 10 minutes each time, and the secondary antibody (horseradish peroxidase-labeled goat anti-mouse IgG) was incubated for 2 hours at room temperature, and the membrane was washed three times with TBST solution for 10 minutes each time.

(3) Result detection

Finally, the color of the ECL coloring solution was added, and the image was taken with an automatic chemiluminometer, and the pictures were collected and analyzed. The degradation of the BRD4 protein by the compounds of the present invention is shown in Figure 1.

It can be seen from Fig. 1 that the compound provided by the present invention has a good inhibitory effect on the proliferation of 22Rv1 cells, and the inhibitory effect is particularly good, indicating that the compound of the present invention can be prepared as an antitumor drug, particularly a drug for treating prostate cancer.

In summary, the present invention provides a chimeric molecule consisting of a small molecule compound unit of a target protein, an E3 ubiquitin ligase binding unit, and a linking unit, which is capable of binding to a BRD protein, thereby facilitating degradation of the BRD protein by proteases. In order to inhibit cell proliferation, it can be used as a drug for the degradation of BRD protein for the treatment of cancer or coronary artery disease.

Claims (34)

1. a compound represented by the formula (I), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof:

   M-L-N (I)

   Wherein L is a linking unit, and one atom on M and N is substituted by one end of the linking group L, respectively;

   Unit M is as shown in equation (a) or (c):

   

   Unit N is as shown in equation (b):

   

   Wherein one of R ₁ and R ₂ is selected from the group consisting of halogen, -NH-SO ₂ -R ₇ , hydrogen, cyano, nitro, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl a heterocyclic aryl group, wherein said alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heterocyclic aryl group is independently independently optionally further selected from one or more selected from the group consisting of ruthenium atoms Substituted by a halogen, cyano, nitro, oxo, alkyl, haloalkyl, hydroxyalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heterocyclic aryl substituent ;

   Wherein R _{7 is} selected from the group consisting of hydrogen, cyano, nitro, alkyl, cycloalkyl, heterocycloalkyl, aryl, heterocyclic aryl;

   Another structure of R ₁ and R ₂ is selected from the group consisting of -A-, wherein A is selected from -O-, -COX- or -XCO-, -NH-, -HNCONH-, -NH-SO ₂ -R ₇ , - SO ₂ - or -CH ₂ -, X is selected from -CH ₂ -, -NH-, -SO ₂ -, -O-, acylamino, ester or carbonyl, and A is bonded to linking unit L;

   R ₃ , R ₄ and R ₆ are each independently selected from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heterocyclic aryl, wherein Said alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heterocyclic aryl group, respectively, optionally further optionally one or more selected from the group consisting of a halogen atom, a halogen, a cyano group, a nitrate Substituted with a substituent of an oxo group, an oxo group, an alkyl group, a haloalkyl group, a hydroxyalkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heterocyclic aryl group;

   R _{5 is} selected from the group consisting of alkyl, alkoxy, amino, amido, ester, carbonyl, and any combination of the above.
2. The compound according to claim 1, wherein one of R ₁ and R ₂ is selected from the group consisting of halogen, -NH-SO ₂ -R _{7 ,} hydrogen, and another of R ₁ and R ₂ is selected from -A-. Wherein A is selected from -O-, -COX- or -XCO-, X is selected from -CH ₂ -, -NH-, -SO ₂ -, -O-, acylamino, ester or carbonyl, and R _{7 is} selected from Hydrogen, cyano, nitro, alkyl, cycloalkyl, heterocycloalkyl, aryl, heterocyclic aryl.
3. The compound according to claim 2, wherein said X is selected from the group consisting of -CH ₂ -, -NH-, -O-, an ester group or a carbonyl group.
4. The compound according to claim 1, wherein said M structure is represented by formula (a1) or formula (a2):

   

   Wherein A _{1 is} selected from -COX- or -XCO-, and X is selected from -CH ₂ -, -NH-, -O-, ester or carbonyl;

   R ₃ and R ₆ are each independently selected from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heterocyclic aryl, wherein The alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heterocyclic aryl groups are each independently optionally further selected from one or more selected from the group consisting of a halogen atom, a halogen, a cyano group, a nitro group, and an oxygen group. Substituted with a substituent of an alkyl group, an alkyl group, a halogenated alkyl group, a hydroxyalkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heterocyclic aryl group;

   R _{7 is} selected from the group consisting of hydrogen, cyano, nitro, alkyl, cycloalkyl, heterocycloalkyl, aryl, heterocyclic aryl.
5. The compound according to claim 4, wherein said R _{6 is} selected from the group consisting of C ₁ -C ₄ alkyl groups; preferably, said R _{6 is} selected from the group consisting of methyl groups.
6. The compound according to claim 4, wherein said R _{7 is} selected from the group consisting of C ₁ -C ₄ alkyl groups; preferably, said R ₇ is an ethyl group.
7. The compound according to claim 1, wherein said M structure is represented by formula (a3) or formula (a4):

   

   Wherein A _{1 is} selected from -COX- or -XCO-, and X is selected from -CH ₂ -, -NH-, -O-, ester or carbonyl;

   R _{3 is} selected from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heterocyclic aryl, wherein said alkyl, alkenyl, The alkynyl, cycloalkyl, heterocycloalkyl, aryl or heterocyclic aryl group, respectively, is optionally further optionally further selected from one or more selected from the group consisting of a halogen atom, a halogen, a cyano group, a nitro group, an oxo group, an alkyl group, Substituted by a substituent of a haloalkyl group, a hydroxyalkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heterocyclic aryl group.
8. The compound according to claim 7, wherein R _{3 is} selected from the group consisting of hydrogen, halogen, and alkyl.
9. The compound according to claim 1, wherein said M structure is represented by (a5) or (a6):

   

   Wherein A _{1 is} selected from -COX- or -XCO-; and X is selected from -CH ₂ -, -NH-, -O-, ester or carbonyl.
10. The compound according to claim 9, wherein said X is selected from the group consisting of -NH- or -O-.
11. The compound according to claim 10, wherein said M structure is:

    
12. The compound according to claim 1, wherein said L is selected from the group consisting of -(CH ₂ CH ₂ OCH ₂ )n-CO-, -(CH ₂ CH ₂ O)n-CH ₂ CO-, -(CH ₂ OCH ₂ CH ₂ CH ₂ O)n-CH ₂ CO-, -(CH ₂ OCH ₂ )n-CO-, -(CH ₂ OCH ₂ CH ₂ )n-CH ₂ CO- or -(CH ₂ CH ₂ OCH ₂ ) n-CH ₂ CO-, wherein n is an integer from 0 to 20.
13. The compound according to claim 12, wherein n is an integer of from 1 to 3.
14. The compound according to claim 1, characterized in that R _{4 is} selected from H or CH ₃ (S or R).
15. The compound according to claim 1, characterized in that said R _{5 is} selected from an alkyl group or an amino group.
16. The compound of claim 1 wherein said N structure is

    

    Wherein R _{4 is} selected from H or CH ₃ (S or R).
17. The compound according to claim 16, wherein said N structure is represented by (b1), (b2) or (b3);

    
18. The compound according to claim 1, wherein the structure of the compound is as shown in formula (II):

    

    L is as defined in claim 12 or 13.
19. The compound according to claim 1, wherein the structure of the compound is as shown in formula (III):

    

    L is as defined in claim 12 or 13.
20. The compound according to claim 1, wherein the structure of the compound is as shown in formula (IV):

    

    L is as defined in claim 12 or 13.
21. The compound according to claim 1, wherein the structure of the compound is as shown in formula (V):

    

    L is as defined in claim 12 or 13.
22. The compound according to claim 1, wherein the structure of the compound is as shown in formula (VI):

    

    L is as defined in claim 12 or 13.
23. The compound according to claim 1, wherein the structure of the compound is as shown in formula (VII):

    

    L is as defined in claim 12 or 13.
24. The compound according to claim 1, wherein the structure of the compound is as shown in formula (VIII):

    

    L is as defined in claim 12 or 13.
25. The compound according to claim 1, wherein the structure of the compound is as shown in formula (IX):

    

    L is as defined in claim 12 or 13.
26. The compound according to claim 1, wherein the structure of the compound is as shown in formula (X):

    

    L is as defined in claim 12 or 13.
27. The compound according to claim 1, wherein the structure of the compound is as shown in the formula (X I):

    

    L is as defined in claim 12 or 13.
28. The compound according to claim 1, wherein the structure of the compound is as shown in formula (XII):

    

    L is as defined in claim 12 or 13.
29. The compound according to claim 1, wherein the structure of the compound is as shown in formula (XIII):

    

    L is as defined in claim 12 or 13.
30. A compound according to claim 1 wherein the compound is one of the following compounds:

    

    

    

    

    

    

    

    

    
31. Use of a compound according to any one of claims 1 to 30, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof, for the manufacture of a BRD4 inhibitor or a protein degradation agent.
32. The use according to claim 31, characterized in that the medicament is a medicament for treating cancer or coronary artery disease.
33. The use according to claim 32, characterized in that the cancer is prostate cancer, non-small cell lung cancer, breast cancer, melanoma, leukemia or rectal cancer.
34. A pharmaceutical composition comprising the compound according to any one of claims 1 to 30, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof, as an active ingredient, Formulations prepared with pharmaceutically acceptable excipients.

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