CN118930520A - Novel histone acetyltransferase p300 bromodomain inhibitor and its pharmaceutical composition and application - Google Patents

Abstract

The present invention discloses a p300 bromodomain inhibitor and its derivatives, which have the structure shown in the following formula I: The present invention also discloses a pharmaceutical composition comprising the compound and its derivatives and its use as a p300 bromodomain inhibitor. The compound and its derivatives of the present invention can significantly inhibit the activity of histone acetyltransferase p300, have strong proliferation inhibition activity on multiple myeloma cells OPM‑2 and prostate cancer cells 22RV1, etc., have good metabolic stability, safety and drug-like properties, and can be used as candidate molecules for the development of anti-tumor drugs.

Description

Novel histone acetyl transferase p300 bromodomain inhibitor and pharmaceutical composition and application thereof

Technical Field

The invention belongs to the field of synthetic pharmaceutical chemistry, and particularly relates to a novel p300 bromodomain inhibitor, a pharmaceutical composition thereof and application thereof.

Background

Acetylation and deacetylation of histone lysine residues is a reversible post-translational modification that plays a critical role in regulating gene expression, and is an epigenetic key study. Histone acetylation is regulated by three enzymes, histone acetyltransferase (Histone acetyltransferase, HAT), histone deacetylase (Histone deacetylase, HDAC) and Bromodomain (BRD) containing protein (Bromodomain-containingprotein, BCP), respectively.

The protein modified by acetylation can carry different genetic information, so as to regulate gene expression. Acetylated lysine residues (ACETYLATED LYSINE, KAc) are specifically recognized by bromodomains. The bromodomain-containing protein can recognize the KAc of the histone through the bromodomain, and reads the genetic information carried by the histone after the posttranslational modification, thereby regulating and controlling the gene expression. Thus, bromodomains are currently very attractive targets for regulating gene expression, and the study of inhibitors thereof is an important direction in the development of oncologic drugs.

The p300/CBP family consisting of the highly homologous 300kDa protein of adenovirus E1A (adenoviral E1A binding protein of kDa, p 300) and the binding protein of the cyclic adenosine monophosphate response element binding protein (CREB binding protein, CBP) is one of the major members of the HAT family, often collectively referred to as p300/CBP due to its structural homology and similarity in function.

The histone acetyl transferase p300/CBP plays a vital role in epigenetic regulation, can participate in the acetylation process of the histone in the organism, and influences the acetylation and deacetylation balance of the histone. The p300/CBP with the bromodomain can specifically recognize KAc so as to read genetic information carried by the histone after the posttranslational modification, thus playing a role in epigenetic regulation of gene expression and being important for maintaining organism steady state.

Studies show that the abnormality of the p300/CBP bromodomain function is closely related to the occurrence and development of various tumors, such as prostate cancer, multiple myeloma, acute myelogenous leukemia, breast cancer and the like, and the targeting of the p300/CBP bromodomain gradually becomes a new method for treating cancer. The p300/CBP bromodomain inhibitors reported to date are of very few structural types and numbers, with only two candidate compounds entering the clinical study stage. One is FT-7051, developed by Forma Theraputics, which is currently in the phase I clinical study for the treatment of metastatic castration-resistant prostate cancer. Another is CCS1477 developed by CELLCENTRIC, inc., currently in the clinical phase I/II study for the treatment of acute myelogenous leukemia, non-Hodgkin's lymphoma, multiple myeloma, and metastatic castration-resistant prostate cancer. CCS1477 suffers from certain drawbacks, firstly the cell membrane permeability of CCS1477 is low and secondly the in vitro and in vivo half-life of CCS1477 is short. Therefore, based on the biological functions of the p300/CBP bromodomain and the biological activity of the inhibitor thereof, the p300/CBP bromodomain inhibitor with stronger activity, higher selectivity and better drug-like property is found to gradually become a research hotspot of various large pharmaceutical enterprises and scientific research institutions. The method has important significance for the research of p300/CBP biological functions and the development of epigenetic medicines.

Disclosure of Invention

The invention designs and synthesizes a series of novel p300 bromodomain inhibitors, provides a compound with a general formula (I) or derivatives such as pharmaceutically acceptable salts thereof as p300 small molecule inhibitors, and carries out biological evaluation on the compound, and discovers that the obtained substance has better inhibition activity at the molecular level and the cellular level than CCS1477 and good drug property and can be used as the p300 bromodomain inhibitors.

Biological experiment results show that the compound and the derivative thereof provided by the invention have better inhibition effect on molecular and cellular level than the clinical compound CCS1477, and the optimal compound shows strong tumor growth inhibition effect in a prostate cancer cell 22RV1 animal model, so that the tumor fading effect can be achieved under high administration dosage. And can significantly reduce the expression level of c-Myc in cells and in mice. Meanwhile, the compounds or the derivatives thereof have higher selectivity and better bioavailability as inhibitors, and have huge development potential of antitumor drugs.

The present invention provides in a first aspect a compound or derivative thereof (including for example deuterated, salts, isomers, crystalline forms or solvates thereof) characterised in that the compound has the structure shown in formula i:

R ₁、R₂、R₃ and R ₄ are each independently selected from hydrogen, deuterium, halogen, haloalkyl, alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, or cyano;

R ₅ is selected from a substituted or unsubstituted 3-10 membered saturated or unsaturated aliphatic ring or aliphatic heterocyclic ring or aliphatic parallel ring;

r ₆ is selected from hydrogen or halogen;

R ₇ is selected from a substituted or unsubstituted aromatic ring, aromatic heterocycle or aromatic parallel ring;

X is selected from a covalent bond, -CH ₂ -, or-CH ₂CH₂ -;

Y ₁ and Y ₂ are each independently selected from CH or N.

The present invention provides in a second aspect a pharmaceutical composition comprising: (1) The compound of the first aspect of the invention or a deuterated, salt, isomer, crystalline form or solvate thereof; and (2) a pharmaceutically acceptable carrier and/or excipient.

The present invention provides in a third aspect the use of a compound according to the first aspect of the invention or a deuterated, salt, isomer, crystalline form or solvate thereof as a p300 bromodomain inhibitor.

Biological experiments prove that the p300 bromodomain inhibitor with novel structure provided by the invention can obviously inhibit the activity of histone acetyltransferase p300, and the compound has excellent inhibition effect on various tumor cells including prostate cancer cells, leukemia cells, breast cancer cells and multiple myeloma cells (such as OPM-2), and the inhibition effect is even better than that of a target clinical in-research medicament CCS1477. The compound and the derivative thereof have good metabolic stability, safety and drug-like property, and can be used as candidate molecules for developing antitumor drugs. Therefore, the compound and the derivative thereof have wide application prospects in preparing p300 inhibitors, preventing and/or treating tumor, marrow hematopoietic stem cell diseases and regulating and controlling regulatory T cells.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be more fully described in connection with the following detailed description. The embodiments described herein are some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Definition of terms used in connection with the present invention: unless otherwise indicated, the initial definitions provided for groups or terms herein apply to the groups or terms throughout the specification; for terms not specifically defined herein, the meanings that one skilled in the art can impart based on the disclosure and the context.

"Alicyclic" as referred to herein means a saturated or unsaturated cyclic hydrocarbon fragment or group, and "aliphatic heterocyclic" refers to a saturated or unsaturated cyclic fragment or group containing heteroatoms (including, but not limited to, nitrogen, oxygen, sulfur).

Reference herein to "pharmaceutically acceptable" means that the carrier, cargo, diluent, adjuvant, and/or salt formed is generally chemically or physically compatible with the other ingredients comprising the pharmaceutical dosage form, and physiologically compatible with the recipient.

Reference herein to "salts" is to acidic and/or basic salts of a compound or stereoisomer thereof with inorganic and/or organic acids and/or bases, and also includes zwitterionic salts (inner salts) and also includes quaternary ammonium salts, for example alkylammonium salts. These salts may be obtained directly in the final isolation and purification of the compounds. Or by mixing the compound, or an isomer thereof, with a certain amount of an acid or a base as appropriate (for example, equivalent). These salts may be obtained by precipitation in solution and collected by filtration, or recovered after evaporation of the solvent, or by lyophilization after reaction in an aqueous medium.

In the present invention, the salt may be the hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoric acid, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, sprinkle or trifluoroacetate salt of the compound.

Reference herein to "solvate" refers to a solvate of a compound of the invention with a solvent, wherein the solvent includes, but is not limited to: water, ethanol, methanol, isopropanol, propylene glycol, tetrahydrofuran, methylene chloride, or the like.

Reference herein to "deuterated" refers to compounds obtained after replacement of one or more hydrogen atoms in the compounds of the invention with deuterium.

As described above, the present invention provides in a first aspect a compound or a deuterated, salt, isomer, crystalline form, or solvate thereof of such a compound characterized in that said compound has the structure according to formula i:

R ₁、R₂、R₃ and R ₄ are each independently selected from hydrogen, deuterium, halogen, haloalkyl, alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, or cyano;

R ₅ is selected from a substituted or unsubstituted 3-10 membered saturated or unsaturated aliphatic ring or aliphatic heterocyclic ring or aliphatic parallel ring;

r ₆ is selected from hydrogen or halogen;

R ₇ is selected from a substituted or unsubstituted aromatic ring, aromatic heterocycle or aromatic parallel ring;

X is selected from a covalent bond, -CH ₂ -, or-CH ₂CH₂ -;

Y ₁ and Y ₂ are each independently selected from CH or N.

It is also preferred or further preferred that R ₁、R₂、R₃ and R ₄ are each independently selected from substituents of C _1-10 alkyl, C _1-10 alkoxy, C _2-10 alkenyl, C _2-10 alkynyl, halo-substituted C _1-10 alkyl, C _3-12 cycloalkyl, halo, difluoromethyl, trifluoromethyl, or cyano.

It is additionally preferred or further preferred that the substituted or unsubstituted 3-10 membered saturated or unsaturated aliphatic or alicyclic substituent of R ₅ is one or more substituents selected from C _1-10 alkyl, C _1-10 alkoxy, C _2-10 alkenyl, C _2-10 alkynyl, halo substituted C _1-10 alkyl, C _3-12 cycloalkyl, C _3-12 cycloalkoxy, hydroxy, C _1-6 alkyl ester or halo.

It is additionally preferred or further preferred that R ₆ is selected from hydrogen or fluorine.

It is additionally preferred or further preferred that the substituent on the substituted aromatic or aromatic ring of R ₇ is one or more substituents selected from the group consisting of C _1-6 alkyl, C _3-6 cycloalkyl, C _1-6 alkoxy, C _3-6 cycloalkyloxy, halogenated C _1-6 alkyl, halogenated C _1-6 alkoxy, hydroxy, C _1-6 alkyl ester, halogen, cyano, nitro, amino, C _1-6 alkylamino or C _1-6 alkylamido.

It is additionally or further preferred that R ₅ is selected from It is additionally or further preferred that R ₇ is selected from

It is additionally preferred or further preferred that the compound is one of the following compounds:

In a second aspect, the present invention provides a pharmaceutical composition comprising: (1) The compound according to the first aspect of the present invention or a deuterated, salt, isomer, crystal form or solvate thereof of the compound (as an active ingredient); and (2) pharmaceutically acceptable carriers and/or excipients (as adjuvants).

In a third aspect the invention provides the use of a compound according to the first aspect of the invention or a deuterated, salt, isomer, crystalline form or solvate thereof as a p300 bromodomain inhibitor.

It is also preferred or further preferred that the p300 bromodomain inhibitor is used as a medicament for preventing and/or treating malignant diseases of hematopoietic stem cells of the tumor, modulating regulatory T cells.

More preferably, the tumor is selected from one or more of hematological malignancy, gastric cancer, intestinal cancer, cervical cancer, bladder cancer, laryngeal cancer, liver cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, lymphoma, or multiple myeloma.

Preparation example: synthesis of (2S, 4R) -4-fluoro-5-oxopyrrolidine-2-carboxylic acid and (2S, 4S) -4-fluoro-5-oxopyrrolidine-2-carboxylic acid

Sodium periodate (3 equiv) and ruthenium trichloride (0.2 equiv) were dissolved in EA: H ₂ o=1:2 solution at room temperature, then commercially available raw material N-BOC-trans-4-fluoro-L-proline methyl ester (1 equiv) or N-BOC-cis-4-fluoro-L-proline methyl ester (1 equiv) was added to the solution, reacted for 5 hours, 100mL of EA was added thereto, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated to obtain crude product, which was washed with saturated sodium bicarbonate solution, saturated sodium dithionite solution and saturated sodium chloride solution, respectively. And (3) dropwise adding a hydrogen chloride dioxane solution with the concentration of 4M into the crude product under the ice bath condition, reacting for 3 hours at room temperature, and spin-drying the reaction solution to obtain the crude product. The above crude product was dissolved in THF: meOH: H ₂ o=1:1:3 solution, lithium hydroxide (1.5 equiv) was added thereto and reacted for 8 hours, and the reaction mixture was dried by spin drying with water to obtain the crude product, which was directly used for the next reaction.

Examples

The above-described aspects of the present invention will be described in further detail by way of examples. It should not be construed that the scope of the above subject matter of the present invention is limited to the following examples. All techniques implemented based on the above description of the invention are within the scope of the invention as claimed.

Example 1: synthesis of 5- (2- ((2S, 4S) -1- (3, 4-difluorophenyl) -4-fluoro-5-oxopyrrolidin-2-yl) -1- ((trans) -4-methoxycyclohexyl) -1H-benzo [ d ] imidazol-5-yl) -1, 3-dimethylpyridin-2 (1H) -one

Step 1: synthesis of intermediate 1-2:

Starting material 1-1 (5 g,22.7 mmol), trans-4-methoxycyclohexyl-1-amine (2.9 g,22.7 mmol), potassium carbonate (4.7 g,34.1 mmol) were dissolved in 70mL of acetonitrile and reacted overnight at 75 ℃. After the completion of the reaction, cooling, filtering, washing the cake with ethyl acetate, drying over anhydrous sodium sulfate, and concentrating to obtain intermediate 1-2 (7.0 g, yield 93.6%) which was used in the next step without further purification.

Step 2: synthesis of intermediate 1-3:

The intermediate 1-2 (7 g,21.3 mmol) obtained in the above step was dissolved in 150mL of 1:1 tetrahydrofuran and aqueous solution, 10mL of aqueous ammonia was added dropwise to the solution, followed by addition of sodium dithionite (18.5 g,106.3 mmol) and reaction was carried out at room temperature for 2 hours. After the reaction, 50mL of water was added, extraction was performed with ethyl acetate (2 x 100 mL), the organic phases were combined, washed with saturated sodium chloride solution (1 x 100 mL), dried over anhydrous sodium sulfate, and concentrated, and the crude product was separated by column chromatography (petroleum ether: ethyl acetate=1:1) to give product intermediate 1-3 (4.5 g, yield 70.7%).

Step 3: synthesis of intermediates 1-4:

The intermediate 1-3 (1 g,3.3 mmol), (2S, 4S) -4-fluoro-5-oxopyrrolidine-2-carboxylic acid (491.6 mg,3.3 mmol), HATU (CAS NO:148893-10-1,1.9g,5.01 mmol) and N, N-diisopropylethylamine (863.9 mg,6.7 mmol) obtained in the above step were dissolved in 10mL of N, N-dimethylformamide and reacted at room temperature for 3 hours. After the reaction, 50mL of water was added, extraction was performed with ethyl acetate (2 x 50 mL), the organic phases were combined, washed with saturated sodium chloride solution (2 x 50 mL), dried over anhydrous sodium sulfate, concentrated, and the crude product (1.5 g) was dissolved in 10mL of acetic acid and reacted overnight at 75 ℃. After the reaction was completed, cooled, dried by spinning, 50mL of saturated sodium bicarbonate solution was added, extracted with ethyl acetate (2×50 mL), the organic phases were combined, washed with saturated sodium chloride solution (2×50 mL), dried over anhydrous sodium sulfate, concentrated, and the crude product was isolated by column chromatography (dichloromethane: methanol=30:1) to give intermediate 1-4 (820 mg, yield 62.7%).

Step 4, synthesizing the intermediate 1-5:

Intermediate 1-4 (200 mg,0.49 mmol) obtained in the previous step, 3, 4-difluorophenylboronic acid (231 mg,1.5 mmol), copper acetate monohydrate (117 mg,0.59 mmol) and pyridine (309 mg,3.9 mmol) were dissolved in 10mL of dichloromethane and reacted at room temperature for 3 hours. After the reaction, the reaction mixture was dried by spin-drying, extracted with 50mL of water, ethyl acetate (1×50 mL), the organic phases were combined, washed with saturated sodium chloride solution (1×50 mL), dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by column chromatography (dichloromethane: methanol=50:1) to give intermediate 1-5 (190 mg, yield 74.6%).

Step 5 Synthesis of 5- (2- ((2S, 4S) -1- (3, 4-difluorophenyl) -4-fluoro-5-oxopyrrolidin-2-yl) -1- ((trans) -4-methoxycyclohexyl) -1H-benzo [ d ] imidazol-5-yl) -1, 3-dimethylpyridin-2 (1H) -one:

Intermediate 1-5 (20 mg, 38.3. Mu. Mol), 1, 3-dimethyl-5- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-ylpyridinium) -2 (1H) -one (11.5 mg, 45.9. Mu. Mol), 1' -bis-diphenylphosphino ferrocene palladium dichloride (2.8 mg, 3.8. Mu. Mol), potassium carbonate (7.9 mg, 57.4. Mu. Mol) were dissolved in 4mL of 3:1 dioxane and water, and the mixture was reacted three times with nitrogen substitution at 85℃for 4 hours. After the reaction, it was cooled, 25mL of water was added, extraction was performed with ethyl acetate (2×25 mL), the organic phases were combined, washed with saturated sodium chloride solution (1×25 mL), dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography to give the objective compound 1 (15.1 mg, yield 69.4%).

Examples 2 to 16 described below were synthesized using the method described in example 1, or using the corresponding intermediates in a similar manner to example 1.

The target compounds synthesized in examples 1 to 16 were synthesized and their molecular structures and ¹ H NMR spectra were obtained.

Example 17: synthesis of 5- (2- ((2S, 4S) -4-fluoro-1- (3-fluoro-4-methoxyphenyl) -5-oxopyrrolidin-2-yl) -1- ((trans) 4-methoxycyclohexyl) -1H-benzo [ d ] imidazol-5-yl) -1, 3-dimethylpyridin-2 (1H) -one

Step 1: synthesis of intermediate 17-1:

Intermediate 1-4 (300 mg, 731.2. Mu. Mol), 1, 3-dimethyl-5- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-ylpyridinium) -2 (1H) -one (218.6 mg, 877.4. Mu. Mol), 1' -bis-diphenylphosphino ferrocene palladium dichloride (53.3 mg, 73.1. Mu. Mol), potassium carbonate (151.6 mg,1.1 mmol) were dissolved in 12mL of 3:1 dioxane and water, nitrogen was replaced three times, and reacted at 85℃for 4 hours. After the reaction, it was cooled, 50mL of water was added, extraction was performed with ethyl acetate (2×50 mL), the organic phases were combined, washed with saturated sodium chloride solution (1×50 mL), dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography (dichloromethane: methanol=10:1) to give intermediate 17-1 (206 mg, yield 62.3%).

Step 2: synthesis of 5- (2- ((2S, 4S) -4-fluoro-1- (3-fluoro-4-methoxyphenyl) -5-oxopyrrolidin-2-yl) -1- ((trans) 4-methoxycyclohexyl) -1H-benzo [ d ] imidazol-5-yl) -1, 3-dimethylpyridin-2 (1H) -one

Intermediate 17-1 (20 mg, 44.2. Mu. Mol), 3-fluoro-4-methoxyphenylboronic acid (22.5 mg, 132.6. Mu. Mol), copper acetate monohydrate (10.6 mg, 53. Mu. Mol) and pyridine (28 mg, 353.6. Mu. Mol) obtained in the above step were dissolved in 3mL of methylene chloride and reacted at room temperature for 3 hours. After the reaction, the reaction mixture was dried by spin-drying, 25mL of water was added, extraction was performed with ethyl acetate (1×25 mL), the organic phases were combined, washed with saturated sodium chloride solution (1×25 mL), dried over anhydrous sodium sulfate, and concentrated, and the crude product was subjected to column chromatography (dichloromethane: methanol=20:1) to give the objective compound 17 (17.3 mg, yield 69.3%).

Examples 18 to 24 described below were synthesized using the procedure described in example 17, or using the corresponding intermediates in a similar manner to example 17.

The target compounds synthesized in examples 17 to 24 were synthesized and their molecular structures and Mass Spectra (MS).

Example 25: synthesis of 5- (2- ((2S, 4R) -1- (3, 4-difluorophenyl) -4-fluoro-5-oxopyrrolidin-2-yl) -1- ((trans) -4-methoxycyclohexyl) -1H-benzo [ d ] imidazol-5-yl) -1, 3-dimethylpyridin-2 (1H) -one

Step 1: synthesis of intermediate 25-1:

Intermediate 1-3 (1 g,3.3 mmol), (2S, 4R) -4-fluoro-5-oxopyrrolidine-2-carboxylic acid (491.6 mg,3.3 mmol), HATU (CAS NO:148893-10-1,1.9g,5.01 mmol), N, N-diisopropylethylamine (863.9 mg,6.7 mmol) was dissolved in 10mL of N, N-dimethylformamide and reacted at room temperature for 3 hours. After the reaction, 50mL of water was added, extraction was performed with ethyl acetate (2 x 50 mL), the organic phases were combined, washed with saturated sodium chloride solution (2 x 50 mL), dried over anhydrous sodium sulfate, concentrated, and the crude product (1.5 g) was dissolved in 10mL of acetic acid and reacted overnight at 75 ℃. After the reaction was completed, cooled, dried by spinning, 50mL of saturated sodium bicarbonate solution was added, extracted with ethyl acetate (2×50 mL), the organic phases were combined, washed with saturated sodium chloride solution (2×50 mL), dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography (dichloromethane: methanol=30:1) to give intermediate 25-1 (820 mg, yield 62.7%).

Step 2, synthesizing an intermediate 25-2:

Intermediate 25-1 (200 mg,0.49 mmol) obtained in the previous step, 3, 4-difluorophenylboronic acid (231 mg,1.5 mmol), copper acetate monohydrate (117 mg,0.59 mmol), pyridine (309 mg,3.9 mmol) were dissolved in 10mL of dichloromethane and reacted at room temperature for 3 hours. After the reaction, the reaction mixture was dried by spin-drying, 50mL of water was added, extraction was performed with ethyl acetate (1×50 mL), the organic phases were combined, washed with saturated sodium chloride solution (1×50 mL), dried over anhydrous sodium sulfate, and concentrated, and the crude product was purified by column chromatography (dichloromethane: methanol=50:1) to give intermediate 25-2 (190 mg, yield 74.6%).

Step 3 Synthesis of 5- (2- ((2S, 4S) -1- (3, 4-difluorophenyl) -4-fluoro-5-oxopyrrolidin-2-yl) -1- ((trans) -4-methoxycyclohexyl) -1H-benzo [ d ] imidazol-5-yl) -1, 3-dimethylpyridin-2 (1H) -one:

Intermediate 25-2 (20 mg, 38.3. Mu. Mol), 1, 3-dimethyl-5- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-ylpyridinium) -2 (1H) -one (11.5 mg, 45.9. Mu. Mol), 1' -bis-diphenylphosphino ferrocene palladium dichloride (2.8 mg, 3.8. Mu. Mol), potassium carbonate (7.9 mg, 57.4. Mu. Mol) were dissolved in 4mL of 3:1 dioxane and water, and the mixture was reacted three times with nitrogen substitution at 85℃for 4 hours. After the reaction, it was cooled, 25mL of water was added, extraction was performed with ethyl acetate (2×25 mL), the organic phases were combined, washed with saturated sodium chloride solution (1×25 mL), dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography to give the objective compound 25 (15.1 mg, yield 69.4%).

Examples 26 to 30 described below were synthesized using the method described in example 25, or using the corresponding intermediates in a similar manner to example 25.

The target compounds synthesized in examples 25 to 30 were synthesized and their molecular structures and Mass Spectra (MS).

Example 31: synthesis of 3-chloro-5- (2- ((S) -1- (3, 4-difluorophenyl) -4-oxoazetidin-2-yl) -1- ((trans) -4-methoxycyclohexyl) -1H-benzo [ d ] imidazol-5-yl) -1-methylpyridin-2 (1H) -one

Step 1: synthesis of intermediate 31-1:

Intermediate 1-3 (100 mg, 334.2. Mu. Mol), (S) -4-oxoazetidine-2-carboxylic acid (38.5 mg, 334.2. Mu. Mol), HATU (CAS NO:148893-10-1,190.5mg, 501.3. Mu. Mol), N, N-diisopropylethylamine (86.39 mg, 668.4. Mu. Mol) was dissolved in 5mL of N, N-dimethylformamide and reacted at room temperature for 3 hours. After the reaction, 25mL of water was added, extraction was performed with ethyl acetate (2×25 mL), the organic phases were combined, washed with saturated sodium chloride solution (2×25 mL), dried over anhydrous sodium sulfate, concentrated, and the crude product (150 mg) was dissolved in 5mL of acetic acid and reacted overnight at 75 ℃. After the reaction was completed, cooling, spin-drying, adding 20mL of saturated sodium bicarbonate solution, extraction with ethyl acetate (2×25 mL), combining the organic phases, washing with saturated sodium chloride solution (2×25 mL), drying over anhydrous sodium sulfate, concentrating, and separating the crude product by column chromatography (dichloromethane: methanol=30:1) to obtain intermediate 31-1 (88.5 mg, yield 70.0%).

Step 2: synthesis of intermediate 31-2:

The intermediate 31-1 (50 mg, 132.2. Mu. Mol), 1, 3-dimethyl-5- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-ylpyridinium) -2 (1H) -one (39.5 mg, 158.6. Mu. Mol), 1' -bis-diphenylphosphino ferrocene palladium dichloride (9.6 mg, 13.2. Mu. Mol), potassium carbonate (27.4 mg, 198.3. Mu. Mol) obtained in the upper part was dissolved in 4mL of 3:1 dioxane and water, and the mixture was replaced with nitrogen three times, and reacted at 85℃for 4 hours. After the reaction, it was cooled, 25mL of water was added, extraction was performed with ethyl acetate (2×25 mL), the organic phases were combined, washed with saturated sodium chloride solution (1×25 mL), dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography (dichloromethane: methanol=10:1) to give intermediate 31-2 (30.3 mg, yield 54.5%).

Step 3: synthesis of 3-chloro-5- (2- ((S) -1- (3, 4-difluorophenyl) -4-oxoazetidin-2-yl) -1- ((trans) -4-methoxycyclohexyl) -1H-benzo [ d ] imidazol-5-yl) -1-methylpyridin-2 (1H) -one

Intermediate 31-2 (30 mg, 71.3. Mu. Mol), 3, 4-difluorophenylboronic acid (33.8 mg, 214.0. Mu. Mol), copper acetate monohydrate (15.7 mg, 78.5. Mu. Mol), pyridine (45.1 mg, 570.7. Mu. Mol) obtained in the above step were dissolved in 3mL of methylene chloride and reacted at room temperature for 3 hours. After the reaction, the reaction mixture was dried by spin-drying, 25mL of water was added, extraction was performed with ethyl acetate (1×25 mL), the organic phases were combined, washed with saturated sodium chloride solution (1×25 mL), dried over anhydrous sodium sulfate, and concentrated, and the crude product was subjected to column chromatography (dichloromethane: methanol=20:1) to give the objective compound 31 (25.2 mg, yield 66.3%).

Examples 32 to 38 and examples 47 to 53 described below were synthesized using the methods described in example 31, or using the corresponding intermediates in a similar manner to example 31.

The target compounds synthesized in examples 31 to 38 and examples 47 to 53 were shown to have their partial structures and ¹ HNMR spectra.

Example 39: synthesis of (trans) -4- (5- (5-chloro-1-methyl-6-oxo-1, 6-dihydropyridin-3-yl) -2- ((S) -1- (3, 4-difluorophenyl) -5-oxopyrrolidin-2-yl) -1H-benzo [ d ] imidazol-1-yl) cyclohexyl acetate

Step 1: synthesis of intermediate 39-1:

Starting material 1-1 (5 g,22.7 mmol), trans-4-aminocyclohexane-1-ol (2.6 g,22.7 mmol), potassium carbonate (4.7 g,34.1 mmol) were dissolved in 70mL of acetonitrile and reacted overnight at 75 ℃. After the reaction was completed, cooling, filtering, washing the cake with ethyl acetate, drying over anhydrous sodium sulfate, and concentrating to obtain intermediate 1-2 (6.8 g, yield 94.9%) which was used in the next step without further purification.

Step 2: synthesis of intermediate 39-2:

intermediate 39-1 (200 mg, 634.6. Mu. Mol) obtained in the above step was dissolved in 10mL of methylene chloride, triethylamine (128.4 mg,27 mmol) was added thereto, acetyl chloride (74.7 mg, 951.9. Mu. Mol) was added dropwise to the reaction system under ice bath conditions, and the reaction was carried out at room temperature for 1 hour. After the completion of the reaction, the reaction system was dried by spinning, and the crude product was separated by column chromatography (petroleum ether: ethyl acetate=5:1) to give product intermediate 39-2 (103.2 mg, yield 45.5%)

Step 3: synthesis of intermediate 39-3:

intermediate 39-2 (103.2 mg, 288.9. Mu. Mol) obtained in the above step was dissolved in 20mL of 1:1 tetrahydrofuran and an aqueous solution, 200. Mu.L of aqueous ammonia was added dropwise to the solution, followed by addition of sodium dithionite (402.4 mg,2.3 mmol), and the reaction was carried out at room temperature for 1 hour. After the reaction, 30mL of water was added, extraction was performed with ethyl acetate (1×50 mL), the organic phase was washed with saturated sodium chloride solution (1×100 mL), dried over anhydrous sodium sulfate, and concentrated, and the crude product was separated by column chromatography (petroleum ether: ethyl acetate=2:1) to obtain product intermediate 39-3 (60.1 mg, yield 63.6%).

Step 4: synthesis of intermediate 39-4:

Intermediate 39-3 (60 mg, 183.4. Mu. Mol) obtained in the above step, (S) -5-oxopyrrolidine-2-carboxylic acid (23.7 mg,183.4 mmol), HATU (CAS NO:148893-10-1,104.5mg, 275. Mu. Mol) and N, N-diisopropylethylamine (47.4 mg, 366.7. Mu. Mol) were dissolved in 3mL of N, N-dimethylformamide and reacted at room temperature for 3 hours. After the reaction, 25mL of water was added, extraction was performed with ethyl acetate (2×25 mL), the organic phases were combined, washed with saturated sodium chloride solution (2×25 mL), dried over anhydrous sodium sulfate, concentrated, and the crude product (90 mg) was dissolved in 5mL of acetic acid and reacted overnight at 75 ℃. After the reaction was completed, cooled, dried by spinning, 25mL of saturated sodium bicarbonate solution was added, extracted with ethyl acetate (2×50 mL), the organic phases were combined, washed with saturated sodium chloride solution (2×50 mL), dried over anhydrous sodium sulfate, concentrated, and the crude product was isolated by column chromatography (dichloromethane: methanol=30:1) to give intermediate 39-4 (40 mg, yield 51.9%).

Step 5, synthesizing an intermediate 39-5:

Intermediate 39-4 (40 mg, 95.2. Mu. Mol), 3, 4-difluorophenylboronic acid (45.1 mg, 285.5. Mu. Mol), copper acetate monohydrate (20.9 mg, 104.7. Mu. Mol), pyridine (60.2 mg, 761.4. Mu. Mol) obtained in the above step were dissolved in 5mL of methylene chloride and reacted at room temperature for 3 hours. After the reaction, the reaction mixture was dried by spin-drying, extracted with 25mL of water, washed with ethyl acetate (1×25 mL), dried over anhydrous sodium sulfate (2×25 mL), and concentrated to give intermediate 39-5 (40.1 mg, yield 79.2%) by column chromatography (dichloromethane: methanol=50:1).

Step 6 (Synthesis of trans) -4- (5- (5-chloro-1-methyl-6-oxo-1, 6-dihydropyridin-3-yl) -2- ((S) -1- (3, 4-difluorophenyl) -5-oxopyrrolidin-2-yl) -1H-benzo [ d ] imidazol-1-yl) cyclohexyl acetate:

Intermediate 39-5 (40 mg, 79.3. Mu. Mol), 3-chloro-1-methyl-5- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-ylpyridinium) -2 (1H) -one (25.7 mg, 95.2. Mu. Mol), 1' -bis-diphenylphosphino ferrocene palladium dichloride (5.8 mg, 7.9. Mu. Mol), potassium carbonate (16.4 mg, 119.0. Mu. Mol) obtained in the above step were dissolved in 4mL of 3:1 dioxane and water, replaced with nitrogen three times, and reacted at 85℃for 4 hours. After the reaction, it was cooled, 25mL of water was added, extraction was performed with ethyl acetate (2×25 mL), the organic phases were combined, washed with saturated sodium chloride solution (1×25 mL), dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography to give the objective compound 39 (29.4 mg, yield 62.3%).

Example 40: synthesis of 3-chloro-5- (2- ((S) -1- (3, 4-difluorophenyl) -5-oxopyrrolidin-2-yl) -1- ((trans) -4- (methoxy-d ₃) cyclohexyl) -1H-benzo [ d ] imidazol-5-yl) -1-methylpyridin-2 (1H) -one

Step 1: synthesis of intermediate 40-1:

intermediate 39-1 (100 mg, 317.3. Mu. Mol) was dissolved in 5mL of N, N-dimethylformamide, sodium hydride (60%) (63.5 mg,1.59 mmol) was slowly added thereto under ice-bath conditions and reacted for 10 minutes, then deuterated iodomethane (55.2 mg, 380.8. Mu. Mol) was added dropwise to the reaction system, and after completion of the dropwise addition, the reaction was carried out at room temperature for 2 hours. After the completion of the reaction, 25mL of water was added, extraction was performed with ethyl acetate (2.25 mL), the organic phases were combined, washed with saturated sodium chloride solution (2.25 mL), dried over anhydrous sodium sulfate, and concentrated, and the crude product was separated by column chromatography to give (petroleum ether: ethyl acetate=4:1) intermediate 40-1 (56.3 mg, yield 53.4%).

Step 2-5 the objective compound 40 was obtained in reference to step 3-6 in example 39.

Examples 41 to 46 and examples 54 to 63 described below were synthesized using the methods described in examples 39 or 40, or using the corresponding intermediates in a similar manner to example 31.

The target compounds synthesized in examples 39 to 46 and examples 54 to 63 were synthesized with their molecular structures and ¹ HNMR spectra.

Comparative example 1 (CCS 1477)

The compound CCS1477 used was purchased from MCE.

Detection example 1: in vitro inhibition activity detection of histone acetyltransferase p300 by compounds

The inhibitory activity of the compounds on the bromodomain of p300 protein was tested using the AlphaLISA technique. AlphaLISA uses microbeads as donor and acceptor for detection of biomolecules. If a detection target exists in the sample, the donor microbead and the receptor microbead are close to each other due to the specific recognition of the antibody, so that the chemiluminescent reaction of cascade amplification on the receptor microbead is excited, and finally, a signal is transmitted to europium to generate a signal.

1X detection buffer was prepared prior to use. The compounds were diluted with DMSO to the desired concentration for the reaction, and then 5 μl of the compounds were transferred from the source plate to the assay plate (OptiPlate-384). Protein and peptide solutions were prepared in 1x assay buffer. 2.5. Mu.L of protein solution and 2.5. Mu.L of peptide solution were added to each well. The assay plates were centrifuged at 1000rpm for 1 min and then incubated at room temperature for 20 min. Acceptor microbead and donor microbead solutions were prepared in 1x detection buffer. To each well of the assay plate 10 μl of acceptor bead solution was added and the assay plate was incubated for 1 hour at room temperature. To each well of the assay plate 10 μl of donor bead solution was added and the assay plate was incubated for 30 minutes at room temperature. The signal is read using the Alpha mode of Tecan. The resulting data was analyzed with GRAPHPAD PRISM. The experimental results are shown in Table 1.

Table 1: in vitro inhibitory Activity of Compounds against p300

++++: Representative IC ₅₀ <30nM

+++: Represents 30 nM.ltoreq.IC ₅₀ < 100nM

++: Represents 100 nM.ltoreq.IC ₅₀ < 1. Mu.M

From the results, the compound can strongly inhibit the activity of histone acetyl transferase p300, and the inhibition activity of part of the compound is superior to that of the target clinical research medicament CCS1477, so that the compound is worthy of further development.

Detection example 2: CCK8 method for detecting proliferation inhibition experiment of sensitive cells

The proliferation inhibition effect of the compound of the present invention on tumor cells was examined, and the compound of the present invention was used to conduct a cell growth inhibition activity test on p300/CBP inhibition-sensitive cell lines (OPM-2 cells were purchased from German collection of microbial strains (DSMZ), and 22RV1 cells were purchased from the family of Bai cell banks) obtained in the previous work. Cells in the logarithmic growth phase were seeded at a concentration of 2.5X10 ³ cells per well in 96-well plates, after 24 hours, the gradient diluted compound was added, flow-cultured for 5 days after the seeding, and then 10. Mu.L of CCK-8 reagent (Cell Counting Kit-8 cell counting reagent) was added to each well, and cultured at 37℃for 2-6 hours. Absorbance at a wavelength of 450nm was read for each well using an microplate reader and analyzed using SoftMax Pro 5.4.1 software. IC ₅₀ values (concentration-response curve fit) were calculated by four-parameter method using GRAPHPAD PRISM statistical software. The experimental results are shown in Table 2.

Table 2: proliferation inhibitory Activity of Compounds against tumor cells

From the above results, it can be seen that the compounds of the present invention exhibit superior antiproliferative effects on multiple myeloma cells OPM-2 and prostate cancer cells, and can be used as candidate p300 bromodomain inhibitors for the treatment of related cancers.

Detection example 3: pharmacokinetic evaluation

Animal information

Species/breed: ICR (CD-1) mice; gender/number: a male; body weight (g): male (25-30 g).

The feeding mode is as follows: standard serrate diet, without limitation to drinking water, started fasting for 12 hours prior to dosing and remained fasting for 2 hours after dosing.

Feeding environment: the animal room environment is controlled (target conditions: temperature 18 to 29 ℃, relative humidity 30 to 70%. The temperature and relative humidity are monitored daily. An electronic time controlled lighting system is used to provide 12 hours light/12 hours dark cycle.

Administration information

Sample collection

ICR mice were orally administered with 10mg/kg of test compound, a volume of administration of 10mL/kg, and blood was collected from the orbital venous plexus after administration in a heparinized EP tube at 0.25h,0.5h,1h,2h,4h,8h,24h, and placed on crushed ice.

Sample processing and analysis

Centrifugation at 8000rpm for 5min, transfer of the upper plasma 15. Mu.L into 96 well plates. To 15. Mu.L of plasma was added 150. Mu.L of methanol: ethylene wax (v/v=l: L) (containing 20ng/mL of tolbutamide), shaking for 3min, centrifuging at 4500rpm for 5min, and taking 100. Mu.L of supernatant to a 2mL deep well plate. 100. Mu.L of diluent (pure water) was added, and the mixture was shaken for 3min and centrifuged at 4500rpm for 5min. 180. Mu.L of the supernatant was transferred to a sample plate, and the content of the compound in the supernatant sample was analyzed by LC-MS/MS, and each pharmacokinetic parameter was calculated using WinNonlin software. Pharmacokinetic parameters of the test compounds are shown in table 3.

Table 3: in vivo PK Properties in mice of Compounds of the invention

Experimental results show that the compound has good oral absorption exposure, higher C _max and AUC, longer half-life T _1/2 and residence time MRT, and the data show that the compound has potential of being an orally effective antitumor drug.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (9)

1.A compound or a deuterated, salt, isomer, crystalline form, or solvate thereof, characterized in that: the compound has a structure shown in the following formula I:

Wherein,

R ₁、R₂、R₃ and R ₄ are each independently selected from hydrogen, deuterium, halogen, haloalkyl, alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, or cyano;

R ₅ is selected from a substituted or unsubstituted 3-10 membered saturated or unsaturated aliphatic ring or aliphatic heterocyclic ring or aliphatic parallel ring;

r ₆ is selected from hydrogen or halogen;

R ₇ is selected from a substituted or unsubstituted aromatic ring, aromatic heterocycle or aromatic parallel ring;

X is selected from a covalent bond, -CH ₂ -, or-CH ₂CH₂ -;

Y ₁ and Y ₂ are each independently selected from CH or N.

2. The compound according to claim 1 or a deuterated, salt, isomer, crystalline form, or solvate thereof, of said compound, wherein:

R ₁、R₂、R₃ and R ₄ are each independently selected from the group consisting of C _1-10 alkyl, C _1-10 alkoxy, C _2-10 alkenyl, C _2-10 alkynyl, halo-substituted C _1-10 alkyl, C _3-12 cycloalkyl, halo, difluoromethyl, trifluoromethyl, or cyano.

3. The compound according to claim 1 or a deuterated, salt, isomer, crystalline form, or solvate thereof, of said compound, wherein:

The substituted or unsubstituted 3-10 membered saturated or unsaturated aliphatic ring or aliphatic heterocyclic ring of R ₅ is one or more substituents selected from C _1-10 alkyl, C _1-10 alkoxy, C _2-10 alkenyl, C _2-10 alkynyl, halo substituted C _1-10 alkyl, C _3-12 cycloalkyl, C _3-12 cycloalkoxy, hydroxy, C _1-6 alkyl ester group or halo.

4. The compound according to claim 1 or a deuterated, salt, isomer, crystalline form, or solvate thereof, of said compound, wherein:

r ₆ is selected from hydrogen or fluorine.

5. The compound according to claim 1 or a deuterated, salt, isomer, crystalline form, or solvate thereof, of said compound, wherein:

The substituent on the substituted aromatic ring or aromatic parallel ring of R ₇ is one or more substituents selected from C _1-6 alkyl, C _3-6 cycloalkyl, C _1-6 alkoxy, C _3-6 cycloalkyloxy, halogenated C _1-6 alkyl, halogenated C _1-6 alkoxy, hydroxy, C _1-6 alkyl ester, halogen, cyano, nitro, amino, C _1-6 alkylamino or C _1-6 alkylamido.

6. The compound according to claim 1 or a deuterated, salt, isomer, crystalline form, or solvate thereof, of said compound, wherein:

R ₅ is selected from And/or R ₇ is selected from

7. The compound according to claim 1, or a deuterated, salt, isomer, crystal form, or solvate thereof, wherein said compound is one of the following compounds:

8. A pharmaceutical composition, characterized in that, the pharmaceutical composition comprises:

(1) The compound of any one of claims 1 to 7, or a deuterated, salt, isomer, crystalline form, or solvate thereof of the compound; and

(2) Pharmaceutically acceptable carriers and/or excipients.

9. Use of a compound according to any one of claims 1 to 7 or a deuterated, salt, isomer, crystalline form, or solvate thereof of said compound in the manufacture of a p300 bromodomain inhibitor medicament according to claim 8;

preferably, the p300 bromodomain inhibitor is used as a medicament for preventing and/or treating malignant diseases of tumor and myeloid hematopoietic stem cells, regulating regulatory T cells;

More preferably, the tumor is selected from one or more of hematological malignancy, gastric cancer, intestinal cancer, cervical cancer, bladder cancer, laryngeal cancer, liver cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, lymphoma, or multiple myeloma.