P300 bromodomain inhibitors, pharmaceutical compositions thereof, and uses thereof

Abstract

The present application relates to p300 bromodomain inhibitors and pharmaceutical compositions thereof and uses thereof. Specifically, the present application discloses a compound and derivatives thereof, which has the following formula I: R1 is selected from substituted or non-substituted five or six-membered aromatic heterocycle; R2 is selected from six-membered substituted or non-substituted aliphatic ring or aliphatic heterocycle; R3 is selected from substituted or non-substituted aromatic ring or aromatic ring; R4 and R5 are independently selected from hydrogen, halogen or C1 to C6 saturated alkyl. The present application also discloses pharmaceutical compositions comprising the compound and derivatives thereof and uses thereof as p300 bromodomain inhibitors. The compound and derivatives thereof of the present application can significantly inhibit the activity of histone acetyltransferase p300, have strong proliferation inhibition activity on multiple myeloma cells OPM-2 and the like, have good metabolic stability, safety and drug-like properties, and can be used as candidate molecules for the development of anti-tumor drugs.

Description

Technical Field

[0001] This invention patent belongs to the field of synthetic medicinal chemistry, specifically relating to a novel p300 bromine domain inhibitor, its pharmaceutical composition, and its applications. Background Technology

[0002] Histone lysine acetylation and deacetylation are reversible post-translational modifications that play a crucial role in regulating gene expression and are a key area of ​​research in epigenetics. Histone acetylation is regulated by three agents: histone acetyltransferase (HAT), histone deacetylase (HDAC), and bromodomain-containing protein (BCP).

[0003] The p300 / CBP family, composed of highly homologous adenovirus E1A-associated 300kDa protein (p300) and cyclic adenosine monophosphate (CREB)-binding protein (CBP), is one of the main members of the HAT family. Due to their structural homology and functional similarity, they are often collectively referred to as p300 / CBP. p300 / CBP possesses both catalytic and bromine domains, enabling it to simultaneously acetylate histones and recognize acetylated lysine (KAc) residues on histones.

[0004] As the second largest family of bromine domains after the BET family, the p300 / CBP bromine domain contains 110 amino acid residues, consisting of four α-helices (αZ, αA, αB, αC) linked by ZA and BC loops, forming a binding pocket for KAc recognition. This pocket contains a large number of water molecules, which can form a network of hydrogen bonds. p300 / CBP with its bromine domain can specifically recognize KAc, thereby reading the genetic information carried by post-translational modified histones. Therefore, it plays a role in the epigenetic regulation of gene expression and is crucial for maintaining homeostasis.

[0005] Studies have shown that abnormalities in the function of the p300 / CBP bromine domain are closely related to the occurrence and development of certain tumors. Therefore, the p300 / CBP bromine domain is a potential target for the discovery of anti-tumor drugs. The design and synthesis of highly active and selective p300 / CBP bromine domain inhibitors are currently a research hotspot, which is of great significance for the study of the biological function of p300 / CBP and the development of epigenetic drugs.

[0006] Of the p300 / CBP bromine domain inhibitors reported so far, only two compounds have entered clinical trials. One is FT-7051 (structure not yet disclosed), developed by Forma Theraputics, currently in Phase I clinical trials for the treatment of metastatic castration-resistant prostate cancer. The other is CCS1477, developed by Cellcentric, currently in Phase I / II clinical trials for the treatment of acute myeloid leukemia, non-Hodgkin's lymphoma, multiple myeloma, and metastatic castration-resistant prostate cancer. However, CCS1477 also has certain limitations. First, its cell permeability needs to be improved to further enhance its inhibitory activity against tumor cell proliferation. Second, CCS1477 has a relatively short half-life both in vitro and in vivo. The superior compound in this invention addresses these issues to some extent, improving its inhibitory activity against tumor cell proliferation and significantly increasing its half-life, thereby enhancing its potential as a cancer therapeutic.

[0007] Currently, the reported structural types and numbers of p300 / CBP bromodomain inhibitors are limited, and they are mainly used for the treatment of prostate cancer and hematological malignancies. A large number of biological functions and mechanisms of action remain to be elucidated. Therefore, it is necessary to discover p300 / CBP bromodomain inhibitors with novel structures, strong activity, high selectivity, and good drug-like properties as probe molecules or candidate molecules for further biological research and drug development of p300 / CBP. Summary of the Invention

[0008] This invention designs and synthesizes a series of novel p300 bromodomain inhibitors, providing compounds with the general formula (Ⅰ) structure or their pharmaceutically acceptable salts and other derivatives as small molecule inhibitors of p300. Biological evaluation reveals that the obtained inhibitors exhibit superior inhibitory activity at both the molecular and cellular levels compared to CCS1477, making them drug-like substances suitable as p300 bromodomain inhibitors.

[0009] Experiments have shown that the compounds and their derivatives of this invention exhibit superior inhibitory effects compared to CCS1477 at the molecular, cellular, and animal levels, and can significantly reduce the expression level of c-Myc in human multiple myeloma OPM-2 cells. Furthermore, these compounds or their derivatives demonstrate high selectivity and good bioavailability as inhibitors, indicating significant potential for the development of antitumor drugs.

[0010] Therefore, in a first aspect, the present invention provides a compound or its derivatives (including, for example, deuterated derivatives, pharmaceutically acceptable salts, isomers, crystal forms, solvates thereof, or solvates of pharmaceutically acceptable salts thereof), characterized in that the compound has a structure as shown in Formula I:

[0011]

[0012] R1 is selected from one or more of substituted or unsubstituted five- or six-membered aromatic heterocycles;

[0013] R2 is selected from one or more of a 6-membered substituted or unsubstituted aliphatic rings or aliphatic heterocycles;

[0014] R3 is selected from one or more of substituted or unsubstituted aromatic rings or aromatic fused rings;

[0015] R4 and R5 are independently selected from one or more of hydrogen, halogens, or saturated alkyl groups from C1 to C6.

[0016] In a second aspect, the present invention provides a general synthetic route for synthetic formula I.

[0017] The present invention provides a pharmaceutical composition in a third aspect, the pharmaceutical composition comprising: (1) the compound of the first aspect of the present invention, its deuterated form, its pharmaceutically acceptable salt, its isomer, its crystal form, its solvate or a solvate of a pharmaceutically acceptable salt thereof; and (2) a pharmaceutically acceptable carrier and / or excipient.

[0018] The present invention provides, in a fourth aspect, the use of the compounds described in the first aspect of the invention, their deuterated derivatives, their pharmaceutically acceptable salts, their isomers, their crystal forms, their solvates, or solvates of their pharmaceutically acceptable salts as p300 bromodomain inhibitors.

[0019] Through biological experiments, the inventors have confirmed that the novel p300 bromodomain inhibitors provided in this invention can significantly inhibit the activity of histone acetyltransferase p300. Furthermore, the compounds of this invention exhibit excellent inhibitory effects on various tumor cells, including prostate cancer cells, leukemia cells, breast cancer cells, and multiple myeloma cells (such as OPM-2), with inhibitory effects even superior to those of the clinically investigated drug CCS1477 targeting the same target. The compounds and their derivatives of this invention also possess good metabolic stability, safety, and drug-like properties, making them potential candidate molecules for anti-tumor drug development. Therefore, the compounds and their derivatives of this invention have broad application prospects in the preparation of p300 inhibitors, as well as in the prevention and / or treatment of tumors, myeloid hematopoietic stem cell diseases, and in the regulation of regulatory T cells. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described more clearly and completely below in conjunction with specific embodiments. However, the embodiments described herein are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0021] Regarding the definition of terms used in this invention: Unless otherwise stated, the initial definitions provided for groups or terms herein apply to the groups or terms used throughout this specification; for terms not specifically defined herein, the meanings that a person skilled in the art would give them should be given based on the disclosure and context.

[0022] The term "aliphatic ring" as used in this article refers to saturated or unsaturated cyclic hydrocarbon segments or groups, while "aliphatic heterocycle" refers to saturated or unsaturated cyclic segments or groups containing heteroatoms (including but not limited to nitrogen, oxygen, and sulfur).

[0023] The term "pharmaceutically acceptable" as used in this article means that a carrier, delivery substance, diluent, excipient, and / or the salt formed therefrom is generally chemically or physically compatible with other components constituting a drug dosage form and physiologically compatible with the receptor.

[0024] The term "salt" as used herein refers to acidic and / or basic salts formed by reacting a compound or its stereoisomers with inorganic and / or organic acids and / or bases, including zwitterionic salts (internal salts) and quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly during the final separation and purification of the compound. Alternatively, they can be obtained by mixing the compound, or its isomers, with an appropriate (e.g., equimolar) amount of acid or base. These salts may precipitate in solution and be collected by filtration, or be recovered after solvent evaporation, or be prepared by freeze-drying after reaction in an aqueous medium.

[0025] In this invention, the salt may be a hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoric acid, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartaric acid, or trifluoroacetate of the compound.

[0026] The term "solvent" as used herein refers to a solvate formed by the compound of this invention and a solvent, wherein the solvent includes, but is not limited to, water, ethanol, methanol, isopropanol, propylene glycol, tetrahydrofuran, or dichloromethane.

[0027] The term "deuterated compound" as used herein refers to a compound obtained by replacing one or more hydrogen atoms in the compound of the present invention with deuterium.

[0028] As described above, the present invention provides, in a first aspect, a compound, its deuterated derivative, its pharmaceutically acceptable salt, its isomer, its crystal form, its solvate, or a solvate of a pharmaceutically acceptable salt thereof, characterized in that the compound has a structure as shown in Formula I:

[0029]

[0030] R1 is selected from one or more of substituted or unsubstituted five- or six-membered aromatic heterocycles;

[0031] R2 is selected from one or more of a 6-membered substituted or unsubstituted aliphatic rings or aliphatic heterocycles;

[0032] R3 is selected from one or more of substituted or unsubstituted aromatic rings or aromatic fused rings;

[0033] R4 and R5 are independently selected from one or more of hydrogen, halogens, or saturated alkyl groups from C1 to C6, wherein the saturated alkyl groups from C1 to C6 may be, for example, methyl, ethyl, propyl, butyl, pentyl, or hexyl, and the halogen may be fluorine, chlorine, bromine, or iodine, preferably fluorine or methyl, and more preferably fluorine.

[0034] It is also preferred or even more preferred that the substituents on the substituted five- or six-membered aromatic heterocycle of R1 are one or more substituents selected from methyl and -F3C.

[0035] Furthermore, and even more preferably, the substituent on the 6-membered substituted aliphatic ring or aliphatic heterocycle of R2 is one or more substituents selected from methoxy, ethoxy, -OCD3, ethyl ester, and hydroxyl groups. Currently, the limited research available focuses primarily on the selection of the substituent for R2.

[0036] Furthermore, preferably, the substituent on the substituted aromatic ring or aromatic cyclic ring of R3 is one or more substituents selected from halogens, methoxy groups, and -CF3. The halogen can be fluorine, chlorine, bromine, or iodine, preferably fluorine or chlorine.

[0037] R4 and R5 are independently selected from hydrogen, halogen, and saturated alkyl groups from C1 to C6. Preferably, one of R4 and R5 is a halogen or methyl, more preferably chlorine or fluorine, most preferably fluorine, and the other is preferably hydrogen.

[0038] The C1 to C6 saturated alkyl groups are, for example, methyl, ethyl, propyl, butyl, pentyl, or hexyl; the halogen can be fluorine, chlorine, bromine, or iodine, preferably fluorine or chlorine, and more preferably fluorine. In the limited number of studies conducted to date, the rings connected to R4 and R5 are mostly six-membered rings, with very few five-membered rings. Moreover, if they are five-membered rings, R4 and R5 are usually both hydrogen or methyl, and in the case of methyl, the atoms on the connected rings are mostly nitrogen (N); it is difficult to simultaneously achieve high inhibitory activity and a long half-life. However, if the rings connected to R4 and R5 are five-membered rings and at least one of R4 and R5 is a halogen, especially fluorine (F), high inhibitory activity and a long half-life can be achieved simultaneously. Therefore, in a particularly preferred embodiment, at least one of R4 and R5 is a halogen, especially fluorine (F).

[0039] Alternatively, or more preferably, R1 is selected from...

[0040] Alternatively, or more preferably, R2 is selected from...

[0041] Alternatively, or more preferably, R3 is selected from...

[0042] Alternatively, or even more preferably, in Formula I... Groups are selected from One of them.

[0043] It is also preferred, or even more preferred, that the compound is one of the following compounds:

[0044]

[0045]

[0046]

[0047]

[0048] In some preferred embodiments, the compound is compound 1, compound 2, compound 8, compound 9, compound 13, compound 15 or compound 26; more preferably, the compound is compound 8, compound 9 or compound 26.

[0049] A second aspect of the present invention provides a method for preparing a compound having the structure shown in Formula I, comprising the following steps:

[0050]

[0051] Wherein, R1, R2, R3, R4 and R5 have the definitions described in Formula I of the above claims;

[0052] The method includes the following steps:

[0053] 1) The raw material 4-bromo-1-fluoro-2-nitrobenzene and the compound of formula (1) are reacted via the Suzuki reaction to produce the compound of formula (2);

[0054] 2) The compound of formula (2) reacts with the compound of formula (3) via a nucleophilic attack reaction to generate the compound of formula (4);

[0055] 3) The compound of formula (4) is reduced to the compound of formula (5);

[0056] 4) The compound of formula (5) and the compound of formula (6) are reacted via an acid-amine condensation reaction to generate the compound of formula (7);

[0057] 5) React the compound of formula (7) under acetic acid conditions to produce the compound of formula (8);

[0058] 6) The compound of formula (8) and the compound of formula (9) are reacted via a Chan-Lam reaction to generate the compound of formula I.

[0059] A third aspect of the present invention provides a pharmaceutical composition comprising: (1) the compound of the first aspect of the present invention, its deuterated form, its pharmaceutically acceptable salt, its isomer, its crystal form, its solvate or a solvate of a pharmaceutically acceptable salt thereof (as an active ingredient); and (2) a pharmaceutically acceptable carrier and / or excipient (as an excipient).

[0060] The fourth aspect of the present invention provides the use of the compounds described in the first aspect of the present invention, their deuterated derivatives, their pharmaceutically acceptable salts, their isomers, their crystal forms, their solvates, or solvates of their pharmaceutically acceptable salts as p300 bromodomain inhibitors.

[0061] Furthermore, it is preferred or even more preferred that the p300 bromine domain inhibitor be used to prepare drugs for the prevention and / or treatment of tumors, malignant diseases of myeloid hematopoietic stem cells, or regulation of regulatory T cells.

[0062] More preferably, the tumor is selected from one or more of the following: hematologic malignancies, gastric cancer, intestinal cancer, cervical cancer, bladder cancer, laryngeal cancer, liver cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, lymphoma, or multiple myeloma.

[0063] Preparation examples: Synthesis of (2S,4R)-4-fluoro-5-oxopyrrolidine-2-carboxylic acid and (2S,4S)-4-fluoro-5-oxopyrrolidine-2-carboxylic acid

[0064]

[0065] Sodium periodate (3 equiv) and ruthenium trichloride (0.2 equiv) were dissolved in a solution of EA:H₂O = 1:2 at room temperature. Then, commercially available raw materials N-BOC-trans-4-fluoro-L-proline methyl ester (1 equiv) or N-BOC-cis-4-fluoro-L-proline methyl ester (1 equiv) were added to the solution. The reaction was allowed to proceed for 5 hours. 100 mL of EA was added, and the mixture was washed with saturated sodium bicarbonate solution, saturated sodium dithionite solution, and saturated sodium chloride solution, respectively. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product. Under ice bath conditions, a 4M dioxane chloride solution was added dropwise to the crude product, and the reaction was allowed to proceed for 3 hours at room temperature. The reaction solution was then evaporated to dryness to obtain the crude product. The crude product was then dissolved in a solution of THF:MeOH:H₂O = 1:1:3, and lithium hydroxide (1.5 equiv) was added. The reaction was allowed to proceed for 8 hours, and the reaction solution was then evaporated to dryness to obtain the crude product, which can be directly used in the next reaction step.

[0066] Example

[0067] The present invention will be further described in detail below through embodiments. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of protection claimed by the present invention.

[0068] Example 1: Synthesis of (3R,5S)-1-(3,4-difluorophenyl)-5-(5-(3,5-dimethylisoxazol-4-yl)-1-((trans)-4-methoxycyclohexyl)-1H-benzo[d]imidazol-2-yl)-3-fluoropyrrolidone-2-one

[0069] Step 1: Synthesis of intermediate 1-1:

[0070]

[0071] 4-Bromo-1-fluoro-2-nitrobenzene (10 g, 45.46 mmol), 3,5-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxorane-2-yl)isoxazole (11.15 g, 50.00 mmol), 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride (351 mg, 0.45 mmol), and potassium carbonate (9.42 g, 68.18 mmol) were dissolved in 50 mL of N,N-dimethylformamide. 5 mL of water was added dropwise to the solution, and the mixture was purged with nitrogen three times. The reaction was carried out overnight at 100 °C. After the reaction was complete, the mixture was cooled, 200 mL of water was added, and the mixture was extracted with ethyl acetate (3 x 200 mL). The organic phases were combined, washed with saturated sodium chloride solution (2 x 100 mL), and the organic phase was collected. The mixture was dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography (petroleum ether: ethyl acetate = 5:1) to obtain 8.1 g of the product (i.e., intermediate 1-1), with a yield of 75.4%. 1 HNMR (400MHz, CDCl3) δ7.96(dd,J=7.0,2.5Hz,1H),7.54(ddd,J=8.5,4.0,2.5Hz,1H),7.41(dd,J=10.5,8.5Hz,1H),2.44(s,3H),2.29(s,3H).

[0072] Step 2: Synthesis of intermediates 1-2:

[0073]

[0074] The intermediate 1-1 (300 mg, 1.27 mmol) obtained in the previous step, trans-4-methoxycyclohexyl-1-amine (180.51 mg, 1.40 mmol), and potassium carbonate (438.83 mg, 3.18 mmol) were dissolved in 5 mL of N,N-dimethylformamide and reacted overnight at 70 °C. After the reaction was completed, the mixture was cooled, 50 mL of water was added, and the mixture was extracted with ethyl acetate (3 x 50 mL). The organic phases were combined, washed with saturated sodium chloride solution (2 x 25 mL), and the organic phase was collected, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography (petroleum ether: ethyl acetate = 3:1) to obtain the product (i.e., intermediate 1-2) 310 mg, with a yield of 70.6%.

[0075] Step 3: Synthesis of intermediates 1-3:

[0076]

[0077] The intermediates 1-2 obtained in the previous step (200 mg, 0.58 mmol) were dissolved in 20 mL of a 1:1 solution of tetrahydrofuran and aqueous solution. 1 mL of ammonia was added dropwise to the solution, followed by sodium dithionite (1.01 g, 5.79 mmol). The reaction was carried out at room temperature for 2 hours. After the reaction was complete, 50 mL of water was added, and the mixture was extracted with ethyl acetate (1 x 50 mL). The organic phases were combined, washed with saturated sodium chloride solution (2 x 25 mL), and collected. The organic phases were dried over anhydrous sodium sulfate, concentrated, and the crude product (containing intermediates 1-3) was directly proceeded to the next step without purification.

[0078] Step 4: Synthesis of intermediates 1-4:

[0079]

[0080] Intermediates 1-3 (110 mg, 0.35 mmol), (2S,4R)-4-fluoro-5-oxopyrrolidine-2-carboxylic acid (49.92 mg, 0.35 mmol), HATU (CAS NO: 148893-10-1, 133 mg, 0.52 mmol), and N,N-diisopropylethylamine (90.15 mg, 0.70 mmol) obtained in the previous step were dissolved in 5 mL of N,N-dimethylformamide and reacted at room temperature for 3 hours. After the reaction was completed, 50 mL of water was added, and the mixture was extracted with ethyl acetate (2 x 50 mL). The organic phases were combined, washed with saturated sodium chloride solution (2 x 25 mL), and the organic phase was collected, dried over anhydrous sodium sulfate, concentrated, and the resulting crude product (100 mg, 0.23 mmol) was dissolved in 5 mL of acetic acid and reacted overnight at 70 °C. After the reaction was complete, the mixture was cooled, evaporated to dryness, and 50 mL of saturated sodium bicarbonate solution was added. The mixture was extracted with ethyl acetate (2 x 25 mL). The organic phases were combined, washed with saturated sodium chloride solution (2 x 25 mL), collected, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography (dichloromethane:methanol = 30:1) to obtain 60 mg of the product (i.e., intermediate 1-4), with a yield of 62.7%. 1 H NMR (400MHz, CDCl3) δ7.60 (s, 1H), 7.56 (d, J = 8.4Hz, 1H), 7.31 (s, 1H), 7.13 (d, J=8.4Hz,1H),5.35(dt,J=52.5,5.9Hz,1H),5.26–5.20(m,1H),4.23(s,1H),3.4 3(s,3H),3.37(t,J=11.0Hz,1H),3.03–2.87(m,1H),2.87–2.71(m,1H),2.44–2 .29(m,7H),2.26(s,3H),2.06(dd,J=23.6,13.2Hz,2H),1.51(t,J=12.7Hz,2H).

[0081] Step 5: Synthesis of (3R,5S)-1-(3,4-difluorophenyl)-5-(5-(3,5-dimethylisoxazol-4-yl)-1-((trans)-4-methoxycyclohexyl)-1H-benzo[d]imidazol-2-yl)-3-fluoropyrrolidone-2-one:

[0082]

[0083] Intermediates 1-4 (60 mg, 0.14 mmol), 3,4-difluorophenylboronic acid (44.85 mg, 0.28 mmol), copper acetate monohydrate (34.02 mg, 0.17 mmol), and pyridine (112.33 mg, 1.42 mmol) obtained in the previous step were dissolved in 5 mL of dichloromethane and reacted at room temperature for 5 hours. After the reaction was completed, the reaction solution was evaporated to dryness, 50 mL of water was added, and the mixture was extracted with ethyl acetate (1 x 50 mL). The organic phases were combined, washed with saturated sodium chloride solution (2 x 25 mL), and the organic phase was collected, dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography (dichloromethane:methanol = 50:1) to give product 1, with a yield of 71.2%.

[0084] Examples 2 to 7 described below are synthesized using the method described in Example 1, or synthesized using corresponding intermediates in a method similar to that in Example 1.

[0085]

[0086]

[0087] The target compounds synthesized in Examples 1 to 7 and their molecular structures and 1 H NMR spectrum.

[0088]

[0089]

[0090] Example 8: Synthesis of (3S,5S)-1-(3,4-difluorophenyl)-5-(5-(3,5-dimethylisoxazol-4-yl)-1-((trans)-4-methoxycyclohexyl)-1H-benzo[d]imidazol-2-yl)-3-fluoropyrrolidone-2-one

[0091] Step 1: Synthesis of intermediate 8-1

[0092]

[0093] The intermediates 1-3 (110 mg, 0.35 mmol), (2S,4S)-4-fluoro-5-oxopyrrolidine-2-carboxylic acid (49.92 mg, 0.35 mmol), HATU (CAS NO: 148893-10-1, 133 mg, 0.52 mmol), and N,N-diisopropylethylamine (90.15 mg, 0.70 mmol) obtained above were dissolved in 5 mL of N,N-dimethylformamide and reacted at room temperature for 3 hours. After the reaction was completed, 50 mL of water was added, and the mixture was extracted with ethyl acetate (2 x 50 mL). The organic phases were combined, washed with saturated sodium chloride solution (2 x 25 mL), and the organic phases were collected, dried over anhydrous sodium sulfate, concentrated, and the resulting crude product (100 mg, 0.23 mmol) was dissolved in 5 mL of acetic acid and reacted overnight at 70 °C. After the reaction was complete, the mixture was cooled, evaporated to dryness, and 50 mL of saturated sodium bicarbonate solution was added. The mixture was extracted with ethyl acetate (2 x 25 mL). The organic phases were combined, washed with saturated sodium chloride solution (2 x 25 mL), collected, dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by column chromatography (dichloromethane:methanol = 30:1) to obtain 62 mg of the product (i.e., intermediate 8-1), with a yield of 65.3%. 1 H NMR (400MHz, CDCl3) δ7.63(s,1H),7.57(s,1H),7.15(s,1H),6.90(s,1H),5.43–5.19(m,1H),5.06(s,1H),4.23(s,1H), 3.43(s,3H),3.37(s,1H),3.14(s,1H),2.63(s,1H),2.50–2.30(m,7H),2.27(d,J=4.3Hz,4H),2.04(s,2H),1.48(s,2H).

[0094] Step 2: Synthesis of (3S,5S)-1-(3,4-difluorophenyl)-5-(5-(3,5-dimethylisoxazol-4-yl)-1-((trans)-4-methoxycyclohexyl)-1H-benzo[d]imidazol-2-yl)-3-fluoropyrrolidone-2-one

[0095]

[0096] The intermediate 8-1 (60 mg, 0.14 mmol) obtained in the previous step, 3,4-difluorophenylboronic acid (44.85 mg, 0.28 mmol), copper acetate monohydrate (34.02 mg, 0.17 mmol), and pyridine (112.33 mg, 1.42 mmol) were dissolved in 5 mL of dichloromethane and reacted at room temperature for 5 hours. After the reaction was completed, the reaction solution was evaporated to dryness, 50 mL of water was added, and the mixture was extracted with ethyl acetate (1 x 50 mL). The organic phases were combined, washed with saturated sodium chloride solution (2 x 25 mL), and the organic phase was collected, dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography (dichloromethane:methanol = 50:1) to give product 8, with a yield of 72.6%.

[0097] The following embodiments are synthesized using the above method, or using a similar method with corresponding intermediates.

[0098]

[0099]

[0100] The target compounds synthesized in Examples 8 to 17 and their molecular structures and 1 H NMR spectrum.

[0101]

[0102]

[0103]

[0104]

[0105] Example 18: (3S,5S)-1-(3,4-difluorophenyl)-5-(5-(1,3-dimethyl-1H-pyrazol-5-yl)-1-((trans)-4-methoxycyclohexyl)-1H-benzo[d]imidazol-2-yl)-3-fluoropyrrolidone-2-one

[0106] Step 1: Synthesis of intermediate 18-1

[0107] The synthetic route and operation steps are the same as those for intermediates 1-4.

[0108] Step 2: Synthesis of intermediate 18-2

[0109]

[0110] Intermediate 18-1 (60 mg, 0.14 mmol), 3,4-difluorophenylboronic acid (44.85 mg, 0.28 mmol), copper acetate monohydrate (34.02 mg, 0.17 mmol), and pyridine (112.33 mg, 1.42 mmol) were dissolved in 5 mL of dichloromethane and reacted at room temperature for 5 hours. After the reaction was complete, the reaction mixture was evaporated to dryness, 50 mL of water was added, and the mixture was extracted with ethyl acetate (1 x 50 mL). The organic phases were combined, washed with saturated sodium chloride solution (2 x 25 mL), and collected. The organic phases were dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography (dichloromethane:methanol = 50:1) to give the product (intermediate 18-2) in 80.3% yield.

[0111] Step 3: Synthesis of (3S,5S)-1-(3,4-difluorophenyl)-5-(5-(1,3-dimethyl-1H-pyrazol-5-yl)-1-((trans)-4-methoxycyclohexyl)-1H-benzo[d]imidazol-2-yl)-3-fluoropyrrolidone-2-one

[0112]

[0113] For detailed operating procedures, please refer to the synthesis of intermediate 1-1. The difference is that the starting material 4bromo-1-fluoro-2-nitrobenzene is replaced with intermediate 18-2, and 3,5-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxorane-2-yl)isoxazole is replaced with 1,3-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxorane-2-yl)-1H-pyrazole.

[0114] The following embodiments are synthesized using the above method, or using a similar method with corresponding intermediates.

[0115]

[0116] The target compounds synthesized in Examples 18 to 31 and their molecular structures and 1 H NMR spectrum.

[0117]

[0118]

[0119]

[0120] Synthesis of Examples 24 to 31

[0121]

[0122]

[0123] The target compounds synthesized in Examples 24 to 31 and their molecular structures and 1 H NMR spectrum.

[0124]

[0125]

[0126]

[0127]

[0128] Synthesis of Examples 32 to 33

[0129]

[0130] The target compounds synthesized in Examples 32 and 33 and their molecular structures and 1 H NMR spectrum.

[0131]

[0132]

[0133] Comparative Example 1 (CCS1477)

[0134]

[0135] The compound CCS1477 used was purchased from MCE.

[0136] Comparative Example 2

[0137] The synthesis was carried out using a method essentially the same as in Example 1, except that the starting material (2S,4R)-4-fluoro-5-oxopyrrolidine-2-carboxylic acid in step 4 was replaced with (R)-2-methyl-5-oxopyrrolidine-2-carboxylic acid.

[0138]

[0139] Example 1: Detection of the in vitro inhibitory activity of the compound against histone acetyltransferase p300

[0140] The inhibitory activity of the compound on the bromine domain of the p300 protein was detected using AlphaLISA technology. AlphaLISA utilizes microbeads as both donor and acceptor for the detection of biomolecules. If the target is present in the sample, the donor and acceptor microbeads will approach each other due to the specific recognition by the antibody, thereby stimulating a cascaded amplified chemiluminescent reaction on the acceptor microbeads, and finally transmitting the signal to europium to generate a signal.

[0141] Prepare 1x detection buffer before use. Dilute the compound with DMSO to the desired concentration for the reaction, then transfer 5 μL of the compound from the source plate to the assay plate (OptiPlate-384). Prepare protein and peptide solutions in 1x detection buffer. Add 2.5 μL of protein solution and 2.5 μL of peptide solution to each well. Centrifuge the assay plate at 1000 rpm for 1 minute, then incubate at room temperature for 60 minutes. Prepare receptor and donor bead solutions in 1x detection buffer. Add 10 μL of receptor bead solution to each well of the assay plate and incubate the assay plate at room temperature for 1 hour. Add 10 μL of donor bead solution to each well of the assay plate and incubate the assay plate at room temperature for 30 minutes. Read the signal using the Alpha mode of Tecan. Analyze the obtained data using GraphPad Prism. The experimental results are shown in Table 1.

[0142] Table 1: In vitro inhibitory activity of the compounds against p300

[0143]

[0144]

[0145] ++++: Represents IC 50 <10nM

[0146] +++: represents 10nM≤IC 50 <100nM

[0147] ++: represents 100nM≤IC 50 <1μM

[0148] The results above show that, compared with the introduction of methyl groups, the introduction of fluorine atoms at the carbonyl α-position of the five-membered lactam ring in the compounds of the present invention can improve the inhibitory activity against histone acetyltransferase p300, and the inhibitory activity of some compounds is comparable to that of the clinical investigational drug CCS1477 targeting this target.

[0149] Example 2: CCK8 assay for detecting inhibition of sensitive cell proliferation

[0150] To investigate the inhibitory effect of the compounds of this invention on tumor cell proliferation, the compounds were used to test the cell growth inhibitory activity of p300 / CBP-sensitive cell lines (OPM-2 cells purchased from the German Microbiological Culture Collection (DSMZ) and 22RV1 cells purchased from the Koebner Cell Bank) obtained in previous work. Cells in the logarithmic growth phase were spaced at 2.5 × 10⁶ cells per well. 3Cells were seeded at a concentration of [number] cells per well in 96-well plates. After 24 hours, serially diluted compounds were added. The plates were then flow-cultured for 5 days. Then, 10 μL of CCK-8 reagent (Cell Counting Kit-8) was added to each well, and the plates were incubated at 37°C for 2–6 hours. The absorbance of each well at 450 nm was read using a microplate reader, and analysis was performed using SoftMax Pro 5.4.1 software. The IC50 was calculated using a four-parameter method with GraphPad Prism 5 statistical software. 50 Values ​​(concentration-response curve fitting). Experimental results are shown in Table 2.

[0151] Table 2: Inhibitory activity of compounds against tumor cell proliferation

[0152]

[0153] The results above show that the compound of the present invention exhibits good anti-proliferative activity against OPM-2 multiple myeloma cells and can be used as a superior p300 bromodomain inhibitor for cancer treatment.

[0154] Example 3: Pharmacokinetic Evaluation

[0155] Animal Information

[0156] Species / breed: ICR (CD-1) mouse; sex / number: male; weight (g): male (25-30g).

[0157] Feeding method: Standard diet for serpentine animals, no restriction on drinking water, fasting for 12 hours before administration and fasting for 2 hours after administration.

[0158] Husbandry environment: Control the animal room environment (target conditions: temperature 18 to 29°C, relative humidity 30 to 70%). Monitor temperature and relative humidity daily. An electronic time-controlled lighting system is used to provide a 12-hour light / 12-hour dark cycle.

[0159] Dosage information

[0160]

[0161]

[0162] Sample collection

[0163] ICR mice were orally administered the test compound at a dose of 10 mg / kg, with an administration volume of 10 mL / kg. Blood samples were collected from the orbital venous plexus at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h after administration and placed in heparinized EP tubes, which were then temporarily placed on crushed ice.

[0164] Sample processing and analysis

[0165] Centrifuge at 8000 rpm for 5 min, and transfer 15 μL of the supernatant plasma to a 96-well plate. Add 150 μL of methanol:acetylene (v / v = 1:1) (containing 20 ng / mL tolbutamide) to the 15 μL plasma, shake for 3 min, centrifuge at 4500 rpm for 5 min, and transfer 100 μL of the supernatant to a 2 mL deep-well plate. Add 100 μL of diluent (pure water), shake for 3 min, and centrifuge at 4500 rpm for 5 min. Transfer 180 μL of the supernatant to the injection plate, analyze the compound content in the supernatant sample by LC-MS / MS, and calculate various pharmacokinetic parameters using WinNonlin software. The pharmacokinetic parameters of the test compounds are shown in Table 3.

[0166] Table 3: PK properties of some invented compounds in mice

[0167]

[0168] Experimental results show that the compound of the present invention has good oral absorption and exposure levels, which are significantly improved compared to Comparative Example 2, and compared to Comparative Example 1, although C max The AUC decreased, but the half-life T... 1 / 2 Both the residence time (MRT) and the duration of residence (MRT) have been improved, suggesting that the compound of this invention has the potential to become an orally effective antitumor drug.

[0169] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, characterized in that, The compound is: (3S,5S)-1-(3,4-difluorophenyl)-5-(5-(3,5-dimethylisoxazol-4-yl)-1-((trans)-4-(methoxy-d3)cyclohexyl)-1H-benzo[d]imidazol-2-yl)-3-fluoropyrrolidone-2-one, its structural formula is as follows: 。 2. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (1) The compound of claim 1 or a pharmaceutically acceptable salt thereof; and (2) Pharmaceutically acceptable carriers and / or excipients.

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