CN110003204B - A kind of BET protein inhibitor, its preparation method and use - Google Patents

Abstract

The invention belongs to the technical field of biomedicine, and in particular relates to a BET protein inhibitor, a preparation method and application thereof. Compared with the prior art, the present invention further improves the structure on the basis of the existing BET protein inhibitor, and obtains a new type of BET protein inhibitor, which has better BET protein inhibitory activity and good pharmacokinetics. It is expected to be developed into a new generation of BET protein inhibitors with high efficiency and low toxicity.

Description

A kind of BET protein inhibitor, its preparation method and use

technical field

The invention belongs to the technical field of biomedicine, and in particular relates to a BET protein inhibitor, a preparation method and application thereof.

Background technique

Epigenetics is one of the hottest topics in drug discovery right now. Histone acetylation is an important part of epigenetic studies. Bromodomain (BRD) is a class of conserved protein domains that can specifically recognize acetylated lysine (KAc) in histones. By combining with acetylated lysine, the protein is enriched in specific gene transcription sites and changes RNA. The activity of polymerase II regulates the transcriptional expression of genes (Kuc and Allis, Bioessays, 1998, 20:615-626).

At present, 61 BRD domains found in the human body exist in 42 proteins. According to the different functions of the parent protein, BRD proteins are divided into 8 families, and the BET protein family is the second type of BRD protein family. BET proteins include four members, BRD2, BRD3, BRD4, and BRDT (Wu and Chiang, J. Biol. chem., 2007, 282: 13141-13145). The first three are widely expressed in all body cells, while the latter is only expressed in testis.

BET proteins play an important role in a variety of tumors. For example, hematopoietic tumors (acute myeloid leukemia, lymphoma, multiple myeloma, B-cell acute lymphoblastic leukemia, etc.) can inhibit the expression of MYC by interfering with the binding of BRD4 to the oncogene MYC, thereby causing tumor cell apoptosis. Fusions between a BET protein (BRD3 or BRD4) and NUT (a protein normally expressed only in the testis) result in an aggressive form of squamous cell carcinoma known as NUT midline carcinoma (French, CancerGenet, Cytogenet., 2010, 203:16-20).

A novel fusion oncogene BRD-NUT was found to involve repeated translocation of NUT (a nuclear protein in the testis) with BRD3 or BRD4 in a highly malignant form of epithelial neoplasia (French et al. Cancer Research 2003, 63:304-307) . Selective reduction of this oncogene restores normal cell differentiation and reverses the tumorigenic phenotype (Filippakopoulos et al., Nature 2010, 468:1068-1073). Knockout of BRD2, BRD3 and BRD4 has been shown to impair the growth and viability of various hematological and solid tumor cells (Zuber et al., Nature 2011, 478:524-528; Delmore et al., Cell 2011, 146:904-917). In addition to a role in cancer, BET proteins also regulate inflammatory responses to bacterial challenge, and BRD2 suballelic mouse models show significantly lower levels of inflammatory cytokines and protection against obesity-induced diabetes (Wang et al., Biochem J. 2009, 425:71-83). Additionally, some viruses use these BET proteins to tether their genomes to host cell chromatin as part of the viral replication process, or use BET proteins to facilitate viral gene transcription and repression (You et al., Ce11 2004, 117:349-360; Zhu et al, Cell Reports 2012, 2:807-816).

Taken together, targeting these proteins may be beneficial for the development of new therapeutic strategies targeting cancer, inflammation and viral infections. At present, small molecule inhibitors targeting this receptor have entered the clinical stage, which are mainly used for the treatment of cancer and autoimmune diseases. Currently, a series of BET inhibitor patent applications have been published, including: WO2013158952, WO2014165127, WO2015075665, WO2016050821, WO2018188047, WO2018130174 and the like.

Abbive Company disclosed a class of BET protein inhibitors in WO2013097052A, wherein the compound ABBV-075 has development prospects and is currently in the phase I clinical trial stage. Incyte Company disclosed the compound INCB-057643 in the CN106414442A patent, which is in the phase I clinical trial stage. Bristol-Myers Squibb Company disclosed another class of BET protein inhibitors in WO2015100282A, and the representative compound BMS-986158 is in the phase I clinical trial stage. GlaxoSmithKline Company disclosed the compound I-BET762 in WO2011054553A, and the compound is in the phase II clinical trial of tumor.

Although there are no drugs on the market, BET protein inhibitors have good application prospects as drug development. At this stage, more novel bromodomain inhibitors for the treatment of diseases and indications involving bromodomain function, including BET domain function, need to be developed. The compounds of the present invention are also helpful to meet such clinical needs, and we hope to develop a new generation of BET protein inhibitors with high efficiency and low toxicity.

SUMMARY OF THE INVENTION

In order to overcome the problems existing in the prior art, the purpose of the present invention is to provide a BET protein inhibitor with high efficiency and low toxicity.

In order to achieve the above purpose and other related purposes, the present invention adopts the following technical solutions:

A BET protein inhibitor, wherein the inhibitor is a compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein,

A is selected from

或被R₁₁和/或R₁₂取代的五元芳杂环；所述的“五元芳杂环”，是指5个原子的单环芳烃，并且含有1个或多个杂原子(例如N，O，S)，包括但不限于呋喃，咪唑，噻吩，吡唑等。其中：Ring B is selected from

Or five-membered aromatic heterocycle substituted by R ₁₁ and/or R ₁₂ ; the "five-membered aromatic heterocycle" refers to a 5-atom monocyclic aromatic hydrocarbon and contains 1 or more heteroatoms (such as N , O, S), including but not limited to furan, imidazole, thiophene, pyrazole, etc. in:

X is O, S or NR ₁₃ ;

Y is CH ₂ , C=O, NR ₁ or Y is absent;

Z is CR ₁₄ or N;

U is CH or N;

W is O or NH;

V is CH or N;

R ₁ is hydrogen, C ₁ -C ₆ alkyl, C ₃ -C ₆ cycloalkyl, substituted C ₁ -C ₆ alkyl, substituted C ₃ -C ₆ cycloalkyl, C ₃ -C ₆ cycloalkyl C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl-SO ₂ - or C ₃ -C ₆ cycloalkyl-SO ₂ -; the substituted C ₁ -C ₆ alkyl refers to C ₁ -C ₆ alkane The hydrogen on the base is replaced by one or more C ₃ -C ₆ cycloalkyl; the substituted C ₃ -C ₆ cycloalkyl means that the hydrogen on the C ₃ -C ₆ cycloalkyl is replaced by one or more C ₁ -C ₆ alkyl substitution;

R ₂ is hydrogen, C ₁ -C ₆ alkyl, C ₁ -C ₃ alkoxy, halogen or cyano;

R ₃ is hydrogen, C ₁ -C ₆ alkyl, C ₁ -C ₃ alkoxy, halogen, cyano, -S(O) ₂ R ₁₅ , -S(O) ₂ NR ₁₆ R ₁₇ or -N( R ₁₆ )S(O) ₂ R ₁₅ ;

R ₄ is hydrogen, C ₁ -C ₆ alkyl, C ₁ -C ₃ alkoxy, halogen or cyano;

R ₅ and R ₆ are each independently hydrogen or C ₁ -C ₆ alkyl;

R ₇ , R ₈ , R ₉ and R ₁₀ are each independently hydrogen, halogen, cyano, C ₁ -C ₃ alkoxy or C ₁ -C ₆ alkyl;

R ₁₁ is hydrogen or C ₁ -C ₆ alkyl;

R ₁₂ is hydrogen or C ₁ -C ₆ alkyl;

R ₁₃ is hydrogen or C ₁ -C ₆ alkyl;

R ₁₄ is hydrogen, C ₁ -C ₆ alkyl, halogen or cyano;

R ₁₅ is C ₁ -C ₆ alkyl;

R ₁₆ is hydrogen or C ₁ -C ₆ alkyl;

R ₁₇ is hydrogen or C ₁ -C ₆ alkyl;

The aforementioned halogen is selected from fluorine, chlorine, bromine or iodine.

Preferably, in the compound of formula I:

A is selected from

Ring B is selected from

in:

X is O, S or NR ₁₃ ;

Y is CH ₂ , C=O, NR ₁ or Y is absent;

Z is CR ₁₄ or N;

U is CH or N;

W is O or NH;

V is CH or N;

R ₁ is hydrogen, C ₁ -C ₄ alkyl, C ₃ -C ₆ cycloalkyl, substituted C ₁ -C ₄ alkyl, substituted C ₃ -C ₆ cycloalkyl, C ₃ -C ₆ cycloalkyl C ₁ -C ₃ alkyl, C ₁ -C ₄ alkyl-SO ₂ - or C ₃ -C ₆ cycloalkyl-SO ₂ -; the substituted C ₁ -C ₄ alkyl refers to C ₁ -C ₄ alkane The hydrogen on the base is replaced by one or more C ₃ -C ₆ cycloalkyl; the substituted C ₃ -C ₆ cycloalkyl means that the hydrogen on the C ₃ -C ₆ cycloalkyl is replaced by one or more C ₁ -C ₄ alkyl substitution;

R ₂ is hydrogen, C ₁ -C ₄ alkyl, C ₁ -C ₂ alkoxy, halogen or cyano;

R ₃ is hydrogen, C ₁ -C ₄ alkyl, C ₁ -C ₂ alkoxy, halogen, cyano, -S(O) ₂ R ₁₅ , -S(O) ₂ NR ₁₆ R ₁₇ or -N( R ₁₆ )S(O) ₂ R ₁₅ ;

R ₄ is hydrogen, C ₁ -C ₄ alkyl, C ₁ -C ₃ alkoxy, halogen or cyano;

R ₅ and R ₆ are each independently hydrogen or C ₁ -C ₄ alkyl;

R ₇ , R ₈ , R ₉ and R ₁₀ are each independently hydrogen, halogen, cyano, C ₁ -C ₃ alkoxy or C ₁ -C ₄ alkyl;

R ₁₁ is hydrogen or C ₁ -C ₄ alkyl;

R ₁₂ is hydrogen or C ₁ -C ₄ alkyl;

R ₁₃ is hydrogen or C ₁ -C ₄ alkyl;

R ₁₄ is hydrogen, C ₁ -C ₄ alkyl, halogen or cyano;

R ₁₅ is C ₁ -C ₄ alkyl;

R ₁₆ is hydrogen or C ₁ -C ₄ alkyl;

R ₁₇ is hydrogen or C ₁ -C ₄ alkyl;

The aforementioned halogen is selected from fluorine, chlorine, bromine or iodine.

Preferably, in the compound of formula I:

A is selected from

Ring B is selected from

in:

X is O, S or NR ₁₃ ;

Y is CH ₂ , C=O, NR ₁ or Y is absent;

Z is CR ₁₄ or N;

U is CH or N;

W is O or NH;

V is CH or N;

R ₁ is hydrogen, C ₁ -C ₃ alkyl, C ₃ -C ₆ cycloalkyl, substituted C ₁ -C ₃ alkyl, substituted C ₃ -C ₆ cycloalkyl, C ₃ -C ₆ cycloalkyl C ₁ -C ₃ alkyl, C ₁ -C ₃ alkyl-SO ₂ - or C ₃ -C ₆ cycloalkyl-SO ₂ -; the substituted C ₁ -C ₃ alkyl refers to C ₁ -C ₃ alkane The hydrogen on the base is replaced by one or more C ₃ -C ₆ cycloalkyl; the substituted C ₃ -C ₆ cycloalkyl means that the hydrogen on the C ₃ -C ₆ cycloalkyl is replaced by one or more C ₁ -C ₃ alkyl substitution;

R ₂ is hydrogen, C ₁ -C ₃ alkyl, C ₁ -C ₂ alkoxy, halogen or cyano;

R ₃ is hydrogen, C ₁ -C ₃ alkyl, C ₁ -C ₂ alkoxy, halogen, cyano, -S(O) ₂ R ₁₅ , -S(O) ₂ NR ₁₆ R ₁₇ or -N( R ₁₆ )S(O) ₂ R ₁₅ ;

R ₄ is hydrogen, C ₁ -C ₃ alkyl, C ₁ -C ₂ alkoxy, halogen or cyano;

R ₅ and R ₆ are each independently hydrogen or C ₁ -C ₃ alkyl;

R ₇ , R ₈ , R ₉ and R ₁₀ are each independently hydrogen, halogen, cyano, C ₁ -C ₂ alkoxy or C ₁ -C ₃ alkyl;

R ₁₁ is hydrogen or C ₁ -C ₃ alkyl;

R ₁₂ is hydrogen or C ₁ -C ₃ alkyl;

R ₁₃ is hydrogen or C ₁ -C ₃ alkyl;

R ₁₄ is hydrogen, C ₁ -C ₃ alkyl, halogen or cyano;

R ₁₅ is C ₁ -C ₃ alkyl;

R ₁₆ is hydrogen or C ₁ -C ₃ alkyl;

R ₁₇ is hydrogen or C ₁ -C ₃ alkyl;

The aforementioned halogen is selected from fluorine, chlorine, bromine or iodine.

The "halogen" in this application refers to F, Cl, Br or I; the "C ₁ -C ₃ alkyl group" refers to methyl, ethyl, n-propyl or isopropyl; the "C ₁ -C ₂ alkoxy" is methoxy or ethoxy; the "cycloalkyl", unless otherwise specified, refers to saturated or partially unsaturated carbon atoms containing 3 to 6 carbon atoms. Cyclic hydrocarbons. Cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl.

The compounds of the present invention can be prepared in the form of pharmaceutically acceptable salts according to conventional methods; including their organic acid salts and inorganic acid salts: inorganic acids include (but not limited to) hydrochloric acid, sulfuric acid, phosphoric acid, diphosphoric acid, hydrobromic acid, nitric acid, etc., Organic acids include, but are not limited to, acetic acid, maleic acid, fumaric acid, tartaric acid, succinic acid, lactic acid, p-toluenesulfonic acid, salicylic acid, oxalic acid, and the like.

Typical compounds of the present invention include, but are not limited to, the following Table 1 compounds:

The second object of the present invention is to provide the synthetic method of above-mentioned compound:

The compound of the general formula IA and the compound of the general formula IB are subjected to a catalytic coupling reaction to obtain the compound of the general formula I, and the definitions of each group are as described above.

The third object of the present invention is to provide the use of the above compound as a novel BET protein inhibitor in the preparation of a medicament for preventing or treating BET protein-related diseases.

Specifically, the BET protein-related diseases refer to tumor diseases, benign hyperplasia, inflammatory diseases, autoimmune diseases, sepsis, viral infections, vascular diseases and neurological diseases. Further, the tumor diseases include but are not limited to acute myeloid leukemia, lymphoma, multiple myeloma, B-cell acute lymphoblastic leukemia, midline cancer, glioma, solid tumor, breast cancer, colorectal cancer, prostate cancer , cervical cancer, non-small cell lung cancer, melanoma, etc.

During the course of disease treatment, the derivatives of the present invention can be used to treat related cancers and other diseases by oral administration, injection, etc. in the form of compositions. When used for oral administration, it can be prepared into conventional solid preparations such as tablets, powders or capsules; when used for injection, it can be prepared into injection solutions.

The fourth object of the present invention is to provide a composition comprising a therapeutically effective amount of the above compound or a pharmaceutically acceptable salt thereof and a medically acceptable carrier.

The mentioned carrier refers to the conventional carrier in the pharmaceutical field, such as: diluents, excipients such as water, etc.; binders such as cellulose derivatives, gelatin, polyvinylpyrrolidone, etc.; fillers such as starch, etc.; disintegrating agents such as carbonic acid Calcium, sodium bicarbonate; in addition, other adjuvants such as flavors and sweeteners can also be added to the composition.

Various dosage forms of the composition of the present invention can be prepared by conventional methods in the medical field, wherein the content of the active ingredient is 0.1% to 99.5% (weight ratio).

The dosage of the present invention can be changed according to the route of administration, the age of the patient, the body weight, the type and severity of the disease to be treated, etc., and its daily dose is 0.005-30 mg/kg body weight (oral) or 0.005-30 mg/kg body weight ( injection).

Compared with the prior art, the present invention further improves the structure on the basis of the existing BET protein inhibitor, and obtains a new type of BET protein inhibitor, which has better BET protein inhibitory activity and good pharmacokinetics. It is expected to be developed into a new generation of BET protein inhibitors with high efficiency and low toxicity.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Reference Example 1: Synthetic route of fragment A1.

Step 1: Synthesis of compound A1-2.

To a solution of compound A1-1 (400 mg, 1.76 mmol) in DMF (20 mL) was added NaH (141 mg, 3.52 mmol) at 0°C. The mixture was stirred at this temperature for 10 minutes and TsCl (671 mg, 3.52 mmol) was added at 0 °C. The mixture was then stirred at room temperature for 2 hours. The reaction mixture was diluted with _H2O (20 mL) and extracted with EA (30 mL x 2). The organic layers were combined and washed with brine (20 mL x 2), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was subjected to silica gel column chromatography (PE/EA=2/1) to obtain compound A1-2 (800 mg, 95% yield) as a yellow solid. MS: 382.0 [M+H] ⁺ .

Step 2: Synthesis of compound A1-3.

To a solution of compound A1-2 (800 mg, 2.1 mmol) in dioxane (10 mL) was added 4M HCl in dioxane (5 mL) and the reaction mixture was stirred at 50°C for 2 hours. The reaction mixture was cooled to room temperature and concentrated, the residue was diluted with _H2O (20 mL) and extracted with EA (30 mL x 2). The organic layers were combined, washed with brine (20 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated to give compound A1-3 (600 mg, 100% yield) as a yellow solid. MS: 368.0 [M+H] ⁺ .

Step 3: Synthesis of compound A1-4.

To a solution of compound A1-3 (600 mg, 1.63 mmol) in DMF (10 mL) at 0 °C was added NaH (131 mg, 3.26 mmol). The mixture was stirred at this temperature for 10 minutes and _CH3I (671 mg, 3.52 mmol) was added in an ice bath. The mixture was stirred at room temperature for 3 hours. The reaction mixture was diluted with _H2O (20 mL) and extracted with EA (30 mL x 2). The combined organic layers were washed with brine (20 mL x 2), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was subjected to silica gel column chromatography (PE/EA=2/1) to obtain compound A1-4 (450 mg, 72% yield) as a yellow solid. MS: 382.0 [M+H] ⁺ .

Step 4: Synthesis of Compound A1.

Compound A1-4 (300 mg, 0.788 mmol), Pin ₂ B ₂ (400 mg, 1.57 mmol), KOAc (232 mg, 2.36 mmol), X-phos (38 mg, 0.08 mmol), Pd ₂ (dba) were sequentially added to the reaction flask ₃ (73 mg, 0.08 mmol) and dioxane (20 mL). The mixture was stirred at 85°C for 3 hours under N2 protection. The reaction mixture was diluted with _H2O (20 mL) and extracted with DCM (30 mL x 2). The combined organic layers were washed with brine (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated to give crude compound A1-5 (500 mg, 100% yield) as a yellow solid. MS: 429.0 [M+H] ⁺ , ¹ H NMR (400 MHz, DMSO-d ₆ ) δ: 8.11 (s, 1H), 7.99-8.01 (m, 2H), 7.92-7.93 (d, J=4.0 Hz, 1H), 7.30-7.32(m, 2H), 6.50-6.51(d, J=4.0Hz, 1H), 3.50(s, 3H), 2.40(s, 3H), 1.25(s, 12H).

Reference Example 2-7: Fragments A2 to A7.

With reference to the synthetic method of steps 1-4 in reference example 1, synthesize each reference example in table 2 below:

Reference Example 8: Synthetic route of fragment A8.

Compound A8-1 (150 mg, 0.95 mmol), Pin ₂ B ₂ (1.2 g, 4.73 mmol), KOAc (186 mg, 1.9 mmol), X-phos (91 mg, 0.19 mmol), Pd ₂ (dba ) ₃ (87 mg, 0.095 mmol) and dioxane (10 mL). The mixture was stirred at 85°C for 1 hour. The reaction solution was cooled to room temperature, diluted with H ₂ O (20 mL), and extracted with DCM (30 mL×2). The organic layers were combined, washed with brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give crude product A8-1 (200 mg, 94% yield) as a yellow solid. MS: 251.1 [M+H] ⁺ .

Reference Examples 9-11: Fragments A9 to A11.

With reference to the synthetic method of Reference Example 8, synthesize each Reference Example in the following table 3:

Reference Example 12: Synthetic route of fragment C1.

Step 1: Synthesis of compound C1-2.

To a solution of compound C1-1 (11 g, 0.05 mol) in concentrated H ₂ SO ₄ (60 mL) was added Br ₂ (8 g, 0.05 mol) dropwise, followed by HNO ₃ (2.5 mL) in an ice-water bath. The reaction solution was heated to 90° C. and incubated for 5 hours. The reaction was cooled to room temperature, diluted with _H2O (50 mL), and extracted with DCM (50 mL x 2). The two extracts were combined, washed with saturated Na ₂ S ₂ O ₃ (50 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography (PE/EA=2/1) to obtain compound C1-2 (4.3 g, 29% yield) as a white solid. MS: 297.1[MH] ^- .

Step 2: Synthesis of compound C1-4.

Compound C1-2 (4.1 g, 13.8 mmol), 4-fluoro-3-iodophenol (3.45 g, 14.5 mmol) and K ₂ CO ₃ (2 g, 14.5 mmol) were added to DMSO (120 mL), and the reaction solution was heated at 120 The reaction was heated at °C for 3 hours. The reaction was cooled to room temperature, quenched with H ₂ O (30 mL), and extracted with EA (50 mL x 2). The combined organic layers were washed with brine (50 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography (PE/EA=2/1) to obtain compound C1-4 (5.4 g, 76% yield) as a yellow solid. MS: 515.1[MH] ^- .

Step 3: Synthesis of compound C1-5.

Compound C1-4 (5.4 g, 10.5 mmol), Fe (2.0 g, 37 mmol) and NH ₄ Cl (334 mg, 6.3 mmol) were added to EtOH (100 mL) and H ₂ O (20 mL) at 90 °C The reaction was stirred for 3 hours. The reaction was cooled to room temperature, diluted with H ₂ O (50 mL) and extracted with EA (80 mL). The layers were separated, the organic layer was concentrated, and the obtained residue was purified by silica gel column chromatography (PE/EA=2/1) to obtain compound C1-5 (3.3 g, 64.7% yield) as a yellow solid. MS: 485.1[MH] ^- .

Step 4: Synthesis of compound C1-6.

Compound C1-5 (500 mg, 1.03 mmol), Cs ₂ CO ₃ (1 g, 3.09 mmol), BINAP (128 mg, 0.2 mmol) and Pd ₂ (dba) ₃ (192 mg, 0.2 mmol) were added to dioxane (40 mL) , the reaction solution was stirred at 80°C overnight. The reaction solution was cooled to room temperature, diluted with H ₂ O (50 mL), and extracted with EA (50 mL×2). The organic layer was washed with brine (30 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography (PE/EA=2/1) to obtain compound C1-6 (290 mg, 37.6% yield) as a yellow solid. MS: 357.2[MH] ^- .

Step 5: Synthesis of compound C1.

Under ice bath, to a mixed solution of compound 5 (660 mg, 0.02 mol), polyoxymethylene (166 mg, 5.53 mmol) and DCM (20 mL) was added TFA (1.3 mL) and Et ₃ SiH (2.6 mL), and the reaction was stirred at room temperature for 2 hours. . The reaction solution was diluted with H ₂ O (30 mL) and extracted with EA (50 mL×2). The organic layers were combined, washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography (PE/EA=2/1) to obtain compound C1 (300 mg, 41% yield) as a yellow solid. MS: 373.2 [M+H] ⁺ , ¹ H NMR (400 MHz, CDCl ₃ ) δ: 7.35(s, 1H), 7.00(s, 1H), 6.52-6.55(m, 1H), 6.38-6.42(m, 1H), 6.36(s, 1H), 3.35(s, 3H), 3.19(s, 3H).

Reference Examples 13-53: Fragments C2 to C42.

With reference to the synthetic method of steps 1-5 in Reference Example 12, synthesize each Reference Example in the following table 4:

Reference Example 54: Synthetic route of fragments C43 and C44.

Step 1: Synthesis of compound C43-3.

Compound C43-1 (2.51 g, 10.0 mmol), C43-2 (1.65 g, 10.5 mmol) and K ₂ CO ₃ (2.1 g, 15 mmol) were added to DMF (20 mL), and the reaction solution was heated at 100 °C for reaction 3 Hour. The reaction was cooled to room temperature, quenched with H ₂ O (30 mL), and extracted with EA (50 mL x 2). The combined organic layers were washed with brine (50 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography (PE/EA=2/1) to obtain compound C43-3 (2.4 g, 62% yield) as a white solid. MS: 390.2 [M+H] ⁺ .

Step 2: Synthesis of compound C43.

Compound C43-3 (2.0 g, 5.1 mmol) was added to polyphosphoric acid (10 mL), heated to 120° C. under stirring for 3 hours, the reaction solution was cooled to room temperature, ice water (30 mL) was added under stirring, stirred for 30 min, filtered, and the resulting The crude product was recrystallized from methanol (10 mL) and dried under vacuum at 50° C. for 4 h to obtain C43 (1.54 g, 81% yield) as an off-white solid. MS: 358.2 [M+H] ⁺ , ¹ H NMR (400 MHz, CDCl ₃ ) δ: 7.41 (s, 1H), 7.05 (s, 1H), 6.67 (m, 2H), 6.58 (m, 1H), 3.86 (s, 1H), 3.35 (s, 3H).

Step 3: Synthesis of compound C44.

1N borane tetrahydrofuran solution (2 mL) was added dropwise to C43 (1.0 g, 2.7 mmol) in THF solution (10 mL) under an ice-salt bath. After the dropwise addition, the reaction was stirred at room temperature for 1 h, and then heated to reflux for 2 h. Cooled to room temperature, anhydrous ethanol (2 mL) was added dropwise with stirring, after the dropwise addition was completed, the mixture was concentrated to dryness under reduced pressure, the residue was extracted with dichloromethane (10 mL)/H ₂ O (5 mL), and the layers were separated. Washed with saturated brine (5 mL), separated, dried and concentrated to obtain crude C42, which was recrystallized from anhydrous ethanol (5 mL) and dried under vacuum at 50°C for 4 h to obtain C44 (0.82 g, 85% yield), which was off-white solid. MS: 372.2 [M+H] ⁺ , ¹ H NMR (400 MHz, CDCl ₃ ) δ: 8.21(s, 1H), 8.05(s, 1H), 7.15-7.22(m, 3H), 3.34(s, 3H) .

Reference Examples 55-56: Fragments C45 to C46.

With reference to the synthetic method of steps 1-3 in Reference Example 54, synthesize each Reference Example in the following table 5:

Reference Example 57: Synthetic route of fragment C47.

Step 1: Synthesis of compound C47-2.

Compound C47-1 (2.51 g, 10.0 mmol), m-fluorophenylboronic acid (1.53 g, 11.0 mmol) and K ₂ CO ₃ (3.45 g, 25.0 mmol) were added to DMF (20 mL), and the reaction solution was under N ₂ gas protection , heated to 100 °C for 3 hours. The reaction solution was cooled to room temperature, extracted with H ₂ O (30 mL)/EtOAc (60 mL), the organic layer was washed with brine (50 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography (PE/EA=1/1) to obtain compound C47-2 (2.13 g, 80% yield) as an off-white solid. MS: 267.3 [M+H] ⁺ .

Step 2: Synthesis of compound C47.

C47-2 (2.1 g, 7.9 mmol), CuI (2.2 g, 11.8 mmol), pivalic acid (0.8 g, 7.9 mmol) and DMSO (10 mL) were successively added to the open reaction flask, and heated to an internal temperature of 130 ℃ under stirring. ℃ reaction 12h. After the reaction was complete, EtOAc (30 mL) was added to the cooled reaction mixture. Ammonia water (15 mL) and brine (15 mL) were successively added for washing and separation. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to dryness in vacuo. The obtained residue was purified by silica gel column chromatography (PE/EA=10/1) to obtain compound C47 (1.78 g, 58% yield) as an off-white solid. MS: 391.2 [M+H] ⁺ , ¹ H NMR (400 MHz, CDCl ₃ ) δ: 8.24(s, 1H), 8.15(s, 1H), 7.88(m, 1H), 7.13(m, 1H), 6.84 (m, 1H), 3.32 (s, 1H).

Reference Examples 58-60: Fragments C48-C50.

With reference to the synthetic method of steps 1-2 in Reference Example 57, synthesize each Reference Example in the following table 6:

Example 1

synthetic route:

Step 1: Synthesis of Compound I-1-1

Compound A1 (473 mg, 0.97 mmol), C1 (180 mg, 0.485 mmol), CsF (221 mg, 1.45 mmol), Pd(dppf)Cl ₂ (35 mg, 0.05 mmol), dioxane (10 mL), H ₂ O ( 2mL) was added to the reaction flask in turn, heated to 85°C under N2 protection and reacted for 12h. The reaction solution was cooled to room temperature, diluted with H ₂ O (20 mL), extracted with DCM (30 mL x 2), and the organic phases were combined. The organic phase was washed with saturated brine (20 mL) and separated. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to dryness in vacuo. The obtained residue was purified by silica gel column chromatography (PE/EA=1/2) to obtain compound I-1-1 (178 mg, 62% yield) as a pale yellow solid. MS: 594.6 [M+H] ⁺ .

Step 2: Synthesis of Compound I-1

To a methanol solution (10 mL) of I-1-1 (160 mg, 0.27 mmol) was added 6M NaOH aqueous solution (5 mL), and the reaction mixture was heated to 80° C. for 1 h. The reaction solution was cooled to room temperature, diluted with H ₂ O (20 mL), extracted with DCM (30 mL x 2), and the organic phases were combined. The organic phase was washed with saturated brine (20 mL) and separated. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to dryness in vacuo. The obtained residue was purified by silica gel column chromatography (PE/EA=1/2) to obtain compound I-1 (52 mg, 44% yield) as a white solid. MS: 440.5 [M+H] ⁺ . ¹ H NMR (400MHz, DMSO-d6)δ: 12.11(s, 1H), 7.31-7.37(m, 3H), 7.14(s, 1H), 6.74-6.77(m, 1H), 6.44-6.52(m, 2H), 6.16(s, 1H), 3.59(s, 3H), 3.25(s, 3H), 3.17(s, 3H).

Referring to the synthetic methods of steps 1-2 in Example 1, the compounds of each example in Table 7 below were synthesized:

Example 56: I-56

synthetic route:

Compound A12 (200 mg, 2.06 mmol), C1 (1533 mg, 4.12 mmol), K ₂ CO ₃ (568 mg, 4.12 mmol), Pd(OAc) ₂ (46 mg, 0.2 mmol), dioxane (20 mL), H ₂ O (2 mL) was added to the reaction flask successively, and the temperature was heated to 85 °C under the protection of N ₂ for 12 h. The reaction solution was cooled to room temperature, diluted with H ₂ O (20 mL), extracted with DCM (30 mL x 2), and the organic phases were combined. The organic phase was washed with saturated brine (20 mL) and separated. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to dryness in vacuo. The obtained residue was purified by silica gel column chromatography (PE/EA=1/1) to obtain compound I-56 (400 mg, 50% yield) as a pale yellow solid. MS: 389.4 [M+H] ⁺ . ¹ H NMR (400MHz, DMSO-d6)δ: 7.41(s, 1H), 7.05(s, 1H), 6.91(m, 1H), 6.50(m, 2H), 3.35(s, 3H), 3.20(s ,3H),2.25(s,6H).

With reference to the synthetic method in Example 56, each example compound in the following table 8 was synthesized:

The NMR and MS data of each embodiment are summarized in Table 9 below:

Test example 1, BRD4 activity test

Combined reaction process:

(1) Prepare 1×Assay buffer.

(2) Preparation of compound concentration gradient: The final concentration of the test compound and the positive control compounds AbbV-075 and I-BET762 are all starting at 1000 nM, and 10 concentrations are diluted 3 times, and each concentration is tested in duplicate. The compound was firstly diluted with DMSO as solvent to obtain a solution of the corresponding 1000-fold final concentration, and then the DMSO solution of the corresponding concentration was diluted 100-fold with 1×Assay buffer (the DMSO concentration was 1% at this time), and 2 μL of compound 1% was transferred with a discharge gun The DMSO solution was placed in a 384-well reaction plate for testing. Transfer 2 μL of 1% DMSO solution to Max well, and transfer 2 μL of AbbV-075 highest concentration 1% DMSO solution to Min well.

(3) Prepare 5× protein solution with 1× reaction solution.

(4) Prepare 5× polypeptide solution with 1× reaction solution.

(5) Add 4 μL of 5× protein solution to each well, centrifuge at 1000 rpm for 1 min, and incubate at room temperature for 15 minutes.

(6) Add 4 μL of 5× polypeptide solution to each well of the reaction plate, and centrifuge at 1000 rpm for 1 min.

(7) Add 10 μL of detection solution, centrifuge at 1000 rpm for 60 seconds, gently shake and mix, and incubate at room temperature for 60 minutes.

(8) Read with EnVision.

data analysis:

Calculation formula

Fit dose-response curve

Taking the log value of the concentration as the X-axis and the percentage inhibition rate on the Y-axis, the log(inhibitor) vs.response-Variable slope of the analysis software GraphPad Prism 5 was used to fit the dose-response curve to obtain the _IC50 of the compound for protein binding inhibition value.

Test example 2, MV4-11 cell proliferation experiment

试剂(Invitrogen)测定细胞活力。pass

Reagent (Invitrogen) to measure cell viability.

MV4-11 cells (acute myeloid leukemia) were seeded at a concentration of 5000 cells/well in 100 μL of growth medium (RPMI1640, 10% FCS) in 96-well microtiter plates. After overnight incubation at 37°C, fluorescence values (C1 values) were determined. Plates were then treated with various substance dilutions and incubated at 37°C for 72 h before fluorescence values (C0 values) were determined. For data analysis, the C1 value was subtracted from the C0 value, and the results of cells treated with various dilutions of substances or with buffer solution alone were compared. Thereby _IC50 values were calculated. The above experimental results are shown in Table 10.

Table 10. Test results:

Conclusion: The binding activity of the compound of the present invention to BRD4 is obviously better than that of ABBV-075 and INCB-057643; the compound of the present invention has obvious inhibitory activity on the proliferation of leukemia cell MV4-11, and the inhibitory activity is better than that of ABBV-075 and INCB-057643.

Test Example 3. Pharmacokinetic test of the compound of the present invention

Taking SD rats as the test animals, the LC/MS/MS method was used to determine the drug concentrations in the plasma of the rats after intragastric administration of ABBV-075 and the compounds of the preferred embodiments of the present invention, and then to determine the drug concentrations in the plasma at different times, and to study the compounds of the present invention in large rats. Pharmacokinetic characteristics in mice.

SD rat source: Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.

Mode of administration: single gavage administration

Dosage and concentration: 10mg/kg; 1mg/mL

Preparation prescription: 0.5% methylcellulose

Sampling point: 5min, 15min, 30min, 1h, 2h, 4h, 8h, 24h.

8.0计算实验结果。Standard curve and quality control sample preparation and processing: take an appropriate amount of stock solution and dilute it with 50% acetonitrile water to 0.04, 0.10, 0.20, 0.40, 1.00, 2.00, 4.00 μg/mL standard working solution, 0.10, 1.00, 3.00 μg/mL Quality control working solution. Take 47.5μL of blank rat plasma and add 2.50μL of standard curve working solution and quality control working solution, respectively, and configure the standard curve containing the analyte concentration of 2.00, 5.00, 10.00, 20.00, 50.00, 100.00, 200.00ng/mL and quality control samples with concentrations of 5.00, 50.00 and 150.00 ng/mL, respectively, add 200 μL of acetonitrile (including internal standard loratadine 5 ng/mL), vortex for 3 min, centrifuge at 15000 rpm for 15 min at 4°C, and take the supernatant. Liquid 100L was analyzed by LC-MS/MS. use

8.0 Computational experimental results.

The pharmacokinetic parameters of preferred compounds of the present invention are shown in Table 11.

Table 11: Preferred Compound Pharmacokinetic Parameters

Conclusion: The compounds of the examples of the present invention show good pharmacokinetic properties. Compared with ABBV-075, they have higher blood concentration and curve area, longer half-life and shorter residence time.

Test Example 4. In vivo efficacy test of the compound of the present invention

Objective: To test the inhibitory effect of the test compounds on the growth of MV4-11 leukemia xenograft tumor in nude mice.

Methods: BALB/c nude mice were subcutaneously inoculated with MV4-11 cells to establish MV4-11 nude mice xenograft model. 13 days after inoculation (d13), the average tumor volume was about 215mm3. According to the size of the tumor volume, the tumor-bearing mice were divided into groups by random block method, including the solvent control group, the control sample INCB-057643 group, and the test sample group, with 6 in each group. Only. Each group was administered by intragastric administration, the administration dose was 30 mg/kg, and the administration volume was 10 ml/kg, administered once a day for 14 consecutive days, and the solvent control group was intragastrically administered with a blank solvent (50mM sodium lactate aqueous solution, PH4.0). Tumors were measured and weighed twice a week after the test drug was started. The animals were euthanized after the experiment.

Table 12 shows the in vivo pharmacodynamic results of the preferred compounds of the present invention in animals.

Table 12: In vivo efficacy of preferred compounds in animals

Conclusion: The compounds of the examples of the present invention show good antitumor activity. Compared with the control group INCB-057643, the antitumor activity was more significant.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the scope of the present invention. within the scope of protection.

Claims (9)

1. a BET protein inhibitor, is characterized in that, its structure is as shown in formula I:

or a pharmaceutically acceptable salt thereof, wherein, A is selected from

Ring B is selected from

or a five-membered aromatic heterocycle substituted by R ₁₁ and/or R ₁₂ ; in: X is O, S or NR ₁₃ ; Y is CH ₂ , C=O or NR ₁ ; Z is CR ₁₄ or N; U is CH or N; V is CH or N; R ₁ is hydrogen, C ₁ -C ₆ alkyl, C ₃ -C ₆ cycloalkyl, substituted C ₁ -C ₆ alkyl, substituted C ₃ -C ₆ cycloalkyl, C ₁ -C ₆ alkyl -SO ₂ - or C ₃ -C ₆ cycloalkyl-SO ₂ -; the substituted C ₁ -C ₆ alkyl means that the hydrogen on the C ₁ -C ₆ alkyl group is replaced by one or more C ₃ -C ₆ cycloalkanes group substitution; the substituted C ₃ -C ₆ cycloalkyl means that the hydrogen on the C ₃ -C ₆ cycloalkyl group is substituted by one or more C ₁ -C ₆ alkyl groups; R ₂ is hydrogen, C ₁ -C ₆ alkyl, C ₁ -C ₃ alkoxy, halogen or cyano; R ₃ is hydrogen, C ₁ -C ₆ alkyl, C ₁ -C ₃ alkoxy, halogen, cyano, -S(O) ₂ R ₁₅ , -S(O) ₂ NR ₁₆ R ₁₇ or -N( R ₁₆ )S(O) ₂ R ₁₅ ; R ₄ is hydrogen, C ₁ -C ₆ alkyl, C ₁ -C ₃ alkoxy, halogen or cyano; R ₅ and R ₆ are each independently hydrogen or C ₁ -C ₆ alkyl; R ₇ , R ₈ , R ₉ and R ₁₀ are each independently hydrogen, halogen, cyano, C ₁ -C ₃ alkoxy or C ₁ -C ₆ alkyl; R ₁₁ is hydrogen or C ₁ -C ₆ alkyl; R ₁₂ is hydrogen or C ₁ -C ₆ alkyl; R ₁₃ is hydrogen or C ₁ -C ₆ alkyl; R ₁₄ is hydrogen, C ₁ -C ₆ alkyl, halogen or cyano; R ₁₅ is C ₁ -C ₆ alkyl; R ₁₆ is hydrogen or C ₁ -C ₆ alkyl; R ₁₇ is hydrogen or C ₁ -C ₆ alkyl; The above-mentioned halogen is selected from fluorine, chlorine, bromine or iodine. 2. BET protein inhibitor as claimed in claim 1, is characterized in that, A is selected from

Ring B is selected from

in: X is O, S or NR ₁₃ ; Y is CH ₂ , C=O or NR ₁ ; Z is CR ₁₄ or N; U is CH or N; V is CH or N; R ₁ is hydrogen, C ₁ -C ₄ alkyl, C ₃ -C ₆ cycloalkyl, substituted C ₁ -C ₄ alkyl, substituted C ₃ -C ₆ cycloalkyl, C ₁ -C ₄ alkyl -SO ₂ - or C ₃ -C ₆ cycloalkyl-SO ₂ -; the substituted C ₁ -C ₄ alkyl means that the hydrogen on the C ₁ -C ₄ alkyl group is replaced by one or more C ₃ -C ₆ cycloalkanes Substituted C ₃ -C ₆ cycloalkyl group means that the hydrogen on the C ₃ -C ₆ cycloalkyl group is replaced by one or more C ₁ -C ₄ alkyl groups; R ₂ is hydrogen, C ₁ -C ₄ alkyl, C ₁ -C ₂ alkoxy, halogen or cyano; R ₃ is hydrogen, C ₁ -C ₄ alkyl, C ₁ -C ₂ alkoxy, halogen, cyano, -S(O) ₂ R ₁₅ , -S(O) ₂ NR ₁₆ R ₁₇ or -N( R ₁₆ )S(O) ₂ R ₁₅ ; R ₄ is hydrogen, C ₁ -C ₄ alkyl, C ₁ -C ₃ alkoxy, halogen or cyano; R ₅ and R ₆ are each independently hydrogen or C ₁ -C ₄ alkyl; R ₇ , R ₈ , R ₉ and R ₁₀ are each independently hydrogen, halogen, cyano, C ₁ -C ₃ alkoxy or C ₁ -C ₄ alkyl; R ₁₁ is hydrogen or C ₁ -C ₄ alkyl; R ₁₂ is hydrogen or C ₁ -C ₄ alkyl; R ₁₃ is hydrogen or C ₁ -C ₄ alkyl; R ₁₄ is hydrogen, C ₁ -C ₄ alkyl, halogen or cyano; R ₁₅ is C ₁ -C ₄ alkyl; R ₁₆ is hydrogen or C ₁ -C ₄ alkyl; R ₁₇ is hydrogen or C ₁ -C ₄ alkyl; The above-mentioned halogen is selected from fluorine, chlorine, bromine or iodine. 3. BET protein inhibitor as claimed in claim 1, is characterized in that, A is selected from

Ring B is selected from

in: X is O, S or NR ₁₃ ; Y is CH ₂ , C=O or NR ₁ ; Z is CR ₁₄ or N; U is CH or N; V is CH or N; R ₁ is hydrogen, C ₁ -C ₃ alkyl, C ₃ -C ₆ cycloalkyl, substituted C ₁ -C ₃ alkyl, substituted C ₃ -C ₆ cycloalkyl, C ₁ -C ₃ alkyl -SO ₂ - or C ₃ -C ₆ cycloalkyl-SO ₂ -; the substituted C ₁ -C ₃ alkyl means that the hydrogen on the C ₁ -C ₃ alkyl group is replaced by one or more C ₃ -C ₆ cycloalkanes Substituted C ₃ -C ₆ cycloalkyl group means that the hydrogen on the C ₃ -C ₆ cycloalkyl group is replaced by one or more C ₁ -C ₃ alkyl groups; R ₂ is hydrogen, C ₁ -C ₃ alkyl, C ₁ -C ₂ alkoxy, halogen or cyano; R ₃ is hydrogen, C ₁ -C ₃ alkyl, C ₁ -C ₂ alkoxy, halogen, cyano, -S(O) ₂ R ₁₅ , -S(O) ₂ NR ₁₆ R ₁₇ or -N( R ₁₆ )S(O) ₂ R ₁₅ ; R ₄ is hydrogen, C ₁ -C ₃ alkyl, C ₁ -C ₂ alkoxy, halogen or cyano; R ₅ and R ₆ are each independently hydrogen or C ₁ -C ₃ alkyl; R ₇ , R ₈ , R ₉ and R ₁₀ are each independently hydrogen, halogen, cyano, C ₁ -C ₂ alkoxy or C ₁ -C ₃ alkyl; R ₁₁ is hydrogen or C ₁ -C ₃ alkyl; R ₁₂ is hydrogen or C ₁ -C ₃ alkyl; R ₁₃ is hydrogen or C ₁ -C ₃ alkyl; R ₁₄ is hydrogen, C ₁ -C ₃ alkyl, halogen or cyano; R ₁₅ is C ₁ -C ₃ alkyl; R ₁₆ is hydrogen or C ₁ -C ₃ alkyl; R ₁₇ is hydrogen or C ₁ -C ₃ alkyl; The above-mentioned halogen is selected from fluorine, chlorine, bromine or iodine. 4. BET protein inhibitor as claimed in claim 1, is characterized in that, is selected from following compound:

or its pharmaceutically acceptable salts. 5. a method for synthesizing the compound described in any one of claims 1 to 4 is characterized in that the reaction formula is as follows:

The compound of the general formula IA and the compound of the general formula IB are subjected to a catalytic coupling reaction to obtain the compound of the general formula I, and the definitions of each group are as described in any one of claims 1-4. 6. Use of the compound according to any one of claims 1 to 4 in the preparation of a medicament for preventing or treating BET protein-related diseases. 7. The use according to claim 6, wherein the disease associated with BET protein refers to tumor disease, benign hyperplasia, inflammatory disease, autoimmune disease, sepsis, viral infection, vascular disease or neurological disease. 8. The use according to claim 7, wherein the tumor disease refers to acute myeloid leukemia, lymphoma, multiple myeloma, B-cell acute lymphoblastic leukemia, midline carcinoma, glioma, breast cancer, Colorectal cancer, prostate cancer, cervical cancer, non-small cell lung cancer, or melanoma. 9. A composition comprising a therapeutically effective amount of the compound of any one of claims 1-4, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.