A BET protein bromodomain BD1 selective covalent inhibitor and its preparation method and application

Abstract

The present invention relates to the field of pharmaceutical chemistry technology, and specifically to a BET protein bromodomain BD1 selective covalent inhibitor and its preparation method and application. The present invention utilizes the unique reactivity of lysine and 2-ethynylbenzaldehyde, introduces a 2-ethynylbenzaldehyde warhead into the compound structure shown in formula (I), and obtains a covalent inhibitor of BET protein BD1, whose inhibition rate on BRD4-BD1 is 104 times that of BRD4-BD2, and can effectively inhibit the proliferation of acute monocytic leukemia cells (MV4-11). Formula (I).

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical chemistry technology, and in particular to a BET protein bromodomain BD1 selective covalent inhibitor and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Bromodomain (BRD) is a key "reader" of histone acetylated lysine (Kac), regulating chromatin remodeling and gene expression. To date, 61 BRDs have been found in 46 proteins, which can be divided into eight subfamilies (I-VIII), including BET family proteins containing BD1 and BD2 domains. The abnormal function of BRD is closely related to the occurrence and development of various diseases such as inflammation, fibrosis, cancer and cardiovascular disease. Therefore, the development of efficient BRD targeted inhibitors has important clinical significance.

[0004] Since the reports of JQ1 and I-BET, the development of BRD-targeted inhibitors has flourished. However, there are still the following problems: first, traditional BRD pan-inhibitors are often accompanied by side effects such as fatigue, nausea, gastrointestinal toxicity (such as diarrhea) and thrombocytopenia; second, tumor subtypes sensitive to BRD inhibitors need to be further clarified; third, drug resistance has already appeared in clinical trials. Because there are many members of the BRD family protein and their functions are different, the development of inhibitors of BRD single domains (especially BD1 and BD2) may provide key answers to solving the above problems.

[0005] Compared with noncovalent inhibitors, targeted covalent inhibitors (TCI) have longer drug action time, higher affinity and selectivity, and may also overcome drug resistance caused by point mutations. The structure of TCI generally contains a protein targeting portion and a covalent warhead that modifies the target protein. Currently, only a few BRD covalent inhibitors targeting methionine and cysteine ​​residues have been reported. However, there are no targetable methionine and cysteine ​​near the Kac binding pocket of most BRDs, making this strategy not universal; at the same time, the activity of these inhibitors in cells has not been fully demonstrated. Therefore, the development of covalent inhibitors targeting other nucleophilic amino acid residues is expected to provide a new strategy for the development of BRD covalent inhibitors, and also help solve problems such as drug resistance of current BRD inhibitors. Summary of the invention

[0006] In order to overcome the above problems, the present invention provides a BET protein bromodomain BD1 selective covalent inhibitor and a preparation method and application thereof.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0008] The first aspect of the present invention provides a compound represented by the following formula (I), a stereoisomer thereof or a physiologically acceptable salt thereof:

[0009]

[0010] Formula (I).

[0011] The first aspect of the present invention provides an intermediate of the compound represented by formula (I) described in the first aspect, the structural formula of which is shown in formula (II);

[0012]

[0013] Formula (II).

[0014] The third aspect of the present invention provides a method for preparing the compound represented by formula (I) described in the first aspect, comprising the following steps:

[0015] (1) Under alkaline conditions, the compound represented by formula (II) reacts with compound 4 in the presence of a catalyst to synthesize compound 2;

[0016] (2) Compound 2 is reacted with tetrabutylammonium fluoride to synthesize a compound represented by formula (I);

[0017] The structural formula of compound 4 is shown below:

[0018] ;

[0019] The structural formula of compound 2 is shown below:

[0020] .

[0021] The TIPS group described in the present invention is a triisopropylsilyl group.

[0022] In one or more embodiments, in step (1), the molar ratio of the compound represented by formula (II) to compound 4 is 1:(1-1.5).

[0023] In one or more embodiments, in step (1), the catalyst is 1,1'-bis(diphenylphosphinoferrocene)palladium dichloride.

[0024] In one or more embodiments, in step (1), the temperature for the reaction to synthesize compound 2 is 65-80°C, preferably 70°C; and the reaction time is 5-7 h, preferably 6 h.

[0025] In one or more embodiments, in step (2), the molar ratio of compound 2 to tetrabutylammonium fluoride is 1:(3-5).

[0026] In one or more embodiments, in step (1), the reaction conditions for synthesizing compound 2 are stirring at room temperature; the stirring time is 1 to 2 h, preferably 1.5 h.

[0027] The fourth aspect of the present invention provides the use of the compound represented by formula (I) described in the first aspect, its stereoisomers or physiologically acceptable salts thereof in the preparation of BET covalent inhibitor drugs.

[0028] In one or more embodiments, the BET covalent inhibitor drug selectively inhibits the BD1 domain of the BET protein.

[0029] The fifth aspect of the present invention provides a pharmaceutical composition comprising the compound represented by formula (I) described in the first aspect, its stereoisomers or physiologically acceptable salts thereof.

[0030] In one or more embodiments, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient and / or carrier.

[0031] The excipients of the present invention refer to ingredients other than the active ingredients in a pharmaceutical composition or pharmaceutical preparation, which are non-toxic to the subject. Commonly used excipients in the art include buffers, stabilizers, preservatives or excipients, and commonly used excipients include binders, fillers, wetting agents, disintegrants, etc.

[0032] As an example, the excipients that can be used in the preparation of the present invention include but are not limited to: the excipient is selected from calcium phosphate, magnesium stearate, talc, dextrin, starch, gel cellulose, methyl cellulose, carboxymethyl cellulose sodium salt and polyvinyl pyrrolidone.

[0033] The drug carrier of the present invention can be a pharmaceutically acceptable solvent, suspending agent, vesicle, nanomaterial, etc., which is used to deliver the compound described in the first aspect of the present invention to an animal or human body. The carrier can be liquid or solid and is selected according to the planned administration method. Proteins and liposomes are also drug carriers.

[0034] Those skilled in the art can formulate the compounds of the present invention into pharmaceutical compositions or preparations using known techniques. For example, any compound (at least one compound) disclosed in the fourth aspect of the present invention is mixed with a pharmaceutical excipient, and then, if necessary, the resulting mixture is formed into a desired shape. Except for those mentioned in the present invention, pharmaceutical preparations can also be prepared according to known pharmaceutical preparations. And, except for those mentioned in the present invention, suitable pharmaceutical excipients are known in the art, for example, see the 2005 edition of the Handbook of Pharmaceutical Excipients (Original Fourth Edition).

[0035] The sixth aspect of the present invention provides the use of the compound represented by formula (I) described in the first aspect, its stereoisomers or physiologically acceptable salts thereof, or the pharmaceutical composition described in the fifth aspect in the preparation of drugs for preventing or treating cancer or anti-inflammation.

[0036] The cancer includes any one of acute monocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia mixed lineage leukemia, NUT midline carcinoma, multiple myeloma, glioma, lung cancer, neuroblastoma, Burkitt's lymphoma, cervical cancer, esophageal cancer, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer or breast cancer, preferably acute monocytic leukemia.

[0037] The inflammation is selected from any one of pharyngitis, prostatitis, vaginitis, cervicitis, scapulohumeral periarthritis, pelvic inflammatory disease, urethritis, pneumonia, conjunctivitis, otitis media, meningitis, myocarditis, ulcerative colitis, asthma, allergic rhinitis, chronic obstructive pulmonary disease, thyroiditis or allergic dermatitis.

[0038] The beneficial effects of the present invention are:

[0039] Lysine is a highly abundant amino acid in the human proteome. More notably, the ZA loop of BRD contains multiple conserved lysines, such as K91 of BRD4-BD1 and K378 of BRD4-BD2. Although the nucleophilicity of lysine exposed to physiological conditions is relatively low, these lysines exhibit strong nucleophilicity due to the hydrophobic microenvironment of the acetylated lysine binding pocket in BRD; and these conserved lysines are not prone to mutation, making them more suitable as covalent binding sites for the development of TCI. The present invention utilizes the unique reactivity of lysine and 2-ethynylbenzaldehyde, and introduces a 2-ethynylbenzaldehyde warhead into the compound structure shown in formula (I) to obtain a covalent inhibitor of the BET protein BD1, whose inhibition rate against BRD4-BD1 is 104 times that of BRD4-BD2 (IC 50 : 21.24 nM VS 2204 nM), and can effectively inhibit the proliferation of acute monocytic leukemia cells (MV4-11). BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0041] Figure 1 The preparation route of the compound represented by formula (I);

[0042] Figure 2 is the H NMR spectrum of compound 1;

[0043] Figure 3 is the NMR carbon spectrum of compound 1;

[0044] Figure 4 is the H NMR spectrum of compound 3;

[0045] Figure 5 is the NMR carbon spectrum of compound 3;

[0046] Figure 6 It is the half-maximal inhibition concentration curve of compound 1 on BRD4-BD1 and BRD4-BD2;

[0047] Figure 7 The figure is the covalent reaction kinetic analysis of compound 1 with BRD4-BD1 and BRD4-BD2, wherein A is the covalent modification yield of BRD4-BD1 at different concentrations of compound 1 within the corresponding time, B is the covalent modification yield of BRD4-BD2 at different concentrations of compound 1 within the corresponding time, C is the fitting curve of the rate constant Kobs of compound 1 at different concentrations with BRD4-BD1, and D is the fitting curve of the rate constant Kobs of compound 1 at different concentrations with BRD4-BD2;

[0048] Figure 8 The complete protein spectrum and site analysis diagram after the reaction of compound 1 with BRD4-BD1 and BRD4-BD2, where A is the complete protein spectrum and B is the site analysis;

[0049] Fig. 9 is the antiproliferative activity of compound 1 in different cell lines;

[0050] Fig.10 is the antiproliferative activity of compound 1 against MV4-11 cells under wash-out and no-wash conditions;

[0051] Fig.11 The effect of compound 1 on c-Myc expression in MV4-11 cells under wash-out and no-wash conditions. DETAILED DESCRIPTION

[0052] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0053] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0054] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0055] Example 1

[0056] Figure 1 For the preparation route of the compound (Compound 1) represented by formula (I), refer to Figure 1 , synthesize the compound represented by formula (I).

[0057] 11.4 mmol 2-bromo-6-chlorobenzaldehyde, 13.68 mmol (triisopropylsilyl) acetylene, 0.285 mmol cuprous iodide, and 0.57 mmol bistriphenylphosphine palladium dichloride were added to a reaction bottle containing 20 mL triethylamine and reacted at 80 °C for 12 h. After evaporating the solvent under reduced pressure, the crude product was purified by silica gel column chromatography with an eluent ratio of dichloromethane: petroleum ether = 1: (4-10) to obtain a white solid, namely compound 5, with a yield of 98%.

[0058] 5.623 mmol of bis(pinacol)diboron, 0.141 mmol of tris(dibenzylideneacetone)dipalladium, 0.5622 mmol of 2-dicyclohexylphosphine-2′,4′,6′-triisopropylbiphenyl, 14.055 mmol of potassium acetate and 4.685 mmol of compound 5 were added to a reaction bottle containing 10 mL of 1,4-dioxane and reacted at 110 °C for 8 h. 5 mL of water was added to quench the reaction and the mixture was extracted with ethyl acetate three times, 10 mL each time. The combined organic phase was dried over sodium sulfate and the solvent was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography with an eluent ratio of dichloromethane: petroleum ether = 1: (5-20) to obtain a white solid, namely compound 4, with a yield of 58%.

[0059] 5.076 mmol 6-bromo-4-azaindole, 6.276 mmol 3,5-dimethylisoxazole-4-boronic acid pinacol ester, 0.5622 mmol [1,1'-bis(diphenylphosphino)ferrocene] dichloropalladium and 12 mL 1M potassium carbonate aqueous solution were added to a reaction bottle containing 24 mL acetonitrile and reacted at 160 °C for 12 h. The reaction was monitored by TLC until it was complete, and extracted with ethyl acetate three times, 10 mL each time. After the combined organic phase was dried over sodium sulfate, the solvent was evaporated under reduced pressure, and the crude product was purified by silica gel column chromatography with an eluent ratio of methanol: dichloromethane = 1: (50~150) to obtain a white solid, namely compound 6, with a yield of 70%.

[0060] 2.1 mmol of compound 6, 2.22 mmol of N-iodosuccinimide and 1.37 mL of N,N-dimethylformamide were added to a reaction bottle containing 30 mL of acetonitrile and reacted at room temperature for 4 h. The reaction was monitored by TLC until it was complete, and extracted with ethyl acetate three times, 10 mL each time. The combined organic phase was dried over sodium sulfate, and the solvent was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography with an eluent ratio of methanol: dichloromethane = 1: (50~150) to obtain a white solid, which was compound 7, with a yield of 95%.

[0061] At 0 ° C, 3.8 mmol of compound 7, 4.37 mmol (R)-2-(1-hydroxyethyl)pyridine, 5.13 mmol of triphenylphosphine and 3.34 mmol of diisopropyl azodicarboxylate were added to a reaction bottle containing 20 mL of tetrahydrofuran and reacted at room temperature for 1.5 h. After evaporating the solvent under reduced pressure, the crude product was purified by silica gel column chromatography with an eluent ratio of methanol: dichloromethane = 1: (50-150) to obtain a white solid, and the compound represented by formula (II), namely compound 3, was obtained with a yield of 65%.

[0062] 0.44 mmol of compound 4, 0.02 mmol of 1,1'-bis(diphenylphosphino)ferrocenepalladium dichloride (PdCl2(dppf)), 2.4 mL of 1M potassium carbonate aqueous solution and 0.338 mmol of compound 3 were added to a reaction bottle containing 4.8 mL of tetrahydrofuran, stirred at 70 °C for 6 hours, and the reaction was monitored by TLC until it was complete. 5 mL of water was added to quench the reaction, and 10 mL of ethyl acetate was used for extraction three times. The combined organic phase was dried over sodium sulfate, and the solvent was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography with an eluent ratio of methanol: dichloromethane = 1: (30~100) to obtain compound 2 with a yield of 42%.

[0063] 0.573 mmol tetrabutylammonium fluoride (TBAF) and 0.143 mmol compound 2 were added to a reaction bottle containing 5 mL tetrahydrofuran at 0 °C, stirred at room temperature for 1.5 hours, and the reaction was monitored by TLC until it was complete. 5 mL of water was added to quench the reaction, and extracted with ethyl acetate three times, 10 mL each time. After the combined organic phase was dried over sodium sulfate, the solvent was evaporated under reduced pressure, and the crude product was purified by silica gel column chromatography, the eluent ratio was methanol: dichloromethane = 1: (15~50), and a light yellow solid was obtained to obtain the compound represented by formula (I), namely compound 1, with a yield of 72%.

[0064] The H NMR spectrum of compound 1 is as follows Figure 2 As shown, the NMR carbon spectrum of compound 1 is as follows Figure 3 shown.

[0065] The H NMR spectrum of compound 3 is as follows Figure 4 As shown, the NMR carbon spectrum of compound 3 is as follows Figure 5 shown.

[0066] Characterization of Compound 1:

[0067] 1 H-NMR (400 MHz, CDCl3) δ 10.34 (s, 1H), 8.66-8.56 (m, 1H), 8.38 (d,J = 1.8 Hz, 1H), 7.85-7.79 (m, 2H), 7.67-7.60 (m, 3H), 7.45 (d, J = 1.8 Hz,1H), 7.25-7.20 (m, 1H), 7.01 (d, J = 7.9 Hz, 1H), 5.76 (q, J = 7.1 Hz, 1H), 3.43(s, 1H), 2.34 (s, 3H), 2.17 (s, 3H), 2.08 (d, J = 7.1 Hz, 3H).

[0068] 13C-NMR (101 MHz, CDCl3) δ 192.34, 165.89, 160.16, 158.91, 149.73,144.73, 144.09, 137.57, 136.14, 135.68, 133.57, 132.48, 132.32, 129.71,129.23, 128.70, 123.20, 120.43, 120.12, 118.42, 114.43, 113.88, 83.00, 81.60,58.25, 20.18, 11.64, 10.83.

[0069] HRMS (ESI) calculated m / z for [M+H] + 447.1816 ; Found 447.1857.

[0070] Example 2

[0071] Inhibitory effect of compound 1 on BRD4-BD1 and BRD4-BD2 (IC 50 ):

[0072] Prepare 24.27 μM BRD4-BD1 stock solution dissolved in HTRF PPI terbium detection buffer (brand: Cisbio, catalog number: 61DB10RDF), 48.66 μM BRD4-BD2 stock solution dissolved in HTRF PPI terbium detection buffer (Cisbio, 61DB10RDF), and 1000 μM acetylated lysine peptide stock solution dissolved in HTRF PPI terbium detection buffer (Cisbio, 61DB10RDF) (for BRD4-BD1 IC 50 ), 2500 μM acetylated lysine peptide stock solution dissolved in HTRFPPI terbium detection buffer (Cisbio, 61DB10RDF) (for BRD4-BD2 IC 5050 nL of the compound 1 stock solution was drawn from each of the above stock solutions and added to the 2.5 μL flask containing 2 M SA-Tb & GST-XL665 (the brand of the reagents is revvity, the English names are 610SATLA and 61GSTXLA, and the molar ratio of SA-Tb and GST-XL665 is 1:40) stock solutions dissolved in HTRF PPI terbium detection buffer (Cisbio, 61DB10RDF) and 60 mM, 15 mM, 3.75 mM, 0.9375 mM, 0.2344 mM, 0.0586 mM, 0.01464 mM, 0.0037 mM, 0.00092 mM, 0.00023 mM, 0.000057 mM, 0.000014 mM and 0.0000036 mM compound 1 stock solutions dissolved in DMSO. 2 μL BRD4-BD1 stock solution or 2.5 μL BRD4-BD2 stock solution and 197.45 μL PPI terbium detection buffer were added to a 384-well plate. After the sample was mixed, it was reacted at room temperature for 60 min. After the reaction, 2.5 μL acetylated lysine peptide stock solution and 5 μL SA-Tb&GST-XL665 detection working stock solution were added, and the sample was mixed and reacted at room temperature for 60 min. After the reaction, the absorbance ratio of each well reaction mixture at 665nm and 620nm was detected by a microplate reader. The inhibitory activity against the protein was calculated by the following formula: the value of the negative control (0.5% DMSO well) was set to 0% inhibition rate, and the value of the positive control (positive drug highest concentration well or background well without enzyme) was set to 100% inhibition rate, and the inhibition rate of each test solution was calculated. The inhibitory activity against the protein was calculated by the following formula: Inhibition rate = (negative control signal-compound signal) / (negative control signal-positive control signal)×100. The inhibitory activity was fitted with the corresponding concentration of compound 1 to obtain the half inhibitory concentration IC 50 .

[0073] The results are as follows Figure 6 As shown, the IC of compound 1 against BRD4-BD1 50 The IC value for BRD4-BD2 is 21.24 nM. 50 The inhibitory rate of compound 1 on BRD4-BD1 was 104 times that of BRD4-BD2.

[0074] Example 3

[0075] Kinetic analysis of the covalent reaction of compound 1 with BRD4-BD1 and BRD4-BD2 (K inact / K I Determination):

[0076] BRD4-BD1: Prepare 5 mg / mL BRD4-BD1 stock solution dissolved in PBS (10 mM, pH = 7.4) and 20 mM, 10 mM, 6 mM, 4.5 mM, 3 mM, 2 mM, 1 mM, 0.5 mM, and 0.1 mM compound 1 stock solutions dissolved in DMSO. Take 1 μL of each of the above compound 1 stock solutions and add it to a 1.5 mL centrifuge tube containing 3.5 μL BRD4-BD1 stock solution and 95.5 μL PBS (pH = 7.4) buffer (the molar ratio of compound 1 to BRD4-BD1 is 200, 100, 60, 45, 30, 20, 10, 5, and 1 μM:10 μM). After mixing, react in a metal bath at 37 °C for 5 min, 10 min, 15 min, 20 min, 30 min, 50 min, and 60 min, respectively. After the reaction, 1% formic acid (volume ratio) was added to quench the reaction, and the reaction solution was transferred to an Amicon® Ultra filter (10 kDa), and 300 μL of ammonium acetate solution (pH = 7.0, 20 mM) was added, and centrifuged at 4 °C for 10 min (13800 g). Another 300 μL of ammonium acetate solution (pH = 7.0, 20 mM) was added, and centrifuged at 4 °C for 10 min (13800 g), and this step was repeated 3 times. After the reaction solution was replaced with ammonium acetate, 1% formic acid (volume ratio) was added, mixed, and used for LC-MS / MS analysis, and the peak area was used to calculate the covalent modification yield. The covalent modification yield of different concentrations of compound 1 after reacting with BRD4-BD1 at different times was fitted with the reaction time, and the rate constant K of compound 1 on BRD4-BD1 was obtained by the One-phase association equation. obs ; K obs The concentration of compound 1 was fitted and the rate constant K was obtained by the Michaelis-Menten equation. I and K inact .

[0077] BRD4-BD2: Prepare 5 mg / mL BRD4-BD2 stock solution dissolved in PBS (10 mM, pH = 7.4) and 20 mM, 10 mM, 5 mM, 3 mM, 2 mM and 0.5 mM stock solutions of compound 1 dissolved in DMSO. Take 1 μL of the above compound 1 stock solution and add it to a 1.5 mL centrifuge tube containing 3.5 μL BRD4-BD2 stock solution and 95.5 μL PBS (10 mM, pH = 7.4) buffer (the molar ratio of compound 1 to BRD4-BD2 is 200, 100, 50, 30, 20 and 5 μM:10 μM). After mixing, react in a metal bath at 37 °C for 30 min, 60 min, 90 min, 120 min, 180 min, 240 min and 360 min, respectively. After the reaction, 1% formic acid (volume ratio) was added to quench the reaction, and the reaction solution was transferred to an Amicon® Ultra filter (10 kDa), and 300 μL of ammonium acetate solution (pH = 7.0, 20 mM) was added, and centrifuged at 4 °C for 10 min (13800 g). Another 300 μL of ammonium acetate solution (pH = 7.0, 20 mM) was added, and centrifuged at 4 °C for 10 min (13800 g), and this step was repeated 3 times. After the reaction solution was replaced with ammonium acetate, 1% formic acid (volume ratio) was added, mixed, and used for LC-MS / MS analysis, and the peak area was used to calculate the covalent modification yield. The covalent modification yield of different concentrations of compound 1 after reacting with BRD4-BD2 at different times was fitted with the reaction time, and the rate constant K of compound 1 on BRD4-BD2 was obtained by the One-phase association equation. obs ; K obs The concentration of compound 1 was fitted and the rate constant K was obtained by the Michaelis-Menten equation. I and K inact .

[0078] The results are as follows Figure 7 As shown, compound 1 has a K inact / K I 0.11×10 6 min -1 M -1 , K of BRD4-BD2 inact / K I 0.19×10 3 min -1 M -1 The covalent reaction efficiency of the two differs by 579 times.

[0079] Example 4

[0080] Complete protein profiling analysis of compound 1 with BRD4-BD1 and BRD4-BD2:

[0081] Prepare 500 μM BRD4-BD1 stock solution in PBS (10 mM, pH = 7.4), 500 μM BRD4-BD2 stock solution in PBS (10 mM, pH = 7.4), and 10 mM compound 1 stock solution in DMSO. Pipette 2 μL of BRD4-BD1 or BRD4-BD2 stock solution and 1 μL of compound 1 stock solution into 1.5 mL centrifuge tubes containing 97 μL PBS (10 mM, pH = 7.4) buffer (the molar ratio of compound 1 to BRD4 is 10:1), and react in a metal bath at 37 °C for 12 h. After adding 1% formic acid (volume ratio) to quench the reaction, transfer the reaction solution to an Amicon® Ultra filter (10 kDa), add 300 μL of ammonium acetate solution (pH = 7.0, 20 mM), and centrifuge at 4 °C for 10 min (13800 g). Then add 300 μL of ammonium acetate solution (pH = 7.0, 20 mM), centrifuge at 4 °C for 10 min (13800 g), and repeat this step three times. After the reaction solution was replaced with ammonium acetate, add 1% formic acid (volume ratio), mix well, and use for LC-MS / MS analysis.

[0082] The results are as follows Figure 8 As shown in Figure A, the covalent reaction efficiency of compound 1 with BRD4-BD1 is 92.21%, while that with BRD4-BD2 is only 23.18%.

[0083] Example 5

[0084] Analysis of the covalent binding site between compound 1 and BRD4-BD1:

[0085] Prepare 5 mg / mL BRD4-BD1 stock solution dissolved in PBS (10 mM, pH = 7.4), 3 mM compound 1 stock solution dissolved in DMSO, 1 M DTT (dithiothreitol) stock solution dissolved in ABC (triethylammonium bicarbonate) buffer (pH = 8.0, 0.1 M), 500 mM IAA (iodoacetamide) stock solution dissolved in ABC (triethylammonium bicarbonate) buffer (pH = 8.0, 0.1 M), and 0.1 mg / mL trypsin stock solution dissolved in ABC (triethylammonium bicarbonate) buffer (pH = 8.0, 0.1 M). 3.5 μL of BRD4-BD1 stock solution and 1 μL of compound 1 stock solution were taken separately and added to a 1.5 mL centrifuge tube containing 95.5 μL of PBS (10 mM, pH = 7.4) buffer (the molar ratio of compound 1 to BRD4(1) was 3:1), and reacted in a metal bath at 37 °C for 4 h. After the reaction, the reaction solution was transferred to an Amicon® Ultra filter (10 kDa). 350 μL of PBS buffer (pH = 7.4, 10 mM) was added, and centrifuged at 4 °C for 10 min (13800 g), and this step was repeated 3 times; 300 μL of ABC (triethylammonium bicarbonate) buffer (pH = 8.0, 100 mM) was added, and centrifuged at 4 °C for 10 min (13800 g), and this step was repeated 2 times to obtain 150 μL of the reaction mixture dissolved in ABC buffer. 1.5 μL DTT stock solution was taken and added to the reaction mixture dissolved in ABC buffer, and the mixture was reacted in a metal bath at 37 °C for 45 min. 6 μL IAA stock solution was taken and added to the reaction mixture dissolved in ABC buffer, and the mixture was reacted in the dark at room temperature for 1 h. After adding 1.5 μL DTT stock solution, the reaction mixture was directly transferred to an Amicon® Ultra filter (10 kDa). 300 μL ultrapure water was added, and the mixture was centrifuged at 4 °C for 10 min (13800 g), and this step was repeated 3 times. After centrifugation, the reaction mixture was transferred to a 1.5 mL centrifuge tube, and 150 μL ABC buffer and 5 μL trypsin stock solution were added, and the mixture was reacted in a metal bath at 37 °C overnight. The reaction mixture was transferred to an Amicon® Ultra filter (30 kDa), and the mixture was centrifuged at 4 °C for 10 min (13800 g). The collected filtrate was filtered through a Merck C 18 Microchromatography column (0.6 µL C 18Resin) desalting and elution: (1) Wetting solution (100% acetonitrile) was drawn to wet the column, and this step was repeated twice; (2) Equilibration solution (0.1% trifluoroacetic acid aqueous solution) was drawn to equilibrate the Ziptip, and this step was repeated twice; (3) The sample was repeatedly drawn 7 to 10 times to allow the sample to bind to the column; (4) The microchromatographic column was cleaned with washing solution (0.1% trifluoroacetic acid aqueous solution), and this step was repeated twice; (5) The peptides were eluted with eluent (0.1% trifluoroacetic acid aqueous solution / 50% acetonitrile) and collected in a centrifuge tube. The collected eluent was dried in a vacuum centrifugal concentrator, redissolved in 10 μL 0.1% formic acid aqueous solution and directly used for LC-MS / MS analysis.

[0086] The results are as follows Figure 8 As shown in B, the covalent binding site of compound 1 and BRD4-BD1 occurs at K91.

[0087] Example 6

[0088] Antiproliferative activity of compound 1 in different cell lines:

[0089] Prepare a 10 μM stock solution of compound 1 in DMEM / DMSO (9:1 volume ratio for U2OS, MCF7, Hep G2, Huh7, U87MG, and HEK293T cells) or IMDM / DMSO (9:1 volume ratio for K562, 786-O, and MV4-11 cells). Cells were cultured in DMEM or IMDM medium containing 10% FBS and 1% antibiotics (penicillin and streptomycin) in a humidified incubator at 37 °C. 1×10 4 U2OS, MCF7, K562, 786-O, MV4-11, Hep G2, Huh7, U87 MG and HEK293T cells were seeded in 96-well plates (90 μL culture medium per well) in a humidified incubator at 37 °C for 12 hours according to the test group (treated group) and control group (control group). 10 μL DMSO (control group) and 10 μL compound 1 stock solution (treated group) were respectively taken and added to each cell in the control group and the test group. After further incubation in a humidified incubator at 37 °C for 72 h, 10 μL CCK-8 reagent (BS350B, Biosharp) was added to the cells of the control group and the test group corresponding to each cell line, and reacted for 2 h. The absorption of the reaction mixture in each well at 450 nm was detected by an ELISA reader. The antiproliferative activity of compound 1 against different cells was calculated by the following formula:

[0090] Cell viability (%) = (OD treated – OD blank ) / (OD control – OD blank ) × 100.

[0091] OD treated Refers to the absorbance of the test group at 450 nm, OD control Refers to the absorbance of the control group at 450 nm, OD blank Refers to the absorption of culture medium without cells at 450 nm.

[0092] The results are as follows Fig. 9 As shown, compound 1 exhibited selective inhibition of the proliferation of acute monocytic leukemia MV4-11 cells.

[0093] Example 7

[0094] Antiproliferative activity of compound 1 on MV4-11 cells under wash-out and no-wash conditions:

[0095] Prepare 10 mM stock solutions of compound 1 in DMSO, 10 mM stock solutions of PLX51107 (non-covalent BET inhibitor) in DMSO, 10 mM stock solutions of JQ1 (non-covalent BET inhibitor) in DMSO, and 10 mM stock solutions of RVX-208 (non-covalent BD2 inhibitor) in DMSO. Cells were cultured in IMDM medium containing 10% FBS and 1% antibiotics (penicillin and streptomycin) in a humidified incubator at 37 °C. 1×10 5 MV4-11 cells were divided into wash-out group, DMSO for wash-out group, no-wash group and DMSO for no-wash group, respectively, and inoculated into 24-well plates (1 mL IMDM culture medium per well) and cultured in a humidified incubator at 37 °C for 12 h.

[0096] 1 μL of compound 1, 1 μL of PLX51107, 1 μL of JQ1, and 1 μL of RVX-208 were added to the washing group and the no-wash group (compound 1 working concentration was 10 μM), and 1 μL of DMSO was added to the washing control group and the no-wash control group, and incubated in a humidified incubator at 37°C for 4 h. After incubation, for the no-wash group, 1 mL of cells was divided into 10 portions, and 100 μL of each portion was added to a 96-well cell culture plate; for the washing group, 1 mL of cells was transferred to a 1.5 mL centrifuge tube, centrifuged at room temperature for 3 min (800 rpm), and the collected cells were resuspended in 1 mL of IMDM culture medium and cultured for 10 min. The culture medium was removed by centrifugation at room temperature for 3 min (800 rpm). This process is the washing step, which was repeated twice. After the washed cells were resuspended in 1 mL of DMEM culture medium, they were divided into 10 portions, and 100 μL of each portion was added to a 96-well cell culture plate. After incubation for 68 h in a humidified incubator at 37 °C, 10 μL of CCK-8 reagent (BS350B, Biosharp) was added to the cells in the washing group and the non-washing group, respectively, and the reaction was continued for 2 h. The absorption of the reaction mixture in each well at 450 nm was detected by an ELISA instrument. The antiproliferative activity of the cells was calculated by the following formula:

[0097] Cell viability (%) = (OD treated – OD blank ) / (OD control – OD blank ) × 100.

[0098] OD treated OD refers to the absorbance at 450 nm of cells with inhibitor added. control Refers to the absorbance at 450 nm of the control group with only DMSO added, OD blank Refers to the absorbance of IMDM medium without cells at 450 nm.

[0099] The results are as follows Fig.10 As shown, under no-wash conditions, compound 1 provided by the present invention exhibited inhibitory effects close to or better than those of the control compounds PLX51107 and JQ1, while the BD2 inhibitor RVX-208 could not inhibit the proliferation of MV4-11 cells; more importantly, compound 1 exhibited excellent inhibitory effects even under washing conditions, while PLX51107, JQ1, and RVX-208 were not sufficient to resist washing, indicating that compound 1 exhibited a more lasting drug effect.

[0100] Example 8

[0101] Effect of compound 1 on c-Myc expression in MV4-11 cells under wash-out and no-wash conditions:

[0102] Prepare 10 mM stock solutions of compound 1 in DMSO, 10 mM stock solutions of PLX51107 (non-covalent BET inhibitor) in DMSO, 10 mM stock solutions of JQ1 (non-covalent BET inhibitor) in DMSO, and 10 mM stock solutions of RVX-208 (non-covalent BD2 inhibitor) in DMSO. Cells were cultured in IMDM medium containing 10% FBS and 1% antibiotics (penicillin and streptomycin) in a humidified incubator at 37 °C. 12 × 10 6 MV4-11 cells were seeded into 6-well cell culture plates (1 mL IMDM culture medium per well) according to the wash-out group, DMSO for wash-out control group, no-wash group, and DMSO for no-wash control group, and cultured in a humidified incubator at 37 °C for 12 h.

[0103] 1 μL of compound 1 stock solution, 1 μL of PLX51107 stock solution, 1 μL of JQ1 stock solution, and 1 μL of RVX-208 stock solution were added to the cells of the washing group and the no-wash group (inhibitor working concentration was 10 μM), and 1 μL of DMSO was added to the cells of the washing control group and the no-wash control group. For the no-wash group, the cells were incubated in a 37 ℃ humidified incubator for 6 hours; for the washing group, after incubation in a 37 ℃ humidified incubator for 4 hours, 1 mL of cells were transferred to a 1.5 mL centrifuge tube, centrifuged at room temperature for 3 minutes (800 rpm), and the collected cells were resuspended in 1 mL of IMDM culture medium and cultured for 10 minutes. The culture medium was removed by centrifugation at room temperature for 3 minutes (800 rpm). This process was the washing step. This step was repeated twice. After the washed cells were resuspended in 1 mL of IMDM culture medium, they were added to a 6-well cell culture plate and incubated in a 37 ℃ humidified incubator for 2 hours.

[0104] After incubation, cells were collected by centrifugation, cell lysis buffer (10 mM PBS, 2.7 mM KCl, 137 mMNaCl, pH 7.4) was added, cells were disrupted by ultrasound, and supernatant was collected by centrifugation. 2× SDS loading buffer was added and heated at 95°C for 10 minutes to denature the protein. After equal amounts of protein were loaded, the proteins were separated by SDS-PAGE and transferred from the gel to a PVDF blotting membrane. The PVDF blotting membrane was incubated with PBST containing 5% BSA (PBS + 0.1% Tween 20) at room temperature for 2 h to block the nonspecific binding of the antibody. The blocking solution was poured out, and a 1:2000 dilution of c-Myc antibody (dissolved in 5% BSA in PBST) was added and incubated overnight at 4°C in a wet box. The solution was poured out and the PVDF blotting membrane was washed 3 times with PBST for 5 minutes each time. Add HRP-conjugated anti-rabbit secondary antibody (dissolved in 5% BSA in PBST, 1:5000 dilution) and incubate the PVDF blot for 1 hour at room temperature in the dark. Pour out the solution and wash the PVDF blot 3 times with PBST, 5 minutes each time. Use the Bio-Rad ChemiDoc XRS+ Chemiluminescent Gel Imaging System to capture images.

[0105] The results are as follows Fig.11 As shown, under no-wash conditions, the compound 1 provided by the present invention exhibited better c-Myc inhibitory effects than the control compounds PLX51107 and JQ1, respectively, while the BD2 inhibitor RVX-208 exhibited lower inhibitory ability; more importantly, compound 1 exhibited excellent inhibitory effects even under washing conditions, while PLX51107, JQ1, and RVX-208 were not sufficient to resist washing, indicating that the covalent compound 1 exhibited a more lasting drug effect.

[0106] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A compound represented by the following formula (I) or a physiologically acceptable salt thereof: Formula (I).

2. The method for preparing the compound represented by formula (I) according to claim 1, characterized in that: The steps include: (1) Under alkaline conditions, the compound represented by formula (II) reacts with compound 4 in the presence of a catalyst to synthesize compound 2; the catalyst is 1,1'-bis(diphenylphosphino)ferrocenepalladium dichloride; (2) Compound 2 is reacted with tetrabutylammonium fluoride to synthesize a compound represented by formula (I); Among them, the structural formula of compound 4 is as follows: ； The structural formula of compound 2 is shown below: ； The compound represented by formula (II) is as follows: Formula (II).

3. The method for preparing the compound represented by formula (I) according to claim 2, characterized in that: The molar ratio of the compound represented by formula (II) to compound 4 is 1:(1-1.5); In step (1), the reaction temperature for synthesizing compound 2 is 65-80 °C and the reaction time is 5-7 h; In step (2), the molar ratio of compound 2 to tetrabutylammonium fluoride is 1:(3-5).

4. A pharmaceutical composition, characterized in that It includes the compound represented by formula (I) as claimed in claim 1 or a physiologically acceptable salt thereof.

5. Use of the compound represented by formula (I) according to claim 1 or a physiologically acceptable salt thereof or the pharmaceutical composition according to claim 4 in the preparation of a medicament for preventing or treating acute monocytic leukemia.

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