Bromodomain activity-based probe as well as preparation method and application thereof

Abstract

The invention relates to the technical field of chemical biology, in particular to an activity-based bromodomain probe as well as a preparation method and application thereof. According to the invention, the high-selectivity and high-efficiency cyclization reactivity of lysine and 2-ethynyl benzaldehyde is utilized, and an EBA bullet and an alkynyl handle capable of clicking a chemical reaction are introduced into the structure of a compound as shown in a formula (I), so that an activity-based probe capable of systematically analyzing the ligand property of BRD lysine is obtained. In chemical proteomics experiments carried out by MV4-11 and U2OS cells, 18 endogenous BRDs are captured in total, and the BRD covalent inhibitor crosses 6 subfamilies, so that extremely valuable information is provided for the development of a BRD covalent inhibitor of targeted lysine. # imgabs0 # is represented by formula (I).

Description

Technical Field

[0001] The present invention relates to the field of chemical biology technology, and in particular to a bromodomain activity-based probe 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) can recognize histone acetylated lysine (Kac) through protein-protein interaction (PPI) to regulate chromatin remodeling, gene expression and DNA repair. To date, 61 BRDs have been found in the human proteome, distributed in 46 proteins, which can be subdivided into eight subfamilies I-VIII. The abnormal function of BRD is closely related to the occurrence and development of various diseases such as inflammation and cancer, and has become a potential new strategy for the treatment of diseases such as prostate cancer and hematological malignancies.

[0004] Targeted covalent inhibitors (TCIs) can covalently bind to specific target protein amino acids to inhibit the biological function of the target, and have received increasing attention in recent years. TCIs have the advantages of long drug action time, strong affinity and high selectivity, and also have the potential to overcome drug resistance caused by point mutations. Compared with the booming development of kinase TCIs, there are relatively few reports on TCIs targeting BRD, and these TCIs mainly target methionine and cysteine ​​residues, but methionine and cysteine ​​are not abundant in the Kac binding pocket of BRD; on the contrary, lysine is conservatively present in the active pocket of BRD. To design TCIs targeting lysine, it is necessary to develop chemical probes that can evaluate the ligandability of BRD lysine.

[0005] The current probes that can be used for broad-spectrum BRD protein spectrum analysis, such as Photo-BS and bromotriazine, have obvious disadvantages: Photo-BS is not suitable for direct analysis of nucleophilic amino acid ligandability; and bromotriazine has a weak ability to label endogenous BRD in the cell environment, and only captured 4 BRDs from cell lysate. Currently, there is a lack of strong, broad-spectrum probes that can be used for lysine ligandability analysis. Summary of the invention

[0006] In order to overcome the above problems, the present invention provides a bromodomain activity-based probe and a preparation method and application thereof.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical solutions: The first aspect of the present invention provides a compound represented by the following formula (I), its stereoisomer or a physiologically acceptable salt thereof;

[0008] Formula (I).

[0009] The second aspect of the present invention provides an intermediate of the compound represented by formula (I) of the first aspect, whose structural formula is shown in formula (II);

[0010] Formula (II).

[0011] The TIPS group described in the present invention is a triisopropylsilyl group.

[0012] The third aspect of the present invention provides a method for preparing the compound represented by formula (I) according to the first aspect, comprising the following steps: (1) Under alkaline conditions, the compound represented by formula (II) reacts with compound 4 in the presence of a catalyst to synthesize compound 2; (2) Compound 2 is reacted with tetrabutylammonium fluoride to synthesize a compound represented by formula (I); The structural formula of compound 4 is shown below: ; The structural formula of compound 2 is shown below: .

[0013] In one or more embodiments, in step (1), the catalyst is chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II).

[0014] In one or more embodiments, in step (1), the molar ratio of the compound represented by formula (II) to compound 4 is 1:1.8-2.2, preferably 1:2.

[0015] In one or more embodiments, in step (1), the solvent used in the reaction is a mixed solution of 1,4-dioxane and water; the volume ratio of 1,4-dioxane to water is 9 to 12:1, preferably 10:1.

[0016] In one or more embodiments, in step (1), the temperature for the reaction to synthesize compound 2 is 110-130° C., preferably 120° C.; and the reaction time is 4-6 h, preferably 5 h.

[0017] In one or more embodiments, in step (2), the molar ratio of compound 2 to tetrabutylammonium fluoride is 1:5.8-6.5, preferably 1:6.1.

[0018] In one or more embodiments, in step (2), the reaction conditions for synthesizing the compound represented by formula (I) are stirring at room temperature; the stirring time is 1 to 2 h, preferably 1.5 h.

[0019] In one or more embodiments, in step (2), the solvent for the reaction to synthesize the compound represented by formula (I) is tetrahydrofuran.

[0020] 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 as a bromodomain activity-based probe.

[0021] In one or more embodiments, the bromodomain activity-based probe targets binding to a lysine in a bromodomain.

[0022] The fifth aspect of the present invention provides a bromodomain activity-based probe, wherein the bromodomain activity-based probe comprises the compound represented by formula (I) according to the first aspect, a stereoisomer thereof or a physiologically acceptable salt thereof.

[0023] The sixth aspect of the present invention provides a composition comprising a compound represented by formula (I), a stereoisomer thereof or a physiologically acceptable salt thereof, and an auxiliary material.

[0024] The excipients described in the present invention are pharmaceutical excipients, such as solubilizers, emulsifiers, stabilizers, preservatives, etc.

[0025] The beneficial effects of the present invention are: The present invention utilizes the highly selective and efficient cyclization reactivity of lysine and 2-ethynylbenzaldehyde, and introduces a 2-ethynylbenzaldehyde warhead and an alkyne handle capable of clickable chemical reactions into the structure of the compound represented by formula (I), thereby obtaining an activity-based probe that can systematically analyze the lysine ligandability of BRD. In chemical proteomics experiments conducted in MV4-11 and U2OS cells, a total of 18 endogenous BRDs spanning 6 subfamilies were captured, providing extremely valuable information for the development of BRD covalent inhibitors targeting lysine. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] 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.

[0027] Figure 1 is a flow chart for the preparation of the compound represented by formula (I); Figure 2 is the H NMR spectrum of compound 1; Figure 3 is the NMR carbon spectrum of compound 1; Figure 4 is the H NMR spectrum of compound 3; Figure 5 is the NMR carbon spectrum of compound 3; Figure 6 is the H NMR spectrum of compound 4; Figure 7 is the NMR carbon spectrum of compound 5; Figure 8 This is the fluorescence imaging image of the BRD protein gel labeling experiment of compound 1 in Example 2; Fig. 9 This is the fluorescence imaging diagram of the gel labeling experiment of the concentration dependence of compound 1 on BRD4 (1) in Example 3; Fig.10 This is the fluorescence imaging diagram of the incubation time dependence of compound 1 on BRD4 (1) in Example 4; Fig.11 The complete protein spectrum and site analysis after the reaction of compound 1 with BRD4 (1) in Examples 5 and 6, wherein A is the complete protein spectrum and B is the site analysis; Fig.12 Fluorescence imaging images of the gel labeling experiment of compound 1 in MV4-11 and U2OS cells in Examples 7 and 8; Fig.13 Western blotting (WB) analysis of the pull-down (PD) experiment of compound 1 in MV4-11 and U2OS cells in Examples 9 and 10; Fig.14 These are LC-MS / MS quantitative analysis graphs of the pull-down (PD) experiments of compound 1 in Examples 9 and 10 in MV4-11 and U2OS cells, wherein A is the LC-MS / MS quantitative analysis graph of the pull-down (PD) experiment carried out in MV4-11, B is the LC-MS / MS quantitative analysis graph of the pull-down (PD) experiment carried out in U2OS, and C is 18 BRDs captured from the two cell lines MV4-11 and U2OS. DETAILED DESCRIPTION

[0028] 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.

[0029] 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.

[0030] 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.

[0031] Example 1 Figure 1 is a flow chart for the preparation of the compound represented by formula (I), refer to Figure 1 Synthesize the compound represented by formula (I).

[0032] 54 mmol 3,4,6-trichloropyrazine and 100 mL 28% (mass fraction) ammonia water were reacted in a high pressure reactor at 95 °C for 8 h. After the reaction was completed and cooled to room temperature, 100 mL water was added to precipitate and filtered to obtain an off-white solid, which was compound 5 with a yield of 67%.

[0033] 36.6 mmol of compound 5 and 60 mL of hydrazine hydrate were reacted in a high pressure reactor at 95 °C for 8 h. After the reaction was completed and cooled to room temperature, 100 mL of ice water was added to precipitate, and a light yellow solid was obtained by filtration, namely compound 6, with a yield of 60%.

[0034] 22 mmol of compound 6 and 29 mL of acetic acid were reacted in a round-bottom flask at 95 °C for 3 h. After the reaction was completed, the mixture was cooled to room temperature, 100 mL of ice water was added, and the precipitate was collected by filtration. 10 mL of saturated sodium carbonate aqueous solution was added to the precipitate, stirred at room temperature for 0.5 h, and filtered to obtain an off-white solid, which was compound 7, with a yield of 43%.

[0035] 14.33 mmol 3-butyn-1-ol, 35.83 mmol 3,4-dihydro-2H-pyran and 0.72 mmol p-toluenesulfonic acid were added to a reaction bottle containing 40 mL dichloromethane, and the reaction was allowed to proceed at room temperature for 1.5 h. 10 mL saturated sodium bicarbonate aqueous solution was added to quench the reaction, and the mixture was extracted with ether for 3 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, and the eluent ratio was ethyl acetate: petroleum ether = 1: (2~15) to obtain the compound 2-(3-butynyloxy)tetrahydro-2H-pyran. 28 mmol n-butyl lithium was slowly added dropwise to a reaction bottle containing 25.32 mmol 2-(3-butynyloxy)tetrahydro-2H-pyran and 25 mL tetrahydrofuran at -78 °C, and the mixture was stirred for 1 h at -78 °C. Triisopropylsilyl chloride dissolved in 12 mL of tetrahydrofuran was slowly added dropwise to the reaction flask, and the reaction was naturally restored to room temperature overnight. 10 mL of saturated sodium bicarbonate aqueous solution was added to quench the reaction, and 10 mL of ether was extracted three times each. After the combined organic phase was dried over sodium sulfate, the solvent was evaporated under reduced pressure without further separation, and the intermediate was added to a methanol (5 mL) solution containing 0.4 mmol of p-toluenesulfonic acid and reacted at room temperature for 1.5 h. 10 mL of saturated sodium bicarbonate aqueous solution was added to quench the reaction, and 10 mL of ether was extracted three times each. 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, and the eluent ratio was ethyl acetate: petroleum ether = 1: (30~50) to obtain compound 10, and the total yield of the three-step reaction was 67%.

[0036] Under ice bath and nitrogen protection, 12.6 mmol of compound 10 was slowly added dropwise to a reaction bottle containing 6.3 mmol of triphosgene and 5.5 mL of tetrahydrofuran. After the addition was completed, the mixture was naturally returned to room temperature and stirred overnight. TLC detected that the reaction was complete, and 18.9 mmol of anhydrous pyridine was added under ice bath and nitrogen protection, and stirred at room temperature for 1 h. After filtering the reaction solution, the filtrate was concentrated under reduced pressure to obtain compound 11 with a yield of 72%.

[0037] 2 mmol of compound 11 was slowly added dropwise to a reaction bottle containing 2 mmol of triethylamine, 1 mmol of compound 7 and 9.5 mL of anhydrous dichloromethane, and the reaction was stirred at room temperature overnight. The reaction was monitored by TLC until it was complete, and the solvent was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography, and the eluent ratio was ethyl acetate: petroleum ether = 1: (1-3) to obtain the compound represented by formula (II), namely compound 3, with a yield of 93%.

[0038] 12 mL of triethylamine was added to a reaction bottle containing 3.22 mmol of 5-bromo-2-iodobenzaldehyde, 3.22 mmol of (triisopropylsilyl)acetylene, 0.081 mmol of cuprous iodide, and 0.04 mmol of bistriphenylphosphine palladium dichloride, and the reaction was carried out at room temperature for 24 h. The solvent was evaporated under reduced pressure, 20 mL of dichloromethane was added to dissolve the reaction mixture, and 3 mL of 10% (mass fraction) hydrochloric acid solution and 5 mL of brine were added to wash the organic phase. 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: (4~10) to obtain compound 8 with a yield of 91%.

[0039] 2.6 mmol of bis(pinacol)diboron, 0.065 mmol of [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) dichloromethane complex, 6.48 mmol of potassium acetate and 2.16 mmol of compound 8 were added to a reaction bottle containing 5.4 mL of 1,4-dioxane and reacted at 100 °C for 7 h under nitrogen protection. The reaction solution was concentrated under reduced pressure and the crude product was purified by silica gel column chromatography with an eluent ratio of dichloromethane: petroleum ether = 1: (5-20) to obtain compound 9 with a yield of 62%.

[0040] 0.74 mmol of compound 9, 3.68 mmol of ammonium acetate, and 2.94 mmol of sodium periodate were added to a reaction bottle containing 7.2 ml of a mixed solvent of acetone and water (volume ratio 1:1), and the reaction was stirred at room temperature overnight. TLC was monitored until the reaction was complete, 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, and the eluent ratio was dichloromethane: petroleum ether = 1: (5~20) to obtain compound 4 with a yield of 33%.

[0041] 0.115 mmol of compound 3, 0.014 mmol of chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II) (XPhos Pd G2), 0.35 mmol of potassium phosphate and 0.23 mmol of compound 4 were added to a reaction bottle containing 3.7 mL of 1,4-dioxane and 0.37 mL of water, stirred at 120 °C for 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. 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, and the eluent ratio was methanol: dichloromethane = 1: (50~150) to obtain compound 2 with a yield of 41%.

[0042] 0.25 mmol tetrabutylammonium fluoride (TBAF) and 0.041 mmol compound 2 were added to a reaction bottle containing 1.5 mL tetrahydrofuran at 0 °C, and stirred at room temperature for 1.5 hours. The reaction was monitored by TLC until completion. The reaction solution was concentrated by evaporation 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 compound represented by formula (I), namely compound 1, with a yield of 60%.

[0043] The H NMR spectrum of compound 1 is shown in Figure 2 As shown, the NMR carbon spectrum is as follows Figure 3 shown.

[0044] The H NMR spectrum of compound 3 is shown in Figure 4 As shown, the NMR carbon spectrum is as follows Figure 5 shown.

[0045] The H NMR spectrum of compound 4 is shown in Figure 6 As shown, the NMR carbon spectrum is as follows Figure 7 shown.

[0046] Characterization of Compound 1: 1 H-NMR (400 MHz, DMSO- d 6) δ 11.18 (s, 1H), 10.43 (s, 1H), 8.37 (d, J =1.9 Hz, 1H), 8.30 (dd, J = 8.1, 1.9 Hz, 1H), 8.15 (s, 1H), 7.89 (d, J = 8.1 Hz,1H), 4.93 (s, 1H), 4.30 (t, J= 6.6 Hz, 2H), 2.94 (t, J = 2.6 Hz, 1H), 2.75 (s,3H), 2.63 (td, J = 6.6, 2.6 Hz, 2H). 13 C-NMR (101 MHz, DMSO- d 6) δ 190.67, 153.56, 152.36, 147.74, 138.79,136.54, 135.67, 135.27, 134.88, 132.21, 126.25, 125.61, 100.79, 89.91, 80.73,78.92, 72.88, 63.61, 18.57, 9.63. ESI-MS calculation for [M+H] + 374.12; Found 374.3. HRMS (ESI) calculated m / z for [M+H] + 374.1248, found 374.1239. Example 2 BRD protein gel labeling experiment of compound 1: A 0.5 mM stock solution of compound 1 dissolved in DMSO and a 1 mg / mL stock solution of recombinant proteins of KAT2B, BRD2(2), BRD3(1), BRD4(1), BRD4(2), BRDT(1), CBP, BRD7, BRD9, TAF1(2) and TAF1L(2) dissolved in PBS buffer (pH = 7.4, composition: 2.7 mM KCl, 2.0 mM KH2PO4, 137 mM NaCl, 10 mM Na2HPO4) were prepared respectively. 1 μL of stock solutions of different BRD proteins (working concentration 0.02 mg / mL) and 1 μL of stock solution of compound 1 (working concentration 10 µM) were taken respectively and added to a 1.5 mL centrifuge tube containing 48 μL PBS buffer (pH = 7.4, components: 2.7 mM KCl, 2.0 mMKH2PO4, 137 mM NaCl, 10 mM Na2HPO4), and reacted in a metal bath at 37 °C for 2 h. Click chemistry reagent (Cyanine5.5-N3, 40 µM) was added and reacted at room temperature for 2 hours. Proteins were precipitated by adding chilled acetone and methanol respectively. The proteins were collected and separated by gel electrophoresis (SDS-PAGE). Fluorescence imaging was taken using an Odyssey CLx imager (Ex = 680 nm). The results are shown in Figure 2. Figure 8 As shown, BRD proteins can be covalently labeled by compound 1 to varying degrees. More importantly, compared with other subfamilies, compound 1 presents significantly stronger fluorescent labeling signals for BET family proteins (BRD2, BRD3 and BRD4).

[0047] Example 3 Gel labeling experiment of compound 1 on BRD4(1) concentration dependence: Compound 1 stock solutions were prepared at 0.015625 mM, 0.03125 mM, 0.0625 mM, 0.125 mM, 0.25 mM, 0.5 mM and 1 mM in DMSO; and BRD4(1) recombinant protein stock solution was prepared at 1 mg / mL in PBS buffer (pH = 7.4, composition: 2.7 mM KCl, 2.0 mM KH2PO4, 137 mM NaCl, 10 mM Na2HPO4). 1 μL of BRD4(1) stock solution (working concentration 0.02 mg / mL) and 1 μL of compound 1 stock solution of different concentrations (working concentrations of 0.3125 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM and 20 μM) were respectively taken and added to a 1.5 mL centrifuge tube containing 48 μL PBS buffer (pH = 7.4, composition: 2.7 mM KCl, 2.0 mM KH2PO4, 137 mM NaCl, 10 mMNa2HPO4) and reacted in a metal bath at 37 °C for 2 h. Click chemistry reagent (Cyanine5.5-N3, 40 μM) was added and reacted at room temperature for 2 hours. Chilled acetone and methanol were added to precipitate proteins respectively. The proteins were collected and separated by gel electrophoresis (SDS-PAGE). Fluorescence imaging was captured using an Odyssey CLx imager (Ex = 680 nm). The results are shown in Figure 2. Fig. 9 As shown in Figure 2, compound 1 labeling of BRD4(1) showed a probe concentration-dependent phenomenon, and the EC 50 is 1.21 µM.

[0048] Example 4 Gel labeling experiment of compound 1 on BRD4(1) incubation time dependence: A 0.5 mM stock solution of compound 1 dissolved in DMSO and a 1 mg / mL stock solution of BRD4(1) recombinant protein dissolved in PBS buffer (pH = 7.4, composition: 2.7 mM KCl, 2.0 mM KH2PO4, 137 mM NaCl, 10 mM Na2HPO4) were prepared. 1 μL of BRD4(1) stock solution (working concentration 0.02 mg / mL) and 1 μL of compound 1 stock solution (working concentration 10 µM) were added to 48 μL of PBS buffer (pH = 7.4, composition: 2.7 mM KCl, 2.0 mM KH2PO4, 137 mM NaCl, 10 mM Na2HPO4) in a 1.5 mL centrifuge tube and reacted in a metal bath at 37 °C for 15 min, 30 min, 45 min, 60 min, and 90 min, respectively. Click chemistry reagent (Cyanine5.5-N3, 40 µM) was added and reacted at room temperature for 2 hours. Proteins were precipitated by adding chilled acetone and methanol, respectively. Proteins were collected and separated by gel electrophoresis (SDS-PAGE). Fluorescence imaging was performed using an Odyssey CLx imager (Ex = 680 nm). The results are shown in Fig.10 As shown in Figure 2, compound 1 showed an incubation time-dependent labeling phenomenon on BRD4(1), and t 1 / 2 It is 113.4min.

[0049] Example 5 Intact protein profiling analysis of compound 1 after reaction with BRD4(1): Prepare a 10 mM stock solution of compound 1 dissolved in DMSO and a 500 µM stock solution of BRD4(1) recombinant protein dissolved in PBS buffer (pH = 7.4, composition: 2.7 mM KCl, 2.0 mM KH2PO4, 137 mM NaCl, 10 mM Na2HPO4). Pipette 2 μL of BRD4(1) stock solution (working concentration 10 μM) and 1 μL of compound 1 stock solution (working concentration 100 μM) into a 1.5 mL centrifuge tube containing 97 μL of PBS buffer (pH = 7.4, composition: 2.7 mM KCl, 2.0 mM KH2PO4, 137 mM NaCl, 10 mM Na2HPO4), mix well, and react in a metal bath at 37 °C for 12 h. Add 1 μL of formic acid to quench the reaction and transfer the reaction solution to an Amicon® Ultra filter (10 kDa). 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. The reaction volume after centrifugation is 100 μL, add 1 μL of formic acid, mix well, and use for LC-MS / MS analysis.

[0050] The results are as follows Fig.11 As shown in Figure A, the covalent reaction efficiency of compound 1 and BRD4 (1) is 90.74%.

[0051] Example 6 Analysis of the covalent binding site between compound 1 and BRD4(1): Prepare a 10 mM stock solution of compound 1 in DMSO, a 5 mg / mL stock solution of BRD4-BD1 in PBS (pH = 7.4, composition: 2.7 mM KCl, 2.0 mM KH2PO4, 137 mM NaCl, 10 mM Na2HPO4), a 1 M stock solution of DTT (dithiothreitol) in ABC (triethylammonium bicarbonate) buffer (pH = 8.0, 0.1 M), a 500 mM stock solution of IAA (iodoacetamide) in ABC (triethylammonium bicarbonate) buffer (pH = 8.0, 0.1 M), and a 0.1 mg / mL stock solution of trypsin in ABC (triethylammonium bicarbonate) buffer (pH = 8.0, 0.1 M). 1 μL of compound 1 stock solution and 3.5 μL of BRD4(1) stock solution were taken and added to a 1.5 mL centrifuge tube containing 95.5 μL PBS (pH = 7.4, composition: 2.7 mM KCl, 2.0 mM KH2PO4, 137 mM NaCl, 10 mM Na2HPO4) buffer (compound 1 working concentration 100 μM, protein working concentration 10 μM), 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). Add 350 μL PBS buffer (pH = 7.4, composition: 2.7 mM KCl, 2.0 mM KH2PO4, 137 mM NaCl, 10 mM Na2HPO4), centrifuge at 4 °C for 10 min (13800 g), repeat this step 3 times; add 300 μL ABC (triethylammonium bicarbonate) buffer (pH = 8.0, 100mM), centrifuge at 4 °C for 10 min (13800 g), repeat this step 2 times, the reaction volume after centrifugation is 150 μL. Pipette 1.5 μL DTT stock solution and add it to the reaction mixture dissolved in ABC buffer, react in a metal bath at 37 °C for 45 min; Pipette 6 μL IAA stock solution and add it to the reaction mixture dissolved in ABC buffer, react at room temperature in the dark for 1 h; after adding 1.5 μL DTT stock solution, directly transfer the reaction solution to an Amicon® Ultra filter (10 kDa). Add 300 μL ultrapure water, centrifuge at 4 °C for 10 min (13800 g), and repeat this step 3 times. After centrifugation, transfer the reaction solution to a 1.5 mL centrifuge tube, add 150 μL ABC buffer and 5 μL trypsin stock solution, and react in a metal bath at 37 °C overnight.The reaction solution was transferred to an Amicon® Ultra filter (30 kDa) and centrifuged at 4 °C for 10 min (13800 g). The collected filtrate was desalted and eluted through a Merck C18 microchromatographic column (0.6 µL C18 resin): (1) Wetting the column with aspirated wetting solution (100% acetonitrile) and repeated twice; (2) Equilibrating the Ziptip with aspirated equilibration solution (0.1% trifluoroacetic acid aqueous solution) and repeated twice; (3) Repeatedly aspirating the sample 7-10 times to allow the sample to bind to the column; (4) Washing the microchromatographic column with a washing solution (0.1% trifluoroacetic acid aqueous solution) and repeated twice; (5) Eluting with an eluent (0.1% trifluoroacetic acid aqueous solution / 50% acetonitrile) and collecting the eluent in a centrifuge tube; (6) Drying the eluent with a vacuum centrifugal concentrator, redissolved in 10 µL 0.1% formic acid aqueous solution and directly used for LC-MS / MS analysis.

[0052] The results are as follows Fig.11 As shown in B, the covalent binding site of compound 1 to BRD4 (1) occurs at K91.

[0053] Example 7 Protein gel labeling experiment of compound 1 in MV4-11 cells: Cells were cultured in IMDM medium containing 10% FBS and 1% antibiotics (penicillin and streptomycin) in a humidified incubator at 37 °C. MV4-11 cells were seeded in a 6-well cell culture plate and cultured in a humidified incubator at 37 °C for 12 h. Compound 1 (working concentration: 10 µM) was added and the cells were incubated at 37 °C for 6 h. The IMDM medium containing cells was collected, centrifuged, and washed twice with PBS to collect the cells. Cell lysis buffer (20 mM HEPES, 100 mM KCl, 2 mM MgCl2, pH7.4) was added, the cells were ultrasonically disrupted, and the supernatant was collected by centrifugation. The protein concentration was adjusted to 2 mg / mL, and click chemistry reagent (Cyanine5.5-N3, 40 µM) was added. The reaction was reacted at room temperature in the dark for 2 h. The proteins were precipitated / washed by adding chilled acetone / methanol, respectively. The proteins were collected and separated by gel electrophoresis (SDS-PAGE). Fluorescence imaging was performed using an Odyssey CLx imager (Ex = 680 nm). Fig.12 As shown, compound 1 successfully labeled several proteins in MV4-11 cells, while no obvious signal was detected with DMSO.

[0054] Example 8 Protein gel labeling experiment of compound 1 in U2OS cells: The cells were cultured in DMEM medium containing 10% FBS and 1% antibiotics (penicillin and streptomycin) in a humidified incubator at 37 °C. U2OS cells were seeded in a 6-well cell culture plate and cultured in a humidified incubator at 37 °C for 12 hours. The DMEM medium was poured out, the DMEM was washed three times, and fresh DMEM medium containing compound 1 (working concentration 10 µM) was added, and the cells were incubated at 37 °C for 6 h. The DMEM medium containing compound 1 was poured out, the cells were washed twice with PBS, trypsin was added for 5 min, and the cells were collected by centrifugation. Cell lysis buffer (20 mM HEPES, 100 mM KCl, 2 mM MgCl2, pH 7.4) was added, the cells were ultrasonically disrupted, and the supernatant was collected by centrifugation. The protein concentration was adjusted to 2 mg / ml, and the click chemistry reagent (Cyanine5.5-N3, 40 µM) was added, and the reaction was carried out at room temperature in the dark for 2 hours. Frozen acetone and methanol were added to precipitate and wash the protein, respectively. Proteins were collected and separated by gel electrophoresis (SDS-PAGE). Fluorescence imaging was performed using an Odyssey CLx imager (Ex = 680 nm). Fig.12 As shown, compound 1 successfully labeled several proteins in U2OS cells, while no obvious signal was detected with DMSO.

[0055] Example 9 Pull-down experiment of compound 1 in MV4-11: MV4-11 cells were cultured in IMDM medium containing 10% FBS and 1% antibiotics (penicillin and streptomycin). MV4-11 cells were seeded in 10 cm cell culture dishes and cultured in a humidified incubator at 37 °C for 12 h. Compound 1 (working concentration 10 µM) was added and the cells were incubated at 37 °C for 6 h. Cells were directly collected by centrifugation and washed twice with PBS. Cell lysis buffer (20 mM HEPES, 100 mM KCl, 2 mM MgCl2, pH 7.4) was added, cells were disrupted by ultrasound, and the supernatant was collected by centrifugation. The protein concentration was adjusted to 2 mg / ml, and click chemistry reagent (Bio-N3, 40 µM) was added. The reaction was carried out at room temperature in the dark for 2 h. Chilled acetone and methanol were added to precipitate and wash the protein, and the protein pellet was collected by centrifugation. PBS containing 0.2% SDS was added to redissolve the protein pellet obtained by precipitation. Streptavidin magnetic beads were added to enrich the protein captured by compound 1 and incubated overnight at 4 °C. The magnetic beads were collected by magnet and washed with 1% SDS / PBS. The magnetic beads were collected by magnet and SDS-PAGE protein loading buffer was added. The samples were heated at 90°C for 20 minutes and the upper buffer containing the pulled-down proteins was collected by centrifugation. Western blotting analysis ( Fig.13), in MV4-11 cells, BRD2, BRD3 and BRD4 were successfully captured by compound 1; further quantitative analysis by LC-MS / MS showed that Fig.14 As shown in A and C, in MV4-11 cells, compound 1 captured a total of 4704 proteins (fold change>2, p<0.05), including BRD4, BRD2, BRD3, BAZ1A, EP300, BRD8, BAZ1B, ATAD2, BRD7, ATAD2B, BRD1, TRIM24, SP100, BAZ2A, TRIM28 and SMARCA4 (16 BRDs).

[0056] Example 10 Pull-down experiment of compound 1 in U2OS: U2OS cells were cultured in DMEM medium containing 10% FBS and 1% antibiotics (penicillin and streptomycin). U2OS cells were seeded in 10 cm cell culture dishes and cultured in a humidified incubator at 37 °C for 12 h. The DMEM medium was poured out, the cells were washed 3 times with DMEM, and fresh DMEM medium containing compound 1 (10 µM) was added, and the cells were incubated at 37 °C for 6 h. The DMEM medium containing the probe was poured out, the cells were washed twice with PBS, trypsin was added for 5 min, and the cells were collected by centrifugation. Cell lysis buffer (20mM HEPES, 100 mM KCl, 2 mM MgCl2, pH 7.4) was added, the cells were ultrasonically disrupted, and the supernatant was collected by centrifugation. The protein concentration was adjusted to 2 mg / mL, and the click chemistry reagent (Bio-N3, 40 µM) was added, and the reaction was carried out at room temperature in the dark for 2 h. Frozen acetone and methanol were added to precipitate and wash the protein, and the protein particles were collected by centrifugation. Add PBS containing 0.2% SDS to re-dissolve the protein particles obtained by precipitation. Add streptavidin magnetic beads to enrich the protein captured by compound 1 and incubate at 4°C overnight. Collect the magnetic beads with a magnet and wash them with 1% SDS / PBS. Collect the magnetic beads with a magnet, add SDS-PAGE protein loading buffer, heat at 90°C for 20 minutes, and centrifuge to collect the upper buffer containing the pulled-down protein. Analyze by Western blotting ( Fig.13 ), in U2OS cells, BRD2 / 3 / 4 were successfully captured by compound 1; further quantitative analysis by LC-MS / MS showed that Fig.14As shown in B and C, in U2OS cells, compound 1 captured a total of 4835 proteins (fold change>2, p<0.05), including BRD4, BRD2, BRD3, BAZ1A, EP300, CREBBP, PHIP, BRD8, BAZ1B, ATAD2, BRD7, BRD1, TRIM24, SP100, BAZ2A, TRIM28 and SMARCA4 (17 BRDs).

[0057] Compound 1 captured a total of 18 BRDs across 6 subfamilies from MV4-11 and U2OS cells. These results indicate that compound 1 designed based on the EBA warhead has the ability to comprehensively analyze the ligandability of BRD lysine.

[0058] 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), its stereoisomer or a physiologically acceptable salt thereof; Formula (I).

2. An intermediate of the compound represented by formula (I) according to claim 1, characterized in that: Its structural formula is shown in formula (II); Formula (II).

3. 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 chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II); (2) Compound 2 is reacted with tetrabutylammonium fluoride to synthesize a compound represented by formula (I); The structural formula of compound 4 is shown below: ； The structural formula of compound 2 is shown below: 。 4. The preparation method according to claim 3, characterized in that: In step (1), in step (1), the molar ratio of the compound represented by formula (II) to compound 4 is 1:1.8-2.

2.

5. The preparation method according to claim 3, characterized in that: In step (1), the solvent used in the reaction is a mixed solution of 1,4-dioxane and water; the volume ratio of 1,4-dioxane to water is 9-12:1; In step (1), the reaction temperature for synthesizing compound 2 is 110-130° C. and the reaction time is 4-6 h.

6. The preparation method according to claim 3, characterized in that: In step (2), the molar ratio of compound 2 to tetrabutylammonium fluoride is 1:5.8-6.5; In step (2), the reaction conditions for synthesizing the compound represented by formula (I) are stirring at room temperature; the stirring time is 1 to 2 h; In step (2), the solvent for the reaction to synthesize the compound represented by formula (I) is tetrahydrofuran.

7. Use of the compound represented by formula (I) according to claim 1, its stereoisomers or physiologically acceptable salts thereof as a bromodomain activity-based probe.

8. The use according to claim 7, characterized in that The bromodomain activity-based probe targets lysines in bromodomains.

9. A bromodomain activity-based probe, characterized in that The bromodomain activity-based probe comprises the compound represented by formula (I) of claim 1, its stereoisomers or physiologically acceptable salts thereof.

10. A composition, characterized in that The composition comprises a compound represented by formula (I), a stereoisomer thereof or a physiologically acceptable salt thereof and auxiliary materials.

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