Use of diphenylacetonitrile compounds and protein target hydrolyzable chimeric compounds

Abstract

The application relates to the field of medicinal chemistry and provides a use of a diphenylacetonitrile compound and a protein-targeting hydrolytic chimeric compound. The application finds that a diphenylacetonitrile compound shown in formula (I) directly interacts with FEM1B, thereby serving as an inhibitor of a human E3 ubiquitin ligase substrate recognition receptor FEM1B. The application also provides a compound shown in formula (II), and the compound shown in formula (II) is a protein-targeting hydrolytic chimeric (PROTAC) drug, wherein Q2 is a target protein ligand molecule, Q1 is a FEM1B inhibitor, Q2 is combined with a target protein, thereby inducing E3 ligase FEM1B combined with Q1 to approach the target protein, leading to ubiquitination and degradation of the target protein. Experimental results show that the PROTAC drug shown in formula (II) provided by the application can specifically degrade a target protein in cells. Formula (I); formula (II).

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical chemistry, and particularly relates to a use of a diphenylacetonitrile compound and a protein-targeted proteolysis chimera compound. BACKGROUND

[0002] Targeted protein degradation (TPD) is a breakthrough drug development method that selectively eliminates specific target proteins by utilizing intracellular protein degradation mechanisms, thereby solving the problem that certain proteins cannot be drug targets. This technology usually involves the use of molecular glue or protein-targeted proteolysis chimeras (PROTACs) to induce E3 ubiquitin ligases to approach target proteins, thereby leading to ubiquitination and proteasome-mediated degradation. In theory, TPD has the potential to selectively eliminate any pathogenic protein in cells. However, although there are about 600 different E3 ubiquitin ligases, only a few E3 ubiquitin ligases have been successfully utilized in this strategy, such as cereblon and VHL.

[0003] Currently, there are few reports on PROTAC drug development for human E3 ubiquitin ligase substrate recognition receptor FEM1B. The only report is an article published by Nathaniel J. et al. in the Journal of ACS in 2022. The article discovered a covalent inhibitor EN106 of FEM1B, which can form a covalent bond with C186 of FEM1B, inhibit the interaction between FEM1B and the substrate folliculin interacting protein 1 (FNIP1), and based on this, developed two PROTAC drugs NJH-1-106 and NJH-2-142. Developing more inhibitors of FEM1B is one of the needs in the field of targeted protein degradation research. SUMMARY

[0004] Therefore, the present application aims to solve the technical problem of providing a use of a diphenylacetonitrile compound and a protein-targeted proteolysis chimera compound. The compound provided in the present application can achieve FEM1B-dependent targeted degradation of the substrate.

[0005] Diclazuril (DIC) is also known as clozylacon, which belongs to triazine phenylacetonitrile compounds, and its structural formula is shown as formula (d). It is a broad-spectrum anticoccidial drug developed by Janssen Company in Belgium, and is currently more applied in the prevention and treatment of coccidiosis in horses, sheep, rabbits, chickens and other livestock and poultry. Studies have shown that the drug can affect the synthesis of coccidial nucleic acid, inhibit the formation of coccidial meronts and microgamonts, and interfere with coccidial nuclear division, thereby killing coccidia. Existing studies mainly focus on the prevention and treatment effects of diclazuril on animal coccidiosis, and the application research of diclazuril in the field of human medicine is still in the initial stage. The present application found that diclazuril can interact with the substrate recognition subunit FEM1B protein of human multi-subunit E3 ubiquitin ligase CRL2 FEM1B in vitro, so as to prepare a PROTAC drug to achieve FEM1B-dependent targeted degradation of the substrate.

[0006] Formula (d).

[0007] Based on this, the present application provides the use of a compound shown as formula (I) in the preparation of a FEM1B inhibitor:

[0008] Formula (I);

[0009] In formula (I), X is selected from halogen;

[0010] R1 is selected from a group containing an amino group or an imino group.

[0011] The present application found that the compound shown as formula (I) has a direct interaction with FEM1B, so as to be used as an inhibitor of the substrate recognition receptor FEM1B of human E3 ubiquitin ligase.

[0012] In some specific implementation manners, the compound has a structure of formula (I-1):

[0013] Formula (I-1).

[0014] In some specific implementation manners, X is halogen, including but not limited to Cl or Br, and preferably Cl.

[0015] In some specific implementation manners, R1 is selected from the structures shown as formula (a) or formula (b):

[0016] Formula (a);

[0017] Formula (b);

[0018] In formula (a), n is 0 or an integer from 1 to 10, preferably 0 or an integer from 1 to 5, more preferably 0 or an integer from 1 to 3, for example 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0019] In formula (b), ring A is a 5- to 10-membered aromatic ring, preferably a 5- to 8-membered aromatic ring, more preferably a 6- to 7-membered aromatic ring. In addition to N, the ring A preferably further comprises 0 to 3 heteroatoms, for example 0, 1, 2 or 3 heteroatoms, selected from N, O or S, preferably N. In some specific implementations, the ring A can be substituted with a substituent selected from an alkyl group or a keto group, for example a C1-C6 alkyl group or a keto group directly formed on the ring A.

[0020] In some specific implementations, R1 is selected from a structure shown in formula (b-1):

[0021] Formula (b-1);

[0022] In formula (b-1), X1 is selected from C or N; X2 or X3 is independently selected from C, N, O or S; and r is an integer from 1 to 5, for example 1, 2, 3, 4, 5, etc. The compound shown in formula (b-1) is an aromatic compound, and the number of double bonds is related to the values of X1, X2 and X3 and r, for example when r is 1, X2 is not O or S.

[0023] In some specific implementations, the compound of formula (I) has a structure of formula (I-1-a) or formula (I-1-b):

[0024] Formula (I-1-a); Formula (I-1-b).

[0025] The present application also provides a compound shown in formula (II):

[0026] Formula (II);

[0027] In formula (II), Q1 is a residue of a structure of formula (I):

[0028] Formula (I);

[0029] In formula (I), X is selected from a halogen;

[0030] R1 is selected from a group containing an amino group or an imino group;

[0031] Linker is a linking group;

[0032] Q2 is a residue of a target protein ligand molecule.

[0033] The compound shown in formula (II) is a protein targeting hydrolysis chimera (PROTAC) drug, wherein Q2 is a target protein ligand molecule, Q1 is an FEM1B inhibitor, Q2 binds to the target protein, thereby inducing the E3 ligase FEM1B combined with Q1 to approach the target protein, resulting in ubiquitination and degradation of the target protein, and the principle is as shown in Figure 1 Figure 1 The principle diagram of the compound shown in formula (II) is shown in the figure.

[0034] In some specific implementations, Q1 is a residue of the compound shown in formula (I):

[0035] Formula (I).

[0036] The compound shown in formula (I) is described above, and will not be repeated here.

[0037] In formula (II), Q1 is a residue of the compound shown in formula (I), which can be a residue after removing one hydrogen from an amino group or an imino group, or a residue after removing X, or a residue after removing a cyano group, preferably a residue after removing one hydrogen from an amino group or an imino group.

[0038] In formula (II), Q2 is a residue of a target protein ligand molecule, including but not limited to a residue of a bromodomain protein 4 (BRD4) ligand molecule, a residue of a bromodomain protein 9 (BRD9) ligand molecule, a residue of a Bruton's tyrosine protein kinase (BTK) ligand molecule, a residue of a cyclin-dependent kinase (CDK) ligand molecule, a residue of an androgen receptor (AR) ligand molecule, a residue of an estrogen receptor (ER) ligand molecule, or a residue of a tyrosine kinase (RTK) ligand molecule.

[0039] Specifically, the BRD4 ligand molecule can be JQ-1, which has the structure of formula (1):

[0040] Formula (1);

[0041] The BTK ligand molecule can be Ibrutinib, which has the structure of formula (2):

[0042] Formula (2);

[0043] The CDK ligand molecule can be Ribociclib (LEE011), which has the structure of formula (3):

[0044] Formula (3).

[0045] ​Q2 is a residue of the target protein ligand molecule, which can be a residue after removing a hydroxyl group from a carboxyl group of the target protein ligand molecule, or a residue after removing a hydrogen atom from an amino group of the target protein ligand molecule, and can be selected according to the structure of the target protein ligand molecule.

[0046] In formula (II), the linker is used to connect Q1 and Q2. In some specific embodiments, the linker has a structure of formula (c):

[0047] Formula (c);

[0048] In formula (c), m and q are independently selected from integers from 1 to 6.

[0049] In some specific embodiments, the compound has a structure of formula (II-1) or formula (II-2):

[0050] Formula (II-1);

[0051] Formula (II-2).

[0052] The structural composition of the compound shown in formula (II-1) and the compound shown in formula (II-2) is as shown in Figure 2 and Figure 3 , Figure 2 The structural composition of the compound shown in formula (II-1) is Figure 3 The structural composition of the compound shown in formula (II-2) is. FEM1B specifically interacts with the structure shown in formula (I), and the structure shown in formula (I) is connected to the target protein ligand molecule through a linker to form a PROTAC molecule, which specifically mediates the polyubiquitination and degradation of BRD4.

[0053] Experimental results show that the protein targeting hydrolysis chimera (PROTAC) drug shown in formula (II) provided by the present application can specifically degrade target proteins in cells. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 is a schematic diagram of the compound shown in formula (II);

[0055] Figure 2 is the structural composition of the compound shown in formula (II-1);

[0056] Figure 3 is the structural composition of the compound shown in formula (II-2);

[0057] Figure 4 is the nuclear magnetic resonance spectrum of the compound prepared in Example 1;

[0058] Figure 5 LCMS spectrum of the compound prepared in Example 1; wherein, Figure 5 (a) is the chromatogram at 220 nm, Figure 5 (b) is the chromatogram at 254 nm, Figure 5 (c) is the total ion current chromatogram;

[0059] Figure 6 NMR spectrum of the compound prepared in Example 2;

[0060] Figure 7 LCMS spectrum of the compound prepared in Example 2; wherein, Figure 7 (a) is the chromatogram at 220 nm, Figure 7 (b) is the chromatogram at 254 nm, Figure 7 (c) is the total ion current chromatogram;

[0061] Figure 8 Gel filtration chromatogram of SUMO-FEM1B 1-243 protein;

[0062] Figure 9 SDS-PAGE gel of SUMO-FEM1B 1-243 protein;

[0063] Figure 10 ITC experiment results of FEM1B 1-243 and dechlorcholine; wherein, Figure 10 (a) is the heat change curve, the horizontal coordinate is time (min), and the vertical coordinate is heat flow rate (μcal / s); Figure 10 (b) is the binding isotherm, obtained by heat integration of (a) figure, after fitting by unit point binding model, the horizontal coordinate is molar ratio, and the vertical coordinate is binding enthalpy ΔH (kcal / mol);

[0064] Figure 11 In vitro ubiquitination experiment results of dechlorcholine;

[0065] Figure 12 In vitro ubiquitination experiment results of Target 3 compound prepared in Example 1;

[0066] Figure 13 In vitro ubiquitination experiment results of Target 4 compound prepared in Example 2;

[0067] Figure 14 Immunofluorescence figure of the compound provided in the examples of the present application in specific degradation of BRD4 in cells;

[0068] Figure 15Quantification plots of the amount of BRD4 specifically degraded by the compounds provided in the examples of the present application in cells, wherein, Figure 15 (a) a plot of the number of BRD4 foci in each nucleus, Figure 15 (b) a plot of the number of SREBP2 foci in each nucleus. DETAILED DESCRIPTION

[0069] The present application provides a use of a diphenylacetonitrile compound and a protein-targeting hydrolytic chimera compound. Those skilled in the art can refer to the content herein, and appropriately modify the process parameters to achieve the present application. The method and application of the present application have been described by preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the method and application herein without departing from the content, spirit and scope of the present application, to achieve and apply the present application.

[0070] The present application provides a use of a compound represented by formula (I) in the preparation of a FEM1B inhibitor:

[0071] Formula (I);

[0072] In formula (I), X is selected from halogen;

[0073] R1 is selected from a group containing an amino or imino group.

[0074] The present application researches and finds that the compound represented by formula (I) has a direct interaction with FEM1B, and can be used as an inhibitor of the human E3 ubiquitin ligase substrate recognition receptor FEM1B.

[0075] The present application also provides a compound represented by formula (II):

[0076] Formula (II);

[0077] In formula (II), Q1 is a residue of the structure of formula (I):

[0078] Formula (I);

[0079] In formula (I), X is selected from halogen;

[0080] R1 is selected from a group containing an amino or imino group.

[0081] Linker is a linking group;

[0082] Q2 is a residue of a target protein ligand molecule.

[0083] The compound shown in formula (II) is a protein targeted hydrolysis chimera (PROTAC) drug, wherein Q2 is a target protein ligand molecule, Q1 is an FEM1B inhibitor, Q2 binds to the target protein, and then induces the E3 ligase FEM1B combined with Q1 to approach the target protein, resulting in ubiquitination and degradation of the target protein. Experimental results show that the protein targeted hydrolysis chimera (PROTAC) drug provided in formula (II) can specifically degrade the target protein in cells.

[0084] The present application is further illustrated below in conjunction with examples.

[0085] Example 1

[0086] The compound shown in formula (II-1) was synthesized by a contract drug company, and the specific process is as follows:

[0087] 3,4,5-trichloronitrobenzene (CAS No.: 20098-48-0) and p-chlorophenylacetonitrile (CAS No.: 140-53-4) were reacted in the presence of appropriate NaOH and tetrabutylammonium bromide (TBAB) in H2O / tetrahydrofuran (THF) mixed solvent at 60°C for 12 hours to obtain intermediate Target 3_1;

[0088] In the presence of NH4Cl, intermediate Target 3_1 was reacted with appropriate Fe as a reducing agent in EtOH / H2O (ethanol / water) mixed solvent at 80°C for 12 hours to obtain intermediate Target 3_2;

[0089] Intermediate Target 3_2 and glyoxylic acid (CAS No.: 298-12-4) were reacted in the presence of appropriate sodium cyanoborohydride (NaBH3CN) and acetic acid (AcOH) in methanol (MeOH) solvent at 25°C for 12 hours to obtain intermediate product Target 3_3;

[0090] Under the activation of 1-hydroxybenzotriazole (HOBT), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and N,N-diisopropylethylamine (DIEA), intermediate product Target 3_3 was mixed with N-tert-butoxycarbonyl-1,4-butanediamine (CAS No.: 68076-36-8) in N,N-dimethylformamide (DMF) solvent at 25°C for 12 hours to obtain intermediate product Target 3_4;

[0091] Under the action of HCl and dioxane, intermediate product Target 3_4 was deprotected in methanol (MeOH) solvent at 25°C for 12 hours to obtain intermediate Target 3_5;

[0092] Under the activation of HOBT, EDCI and DIEA, intermediate Target 3_5 was mixed with JQ-1 (CAS No: 202592-23-2) in N,N-dimethylformamide (DMF) solvent, and reacted at 25°C for 12 hours to obtain the final product Target 3.

[0093] The reaction scheme is as follows:

[0094]

[0095] The Target 3 was analyzed by nuclear magnetic resonance and liquid chromatography-mass spectrometry (LCMS), and the results are shown in Figure 4 and Figure 5 , Figure 4 The nuclear magnetic spectrum of the compound prepared in Example 1 is shown in Figure 5 The LCMS spectrum of the compound prepared in Example 1 is shown in, wherein Figure 5 (a) is the chromatogram at 220 nm, Figure 5 (b) is the chromatogram at 254 nm, Figure 5 (c) is the total ion flow chromatogram. It can be seen from Figure 4 and Figure 5 that the compound prepared in Example 1 has a structure as shown in formula (II-1) and a purity of 95%.

[0096] Example 2

[0097] The compound shown in formula (II-2) was synthesized by entrusting WuXi Biotechnology Co., Ltd., and the specific process is as follows:

[0098] Under the action of potassium carbonate (K2CO3), diclazuril (CAS No: 101831-37-2) was mixed with 10 g of 3-bromopropionic acid (CAS No: 590-92-1) compound in DMF solvent, and reacted at 60°C for 24 hours to obtain the intermediate product Target 4_1;

[0099] Under the activation of HOBT, EDCI and DIEA, Target 4_1 was mixed with N-tert-butoxycarbonyl-1,4-butanediamine (CAS No: 68076-36-8) in DMF solvent, and reacted at 25°C for 12 hours to obtain the intermediate product Target 4_2;

[0100] Under the action of HCl and dioxane, the intermediate product Target 4_2 was reacted in methanol (MeOH) solvent at 25°C for 12 hours to obtain the intermediate Target 4_3;

[0101] Target 4_5 was mixed with JQ-1 (CAS No.: 202592-23-2) in DMF solvent under the activation of HOBT, EDCI and DIEA, and reacted at 25°C for 12 hours to obtain the final product Target 4.

[0102] The reaction scheme is as follows:

[0103]

[0104] Target4

[0105] 100 mg, 95% purity, 98% ee.

[0106] The Target 3 was subjected to nuclear magnetic resonance and liquid chromatography-mass spectrometry (LCMS) analysis, and the results are shown in Figure 6 and Figure 7 , Figure 6 the nuclear magnetic spectrum of the compound prepared in Example 2, Figure 7 the LCMS spectrum of the compound prepared in Example 2, wherein, Figure 7 (a) is the chromatogram at 220 nm, Figure 7 (b) is the chromatogram at 254 nm, Figure 7 (c) is the total ion flow chromatogram. It can be seen from Figure 6 and Figure 7 that the compound prepared in Example 2 has a structure as shown in formula (II-2) and a purity of 95%.

[0107] Test Example 1

[0108] The isothermal titration calorimetry (ITC) experiment was carried out on a PEAQ-ITC (MicroCal) instrument, and the specific experimental steps were as follows:

[0109] (a) Sample preparation: according to the gene cloning, protein expression and purification method disclosed in the literature “Molecular basis for arginine C-terminal degron recognition by Cul2 FEM1 E3 ligase”, FEM1B 1-356 was replaced by FEM1B 1-243 , and then SUMO-FEM1B 1-243 protein was expressed in E. coli; and 1-243The product was first purified by nickel column and then further purified by gel filtration chromatography in a buffer of 200 mM NaCl, 20 mM Tris-HCl, pH 7.5. Figure 8 and Figure 9 , Figure 8 SUMO-FEM1B 1-243 Gel filtration chromatography of protein, where the absorption peak is the absorption peak of protein at 280nm, Figure 9 SUMO-FEM1B 1-243 SDS-PAGE gel image of protein.

[0110] Purified SUMO-FEM1B 1-243 Concentrate to 0.7 mM and diclazuril to 50 μM solution, keeping the buffer conditions consistent.

[0111] (b) Loading: SUMO-FEM1B 1-243 Load the sample (60 μL) into the titration needle and diclazuril (350 μL) into the sample pool. For the control group, replace the sample pool with buffer.

[0112] (c) Parameter settings: The temperature is generally 25°C, the reference power is 5 μCal / s, and there are 19 drops in total. Except for the first drop which is a pre-drop with a volume of 1 μL, the remaining 18 drops are all 2 μL. The interval between each drop is 120 s.

[0113] (d) Analysis and plotting of experimental results: The ITC experimental results were fitted using the “single-site binding model” in MicroCal PEAQ-ITC Analysis software.

[0114] Results see Figure 10 , Figure 10 For FEM1B 1-243 The ITC experimental results with diclazuril, among which, Figure 10 (a) is the heat change curve, the horizontal axis is time (min), and the vertical axis is heat flow rate (μcal / s); Figure 10 (b) is the binding isotherm, which is obtained by integrating the heat of Figure (a) and fitting the single-site binding model. The horizontal axis is the molar ratio and the vertical axis is the binding enthalpy ΔH (kcal / mol). Figure 10 It can be seen that diclazuril can bind to human multi-subunit E3 ubiquitin ligase CRL2 in vitro FEM1B Diclazuril interacts with the substrate recognition subunit FEM1B protein 1-243 The dissociation constant between them is 3.5 μM (K D =3.5 μM).

[0115] Test Example 2

[0116] The specific experimental steps of the in vitro ubiquitination reaction are as follows:

[0117] All components required for the in vitro ubiquitination reaction were prepared by in vitro purification, including E1 ubiquitin activating enzyme UBA1, E2 ubiquitin binding enzyme UBE2R1, E3 ligase N8 CRL2 FEM1B , ubiquitin (Ub), and substrate CCDC89. Each component was added according to the following reaction system: 0.2 µM UBA1, 1 µM UBE2R1, 1 µM N8 CRL2 FEM1B , 1 µM CCDC89, 30 µM Ub, 0 µM, 16 µM, or 100 µM diclazuril, 1 mM dithiothreitol (DTT), 10 mM MgCl2, 5 mM adenosine triphosphate (ATP). The total reaction system was 40 μL, and the reaction buffer was: 100 mM NaCl, 20 mM HEPES buffer, pH 7.5, 1% dimethyl sulfoxide (DMSO). The reaction system was placed in a 37 ℃ environment for a corresponding period of time (0-60 min), and then the reaction was terminated by adding 5×sodium dodecyl sulfate loading buffer (SDS loading buffer).

[0118] After the in vitro ubiquitination reaction, the generation of ubiquitination bands was detected by immunoblotting, and the specific operation steps were as follows:

[0119] (a) Electrophoresis: load the protein sample into a 4%-20% precast gel for gel electrophoresis, with pre-stained protein Mark as the marker, and MOPS-SDS running buffer in the inner and outer slots (Running Buffer), voltage 120 V, current 80 mA, time 70-90 min, and the specific electrophoresis time can be adjusted according to the position of the pre-stained Mark band.

[0120] (b) Gel cutting: after electrophoresis, peel off the gel from the gel plate, cut the position of the sample well and the un-sampled lanes with a small knife, and only keep the sampled lanes, and the bromophenol blue and the part below need to be cut off.

[0121] (c) Transfer: wet transfer method, transfer buffer: 25 mM Tris base, 190 mM Glycine, 20% methanol. Generally, first prepare 10x transfer solution (250 mM Tris base, 1900 mM Glycine), 1x transfer solution needs 100 mL 10x transfer solution, 700 mL ultrapure water, 200 mL methanol (100%). Transfer solution needs to be pre-cooled at -20°C. The polyvinylidene fluoride (PVDF) membrane used for transfer needs to be cut according to the size of the gel, and then placed in 100% methanol for 1 min to activate the PVDF membrane. Pour the pre-cooled transfer solution into the transfer disc, and at the same time open the transfer clamp, black down, white up. Place a layer of sponge on the black side of the clamp, cover the top with three layers of filter paper, then place the cut gel on top, cover the gel with a PVDF membrane, carefully chase away the air bubbles between the gel and the membrane with a roller to ensure the flatness of the membrane, and finally cover the PVDF membrane with another three layers of filter paper and a piece of sponge, thus forming a "sandwich" structure. Close the transfer clamp and place it in the transfer tank, pour in the transfer solution and place it in the ice box prepared in advance, constant voltage 80 V, 30 min, then change to 100 V, 90 min. Throughout the ice water bath to offset the large amount of heat released during the transfer.

[0122] (d) Blocking: after the transfer is completed, separate the membrane from the clamp and soak it in blocking solution, place it on a horizontal shaker and block at room temperature for 2 h. The blocking solution is prepared as follows: 2.5 g of skimmed milk powder is dissolved in 150 mM NaCl, 0.1% Tween 20, 20 mM Tris-HCl, pH 7.5 Tween 20-containing Tris buffer saline (TBST) solution.

[0123] (e) Primary antibody incubation: after blocking is completed, pour out the blocking solution, add 5-7 mL of fresh and pre-cooled blocking solution at 4°C, then add 5 μL of anti-FLAG antibody (primary antibody dilution ratio 1:1000) to it, place it on a horizontal shaker and incubate overnight at 4°C.

[0124] (f) Secondary antibody incubation: After the primary antibody incubation, the blocking solution was recovered into a 15 mL conical centrifuge tube and stored at -80 °C (the recovered primary antibody can be reused). Then 15 mL of TBST solution was added for membrane washing, this step was repeated 3 times for 10 min each time, to completely remove the unbound primary antibody. Then 5 mL of fresh TBST solution was added, and 5 μL of secondary antibody (anti-rabbit IgG, HRP-linked antibody, secondary antibody dilution ratio 1:1000) was added, and incubated at room temperature for 2 h.

[0125] (g) Development and imaging: After the secondary antibody incubation, the TBST solution containing the secondary antibody was recovered into a 15 mL conical centrifuge tube and stored at -80 °C (the recovered secondary antibody can be reused). Then 15 mL of TBST solution was added for membrane washing, this step was repeated 5 times for 10 min each time, to completely remove the unbound primary antibody. The water on the membrane was dried as much as possible, and the membrane was laid in a transparent plastic dish, attention should be paid to avoid the generation of bubbles, and then 1 mL of developing solution (500 μL of A solution + 500 μL of B solution) was prepared. The developing solution was evenly dropped onto the membrane, and after 1-2 min, imaging was performed in a molecular imager LAS4000 (including chemiluminescence imaging and white light imaging).

[0126] Results are shown in Figure 11 , Figure 11 The results of the in vitro ubiquitination experiment of diclazuril are shown, wherein the control group (control) does not add diclazuril. The results show that diclazuril competes with FEM1B to inhibit the binding of the substrate, thereby hindering the polyubiquitination modification of FEM1B to the physiological substrate CCDC89 and the degradation process thereof.

[0127] Test Example 3

[0128] The difference from Test Example 2 is that the Target 3 compound prepared in Example 1 is used instead of diclazuril, and the substrate BRD4 is used instead of the substrate CCDC89 to perform an in vitro ubiquitination experiment. The addition concentration of the Target 3 compound is 0, 4, 8, 16, 32, 64, 125, 250, 500, 1000, 2000 and 4000 nM, respectively, and the reaction is performed for 60 min. Results are shown in Figure 12 , Figure 12 The results of the in vitro ubiquitination experiment of the Target 3 compound prepared in Example 1 are shown, and the results show that the Target 3 compound can induce the polyubiquitination of BRD4 in vitro.

[0129] Test Example 4

[0130] The difference from Test Example 2 is that the in vitro ubiquitination experiment is carried out using the Target 4 compound prepared in Example 2 instead of the griesofulvin, and the substrate BRD4 instead of the substrate CCDC89, and the addition concentration of the Target 4 compound is 0, 4, 8, 16, 32, 64, 125, 250, 500, 1000, 2000 and 4000 nM, respectively, and the reaction is carried out for 60 min, and the results are shown in Table 2. Figure 13 , Figure 13 The in vitro ubiquitination experiment results of the Target 4 compound prepared in Example 2 show that the Target 4 compound can induce the polyubiquitination of BRD4 in vitro.

[0131] Test Example 5

[0132] The Target 3 compound prepared in Example 1 and the Target 4 compound prepared in Example 2 are used to carry out the experiment of specific degradation of BRD4 in cells, and the specific experimental steps are as follows:

[0133] Day 1: Coat the coverslips, add 1 mL of collagen solution to the 12-well plate, and place the round coverslips with a thickness of 0.17 mm into the well plate, and place it in the cell culture incubator overnight for coating.

[0134] Day 2: Pave the cells. At 9:00 am, recover the collagen solution, wash the coverslips twice with phosphate buffer solution (PBS), and aspirate the residual PBS. Pave 6×10 4

[0135] Day 3: Cholesterol starvation culture. At 17:00, aspirate the culture medium, wash twice with PBS, add preheated cholesterol starvation medium, and starve for 16 h.

[0136] Day 4: Compound treatment. At 9:00 am, aspirate the culture medium, add fresh cholesterol starvation medium containing the corresponding concentration of compound (0 μmol, 5 μmol JQ-1, 10 μmol JQ-1, 5 μmol Target 3, 10 μmol Target 3, 5 μmol Target 4, 10 μmol Target 4) to treat the cells for 4 h.

[0137] At 13:00, collect the cells for immunostaining, and the specific steps are as follows:

[0138] (a) Aspirate the culture medium in the well plate, and wash once with PBS.

[0139] (b) Fixing. Add pre-cooled 4% paraformaldehyde (PFA) solution to the well plate, and fix for 10 min.​

[0140] (c) Permeabilization. Wash three times with PBS and add 0.5% Triton X-100 solution to the plate for 10 min.

[0141] (d) Blocking. Wash three times with PBS. Remove the coverslip and place the front side on the well plate lid. Place the well plate lid in a light-proof incubation box with an appropriate amount of water. Add 100 μL of 3% bovine serum albumin (BSA) solution to the coverslip and block for 30 min.

[0142] (e) Incubation with primary antibody (anti-FLAG DYKDDDDK tag antibody (Cell Signaling Technology, Catalog #2368, dilution 1:1000)). 100 μL of primary antibody solution (7D4 / Ms + anti-BRD4 / Rb, 2 μg / mL) was added to the coverslip and incubated at room temperature in the dark for 2 h.

[0143] (f) Wash with PBS five times, 3 min each time.

[0144] (g) Incubate with secondary antibody (HRP-conjugated anti-rabbit IgG secondary antibody (Cell Signaling Technology, Catalog #7074, dilution 1:1000)). Add 100 μL of secondary antibody solution (Ms-488 + Rb-647) to the coverslip and incubate at room temperature in the dark for 2 h.

[0145] (h) Wash with PBS five times, 3 min each time.

[0146] (i) Nuclear staining: Add 100 μL of 4',6-diamidino-2-phenylindole (DAPI) solution prepared in PBS to the coverslip and incubate at room temperature in the dark for 10 min.

[0147] (j) Wash three times with PBS, 3 min each time.

[0148] (k) Mounting. Add a small amount of mounting medium to the slide. Place the coverslip upside down on the slide containing the mounting medium. Protect from light and wait for it to dry before photographing.

[0149] (l) Imaged using a Zeiss Lattice SIM super-resolution microscope with a 63x objective.

[0150] Statistics and Analysis:

[0151] The slides were observed using a Zeiss Lattice SIM microscope, and random fields were selected for imaging. The exposure time and other shooting conditions were uniform. The ZEN Microscopy Software 3.8 (Zeiss) software was used to reduce the noise of the images, and then the Analyze Particles plug-in of Image J (NIH) was used to automatically quantify the number of SREBP2 and BRD4 condensates in each nucleus (n = 24-27). The analyzed data were statistically analyzed using GraphPad Prism 9.0.0 software, and the one-way ANOVA followed by Turkey's multiple comparison method was used for difference analysis.

[0152] The results are shown in Figure 14 and Figure 15 , Figure 14 The immunofluorescence images of the compound provided in the embodiments of the present application for specific degradation of BRD4 in cells are shown in Figure 14 from left to right are the fluorescence images of sterol regulatory element binding protein 2 (SREBP2), bromodomain-containing protein 4 (BRD4), and the fluorescence images of the two signals superimposed (merge); Figure 15 The quantitative statistical graphs of the compound provided in the embodiments of the present application for specific degradation of BRD4 in cells are shown in Figure 15 (a) is a statistical graph of the number of BRD4 condensates in each nucleus, Figure 15 (b) is a statistical graph of the number of SREBP2 condensates in each nucleus. From Figure 14 and Figure 15 it can be seen that the compounds Target 3 and Target 4 provided in the embodiments of the present application can effectively degrade BRD4 specifically in cells at a concentration of 5-10 μM. The degradation of BRD4 leads to a decrease in the expression of SREBP2 protein downstream thereof.

[0153] The above is only a preferred embodiment of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, and these improvements and refinements should also be considered within the scope of protection of the present application.

Claims

1. A compound characterized by, having the structure of formula (II-1) or formula (II-2): Formula (II-1); Formula (II-2).

Images