Bivalent ligand molecules targeting bcl6 protein degradation and uses thereof

Abstract

The application discloses a kind of bivalent ligand molecules for targeting BCL6 protein degradation and application thereof, it is related to the technical field of drug development;The structure general formula of the bivalent ligand molecules for targeting BCL6 protein degradation is as follows: M1-L-M2;Wherein, M1 and M2 are independent structure same or different BCL6 protein ligand;L is any chain or cyclic hydrocarbon fragment capable of forming covalent bond with BCL6 ligand.The application also provides the application of the bivalent ligand molecules in the preparation of anti-tumor and / or immune disease drugs.The bivalent ligand molecules can exert good anti-tumor activity on a variety of BCL6-dependent tumors by selectively inducing BCL6 protein degradation, and are also related to the occurrence of a variety of immune diseases, and can be used in related tumor and immune disease drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of drug development, in particular to a bivalent ligand molecule targeting BCL6 protein degradation and application thereof. BACKGROUND

[0002] B-cell lymphoma 6 (BCL6) is widely expressed in various cancer cells, and its research mainly focuses on lymphoma and breast cancer. Existing BCL6 inhibitors are mainly designed for the BTB domain of BCL6, and play an inhibitory activity by competitively binding with a co-repressor. However, there are relatively few studies on BCL6 degradation agents, among which the BCL6 PROTAC protein degradation activity is poor, and other degradation agents are BCL6 molecular glue, which are accidentally obtained when screening inhibitors and cannot be rationally designed. These molecular glues have better effects than inhibitors of the same type.

[0003] Targeted protein degradation (TPD) technology utilizes the ubiquitin proteasome system and lysosomal degradation system existing in cells to achieve specific and efficient degradation of disease-related proteins, thereby achieving disease treatment effects. Compared with traditional small molecule inhibitors, TPD has more advantages: event-driven, lower drug dosage, only a catalytic dose is needed to play a role; can target some undruggable proteins, etc.

[0004] There are many types of existing protein degradation molecules such as PROTAC, molecular glue, AUTAC, LYTAC, etc., each of which has its own characteristics. At present, PROTAC and molecular glue are more in-depth studied, and some PROTAC and molecular glue have entered the clinical research stage. PROTAC still has some problems in drugability, such as larger molecular weight and poor target selectivity. The existing technology of molecular glue cannot be rationally designed at present, and can only be obtained accidentally. SUMMARY

[0005] In order to solve the above technical problems, the purpose of the present application is to provide a bivalent ligand molecule targeting BCL6 protein degradation and application thereof, which can induce selective degradation of BCL6 protein and exhibit good antitumor activity against various BCL6-dependent tumors. In addition, it is also related to the occurrence of various immune diseases and can be used in drugs for related tumors and immune diseases.

[0006] The technical solution of the present application to solve the above technical problems is as follows: a bivalent ligand molecule targeting BCL6 protein degradation is provided, and the structural general formula thereof is:

[0007] M1-L-M2;

[0008] wherein M1and M2are independently the same or different BCL6 protein ligand;

[0009] L is any linear or cyclic hydrocarbon fragment capable of forming a covalent bond with the BCL6 ligand.

[0010] Further, L is

[0011] L2is -(CH2) n - or -(CH2CH2O) n -;

[0012] X1and X2are independently -CH2-, -CH2NH-, -CO-, -CONR1-, -O-, -S-, -NR1-, a single, double or triple bond;

[0013] X3and X4are independently

[0014] R1is hydrogen, C1-C6alkyl or C3-C6cycloalkyl;

[0015] n is any integer between 1 and 10;

[0016] n1, n2, n3and n4are independently any integer between 1 and 4.

[0017] Further, the BCL6 protein ligand is

[0018]

[0019] wherein X is hydrogen or halogen;

[0020] L1is

[0021] L3is -(CH2) n - or -(CH2CH2O) n -;

[0022] X5and X6are independently -CH2-, -CH2NH-, -CO-, -CONR4-, -O-, -S-, -NR4- or a single bond;

[0023] X7and X8are independently

[0024] R2is hydrogen or alkoxy;

[0025] R3is hydrogen, COOH or COOR5;

[0026] R4is hydrogen, C1-C6alkyl or C3-C6cycloalkyl

[0027] R5 is hydrogen, C1-C6 alkyl or C3-C6 cycloalkyl;

[0028] n' is any integer between 0-4;

[0029] n5, n6, n7 and n8 are independently any integer between 1-4.

[0030] Further, the BCL6 protein ligand is

[0031]

[0032] wherein X7 and X8 are independently

[0033] R2 is hydrogen or methoxy.

[0034] Further, the BCL6 protein ligand is

[0035] Further, the structure of the bivalent ligand molecule targeting BCL6 protein degradation is:

[0036]

[0037]

[0038]

[0039]

[0040]

[0041] The bivalent ligand molecule targeting BCL6 protein degradation described above in the preparation of an anti-tumor and / or immune disease drug.

[0042] The bivalent ligand molecule targeting BCL6 protein degradation described above, its stereoisomer, its pharmaceutically acceptable salt or its solvate in the preparation of an anti-tumor and / or immune disease drug.

[0043] An anti-tumor and / or immune disease drug comprising the bivalent ligand molecule targeting BCL6 protein degradation described above.

[0044] The present application has the following beneficial effects:

[0045] 1、The BCL6 bivalent ligand in the application can bind to two BCL6 dimers at the same time, induce BCL6 dimers to form multimers through chain assembly, and finally be degraded by the intracellular protein quality control system. By selectively inducing the degradation of BCL6 protein, the bivalent ligand molecule can exert good antitumor activity on various BCL6-dependent tumors, and is also related to the occurrence of various immune diseases, and can be used in drugs related to tumors and immune diseases.

[0046] 2、The bivalent ligand molecule of the application, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof can induce BCL6 protein degradation, thereby exerting a therapeutic effect on BCL6-dependent tumors or other diseases. Such compounds are expected to be used as one of the components of a pharmaceutical preparation for the treatment of BCL6-dependent tumors or other diseases. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 Results of degradation activity on BCL6 at different concentrations (cell line: MDA-MB-468);

[0048] Figure 2 Results of degradation activity on BCL6 at different concentrations (cell line: SU-DHL-4);

[0049] Figure 3 Results of degradation activity on BCL6 at different concentrations and different action times of the target compound of Example 4 (cell line: MDA-MB-468). DETAILED DESCRIPTION

[0050] The principles and characteristics of the application are described below, and the examples are only used to explain the application and not to limit the scope of the application. If no specific conditions are specified in the examples, conventional conditions or manufacturer's recommended conditions are used. If no manufacturer of the reagent or instrument is specified, it is a conventional product that can be purchased on the market.

[0051] Example 1

[0052] A bivalent ligand molecule targeting BCL6 protein degradation, the preparation process is as follows:

[0053]

[0054] Specifically includes the following steps:

[0055] Intermediate 1 (106 mg, 2 eq) and piperazine (1 eq), HATU (76 mg, 2 eq) and DIEA (38 μL, 2.5 eq) were dissolved in DMSO, heated to 75 °C to make the raw materials fully dissolved, then reacted at room temperature for 6 h, TLC monitored the end of the reaction, stopped the reaction, added water, filtered, dried, purified by column chromatography to obtain the target compound.

[0056] The preparation methods of Examples 2-5, Example 7, Example 9, Example 10 and Example 12 are the same as above.

[0057] Example 6

[0058] A bivalent ligand molecule targeting BCL6 protein degradation, its preparation process is as follows:

[0059]

[0060] Specifically includes the following steps:

[0061] Intermediate 2 (106 mg, 2 eq) and suberic acid (1 eq), HATU (76 mg, 2 eq) and DIEA (38 μL, 2.5 eq) were dissolved in DMSO, heated to make the raw materials fully dissolved, then slowly cooled to room temperature after 6 h, TLC monitored the end of the reaction, stopped the reaction, added water, filtered, dried, purified by column chromatography to obtain the target compound.

[0062] The preparation method of Example 11 is the same as above.

[0063] Example 13

[0064] A bivalent ligand molecule targeting BCL6 protein degradation, its preparation process is as follows:

[0065]

[0066] Specifically includes the following steps:

[0067] Intermediate 1 (159 mg, 0.3 mmol, 1 eq) and Boc-piperazine (67 mg, 0.36 mmol, 1.2 eq) were dissolved in DMSO. Then, HATU (228 mg, 0.6 mmol, 2 eq) and DIEA (130 μL, 0.75 mmol, 2.5 eq) were added. The reaction was carried out at room temperature. After the reaction was completed as monitored by TLC, the reaction was stopped, water was added, the mixture was filtered, dried, and purified by column chromatography to obtain intermediate 2. Intermediate 2 was then dissolved in 10 mL of DCM. Add trifluoroacetic acid (5 mL) to the mixture and stir at room temperature. After the reaction is completed by TLC monitoring, stop stirring and concentrate to obtain intermediate 3. Then, dissolve intermediate 3, FX-1 (37 mg, 0.1 mmol, 1 eq), and HATU (76 mg, 0.2 mmol, 2 eq) in DMSO (3 mL), add DIEA (86 μL, 0.25 mmol, 2.5 eq), and stir at room temperature. After the reaction is completed by TLC monitoring, add water, filter, and purify by column chromatography to obtain the target compound.

[0068] The structural formulas of the target compounds obtained in Examples 1-13 above are shown in Table 1, and their structural characterization was performed, with the results shown in Table 2.

[0069] Table 1. Structural formulas of target compounds in Examples 1-13

[0070]

[0071]

[0072]

[0073]

[0074] Table 2 Structural characterization data of Examples 1-13

[0075]

[0076]

[0077]

[0078] Experimental Example 1: BCL6 Protein Degradation Assay

[0079] The steps for the BCL6 protein degradation assay are as follows:

[0080] ① Cell culture and drug treatment: MDA-MB-468 and SU-DHL-4 cells in logarithmic growth phase were cultured at 5 × 10⁶ cells per dish. 5The cells were seeded in 60 mm dishes and cultured in a 37°C, 5% CO2 incubator. Once the cells had basically adhered to the wall and reached the logarithmic growth phase, the culture medium was removed, and the cells were treated with different concentrations of compounds for 24 h.

[0081] ② Protein Extraction and Quantification: After tryingpsin digestion and culture to terminate the digestion, the cells were centrifuged and the precipitate was collected in EP tubes. The cells were washed 2-3 times with pre-chilled PBS on ice, and a certain amount of cell lysis buffer was added for lysis for about 30 minutes. The sample was then sonicated on ice until the solution was clear, and then lysed on ice for about 30 minutes, followed by centrifugation on ice for 15 minutes. The supernatant was collected into a new EP tube. The protein supernatant collected in the above process was used to quantify the total protein using a BCA protein quantification kit. The sample was diluted with RIPA to ensure the same protein concentration in each tube. Then, 1 / 4 of the protein loading buffer (5×) was added, vortexed until mixed, and placed in a metal bath to denature the protein. After natural cooling, the samples were stored at -20°C.

[0082] ③ Protein Immunoblot Analysis: Select an appropriate concentration of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel based on the molecular weight of the target protein, and prepare the required amount of gel according to the instructions. After checking for leaks in the solidified gel, add the pre-stained protein marker and the prepared protein sample to the gel wells according to the loading order and volume. Run the sample out of the stacking gel area using 80V, then adjust the voltage to 120V and continue running the gel. Stop electrophoresis when the sample reaches the bottom of the gel. Transfer the sample using a methanol-activated PVDF membrane. Place the prepared transfer clamp into the electrophoresis tank, place it in an ice box, and select an appropriate transfer voltage and time for transfer. After transfer, remove the PVDF membrane and place it in milk for blocking. After blocking, incubate with primary antibody overnight. After primary antibody incubation, wash (3 times, 10 min each time), then incubate with secondary antibody at room temperature for 1 hour, followed by washing (3 times, 10 min each time). After cleaning, the PDVF film was immersed in the ultrasensitive ECL chemiluminescent substrate for a few seconds, and then placed in an exposure machine for exposure to obtain a WB image, which was then analyzed for grayscale using ImageJ.

[0083] The result is as follows Figures 1-3 As shown. Among them, Figure 3 The top image shows the cell line: MDA-MB-468; treatment time: 24h; the bottom image shows the cell line: MDA-MB-468; drug concentration: 1μM.

[0084] Depend on Figures 1-2It was found that the efficacy of the drug in inducing BCL6 protein degradation was tested 24 hours after drug administration. The results showed that some divalent ligands could induce BCL6 protein degradation to varying degrees in the tested cells.

[0085] Depend on Figure 3 It is known that the divalent ligand molecule targeting BCL6 protein degradation of the present invention can degrade BCL6 protein in a concentration-dependent manner, and its DC 50 The concentration was 0.27 nM. Furthermore, this divalent ligand molecule significantly degraded BCL6 protein within 1 hour of application, and its degradative effect on BCL6 persisted even after 24 hours of application.

[0086] Experimental Example 2: Tumor Cell Proliferation Inhibition Activity Test

[0087] The method for tumor cell proliferation inhibition activity assay is as follows:

[0088] Tumor cells in the logarithmic growth phase were collected and resuspended in appropriate cell culture medium to a concentration of 1-9 × 10⁶ cells / mL. 5 Cell suspension was added to 96-well plates at a rate of 100 μL per well, and the side wells were filled with 200 μL of sterile physiological saline. Adherent cells were incubated at 37°C for 24-48 hours to allow cell adhesion; suspension cells were immediately added to the plate. The specified compound was prepared into a drug-containing medium at the specified concentration using the appropriate culture medium in the deep-well plate dosing tank, and 100 μL was added to each well. The plates were incubated at 37°C for 4 days. Then, under dark conditions, 20 μL of 5% MTT solution was added to each well, and the plates were incubated for another 2-4 hours. Afterward, for suspension cells, 50 μL of 20% SDS solution was added to each well, and the plates were incubated overnight. For adherent cells, the liquid in the wells was carefully discarded, and 150 μL of dimethyl sulfoxide was added to each well. The absorbance at 570 nM was measured using a microplate reader, and cell growth curves were plotted using GraphPad 8.0 software to calculate the IC50. 50 value.

[0089] The results are shown in Table 3. "-" indicates IC 50 >10μM; "+" indicates 10μM >IC 50 >1μM; "++" indicates 1μM >1000μM 50 >0.1μM; "+++" indicates IC 50 <0.1μM).

[0090] Table 3 Inhibitory activity against tumor cell proliferation (IC50) 50 :μM)

[0091] Compound No. Example 1 Example 2 Example 3 Example 4 Example 12 Farage ++ - +++ +++ ++ SU-DHL-4 + + - + +

[0092] As shown in Table 3, the bivalent ligand molecules for targeting BCL6 protein degradation of the present application can exhibit proliferation inhibition activity on Farage and Su-DHL-4 cells, wherein the target compounds of Example 3-4 exhibit good inhibition activity on Farage cells.

[0093] The above description is merely preferred embodiments of the present application, and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall fall within the protection scope of the present application.

Claims

1. A bivalent ligand molecule targeting degradation of BCL6 protein, characterized in that, A structure general formula is: M1-L-M2; Wherein, M1 and M2 are the following BCL6 protein ligand: ； R2 is methoxy; X7 is ; X8is ; L is ; L2 is , , -(CH2) n -; X1 and X2 are -CO-, -CONH-; X3and X4are ; n is any integer between 2-8; n1, n2, n3 and n4 are independent integers between 1-2.

2. The bivalent ligand molecule targeted to BCL6 protein degradation of claim 1, wherein, A structure formula is: , , or .

3. Use of the bivalent ligand molecule targeting BCL6 protein degradation according to any one of claims 1-2 in the preparation of an antitumor and / or immune disease drug.

4. Use of the bivalent ligand molecule targeting BCL6 protein degradation according to any one of claims 1-2, stereoisomer thereof, pharmaceutically acceptable salt thereof in the preparation of an antitumor and / or immune disease drug.

5. An antitumor and / or immunological disease drug, characterized by, The bivalent ligand molecule targeting BCL6 protein degradation according to any one of claims 1-2.

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