3-(5-(aminomethyl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione derivatives, process for their synthesis, use

Abstract

The application discloses a 3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)piperidine-2,6-dione derivative and a synthesis method and application thereof, and relates to a 3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)piperidine-2,6-dione derivative which is a compound with the following general formula (I): wherein R1 is selected from,,,,,,,,, and R2 is selected from,,, and. The compound can significantly inhibit the proliferation of leukemia, multiple myeloma, lymphoma, breast cancer, liver cancer and the like at a low dose (nanomole), can effectively degrade IKZF1, IKZF3, BRD4, GSPT1 and CK1 alpha, and has the prospect of being developed into an anti-tumor drug. The application solves the problem that the existing malignant hematological disease treatment drugs have a high safety risk.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a 3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)piperidine-2,6-dione derivative, its synthesis method, and its applications. Background Technology

[0002] The incidence of hematological malignancies is on the rise due to factors such as environmental pollution and changes in lifestyle. In 2024, the global market size for hematological malignancies was US$88.87 billion. It is projected to grow at a CAGR of 10.7% during the forecast period of 2024-2029. With advancements in medical technology, new drugs and treatments are constantly emerging, providing more options for the treatment of hematological malignancies. Current treatment methods for hematological malignancies include chemotherapy, radiotherapy, immunotherapy, and hematopoietic stem cell transplantation, but the treatment effects and prognoses vary considerably. The diagnosis and treatment of hematological malignancies face many challenges, such as high relapse rates, strong drug resistance, and significant treatment side effects, necessitating research into novel treatment strategies.

[0003] The Ikaros family of zinc finger DNA-binding proteins belongs to the Kruppel transcription factor superfamily. Ikaros proteins are characterized by a zinc finger DNA-binding domain at their N-terminus and a dimerizing domain at their C-terminus. Members of the Ikaros family include Ikaros, Aiolos, Helios, EOS, and Pegasus. All members of this family can form homodimers and heterodimers with other Ikaros family members. Most family members also have multiple isoforms, which arise from differential splicing, and some of these isoforms exhibit dominant-negative effects during dimerization. Ikaros (IKZF1, LYF1) is a typical Ikaros family zinc finger transcription factor, highly expressed in lymphocytes. Genetic studies in mice have shown that Ikaros is a tumor suppressor, crucial for the normal development of B cells, T cells, natural killer cells, and dendritic cells. Further research has indicated that imbalances in the expression of different Ikaros isoforms and mutations in the corresponding genes are associated with various hematologic malignancies in humans.

[0004] Eukaryotic releasing factor 3 (eRF3, GSPT1) is an evolutionarily conserved class II releasing factor belonging to the GTPase superfamily, and it cooperates with eRF1 in peptide translation termination. Studies have confirmed that eRF3 undergoes caspase-mediated cleavage and degradation during DNA damage-induced apoptosis, which is associated with reduced protein synthesis. Further research has shown that the polyglycine extension at the N-terminus of eRF3a is associated with increased susceptibility to breast and gastric cancer.

[0005] Lenalidomide and pomalidomide, among other drugs, have been marketed for many years and have been proven to be degraders of IKZF1 / 3 (important transcription factors in the growth of multiple myeloma tumor cells). Lenalidomide is a ligand for the ubiquitin E3 ligase cereblon (CRBN), and can selectively ubiquitinate and degrade two lymphocyte transcription factors, IKZF1 and IKZF3, via the CRBN-CRL4 ubiquitin ligase. The basic principle of immunomodulatory drugs is to inhibit the growth of blood cells by inhibiting or degrading these two transcription factors, IKZF1 and IKZF3. Inhibition of IKZF1 can treat peripheral T-cell lymphoma, mantle cell lymphoma, and diffuse large B-cell lymphoma, while inhibition of IKZF3 can treat multiple myeloma. Currently, there are many immunomodulatory drugs on the market with similar mechanisms of action, but their clinical application carries some significant safety risks, including neutropenia, thrombocytopenia, pancytopenia, and increased risk of infection. These safety risks may be related to its high half-maximal inhibitory concentration (IC50), such as the fact that lenalidomide has an IC50 in the micromolar (μM) range for the vast majority of tumors. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, a 3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)piperidine-2,6-dione derivative, its synthesis method, and its application are provided to address the issue of high safety risks associated with commercially available treatments for malignant hematological diseases.

[0007] To achieve the above objective, a 3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)piperidine-2,6-dione derivative is provided, wherein the 3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)piperidine-2,6-dione derivative is a compound having the following general formula (I):

[0008] (I) Wherein, R1 is selected from , , , , , , , , , , , R2 is selected from , , , , , .

[0009] Furthermore, the 3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)piperidine-2,6-dione derivative is selected from one of the following compounds:

[0010] The present invention provides a pharmaceutically acceptable salt formed by the aforementioned 3-(5-(aminomethyl)-1-oxoisoindololin-2-yl)piperidine-2,6-dione derivative with at least one of acetic acid, dihydrofolate, benzoic acid, citric acid, sorbic acid, propionic acid, oxalic acid, fumaric acid, maleic acid, hydrochloric acid, malic acid, phosphoric acid, sulfurous acid, sulfuric acid, vanillic acid, tartaric acid, ascorbic acid, boric acid, lactic acid, and ethylenediaminetetraacetic acid.

[0011] This invention provides a method for synthesizing a 3-(5-(aminomethyl)-1-oxoisoindololin-2-yl)piperidine-2,6-dione derivative, the reaction route of which is as follows:

[0012] The synthesis method includes the following steps: Compound (1), compound (2) and potassium carbonate were dissolved in DMF and reacted. The reaction solution was diluted with ethyl acetate, washed with saturated brine, and the organic phase was evaporated to dryness. After purification, compound (3) was obtained. The compound (3) and DIEA were dissolved in DCM, and compound (4) was added dropwise. After the reaction was carried out, the reaction solution was diluted with DCM, washed with water, and the organic phase was dried by rotary evaporation to obtain compound (5). The compound (5) was dissolved in a mixed solvent of methanol and water, and NaOH was added to react. The methanol in the reaction solution was removed by vortexing, the solid was filtered off, and the solution was dried by vortexing to obtain compound (6). Compound (6), compound (7) and DIEA were dissolved in DMF, HBTU was added, and after the reaction, the reaction solution was diluted with ethyl acetate, washed with brine, the organic phase was evaporated to dryness, and then purified to obtain the 3-(5-(aminomethyl)-1-oxoisoindololin-2-yl)piperidine-2,6-dione derivative.

[0013] Furthermore, the molar ratio of the compound (1), the compound (2), and the potassium carbonate is 1:1~1.5:1~1.5; The molar ratio of compound (3), compound (4) and DIEA is 1:1~1.5:1~1.5; The molar ratio of the compound (5) to NaOH is 1:2~4; The molar ratio of compound (6), compound (7), DIEA and HBTU is 1:1~1.5:3~4:1~1.5.

[0014] This invention provides the use of a diketone derivative or a pharmaceutically acceptable salt thereof in the preparation of a medicament for diseases mediated by IKZF1, IKZF3, BRD4, GSPT1 and / or CK1α.

[0015] Furthermore, the disease in question is cancer.

[0016] Furthermore, the cancer is leukemia, multiple myeloma, lymphoma, breast cancer, and / or liver cancer.

[0017] This invention provides the use of a diketone derivative or a pharmaceutically acceptable salt thereof as a protein degrader for IKZF1, IKZF3, BRD4, GSPT1 and / or CK1α.

[0018] The beneficial effects of this invention lie in the fact that the 3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)piperidine-2,6-dione derivatives and their pharmaceutically acceptable salts of this invention, through biological analysis, have demonstrated that they can significantly inhibit the proliferation of leukemia, multiple myeloma, lymphoma, and other cell types at extremely low doses (nanomolar levels, approximately 1000 times lower than conventional drugs), and can effectively degrade IKZF1, IKZF3, BRD4, GSPT1, and CK1α, potentially significantly reducing toxic side effects. Therefore, further development of such compounds will be of great significance in the application of tumor treatment. Attached Figure Description

[0019] Other features, objects, and advantages of this application will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 The figure shows the effect of different concentrations of compound LNB018 on protein expression in MM1.S cells.

[0020] Figure 2 This is the 1H NMR spectrum of compound LNB03 in this embodiment.

[0021] Figure 3 The image shows the 1H NMR spectrum of compound LNB05 in this embodiment.

[0022] Figure 4 The image shows the 1H NMR spectrum of compound LNB06 in this embodiment.

[0023] Figure 5 The image shows the 1H NMR spectrum of compound LNB018 in this embodiment. Detailed Implementation

[0024] The present application will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. Furthermore, it should be noted that, for ease of description, only the parts relevant to the invention are shown in the accompanying drawings.

[0025] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0026] This invention provides a 3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)piperidine-2,6-dione derivative, wherein the 3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)piperidine-2,6-dione derivative is a compound having the following general formula (I): (1) Wherein, R1 is selected from , , , , , , , , , , , R2 is selected from , , , , , .

[0028] In this embodiment, the 3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)piperidine-2,6-dione derivative is selected from one of the following compounds: LNB001, LNB002, LNB003, LNB004, LNB005, LNB006, LNB007, LNB008, LNB009, LN2B010, LNB011, LNB012, LNB013, LNB014, LNB015, LNB016, LNB017, LNB018, LNB019 (hereinafter referred to as LNB001~019).

[0029] Specifically, the structural formula of the compound is shown below:

[0030] The present invention provides a pharmaceutically acceptable salt formed by the aforementioned 3-(5-(aminomethyl)-1-oxoisoindololin-2-yl)piperidine-2,6-dione derivative with at least one of acetic acid, dihydrofolate, benzoic acid, citric acid, sorbic acid, propionic acid, oxalic acid, fumaric acid, maleic acid, hydrochloric acid, malic acid, phosphoric acid, sulfurous acid, sulfuric acid, vanillic acid, tartaric acid, ascorbic acid, boric acid, lactic acid, and ethylenediaminetetraacetic acid.

[0031] This invention provides a method for synthesizing 3-(5-(aminomethyl)-1-oxoisoindololin-2-yl)piperidine-2,6-dione derivatives. The reaction route of the synthetic method is as follows: ; Specifically, the synthesis method includes the following steps: Step S1: Compound 1, Compound 2 and potassium carbonate were dissolved in DMF (N,N-dimethylformamide). After the reaction was completed at 80°C, the reaction solution was diluted with ethyl acetate and washed with saturated brine. The organic phase was evaporated to dryness, and the crude product was purified by column chromatography to obtain Compound 3.

[0032] The molar ratio of compound 1, compound 2 and potassium carbonate is 1:(1~1.5):(1~1.5).

[0033] Step S2: Dissolve compound 3 and DIEA in DCM, add compound 4 dropwise, stir at room temperature until the reaction is complete, dilute the reaction solution with DCM, wash twice with water, and evaporate the organic phase to obtain compound 5.

[0034] The molar ratio of compound 3, compound 4 and DIEA is 1:(1~1.5):(1~1.5).

[0035] Step S3: Dissolve compound 5 in a mixed solvent of methanol and water, add NaOH, stir at room temperature until the reaction is complete, remove the methanol from the reaction solution by vortexing, adjust the pH to 3 with hydrochloric acid, filter the solid, and evaporate to dryness to obtain compound 6.

[0036] The molar ratio of compound 5 to NaOH is 1:(2~4).

[0037] Step S4: Dissolve compound 6, compound 7 and DIEA in DMF, add HBTU, stir at room temperature until the reaction is complete, dilute the reaction solution with ethyl acetate, wash three times with brine, evaporate the organic phase to dryness, and pass it through a column to obtain the target compound.

[0038] The molar ratio of compound 6, compound 7, DIEA and HBTU is 1:(1~1.5):(3~4):(1~1.5).

[0039] In this invention, the inhibitory effects of 3-(5-(aminomethyl)-1-oxoisoindololin-2-yl)piperidine-2,6-dione derivatives on the proliferation of various cancer cells were detected by the CCK-8 assay; and these compounds can effectively degrade IKZF1, IKZF3, BRD4, GSPT1, and CK1α proteins. Therefore, this invention also provides the use of dione derivatives or pharmaceutically acceptable salts thereof in the preparation of medicaments for diseases mediated by IKZF1, IKZF3, BRD4, GSPT1, and / or CK1α.

[0040] In this embodiment, the disease is cancer. More specifically, the cancer may be leukemia, multiple myeloma, lymphoma, breast cancer, and / or liver cancer.

[0041] In this invention, we also provide the use of a diketone derivative or a pharmaceutically acceptable salt thereof as a protein degrader for IKZF1, IKZF3, BRD4, GSPT1 and / or CK1α.

[0042] In the method for synthesizing compound I of this invention, the various raw materials used in the reaction can be prepared by those skilled in the art based on existing knowledge, or can be obtained by methods known in the literature, or can be commercially available. The intermediates, raw materials, reagents, reaction conditions, etc., used in the above reaction scheme can be appropriately modified based on the existing knowledge of those skilled in the art.

[0043] To make the technical objectives, technical solutions, and beneficial effects of the present invention clearer, the technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0044] In this invention, unless otherwise stated, wherein: (i) temperature is expressed in degrees Celsius (°C), and the operation is carried out at room temperature; more specifically, room temperature refers to 20–30°C; (ii) the organic solvent is dried using a common drying method, and the solvent is evaporated using a rotary evaporator under reduced pressure, with a bath temperature not exceeding 50°C; the developing solvent and eluent are in volume ratio; (iii) the reaction process is monitored by thin-layer chromatography (TLC); and (iv) the final product exhibits satisfactory proton nuclear magnetic resonance (NMR) values. 1 H-NMR).

[0045] Example 1 The synthesis of compound LNB018, with the following structural formula: .

[0046] The name of compound LNB018 is: 4-(tert-butyl)-N-(2-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)amino)-2-oxoethyl)-N-phenylbenzamide.

[0047] The synthetic route for compound LNB018 is shown below: .

[0048] Specifically, the synthesis method of compound LNB018 is as follows: Step 1: Obtain compound 3 (ethyl phenylglycinate).

[0049] Compound 1 (5.00 g, 53.7 mmol, 1.0 eq), compound 2 (9.86 g, 59.06 mmol, 1.1 eq), and potassium carbonate (8.90 g, 64.43 mmol, 1.2 eq) were dissolved in 100 mL of DMF. The reaction mixture was stirred at 80 °C for 12 hours, and the reaction was monitored by TLC until completion. The reaction solution was diluted with 200 mL of ethyl acetate, washed three times with saturated brine (200 mL × 3), the organic phase was dried by rotary evaporation, and the crude product was purified by column chromatography (PE / EA = 20 / 1~5 / 1) to give 6.85 g of brown oily compound 3, with a yield of 71.2%. 1 H NMR (CDCl3, 400 MHz) δ: 7.22-7.18 (m, 2H), 6.77-6.74 (t, J = 4Hz, 1H), 6.62 (d, J = 8 Hz, 2H), 4.28-4.22 (m, 3H), 3.90(s, 1H), 1.33-1.28(t, J = 8 Hz, 3H).

[0050] Step 2: Obtain compound 5 (ethyl-(4-(tert-butyl)benzoyl)-N-phenylglycinate).

[0051] Compound 3 (2.00 g, 11.16 mmol, 1.0 eq) and DIEA (1.73 g, 13.39 mmol, 1.2 eq) were dissolved in 30 mL of DCM, and compound 4 (2.19 g, 11.16 mmol, 1.0 eq) was added dropwise. The reaction mixture was stirred at room temperature for 4 hours, and the reaction was monitored by TLC until completion. The reaction solution was diluted with 200 mL of DCM and washed twice with 150 mL of water each time. The organic phase was collected, dried over anhydrous sodium sulfate, and then evaporated to dryness to give 3.75 g of yellow solid compound 5, which was used directly in the next step without purification, with a yield of 98.9%. 1 H NMR (CDCl3, 400 MHz) δ: 7.29-7.22 (m, 5H), 7.18-7.14 (m,4H), 4.25 (s, 2H), 4.22 (q, J = 12 Hz, 16 Hz, 2H), 1.31-1.27 (t, J = 8 Hz, 3H), 1.23 (s, 9H).

[0052] Step 3: Obtain compound 6 (N-(4-(tert-butyl)benzoyl)-N-phenylglycine).

[0053] Compound 5 (1.00 g, 2.95 mmol, 1.0 eq) was dissolved in 20 mL of methanol and 5 mL of water. Sodium hydroxide (353.5 mg, 8.84 mmol, 3.0 eq) was added, and the mixture was stirred at room temperature for 12 hours. The reaction was monitored by TLC until completion. The methanol was evaporated to dryness, and the pH of the remaining reaction solution was adjusted to 2 with 1 M dilute hydrochloric acid. A solid precipitated out. The solid was collected by filtration and evaporated to dryness to give 820 mg of a grayish-white solid, compound 6, in 89.4% yield. 1 H NMR (DMSO-d 6 , 400 MHz) δ: 7.28-7.23(m, 4H), 7.20-7.15 (m, 5H), 4.46 (s, 2H), 1.20 (s, 9H).

[0054] Step 4. Synthesis of LNB018 (4-(tert-butyl)-N-(2-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol in-5-yl)methyl)amino)-2-oxoethyl)-N-phenylbenzamide).

[0055] Compound 6 (100 mg, 0.32 mmol, 1.0 eq), compound 7 (99.48 mg, 0.32 mmol, 1.0 eq), and DIEA (124 mg, 0.96 mmol, 3.0 eq) were dissolved in 10 mL of DMF, and HBTU (146 mg, 0.38 mmol, 1.2 eq) was added with stirring. The reaction was carried out at room temperature for 6 hours, and the reaction was monitored by TLC until completion. The reaction solution was diluted with 100 mL of ethyl acetate and washed three times with saturated brine (100 mL × 3). The organic phase was dried under reduced pressure, and the crude product was purified by column chromatography (DCM / MeOH = 60 / 1~20 / 1) to give 110 mg of brown solid compound LNB018, in 60.4% yield. 1 H NMR (DMSO-d 6 , 400 MHz) δ:10.99(s, 1H), 8.66-8.63 (t, J = 4 Hz, 1H), 7.65 (d, J = 4 Hz, 1H), 7.39-7.33(m, 2H), 7.28-7.15 (m, 6H), 5.13-5.08 (m, 1H), 4.45-4.25 (m, 6H), 2.95-2.87(m, 1H), 2.67-2.62(m, 1H), 2.42-2.33 (m, 2H), 2.01-1.98 (m, 1H), 1.20 (s,9H).

[0056] Furthermore, the synthesis methods for LNB001, LNB002, LNB003, LNB004, LNB005, LNB006, LNB007, LNB008, LNB009, LN2B010, LNB011, LNB012, LNB013, LNB014, LNB015, LNB016, LNB017, and LNB019 are the same as those in Example 1, except that compound 1 or compound 4 can be replaced with the corresponding starting materials.

[0057] Specifically, in the synthesis of LNB001, compound 1 is aniline and compound 4 is benzoyl chloride.

[0058] In the synthesis of LNB002, compound 1 is aniline and compound 4 is 4-methoxybenzoyl chloride.

[0059] In the synthesis of LNB003, compound 1 is aniline and compound 4 is 4-chlorobenzoyl chloride.

[0060] In the synthesis of LNB004, compound 1 is aniline and compound 4 is 2-chlorobenzoyl chloride.

[0061] In the synthesis of LNB005, compound 1 is aniline and compound 4 is 3-chlorobenzoyl chloride.

[0062] In the synthesis of LNB006, compound 1 is aniline and compound 4 is 4-methylbenzoyl chloride.

[0063] In the synthesis of LNB007, compound 1 is aniline and compound 4 is 2-methoxybenzoyl chloride.

[0064] In the synthesis of LNB008, compound 1 is aniline and compound 4 is 4-fluorobenzoyl chloride.

[0065] In the synthesis of LNB009, compound 1 is aniline and compound 4 is 2,4-dichlorobenzoyl chloride.

[0066] In the synthesis of LNB010, compound 1 is 3-chloroaniline and compound 4 is 4-chlorobenzoyl chloride.

[0067] In the synthesis of LNB011, compound 1 is 4-fluoroaniline and compound 4 is 4-chlorobenzoyl chloride.

[0068] In the synthesis of LNB012, compound 1 is 4-aminopyridine and compound 4 is 4-methylbenzoyl chloride.

[0069] In the synthesis of LNB013, compound 1 is 4-aminopyridine and compound 4 is benzoyl chloride.

[0070] In the synthesis of LNB014, compound 1 was isopropylamine and compound 4 was benzoyl chloride.

[0071] In the synthesis of LNB015, compound 1 is adamantane-1-amine and compound 4 is benzoyl chloride.

[0072] In the synthesis of LNB016, compound 1 is adamantane-1-amine and compound 4 is 4-methoxybenzoyl chloride.

[0073] In the synthesis of LNB017, compound 1 is aniline and compound 4 is 4-cyanobenzoyl chloride.

[0074] In the synthesis of LNB019, compound 1 was aniline and compound 4 was 4-trifluoromethylbenzoyl chloride. Example

[0075] LNB001~019 inhibits the proliferation of cells in leukemia, multiple myeloma, lymphoma, breast cancer, and liver cancer.

[0076] MM1.S, U266, MV-4-11, TF-1, MOLM-16, HL60, KG-1α, MEC-1, OCI-LY7, OCI-LY3, MDA-MB-468, Huh-7, and SMMC7721 cells in logarithmic growth phase were collected, counted, and the cell suspension concentration was adjusted to 5 × 10⁻⁶ cells / mL. 4 Compounds LNB001-019 were diluted with DMSO and added to 96-well cell culture plates at a concentration of 100 / 200 μL per well. Using DMSO as a solvent control, compounds LNB001-019 were diluted with DMSO and added to the wells to achieve final concentrations of 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 μmol / L. After culturing for 96-168 h, 10 / 20 μL of CCK-8 solvent was added to each well, and the cells were incubated at 37°C for 3 h. The OD values ​​at 450 nm were measured using a microplate reader, and the results were recorded. A cell growth curve was plotted with the compound dosage on the x-axis and the absorbance value on the y-axis. The statistical results of the half-maximal inhibitory rate (IC50 value) of compounds LNB001-019 against tumor cells are shown in Tables 1 and 2 below. Table 1. Structure of compounds LNB001~019 inhibiting tumor cells as detected by CCK-8 assay.

[0077] Table 2. CCK-8 assay results showing the half-maximal inhibitory effect of compounds LNB001~019 on tumor cells.

[0078] Based on Tables 1 and 2, compounds LNB001~019 showed good inhibitory effects on the proliferation of cells from leukemia, multiple myeloma, lymphoma, breast cancer, and liver cancer.

[0079] Example 3 Compound LNB018 was shown to degrade IKZF1, IKZF3, BRD4, GSPT1, and CK1α in MM1.S cells. This example provides a preliminary study of the antitumor mechanism of this type of compound using LNB018 as an example.

[0080] I. Cell Culture and Drug Administration.

[0081] a. Take MM1.S cells in the logarithmic growth phase and adjust their density to 1×10⁻⁶. 6 A single-cell suspension of 2 mL cells per well was seeded into a 6-well plate.

[0082] b. Incubate overnight at 37°C, then add different concentrations (to make the final concentrations 0, 10, 30, 100, and 300 nM) of LNB018, with DMSO as a negative control and 30 nM C27 as a positive control.

[0083] c. After culturing for another 16 hours, lyse the cells with RIPA lysis buffer and collect the proteins.

[0084] II. Cell collection and lysis.

[0085] a. Discard the upper culture medium and wash the cells twice with pre-cooled PBS. Add 100 μL of pre-cooled RIPA cell lysis buffer (the protease inhibitor and PMSF are added to the lysis buffer at a ratio of 1:100 and mixed well in advance) to each well.

[0086] b. Lyse on ice for 3 min, scrape the cells off with a cell scraper and collect them into a 1.5 mL EP tube; lyse on ice for 30 min, vortexing once every 6 min.

[0087] c. Centrifuge at 13000 rpm / min for 10 min at 4 ℃.

[0088] d. Transfer the cell supernatant to a new EP tube.

[0089] e. The cell supernatant is divided into two parts: 5 μL is added to a 1.5 mL EP tube for BCA protein content measurement, and then 45 μL of 1×PBS is added and mixed for later use; the remaining cell supernatant is quantified by taking 80 μL, adding 20 μL of 5×SDS Loading Buffer, mixing well, boiling in boiling water for 10 min, centrifuging, and then loading or storing in a -20℃ freezer.

[0090] f. Protein concentration determination procedure: (1) Preparation of BCA working solution: Calculate the total amount of mixed working solution of A and B required based on the number of standards and test samples. Prepare the working solution according to the volume ratio of BCA reagent A to B of 50:1, and vortex to mix well for later use.

[0091] (2) Dilute the protein standard with 1×PBS as shown in Table 3 below.

[0092] Table 3. Preparation of 1×PBS diluted protein standards

[0093] (3) Add 25 μL each of the protein standard solution and the sample supernatant diluted with PBS (10-fold dilution) to a new 96-well plate. Then add 200 μL of the pre-prepared BCA working solution to each well and mix thoroughly. Do not blow air to generate bubbles. Tightly cover the 96-well plate and incubate at 37°C for 30 min.

[0094] (4) Remove the 96-well plate and allow it to return to room temperature for 3-5 minutes. Measure the absorbance of A562 using a microplate reader and save the obtained values ​​in an Excel spreadsheet. Plot a standard curve and calculate the protein content per 1 μL of each sample for protein loading.

[0095] III. SDS-PAGE.

[0096] (1) Fix the glue plate and prepare 10% SDS-PAGE separating glue.

[0097] Prepare the separating gel according to Table 4 below: 10 mL.

[0098] Table 4. Formulation of SDS-PAGE separating gel 30% (m / v) Acrylamide 3.3 mL 1.5 M Tris-HCl (pH 8.8) buffer 2.5 mL 10% (m / v) SDS 0.1 mL 10% (m / v) APS 0.1 mL TEMED 4 μL Total 10 mL (2) Add the mixed separating gel to two gel plates respectively, up to 1.0 cm from the top, fill the gel plates with anhydrous ethanol, and let stand for 30~45 min.

[0099] (3) After the gel is separated, pour out the remaining anhydrous ethanol and use filter paper to absorb the remaining anhydrous ethanol.

[0100] (4) Prepare 5 mL of 5% concentrated gel according to Table 5 below.

[0101] Table 5. Preparation of 5% Concentrated Gel 30% (m / v) Acrylamide 830 μL 0.5 M Tris-HCl (pH 6.8) buffer 1.26 mL 10% (m / v) SDS 50 μL 10% (m / v) APS 50 μL TEMED 5 μL Total 5 mL (5) Slowly add the prepared concentrated glue to the glue plate to avoid air bubbles, insert the comb, and let it stand for 30~45 minutes.

[0102] (6) Take out the protein sample, heat it in a water bath at 100℃ for 5 min, and centrifuge it at 10000 rpm for 5 min.

[0103] (7) Fix the gel plate into the electrophoresis tank, add SDS-PAGE electrophoresis buffer, remove the comb, and add the processed protein samples into the sample well in sequence, 20 µg of protein per well.

[0104] (8) Electrophoresis at 80 V for 40 min.

[0105] (9) Change the voltage to 120 V and perform electrophoresis for about 1.5 h until bromophenol blue runs out of the colloid; IV. Western blot.

[0106] (1) After electrophoresis, the SDS-PAGE gel is rinsed once in TBST buffer, and the protein gel is soaked in transfer buffer.

[0107] (2) Moisten a layer of cotton pad in membrane transfer buffer, and place it on the transfer apparatus with tweezers. Arrange the layers in the following order: blackboard, cotton pad, filter paper, protein gel, PVDF membrane, filter paper, cotton pad, and whiteboard. Clamp the layers together and place them in the transfer apparatus. If there are air bubbles between layers, gently roll them out with a glass tube.

[0108] (3) Turn on the transfer apparatus and transfer at a constant current of 300 mA for 80 min.

[0109] (4) Place the membrane in TBST buffer and rinse 3 times, 8 min each time.

[0110] (5) Block with 20 mL of 5% BSA-TBST blocking solution at room temperature for 2 h.

[0111] (6) Add primary antibody and incubate overnight at 4°C and 60 rpm.

[0112] (7) At room temperature, shake at 60 rpm and wash the membrane three times with TBST for 10 min each time.

[0113] (8) Add secondary antibody and incubate at room temperature for 1 h.

[0114] (9) At room temperature, shake at 60 rpm and wash the membrane three times with TBST for 10 min each time.

[0115] (10) Take 1 mL each of chemiluminescent substrate solution A and solution B, and develop color at room temperature for 2 min.

[0116] (11) Use filter paper to absorb the liquid on the membrane and expose it on the machine.

[0117] V. Reagent preparation.

[0118] (1) 10% SDS: Weigh 1g of high-purity (electrophoresis grade) SDS into a 10 mL centrifuge tube, add about 8 mL of deionized water, heat to dissolve, and make up to 10 mL. Store at room temperature.

[0119] (2) 10% ammonium persulfate (AP): Weigh 1g of ammonium persulfate, add about 10mL of deionized water and stir to dissolve, then store at 4℃.

[0120] (3) 5× Electrophoresis buffer: Weigh 15.1 g of Tris, 94 g of Glycine and 5.0 g of SDS into a beaker, add 1L of double-distilled water to dissolve, store at room temperature, and dilute 5 times when needed.

[0121] (4) Transfer buffer: Weigh 5.8 g of Tris, 11.6 g of glycine and 0.75 g of SDS into a beaker, add 700 mL of double-distilled water, dissolve and bring the volume up to 800 mL, and finally add 200 mL of methanol.

[0122] (5) 1.5 mol / L Tris-HCl, 100 mL: Dissolve 18.15 g of Tris base in 80 mL of water, adjust the pH to 8.8 with 4 N HCl, and make up to 100 mL.

[0123] (6) 0.5 mol / L Tris-HCl, 1000 mL: Weigh 60.5 g Tris base, add water to 850 mL, add concentrated hydrochloric acid and stir until completely dissolved, then adjust the pH to 6.8 and add water to 1 L.

[0124] (7) TBS buffer: Weigh 8.8 g of NaCl into 800 mL of distilled water, dissolve it, add 10 mL of 1 mol / L Tris-HCl (pH 7.5), and bring the volume up to 1 L. Store at room temperature.

[0125] (8) TBST buffer: Add 500 μL of 20% Tween20 to 1L of TBS buffer to make the final concentration of Tween20 0.1%. Prepare fresh before use.

[0126] (9) Blocking buffer, antibody dilution buffer: Add 5% skim milk powder or BSA to TBST buffer, prepare fresh before use.

[0127] For details of the above test results, please refer to [link / reference]. Figure 1 ,like Figure 1 The results showed that treatment with LNB018 at 100 nM and 300 nM could effectively degrade IKZF1, IKZF3, BRD4, GSPT1 and CK1α proteins, especially the degradation of GSPT1 and BRD4, which produced significant effects at 10 nM.

[0128] In summary, the results indicate that compounds LNB001-019 can significantly inhibit the proliferation of leukemia, multiple myeloma, lymphoma, breast cancer, and liver cancer cells, and can effectively degrade IKZF1, IKZF3, BRD4, GSPT1, and CK1α proteins. Therefore, this type of drug has good anti-cancer effects and development potential.

[0129] Following the general approach to drug development (first conducting routine in vitro screening for antitumor drugs, and then conducting targeted research), the compounds of this invention can be applied to cancer treatment drugs related to abnormal cell proliferation, and can be prepared as antitumor drugs by mixing with human-acceptable salts or with pharmaceutical carriers.

[0130] The compounds LNB001~019 of this invention, through further chemical modification of the biologically active 3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)piperidine-2,6-dione core group, yield numerous compounds with even higher biological activity, expanding the wide application of such compounds in biomedicine and the prospects for drug formulation development. These compounds can significantly increase the proliferation of leukemia, multiple myeloma, lymphoma, breast cancer, and liver cancer cells at low doses (nanomolar), and can effectively degrade IKZF1, IKZF3, BRD4, GSPT1, and CK1α, showing promise for development as anti-tumor drugs.

[0131] The above description is merely a preferred embodiment of this application and an explanation of the technical principles employed. Those skilled in the art should understand that the scope of the invention involved in this application is not limited to technical solutions formed by specific combinations of the above-described technical features, but should also cover other technical solutions formed by arbitrary combinations of the above-described technical features or their equivalents without departing from the inventive concept. For example, technical solutions formed by substituting the above features with (but not limited to) technical features with similar functions disclosed in this application.

Claims

1. A 3-(5-(aminomethyl)-1-oxoisoindololin-2-yl)piperidine-2,6-dione derivative, characterized in that, The 3-(5-(aminomethyl)-1-oxoisoin Dolin-2-yl)piperidine-2,6-dione derivatives are compounds having the following general formula (I): ； （I） Wherein, R1 is selected from , , , , , , , , , , , ; R2 is selected from , , , , , .

2. The 3-(5-(aminomethyl)-1-oxoisoindololin-2-yl)piperidine-2,6-dione derivative according to claim 1, characterized in that, The 3-(5-(aminomethyl)-1-oxoisoindololin-2-yl)piperidine-2,6-dione derivative is selected from one of the following compounds: 。 3. A pharmaceutically acceptable salt, characterized in that, The product is formed by using the 3-(5-(aminomethyl)-1-oxoisoindoline-2-yl)piperidine-2,6-dione derivative as described in claim 1 or 2 with at least one of acetic acid, dihydrofolate, benzoic acid, citric acid, sorbic acid, propionic acid, oxalic acid, fumaric acid, maleic acid, hydrochloric acid, malic acid, phosphoric acid, sulfurous acid, sulfuric acid, vanillic acid, tartaric acid, ascorbic acid, boric acid, lactic acid, and ethylenediaminetetraacetic acid.

4. A method for synthesizing the 3-(5-(aminomethyl)-1-oxoisoindololin-2-yl)piperidine-2,6-dione derivative as described in any one of claims 1 to 2, characterized in that, The reaction route of the synthesis method is as follows: ； The synthesis method includes the following steps: Compound (1), compound (2) and potassium carbonate were dissolved in DMF and reacted. The reaction solution was diluted with ethyl acetate, washed with saturated brine, and the organic phase was evaporated to dryness. After purification, compound (3) was obtained. The compound (3) and DIEA were dissolved in DCM, and compound (4) was added dropwise. After the reaction, the reaction solution was diluted with DCM, washed with water, and the organic phase was dried by rotary evaporation to obtain compound (5). The compound (5) was dissolved in a mixed solvent of methanol and water, and NaOH was added to react. The methanol in the reaction solution was then removed by vortexing, the solid was filtered off, and the mixture was dried by vortexing to obtain compound (6). Compound (6), compound (7) and DIEA were dissolved in DMF, HBTU was added, and after the reaction, the reaction solution was diluted with ethyl acetate, washed with brine, the organic phase was evaporated to dryness, and then purified to obtain the 3-(5-(aminomethyl)-1-oxoisoindololin-2-yl)piperidine-2,6-dione derivative.

5. The method according to claim 4, characterized in that, The molar ratio of the compound (1), the compound (2) and the potassium carbonate is 1:1~1.5:1~1.5; The molar ratio of the compound (3), the compound (4) and the DIEA is 1:1~1.5:1~1.5; The molar ratio of the compound (5) to NaOH is 1:2~4; The molar ratio of compound (6), compound (7), DIEA and HBTU is 1:1~1.5:3~4:1~1.

5.

6. Use of the diketone derivative or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 3 in the preparation of medicaments for diseases mediated by IKZF1, IKZF3, BRD4, GSPT1 and / or CK1α.

7. The application according to claim 6, characterized in that, The disease in question is cancer.

8. The application according to claim 7, characterized in that, The cancers mentioned are leukemia, multiple myeloma, lymphoma, breast cancer, and / or liver cancer.

9. The use of the diketone derivative or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 3 as a protein degrader of IKZF1, IKZF3, BRD4, GSPT1 and / or CK1α.

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