A glutarimide GSPT1 molecular glue and a preparation method and application thereof

Abstract

The application provides a glutarimide GSPT1 molecular glue as well as a preparation method and application thereof, and belongs to the technical field of organic compound synthesis. In the application, 4-((4-nitrophenyl)ethynyl)phenol is used as a starting material, and nucleophilic substitution reaction is carried out with N-(3-chloropropyl)morpholine to obtain an intermediate A3; the intermediate A3 is subjected to a reduction reaction to obtain an intermediate A4; the intermediate A4 is subjected to a urea reaction with 3-[5-(aminomethyl)-1-oxoisoindolin-2-yl]piperidine-2,6-dione to synthesize a compound GT-1, and the molecular structure of the compound GT-1 is confirmed. The compound GT-1 can significantly degrade GSPT1, has a significant acute myeloid leukemia effect, and is an effective GSPT1 degrading agent.

Description

Technical Field

[0001] This invention relates to the field of organic compound synthesis technology, and more particularly to the field of chemical pharmaceutical raw material preparation technology, specifically to a glutarimide-based GSPT1 molecular gel, its preparation method, and its application. Background Technology

[0002] Hematologic malignancies mainly include three categories: leukemia, malignant lymphoma, and multiple myeloma. According to GLOBOCAN 2020 epidemiological data and domestic cancer statistics, non-Hodgkin's lymphoma (NHL) and leukemia are the two leading causes of death and morbidity in hematologic malignancies. In 2020, the global incidence and mortality rates for NHL were 2.8% and 2.6%, respectively, while those for leukemia were 2.5% and 3.1%, respectively. In the same year, my country reported 97,788 new cases of NHL and 88,249 new cases of leukemia. With the widespread clinical application of targeted anti-tumor drugs, the five-year survival rate for patients with hematologic malignancies has increased by more than 20% compared to half a century ago; however, the overall survival prognosis for patients with acute myeloid leukemia (AML) remains significantly low. Currently, while targeted drugs used clinically to treat non-Hodgkin's lymphoma and acute myeloid leukemia can reduce chemotherapy-related toxicities, they generally suffer from the drawback of developing drug resistance after use. Furthermore, most of the existing mainstream drugs are developed by foreign pharmaceutical companies, resulting in high drug prices and a heavy economic burden on patients. There is also a shortage of domestically developed innovative drugs of the same type, creating an urgent need for new drug development in clinical practice.

[0003] GSPT1 (G1 to S phase transition 1), formerly known as eukaryotic translation termination factor eRF3a, is a key gene regulating the cell cycle transition from G1 to S phase. As a core component of eukaryotic peptide chain releasing factors, eRF3a is widely involved in multiple physiological and biochemical processes, including protein translation termination, intracellular mRNA degradation, cell cycle regulation, apoptosis, and cytoskeleton formation. Abnormal expression and function of eRF3a are highly associated with the occurrence and progression of various hematological malignancies, such as non-Hodgkin's lymphoma and acute myeloid leukemia. Currently, the world's first GSPT1-targeted degradation agent to enter clinical development is CC, developed by Bristol-Myers Squibb (BMS, formerly Celgene). 90009 (generic name: Ergidomide; chemical name: 2-(4-chlorophenyl)-N-[[2-(2,6-dioxopiperidin-3-yl)-1-oxo-3H-isoindol-5-yl]methyl]-2,2-difluoroacetamide, 2-(4-chlorophenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide) has been advanced to Phase II clinical trials, with indications covering acute myeloid leukemia (clinical trial number NCT04336982) and myelodysplastic syndromes (clinical trial number NCT02848001). However, the lead compound has a simple core structure, and there is still considerable room for optimization of its in vivo antitumor activity against hematologic malignancies, thus limiting its clinical application. Given the aforementioned shortcomings of existing technologies, there is an urgent need to develop novel GSPT1-targeting small molecule inhibitors with novel structures and superior anti-tumor activity. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a glutarimide-based GSPT1 molecular gel, its preparation method, and its applications. This glutarimide-based GSPT1 molecular gel can significantly degrade GSPT1 and exhibits a significant anti-acute myeloid leukemia (AML) effect.

[0005] Specifically, the first objective of this invention is to provide a glutarimide-based GSPT1 molecular gel, wherein the glutarimide-based GSPT1 molecular gel is a compound represented by formula (I), or a stereoisomer of the compound represented by formula (I), or a pharmaceutically acceptable salt of the compound represented by formula (I), or a pharmaceutically acceptable salt of a stereoisomer of the compound represented by formula (I), and the structural formula of the compound represented by formula (I) is as follows: (I).

[0006] A second objective of this invention is to provide a method for preparing the glutarimide-based GSPT1 molecular adhesive, comprising the following steps: S1, Compound A1 and Compound A2 undergo a nucleophilic substitution reaction to generate intermediate A3; S2 and intermediate A3 undergo a reduction reaction in the presence of reduced iron powder to generate intermediate A4; S3, intermediate A4 and compound A5 undergo a urea-forming reaction to generate compound GT-1, which is the glutarimide-based GSPT1 molecular glue; The synthetic route for the glutarimide-based GSPT1 molecular gel is as follows: .

[0007] In the preferred embodiment, the molar ratio of compound A1 to compound A2 in step S1 is 1:(1~2).

[0008] In a further preferred embodiment, the molar ratio of compound A1 to compound A2 in step S1 is 1:1.5.

[0009] In a preferred embodiment, in step S1, compound A1 and compound A2 react in the presence of an inorganic base.

[0010] In a further preferred embodiment, the inorganic base in step S1 is at least one of potassium carbonate, sodium carbonate, and cesium carbonate.

[0011] In a preferred embodiment, in step S1, compound A1 and compound A2 react in an ether solvent.

[0012] In a further preferred embodiment, the ether solvent in step S1 is 1,4-dioxane.

[0013] In the preferred embodiment, the reaction conditions in step S1 are: reacting at 70℃~90℃ for 8~12 hours.

[0014] In the preferred embodiment, the molar ratio of intermediate A3 to reduced iron powder in step S2 is 1:(2~3).

[0015] In a further preferred embodiment, the molar ratio of intermediate A3 to reduced iron powder in step S2 is 1:2.

[0016] In the preferred embodiment, in step S2, intermediate A3 undergoes a reduction reaction in the presence of ammonium chloride.

[0017] In a preferred embodiment, intermediate A3 in step S2 reacts in a mixed solvent consisting of an alcohol solvent and water.

[0018] In a further preferred embodiment, the alcohol solvent in step S2 is ethanol.

[0019] In a further preferred embodiment, the volume ratio of ethanol to water in step S2 is 10:1.

[0020] In the preferred embodiment, the reaction conditions in step S2 are: reacting at 70℃~90℃ for 8~12 hours.

[0021] In a preferred embodiment, the molar ratio of intermediate A4 to compound A5 in step S3 is 1:(1~2).

[0022] In a further preferred embodiment, the molar ratio of intermediate A4 to compound A5 in step S3 is 1:1.

[0023] In a preferred embodiment, in step S3, intermediate A4 reacts with compound A5 in the presence of N,N'-carbonyldiimidazole (CDI).

[0024] In a preferred embodiment, intermediate A4 reacts with compound A5 in an ether solvent during step S3.

[0025] In a further preferred embodiment, the ether solvent in step S3 is 1,4-dioxane.

[0026] In the preferred embodiment, the reaction conditions in step S3 are: reacting at 70℃~90℃ for 8~12 hours.

[0027] A third objective of this invention is to provide the use of the glutarimide-based GSPT1 molecular gel in the preparation of antitumor drugs for treating GSPT1 targets.

[0028] In a preferred embodiment, the antitumor drug associated with the GSPT1 target is a GSPT1 degrader.

[0029] In a preferred embodiment, the antitumor drug targets acute myeloid leukemia.

[0030] A fourth object of the present invention is to provide a pharmaceutical composition comprising the glutarimide-based GSPT1 molecular gel.

[0031] In a preferred embodiment, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipient.

[0032] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: (1) The compound GT-1 provided by the present invention is a GSPT1 molecular glue. The compound GT-1 can significantly degrade GSPT1 protein and has excellent in vivo anti-AML tumor pharmacological effects.

[0033] (2) The method for preparing compound GT-1 in this invention has high atom utilization and generates less waste, which is conducive to environmental protection and industrial production requirements. Attached Figure Description

[0034] Figure 1 The proton NMR spectrum of compound GT-1 prepared in Example 1 of this invention; Figure 2 The HPLC chromatogram of compound GT-1 prepared in Example 1 of this invention; Figure 3 This is a Western blot result of the degradation effect of compound GT-1 on GSPT1 protein in HL-60 cells in Example 2 of the present invention. Detailed Implementation

[0035] The following description, in conjunction with embodiments, clearly and completely describes the technical solutions of this application, so that those skilled in the art can fully understand this application. Obviously, the described embodiments are merely some preferred embodiments of this application, and not all embodiments. Any equivalent modifications or substitutions made by those skilled in the art to the following embodiments without creative effort are within the protection scope of this application.

[0036] The reaction process of this invention is monitored using conventional monitoring methods (such as TLC (thin-layer chromatography), LCMS (liquid chromatography-mass spectrometry) or NMR (nuclear magnetic resonance)). The reaction endpoint is generally defined as the disappearance of the reaction substrate.

[0037] In the following specific embodiments, the high-performance liquid chromatography (HPLC) conditions used for the detection and identification of compound GT-1 were as follows: Shimadzu LC-20AD / T, DGU-20A5R degasser, CTO-20A column oven, and SPD-20A UV-Vis detector. The chromatographic column was an Xbridge C18 (50 mm × 4.6 mm, 5.0 μm), with deionized water as mobile phase A and acetonitrile containing 0.1% (v / v) trifluoroacetic acid as mobile phase B, using gradient elution. The gradient elution program was as follows: 0–1 min, 20% (v / v) mobile phase B; 1–23 min, mobile phase B linearly increased to 70% (v / v); 23–24 min, maintained at 70% mobile phase B; 24–25 min, mobile phase B linearly decreased to 20% (v / v); 25–30 min, maintained at 20% (v / v) mobile phase B. The flow rate was 0.65 mL / min, the column temperature was 30 °C, the injection volume was 5 μL, and the detection wavelength was 254 nm.

[0038] Unless otherwise stated, the following terms appearing in this specification and claims have the following meanings: The present invention also relates to available forms of the compounds disclosed herein, such as metabolites, hydrates, solvates, prodrugs, salts, especially pharmaceutically acceptable salts, and coprecipitates.

[0039] Furthermore, the compounds of the present invention exist in a free form, for example, as a free base or free acid or zwitterion, or in the form of a salt. The salt can be any pharmaceutically acceptable salt, organic or inorganic addition salt, especially any pharmaceutically acceptable organic or inorganic addition salt.

[0040] Pharmaceutically acceptable salts of the compounds of the present invention may be, for example, acid addition salts of the compounds of the present invention carrying nitrogen atoms in the chain or ring, or acid addition salts of sufficiently basic compounds of the present invention, or acid addition salts formed with inorganic acids, or acid addition salts formed with organic acids. For example, the acids that add to the compounds of the present invention are selected from hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, nitric acid, formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)-benzoic acid, camphoric acid, cinnamic acid, cyclopentanoic acid, digluconic acid, 3-hydroxy-2-naphthic acid, nicotinic acid, dihydroxynaphthic acid, pectinic acid, persulfate, 3-phenylpropionic acid, bitter acid, etc. Acids, neopentanoic acid, 2-hydroxyethanesulfonic acid, itaconic acid, aminosulfonic acid, trifluoromethanesulfonic acid, dodecyl sulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheponic acid, glycerophosphate, aspartic acid, sulfosalicylic acid, hemisulfonic acid, or thiocyanate.

[0041] The compounds of the present invention may contain non-natural proportions of isotopes on one or more atoms constituting the compound, for example, replacing hydrogen with deuterium to form deuterated drugs.

[0042] In this invention, the term "stereoisomer" refers to compounds having the same chemical composition but differing in the spatial arrangement of atoms or groups. Stereoisomers include enantiomers, diastereomers, and conformational isomers. The term "enantiomer" refers to two stereoisomers of a compound that are non-overlapping mirror images of each other. The term "diastereomer" refers to stereoisomers having two or more chiral centers whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting point, boiling point, spectral properties, or biological activity. Mixtures of diastereomers can be separated using chiral HPLC.

[0043] Those skilled in the art will understand that compounds of formula (I) may contain one or more chiral centers, and thus have two or more stereoisomers. Therefore, the compounds of the present invention may be a single stereoisomer (e.g., an enantiomer, a diastereomer) or a mixture of multiple stereoisomers in any proportion. For example, they may exist in racemic form, and, where appropriate, in the form of their tautomers or geometric isomers.

[0044] Example 1 This embodiment provides a glutarimide-based GSPT1 molecular gel, with the chemical name 1-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-3-(4-((4-(3-morpholinopropoxy)phenyl)ethynyl)phenyl)urea (hereinafter referred to as compound GT-1), and its chemical structural formula is as follows: .

[0045] The preparation method of compound GT-1 includes the following steps: S1. Compound A1 (1 g, 4.18 mmol), compound A2 (1.03 g, 6.27 mmol), potassium carbonate (866.56 mg, 6.27 mmol), and 1,4-dioxane (50 mL) solvent were added to the reaction flask. The mixture was stirred and heated to 80 °C for 10 hours, monitored by TLC (developing solvent: a mixed solvent of PE and EA in a volume ratio of 2:1). The mixture was then cooled to 25 °C, and the reaction solution was rotary evaporated until the solvent was completely evaporated. The solution was then purified by column chromatography using silica gel (200-300 mesh, Huanghai Chemical Industry Research Institute (Tianjin) Co., Ltd.), eluted with a petroleum ether (PE): ethyl acetate (EA) system (PE to EA volume ratio of 2:1) to give yellow intermediate A3 (1.5 g, yield 97.93%). LCMS: (MS-ESI, m / z): [M+H] + = 367.2.

[0046] S2. At room temperature, intermediate A3 (500 mg, 1.36 mmol), iron powder (152.41 mg, 2.73 mmol), ammonium chloride (145.98 mg, 2.73 mmol), and an ethanol / water mixture (40 mL / 4 mL) were added to a reaction flask. The mixture was stirred and heated to 80 °C for 10 hours, monitored by TLC (developing solvent: a 1:1 volume ratio of PE and EA). After the reactants had reacted completely, the mixture was cooled to 25 °C. The reaction solution was filtered with diatomaceous earth and then rotary evaporated until the solvent was completely evaporated. The solution was then purified by column chromatography using silica gel (200-300 mesh, Huanghai Chemical Industry Research Institute (Tianjin) Co., Ltd.) and eluted with a petroleum ether (PE): ethyl acetate (EA) system (PE to EA volume ratio 1:1) to give a pale yellow intermediate A4 (450 mg, yield 98.02%). LCMS: (MS-ESI, m / z): [M+H] + =337.2.

[0047] S3. Take intermediate A4 (100 mg, 297.23 µmol), compound A5 (81.23 mg, 297.23 µmol), and CDI (53.02 mg, 326.96 µmol) obtained in step S2 and add them to a 100 mL round-bottom flask. The reaction solvent is 1,4-dioxane (20 mL). Stir and heat to 80 °C for 10 hours. After the reaction is complete, the solvent is removed by vacuum distillation. Column chromatography is performed using silica gel (200-300 mesh, Huanghai Chemical Industry Research Institute (Tianjin) Co., Ltd.). Elution is performed using a dichloromethane (DCM):methanol (MeOH) system (DCM:MeOH volume ratio 20:1) to obtain compound GT-1 (180 mg, yield 95.26%, HPLC purity 97.517%, HPLC chromatogram shown in [reference needed]). Figure 2 ).

[0048] The main reaction route of the above preparation method is as follows: .

[0049] The proton NMR spectrum of compound GT-1 is shown below. Figure 1 The detection and characterization results are as follows: 1 H NMR (400 MHz, d 6-DMSO) δ 10.99 (s, 1H), 8.89 (s, 1H), 7.71 (d, J = 7.0 Hz, 1H), 7.53 (s, 1H), 7.44 (m, 4H), 7.38 (d, J = 7.5 Hz, 2H), 6.96 (d, J = 7.8 Hz, 2H), 6.86 (s, 1H), 5.11 (d, J = 8.7 Hz, 1H), 4.46 (d, J = 16.4 Hz, 3H), 4.32 (d, J = 17.0 Hz, 1H), 4.04 (s, 2H), 3.58 (s, 4H), 2.90 (m, 1H), 2.61 (m, 2H), 2.40 (m, 7H), 2.02(s, 1H), 1.88 (s, 2H). HRMS m / z: calcd for C 36 H 38 N5O6[M+H] + 636.2822, found636.2825.

[0050] Example 2 This example investigated the degradation effect of compound GT-1 on GSPT1 and its cytotoxicity to AML cells in Example 1.

[0051] I. Degradation of GSPT1 protein (Western blot method) 1.1 Cell Culture: HL-60 cells (purchased from Wuhan Pronosai Life Science Technology Co., Ltd.) were cultured in RPMI 1640 (purchased from BIOSPECIES / Xinyuan, catalog number: Bios-P4533) + 10% FBS (fetal bovine serum, purchased from Gibco, catalog number: 10099-141) + 1% penicillin. Streptomycin solution was used, and cells were cultured at 37°C and 5% CO2.

[0052] 1.2 Plating: One day in advance, add 2 mL of 1×10⁻⁶ solution to each well of a 6-well plate. 6 Cells were cultured at a density of 2 × 10⁶ / mL overnight, and the cell density reached 2 × 10⁶ / mL the next day. 6 At a concentration of 1 μM / mL, solutions of the test compound (prepared with RPMI 1640 medium) at different final concentrations (1 μM, 0.33 μM, 0.11 μM, 0.037 μM, 0.0123 μM, 0.00411 μM, 0.00137 μM, 0.000457 μM) were added to each well and incubated at 37°C in a 5% CO2 incubator for 24 h. The positive control group was CC-90009 (purchased from Shanghai Bied Pharmaceutical Technology Co., Ltd., catalog number: BD01290362). The negative control group consisted of RPMI 1640 medium without the test compound.

[0053] 1.3 Extraction of Cell Proteins (1) Mix the RIPA lysis buffer (purchased from Solarbio, catalog number R0010) and the protease inhibitor (purchased from Solarbio, catalog number A8260) in the proportion recommended in the reagent instructions.

[0054] (2) Centrifuge the cells and discard the culture medium. Resuspend the cells in PBS buffer (pH 7.2~7.4, 0.01M, cell culture), centrifuge again, and retain the precipitate.

[0055] (3) Add RIPA lysis buffer to each well according to the recommended dosage in the reagent instructions, based on the cell volume, and transfer it to a 1.5 mL EP tube after pipetting.

[0056] (4) Place the 1.5 mL EP tube on ice for 10 min, centrifuge at 12000 g for 10 min at 4 °C, and transfer the protein supernatant to a new 1.5 mL EP tube.

[0057] 1.4 Bradford assay for total protein concentration (1) Preparation of standard curve: Take BSA standard (purchased from Solarbio, catalog number PC0001), and use a dilution solvent (RPMI 1640 medium) that is completely consistent with the supernatant of the cells to be tested to prepare a series of BSA standard solutions with final concentrations of 0 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL, 750 μg / mL and 1000 μg / mL, and set up blank zero concentration wells.

[0058] (2) Dilute the supernatant protein 20 times, and take 3 μL of diluent + 100 μL of Bradford into a new tube.

[0059] (3) After mixing, take 90 μL into a 96-well plate and let it stand for 5 min.

[0060] (4) The absorbance was measured using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 595 nm.

[0061] (5) Concentration calculation: The concentration of BSA standard is plotted on the x-axis, corresponding to the OD value. 595nm The absorbance was used as the ordinate to fit a linear standard curve. The protein concentration after dilution was calculated based on the absorbance of the sample to be tested, and finally multiplied by a dilution factor of 20 to obtain the total protein concentration of the original protein supernatant.

[0062] 1.5 SDS-PAGE (sodium dodecyl sulfate-polyacrylamide) gel electrophoresis (1) Add SDS loading buffer (purchased from Solarbio, catalog number P1040) to the protein sample and denature at 95°C for 5 min.

[0063] (2) Carefully remove the comb from the gel, install the glass plate filled with SDS-PAGE gel (purchased from Solarbio, product number P1321) on the electrophoresis rack with the short plate facing inward, and then place it in the electrophoresis tank. Pour 1× Tris-Gly electrophoresis buffer that has been pre-cooled overnight at 4°C into the electrophoresis tank.

[0064] (3) Add the same concentration of protein sample to each lane, add 3 μL of protein marker (purchased from Solarbio, catalog number PR1910) to both sides, and add an equal volume of 1× SDS loading buffer to lanes without samples. The sample loading volume for each well is 15–20 μL.

[0065] (4) Cover the electrophoresis tank, turn on the power, and perform electrophoresis at a constant voltage of 80V for 20-30 minutes. After the protein sample is pressed into a line, increase the voltage to 120V for about 40-60 minutes and continue electrophoresis until the bromophenol blue reaches the bottom of the separating gel.

[0066] 1.6 Transfer of film (1) Prepare sponge, filter paper, and PVDF membrane. Pour 1× transfer buffer (purchased from Solarbio, catalog number D1060) that has been pre-cooled overnight at 4°C into enamel pans and methanol into 0.45μm PVDF (polyvinylidene fluoride) membrane into methanol for 10 min to activate it. Soak sponge and filter paper in transfer solution.

[0067] (2) Remove the glass plate from the electrophoresis tank, separate the two glass plates, cut off the stacking gel, cut off the excess separating gel according to the size of the target protein, and place it in the transfer buffer.

[0068] (3) Place the PVDF membrane in the transfer fixer in the order of black gel and white membrane (gel at the negative electrode, membrane at the positive electrode). After each layer is placed, gently remove any air bubbles and mark the PVDF membrane. Clamp the membrane and place it in the transfer electrophoresis tank with the black sides facing each other. Pour in the transfer buffer and close the lid.

[0069] (4) Place the transfer tank into a foam box filled with crushed ice. Turn on the power and transfer at a constant current of 260~280mA for 60 minutes.

[0070] 1.7 Immunostaining 1.7.1, Closed (1) Prepare 5% skim milk powder (5g skim milk powder, add 1×TBST buffer to dissolve fully, adjust the volume to 100mL, prepare 5% (w / v) skim milk powder blocking solution), mix well and pour into the incubation box.

[0071] (2) After the transfer is completed, take out the PVDF membrane, rinse it once with TBST buffer (purchased from Solarbio, catalog number T1081) to remove salt, put it into an incubation box containing 5% skim milk powder, and shake it slowly on a shaker for 1 hour.

[0072] 1.7.2 Target protein and primary antibody reaction (GSPT1): (1) Preparation of primary antibody reaction solution: Prepare a 15mL centrifuge tube, add 3mL of primary antibody diluent, and add the primary antibody (Anti-eRF3 / GSPT1 antibody, purchased from Abcam, catalog number AB234433) according to the dilution ratio indicated in the antibody instructions. Invert and mix well, and place on ice. The internal control antibody GAPDH was purchased from Solarbio, catalog number K200057M.

[0073] (2) Transfer the PVDF membrane from the blocking solution to the TBST buffer and cut the bands according to the molecular weight of the target protein.

[0074] (3) Place the strip into a centrifuge tube containing the anti-reaction solution and incubate overnight at 4°C with shaking.

[0075] 1.7.3 Reaction between primary antibody and HRP-labeled secondary antibody: (1) Wash 3 times with 1×TBST buffer, 10 min each time.

[0076] (2) Preparation of secondary antibody (Goat anti-mouse IgG H&L (HRP), purchased from abcam, catalog number AB205719) reaction solution: Prepare a 15mL centrifuge tube, add 3mL of 5% (w / v) skim milk powder, and then add goat anti-mouse IgG-HRP (the secondary antibody is diluted at a volume ratio of 1:5000). The secondary antibody dilution buffer is uniformly 1×TBST buffer containing 5% (w / v) skim milk powder. Invert and mix well.

[0077] (3) Place the strip into a plastic box containing the secondary antibody reaction solution and incubate at room temperature with shaking for 1 hour.

[0078] (4) Exposure: (a) Wash 3 times with 1×TBST buffer, 10 min each time. (b) Add ECL Plus supersensitive luminescent solution (purchased from Solarbio, catalog number PE0010), and protect from light. (c) Place the strip in a black vinyl record, wipe off excess water, add color developer to fully cover the strip, and expose for color development.

[0079] The degradation results of compound GT-1 on GSPT1 protein at various concentrations are as follows: Figure 3 As shown, the half-maximal degradation concentration (DC) 50 See Table 1.

[0080] Table 1. Degradation test results of each compound on GSPT1 protein

[0081] As shown in Table 1, the activity of compound GT-1 in degrading GSPT1 protein was significantly higher than that of the positive control CC-90009.

[0082] II. Inhibitory activity against AML tumor cell proliferation Reagents and consumables: sterile DMSO (dimethyl sulfoxide), cell culture medium (RPMI 1640 + 10% FBS + 1% penicillin) Streptomycin solution), 96-well cell culture plates, 1.5 mL EP tubes, CCK-8 kit (purchased from Abbkine, Inc., catalog number BMU106-CN), HL-60 cells (purchased from Wuhan Pronosai Life Science Technology Co., Ltd.)

[0083] HL-60 cells were cultured in the above-mentioned cell culture medium at 37°C and 5% CO2 until the logarithmic growth phase.

[0084] Experimental steps: The test cells were seeded at a density of 5000 cells per well in 96-well cell culture plates (purchased from Wuhan Pronosai Life Science Technology Co., Ltd.) and incubated overnight at 37°C in a 5% CO2 incubator. The original culture medium was discarded, and 100 μL of serum-free basal culture medium (RPMI 1640) containing gradient concentrations of the target compound (20 μM, 10 μM, 3.3 μM, 1.1 μM, 0.37 μM, 0.12 μM, 0.04 μM, 0.01 μM, 0.004 μM, 0.001 μM, 0.0005 μM, 0.0001 μM, 0 μM) was added to each well. Incubation was continued for another 48 h. Then, 10 μL of CCK was added to each well. 8. Incubate with the chromogenic working solution (included in the CCK-8 kit) in the dark for 1 hour. Measure the absorbance of each well using a microplate reader at a detection wavelength of 490 nm. Calculate the half-maximal inhibitory concentration (IC50) of the compound using a nonlinear regression model fitted with GraphPad Prism software. 50 The cell proliferation inhibitory activity of the compounds was characterized using this method. CC-90009 was used as the positive control. The cytotoxicity test results of each compound on AML tumor cells are shown in Table 2. All the above cell experiments were performed independently in triplicate.

[0085] Table 2. Cytotoxicity test results of each compound against AML tumor cells.

[0086] As shown in Table 2, compound GT-1 can effectively inhibit the proliferation of HL-60 cells, and its effect is better than that of the clinical drug CC-90009.

[0087] III. Testing the Anti-tumor Therapeutic Effects of Compounds 3.1 Experimental Materials The tumor cell line used in this experiment was HL-60 (purchased from Wuhan Pronosei Life Sciences Co., Ltd., catalog number CL-0110). HL-60 cells were adapted for in vivo modeling in NOD-SCID mice. This cell line is a classic tumor model cell line recognized by current standards for evaluating the immunopharmacological efficacy of anti-AML tumors. The model has high stability and good reproducibility, and can objectively evaluate the in vivo anti-AML tumor effects of the test compounds.

[0088] Healthy male NOD-SCID mice aged 6-7 weeks were used as experimental animals. All experimental mice were provided by Spiford (Suzhou) Biotechnology Co., Ltd., and all experimental animals had complete quality certificates. The animal housing environment met the SPF (Specific Pathogen Free) level barrier system housing standards. The temperature, humidity, and diurnal light rhythm of the housing environment were kept constant. The mice had free access to food and water. After one week of acclimatization, formal animal modeling and drug administration experiments were carried out to ensure the stability of the animals' physiological state and to eliminate the interference of environmental and physiological stress on the drug efficacy evaluation results.

[0089] 3.2 Experimental Methods NOD-SCID mouse subcutaneous allogeneic tumor transplantation model construction: HL-60 cells in good logarithmic growth phase and with normal activity were collected and resuspended in sterile PBS buffer (0.01 mol / L, pH 7.2–7.4) to prepare homogeneous single-cell suspensions. Cell viability (>95%) and cell suspension concentration (1×10⁻⁶) were strictly controlled. 8 Cells / mL). Under aseptic conditions, cells were seeded subcutaneously in the axilla of one forelimb of each mouse, with 1 × 10⁻⁶ cells seeded per mouse. 7 Establish a NOD-SCID mouse subcutaneous solid tumor transplantation model using one mouse per mouse.

[0090] Following inoculation, NOD-SCID mice were routinely observed daily for their mental state, eating habits, activity levels, and subcutaneous tumor growth. The long and short diameters of the tumors were measured periodically using calipers, and the tumor volume was recorded in real-time according to the tumor volume calculation formula. The NOD-SCID mice were induced to complete the inoculation when the subcutaneous tumors reached a uniform size of 100 mm. 3 When the modeling of the standard interval was successful, the tumor-bearing NOD-SCID mice were randomly divided into 3 groups (n=5 per group) to ensure that there were no statistically significant differences in the average tumor volume and body weight of NOD-SCID mice in each group, thus excluding the influence of baseline differences between groups on the efficacy evaluation.

[0091] After grouping, NOD-SCID mice bearing tumors in each group were administered the corresponding test compound and control drug for intervention. Throughout the entire drug treatment experiment, changes in body weight, mental state, diet, and activity levels of the mice in each group were continuously monitored and recorded. Simultaneously, changes in tumor volume growth in each group were measured and recorded periodically. The inhibitory effect of the test compound on tumor growth and drug safety in mice were dynamically observed. The entire experiment strictly adhered to animal experimental ethics guidelines and standard operating procedures for antitumor pharmacodynamic evaluation.

[0092] 3.3 Experimental Results The compound GT-1 provided by this invention (administered intraperitoneally at a dose of 10 mg / kg once daily for 15 days, in a solvent composed of 30% (v / v) PEG-300, 5% (v / v) DMSO and 65% (v / v) saline) significantly and effectively inhibited AML tumor weight compared with the control group (administered intraperitoneally with the same volume of solvent once daily for 15 days, in a solvent composed of 30% (v / v) PEG-300, 5% (v / v) DMSO and 65% (v / v) saline), achieving a tumor growth inhibition rate (TGI) of 100% at 10 mg / kg. The in vivo antitumor activity was superior to that of the selective CC-90009 group (administered via intraperitoneal injection, 10 mg / kg, once daily for 15 days, in a solvent composed of 30% (v / v) PEG-300, 5% (v / v) DMSO and 65% (v / v) saline, with a tumor growth inhibition rate of 80.2%).

[0093] In summary, the compound GT-1 provided by this invention is a GSPT1 molecular gel with excellent anti-AML tumor pharmacological effects and clinical application translational value.

[0094] The embodiments described above are merely preferred embodiments of this application and are not intended to limit the scope of protection of this application. Various modifications and variations can be made to this application by anyone skilled in the art. Any simple equivalent changes and modifications made based on the scope of protection claimed in this application and the content of the specification should be included within the scope of protection of this application.

Claims

1. A glutarimide-based GSPT1 molecular adhesive, characterized in that, The glutarimide-based GSPT1 molecular gel is a compound represented by formula (I), or a stereoisomer of the compound represented by formula (I), or a pharmaceutically acceptable salt of the compound represented by formula (I), or a pharmaceutically acceptable salt of a stereoisomer of the compound represented by formula (I). The structural formula of the compound represented by formula (I) is as follows: （I）。 2. The method for preparing the glutarimide-based GSPT1 molecular adhesive according to claim 1, characterized in that, Includes the following steps: S1, Compound A1 and Compound A2 undergo a nucleophilic substitution reaction to generate intermediate A3; S2 and intermediate A3 undergo a reduction reaction in the presence of reduced iron powder to generate intermediate A4; S3, intermediate A4 and compound A5 undergo a urea-forming reaction to generate compound GT-1, which is the glutarimide-based GSPT1 molecular glue; The synthetic route for the glutarimide-based GSPT1 molecular gel is as follows: 。 3. The preparation method according to claim 2, characterized in that, In step S1, the molar ratio of compound A1 to compound A2 is 1:(1~2); Or / and, in step S2, the molar ratio of intermediate A3 to reduced iron powder is 1:(2~3); Or / and, in step S3, the molar ratio of intermediate A4 to compound A5 is 1:(1~2).

4. The preparation method according to claim 2, characterized in that, In step S2, intermediate A3 undergoes a reduction reaction in the presence of ammonium chloride; Or / and, in step S3, intermediate A4 reacts with compound A5 in the presence of N,N'-carbonyldiimidazole.

5. The preparation method according to claim 2, characterized in that, In step S1, compound A1 and compound A2 react in the presence of an inorganic base; Or / and, in step S1, compound A1 reacts with compound A2 in an ether solvent; Or / and, in step S2, intermediate A3 reacts in a mixed solvent consisting of an alcohol solvent and water; Or / and, in step S3, intermediate A4 reacts with compound A5 in an ether solvent.

6. The preparation method according to claim 5, characterized in that, The inorganic base in step S1 is at least one of potassium carbonate, sodium carbonate, and cesium carbonate; Or / and, the ether solvent in step S1 is 1,4-dioxane; Or / and, the alcohol solvent in step S2 is ethanol; Or / and, the ether solvent in step S3 is 1,4-dioxane.

7. The preparation method according to claim 2, characterized in that, The reaction conditions in step S1 are: reacting at 70℃~90℃ for 8~12 hours; Or / and, the reaction conditions in step S2 are: reacting at 70℃~90℃ for 8~12 hours; Or / and, the reaction conditions in step S3 are: react at 70℃~90℃ for 8~12 hours.

8. The use of the glutarimide-based GSPT1 molecular gel according to claim 1 in the preparation of antitumor drugs for treating GSPT1 targets.

9. The application according to claim 8, characterized in that, The antitumor drug targets acute myeloid leukemia.

10. A pharmaceutical composition, characterized in that, It contains the glutarimide-based GSPT1 molecular adhesive as described in claim 1.

Images