Bcr-Abl targeting molecular glue and preparation method and application thereof

Abstract

The invention belongs to the field of medicinal chemistry, and discloses a molecular glue degradation agent targeting Bcr-Abl protein as well as a preparation method and application of the molecular glue degradation agent. The structural formula of the molecular glue targeting BCR-ABL is as shown in the specification, # imgabs0 #, and R1 is hydrogen or hydroxyl; r2 satisfies R2COOH and is selected from one of (2E)-4-(4-methoxyphenyl)-4-oxo butyl-2-olefine acid, cyclopent-1-en-1-formic acid, (2E)-3-[4-(trifluoromethyl) phenyl] propan-2-olefine acid, (2E)-3-[2-(trifluoromethyl) phenyl] propan-2-olefine acid and acrylic acid. Compared with a third-generation BCR-ABL inhibitor H0 in a clinical stage, the compound H0-mGlu and the compound H1-mGlu have the advantages that the inhibitory activity (IC50) is remarkably improved (the IC50 value can be reduced by 1-2 orders of magnitude), and therefore the compound H0-mGlu and the compound H1-mGlu have good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry, and particularly relates to a molecular glue degrading agent targeting Bcr-Abl protein, and a preparation method and application thereof. Background Art

[0002] Chronic myeloid leukemia (CML) is a malignant myeloproliferative disease caused by a mutation in the gene of hematopoietic stem cells. Studies have shown that 90% to 95% of CML patients have the Philadelphia (Ph) chromosome, which is formed by the reciprocal translocation of the Abelson (ABL) tyrosine kinase gene on chromosome 9 and the Breakpoint Cluster Region (BCR) gene on chromosome 22. This translocation process can produce a BCR-ABL chimeric fusion gene, the encoded Bcr-Abl tyrosine kinase can promote uncontrolled cell proliferation and reduce the adhesion of CML cells to the bone marrow matrix. In addition, studies have also found that the Bcr-Abl fusion protein is also expressed in 30% to 35% of acute lymphoblastic leukemia (ALL) patients, indicating its key role in promoting the pathological process of CML and ALL, and has great potential in the treatment of CML and ALL.

[0003] Before the last century, CML was considered an "incurable disease", but in the past 20 years, the treatment of this disease has undergone a subversive change. Since the launch of the first-generation targeted drug "imatinib", researchers have developed a series of tyrosine kinase inhibitors (TKIs) targeting BCR-ABL kinase, which can be roughly divided into four generations of drugs (Table 1). The first-generation drug imatinib competitively blocks BCR-ABL kinase (binding to the ATP site), specifically inhibits the abnormal activation of leukemia-related signal transduction pathways, thereby inhibiting cancer cell proliferation and greatly reducing toxicity to normal cells. Although imatinib can effectively treat CML, it also produces drug-resistant mutations, resulting in relapses in about 50-90% of patients after clinical treatment. The most common mutations are T315I, E225K, Y253F, and M351T, accounting for about 60% of all imatinib resistance-related mutations. T315I is a gatekeeper mutant of the ATP binding site, which accounts for 15% to 20% of all clinically observed mutants. Second-generation drugs such as dasatinib have been launched, but they are ineffective against T315I mutations. The third-generation TKIs ponatinib and orebactinib provide solutions to overcome the T315I mutation. The first three generations of TKIs are ATP-competitive inhibitors, which will lead to inevitable mutations and drug resistance and hinder the binding of drugs to proteins. The fourth-generation inhibitors are designed to bind to the myristoyl pocket of the Bcr-Abl allosteric site, but have been shown to induce new myristoyl site mutations.

[0004] In general, the above-listed drugs can achieve therapeutic effects in the short term and prolong the survival of patients to a great extent, but various mutations caused by long-term use lead to drug resistance. Some drugs have also exposed serious cardiovascular and liver toxicity problems in clinical applications and have been issued black box warnings by the US FDA. Therefore, there is still an urgent need to develop new treatment strategies and drugs for the Bcr-Abl-dependent drug resistance caused by Bcr-Abl point mutations and overexpression.

[0005] With the emergence of targeted drugs, cancer treatment has ushered in a new era of precision treatment. However, traditional small molecule inhibitors rely on the "occupancy-driven" model, and about 80% to 85% of proteins do not have binding pockets for small molecule drugs. In addition, maintaining effective drug concentrations may lead to toxic accumulation and drug-resistant mutations. Targeted protein degradation (TPD) technology, which completely eliminates pathological proteins, provides a new strategy for treatment and can effectively avoid the occurrence of drug resistance.

[0006] Protein homeostasis is the basis for the normal life activities of cells. When a certain link is out of balance, the balance of protein homeostasis is broken, which may cause a series of diseases, including cardiovascular disease, cancer, neurodegeneration and viral infection. In order to restore the imbalanced protein homeostasis, the use of endogenous cell degradation pathways to remove pathogenic proteins has become a treatment method that has emerged in recent years.

[0007] Targeted protein degradation technology (TPD) is based on the proteasome and lysosome degradation mechanism in cells, and can achieve specific and efficient removal of target proteins. According to different working principles, TPD can be divided into two categories. One is the targeted degradation technology that relies on the UPS pathway, including PROTAC (Proteolysis targeting chimeras), molecular glue, and hydrophobic tag (HyT); the other is the lysosome-targeted chimera (LYTAC), autophagy-targeted chimera (AUTAC), and autophagosome-binding compounds (ATTEC) that utilize the lysosomal degradation pathway. Undoubtedly, the rise of TPD has opened up a new direction in the field of targeted therapy.

[0008] The advantage of targeted protein degradation technology is that, compared with protein inhibitors that need to bind to active sites to inhibit proteins, TPD destroys all protein functions by recruiting targets to participate in multiple protein degradation processes, thereby reducing drug resistance and increasing the possibility of degrading "undruggable" proteins such as non-enzyme proteins. In addition, TPD has a reduced dependence on high-affinity ligand binding, making it easier to discover candidate drugs with low nanomolar potency.

[0009] 1. PROTAC As the main protein degradation pathway in cells, the ubiquitin-proteasome degradation pathway (UPS) plays a decisive role in maintaining protein homeostasis and regulating intracellular metabolism, and can specifically degrade more than 80% of proteins in cells. PROTAC is one of the targeted degradation technologies of UPS, which consists of target protein ligands, E3 ligase ligands and linkers. When PROTAC enters the cell, a "target protein-PROTAC-E3 ligase" ternary complex is quickly formed. In this complex, the E3 ligase can mediate the ubiquitination labeling of the target protein by the ubiquitin-binding enzyme E2, and then the labeled target protein is recognized and degraded by the proteasome.

[0010] At present, researchers have developed various types of Bcr-Abl PROTACs. For example, Nagar and Lai's teams developed corresponding PROTACs based on the first-generation Bcr-Abl inhibitor imatinib and the second-generation inhibitors dasatinib and bosutinib, respectively, and successfully achieved the degradation of Bcr-Abl protein. Crews' team, Jiang's team, Rao's team and Lu's team have solved the drug resistance dilemma caused by Bcr-Abl point mutations to a certain extent by developing diverse PROTACs, providing effective synthetic strategies and drugs for the treatment of CML and its drug resistance.

[0011] 2. Molecular glue The concept of "molecular glue" was first proposed in the 1990s. It describes a class of small molecule compounds that can induce or stabilize protein-protein interactions (PPI). The immunosuppressants cyclosporine A (CsA) and FK506 were the first molecular glues reported. As the research on the mechanism of action of CsA and FK506 has gradually deepened, this new mechanism of chemically induced protein binding has gradually become an effective strategy for exploring biological mechanisms and drug development. In particular, the use of molecular glues to induce protein degradation seems to be an effective means of treating diseases. Therefore, the concept of molecular glue degraders has gradually taken shape. They regulate protein ubiquitination by inducing or stabilizing PPIs between ubiquitin ligases and target proteins, and rely on UPS to degrade target proteins.

[0012] PROTAC and molecular glue are the two most mature types of TPD currently in terms of widespread research popularity, number of companies and pipelines, and progress of clinical trials.

[0013] In PROTAC, two ligands are connected by a linker with a molecular weight between 700-1100 Da. Its flexible linker can bend and twist, allowing the two proteins to establish a connection. However, the current problems with this technology are that due to its large molecular weight, poor solubility and cell permeability, it cannot be designed as an oral drug, has low bioavailability, and has off-target problems. These factors have limited the clinical transformation of PROTAC drugs to a certain extent.

[0014] The difference is that molecular glues are monovalent small molecules (smaller molecular weight than PROTACs) that reshape the surface of E3 ligase receptors and promote new protein-protein interactions (PPIs). Molecular glues can more directly enhance the formation of complexes between E3 ligases and target proteins by squeezing into the protein-protein interface. Molecular glues are generally defined as small molecules that interact with the surfaces of two proteins to induce or enhance the affinity between the two proteins. The physicochemical properties of molecular glues are more similar to traditional small molecule drugs and generally follow the "five principles of drug development". In theory, they have better drug properties, such as lower molecular weight, higher oral bioavailability, and good pharmacokinetic (PK) and pharmacodynamic (PD) performance.

[0015] Currently, there are many mature design and optimization strategies that can be applied to the research and development of PROTACs, but the discovery of molecular glues still mainly relies on accidental discoveries, and lacks unified discovery principles and systematic evaluation methods. Summary of the invention

[0016] In view of the pain point that the first four generations of BCR-ABL small molecule inhibitors in the clinic all have drug-resistant mutations and thus cannot effectively treat CML, the primary purpose of the present invention is to provide a molecular glue targeting BCR-ABL. In view of the fact that the current molecular glue protein degradation technology is mostly discovered by chance and lacks reasonable design, the present invention adopts a "modular design strategy" to develop multiple BCR-ABL molecular glues, which are composed of a molecular glue handle targeting RNF126 E3 ligase and a BCR-ABL inhibitor H0 in the clinical stage.

[0017] Another object of the present invention is to provide a method for preparing the above-mentioned molecular glue targeting BCR-ABL.

[0018] Another object of the present invention is to provide the application of the above-mentioned molecular glue targeting BCR-ABL.

[0019] The purpose of the present invention is achieved through the following solutions: A molecular glue targeting BCR-ABL has a general formula as shown in the following formula (I): Formula (I) In the formula, R1 is hydrogen or hydroxyl; R2 satisfies R2COOH and is selected from one of (2E)-4-(4-methoxyphenyl)-4-oxyidenebut-2-enoic acid, cyclopent-1-ene-1-carboxylic acid, (2E)-3-[4-(trifluoromethyl)phenyl]prop-2-enoic acid, (2E)-3-[2-(trifluoromethyl)phenyl]prop-2-enoic acid, and acrylic acid.

[0020] Preferably, the BCR-ABL targeting molecular glue has one of the structures shown below: More preferably, the BCR-ABL targeting molecular glue has one of the structures shown below: A method for preparing the above-mentioned BCR-ABL targeting molecular glue comprises the following steps: When R1 is H, it comprises the following steps: (1) 6-bromobenzo[d]thiazol-2-amine and 2-methoxyphenylboronic acid pinacol ester undergo Suzuki coupling reaction to obtain compound 1; (2) Activating the amino group of compound 1 with carbonyldiimidazole to generate an intermediate, and then adding N-Boc-piperazine to further react to obtain compound 2 through a one-pot method; (3) Compound 2 is deprotected by the tert-butyloxycarbonyl group by trifluoroacetic acid, and then reacts with R2COOH to undergo an amide condensation reaction to obtain a targeted BCR-ABL molecular glue as shown in formula (I); The reaction route is as follows: When R1 is hydroxyl, it comprises the following steps: (4) 6-Bromobenzo[d]thiazol-2-amine was first activated with carbonyldiimidazole to obtain an intermediate, and then N-Boc-piperazine was added to further react to obtain compound 3 through a one-pot method; (5) Compound 3 undergoes a Suzuki coupling reaction with 3-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol to obtain compound 4; (6) Compound 4 is deprotected by the tert-butyloxycarbonyl group by trifluoroacetic acid, and then reacts with R2COOH to undergo an amide condensation reaction to obtain a targeted BCR-ABL molecular glue as shown in formula (I); The reaction route is as follows: The Suzuki coupling reaction in step (1) is carried out at 100-120°C for 4-6 hours; The activation in step (2) refers to reacting at room temperature for 8-12 hours under the protection of inert gas or nitrogen, and the reaction solvent is at least one of DMF and DCM; the amount of carbonyldiimidazole used in the activation process is 1.5-3 times, preferably 2 times, the molar amount of the compound; The one-pot method described in step (2) refers to the reaction of the intermediate product with N-Boc-piperazine in the presence of a base at 60-80° C. for 2-5 hours, wherein the base is preferably triethylamine.

[0021] The removal of the tert-butyloxycarbonyl group in step (3) is to add compound 2 to a mixed solution of dichloromethane and trifluoroacetic acid, and react at room temperature for 1-2.5 h, wherein the volume ratio of trifluoroacetic acid to dichloromethane is 1:(2-3); The amide condensation reaction described in step (3) refers to the reaction at room temperature-60°C for 5-12 hours in the presence of a condensing agent and a base. The condensing agent is preferably 2-(7-azobenzotriazole)-tetramethyluronium hexafluorophosphate (HATU), and its amount is 1.1-2.5 times, preferably 1.2 times, the molar amount of the product after the tert-butyloxycarbonyl protection of compound 2 is removed by trifluoroacetic acid; the base is preferably at least one of triethylamine and N,N-diisopropylethylamine, and the amount of the base is 2-4 times, preferably 2 times, the molar amount of the product after the tert-butyloxycarbonyl protection of compound 2 is removed by trifluoroacetic acid.

[0022] The activation in step (4) refers to reacting at room temperature for 8-10 hours under the protection of inert gas or nitrogen, and the reaction solvent is at least one of DMF and DCM; the amount of carbonyldiimidazole used in the activation process is 1.5-3 times the molar amount of 6-bromobenzo[d]thiazol-2-amine, preferably 2 times; The one-pot method described in step (4) refers to the reaction of the intermediate product with N-Boc-piperazine in the presence of a base at 60-80° C. for 2-4 hours, wherein the base is preferably triethylamine.

[0023] The Suzuki coupling reaction described in step (5) is carried out at 100-120° C. for 5-7 hours.

[0024] The removal of the tert-butyloxycarbonyl group in step (6) is to add compound 4 to a mixed solution of dichloromethane and trifluoroacetic acid, and react at room temperature for 1-2.5 hours, wherein the volume ratio of trifluoroacetic acid to dichloromethane is 1:2-3; The amide condensation reaction described in step (6) refers to the reaction at room temperature-60°C for 5-12 hours in the presence of a condensing agent and a base; wherein the condensing agent is preferably 2-(7-azobenzotriazole)-tetramethyluronium hexafluorophosphate (HATU), and its amount is 1.1-2.5 times, preferably 1.2 times, the molar amount of the product after the tert-butyloxycarbonyl protection of compound 4 is removed by trifluoroacetic acid; the base is preferably at least one of triethylamine and N,N-diisopropylethylamine, and the amount of the base is 2-4 times, preferably 2 times, the molar amount of the product after the tert-butyloxycarbonyl protection of compound 4 is removed by trifluoroacetic acid.

[0025] The specific reaction routes of H0-mGlu, H0-mGlu1, H0-mGlu2, H0-mGlu3, H0-mGlu4, and H1-mGlu are as follows: Application of the above-mentioned targeted BCR-ABL molecular glue in the preparation of anti-chronic myeloid leukemia drugs.

[0026] Compared with the prior art, the present invention has the following advantages and beneficial effects: Compared with the third-generation BCR-ABL inhibitor H0 in clinical stage, the inhibitory activity of compound H0-mGlu and H1-mGlu (IC 50 ) has a very significant improvement (IC 50 The value can be reduced by 1-2 orders of magnitude), so it has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 2 is a graph showing the degradation ability of different Bcr-Abl molecular glues of the present invention on Bcr-Abl and Abl proteins in K562 cells. In the graph, labels A and B are the degradation and half degradation rate DC of Bcr-Abl and Abl proteins in K562 cells by H0-mGlu. 50 ; C and D are the degradation and half degradation rate of Bcr-Abl and Abl proteins in K562 cells by H1-mGlu. 50 ; E, F, G, and H show the degradation of Bcr-Abl and Abl proteins in K562 cells by H0-mGlu1 / 2 / 3 / 4.

[0028] Figure 2 The figure is the result of the time-dependent degradation ability of different Bcr-Abl molecular glues of the present invention on Bcr-Abl protein, wherein A is the protein development band of the H0-mGlu and H1-mGlu degradation experiments, and B is the quantification of the expression amount of Bcr-Abl protein.

[0029] Figure 3 This is the verification of the degradation mechanism of Bcr-Abl protein by the Bcr-Abl molecular glue H1-mGlu of the present invention.

[0030] Figure 4 It is a verification of the in vitro reactivity of all compounds of the present invention.

[0031] Figure 5 The cytotoxicity of all compounds of the present invention on CML cells (IC 50In the figure, label A is the anti-proliferation curve of Bcr-Abl molecular glue on K562 cells; B and C are the anti-proliferation curves of Bcr-Abl molecular glue on BaF3 cells transfected with Bcr-Abl WT and Bcr-AblT315I.

[0032] Figure 6 This is the result of the effects of all compounds of the present invention on the Bcr-Abl signaling pathway.

[0033] Figure 7 This is the result of the effects of all the compounds of the present invention on the apoptosis of K562 cells.

[0034] Figure 8 This is the result of the effects of all the compounds of the present invention on the apoptosis signaling pathway of K562 cells. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below in conjunction with the examples and drawings, but the embodiments of the present invention are not limited thereto. If the specific conditions are not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are followed. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially. The cells used include K562 (ATCC No.: CCL-243) and BaF3 cells (ATCC No.: HB-283). The eq in the examples refers to molar equivalent.

[0036] Unless otherwise specified, all compounds of the present invention include all optical isomers or tautomeric forms.

[0037] Example 1 Preparation of N-[6-(2-methoxyphenyl)benzo[d][1,3]thiazol-2-yl]-4-[(2E)-4-(4-methoxyphenyl)-1,4-dioxybut-2-enyl]piperazine-1-carboxamide 1.1 Synthesis of compound 1 A mixture of 6-bromobenzo[d]thiazol-2-amine (1 g, 1 eq), 2-methoxyphenylboronic acid pinacol ester (1.43 g, 1.4 eq), Pd(dppf)Cl2·CH2Cl2 (354.22 mg, 0.1 eq), and K2CO3 (1.81 g, 3 eq) was dissolved in 6 mL of 1,4-dioxane and 2 mL of H2O, then stirred in a sealed tube at 120 °C for 5 h, and the formation of the product was confirmed by TLC and LC-MS.

[0038] After evaporation of the solvent, the crude product was extracted between EA (3 × 30 mL) and water, and the combined organic extracts were washed with saturated NaCl solution, dried over Na2SO4, and distilled under reduced pressure. The crude product was purified by column chromatography (EA / hexane 0 - 33%) to give compound 1 (1.05 g, 93%) as a white solid.

[0039] LC-MS: m / z 257.1 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 7.74 (s, 1H), 7.50 (s, 2H), 7.35-7.28 (m, 4H), 7.09 (d, J = 8.0 Hz, 1H), 7.01 (t, J = 8.0 Hz, 1H), 3.76 (s, 3H). 13 CNMR (101 MHz, CDCl3) δ 165.86, 155.45, 150.86, 132.96, 131.49, 130.95, 130.3, 128.52, 127.83, 121.84, 120.90, 118.70, 111.26, 55.61. 1.2 Synthesis of compound 2 Compound 1 (6-(2-methoxyphenyl)benzo[d]thiazol-2-amine, 1 g, 3.90 mmol, 1 eq) and carbonyldiimidazole (1.27 g, 7.80 mmol, 2 eq) were dissolved in anhydrous DMF and stirred at room temperature for 8 h under N2 protection, followed by addition of N-Boc-piperazine (1.46 g, 7.82 mmol) and triethylamine (1.19 g, 3 eq) and stirring in a sealed tube at 80 °C for 2 h.

[0040] After the reaction was completed, it was concentrated in vacuo. The combined organic extracts were washed with saturated NaCl solution, dried over Na2SO4, filtered, and distilled under reduced pressure. The crude product was purified by column chromatography (EA / hexane 0-25%) to obtain the target compound 2 (1.60 g, yield 87%) as a white solid.

[0041] LC-MS: m / z 468.2 [M+H] + . 1H NMR (400 MHz, CDCl3) δ 7.87 (s, 1H), 7.55 (s, 2H), 7.36-7.26 (m, 2H), 7.04 (t, J = 8.0 Hz, 1H), 7.00 (d, J = 8.0 Hz, 1H), 3.83 (s, 3H), 3.61 (s, 4H), 3.49 (s, 4H), 1.48 (s, 9H). 13 C NMR (101 MHz, DMSO) δ 156.40, 154.57, 134.03, 130.98, 129.94, 128.78, 128.53, 128.07, 122.48, 121.88, 120.94, 120.89, 111.23, 80.41, 55.60, 43.83, 28.39, 22.62, 11.44. 1.3 Synthesis of target product H0-mGlu A mixture of (2E)-4-(4-methoxyphenyl)-4-oxyylidenebut-2-enoic acid (CAS No. 5711-41-1, Bidex Pharmaceuticals) (1.1 eq) and 2-(7-azobenzotriazole)-tetramethyluronium hexafluorophosphate (HATU) (1.2 eq) was dissolved in N,N-dimethylformamide (DMF) (the concentration of (2E)-4-(4-methoxyphenyl)-4-oxyylidenebut-2-enoic acid in the solution was 0.1 M), then triethylamine (TEA) (2 eq) was added, and the reaction mixture was stirred at room temperature for 30 min. The amine (1.0 eq) obtained after deprotection of compound 2 (specific steps: adding compound 2 to a mixed solution of dichloromethane and trifluoroacetic acid, reacting at room temperature for 1 h, wherein the volume ratio of trifluoroacetic acid to dichloromethane is 1:3) was dissolved in DMF (0.1 M), then added dropwise to the reaction mixture, and stirred at room temperature overnight.

[0042] The reactant was diluted with ethyl acetate (EA), washed three times with saturated NaHCO3 solution, saturated NH4Cl solution and saturated NaCl solution, then dried over anhydrous Na2SO4, filtered and distilled under reduced pressure. The crude product was purified by silica gel chromatography (MeOH / DCM = 0%-5%) to obtain yellow powder H0-mGlu (60 mg, yield 79.73%).

[0043] HRMS (ESI+) calculation for C 30 H 28 N4O5S([M+H] + ): 557.1780, found 557.1870. 1H NMR (400 MHz, DMSO-d6) δ 11.43 (s, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.89 (s, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.48-7.45 (m, 3H), 7.36-7.31 (m, 3H), 7.13-7.10 (m, 3H), 7.03 (t, J = 8.0 Hz, 1H), 3.87 (s, 3H), 3.77 (s, 3H), 3.66 (d, J = 8.0 Hz, 8H). 13 C NMR (101 MHz, DMSO) δ166.07, 164.23, 156.58, 134.12, 133.31, 133.31, 133.09, 131.67, 130.99, 129.99, 129 .92, 129.22, 128.06, 122.65, 121.25, 114.80, 112.20, 56.14, 55.96, 45.92, 42.00, 8.95.

[0044] Example 2 Synthesis of 4-(cyclopent-1-enylcarbonyl)-N-[6-(2-methoxyphenyl)benzo[d][1,3]thiazol-2-yl]piperazine-1-carboxamide A mixture of cyclopent-1-ene-1-carboxylic acid (36.52 mg, 1.2 eq) and HATU (1.2 eq) was dissolved in N,N-dimethylformamide (DMF) (cyclopent-1-ene-1-carboxylic acid concentration was 0.1 M), N,N-diisopropylethylamine (DIPEA) (2 eq) was added and the reaction mixture was stirred at room temperature for 30 minutes. Compound 2 (100 mg, 271.41 μmol, 1 eq) was deprotected at room temperature (DCM:TFA = 3:1) and dissolved in DMF (0.1 M), then added dropwise, and the reaction mixture was stirred at 60°C for 5 h.

[0045] The reaction mixture was diluted with EA, washed three times with a saturated NaHCO3 solution, a saturated NH4Cl solution, and a saturated NaCl solution, then dried over Na2SO4, filtered, and distilled under reduced pressure. The crude product was purified by silica gel column chromatography (MeOH / DCM = 0%-7%) to obtain a white powder (58.0 mg, yield 46.20%).

[0046] HRMS (ESI+) calculation for C 25 H 26 N4O3S([M+H] +): 463.1726, found 463.1782. 1 H NMR (400 MHz, DMSO-d6) δ 11.74 (s, 1H), 7.88 (s, 1H), 7.51 (d, J = 8.0 Hz, 2H), 7.46 (s, 1H), 7.34-7.31 (m, 2H), 7.11 (d, J = 8.0 Hz, 1H), 7.03 (t, J =8.0 Hz, 1H), 3.77 (s, 3H), 3.57 (dd, J = 20.0, 4.0 Hz, 8H), 2.50 (m, 2H), 2.44 (t, J =8.0 Hz, 2H), 1.90-1.83 (m, 2H). 13 C NMR (101 MHz, DMSO) δ 167.66, 156.59, 138.16, 133.30, 132.74, 131.00, 130.01, 129.20, 128.04, 122.62, 121.24, 112.20, 55.98, 34.46, 33.36, 22.73. Example 3 Synthesis of N-[6-(2-methoxyphenyl)benzo[d][1,3]thiazol-2-yl]-4-[(2E)-1-oxyylidene-3-[4-(trifluoromethyl)phenyl]prop-2-enyl]piperazine-1-carboxamide A mixture of (2E)-3-[4-(trifluoromethyl)phenyl]prop-2-enoic acid (CAS No.: 5711-41-1, Bidex Pharmaceuticals) (70.40 mg, 1.2 eq) and HATU (1.2 eq) was dissolved in N,N-dimethylformamide (DMF) (the concentration of (2E)-3-[4-(trifluoromethyl)phenyl]prop-2-enoic acid was 0.1 M), N,N-diisopropylethylamine (DIPEA) (2 eq) was added and the reaction mixture was stirred at room temperature for 30 minutes. Compound 2 (100 mg, 271.41 μmol, 1 eq) was deprotected at room temperature (DCM:TFA = 3:1) and dissolved in DMF (0.1 M), then added dropwise, and the reaction mixture was stirred at 60°C for 5 h.

[0047] The reaction mixture was diluted with EA, washed three times with saturated NaHCO3 solution, saturated NH4Cl solution and saturated NaCl solution, then dried with Na2SO4, filtered and distilled under reduced pressure. The crude product was purified by silica gel column chromatography (PE / EA = 0% ~ 50%) to obtain a white powder (71.30 mg, yield 46.37%).

[0048] HRMS (ESI+) calculation for C 29 H 25 F3N4O3S([M+H] + ): 567.1599, found 567.1710. 1 HNMR (400 MHz, DMSO-d6) δ 11.40 (s, 1H), 7.99 (d, J = 8.0 Hz, 2H), 7.90 (s, 1H), 7.78 (d, J = 8.0 Hz, 1H), 7.62-7.45 (m, 4H), 7.37-7.31 (m, 2H), 7.39 (dd, J = 8.3, 1.8 Hz, 1H), 7.12 (d, J = 8.0 Hz, 1H), 7.035 (td, J = 4.0, 8.0 Hz, 1H), 3.81 (s, 2H), 3.77 (s, 3H), 3.65 (s, 6H). 13 C NMR (101 MHz, DMSO) δ 164.68, 156.59, 140.45, 139.67, 133.31, 131.00, 130.03, 129.91, 128.03, 126.07, 126.03, 121.63, 121.25, 112.20, 55.98.

[0049] Example 4 Preparation of N-[6-(2-methoxyphenyl)benzo[d][1,3]thiazol-2-yl]-4-[(2E)-1-oxyylidene-3-[2-(trifluoromethyl)phenyl]prop-2-enyl]piperazine-1-carboxamide A mixture of (2E)-3-[2-(trifluoromethyl)phenyl]prop-2-enoic acid (70.42 mg, 325.68 μmol, 1.1 eq) and HATU (1.2 eq) was dissolved in N,N-dimethylformamide (DMF) (the concentration of (2E)-3-[2-(trifluoromethyl)phenyl]prop-2-enoic acid was 0.1 M), N,N-diisopropylethylamine (DIPEA) (2 eq) was added and the reaction mixture was stirred at room temperature for 30 minutes. Compound 2 (1 eq) was deprotected at room temperature (DCM:TFA = 3:1) and dissolved in DMF (0.1 M), then added dropwise, and the reaction mixture was stirred at 60°C for 5 h.

[0050] The reaction was diluted with EA, washed three times with saturated NaHCO3 solution, saturated NH4Cl solution and saturated NaCl solution, then dried with Na2SO4, filtered and distilled under reduced pressure. The crude product was purified by silica gel column chromatography (PE / EA = 0% ~ 50%) to obtain the target compound as a white powder (68.38 mg, yield 44.47%).

[0051] HRMS (ESI+) calculation for C 29 H 25 F3N4O3S([M+H] + ): 567.1599, found 567.1687. 1 HNMR (400 MHz, DMSO-d6) δ 11.42 (s, 1H), 8.17 (d, J = 1.8 Hz, 1H), 7.84-7.78 (m, 3H), 7.75 (t, J = 8.0 Hz, 1H), 7.60 (t, J = 8.0 Hz, 1H), 7.47-7.45 (m, 2H), 7.42 (d, J = 16.0 Hz, 1H), 7.36-7.31 (m, 2H), 7.11 (dd, J = 8.4, 1.2 Hz, 1H), 7.03(dt, J = 7.2, 0.8 Hz, 1H), 3.81 (d, J = 4.0 Hz, 2H), 3.77 (s, 3H), 3.65 (s, 6H). 13 C NMR (101 MHz, DMSO) δ 164.31, 156.59, 136.65, 133.90, 133.32, 133.29, 131.00, 130.13, 130.02, 129.20, 129.04 , 128.04, 127.45, 127.16, 126.47, 126.41, 126.06, 123.34, 123.11, 121.24, 112.19, 55.97. Example 5 Synthesis of N-[6-(2-methoxyphenyl)benzo[d][1,3]thiazol-2-yl]-4-(1-oxyylideneprop-2-enyl)piperazine-1-carboxamide A mixture of acrylic acid ((23.47 mg, 1.2 eq)) and HATU (1.2 eq) was dissolved in N,N-dimethylformamide (DMF) ((2E)-3-[2-(trifluoromethyl)phenyl]prop-2-enoic acid concentration was 0.1 M), N,N-diisopropylethylamine (DIPEA) (2 eq) was added and the reaction mixture was stirred at room temperature for 30 minutes. Compound 2 (1 eq) was deprotected at room temperature (DCM:TFA = 3:1) and then dissolved in DMF (0.1 M), then added dropwise, and the reaction mixture was stirred at 60°C for 5 hours.

[0052] The reaction was diluted with EA, washed three times with saturated NaHCO3 solution, saturated NH4Cl solution and saturated NaCl solution, then dried with Na2SO4, filtered and distilled under reduced pressure. The crude product was purified by silica gel column chromatography (EA / PEA = 0%-6%) to obtain the target compound as a white powder (78.30 mg, yield 68.28%).

[0053] HRMS (ESI+) calculation for C 22 H 22 N4O3S([M+H] + ): 423.1413, found 423.1489. 1 H NMR (400 MHz, DMSO-d6) δ 11.77 (s, 1H), 7.88 (s, 1H), 7.52 (t, J = 8.0 Hz, 1H), 7.455 (dd, J = 4.0, 8.0 Hz, 1H), 7.36-7.30 (m, 2H), 7.11 (d, J = 8.0 Hz, 1H), 7.03 (t, J = 8.0 Hz, 1H), 6.84 (dd, J = 12.0, 16.0 Hz, 1H), 6.15 (dd, J = 16.4, 2 Hz, 1H), 5.71 (dd, J = 10.4, 2.4 Hz, 1H), 3.76 (s, 3H), 3.61 (d, J = 12.0 Hz, 8H). 13 C NMR (101 MHz, DMSO) δ 164.45, 156.14, 132.85, 130.55, 129.56, 128.75, 128.68, 128.18, 127.65, 127.59, 122.16, 120.80, 111.75, 55.53. Example 6 Synthesis of N-[6-(4-hydroxy-2-methoxyphenyl)benzo[d][1,3]thiazol-2-yl]-4-[(2E)-4-(4-methoxyphenyl)-1,4-dioxybut-2-enyl]piperazine-1-carboxamide 6.1 Synthesis of compound 3 6-Bromobenzo[d][1,3]thiazol-2-amine (1 g, 4.37 mmol, 1 eq) and carbonyldiimidazole (2 eq) were dissolved in anhydrous DMF and stirred at room temperature for 8 h under N2 protection. Subsequently, N-Boc-piperazine (2 eq) and triethylamine (3 eq) were added and stirred at 80 °C in a sealed tube for 2 h.

[0054] After the reaction was completed, it was concentrated in vacuo. The combined organic extracts were washed with saturated NaCl solution, dried over Na2SO4, filtered, and distilled under reduced pressure. The crude product was purified by column chromatography (EA / hexane 0 - 33%) to obtain a white solid (1.72 g, yield 89.28%).

[0055] LC-MS: m / z 441.0 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 11.65 (s, 1H), 8.09 (s, 1H), 7.50-7.47 (m, 2H), 3.54 (t, J = 4.0 Hz, 4H), 3.37-3.36 (m, 4H), 1.41 (s, 9H). 13 C NMR (101 MHz, DMSO) δ 162.71, 154.51, 129.43, 124.07, 116.20, 80.57, 43.85, 28.38. 6.2 Synthesis of compound 4 A mixture of compound 3 (tert-butyl 4-((6-bromobenzo[d]thiazol-2-yl)carbamoyl)piperazine-1-carboxylate, 580 mg, 1.31 mmol, 1 eq), 3-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (1.4 eq), Pd(dppf)Cl2·CH2Cl2 (0.1 eq), and K2CO3 (3 eq) was dissolved in 6 mL of 1,4-dioxane and 2 mL of H2O, then stirred in a sealed tube at 120°C for 5 h, and the formation of the product was confirmed by TLC and LC-MS.

[0056] After evaporation of the solvent, the crude product was extracted between EA (3 × 30 mL) and water, and the combined organic extracts were washed with saturated NaCl solution, dried over Na2SO4, and distilled under reduced pressure. The crude product was purified by column chromatography (EA / hexane 0 - 33%) to give a white solid (308 mg, 48.37% yield).

[0057] 6.3 Synthesis of the target product H1-mGlu A mixture of (2E)-4-(4-methoxyphenyl)-4-oxyylidenebut-2-enoic acid (1.1 eq) and HATU (1.2 eq) was dissolved in N,N-dimethylformamide (DMF) (2E)-4-(4-methoxyphenyl)-4-oxyylidenebut-2-enoic acid concentration was 0.1 M), and then triethylamine (TEA) (2 eq) was added, and the reaction mixture was stirred at room temperature for 30 minutes. The amine (1 eq) obtained after deprotection (DCM:TFA=3:1) of compound 4 (460 mg, 156.07 μmol) at room temperature for 1 hour was dissolved in DMF (0.1 M) and then added dropwise to the reaction mixture, which was stirred at room temperature overnight.

[0058] The reactant was diluted with EA, washed three times with a saturated NaHCO3 solution, a saturated NH4Cl solution, and a saturated NaCl solution, then dried over anhydrous Na2SO4, filtered, and distilled under reduced pressure. The crude product was purified by silica gel column chromatography (DCM / MeOH = 0%-5%) to obtain a yellow powder (44.32 mg, yield 49.59%).

[0059] HRMS (ESI+) calculation for C 30 H 28 N4O6S([M+H] + ): 573.1730, found 573.1783. 1 H NMR (400 MHz, DMSO-d6) δ 11.58 (s, 1H), 9.56 (s, 1H), 8.05 (d, J = 8.0 Hz, 2H), 7.81 (d, J = 12.0 Hz, 1H), 7.48-7.38 (m, 3H), 7.12-7.09 (m, 3H), 6.51 (s, 1H), 6.44 (dd, J = 8.0, 8.0Hz, 1H), 3.87 (s, 3H), 3.71 (s, 3H), 3.65 (d, J = 8.0 Hz, 8H), 3.24 (d, J = 5.6 Hz, 2H). 13CNMR (101 MHz, DMSO) δ 188.05, 164.22, 158.63, 157.54, 134.12, 133.71, 133.08, 131.66, 131.50, 1 29.93, 127.95, 120.89, 114.79, 107.95, 99.93, 56.12, 55.78, 45.84, 42.01. Comparative Example 1 Synthesis of H0: S.-M. Yun et al. / Cancer Letters 348 (2014) 50–60.

[0060] The structure of H0 is as follows: Comparative Example 2 Synthesis of H1 The synthesis of compound 5 was based on S.-M. Yun et al. / Cancer Letters 348 (2014) 50–60.

[0061] Synthesis of target compound H1 A mixture of compound 5 (1-(6-bromobenzo[d]thiazol-2-yl)-3-(2-hydroxyethyl)urea, 300 mg, 1 eq), 3-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (284.7 mg, 1.2 eq), XPhos Pd G2 (0.1 eq), and K3PO4 (2 eq) was dissolved in 10 mL 1,4-dioxane and 1 mL H2O, and then stirred in a sealed tube at 90 °C for 4 h. The formation of the product was confirmed by TLC and LC-MS. After evaporating the solvent, EA (3 × 30 mL) was used for extraction, and the combined organic extracts were washed with saturated NaCl solution, dried over Na2SO4, filtered, and distilled under reduced pressure. The crude product was purified by column chromatography (DCM / MeOH 0% ~ 3%) to obtain the target compound H1 (234 mg, yield 68.62%). HRMS (ESI+) calcdfor C 17 H 17 N3O4S([M+H] + ): 360.0940, found 360.1021. 1H NMR (400 MHz, DMSO-d6) δ 10.63 (s, 1H), 9.54 (s, 1H), 7.86 (d, J = 1.8 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.39 (dd, J = 8.3, 1.8 Hz, 1H), 7.12 (d, J = 8.2 Hz, 1H), 6.85 (t, J = 5.6 Hz, 1H), 6.51 (d, J = 2.3 Hz, 1H), 6.45 (dd, J = 8.2, 2.3 Hz, 1H), 4.84 (d, J = 5.2 Hz, 1H), 3.72 (s, 3H), 3.49 (d, J =5.2 Hz, 2H), 3.24 (d, J = 5.6 Hz, 2H). 13 C NMR (101 MHz, DMSO) δ 160.01, 158.57, 157.57, 133.60, 131.77, 131.53, 127.71, 121.85, 121.07, 119.32, 107.95, 99.96, 60.38, 55.79, 55.37, 42.47. Application Examples (I) Degradation effects of six molecular glues on Bcr-Abl and Abl proteins in K562 cells The reported proteomics data showed that the degraders developed based on the fumaric acid molecular glue handle can covalently target the E3 ligase RNF126, thereby ubiquitin-labeling the target protein and achieving protein degradation. Therefore, we first preliminarily verified the degradation of Bcr-Abl and Abl proteins in the K562 cell line by different molecular glues through WB. The specific steps are as follows: (1) Cell seeding: 1×10 6 Cells were seeded in 6-well plates at a number of cells / well, 2 mL of culture medium (Keygen Biotechnology) was added to each well, and the 6-well plates were placed in an incubator (37°C, 5% CO2) for overnight culture. (2) Cell administration: After incubating cells with compound doses at final concentrations of 0, 5, 25, 125, 625, and 3125 nM for different time periods, the cells were collected. 100 μL of RIPA lysis buffer containing protease / phosphatase inhibitors was added to each cell pellet. For the Bcr-Abl signaling pathway, the drug was incubated for 6 hours; for the apoptosis signaling pathway, the drug was incubated for 16 hours. (3) Protein extraction and quantification: Cells were disrupted by ultrasound, and the protein supernatant was collected by centrifugation (13500 rpm × 20 min, 4°C). The protein concentration was then quantified using the Bradford protein concentration assay kit. Finally, the loading buffer was added to the homogenized protein sample and the sample was incubated in a metal bath for 100 min. oHeat at 400 °C for 10 min. (4) Electrophoresis: Fix the prepared SDS-PAGE in the electrophoresis tank, and add the same volume and concentration of protein samples and protein markers (Biorad) to the lanes in the predetermined loading order. Turn on the electrophoresis, first perform slow electrophoresis at a constant voltage of 70 V. After the protein sample in the lane is compressed into a thin line, adjust the voltage to 110 V and continue fast electrophoresis. Using the protein marker band as an indicator, stop the electrophoresis after the area where the target band is located is completely separated from the internal reference protein area. (5) Transfer: Remove the above gel from the gel plate, and place the gel and nitrocellulose membrane (NC membrane) together in a "sandwich" transfer cassette. Place the transfer cassette in the transfer tank, turn on the power supply, and start transfer at a constant current of 300 mA. The transfer time can be appropriately adjusted according to the molecular weight. For proteins with larger molecular weights such as Bcr-Abl, the transfer time can be appropriately extended to 150 min. For proteins with smaller molecular weights such as CRKL, the transfer time can be fixed at 120 min. The entire transfer process needs to be performed under ice bath conditions. (6) Blocking: After the transfer is completed, place the NC membrane in a TBST solution containing 5% skim milk powder and incubate on a shaker at room temperature for 2 h. (7) Incubation with primary antibody: According to the protein to be detected, place the NC membrane in the prepared primary antibody solution (c-ABL antibody (2862T, Cell Signaling Technology), phospho-STAT5 (Tyr694) antibody (AF3305, Affinity Biosciences), phospho-CRKL (Tyr207) antibody (AF2332, Affinity Biosciences), β-Tubulin antibody (AF7011, Affinity Biosciences), dilute according to the instructions, and dilute with primary antibody diluent (Biyuntian)), and incubate overnight at 4°C on a shaker. (8) Incubate with secondary antibody (Qinke Biotechnology, dilute according to the instructions, use TBST containing 5% skim milk powder): Use TBST to wash away the primary antibody free on the NC membrane, and place the membrane in the prepared secondary antibody solution, and incubate on a shaker at room temperature for 1 h. (9) Chemiluminescent imaging: After using TBST to wash away the secondary antibody free on the NC membrane, place the membrane in a chemiluminometer and use ECL chemiluminescent liquid for imaging. Finally, use ImageJ software to analyze the gray value of the exposed bands to semi-quantitate the expression of the protein.

[0062] like Figure 1 As shown in Figure 2, among the six molecular glues, H0-mGlu and H1-mGlu showed obvious degradation ability of Bcr-Abl and Abl proteins in a concentration-dependent manner.50 The results showed that H1-mGlu has an inhibitory effect on the expression of Bcr-Abl and Abl proteins in DC. 50 At the same time, we found that H0-mGlu1, H0-mGlu2, H0-mGlu3, and H0-mGlu4 had poor degradation effects on Bcr-Abl and Abl proteins, which was consistent with the in vitro reactivity data and may be related to the reactivity of the molecular glue handle with the E3 ligase. In addition, after treating the molecular glue with cells for different time periods, we found that both molecular glues H0-mGlu and H1-mGlu degraded Bcr-Abl and Abl proteins in a time-dependent manner. H0-mGlu degradation of Bcr-Abl and Abl proteins T 1 / 2 About 10 h; H1-mGlu degradation of Bcr-Abl and Abl proteins T 1 / 2 About 8 h ( Figure 2 ).

[0063] However, when MG132 (a proteasome inhibitor) was added to the cells, WB experiments revealed that Bcr-Abl / Abl could not be degraded ( Figure 3 ), indicating that the degradation of Bcr-Abl / Abl protein depends on the ubiquitin-proteasome system (UPS).

[0064] 2. In vitro reactivity of the six molecular glues Daniel et al. showed that electrophilic molecular glue handles can covalently target the cysteine ​​of the E3 ligase RNF126. Therefore, we used HPLC to analyze the in vitro reactivity of different molecular glues, molecular glue handle structural units, and multiple compounds containing α,β-unsaturated carbonyl structures. The specific steps are to add 1 μL of compound (10 mM) to 99 μL of GSH PBS buffer containing 20% ​​DMSO, the reaction concentration of GSH is 2 mM, and incubate at 37°C for different times. Then, HPLC analysis is performed using acetonitrile aqueous solution containing 0.1% trifluoroacetic acid as the mobile phase, and the compound is gradient eluted in the C18 column for 30 min. The characteristic peak of the product is monitored at a UV wavelength of 254 nm, and the product collected at the peak time of the characteristic peak is confirmed by LC-MS ( Figure 4). As shown in the figure, compound C1 reacts fastest with GSH, followed by C2, KN1021 (the smallest structural unit of the molecular glue handle, the synthesis route refers to ACS Cent. Sci. 2023, 9, 5, 915–926), H0-mGlu, and H1-mGlu, which can react completely with GSH within 30 minutes. C3 can react completely within 60 minutes. The reaction time of H0-mGlu1, H0-mGlu2, and H0-mGlu4 is greater than 24 hours. The above data provide a theoretical basis for the evaluation of the biological activity of molecular glue in cells.

[0065] (III) Inhibitory effects of six molecular glues on chronic myeloid leukemia cell lines Next, we further explored the anticancer activity of molecular glue through antiproliferation experiments ( Figure 5 ). The specific steps are as follows: (1) Cell seeding: 5000 cells / well were seeded in a 96-well plate, with each well containing 90 μL of culture medium (Keygen Biotechnology). The 96-well plate was placed in an incubator (37°C, 5% CO2) for overnight culture.

[0066] (2) Cell administration: The compound was gradiently diluted to obtain 8 concentrations of drug solution, namely 500, 100, 20, 4, 0.8, 0.16, 0.032, and 0.0064 μM. 10 μL of each drug solution was added to a well plate containing 90 μL of cell culture medium. The final test concentrations of the compound were 50, 10, 2, 0.4, 0.08, 0.016, 0.0032, and 0.00064 μM, respectively.

[0067] (3) MTT assay for cell viability: After 72 h of drug incubation, 10 μL of 5 mg / mL MTT solution (Sigma-Aldrich) was added to each well and incubated for 4 h. Next, 100 μL of a triplicate solution (10% SDS-5% isobutanol-0.012 M hydrochloric acid solution) was added to each well and incubated for 4 h to dissolve the generated formazan. Finally, the absorbance (OD) was measured at a wavelength of 570 nm using a microplate reader (BioTek), and the cell viability at each drug concentration was calculated according to the formula.

[0068] The data of K562 cells showed that molecular glues were more cytotoxic than the corresponding inhibitors. Among them, H0-mGlu was 18 times more cytotoxic than the inhibitor H0 to K562 cells (IC 50 : 166.4 nM vs 9.0 nM); H1-mGlu was 45 times that of H1 (145.3 nM vs 3.2 nM). Consistent with the degradation ability, the IC50 All of them were greater than 1 μM, and did not show enhanced cytotoxicity. At the same time, we used cell lines transfected with Bcr-Abl and Bcr-AblT315I mutations to test the molecular glue H0-mGlu and H1-mGlu for anti-proliferation experiments. The results showed that H0-mGlu and H1-mGlu not only had obvious toxicity to Bcr-AblWT cells, but also showed strong killing effects on the T315I mutation cell line, which is currently the most difficult to overcome in clinical practice, and both were superior to inhibitors H0 and H1.

[0069] (IV) Study on the anticancer mechanism of compound H1-mGlu Bcr-Abl affects cell proliferation, migration and drug resistance by continuously inducing phosphorylation of downstream signaling pathway proteins, thus leading to unlimited cell proliferation. In order to further determine whether the anti-proliferation effect of molecular glue depends on the degradation of Bcr-Abl protein and the inhibition of downstream protein activity, we detected the expression levels of Bcr-Abl and its downstream signals Stat5 and Crkl and their phosphorylated proteins through the above-mentioned WB experiment. Figure 6 As shown in the figure, similar to the corresponding inhibitors, H0-mGlu and H1-mGlu can effectively inhibit the expression of Stat5 and Crk1 phosphorylated proteins, but in addition to inhibiting phosphorylated proteins, H0-mGlu and H1-mGlu can also inhibit the expression of Stat5 and Crk1 proteins. This fully demonstrates that H0-mGlu and H1-mGlu can degrade Bcr-Abl protein in a concentration-dependent manner and effectively inhibit the signal transduction of Bcr-Abl.

[0070] Subsequently, in order to verify whether the toxicity of molecular glue is related to its induction of cell apoptosis, we detected cell apoptosis by flow cytometry. The specific steps were as follows: the drug was incubated with K562 cells for 16 h, and then the cells were collected. According to the instructions of the apoptosis kit (KTA0010, Abbkine), the cells were stained with Annexin V-FITC and PI, respectively. Next, the stained cells were analyzed using a flow cytometer (Beckman), and the data were processed using the FlowJo software package.

[0071] like Figure 7As shown, both H0-mGlu and H1-mGlu induced cell apoptosis in a concentration-dependent manner, and at a concentration of 10 μM, the apoptosis rate reached more than 90%. At the same time, we detected the expression of apoptosis-related proteins Caspase 3, Cleaved-Caspase 3, PARP, Cleaved-PARP protein, Bcl2 and Mcl1 through the above WB experiment. The data showed that H0-mGlu and H1-mGlu could induce the production of Cleaved-Caspase 3 and Cleaved-PARP protein, and inhibit the expression of Bcl2 and Mcl1 in a concentration-dependent manner ( Figure 8 ), which suggests that H0-mGlu and H1-mGlu can induce apoptosis through a mitochondria-dependent pathway.

[0072] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A molecular glue targeting BCR-ABL, characterized in that It has the general formula shown in the following formula (I): Formula (I) In the formula, R1 is hydrogen or hydroxyl; R2 satisfies R2COOH and is selected from one of (2E)-4-(4-methoxyphenyl)-4-oxyidenebut-2-enoic acid, cyclopent-1-ene-1-carboxylic acid, (2E)-3-[4-(trifluoromethyl)phenyl]prop-2-enoic acid, (2E)-3-[2-(trifluoromethyl)phenyl]prop-2-enoic acid, and acrylic acid.

2. The molecular glue targeting BCR-ABL according to claim 1, characterized in that It has one of the following structures: 。 3. A method for preparing the targeted BCR-ABL molecular glue according to claim 1 or 2, characterized in that The following steps are involved: When R1 is H, it comprises the following steps: (1) 6-bromobenzo[d]thiazol-2-amine and 2-methoxyphenylboronic acid pinacol ester undergo Suzuki coupling reaction to obtain compound 1; (2) Activating the amino group of compound 1 with carbonyldiimidazole to generate an intermediate, and then adding N-Boc-piperazine to further react to obtain compound 2 through a one-pot method; (3) Compound 2 is deprotected by the tert-butyloxycarbonyl group by trifluoroacetic acid, and then reacts with R2COOH to undergo an amide condensation reaction to obtain a targeted BCR-ABL molecular glue as shown in formula (I); The reaction route is as follows: When R1 is hydroxyl, it comprises the following steps: (4) 6-Bromobenzo[d]thiazol-2-amine was first activated with carbonyldiimidazole to obtain an intermediate, and then N-Boc-piperazine was added to further react to obtain compound 3 through a one-pot method; (5) Compound 3 undergoes a Suzuki coupling reaction with 3-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol to obtain compound 4; (6) Compound 4 is deprotected by the tert-butyloxycarbonyl group by trifluoroacetic acid, and then reacts with R2COOH to undergo an amide condensation reaction to obtain a targeted BCR-ABL molecular glue as shown in formula (I); The reaction route is as follows: 。 4. The method for preparing the BCR-ABL targeting molecular glue according to claim 3, characterized in that: The Suzuki coupling reaction described in step (1) refers to a reaction at 100-120° C. for 4-6 hours.

5. The method for preparing the BCR-ABL targeting molecular glue according to claim 3, characterized in that: The activation in step (2) refers to reacting at room temperature for 8-12 hours under the protection of inert gas or nitrogen, and the reaction solvent is at least one of DMF and DCM; the amount of carbonyldiimidazole used in the activation process is 1.5-3 times the molar amount of the compound; The one-pot method described in step (2) refers to the reaction of the intermediate product with N-Boc-piperazine in the presence of a base at 60-80° C. for 2-5 hours, wherein the base is triethylamine.

6. The method for preparing the BCR-ABL targeting molecular glue according to claim 3, characterized in that: The removal of the tert-butyloxycarbonyl group in step (3) is to add compound 2 to a mixed solution of dichloromethane and trifluoroacetic acid, and react at room temperature for 1-2.5 hours, wherein the volume ratio of trifluoroacetic acid to dichloromethane is 1:(2-3); The amide condensation reaction described in step (3) refers to the reaction at room temperature-60°C for 5-12 hours in the presence of a condensing agent and a base; wherein the condensing agent is 2-(7-azobenzotriazole)-tetramethyluronium hexafluorophosphate, and its amount is 1.1-2.5 times the molar amount of the product after the tert-butyloxycarbonyl protection of compound 2 is removed by trifluoroacetic acid; the base is at least one of triethylamine and N,N-diisopropylethylamine, and the amount of the base is 2-4 times the molar amount of the product after the tert-butyloxycarbonyl protection of compound 2 is removed by trifluoroacetic acid.

7. The method for preparing the BCR-ABL targeting molecular glue according to claim 3, characterized in that: The activation in step (4) refers to reacting at room temperature for 8-10 hours under the protection of inert gas or nitrogen, and the reaction solvent is at least one of DMF and DCM; the amount of carbonyldiimidazole used in the activation process is 1.5-3 times the molar amount of 6-bromobenzo[d]thiazol-2-amine; The one-pot method described in step (4) refers to the reaction of the intermediate product with N-Boc-piperazine in the presence of a base at 60-80° C. for 2-4 hours, wherein the base is triethylamine.

8. The method for preparing the BCR-ABL targeting molecular glue according to claim 3, characterized in that: The Suzuki coupling reaction described in step (5) refers to the reaction at 100-120° C. for 5-7 hours.

9. The method for preparing the BCR-ABL targeting molecular glue according to claim 3, characterized in that: The removal of the tert-butyloxycarbonyl group in step (6) is to add compound 4 to a mixed solution of dichloromethane and trifluoroacetic acid, and react at room temperature for 1-2.5 hours, wherein the volume ratio of trifluoroacetic acid to dichloromethane is 1:2-3; The amide condensation reaction described in step (6) refers to the reaction at room temperature-60°C for 5-12 hours in the presence of a condensing agent and a base; wherein the condensing agent is 2-(7-azobenzotriazole)-tetramethyluronium hexafluorophosphate, and its amount is 1.1-2.5 times the molar amount of the product after the tert-butyloxycarbonyl protection of compound 4 is removed by trifluoroacetic acid; the base is at least one of triethylamine and N,N-diisopropylethylamine, and the amount of the base is 2-4 times the molar amount of the product after the tert-butyloxycarbonyl protection of compound 4 is removed by trifluoroacetic acid.

10. Use of the BCR-ABL targeting molecular glue according to claim 1 or 2 in the preparation of anti-chronic myeloid leukemia drugs.

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