A molecular glue targeting Bcr-Abl and its preparation method and use

Abstract

The present invention belongs to the field of medicinal chemistry, and discloses a molecular glue degrader targeting Bcr-Abl protein, its preparation method and use. The structural formula of the molecular glue targeting BCR-ABL is as follows, wherein R1 is hydrogen or hydroxyl; R2 satisfies R2COOH and is selected from one of (2E)-4-(4-methoxyphenyl)-4-oxydibutyl-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. Compared with the third-generation BCR-ABL inhibitor H0 in the clinical stage, the compound H0-mGlu and H1-mGlu inhibitory activity (IC 50 ) has shown a significant improvement (IC 50 The value can be reduced by 1-2 orders of magnitude), so it has good application prospects.

Description

Technical Field

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

[0002] Chronic myeloid leukemia (CML) is a malignant myeloproliferative disorder caused by genetic mutations in hematopoietic stem cells. Studies have shown that 90% to 95% of CML patients harbor the Philadelphia chromosome (Ph), which is formed by a reciprocal translocation between the Abelson (ABL) tyrosine kinase gene on chromosome 9 and the Breakpoint Cluster Region (BCR) gene on chromosome 22. This translocation generates a chimeric BCR-ABL fusion gene. The encoded Bcr-Abl tyrosine kinase promotes uncontrolled cell proliferation and reduces the adhesion of CML cells to the bone marrow stroma. Furthermore, studies have found that the Bcr-Abl fusion protein is also expressed in 30% to 35% of patients with acute lymphoblastic leukemia (ALL), demonstrating its key role in driving the pathological progression of CML and ALL and its great potential for treatment.

[0003] Before the last century, CML was considered an incurable disease, but the past two decades have seen a dramatic shift in treatment. Since the launch of the first-generation targeted drug, imatinib, researchers have developed a series of tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL kinase, broadly categorized into four generations (Table 1). The first-generation drug, imatinib, competitively blocks BCR-ABL kinase (binding to the ATP site), specifically inhibiting the aberrant activation of leukemia-related signaling pathways, thereby suppressing cancer cell proliferation and significantly reducing toxicity to normal cells. While imatinib is effective in treating CML, it can also induce drug-resistant mutations, resulting in relapse in approximately 50-90% of patients after treatment. The most common mutations are T315I, E225K, Y253F, and M351T, accounting for approximately 60% of all imatinib-resistance mutations. T315I is a gatekeeper mutant in the ATP-binding site, accounting for 15% to 20% of all clinically observed mutations. Second-generation drugs such as dasatinib have been introduced, but none are effective against the T315I mutation. Third-generation TKIs, ponatinib and orebactinib, offer a solution to overcoming the T315I mutation. The first three generations of TKIs are ATP-competitive inhibitors, which inevitably lead to mutations and drug resistance, hindering drug-protein binding. Fourth-generation inhibitors, designed to bind to the myristoyl pocket of the Bcr-Abl allosteric site, have been shown to induce new myristoyl site mutations.

[0004] In general, these marketed drugs can achieve short-term therapeutic effects and significantly prolong patient survival. However, long-term use can lead to mutations that can lead to drug resistance. Some drugs have also been associated with serious cardiovascular and hepatotoxicity in clinical practice, resulting in black box warnings from the US FDA. Therefore, there remains an urgent need to develop novel therapeutic strategies and drugs to address Bcr-Abl-dependent drug resistance caused by Bcr-Abl point mutations and overexpression.

[0005] With the advent of targeted drugs, cancer treatment has ushered in a new era of precision medicine. However, traditional small molecule inhibitors rely on an "occupancy-driven" model, and approximately 80% to 85% of proteins lack binding pockets for small molecule drugs. Furthermore, maintaining effective drug concentrations can lead to toxic accumulation and drug-resistant mutations. Targeted protein degradation (TPD), which completely eliminates pathological proteins, offers a new therapeutic strategy that can effectively prevent the development of drug resistance.

[0006] Protein homeostasis (proteostasis) is fundamental to the normal functioning of cells. When any of these processes become dysregulated, the balance of proteostasis is disrupted, potentially leading to a range of diseases, including cardiovascular disease, cancer, neurodegeneration, and viral infections. To restore this imbalance, utilizing endogenous cellular degradation pathways to eliminate pathogenic proteins has become a newly emerging therapeutic approach in recent years.

[0007] Targeted protein degradation (TPD) technology is based on the intracellular proteasome and lysosome degradation mechanisms, enabling specific and efficient removal of target proteins. Based on their mechanism of action, TPD can be divided into two major categories: one is targeted degradation technology that relies on the UPS pathway, including PROTAC (Proteolysis targeting chimeras), molecular glue, and hydrophobic tags (HyT); the other is lysosomal-targeted chimeras (LYTAC), autophagy-targeted chimeras (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, unlike protein inhibitors that require binding to active sites to inhibit proteins, TPD disrupts all protein functions by recruiting targets to participate in multiple protein degradation processes, thereby reducing the development of drug resistance and increasing the potential for degradation of "undruggable" proteins such as non-enzymatic proteins. Furthermore, TPD's reduced reliance on high-affinity ligand binding makes it easier to discover drug candidates with low nanomolar potency.

[0009] 1. PROTAC

[0010] The ubiquitin-proteasome degradation pathway (UPS), as the main protein degradation pathway in cells, plays a decisive role in maintaining protein homeostasis and regulating intracellular metabolism. It can specifically degrade more than 80% of proteins in cells. PROTAC is one of the targeted degradation technologies of UPS, consisting of a target protein ligand, an E3 ligase ligand, and a linker. 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 tagging of the target protein by the ubiquitin-binding enzyme E2, and then the tagged target protein is recognized and degraded by the proteasome.

[0011] Currently, researchers have developed a variety of Bcr-Abl PROTACs. For example, the Nagar and Lai teams developed corresponding PROTACs based on the first-generation Bcr-Abl inhibitor imatinib and the second-generation inhibitors dasatinib and bosutinib, respectively, successfully achieving Bcr-Abl protein degradation. The Crews team, the Jiang team, the Rao team, and the Lu team have, to a certain extent, solved the drug resistance dilemma caused by Bcr-Abl point mutations by developing diverse PROTACs, providing effective synthetic strategies and drugs for the treatment of CML and its resistance.

[0012] 2. Molecular glue

[0013] The concept of "molecular glue" was first proposed in the 1990s, describing a class of small molecule compounds capable of inducing or stabilizing protein-protein interactions (PPIs). The immunosuppressants cyclosporine A (CsA) and FK506 were the first reported molecular glues. As research into the mechanisms of action of CsA and FK506 deepened, this novel mechanism of chemically inducing protein binding gradually became an effective strategy for exploring biological mechanisms and drug development. In particular, molecular glue-induced protein degradation appears to be an effective means of treating diseases. Consequently, the concept of molecular glue degraders gradually took shape. These agents regulate protein ubiquitination by inducing or stabilizing PPIs between ubiquitin ligases and target proteins, and then rely on the UPS to degrade target proteins.

[0014] PROTAC and molecular glue are the two most mature types of TPD currently in terms of widespread research interest, number of companies and pipelines, and progress of clinical trials.

[0015] 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, it has poor solubility and cell permeability, making it impossible to design it as an oral drug. It also has low bioavailability and off-target issues. These factors have, to a certain extent, limited the clinical transformation of PROTAC drugs.

[0016] In contrast, molecular glues are monovalent small molecules (with a 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) properties.

[0017] 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

[0018] The primary purpose of this invention is to provide a molecular glue targeting BCR-ABL, addressing the pain point that the first four generations of BCR-ABL small molecule inhibitors currently in clinical use all harbor drug-resistant mutations, making them ineffective in the sustained and effective treatment of CML. Given that current molecular glue protein degradation technologies are mostly serendipitous discoveries and lack rational design, this invention employs a "modular design strategy" to develop multiple BCR-ABL molecular glues, consisting of a molecular glue handle targeting the RNF126 E3 ligase and the clinical-stage BCR-ABL inhibitor H0.

[0019] Another object of the present invention is to provide a method for preparing the above-mentioned molecular glue targeting BCR-ABL.

[0020] Another object of the present invention is to provide applications of the above-mentioned molecular glue targeting BCR-ABL.

[0021] The purpose of the present invention is achieved through the following solutions:

[0022] A molecular glue targeting BCR-ABL has the general formula shown in the following formula (I):

[0023]

[0024] Formula (I)

[0025] 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.

[0026] Preferably, the BCR-ABL targeting molecular glue has one of the following structures:

[0027]

[0028] More preferably, the BCR-ABL targeting molecular glue has one of the following structures:

[0029]

[0030] A method for preparing the above-mentioned BCR-ABL targeting molecular glue comprises the following steps:

[0031] When R1 is H, it comprises the following steps:

[0032] (1) 6-Bromobenzo[d]thiazol-2-amine and 2-methoxyphenylboronic acid pinacol ester undergo Suzuki coupling reaction to obtain compound 1;

[0033] (2) Carbonyldiimidazole was used to activate the amino group of compound 1 to generate an intermediate, which was then further reacted with N-Boc-piperazine to obtain compound 2 through a one-pot reaction.

[0034] (3) Compound 2 is deprotected by the tert-butyloxycarbonyl group using trifluoroacetic acid, and then undergoes an amide condensation reaction with R2COOH to obtain the targeted BCR-ABL molecular glue shown in formula (I);

[0035] The reaction route is as follows:

[0036]

[0037] When R1 is hydroxyl, it comprises the following steps:

[0038] (4) 6-Bromobenzo[d]thiazol-2-amine was first activated with carbonyldiimidazole to obtain an intermediate, which was then further reacted with N-Boc-piperazine to obtain compound 3 in a one-pot reaction.

[0039] (5) Compound 3 undergoes Suzuki coupling reaction with 3-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol to obtain compound 4;

[0040] (6) Compound 4 is deprotected by the tert-butyloxycarbonyl group using trifluoroacetic acid, and then undergoes an amide condensation reaction with R2COOH to obtain the targeted BCR-ABL molecular glue shown in formula (I);

[0041] The reaction route is as follows:

[0042]

[0043] The Suzuki coupling reaction in step (1) is carried out at 100-120°C for 4-6 hours;

[0044] The activation in step (2) refers to the reaction 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, preferably 2 times;

[0045] The one-pot method 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 h, wherein the base is preferably triethylamine.

[0046] The removal of the tert-butyloxycarbonyl group in step (3) is performed by adding compound 2 to a mixed solution of dichloromethane and trifluoroacetic acid, and reacting at room temperature for 1-2.5 h, wherein the volume ratio of trifluoroacetic acid to dichloromethane is 1:(2-3);

[0047] The amide condensation reaction described in step (3) refers to a 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.

[0048] The activation in step (4) refers to a reaction at room temperature for 8-10 hours under the protection of an 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;

[0049] 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 h, wherein the base is preferably triethylamine.

[0050] The Suzuki coupling reaction described in step (5) is carried out at 100-120°C for 5-7 hours.

[0051] The removal of the tert-butyloxycarbonyl group in step (6) is performed by adding compound 4 to a mixed solution of dichloromethane and trifluoroacetic acid, and reacting at room temperature for 1-2.5 hours, wherein the volume ratio of trifluoroacetic acid to dichloromethane is 1:2-3;

[0052] 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.

[0053] The specific reaction routes of H0-mGlu, H0-mGlu1, H0-mGlu2, H0-mGlu3, H0-mGlu4, and H1-mGlu are as follows:

[0054] Application of the above-mentioned targeted BCR-ABL molecular glue in the preparation of anti-chronic myeloid leukemia drugs.

[0055] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0056] 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 shown a 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

[0057] Figure 1 The figure shows the degradation ability of Bcr-Abl and Abl proteins in K562 cells by different Bcr-Abl molecular glues of the present invention. In the figure, labels A and B are the degradation and half degradation rate DC of Bcr-Abl and Abl proteins in K562 cells by HO-mGlu. 50 ; C, D are the degradation and half degradation rate DC of Bcr-Abl and Abl proteins in K562 cells induced 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.

[0058] Figure 2 This figure shows the time-dependent degradation of Bcr-Abl protein by different Bcr-Abl molecular glues of the present invention. A shows the protein imaging bands from the H0-mGlu and H1-mGlu degradation experiments, and B shows the quantification of Bcr-Abl protein expression.

[0059] 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.

[0060] Figure 4 This is a test to verify the in vitro reactivity of all compounds of the present invention.

[0061] Figure 5 The cytotoxicity of all compounds of the present invention on CML cells (IC 50 ) Results. In the figure, label A shows the anti-proliferation curve of Bcr-Abl molecular glue on K562 cells; B and C show the anti-proliferation curves of Bcr-Abl molecular glue on BaF3 cells transfected with Bcr-Abl WT and Bcr-AblT315I, respectively.

[0062] Figure 6 This is the result of the effects of all compounds of the present invention on the Bcr-Abl signaling pathway.

[0063] Figure 7 These are the results of the effects of all the compounds of the present invention on the apoptosis of K562 cells.

[0064] 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

[0065] The present invention is further described in detail below with reference to the examples and accompanying drawings, but the embodiments of the present invention are not limited thereto. In the examples, where specific conditions are not specified, conventional conditions or those recommended by the manufacturer were followed. Reagents and instruments used, where the manufacturer is not specified, are commercially available conventional products. Cells used include K562 (ATCC No. CCL-243) and BaF3 cells (ATCC No. HB-283). "eq" in the examples refers to molar equivalent.

[0066] Unless otherwise specified, all compounds of the present invention include all optical isomers or tautomeric forms.

[0067] Example 1

[0068] Preparation of N-[6-(2-methoxyphenyl)benzo[d][1,3]thiazol-2-yl]-4-[(2E)-4-(4-methoxyphenyl)-1,4-dioxyidenebut-2-enyl]piperazine-1-carboxamide

[0069]

[0070] 1.1 Synthesis of compound 1

[0071] 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, and then stirred in a sealed tube at 120°C for 5 h. The formation of the product was confirmed by TLC and LC-MS.

[0072] After evaporation of the solvent, the crude product was extracted between EA (3 × 30 mL) and water. The combined organic extracts were washed with saturated NaCl solution, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by column chromatography (EA / hexane 0-33%) to afford compound 1 (1.05 g, 93%) as a white solid.

[0073] 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.

[0074] 1.2 Synthesis of compound 2

[0075] 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. Subsequently, N-Boc-piperazine (1.46 g, 7.82 mmol) and triethylamine (1.19 g, 3 eq) were added and stirred at 80 °C in a sealed tube for 2 h.

[0076] After completion of the reaction, the mixture was concentrated in vacuo. The combined organic extracts were washed with saturated NaCl solution, dried over Na2SO4, filtered, and evaporated under reduced pressure. The crude product was purified by column chromatography (EA / hexane 0-25%) to afford the title compound 2 (1.60 g, 87% yield) as a white solid.

[0077] LC-MS: m / z 468.2 [M+H] + . 1 H 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.

[0078] 1.3 Synthesis of the target product H0-mGlu

[0079] 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). Triethylamine (TEA) (2 eq) was then added, and the reaction mixture was stirred at room temperature for 30 min. Compound 2 was deprotected (specifically, compound 2 was added to a mixed solution of dichloromethane and trifluoroacetic acid and reacted at room temperature for 1 h, where the volume ratio of trifluoroacetic acid to dichloromethane was 1:3). The resulting amine (1.0 eq) was dissolved in DMF (0.1 M) and then added dropwise to the reaction mixture, which was stirred at room temperature overnight.

[0080] The reaction mixture was diluted with ethyl acetate (EA) and washed three times with a saturated NaHCO₃ solution, a saturated NH₄Cl solution, and a saturated NaCl solution. The mixture was then dried over anhydrous Na₂SO₄, filtered, and evaporated under reduced pressure. The crude product was purified by silica gel chromatography (MeOH / DCM = 0%-5%) to afford H₀-mGlu (60 mg, 79.73% yield), a yellow powder.

[0081] HRMS (ESI+) calculation for C 30 H 28 N4O5S([M+H] + ): 557.1780, found 557.1870. 1 H 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.

[0082] Example 2

[0083] Synthesis of 4-(cyclopent-1-enylcarbonyl)-N-[6-(2-methoxyphenyl)benzo[d][1,3]thiazol-2-yl]piperazine-1-carboxamide

[0084]

[0085] 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). The mixture was then added dropwise and the reaction mixture was stirred at 60°C for 5 hours.

[0086] The reaction mixture was diluted with EA and washed three times with a saturated NaHCO solution, a saturated NH4Cl solution, and a saturated NaCl solution, respectively. The mixture was then dried over Na2SO4, filtered, and evaporated 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, 46.20% yield).

[0087] 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). 13C 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.

[0088] Example 3

[0089] 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

[0090]

[0091] 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). The mixture was then added dropwise, and the reaction mixture was stirred at 60°C for 5 hours.

[0092] The reaction mixture was diluted with EA and washed three times with a saturated NaHCO₃ solution, a saturated NH₄Cl solution, and a saturated NaCl solution. The mixture was then dried over Na₂SO₄, filtered, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (PE / EA = 0% to 50%) to obtain a white powder (71.30 mg, 46.37% yield).

[0093] HRMS (ESI+) calculation for C 29 H 25 F3N4O3S([M+H] + ): 567.1599, found 567.1710. 1HNMR (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.

[0094] Example 4

[0095] 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

[0096]

[0097] 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). This was then added dropwise, and the reaction mixture was stirred at 60°C for 5 hours.

[0098] The reaction mixture was diluted with EA and washed three times with a saturated NaHCO₃ solution, a saturated NH₄Cl solution, and a saturated NaCl solution, respectively. The mixture was then dried over Na₂SO₄, filtered, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (PE / EA = 0% to 50%) to afford the title compound as a white powder (68.38 mg, 44.47% yield).

[0099] 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.

[0100] Example 5

[0101] Synthesis of N-[6-(2-methoxyphenyl)benzo[d][1,3]thiazol-2-yl]-4-(1-oxyylideneprop-2-enyl)piperazine-1-carboxamide

[0102]

[0103] 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, 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). This was then added dropwise, and the reaction mixture was stirred at 60°C for 5 hours.

[0104] The reaction mixture was diluted with EA and washed three times with a saturated NaHCO₃ solution, a saturated NH₄Cl solution, and a saturated NaCl solution, respectively. The mixture was then dried over Na₂SO₄, filtered, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (EA / PEA = 0%-6%) to afford the title compound as a white powder (78.30 mg, 68.28% yield).

[0105] 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.

[0106] Example 6

[0107] Synthesis of N-[6-(4-hydroxy-2-methoxyphenyl)benzo[d][1,3]thiazol-2-yl]-4-[(2E)-4-(4-methoxyphenyl)-1,4-dioxyidenebut-2-enyl]piperazine-1-carboxamide

[0108]

[0109] 6.1 Synthesis of Compound 3

[0110] 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.

[0111] After completion of the reaction, the mixture was concentrated in vacuo. The combined organic extracts were washed with saturated NaCl solution, dried over Na2SO4, filtered, and evaporated under reduced pressure. The crude product was purified by column chromatography (EA / hexane 0-33%) to afford a white solid (1.72 g, 89.28% yield).

[0112] 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.

[0113] 6.2 Synthesis of compound 4

[0114] 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. The formation of the product was confirmed by TLC and LC-MS.

[0115] After evaporation of the solvent, the crude product was extracted between EA (3 × 30 mL) and water. The combined organic extracts were washed with saturated NaCl solution, dried over NaSO, and evaporated under reduced pressure. The crude product was purified by column chromatography (EA / hexane 0-33%) to afford a white solid (308 mg, 48.37% yield).

[0116] 6.3 Synthesis of the target product H1-mGlu

[0117] 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 at a concentration of 0.1 M). Triethylamine (TEA) (2 eq) was then added, and the reaction mixture was stirred at room temperature for 30 minutes. Compound 4 (460 mg, 156.07 μmol) was deprotected (DCM:TFA = 3:1) at room temperature for 1 hour to obtain the amine (1 eq), which was dissolved in DMF (0.1 M) and added dropwise to the reaction mixture, which was stirred at room temperature overnight.

[0118] The reaction mixture was diluted with EA and washed three times with a saturated NaHCO₃ solution, a saturated NH₄Cl solution, and a saturated NaCl solution. The mixture was then dried over anhydrous Na₂SO₄, filtered, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (DCM / MeOH = 0%-5%) to give a yellow powder (44.32 mg, 49.59% yield).

[0119] 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). 13 CNMR (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.

[0120] Comparative Example 1

[0121] H0 was synthesized according to S.-M. Yun et al. / Cancer Letters 348 (2014) 50–60.

[0122] The structure of H0 is as follows:

[0123]

[0124] Comparative Example 2

[0125] Synthesis of H1

[0126]

[0127] The synthesis of compound 5 was based on S.-M. Yun et al. / Cancer Letters 348 (2014) 50–60.

[0128] Synthesis of target compound H1

[0129] 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 of 1,4-dioxane and 1 mL of H2O and stirred in a sealed tube at 90°C for 4 h. Product formation was confirmed by TLC and LC-MS. After evaporation of the solvent, the mixture was extracted with EA (3 × 30 mL). The combined organic extracts were washed with saturated NaCl solution, dried over Na2SO4, filtered, and evaporated under reduced pressure. The crude product was purified by column chromatography (DCM / MeOH 0% to 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.

[0130] Application Examples

[0131] (I) Degradation effects of six molecular glues on Bcr-Abl and Abl proteins in K562 cells

[0132] Reported proteomics data show 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 plate: according to 1×10 6Cells were seeded in 6-well plates with a cell number of 100 cells / well. 2 mL of culture medium (Keygene Biotechnology) was added to each well. The 6-well plates were placed in an incubator (37°C, 5% CO2) and cultured overnight. (2) Cell administration: The cells were incubated with compounds at final concentrations of 0, 5, 25, 125, 625, and 3125 nM for different time periods, and then 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: The 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 a 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 established loading order. Turn on the electrophoresis and 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. Use the protein marker band as an indicator and stop the electrophoresis after the target band 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 and turn on the power supply. Start transfer at a constant current of 300 mA. The transfer time is adjusted appropriately 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 antibodies: 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), diluted according to the instructions, using primary antibody diluent (Biyuntian)) and incubate on a shaker at 4°C overnight. (8) Incubation with secondary antibody (Qinke Bio, diluted according to the instructions, using TBST containing 5% skim milk powder): Use TBST to wash away the free primary antibody 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) Chemiluminescence imaging: After washing away the free secondary antibody on the NC membrane with TBST, place the membrane in a chemiluminescence instrument and image it with ECL chemiluminescence solution. Finally, grayscale value analysis of the exposed bands was performed using ImageJ software to semi-quantitate protein expression.

[0133] 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 effects of H1-mGlu on DC expression of Bcr-Abl and Abl proteins were 0.79 μM and 0.64 μM, respectively. 50 were 0.51 μM and 0.45 μM, respectively. 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 hours ( Figure 2 ).

[0134] However, when MG132 (a proteasome inhibitor) was added to the cells, WB experiments showed 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).

[0135] (II) In vitro reactivity of six molecular glues

[0136] Daniel et al. have shown 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 as follows: 1 μL of compound (10 mM) was added to 99 μL of GSH PBS buffer containing 20% ​​DMSO, and the reaction concentration of GSH was 2 mM. The mixture was incubated at 37°C for different times, and then HPLC analysis was performed using an acetonitrile aqueous solution containing 0.1% trifluoroacetic acid as the mobile phase. The compound was gradient eluted on a C18 column for 30 minutes, and the characteristic peak of the product was monitored at a UV wavelength of 254 nm. The product collected at the time of the characteristic peak was confirmed by LC-MS ( Figure 4As shown in the figure, compound C1 reacts most quickly with GSH, followed by C2, KN1021 (the smallest structural unit of the molecular glue handle; synthesis route referenced in ACS Cent. Sci. 2023, 9, 5, 915–926), H0-mGlu, and H1-mGlu, all reacting completely with GSH within 30 minutes. C3 reacts completely within 60 minutes. H0-mGlu1, H0-mGlu2, and H0-mGlu4, on the other hand, react in excess of 24 hours. These data provide a theoretical basis for evaluating the bioactivity of molecular glues in cells.

[0137] (III) Inhibitory effects of six molecular glues on chronic myeloid leukemia cell lines

[0138] Next, we further explored the anticancer activity of molecular glue through anti-proliferation experiments ( Figure 5 The specific steps are as follows: (1) Cell seeding: Cells were seeded into 96-well plates at a density of 5000 cells per well, with each well containing 90 μL of culture medium (Keygen Biotechnology). The 96-well plates were placed in an incubator (37°C, 5% CO2) and cultured overnight.

[0139] (2) Cell administration: The compound was diluted gradiently 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.

[0140] (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.

[0141] 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 IC values ​​of H0-mGlu1, H0-mGlu2, H0-mGlu3 and H0-mGlu4 were 50 The concentrations of H0-mGlu and H1-mGlu were both greater than 1 μM, indicating no enhanced cytotoxicity. Furthermore, we used cell lines transfected with Bcr-Abl and Bcr-AblT315I mutations to conduct anti-proliferation experiments on molecular glues H0-mGlu and H1-mGlu. The results showed that H0-mGlu and H1-mGlu not only had significant toxicity to Bcr-AblWT cells, but also exhibited a strong killing effect on the T315I mutation cell line, which is currently the most difficult to overcome in clinical practice. Both were superior to inhibitors H0 and H1.

[0142] (IV) Study on the anticancer mechanism of compound H1-mGlu

[0143] Bcr-Abl affects cell proliferation, migration and drug resistance by continuously inducing phosphorylation of downstream signaling pathway proteins, thereby leading to unlimited cell proliferation. To further determine whether the anti-proliferative 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 results, similar to their corresponding inhibitors, both H0-mGlu and H1-mGlu effectively inhibited the expression of phosphorylated Stat5 and Crk1 proteins. However, in addition to inhibiting phosphorylated proteins, H0-mGlu and H1-mGlu also inhibited the expression of Stat5 and Crk1 proteins. This clearly demonstrates that H0-mGlu and H1-mGlu can degrade Bcr-Abl protein in a concentration-dependent manner and effectively inhibit Bcr-Abl signaling.

[0144] To verify whether the toxicity of the molecular glue was related to its induction of apoptosis, we examined apoptosis by flow cytometry. K562 cells were incubated with the drug for 16 hours, after which the cells were harvested. Following the instructions for the apoptosis kit (KTA0010, Abbkine), the cells were stained with Annexin V-FITC and PI, respectively. The stained cells were then analyzed using a flow cytometer (Beckman), and the data were processed using the FlowJo software package.

[0145] 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 over 90%. At the same time, we used the above-mentioned WB experiment to detect the expression of apoptosis-related proteins Caspase 3, Cleaved-Caspase 3, PARP, Cleaved-PARP protein, Bcl2, and Mcl1. 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 indicates that H0-mGlu and H1-mGlu can induce cell apoptosis through mitochondria-dependent pathways.

[0146] 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 considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A molecular glue targeting BCR-ABL, characterized in that It has the general formula shown below: 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 a BCR-ABL targeting 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) Carbonyldiimidazole was used to activate the amino group of compound 1 to generate an intermediate, which was then further reacted with N-Boc-piperazine to obtain compound 2 through a one-pot reaction. (3) Compound 2 is deprotected by the tert-butyloxycarbonyl group using trifluoroacetic acid, and then undergoes an amide condensation reaction with R2COOH to obtain the targeted BCR-ABL molecular glue shown in formula (I); The reaction scheme 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, which was then further reacted with N-Boc-piperazine to obtain compound 3 in a one-pot reaction. (5) Compound 3 undergoes 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 using trifluoroacetic acid, and then undergoes an amide condensation reaction with R2COOH to obtain the targeted BCR-ABL molecular glue shown in formula (I); The reaction scheme 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) is carried out 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 the reaction 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 performed by adding compound 2 to a mixed solution of dichloromethane and trifluoroacetic acid, and reacting 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 a 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 a reaction at room temperature for 8-10 hours under the protection of an 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 h, 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) is carried out 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 performed by adding compound 4 to a mixed solution of dichloromethane and trifluoroacetic acid, and reacting 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 a 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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