Preparation method and application of self-assembled nano material targeting Bcr-Abl

Abstract

The invention belongs to the field of medicinal chemistry, and discloses a preparation method and application of a Bcr-Abl targeting self-assembled nano material. The structure of the Bcr-Abl targeting self-assembled nano material is shown in the specification, and according to the BCR-ABL targeting self-assembled nano material provided by the invention, the accumulation of a drug at a tumor part is improved and the toxicity of the drug to normal tissues is reduced while the high toxicity of the drug to tumors is retained, so that the occurrence of side effects is reduced. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry, and particularly relates to a preparation method and application of a self-assembly nanomaterial targeting Bcr-Abl. Background Art

[0002] Chronic myeloid leukemia (CML) is a malignant myeloproliferative disease caused by a genetic mutation in 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 that it plays a key role in promoting the pathological process of CML and ALL.

[0003] With the emergence of targeted drugs, cancer treatment has ushered in a new era of precision treatment. Targeted protein degradation technology (TPD) that completely eliminates pathological proteins provides a new strategy for treatment and can effectively avoid the occurrence of drug resistance. Targeted protein degradation technology (TPD) is based on the proteasome and lysosome degradation mechanism in cells, which 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, molecular glue, and hydrophobic tags (HyT); the other is the lysosomal chimera (LYTAC), autophagy-targeted chimera (AUTAC), and autophagosome binding compounds (ATTEC) that utilize the lysosomal degradation pathway.

[0004] 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 (PPIs). By inducing or stabilizing PPIs between ubiquitin ligases and target proteins, it regulates protein ubiquitination and relies on UPS to degrade target proteins. The discovery of molecular glues currently still relies mainly on accidental discoveries, and there is a lack of unified discovery principles and systematic evaluation methods. In addition, molecular glues have poor targeting selectivity and often bring greater toxicity.

[0005] In recent years, nano drug delivery systems have developed rapidly. Compared with traditional small molecule drugs, they have obvious advantages in improving drug solubility, stability, targeting and controlled release. By encapsulating small molecule drugs, nano delivery systems can enhance the in vivo retention time and tumor accumulation of drugs, achieve passive targeting with the help of high permeability and long retention (EPR) effect, and achieve active targeting with the help of tumor-specific ligands, thereby improving the efficacy. In addition, nano drugs also have the ability to responsively release drugs in the tumor microenvironment and reduce toxic side effects. Therefore, it is necessary to provide a self-assembled nanomaterial targeting Bcr-Abl, which can improve tumor accumulation and reduce the toxicity of drugs to normal tissues while retaining the high toxicity of drugs to tumors, thereby reducing the occurrence of side effects. Summary of the invention

[0006] The adverse reactions and side effects of drugs will seriously affect the survival rate and compliance of patients taking the drugs. In order to overcome the shortcomings and deficiencies of the above-mentioned prior art, the primary purpose of the present invention is to provide a self-assembled nanomaterial targeting BCR-ABL, which can increase the accumulation of drugs in tumor sites while retaining the high toxicity of drugs to tumors, reduce the toxicity of drugs to normal tissues, and thus reduce the occurrence of side effects.

[0007] Another object of the present invention is to provide the preparation of the above-mentioned self-assembled nanomaterial targeting BCR-ABL.

[0008] Another object of the present invention is to provide applications of the above-mentioned self-assembled nanomaterial targeting BCR-ABL.

[0009] The purpose of the present invention is achieved through the following solutions:

[0010] A self-assembled nanomaterial targeting BCR-ABL has the following structural formula:

[0011]

[0012] A method for preparing the above-mentioned self-assembled nanomaterial targeting BCR-ABL comprises the following steps:

[0013] (1) 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 process;

[0014] (2) 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;

[0015] (3) Compound 4 was deprotected by the tert-butyloxycarbonyl group by trifluoroacetic acid, and then reacted with (2E)-4-(4-methoxyphenyl)-4-oxyylidenebut-2-enoic acid to obtain the BCR-ABL targeting molecular glue H1-mGlu;

[0016] (4) H1-mGlu is reacted with 2-({3-[(2-methylprop-2-yl)oxy]-3-oxyylidenepropyl}thio)propionic acid to generate compound 5, and then the tert-butyl ester is removed to obtain H1-mGlu-linker;

[0017] (5) H1-mGlu-linker undergoes amide condensation reaction with mPEG-NH2 to obtain cle-NP;

[0018]

[0019] The activation in step (1) 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 6-bromobenzo[d]thiazol-2-amine, preferably 2 times;

[0020] The one-pot method described in step (1) 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 Suzuki coupling reaction in step (2) is carried out at 100-120° C. for 5-7 hours;

[0022] The step (3) of removing the tert-butyloxycarbonyl protection by trifluoroacetic acid refers to dissolving compound 4 in DCM, and then adding trifluoroacetic acid to react at room temperature for 1-2 hours to remove the tert-butyloxycarbonyl group; wherein the volume ratio of trifluoroacetic acid to dichloromethane is 1:2-3;

[0023] The amide condensation reaction described in step (3) refers to a reaction at room temperature for 8-12 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 the molar amount of the product after the tert-butyloxycarbonyl protection of compound 4 is removed by trifluoroacetic acid, and preferably 1.2 times; the base is preferably 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, and preferably 2 times.

[0024] The condensation reaction in step (4) refers to reacting at room temperature for 12-24 hours in the presence of EDC-HCl and DMAP; the removal of tert-butyl ester in step (4) refers to dissolving compound 5 in DCM, and then adding trifluoroacetic acid to react at room temperature for 1-2 hours to remove the tert-butyl group; wherein the volume ratio of trifluoroacetic acid to dichloromethane is 1:2-3;

[0025] The amide condensation reaction described in step (5) refers to the reaction at room temperature for 20-24 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 the molar amount of the product after the tert-butyloxycarbonyl protection of H1-mGlu-linker is removed by trifluoroacetic acid, preferably 1.1 times; the base is preferably at least one of triethylamine and N,N-diisopropylethylamine, and the amount of the base is 2-4 times the molar amount of H1-mGlu-linker, preferably 2 times.

[0026] Application of the above-mentioned self-assembled nanomaterial targeting BCR-ABL in the preparation of anti-CML drugs.

[0027] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0028] The use of polymers to encapsulate drugs to form self-assembled nanomaterials has the advantages of extending drug retention time and enhancing drug accumulation in tumor sites. The use of ROS / GSH dual-responsive linkers in the tumor microenvironment has the advantages of tumor-specific targeting, enhanced cell uptake, improved safety, and reduced toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The cytotoxicity (IC 50 In 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.

[0030] Figure 2 It is the toxicity of Bcr-Abl molecular glue to Bcr-Abl negative cell lines.

[0031] Figure 3 It is the cytotoxicity and degradation ability of KN1021 and Bcr-Abl molecular glue rH0-mGlu.

[0032] Figure 4 It is the CMC of cle-NP, non-NP and 780-NP measured by pyrene fluorescence probe method.

[0033] Figure 5 It is the characterization and stability test of self-assembled nanoparticles. In the figure, label AC is the TEM image of self-assembled nanoparticles cle-NP, non-NP and 780-NP; DF is the particle size of the three nanoparticles; G is the Zeta potential of the three nanoparticles; H is the change of particle size of cle-NP and non-NP over time; I is the change of characteristic peak of cle-NP over time determined by HPLC.

[0034] Figure 6 is the responsiveness change of self-assembled nanoparticles; in the figure, labels A and B are the particle size changes of the nanoparticles after incubation with H2O2; C and D are TEM images of the nanoparticles after incubation with H2O2; E and F are the particle size changes of the nanoparticles after incubation with GSH; G and H are TEM images of the nanoparticles after incubation with GSH.

[0035] Figure 7 It is the responsive release of self-assembled nanoparticles; in the figure, label A is HPLC showing the degradation of nanoparticles Cle-NP after incubation with H2O2; B is HPLC showing the changes of nanoparticles Cle-NP after incubation with GSH for different time periods.

[0036] Figure 8 It is the cellular uptake of self-assembled nanoparticles; in the figure, label A is the confocal microscopy observation of K562 cells for the uptake of IR-780 and 780-NP; B is the flow cytometry analysis of K562 cells for the uptake of IR-780 and 780-NP, and ctrl in B is the pure cell group.

[0037] Fig. 9 are the cytotoxicity and degradation ability of self-assembled nanoparticles; in the figure, label A is the anti-proliferation curve of cle-NP and non-NP in K562 cells; BF is the degradation of Bcr-Abl and Abl proteins in K562 cells by cle-NP and non-NP.

[0038] Fig.10 It is WB detection of self-assembled nanoparticles inducing cell apoptosis; in the figure, label A is flow cytometry detection of the effect of Cle-NP on K562 cell apoptosis; label B is WB detection of the effect of Cle-NP and Non-NP on the expression of apoptosis signaling proteins in K562 cells.

[0039] Fig.11 : is the effect of self-assembled nanoparticles on erythrocyte hemolysis and agglutination; in the figure, labels A and B are the erythrocyte hemolysis rate and image of cle-NP and H1-mGlu; C is the effect of cle-NP on erythrocyte agglutination.

[0040] Fig.12is the in vivo biodistribution of IR-780 and 780-NP; in the figure, labels A and B are the in vivo fluorescence images of small animals at different time points after tail vein injection of IR-780 and 780-NP; C and D are the in vitro tissue fluorescence images 72 hours after tail vein injection of IR-780 and 780-NP; E is the fluorescence change curve of blood at different time points after tail vein injection of IR-780 and 780-NP. Data are expressed as mean ± SD values, **P < 0.01 compared with IR-780 group in 780-NP group, n = 4.

[0041] Fig.13 This is an in vivo anti-tumor study of self-assembled nanoparticles. In the figure, label A is a flow chart of in vivo anti-tumor study of self-assembled nanoparticles; B is a picture of in vitro tumors of mice in each group after drug treatment; C is the weight of in vitro tumors of mice in each group after drug treatment; D is the growth curve of mouse tumor volume during drug treatment; E is the curve of changes in mouse weight during drug treatment. Data are expressed as mean ± SD, *P < 0.05, **P < 0.01, **P < 0.001 compared with the saline group, n = 5.

[0042] Fig.14 is the tumor inhibition rate of self-assembled nanoparticles.

[0043] Fig.15 The self-assembled nanoparticles degrade Bcr-Abl protein in tumors and induce apoptosis. In the figure, label A is the expression of Abl protein in mouse tumor tissue; B is Tunel staining of mouse tumor sections.

[0044] Fig.16 The effect of self-assembled nanoparticles on blood biochemical indicators of mice.

[0045] Fig.17 For the self-assembled nanoparticles cle-NP 1 H NMR data plot.

[0046] Fig.18 For self-assembled non-NP nanoparticles 1 H NMR data plot.

[0047] Fig.19 For the self-assembled nanoparticles 780-NP 1 H NMR data plot. DETAILED DESCRIPTION

[0048] The present invention is further described in detail below in conjunction with examples and drawings, but the embodiments of the present invention are not limited thereto. If specific conditions are not specified in the examples, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially. The cells used include K562 (ATCC number: CCL-243), BaF3 cells (ATCC number: HB-283), HEK 293T cells (ATCC number: CRL-3216), HL60 cells (ATCC number: CCL-240), molm3 cells (ATCC number: CRL-1552), and MV4-11 cells (ATCC number: CRL-9591).

[0049] Example 1 Synthesis of H1-mGlu

[0050]

[0051] 1.1 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, followed by addition of N-Boc-piperazine (2 eq) and triethylamine (3 eq) and stirring at 80 ° C in a sealed tube for 2 h. 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 give a white solid (1.72 g, yield 89.28%). LC-MS: m / z 441.0 [M+H] +.1 H NMR (400MHz, DMSO-d6) δ11.65 (s, 1H), 8.09 (s, 1H), 7.50-7.47 (m, 2H), 3.54 (t, J=4.0Hz, 4H), 3.37-3.36 (m, 4H), 1.41 (s, 9H). 13 C NMR (101MHz, DMSO) δ162.71, 154.51, 129.43, 124.07, 116.20, 80.57, 43.85, 28.38.

[0052] 1.2 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), 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, and then stirred in a sealed tube at 120°C for 5 h, and the product was confirmed by TLC and LC-MS. 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, yield 48.37%).

[0053] 1.3 Synthesis of molecular glue H1-mGlu: A mixture of (2E)-4-(4-methoxyphenyl)-4-oxybut-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-oxybut-2-enoic acid in the solution 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 intermediate 4 (460 mg, 156.07 μmol, 1 eq) was deprotected (DCM:TFA=3:1 (V / V)) for 1 hour, and the resulting amine (1 eq) was dissolved in DMF (0.1 M), and then added dropwise to the reaction mixture, and stirred overnight at room temperature. The reactant was diluted with EA, washed three times with a saturated solution of NaHCO3, a saturated solution of NH4Cl and a saturated solution of NaCl, 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%). HRMS (ESI+) calcd for C 30 H 28 N4O6S([M+H] + ):573.1730, found573.1783. 1H NMR (400MHz, DMSO-d6) δ11.58 (s, 1H), 9.56 (s, 1H), 8.05 (d, J = 8.0Hz, 2H), 7.81 (d, J = 12.0Hz, 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=8.0Hz, 8H), 3.24 (d, J=5.6Hz, 2H). 13 C NMR (101MHz, DMSO) δ188.05, 164.22, 158.63, 157.54, 134.12, 133.71, 133.08, 131.66 , 131.50, 129.93, 127.95, 120.89, 114.79, 107.95, 99.93, 56.12, 55.78, 45.84, 42.01.

[0054] Example 2 Preparation of cle-NP

[0055]

[0056] 2.1 Synthesis of H1-mGlu-linker and compound 5: H1-mGlu (200 mg, 349.26 μmol), 2-({3-[(2-methylpropan-2-yl)oxy]-3-oxyylidenepropyl}thio)propanoic acid (preparation method reference: J.Am.Chem.Soc.2019,141,15611-15618; DOI:10.1021 / jacs.9b07171)) (98 mg, 1.2 eq), DMAP (51.2 mg, 1.2 eq) were dissolved in anhydrous DMF (2.0 mL) and the solution was cooled to 0 ° C. EDC-HCl (87.04 mg, 1.3 mmol) was added dropwise to the solution and stirred at room temperature for 12 h. After completion, the mixture was diluted with EA (100 mL) and washed with aqueous sodium bicarbonate solution (100 mL×3). The crude product was purified by column chromatography (DCM / MeOH 0%-20%) to give compound 5 (151.20 mg, yield 54.87%). LC-MS found for C 17 H 17 N3O4S([M+H] + ):790.3. 1H NMR (400MHz, CDCl3) δ8.04 (d, J=8.0Hz, 2H), 7.995 (d, J=12.0Hz, 1H), 7.80 ( s, 1H), 7.50-7.47 (m, 3H), 7.33 (d, J=8.0Hz, 1H), 6.97 (d, J=8.0Hz, 2H), 6.8 2-6.79 (m, 3H), 3.89 (s, 3H), 3.82 (s, 3H), 3.73 (d, J=8.0Hz, 8H), 3.05-2.96 (m, 2H), 2.605 (dd, J=8.0, 4.0Hz, 2H), 1.595 (d, J=4.0Hz, 3H), 1.46 (s, 9H). 13 C NMR (101MHz, CDCl3) δ187.58, 171.55, 170.91, 164.38, 164.27, 157.10, 151.05, 135.05, 133.37, 131.36 , 131.24, 130.99, 129.87, 128.16, 122.63, 114.16, 113.52, 105.08, 81.09, 55.86, 55.60, 45.79, 42.09, 4 Compound 5 was dissolved in anhydrous dichloromethane (3 mL), trifluoroacetic acid (TFA) (1 mL) was added dropwise to the solution and the resulting mixture was stirred at room temperature for 2 h, and then the solvent was evaporated under low pressure to give H1-mGlu-linker, which was directly used in the next step after verification by thin layer chromatography.

[0057] 2.2 The mixture of the above carboxylic acid H1-mGlu-linker (60 mg, 1 eq) and HATU (34.25 mg, 1.1 eq) was dissolved in N, N-dimethylformamide (DMF) (0.1 M), triethylamine (TEA) (34.14 ul, 2 eq) was added and the reaction mixture was stirred at room temperature for 30 minutes, and mPEG-NH2 (n = 44, molecular weight: 2000 MWCO, Aladdin) (82.43 mg, 0.5 eq) was added dropwise, and the reaction mixture was stirred at room temperature for 24 h. After the coupling reaction was completed, the polymer solution was dialyzed with distilled water through a dialysis bag with a cutoff molecular weight of 2000 for 48 h, and then freeze-dried to obtain cle-NP (yield 40.29%), and then 1 H NMR analysis confirmed that the product ( Fig.17 ).

[0058] Example 3 Preparation of non-NP

[0059] H1-mGlu (60 mg, 104.78 μmol), mPEG-OTs (n=44, 111.30 mg, 0.5 eq, Meiluo Technology) and K2CO3 (28.96 mg, 2 eq) were dissolved in anhydrous acetonitrile (4 ml) and then stirred at 80°C under N2 protection for 12 h. The product was dissolved in distilled water, dialyzed against distilled water for 48 h using a dialysis bag with a molecular weight cutoff of 2000, and then freeze-dried to obtain non-NP (yield 46.11%). 1 H NMR analysis confirmed that the product ( Fig.18 ).

[0060] Example 4 Synthesis of IR780-NH

[0061]

[0062] IR-780 iodine (200 mg, 299.80 μmol) and piperazine (103.3 mg, 4 eq, 1.2 mmol) were dissolved in anhydrous DMF (15 mL) under nitrogen protection and stirred at 80 ° C for 4 h under light-proof conditions. After dilution with ethyl acetate (100 mL), it was washed with water (100 mL×3), and the crude product was purified by flash chromatography (DCM / MeOH=0-20%) to obtain IR780-NH (130 mg, yield 73.51%). 1 H NMR (400MHz, DMSO-d6) δ7.60(d,J=13.5Hz,2H),7.52(d,J=7.3Hz,2H),7.36-7.30(m,2H),7.28(d,J=7.8Hz,2H),7.13(t,J=7.3Hz,2H),5.99(d,J=1 3.6Hz,2H),4.03(t,J=7.0Hz,4H),3.71-3.64(m,4H),3.22-3.15(m,4H),2 .49-2.45(m,4H),1.76-1.65(m,6H),1.62(s,12H),0.92(t,J=7.4Hz,6H).

[0063] Example 5 Preparation of 780-NP

[0064] IR780-NH (30 mg, 50.86 μmol, 1 eq) and carbonyldiimidazole (12.37 mg, 1.5 eq) were dissolved in anhydrous DMF and stirred for 8 h at room temperature, under N2 protection and in the dark. mPEG-NH2 (n=44, 51.19 mg, 0.5 eq) and triethylamine (10.29 mg, 2 eq) were added and stirred at room temperature for 24 h. After the coupling reaction was completed, it was precipitated with excess ether, and then the polymer solution was dialyzed with distilled water for 48 h through a dialysis bag with a molecular weight cutoff of 2000, and then lyophilized to obtain IR780 NP (yield: 48.76%). 1 H NMR analysis confirmed that the product ( Fig.19 ).

[0065] Comparative Example 1: Synthesis of H0

[0066] The synthesis reference of H0 is S.-M. Yun et al. / Cancer Letters 348 (2014) 50-60.

[0067] The structure of H0 is as follows:

[0068]

[0069] Comparative Example 2: Synthesis of H1

[0070]

[0071] A mixture of compound 6 (1-(6-bromobenzo[d]thiazol-2-yl)-3-(2-hydroxyethyl)urea, 300 mg, 1 eq) (synthesis of compound 6, see S.-M.Yun et al. / Cancer Letters 348 (2014) 50-60), 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 then stirred at 90°C in a sealed tube for 4 h, and 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 a saturated NaCl solution, dried over Na2SO4, filtered, and distilled 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+) calculation for C 17 H 17 N3O4S([M+H] +):360.0940, found 360.1021. 1 H NMR (400MHz, DMSO-d6) δ10.63 (s, 1H), 9.54 (s, 1H), 7.86 (d, J=1.8Hz, 1H), 7 .57 (d, J=8.4Hz, 1H), 7.39 (dd, J=8.3, 1.8Hz, 1H), 7.12 (d, J=8.2Hz, 1H), 6.8 5(t, J=5.6Hz, 1H), 6.51 (d, J=2.3Hz, 1H), 6.45 (dd, J=8.2, 2.3Hz, 1H), 4.84 (d, J=5.2Hz, 1H), 3.72 (s, 3H), 3.49 (d, J=5.2Hz, 2H), 3.24 (d, J=5.6Hz, 2H). 13 C NMR (101MHz, DMSO) δ160.01, 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.

[0072] Comparative Example 3: Preparation of Molecular Glue H0-mGlu and rH0-mGlu

[0073]

[0074] 3.1 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, 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. 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. Purification by column chromatography (EA / hexane 0-33%) gave a white solid (1.05 g, 93%). LC-MS: m / z 257.1 [M+H] +.1 H NMR (400MHz, DMSO-d6) δ7.74 (s, 1H), 7.50 (s, 2H), 7.35-7.28 (m, 4H), 7.09 (d, J=8.0Hz, 1H), 7.01 (t, J=8.0Hz, 1H), 3.76 (s, 3H). 13CNMR (101MHz, CDCl3) δ165.86, 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.

[0075] 3.2 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 at 80 ° C in a sealed tube for 2 h. 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. Purification by column chromatography (EA / hexane 0-25%) gave a white solid 2 (1.60 g, yield 87%). LC-MS: m / z 468.2 [M+H] +.1 H NMR (400MHz, CDCl3) δ7.87 (s, 1H), 7.55 (s, 2H), 7.36-7.26 (m, 2H), 7.04 (t, J=8.0H z, 1H), 7.00 (d, J=8.0Hz, 1H), 3.83 (s, 3H), 3.61 (s, 4H), 3.49 (s, 4H), 1.48 (s, 9H). 13 C NMR (101MHz, DMSO) δ156.40, 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.

[0076] 3.3 Synthesis of H0-mGlu: A mixture of (2E)-4-(4-methoxyphenyl)-4-oxydebut-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-oxydebut-2-enoic acid in the solution 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 (1.0 eq) obtained after deprotection of compound 2 at room temperature (DCM:TFA=3:1) for 1 hour was dissolved in DMF (0.1 M), and then added dropwise to the reaction mixture, and stirred at room temperature overnight. The reactant was diluted with ethyl acetate (EA), washed three times with NaHCO3NH4Cl and NaCl saturated 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%). HRMS (ESI+) calcd for C 30 H 28 N4O5S([M+H] + ):557.1780, found 557.1870. 1 H NMR (400MHz, DMSO-d6) δ11.43 (s, 1H), 8.06 (d, J=8.0Hz, 1H), 7.89 (s, 1H), 7.82 (d, J=8.0Hz, 1H), 7.48-7.45 (m, 3 H), 7.36-7.31 (m, 3H), 7.13-7.10 (m, 3H), 7.03 (t, J=8.0Hz, 1H), 3.87 (s, 3H), 3.77 (s, 3H), 3.66 (d, J=8.0Hz, 8H). 13 C NMR (101MHz, DMSO) δ166.07, 164.23, 156.58, 134.12, 133.31, 133.31, 133.09, 131.67, 130.99, 12 9.99, 129.92, 129.22, 128.06, 122.65, 121.25, 114.80, 112.20, 56.14, 55.96, 45.92, 42.00, 8.95.

[0077] 3.4 Synthesis of rH0-mGlu: Replace the raw material "(2E)-4-(4-methoxyphenyl)-4-oxybut-2-enoic acid" in "Synthesis of 3.3 H0-mGlu" with "4-(4-methoxyphenyl)-4-oxybutyric acid (CAS No.: 3153-44-4, Bidex Pharmaceuticals)", and the remaining steps are the same as "Synthesis of 3.3 H0-mGlu". Yellow powder rH0-mGlu (81.40 mg, yield 89.84%) was obtained. HRMS (ESI+) calcd for C 30 H 30 N4O5S([M+H] + ):559.1937, found 559.2016. 1 HNMR (400MHz, CDCl3) δ7.99 (d, J=8.0Hz, 2H), 7.83 (s, 1H), 7.48 (s, 2H), 7.33 (s, 2H), 7.05-6.98 (m, 2H), 6.93 (d, J=12.0Hz, 2H), 3.85 (s, 3H), 3.82 (s, 3H), 3.75-3.67 (m, 8H), 3.35 (t, J=8.0Hz, 2H), 2.80 (t, J=8.0Hz, 2H). 13 C NMR (101MHz, CDCl3) δ197.49, 170.83, 163.60, 156.40, 134.06, 130.95, 130.40, 129.79, 12 8.81, 128.12, 122.55, 120.95, 113.76, 111.24, 55.60, 55.48, 45.26, 41.56, 33.24, 27.14.

[0078] Application Examples

[0079] (I) Selectivity of Bcr-Abl molecular glue and potential problems

[0080] The anticancer activity of the molecular glue was further explored by anti-proliferation experiments. Figure 1). The specific steps are as follows: (1) Cell seeding: Cells were seeded in 96-well plates at a rate of 5000 cells / well, with each well containing 90 μL of culture medium (Keygen Biotechnology). The well plates were placed in an incubator (37°C, 5% CO2) for overnight culture. (2) Cell administration: The compounds were graded diluted to a final test concentration of 50, 10, 2, 0.4, 0.08, 0.016, 0.0032, and 0.00064 μM, respectively. (3) Thiazolyl blue (MTT) assay for cell viability: After 72 hours of drug incubation, 10 μL of 5 mg / mL MTT solution (Sigma-Aldrich) was added to each well and incubated for 4 hours. Subsequently, 100 μL of a triple solution (10% SDS-5% isobutanol-0.012 M hydrochloric acid solution) was added to each well and incubated for 4 hours 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 survival rate at each concentration was calculated according to the formula.

[0081]

[0082] The data of K562 cells showed that molecular glues were more cytotoxic than the corresponding inhibitors, and H0-mGlu was 18 times more cytotoxic to K562 cells than the inhibitor H0 (IC 50 :166.4nM vs 9.0nM); H1-mGlu is 45 times that of H1 (145.3nM vs 3.2nM). At the same time, anti-proliferation experiments were conducted on H0-mGlu and H1-mGlu using Bcr-Abl and Bcr-AblT315I mutant cell lines. The results showed that H0-mGlu and H1-mGlu not only have obvious toxicity to Bcr-AblWT cells, but also have strong killing effects on T315I mutant cell lines, which are currently the most difficult to overcome in clinical practice, and are superior to H0 and H1.

[0083] The cell selectivity of molecular glue H0-mGlu and H1-mGlu was also verified by anti-proliferation experiments. First, other blood tumors, including Bcr-Abl negative cell lines such as MV4-11, HL-60 and Molm-13, were selected to test toxicity through anti-proliferation experiments. The data showed that the two molecular glues (H0-mGlu and H1-mGlu) also had a strong cell killing effect on Bcr-Abl negative cell lines. 50 All of them are less than 100nM. This shows that the introduced molecular glue handle significantly reduces the cell selectivity of compounds H0 and H1 while increasing cytotoxicity. To further verify whether the molecular glue has potential toxicity to normal cells, HEK293T cells and BaF3 parent cells were selected for toxicity tests. The data showed that the IC of H0-mGlu to HEK293T cells 50The IC of H1-mGlu for HEK293T cells is about 45nM, which is 5 times that of K562 cell line (9nM). 50 About 45nM, 15 times that of K562 cell line (3nM). IC of H0-mGlu and H1-mGlu for BaF3 parental cells 50 The concentrations of 100 nM and 200 nM were also less than 100 nM. This suggests that the molecular glue may have potential off-target toxicity, which will seriously limit its application in the treatment of CML. Figure 2 )

[0084] Inhibitors H0 and H1 have strong cell selectivity, but this selectivity is greatly reduced after the introduction of the molecular glue handle. It is speculated that this widespread cytotoxicity comes from the molecular glue handle itself. Therefore, the reasons for the widespread toxicity of molecular glue were explored in the hope of proposing a method to circumvent potential toxicity.

[0085] First, KN1021 (the synthetic route is known, refer to ACS Cent. Sci. 2023, 9, 5, 915-926) was tested for cytotoxicity through anti-proliferation experiments. The results showed that the compound with only a molecular glue handle introduced on a benzene ring can produce a strong cell-killing effect on blood tumor cells such as K562, MV4-11, HL-60 and Molm-13, and it also has obvious toxicity to normal cells HEK293T and BaF3 parent cells. This further verifies the conjecture that the extensive cytotoxicity comes from the molecular glue handle. Subsequently, the carbon-carbon double bond of the α, β-unsaturated carbonyl group in the molecular glue H0-mGlu handle was replaced with a single bond to obtain the molecular glue rH0-mGlu.

[0086] The protein degradation ability of rH0-mGlu was verified by WB experiment. The specific steps were as follows: (1) Cell seeding: 1×10 6Cells 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 and apoptosis signaling pathway, the drugs were incubated for 6 hours and 16 hours, respectively. (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 protein sample and heated in a metal bath at 100°C for 10 min. (4) Electrophoresis: Fix the prepared SDS-PAGE in the electrophoresis tank, and add the same volume and concentration of the above protein samples and protein markers (Biyuntian) to the lanes in turn. Turn on the electrophoresis, first perform slow electrophoresis at a constant voltage of 70V. After the protein sample in the lane is compressed into a thin line, adjust the voltage to 110V and continue fast electrophoresis. Use the protein marker band as an indicator, and stop the electrophoresis after the area where the target band is located is completely separated from the internal reference protein area. (5) Transfer: Take the above gel out of the gel plate, and place the gel and nitrocellulose membrane (NC membrane) together in a "sandwich" structure transfer clasp. Place the transfer clasp in the transfer tank, turn on the power, and start transfer at a constant current of 300mA. The transfer time is appropriately adjusted according to the molecular weight. For proteins with larger molecular weights such as Bcr-Abl, the transfer time is 150min. For proteins with smaller molecular weights such as CRKL, the transfer time can be fixed at 120min. The whole process is carried out in an ice bath. (6) Blocking: After the transfer is completed, the NC membrane is placed in a TBST solution containing 5% skim milk powder and incubated on a shaker at room temperature for 2 hours. (7) Incubation of primary antibody: According to the protein to be detected, the NC membrane is placed in the prepared primary antibody solution (c-ABL antibody (2862T, Cell Signaling Technology), β-Tubulin antibody (AF7011, Affinity Biosciences, diluted according to the instructions, using primary antibody diluent (Biyuntian)) and incubated on a shaker at 4°C overnight. (8) Incubation of secondary antibody (Qinke Biosciences, diluted according to the instructions, using TBST solution containing 5% skim milk powder): Use TBST to wash off 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 hour. (9) Chemiluminescent imaging: After using TBST to wash off the free secondary antibody on the NC membrane, place the membrane in a chemiluminescence instrument and image it with the help of ECL chemiluminescent solution.Finally, the grayscale value of the exposed bands was analyzed by Image J software to semi-quantitate protein expression.

[0087] Surprisingly, rH0-mGlu lost its degradation ability and could not degrade Bcr-Abl and Abl proteins even at a high concentration of 10 μM. Cytotoxicity showed that rH0-mGlu had no significant toxicity to the above tumor cells or normal cells ( Figure 3 ). Therefore, it is envisioned that the double bonds of the molecular glue handle can be further modified to make it a prodrug, or it can be encapsulated by a nano drug delivery system so that it can be released in a specific tumor microenvironment, thereby exerting a powerful anti-tumor effect.

[0088] In summary, although the new Bcr-Abl molecular glue degrader H1-mGlu can effectively degrade Bcr-Abl protein and has obvious cytotoxicity to CML and its drug-resistant cells, its extensive cytotoxicity may limit the application of this type of molecular glue in the treatment of CML. Therefore, responsive self-assembled nanomaterials are designed to accurately deliver and release the drug, mask the toxicity of the molecular glue to normal cells, and enable it to play a role in specific tumor sites.

[0089] 2. Characterization of self-assembled nanomaterials

[0090] First, in order to test whether the above-mentioned complexes have the ability to self-assemble, cle-NP, non-NP and 780-NP were dissolved in PBS respectively, and they were self-assembled into nanoparticles in aqueous solution. Their critical micelle concentrations (CMC) were determined by the pyrene fluorescent probe method, among which cle-NP was 0.68μM, non-NP was 0.59μM, and 780-NP was 1.48μM ( Figure 4 ), which indicates that the above three complexes are amphiphilic and can be easily self-assembled in aqueous solution.

[0091] In order to further explore the nanoparticles formed by self-assembly, the morphology of the nanoparticles was first observed by transmission electron microscopy (TEM), such as Figure 5 As shown in the figure, the three nanoparticles are spherical, with similar particle sizes and no obvious aggregates, indicating that they are stable in PBS solution. Dynamic light scattering analysis shows that their hydrodynamic diameters are similar, about 200nm, and the PDI values ​​are all around 0.2, indicating that the three complexes have formed stable nanoparticles and have good dispersibility in the solution. In addition, as Figure 5As shown in G, the surfaces of the three nanoparticles obtained by self-assembly are all negatively charged, which can effectively avoid aggregation between nanoparticles and plays an important role in maintaining the stability of the dispersed system. In addition, such nanoparticles can avoid non-specific adsorption with serum proteins that are also negatively charged in the blood circulation. The above data show that hydrophobic molecular glue can be wrapped into nanoparticles with the help of hydrophilic mPEG. The nanoparticles have a smaller nanoparticle size and dispersibility, and have a higher drug loading potential.

[0092] The chemical coupling prodrug system mainly relies on the breaking of the connecting bond. In theory, the self-assembled nanoparticles are stable before reaching the tumor microenvironment. In order to verify the stability of the nanoparticles, Cle-NP and Non-NP were dispersed in PBS buffer (0.1 mg sample dissolved in 1 ml PBS), and the particle size change of the nanoparticles was measured by DLS after being placed for different time periods. Figure 5 It can be seen from the H that the particle size of Cle-NP and Non-NP did not change significantly after being placed at room temperature for 48 hours.

[0093] In addition, high performance liquid chromatography (HPLC) was used to further verify the stability of Cle-NP. The specific steps were as follows: Cle-NP was dispersed in PBS buffer (0.1 mg sample was dissolved in 1 ml PBS), each sample was incubated at room temperature for 0, 12, 24, and 48 h, and HPLC analysis was performed using trifluoroacetic acid in acetonitrile as the mobile phase. The compound was gradient eluted in a C18 column for 30 min, and the changes in the elution time of the characteristic peaks were monitored and compared at an ultraviolet wavelength of 254 nm. Figure 5 As shown in Figure 1, within 48 hours, the peak time of the Cle-NP solution remained consistent, and no other substances were generated in the solution, which proved that the self-assembled nanoparticles had good stability.

[0094] (III) Verification of the responsiveness of responsive self-assembled nanomaterials

[0095] When tumor cells are in a state of hyperproliferation, the redox dynamics inside the cells are out of balance, and the overall state of oxidative stress is present. In tumor cells, the concentration of ROS is usually 100 times that of normal cells, and the concentration of GSH is 7-10 times that of normal cells. Based on the high concentration of GSH and ROS in tumor cells, H1-mGlu and mPEG are combined through a redox-responsive linker and self-assembled into nanoparticles.

[0096] In order to verify the responsive release of self-assembled nanoparticles, cle-NP or non-NP were incubated with GSH or H2O2 (molar ratio NP:GSH = 1:200, NP:H2O2 = 1:6). DLS showed that after incubation with H2O2, the particle size and distribution of Cle-NP changed significantly, while the particle size of Non-NP did not change significantly ( Figure 6 ). Then, the morphological changes of the two nanoparticles after reacting with H2O2 were observed under TEM. Figure 6 C and D show that Cle-NPs have no obvious nanomorphology after incubation with H2O2, while the morphology of Non-NPs does not change significantly.

[0097] Next, HPLC was used to analyze the release of Cle-NP under H2O2 stimulation, and further verify whether the released product was the original drug H1-mGlu. The data showed that after Cle-NP was incubated with H2O2 for 2 hours, a new product peak appeared at the same elution time as free H1-mGlu, indicating that under the action of H2O2, Cle-NP was able to release the original drug H1-mGlu. As the incubation time increased, the peak area of ​​H1-mGlu gradually increased, and at 16h, the characteristic peak of Cle-NP completely disappeared ( Figure 7 A). LC-MS analysis proved that the product was H1-mGlu. The above data fully demonstrate that Cle-NP can react with H2O2 and release H1-mGlu. In the GSH-responsive release experiment, it was found that under the stimulation of GSH, the particle size and morphology of Cle-NP changed significantly, but the morphology of Non-NP also seemed to change. Analysis of the nanoparticle structure revealed that there were two GSH reaction sites in the structure of this type of nanoparticle. In addition to the esterification reaction with the phenyl ester bond, GSH may also react with the carbon-carbon double bond on the molecular glue handle. HPLC data showed that after Cle-NP was incubated with GSH for 1 hour, while releasing a small amount of H1-mGlu, Cle-NP would preferentially react with GSH to form a Cle-NP-GSH complex. As time goes on, Cle-NP gradually reacts completely, and at this time it mainly exists in the form of a Cle-NP-GSH complex. Then, the excess GSH in the reaction system will further react with the Cle-NP-GSH complex to release the H1-mGlu-GSH complex ( Figure 7 Similarly, Non-NP and GSH will also generate Non-NP-GSH composite nanoparticles ( Figure 6 H).

[0098] Cle-NP can release H1-mGlu under both oxidative and reductive conditions because under oxidative stress, the sulfide group in the chemical linker will be oxidized to a sulfone / sulfoxide group, resulting in the conversion of the hydrophobic site into a hydrophilic site, which will release the active drug H1-mGlu after hydrolysis. Under reductive conditions, GSH will undergo sulfurization with the ester bond on the linker to form an intermediate and release the active drug H1-mGlu at the same time.

[0099] (IV) Study on cellular uptake and anticancer mechanism of self-assembled nanoparticles

[0100] Since cle-NP has no spontaneous fluorescence and cell uptake experiments cannot be performed, we replaced it with the same material to encapsulate the fluorescent dye IR780 for cell uptake experiments for verification.

[0101] The effective uptake of nanoparticles by tumor cells is a key factor in determining the anti-tumor effect of self-assembled nanoparticles. Therefore, the uptake of self-assembled nanoparticles by K562 cells was analyzed by confocal microscopy and flow cytometry. Figure 8 As shown in A, after incubating free IR-780 and 780-NP with K562 cells (concentration 5 μM), there was obvious red fluorescence in the cytoplasm of both groups of cells, and the fluorescence intensity increased with the incubation time. This shows that K562 cells can take up IR-780 and 780-NP in a time-dependent manner. At the same time, it was found that at the same incubation time, compared with free IR-780, the cells in the 780-NP group showed stronger fluorescence intensity. Flow cytometry data verified this phenomenon ( Figure 8 B), at the same dose and incubation time, the fluorescence intensity of 780-NP cells was stronger, further indicating that the cells have a stronger uptake ability for 780-NP than free IR-780.

[0102] Subsequently, the cytotoxicity of Cle-NP and Non-NP against K562 cells was evaluated by antiproliferation assay, as Fig. 9 As shown in A, Cle-NP has strong toxicity to K562 cells, IC 50 =74.8nM. Non-NP is almost non-toxic to cells (IC 50 >10μM). Subsequently, the protein degradation ability of the nanoparticles in K562 cells was verified by WB, e.g. Fig. 9 As shown in Figure B, Cle-NP degraded Bcr-Abl and Abl proteins in a concentration-dependent manner. 50 The concentrations of Cle-NP and H2O2 in tumor cells were 1.76 μM and 1.83 μM, respectively, while Non-NP failed to degrade protein even at 10 μM. This indicates that Cle-NP can effectively release H1-mGlu with the help of high concentrations of GSH and H2O2 in tumor cells, thereby exerting protein degradation and cell killing effects.

[0103] Next, in order to verify whether the cell killing effect produced by the nanoparticles is mediated by cell apoptosis. The apoptosis induced by Cle-NP was detected by flow cytometry. The specific steps were as follows: the drug was incubated with K562 cells for 16 hours, and then the cells were collected. According to the instructions of the apoptosis kit (KTA0010, Abbkine), the cells were stained with AnnexinV-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. The results are shown in Figure 2. Fig.10 As shown in A, Cle-NP can effectively induce cell apoptosis in a concentration-dependent manner. WB (WB specific steps are the same as above) data show ( Fig.10 B), Cle-NP can promote the production of Cle-Caspase3 and Cle-PARP proteins in a concentration-dependent manner, while inhibiting the expression of BCL2 and MCL1 proteins. However, Non-NP failed to induce changes in the expression of apoptosis-related proteins even at a high concentration of 10μM. The above results indicate that self-assembled nanoparticles Cle-NP can release H1-mGlu with the help of high concentrations of GSH and H2O2 in tumor cells, inducing mitochondria-dependent cell apoptosis, which may be one of the reasons why Cle-NP exerts strong cytotoxicity.

[0104] (V) In vivo biodistribution of self-assembled nanoparticles

[0105] The above in vitro data show that the self-assembled nanoparticles based on H1-mGlu can hide potential toxicity and have good cell efficacy. Therefore, the biodistribution of nanoparticles in vivo will continue to be explored. The high biocompatibility and low toxicity of nanoparticles are the basis for in vivo experiments. Therefore, the hemolysis and agglutination ability of H1-mGlu and Cle-NP nanoparticles on mouse erythrocytes were first tested. The specific operation of the erythrocyte hemolysis experiment is as follows: fresh blood samples are collected from healthy BALB / c nude mice through the orbital venous sinus, and the blood samples are stored in anticoagulation tubes. The whole blood is then diluted and centrifuged with normal saline (1500rpm, 10min), and the red blood cell pellet is washed 4 times with a physiological saturated NaCl solution to remove white blood cells, platelets and other plasma components. Finally, the red blood cells are diluted to a concentration of 2% (v / v) with normal saline.

[0106] Different concentrations of H1-mGlu or Cle-NP were placed in a constant temperature shaker together with the prepared red blood cell suspension and incubated at 37°C for 1.5 hours. After the incubation, the solution was centrifuged at 2000rpm for 10 minutes. The positive control and negative control were processed similarly, and the same volume of blood was directly mixed with ultrapure water and saline, respectively. Finally, the absorbance (OD) of the supernatant of each group was measured at a wavelength of 540nm using an ELISA reader (BioTek), and the degree of hemolysis was analyzed according to the following hemolysis rate calculation formula:

[0107]

[0108] (2) The specific operation of the red blood cell agglutination experiment is: observe the red blood cells incubated with Cle-NP under a microscope to determine whether Cle-NP can cause red blood cell agglutination. Fig.11 As shown in A and B (the concentration on the horizontal axis refers to the final concentration after H1-mGlu or Cle-NP is mixed with the prepared red blood cell suspension), compared with the saline group, H1-mGlu at 50μM will cause obvious hemolysis of red blood cells (hemolysis rate>5%), while Cle-NP nanoparticles still do not cause hemolysis of red blood cells at a concentration of 800μM (hemolysis rate<5%), indicating that Cle-NP has stronger biosafety than free H1-mGlu and meets the requirements for intravenous injection of preparations. In addition, Cle-NP nanoparticles will not cause red blood cell agglutination ( Fig.11 The above results show that Cle-NP has high biocompatibility and safety and meets the requirements of intravenous administration, which lays the foundation for the next in vivo experiment.

[0109] Next, the biodistribution and tumor targeting of self-assembled nanoparticles in vivo were studied in depth using a K562 tumor-bearing mouse model. First, the mice were given the same dose of IR-780 or 780-NP (3mM, 100μL saline) by tail vein injection. Then, the changes of fluorescence signals in the mice over time were analyzed using a small animal in vivo imaging system. Fig.12As shown in A, 3-6 hours after tail vein injection, the fluorescence signals of 780-NP and free IR-780 can be clearly detected in the mouse tumor tissue, and the signal intensity in the tumor of the 780-NP group is higher than that of the free IR-780 group. 24 hours after injection, the fluorescence values ​​of the tumor sites of both groups reached the maximum value, and then the signal intensity in the tumor tissue gradually decreased with time. Throughout the experiment, the fluorescence signal in the tumor of the mice in the free IR-780 group was always lower than that in the 780-NP group, reflecting that 780-NP has a stronger accumulation and stagnation time at the tumor site. 72 hours after the injection, the mice were dissected, and ex vivo fluorescence imaging was performed on the brain, heart, liver, spleen, lung, kidney and tumor tissues of the mice ( Fig.12 C). Consistent with the results of in vivo imaging, the tumor fluorescence intensity of the 780-NP group was higher than that of the free IR-780 group, indicating that 780-NP has good tumor targeting in vivo. It is worth noting that the lungs of the two groups of mice were also significantly increased. In addition, the blood circulation curves showed ( Fig.12 E), the fluorescence intensity of the IR-780 group dropped to 31% of the initial level within 6 hours, while that of the 780-NP group was 64%, indicating that the IR-780 concentration in the plasma of the 780-NP group decreased slowly, indicating that 780-NP has a longer blood drug half-life and can effectively prolong the blood circulation of the drug in the body.

[0110] (VI) In vivo anti-tumor and biosafety studies of self-assembled nanoparticles

[0111] Based on the excellent in vitro antitumor effect and efficient tumor targeting ability of NPs, the in vivo antitumor effect of nanoparticles was studied in K562 tumor-bearing mice. Considering the poor solubility of H0 and H1-mGlu and the inability to perform tail vein administration, the experiment was conducted using both intraperitoneal injection and tail vein injection. The overall grouping is as follows: Groups 1-5 are intraperitoneal injection groups, namely: (1) saline group, H0 group (25 mg / kg), H1-mGlu group (10 mg / kg), Non-NP group (equivalent to 10 mg / kg of H1-mGlu) and Cle-NP (equivalent to 10 mg / kg of H1-mGlu) group. Groups 6 and 7 are tail vein injection groups, namely: saline group and Cle-NP (equivalent to 5 mg / kg of H1-mGlu) group. Subsequently, the corresponding dosage was injected once every two days, and the tumor volume and mouse weight were recorded. The experimental period was 16 days ( Fig.13 A). Fig.13As shown in D, the tumors of mice in the normal saline group grew rapidly, the growth rate of the Non-NP group was consistent with the normal saline group, and the tumor growth of other drug-treated groups was inhibited to varying degrees. Among them, the tumor growth of the H0 group was slightly inhibited, which is similar to what was reported in the literature. The tumor growth inhibition of the H1-mGlu group was slightly higher than that of the H0 group. Regardless of whether it was an intraperitoneal injection or a tail vein injection group, Cle-NP showed significant tumor inhibition ability. The tumor inhibition rates of each group were: Cle-NP (iv) > Cle-NP (ip) > H1-mGlu > H0 ( Fig.14 ).

[0112] In order to further prove the anti-tumor mechanism of the drug, the expression of Bcr-Abl in tumor tissue was detected by WB (the specific steps are the same as above) Fig.15 A). The results showed that the expression of Bcr-Abl protein in tumors of the Cle-NP and H1-mGlu groups was significantly reduced, while the expression of Bcr-Abl protein in the Non-NP group did not change significantly. It is worth noting that Cle-NP can degrade Bcr-Abl more effectively than H1-mGlu, which may be due to the tumor accumulation effect of NP. The fresh organs were then paraffin-embedded and sectioned, and the sections were programmed to be dewaxed and hydrated. The tissue sections were then stained with TUNEL. Add 50μl of TUNEL detection solution to the sample and incubate at 37°C in the dark for 60 minutes. After washing 3 times with PBS or HBSS, the sections were sealed with anti-fluorescence quenching sealing solution and observed under a fluorescence microscope. The excitation wavelength range that can be used is 450-500nm, and the emission wavelength range is 515-565nm (green fluorescence). The results show that ( Fig.15 B), the cancer cell apoptosis in the Cle-NP group was more obvious than that in the other groups, which was consistent with the cytotoxicity data and further verified the treatment results.

[0113] In addition, it was found that during the treatment, the weight of mice in other groups increased slightly, while the weight of mice in the H1-mGlu group decreased significantly, suggesting that H1-mGlu may have potential toxic side effects. However, the H1-mGlu-based self-assembled nanoparticle group did not lose weight, suggesting that self-assembled nanoparticles have good biosafety ( Fig.16 Subsequently, the hepato-renal toxicity of the drug was detected by blood biochemistry ( Fig.16), the data showed that except for the H1-mGlu group, the biochemical indicators of other groups did not change significantly, further indicating that the nanoparticles have good biosafety. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of mice in the H1-mGlu group increased, suggesting that H1-mGlu may induce liver damage in mice. In addition, the levels of albumin (ALB) and alkaline phosphatase (ALP) related to hepatotoxicity, and urea nitrogen (BUN), urea (UA) and creatinine (CREA) related to renal function did not change significantly. In summary, the in vivo treatment results are consistent with the in vitro data, both proving that Cle-NP can avoid toxic side effects while enhancing the proteolytic ability and tumor killing effect of H1-mGlu, and has good biosafety.

[0114] 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 self-assembled nanomaterial targeting BCR-ABL, characterized in that It has the following structural formula:

2. A method for preparing a self-assembled nanomaterial targeting BCR-ABL according to claim 1, characterized in that The following steps are involved: (1) 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 process; (2) 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; (3) Compound 4 was deprotected by the tert-butyloxycarbonyl group by trifluoroacetic acid, and then reacted with (2E)-4-(4-methoxyphenyl)-4-oxyylidenebut-2-enoic acid to obtain the BCR-ABL targeting molecular glue H1-mGlu; (4) condensing the BCR-ABL-targeting molecular glue H1-mGlu with 2-({3-[(2-methylprop-2-yl)oxy]-3-oxyylidenepropyl}thio)propionic acid to generate compound 5, and then removing the tert-butyl ester to obtain H1-mGlu-linker; (5) H1-mGlu-linker undergoes amide condensation reaction with mPEG-NH2 to obtain cle-NP.

3. The method for preparing the self-assembled nanomaterial targeting BCR-ABL according to claim 2, characterized in that: The activation in step (1) 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 6-bromobenzo[d]thiazol-2-amine; The one-pot method described in step (1) 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.

4. The method for preparing the self-assembled nanomaterial targeting BCR-ABL according to claim 2, characterized in that: The Suzuki coupling reaction described in step (2) refers to a reaction at 100-120° C. for 5-7 hours.

5. The method for preparing the self-assembled nanomaterial targeting BCR-ABL according to claim 2, characterized in that: The step (3) of removing the tert-butyloxycarbonyl protection by trifluoroacetic acid refers to dissolving compound 4 in DCM, and then adding trifluoroacetic acid to react at room temperature for 1-2 hours to remove the tert-butyloxycarbonyl group; 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 for 8-12 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.

6. The method for preparing the self-assembled nanomaterial targeting BCR-ABL according to claim 2, characterized in that: The condensation reaction in step (4) refers to the reaction at room temperature for 12-24 hours in the presence of EDC-HCl and DMAP; The removal of tert-butyl ester by trifluoroacetic acid in step (4) refers to dissolving compound 5 in DCM, then adding trifluoroacetic acid and reacting at room temperature for 1-2 hours to remove the tert-butyl group; wherein the volume ratio of trifluoroacetic acid to dichloromethane is 1:2-3.

7. The method for preparing the self-assembled nanomaterial targeting BCR-ABL according to claim 2, characterized in that: The amide condensation reaction described in step (5) refers to a reaction at room temperature for 20-24 hours in the presence of a condensing agent and a base; wherein the condensing agent is 2-(7-azobenzotriazole)-tetramethyluronium hexafluorophosphate (HATU), and its amount is 1.1-2.5 times the molar amount of the product after H1-mGlu-linker is deprotected by the tert-butyloxycarbonyl group 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 H1-mGlu-linker.

8. Use of the BCR-ABL-targeting self-assembled nanomaterial according to claim 1 in the preparation of an anti-chronic myeloid leukemia drug.

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