Preparation method and use of self-assembled nanomaterials targeting Bcr-Abl

Abstract

The application belongs to the field of pharmaceutical chemistry, and discloses a preparation method and application of a self-assembled nanomaterial targeting Bcr-Abl. The structure of the self-assembled nanomaterial targeting Bcr-Abl is shown in the specification. The self-assembled nanomaterial targeting BCR-ABL provided by the application can retain high toxicity of the drug to tumors, improve accumulation of the drug at the tumor site, reduce toxicity of the drug to normal tissues, and thus reduce the occurrence of side effects.

Description

Technical Field

[0001] The present invention belongs to the field of medicinal 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 genetic mutations in hematopoietic stem cells. Studies have shown that 90% to 95% of CML patients harbor the Philadelphia (Ph) chromosome, 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), indicating its key role in driving the pathological progression of CML and ALL.

[0003] With the emergence of targeted drugs, cancer treatment has ushered in a new era of precision medicine. 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 mechanisms within the cell, and can achieve specific and efficient removal of target proteins. According to different mechanisms of action, 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-targeted 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, they regulate protein ubiquitination and, through the UPS, target protein degradation. The discovery of molecular glues currently relies primarily on serendipitous discoveries and lacks unified discovery principles and systematic evaluation methods. Furthermore, molecular glues have poor target selectivity and often result in significant toxicity.

[0005] In recent years, the nano drug delivery system has developed rapidly, and compared with traditional small molecule drugs, it has obvious advantages in improving drug solubility, stability, targeting and controllable release. By encapsulating small molecule drugs, the nano delivery system can enhance the in vivo retention time and tumor accumulation of drugs, achieve passive targeting by means of high permeability and long retention (EPR) effect, and achieve active targeting by means of tumor-specific ligand, so as to improve the efficacy. In addition, the nano drug also has the ability to respond to the release of drugs in the tumor microenvironment, reducing the toxic side effects. Therefore, it is necessary to provide a self-assembled nano material targeting Bcr-Abl, which can retain the high toxicity of the drug to the tumor, improve the tumor accumulation, reduce the toxicity of the drug to normal tissues, and thus reduce the occurrence of side effects. SUMMARY

[0006] The adverse reactions and side effects of drugs can seriously affect the survival rate and compliance of patients taking the drugs. In order to overcome the shortcomings and deficiencies of the prior art, the primary purpose of the present application is to provide a self-assembled nano material targeting BCR-ABL, which can retain the high toxicity of the drug to the tumor, improve the accumulation of the drug at the tumor site, reduce the toxicity of the drug to normal tissues, and thus reduce the occurrence of side effects.

[0007] Another purpose of the present application is to provide a preparation method of the self-assembled nano material targeting BCR-ABL.

[0008] Another purpose of the present application is to provide a self-assembled nano material targeting BCR-ABL.

[0009] The purposes of the present application are achieved by the following solutions:

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

[0011]

[0012] A preparation method of the self-assembled nano material targeting BCR-ABL, comprising the following steps:

[0013] (1) 6-bromobenzo[d]thiazol-2-amine is first activated by carbonyl diimidazole to obtain an intermediate product, and then N-Boc-piperazine is added to further react by one-pot method to obtain compound 3;

[0014] (2) Compound 3 and 3-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) phenol undergo suzuki coupling reaction to obtain compound 4;

[0015] (3) Compound 4 is subjected to amidation condensation reaction with (2E)-4-(4- methoxyphenyl)-4-oxoacrylate after removal of tert-butyloxycarbonyl protection by trifluoroacetic acid, to obtain the target BCR-ABL molecule glue H1-mGlu;

[0016] (4) H1-mGlu is subjected to condensation reaction with 2-({3-[(2-methylpropan-2- yl)oxy]-3-oxopropyl}thio)propanoic acid to generate compound 5, and then tert-butyl ester is removed to obtain H1-mGlu-linker;

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

[0018]

[0019] The activation in step (1) refers to reaction under inert gas or nitrogen protection at room temperature for 8-12 h, and the reaction solvent is at least one of DMF and DCM; the amount of carbonyldiimidazole used in the activation process is 1.5-3 times, preferably 2 times, the molar amount of 6-bromo-benzo[d]thiazol-2-amine;

[0020] The one-pot method in step (1) refers to 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.

[0021] The suzuki coupling reaction in step (2) refers to reaction at 100-120°C for 5-7 h;

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

[0023] The amidation condensation reaction in step (3) refers to reaction in the presence of a condensing agent and a base at room temperature for 8-12 h; wherein the condensing agent is preferably 2-(7-azobenzo triazole)-tetramethyl urea hexafluorophosphate (HATU), and the amount thereof is 1.1-2.5 times, preferably 1.2 times, the molar amount of the product after removal of tert-butyloxycarbonyl protection of compound 4 by trifluoroacetic acid; the base is preferably at least one of triethylamine and N,N-diisopropyl ethylamine, and the amount of the base is 2-4 times, preferably 2 times, the molar amount of the product after removal of tert-butyloxycarbonyl protection of compound 4 by trifluoroacetic acid.

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

[0025] The amide condensation reaction in step (5) refers to the reaction in the presence of a condensing agent and a base at room temperature for 20-24 h. The condensing agent is preferably 2-(7-azobenzo triazole)-tetramethyl urea hexafluorophosphate (HATU), and the amount of the condensing agent is 1.1-2.5 times, preferably 1.1 times, the molar amount of the product after the removal of tert-butyloxycarbonyl protection of H1-mGlu-linker by trifluoroacetic acid; the base is preferably at least one of triethylamine and N,N-diisopropyl ethylamine, and the amount of the base is 2-4 times, preferably 2 times, the molar amount of H1-mGlu-linker.

[0026] The application of the self-assembled nanomaterial targeting BCR-ABL in the preparation of anti-CML drugs.

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

[0028] The self-assembled nanomaterial formed by wrapping the drug with the polymer has the advantages of prolonging the retention time of the drug and enhancing the accumulation of the drug at the tumor site. The linker responsive to ROS / GSH under the tumor microenvironment has the advantages of tumor-specific targeting, enhanced cell uptake, improved safety and reduced toxic side effects. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 is the cytotoxicity (IC 50 ) results of all compounds of the application on CML cells. In the figure, the symbol 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-Abl T315I.

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

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

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

[0033] Figure 5 Figure 1 is the characterization and stability test of self-assembled nanoparticles; in the figure, labels A-C are TEM images of self-assembled nanoparticles cle-NP, non-NP and 780-NP; D-F are the particle sizes of the three kinds of nanoparticles; G is the Zeta potential of the three kinds of 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 Figure 2 is the responsive change of self-assembled nanoparticles; in the figure, labels A and B are the change of particle size of nanoparticles after incubation with H2O2; C and D are TEM images of nanoparticles after incubation with H2O2; E and F are the change of particle size of nanoparticles after incubation with GSH; G and H are TEM images of nanoparticles after incubation with GSH.

[0035] Figure 7 Figure 3 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 change of nanoparticles Cle-NP after incubation with GSH for different time periods.

[0036] Figure 8 Figure 4 is the cellular uptake of self-assembled nanoparticles; in the figure, label A is the confocal microscope observation of the uptake of IR-780 and 780-NP by K562 cells; B is the flow cytometry analysis of the uptake of IR-780 and 780-NP by K562 cells, and B is the pure cell group.

[0037] Figure 9 Figure 5 is 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; B-F are the degradation of Bcr-Abl and AbI proteins in K562 cells by cle-NP and non-NP.

[0038] Figure 10 Figure 6 is the WB detection of the apoptosis induced by self-assembled nanoparticles; in the figure, label A is the flow cytometry detection of the effect of Cle-NP on K562 cell apoptosis; label B is the WB detection of the effect of Cle-NP and Non-NP on the expression of apoptosis signal proteins in K562 cells.

[0039] Figure 11 Figure 7 is the effect of self-assembled nanoparticles on erythrocyte hemolysis and agglutination; in the figure, labels A, 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] Figure 12is the in vivo biodistribution of IR-780 and 780-NP; in the figure, labels A, B are the small animal live fluorescence images of different time points after tail vein injection of IR-780 and 780-NP; C, D are the ex vivo tissue fluorescence images 72 h 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 value, **P < 0.01 compared with IR-780 group, n = 4.

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

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

[0043] Figure 15 is the degradation of Bcr-Abl protein in tumor and the induction of apoptosis of self-assembled nanoparticles; in the figure, label A is the expression of Abl protein in the tumor tissue of mice; B is the Tunel staining of tumor sections of mice.

[0044] Figure 16 is the effect of self-assembled nanoparticles on the biochemical indicators of mouse blood.

[0045] Figure 17 is the in vivo anti-tumor study of self-assembled nanoparticles cle-NP 1 H NMR data chart.

[0046] Figure 18 is the in vivo anti-tumor study of self-assembled nanoparticles non-NP 1 H NMR data chart.

[0047] Figure 19 is the in vivo anti-tumor study of self-assembled nanoparticles 780-NP 1 H NMR data chart. DETAILED DESCRIPTION

[0048] The present application will be described in further detail below with reference to Examples and the accompanying drawings, but the embodiments of the present application are not limited thereto. In the Examples, unless specific conditions are noted, they are performed under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, unless the manufacturer is noted, are all conventional products that can be obtained by commercial purchase. The cells used include K562 (ATCC No. CCL-243), BaF3 cells (ATCC No. HB-283), HEK 293T cells (ATCC No. CRL-3216), HL60 cells (ATCC No. CCL-240), molm3 cells (ATCC No. CRL-1552), MV4-11 cells (ATCC No. 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 dry DMF and stirred at room temperature under N2protection for 8 h, followed by the addition of N-Boc-piperazine (2 eq) and triethylamine (3 eq) and stirring at 80 °C in a sealed tube for 2 h. After completion of the reaction, it was concentrated under vacuum. The combined organic extracts were washed with saturated NaCl solution, dried over Na2SO4, filtered, and distilled under reduced pressure. The crude product was purified by column chromatography (EA / hexane 0-33%) to obtain a white solid (1.72 g, yield 89.28%). 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.

[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), K2CO3(3 eq) was dissolved in 6 mL 1,4-dioxane and 2 mL H2O, then stirred at 120 °C in a sealed tube for 5 h, the product was confirmed by TLC and LC-MS. After solvent evaporation, the crude product was extracted between EA (3 x 30 mL) and water, the combined organic extracts were washed with saturated NaCl solution, dried over Na2SO4, 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- oximino but-2-enoic acid (CAS number: 5711-41-1, Bide Pharmaceutics, 1.1 eq) and 2-(7-azobenzotriazol)-tetramethyluronium hexafluorophosphate (HATU) (1.2 eq) was dissolved in N,N-dimethylformamide (DMF) (concentration of (2E)-4-(4-methoxyphenyl)-4-oximino but-2-enoic acid in solution was 0.1 M), then triethylamine (TEA) (2 eq) was added, and the reaction mixture was stirred at room temperature for 30 min. The amine (1 eq) obtained after deprotection of intermediate 4 (460 mg, 156.07 pmol, 1 eq) (DCM:TFA = 3:1 (V / V)) for 1 h was dissolved in DMF (0.1 M), then added dropwise to the reaction mixture, stirred at room temperature overnight. The reaction was diluted with EA, washed with saturated NaHCO3 solution, saturated NH4Cl solution and saturated NaCl solution respectively for 3 times, then dried with anhydrous Na2SO4, filtered, distilled 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, yield 49.59%). HRMS (ESI+) calcd for C 30 H 28 N4O6S([M+H] + ): 573.1730, found 573.1783. 1H 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, 8.0 Hz, 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 C NMR (101 MHz, DMSO) δ 188.05, 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 pmol), 2-({3-[(2-methylpropan-2-yl)oxy]-3-oxopropyl}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, to this solution was added EDC-HCl (87.04 mg, 1.3 mmol) dropwise, 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 x 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. The solvent was then 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, 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 hours. After the coupling reaction was completed, the polymer solution was dialyzed with distilled water through a dialysis bag with a molecular weight cutoff of 2000 for 48 hours and then freeze-dried to obtain cle-NP (yield 40.29%). 1 H NMR analysis confirmed that the product ( Figure 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, Meiro Technology) and K2CO3(28.96 mg, 2 eq) were dissolved in anhydrous acetonitrile (4 ml) and then stirred at 80 °C under N2protection for 12 h. The product was dissolved in distilled water, dialyzed against distilled water using a dialysis bag with a molecular weight cut-off of 2000 for 48 h, and then lyophilized to obtain non-NP (yield 46.11%), which was analyzed by 1 H NMR and confirmed the product Figure 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, stirred at 80 °C for 4 h in the dark. After dilution with ethyl acetate (100 mL), washed with water (100 mL x 3), the crude product was purified by flash chromatography (DCM / MeOH=0~20%) to obtain IR780-NH (130 mg, yield 73.51%). 1 H NMR (400 MHz, DMSO-d6) δ 7.60 (d, J = 13.5 Hz, 2H), 7.52 (d, J = 7.3 Hz, 2H), 7.36 - 7.30 (m, 2H), 7.28 (d, J = 7.8 Hz, 2H), 7.13 (t, J = 7.3 Hz, 2H), 5.99 (d, J = 13.6 Hz, 2H), 4.03 (t, J = 7.0 Hz, 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 = 7.4 Hz, 6H).

[0063] Example 5 Preparation of 780-NP

[0064] IR780-NH (30 mg, 50.86 pmol, 1 eq) and carbonyldiimidazole (12.37 mg, 1.5 eq) were dissolved in anhydrous DMF and stirred at room temperature, under N2protection, in the dark for 8 h. 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 completion of the coupling reaction, the polymer solution was precipitated with excess ether, then dialyzed against distilled water through a dialysis bag with a molecular weight cutoff of 2000 for 48 h, and then lyophilized to obtain IR780 NP (yield: 48.76%). The product was analyzed by 1 H NMR analysis and confirmed the product Figure 19 ).

[0065] Comparative Example 1: Synthesis of H0

[0066] Reference for the synthesis of H0 S.-M. Yun et al. / Cancer Letters 348 (2014) 50-60.

[0067] The structure of H0 is shown below:

[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) (reference for the synthesis of compound 6 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), K3PO4(2 eq) was dissolved in 10 mL of 1,4-dioxane and 1 mL of H2O, then stirred in a sealed tube at 90 °C for 4 h, and the product was confirmed by TLC and LC-MS. After evaporating the solvent, EA (3 x 30 mL) was used for extraction, the combined organic extracts were washed with saturated NaCl solution, dried over Na2SO4, filtered, and distilled under reduced pressure. The crude product was purified by column chromatography (DCM / MeOH 0%~3%) to obtain the target compound H1 (234 mg, yield 68.62%). HRMS (ESI+) calcd for C 17 H 17 N3O4S ([M+H] +): 360.0940, found 360.1021. 1 H 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.

[0072] 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), K2CO3(1.81 g, 3 eq) was dissolved in 6 mL of 1,4-dioxane and 2 mL of H2O and then stirred at 120 °C in a sealed tube for 5 h, formation of the product was confirmed by TLC and LC-MS. After evaporation of the solvent, the crude product was extracted between EA (3 x 30 mL) and water, 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%) afforded a white solid (1.05 g, 93%). 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). 13CNMR (101 MHz, CDC13) 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.

[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 dry DMF and stirred at room temperature under N2protection for 8 h, followed by the addition of N-Boc-piperazine (1.46 g, 7.82 mmol) and triethylamine (1.19 g, 3 eq) and stirred at 80 °C in a sealed tube for 2 h. After completion of the reaction, it was concentrated under vacuum. The combined organic extracts were washed with saturated NaCl solution, dried over Na2S04, filtered, and distilled under reduced pressure. Purification by column chromatography (EA / hexane 0-25%) afforded 2 as a white solid (1.60 g, 87% yield). LC-MS: m / z 468.2 [M+H] +.1 H NMR (400 MHz, CDC13) 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.

[0076] 3.3 Synthesis of H0-mGlu: A mixture of (2E)-4-(4-methoxyphenyl)-4- oximino but-2-enoic acid (CAS Number: 5711-41-1, Bide Pharmatech) (1.1 eq) and 2-(7-azobenzotriazol)-tetramethyluronium hexafluorophosphate (HATU) (1.2 eq) was dissolved in N,N-dimethylformamide (DMF) (0.1 M concentration of (2E)-4-(4-methoxyphenyl)-4-oximino but-2-enoic acid in solution) followed by the addition of triethylamine (TEA) (2 eq) and the reaction mixture was stirred at room temperature for 30 minutes. The amine (1.0 eq) obtained after deprotection of compound 2 (DCM:TFA = 3:1) for 1 hour at room temperature was dissolved in DMF (0.1 M) and then added dropwise to the reaction mixture with stirring at room temperature overnight. The reaction was diluted with ethyl acetate (EA), washed with saturated solutions of NaHCO3, NH4Cl and NaCl successively 3 times, then dried with anhydrous Na2SO4, filtered and distilled under reduced pressure. The crude product was purified by silica gel chromatography (MeOH / DCM = 0-5%) to give 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 (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) δ 166.07, 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.

[0077] 3.4 Synthesis of rH0-mGlu: Replace the starting material "(2E)-4-(4- methoxyphenyl)-4-oximino but-2-enoic acid" in "3.3 Synthesis of H0-mGlu" with "4-(4-methoxyphenyl)-4-oximino butanoic acid (CAS No.: 3153-44-4, Bide Pharmatech) ", and the rest of the steps are the same as "3.3 Synthesis of H0-mGlu". rH0-mGlu (81.40 mg, yield 89.84%) was obtained as a yellow powder. HRMS (ESI+) calcd for C 30 H 30 N4O5S([M+H] + ): 559.1937, found 559.2016. 1 HNMR (400 MHz, CDC13) δ 7.99 (d, J = 8.0 Hz, 2H), 7.83 (s, 1H), 7.48 (s, 2H), 7.33 (s, 2H), 7.05-6.98 (m, 2H), 6.93 (d, J = 12.0 Hz, 2H), 3.85 (s, 3H), 3.82 (s, 3H), 3.75-3.67 (m, 8H), 3.35 (t, J = 8.0 Hz, 2H), 2.80 (t, J = 8.0 Hz, 2H). 13 C NMR (101 MHz, CDC13) δ 197.49, 170.83, 163.60, 156.40, 134.06, 130.95, 130.40, 129.79, 128.81, 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) Selective exploration and potential problems of Bcr-Abl molecular glue

[0080] Further explore the anti-cancer activity of molecular glue by anti-proliferation experiment Figure 1Specifically, the steps are as follows: (1) cell plating: cells were plated in 96-well plates at a cell number of 5000 per well, each well containing 90 μL of culture medium (Keygen Biotech). The well plate was placed in an incubator (37°C, 5% CO2) for overnight culture. (2) cell drug administration: the compound was gradiently 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 h of drug incubation, 10 μL of 5 mg / mL MTT solution (Sigma-Aldrich) was added to each well and incubated for 4 h. Subsequently, 100 μL of triplex 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 an enzyme-labeled instrument (BioTek), and the cell survival rate at each concentration was calculated according to the formula.

[0081]

[0082] K562 cell data showed that the molecular glue had stronger cytotoxicity than the corresponding inhibitor, in which H0-mGlu had 18 times of cytotoxicity to K562 than H0 (IC 50 : 166.4 nM vs 9.0 nM); H1-mGlu was 45 times of H1 (145.3 nM vs 3.2 nM). Meanwhile, the anti-proliferation experiment of H0-mGlu and H1-mGlu was performed using the transfected Bcr-Abl and Bcr-Abl T315I mutant cell lines, and the results showed that H0-mGlu and H1-mGlu not only had obvious toxicity to Bcr-Abl WT cells, but also had strong killing effect on the currently most difficult to overcome T315I mutant cell line, and were superior to H0 and H1.

[0083] The cell selectivity of the molecular glue H0-mGlu and H1-mGlu was also verified by the anti-proliferation experiment. First, other blood tumors, including Bcr-Abl negative cell lines such as MV4-11, HL-60 and Molm-13, were selected to detect the toxicity by the anti-proliferation experiment. The data showed that the two molecular glues (H0-mGlu and H1-mGlu) also had strong cell killing effect on the Bcr-Abl negative cell lines, with IC 50 less than 100 nM. This indicated that the introduction of the molecular glue handle not only increased the cytotoxicity, but also significantly reduced the cell selectivity of the compounds H0 and H1. To further verify whether the molecular glue had potential toxicity to normal cells, HEK293T cells and BaF3 parent cells were selected for toxicity test, and the data showed that the IC 50about 45 nM, 5 times of K562 cell line (9 nM), H1-mGlu IC 50 about 45 nM, 15 times of K562 cell line (3 nM). H0-mGlu and H1-mGlu IC 50 are also less than 100 nM. This suggests that the molecular glue may have potential off-target toxicity, which will seriously limit its application in treating CML. Figure 2

[0084] Inhibitors H0 and H1 themselves have strong cell selectivity, but when the molecular glue handle is introduced, this selectivity is greatly reduced. It is speculated that this extensive cytotoxicity comes from the molecular glue handle itself. Therefore, the reason for the extensive toxicity of the molecular glue was explored in order to propose a method to avoid potential toxicity.

[0085] Firstly, KN1021 (synthetic route known, refer to ACS Cent. Sci. 2023, 9, 5, 915-926) was tested for cytotoxicity by antiproliferation experiments, and the results showed that the compound with only a molecular glue handle on one benzene ring could produce strong cell killing effect on K562, MV4-11, HL-60 and Molm-13 blood tumor cells, and also had obvious toxicity to normal cells HEK293T and BaF3 parental cells. This further verifies the guess 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 by a single bond to obtain the molecular glue rH0-mGlu.

[0086] The protein degradation ability of rH0-mGlu was verified by WB experiments, and the specific steps were as follows: (1) cell plating: according to 1×10 6 ​Cells were seeded in 6-well plates with a cell count of 100 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: 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 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 a Bradford protein concentration assay kit. Finally, 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 above protein samples and protein markers (Biyuntian) of the same volume and concentration 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 area of ​​the internal reference protein. (5) Transfer: Remove the above gel from 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 adjusted appropriately 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 entire 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, AffinityBiosciences, diluted according to the instructions, diluted with 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, diluted with 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) Chemiluminescence 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 chemiluminescence solution.Finally, the gray value of the exposed band was analyzed by Image J software to semi-quantitatively analyze the protein expression.

[0087] Surprisingly, rH0-mGlu lost the ability to degrade, even at a high concentration of 10 μM, Bcr-Abl and Abl proteins. Cytotoxicity showed that rH0-mGlu had no significant toxicity to the above tumor cells or normal cells Figure 3 Therefore, it is envisaged that the double bond of the molecular glue handle will be further modified to become a prodrug, or it will be wrapped by a nano drug delivery system, so that it will be released in a specific tumor microenvironment, and thus exert a strong 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 CML treatment, therefore, the design of responsive self-assembled nanomaterials for precise delivery and release of the drug can mask the toxicity of the molecular glue to normal cells, so that it can play a role in a specific tumor site.

[0089] (ii) Characterization of self-assembled nanomaterials

[0090] First, in order to test whether the above complex has self-assembly ability, cle-NP, non-NP and 780-NP were dissolved in PBS to form nanoparticles in aqueous solution. The critical micelle concentration (CMC) of them was determined by pyrene fluorescence probe method, in which cle-NP was 0.68 μM, non-NP was 0.59 μM, and 780-NP was 1.48 μM Figure 4 ), which indicates that the above three complexes have amphiphilic properties and are easy to self-assemble in aqueous solution.

[0091] In order to further explore the nanoparticles formed by self-assembly, first of all, the morphology of the nanoparticles was observed by transmission electron microscopy (TEM), as Figure 5 shown, the three kinds of nanoparticles are spherical, the particle size is similar, and no obvious aggregates are found, indicating that they are stable in PBS solution. Dynamic light scattering analysis showed that their hydrodynamic diameters were similar, about 200 nm, and the PDI values were about 0.2, indicating that the three complexes all formed stable nanoparticles and had good dispersibility in solution. In addition, as Figure 5As shown in FIG. 1G, the three nanoparticles obtained by self-assembly all have negative charges on the surface, which can effectively avoid aggregation between nanoparticles, and plays an important role in maintaining the stability of the dispersion system. Moreover, such nanoparticles can avoid non-specific adsorption with serum proteins with negative charges in blood circulation. The above data show that hydrophilic mPEG can be used to encapsulate hydrophobic molecules into nanoparticles, and the nanoparticles have smaller nanoparticle size and dispersity, and have higher drug loading potential.

[0092] The chemical conjugation prodrug system mainly relies on the cleavage 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 was dissolved in 1 ml PBS), and the particle size change of the nanoparticles was measured by DLS after different time. Figure 5 As shown in FIG. 1H, 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, the stability of Cle-NP was further verified by high performance liquid chromatography (HPLC). The specific steps are as follows: Cle-NP was dispersed in PBS buffer (0.1 mg sample was dissolved in 1 ml PBS), and each sample was incubated at room temperature for 0, 12, 24, and 48 hours, respectively. Trifluoroacetic acid in acetonitrile was used as the mobile phase for HPLC analysis, and the compound was gradient eluted in the C18 column for 30 min. The change of peak time of characteristic peak was monitored and compared under the ultraviolet wavelength of 254 nm. As shown in FIG. 1I, the peak time of Cle-NP solution remained consistent within 48 hours, and no other substances were generated, thereby proving that the self-assembled nanoparticles have good stability. Figure 5

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

[0095] When tumor cells are in a state of excessive proliferation, the internal redox dynamics of the cells is unbalanced, and the overall state is oxidative stress. 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 were combined through an oxidation-reduction responsive linker, and self-assembled into nanoparticles.

[0096] ​To verify the responsive release of self-assembled nanoparticles, cle-NP or non-NP were incubated with GSH or H202, respectively (molar ratio NP:GSH = 1 :200, NP:H202 = 1 :6). DLS showed that the size and distribution of Cle-NP changed significantly after incubation with H202, while the size of Non-NP did not change significantly Figure 6 Subsequently, the morphological changes of the two nanoparticles after reaction with H202 were observed under TEM, Figure 6 Figures C and D show that Cle-NP did not have obvious nano-morphology after incubation with H202, while the morphology of Non-NP did not change significantly.

[0097] Next, the release of Cle-NP under H202 stimulation was analyzed by HPLC, and it was further verified whether the release product was the original drug H1-mGlu. The data showed that after Cle-NP was incubated with H202 for 2 hours, a new product peak appeared at the same elution time as free H1-mGlu, indicating that under the action of H202, Cle-NP could release the original drug H1-mGlu. With the extension of incubation time, the peak area of H1-mGlu gradually increased, and at 16 h, the characteristic peak of Cle-NP completely disappeared Figure 7 LC-MS analysis proved that the product was H1-mGlu. The above data fully showed that Cle-NP could react with H202 and release H1-mGlu. In the GSH-responsive release experiment, it was found that under the stimulation of GSH, the size and morphology of Cle-NP changed significantly, but the morphology of Non-NP also seemed to change. Analysis of the structure of the nanoparticles found that there were two GSH reaction sites in the structure of this type of nanoparticles. 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, a small amount of H1-mGlu was released, and Cle-NP preferentially reacted with GSH to form a Cle-NP-GSH complex. With the extension of time, Cle-NP gradually completely reacted, at this time mainly in the form of 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 will also generate Non-NP-GSH complex nanoparticles after reacting with GSH Figure 6

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

[0099] (IV) Cell uptake of self-assembled nanoparticles and anti-cancer mechanism

[0100] Since cle-NP has no spontaneous fluorescence, it is replaced with the same material to wrap the fluorescent dye IR780 to do cell uptake experiment to verify.

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

[0102] Subsequently, the cytotoxicity of Cle-NP and Non-NP to K562 was evaluated by anti-proliferation experiment, as shown in Figure 9 A, Cle-NP has strong toxicity to K562 cells, IC 50 = 74.8 nM. While Non-NP has almost no toxicity to cells (IC 50 > 10 μM). Subsequently, the protein degradation ability of nanoparticles in K562 cells was verified by WB, as shown in Figure 9 B, Cle-NP degrades Bcr-Abl and Abl proteins in a concentration-dependent manner, DC 50 are 1.76 μM and 1.83 μM, respectively, while Non-NP fails to degrade the protein even at 10 μM. This indicates that Cle-NP can effectively release H1-mGlu by taking advantage of high concentrations of GSH and H2O2 in tumor cells, thereby playing a role in protein degradation and cell killing.

[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 Annexin V-FITC and PI respectively. Next, the stained cells were analyzed using a flow cytometer (Beckman), and the data were processed using the FlowJo software package. The results are shown in Figure 2. Figure 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 ( Figure 10 B) Cle-NPs promoted the production of Cle-Caspase3 and Cle-PARP proteins in a concentration-dependent manner, while inhibiting the expression of BCL2 and MCL1 proteins. However, even at a high concentration of 10 μM, non-NPs failed to induce changes in the expression of apoptosis-related proteins. These results suggest that self-assembled nanoparticles Cle-NPs can utilize high concentrations of GSH and H2O2 in tumor cells to release H1-mGlu, inducing mitochondria-dependent apoptosis. This may be one of the reasons for the potent cytotoxicity of Cle-NPs.

[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 cellular efficacy. Therefore, the biodistribution of nanoparticles in vivo will be further 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 red blood cells were first tested. The specific operation of the red blood cell 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 anticoagulant tubes. The whole blood is then diluted with normal saline, centrifuged (1500 rpm, 10 min), and the red blood cell pellet is washed 4 times with physiological saturated NaCl solution to remove white blood cells, platelets and other plasma components. Finally, the red blood cells are diluted with normal saline to a concentration of 2% (v / v).

[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 2000 rpm for 10 minutes. Among them, the processing method of the positive control and negative control was similar. The same volume of blood was directly mixed with ultrapure water and normal saline, respectively. Finally, the absorbance (OD) of the supernatant of each group was measured at a wavelength of 540 nm using a microplate 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 to observe the red blood cells incubated with Cle-NP under a microscope to determine whether Cle-NP can cause red blood cell agglutination. Figure 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 normal 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 of intravenous injection of preparations. In addition, Cle-NP nanoparticles will not cause red blood cell agglutination ( Figure 11 The above results indicate that Cle-NP has high biocompatibility and safety, meeting 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) via tail vein injection. The changes in fluorescence signals over time in the mice were then analyzed using a small animal in vivo imaging system. Figure 12Figure 6 shows the in vivo fluorescence imaging of the tumor tissues of the mice injected with 780-NP and free IR-780. As shown in Figure 6A, the fluorescence signals of 780-NP and free IR-780 can be detected in the tumor tissues of the mice at 3-6 h after the tail vein injection, and the signal intensity in the tumor tissues of the 780-NP group is higher than that of the free IR-780 group. The fluorescence values of the tumor sites of the two groups reach the maximum at 24 h after the injection, and then gradually decrease with the extension of time. Throughout the experiment, the fluorescence signal in the tumor tissues of the mice in the free IR-780 group is always lower than that in the 780-NP group, which reflects that 780-NP has a stronger accumulation and longer stagnation time in the tumor sites. At 72 h after the injection, the mice are dissected, and the brain, heart, liver, spleen, lung, kidney and tumor tissues of the mice are subjected to ex vivo fluorescence imaging (Figure 6B). As shown in Figure 6C, the fluorescence intensity of the tumor tissues of the 780-NP group is higher than that of the free IR-780 group, which indicates that 780-NP has good tumor targeting in vivo. It is worth noting that the lung tissues of the mice in the two groups have no fluorescence signal, which indicates that 780-NP has no lung targeting. In addition, the blood circulation curve shows (Figure 6E) that the fluorescence intensity of the IR-780 group decreases to 31% of the initial value within 6 h, while that of the 780-NP group is 64% at this time, which indicates that the IR-780 concentration in the plasma of the 780-NP group decreases slowly, and thus 780-NP has a longer plasma half-life and can effectively prolong the blood circulation of the drug in vivo. Figure 12 Figure 12

[0110] (VI) In vivo anti-tumor and biological safety of the self-assembled nanoparticles

[0111] Based on the excellent in vitro anti-tumor effect and high tumor targeting ability of NPs, the in vivo anti-tumor effect of the nanoparticles was studied by K562 tumor-bearing mice. Considering that H0 and H1-mGlu have different solubilities and cannot be administered by tail vein injection, two administration routes, intraperitoneal injection and tail vein injection, were used for the experiment. The total grouping is as follows: groups 1-5 are intraperitoneal injection administration groups, which are respectively: (1) normal 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 administration groups, which are respectively: normal saline group and Cle-NP (equivalent to 5 mg / kg of H1-mGlu) group. Then, the corresponding administration dose is injected twice a day, and the tumor volume and mouse body weight are recorded, and the experimental period is 16 days (Figure 5A). Figure 13 Figure 13 ​​​As 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 the 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 that 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 intraperitoneal injection or tail vein injection, Cle-NP showed significant tumor inhibition ability. The tumor inhibition rates of each group were: Cle-NP (iv) > Cle-NP (ip) > H1-mGlu > H0 ( Figure 14 ).

[0112] To further demonstrate the anti-tumor mechanism of the drug, the expression of Bcr-Abl in tumor tissues was detected by WB (the specific steps are the same as above). Figure 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. 50μl of TUNEL detection solution was added to the sample and incubated 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 showed that ( Figure 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 experience weight loss, indicating that the self-assembled nanoparticles have good biosafety ( Figure 16 Subsequently, the hepatotoxicity and renal toxicity of the drug were detected by blood biochemistry ( Figure 16), the data showed that no significant changes in each biochemical index occurred in each group except for the H1-mGlu group, further indicating that the nanoparticles have good biological safety. The alanine aminotransferase (ALT) and aspartate aminotransferase (AST) contents in the serum of the H1-mGlu group of mice increased, suggesting that H1-mGlu can induce liver damage in mice, in addition, the contents of albumin (ALB) and alkaline phosphatase (ALP) related to hepatotoxicity, and the contents of urea nitrogen (BUN), urea (UA) and creatinine (CREA) related to kidney function did not change significantly. In summary, the in vivo treatment results are consistent with the in vitro data, both of which demonstrate that Cle-NP can enhance the proteolytic ability and tumor killing effect of H1-mGlu while avoiding toxic side effects, and has good biological safety.

[0114] The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are included in the protection scope of the present application.

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 reaction. (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 using 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) The BCR-ABL targeting molecular glue H1-mGlu was condensed with 2-({3-[(2-methylprop-2-yl)oxy]-3-oxyylidenepropyl}thio)propionic acid to generate compound 5, and then the tert-butyl ester was removed 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 described in step (1) 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 6-bromobenzo[d]thiazol-2-amine; The one-pot method 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 in step (2) is carried out 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 removal of the tert-butyloxycarbonyl protection by trifluoroacetic acid in step (3) refers to dissolving compound 4 in DCM, then adding trifluoroacetic acid and reacting 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) is carried out in the presence of EDC-HCl and DMAP at room temperature for 12-24 hours; 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 the tert-butyloxycarbonyl protection of H1-mGlu-linker 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 H1-mGlu-linker.

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

Images