CN116836212A - Redox-responsive nucleolin aptamer-paclitaxel conjugate, preparation method and application thereof - Google Patents

Abstract

The invention discloses a series of new redox-responsive nucleolin aptamer-paclitaxel conjugates represented by formula I, their preparation methods and their applications: R is ‑(CH ₂ ) _m1 S(CH ₂ ) _n1 ‑,‑(CH ₂ ) _m2 SS(CH ₂ ) _n2 ‑,‑(CH ₂ ) _m3 SC(CH ₃ ) ₂ S(CH ₂ ) _n3 ‑中Either way, m1, n1, m2, n2, m3, n3 are 0 or positive integers. The conjugate was designed to respond to the redox heterogeneity of triple-negative breast cancer, has good water solubility and tumor specificity, and exhibits redox dual responsiveness, rapid release of PTX and the highest anti-triple-negative breast cancer ability.

Description

Redox-responsive nucleolin aptamer-paclitaxel conjugate, preparation method thereof and its application

Technical areas:

The invention relates to a redox-responsive nucleolin aptamer-paclitaxel conjugate, its preparation method and its application.

Background technique:

Paclitaxel (PTX) is a tetracyclic diterpene compound that blocks cell mitosis by inhibiting tubulin. Despite decades of discovery, scientists still focus on PTX-based chemotherapy because of its broad-spectrum anticancer activity and high efficiency. However, PTX has adverse reactions, such as bone marrow suppression, gastrointestinal irritation, etc. In addition, PTX has poor solubility in water, which greatly limits its dosage in clinical applications. To overcome these obstacles, various marketed drugs have been developed, such as solvent-based paclitaxel, liposome (Lipsu), albumin-based paclitaxel (Abraxane), etc. However, these drugs reduce rather than eliminate side effects, making it increasingly difficult to meet exact clinical criteriai.

In recent years, attention in the field of cancer treatment is shifting from traditional drugs to targeted drugs. Triple-negative breast cancer (TNBC) is a type of breast cancer (BC) with high malignancy and poor prognosis. Targeted therapies for BC are often ineffective due to lack of receptors (ER/PR/HER2). The current main focus is on improving the specificity of PTX by attaching tumor-targeting ligands. There are currently a large number of ligands involved in PTX-specific strategies, including small molecules, peptides, antibodies, nanoparticles, magnetic iron oxides, and biofilms. Few developed methods have proven to be perfect and are far from ready for use in commercial systems. Antibody-drug conjugates (ADCs) have been positioned at the forefront of chemotherapy, improving the clinical prognosis of patients with specific DNA mutations. However, heavily modified antibodies have risks of difficulty crossing membranes, strong immunogenicity, storage rigidity, reduced target affinity, altered pharmacokinetics, and increased heterogeneity. As a targeting component, aptamers have the advantages of fast screening, fast cell penetration, low immunogenicity, easy synthesis, modification and industrialization, and are a promising cancer treatment method.

Unlike normal cells, nuclear proteins are abundantly expressed on the surface of various cancer cells and have been reported to be an attractive target for triple-negative breast cancer (TNBC) treatment. ACT-GRO-777 (also known as AS1411 ) is a targeting nucleoprotein aptamer (NCT00740441) that has entered clinical phase II research (see Rosenberg JE, Bambury RM, VanAllen EM, et al. A phase II trial of AS1411 (a novel nucleolin-targeted DNAaptamer) in metastatic renal cell carcinoma. Invest New Drugs 2014;32(1):178-187). Notably, AS1411 enhanced cell penetration through the macrocystin pathway, which is beneficial to the development of novel aptamer-drug conjugates (ApDC), such as doxorubicin and melittin. Increasing evidence suggests that tumor microenvironment (TME)-responsive prodrugs have considerable therapeutic value because cancer tissue is clearly differentiated from adjacent normal tissue. In our previous work, we designed a highly water-soluble aptamer-paclitaxel conjugate that is sensitive to cathepsin B and can selectively deliver paclitaxel (PTX) to ovarian tumors (Xue F, Lin X, Cai Z, et al. Doxifluridine-based pharmacosomes delivering miR-122 as tumor microenvironments-activated nanoplatforms for synergistic treatment of hepatocellular carcinoma. ColloidsSurfB Biointerfaces 2021;197:111367). In addition, due to abnormal tumor metabolism, not only the concentration of glutathione (GSH) in tumor cells is significantly higher than that of normal cells (500-1.0×10 ⁴ μM vs. 2-20 μM), but also the level of reactive oxygen species (ROS, especially H ₂ O ₂ ) Significantly higher than normal cells (5-1000μM vs. 0.001-0.7μM). However, current studies have not determined whether oxidative or alternative reducing conditions are the optimal conditions for stimulating ApDCs in the tumor microenvironment (TME).

Contents of the invention:

The purpose of the present invention is to provide a series of new redox-responsive nucleolin aptamer-paclitaxel conjugates, their preparation methods and their applications. The conjugates are designed to respond to redox abnormalities in triple-negative breast cancer (TNBC). It has good water solubility and tumor specificity, shows redox dual responsiveness (oxidation + reduction microenvironment), rapid release of PTX and the highest anti-triple negative breast cancer (TNBC) ability.

The present invention is realized through the following technical solutions:

Redox-responsive nucleolin aptamer-paclitaxel conjugate represented by formula I:

Where, R is -(CH ₂ ) _m1 S(CH ₂ ) _n1 -, -(CH ₂ ) _m2 SS(CH ₂ ) _n2 -, -(CH ₂ ) _m3 SC(CH ₃ ) ₂ S(CH ₂ ) _n3 - any one of them, m1, n1, m2, n2, m3, n3 are 0 or positive integers.

Preferably, m1, n1, m2, n2, m3, n3 are 0 or a positive integer from 1 to 3.

The 5' end and 3' end of unmodified or modified DNA/RNA of any one of aptamer, antisense nucleic acid, siRNA, and CpG.

The preparation method of the redox-responsive nucleolin aptamer-paclitaxel conjugate represented by Formula I includes the following steps: first, a redox-reactive diacid is constructed; then, the redox-reactive diacid is coupled to paclitaxel (PTX) , to obtain the carboxylic acid derivative; finally, the carboxylic acid derivative is further combined with amino DNA to obtain the redox-responsive nucleolin aptamer-paclitaxel conjugate ApDC.

In particular, when R in Formula I is -(CH ₂ ) _m1 S(CH ₂ ) _n1 -monosulfide, m1=0, n1=0, and the synthesis route is as follows:

Reagent description: (i) 1,4-oxythiane-2,6-dione, Py, THF; (ii) NHS, DCC, THF; (iii) Amino-DNA, NaHCO ₃ , H ₂ O, DMF

The synthesis method includes the following steps:

1) Use equal amounts of 1,4-glufosulfane-2,6-dione and pyridine (Py) as raw materials, then add paclitaxel (PTX) and 1,4-glufosulfane to anhydrous tetrahydrofuran (THF) The molar ratio of -2,6-dione and paclitaxel is 1: (0.5-0.9), stir at room temperature for 2-4 days, concentrate the mixture under reduced pressure, and then co-evaporate with toluene and chromatograph to obtain compound 2;

2) Mix compound 2 and N-hydroxysuccinimide (NHS) in anhydrous tetrahydrofuran (THF), add dicyclohexylcarbodiimide (DCC) and stir at room temperature, filter the mixture, and concentrate the filtrate in vacuum. A crude solid was obtained, which was finally cooled to 4°C with diethyl ether to recrystallize, and evaporated to dryness to obtain a white solid compound 3; wherein the molar ratio of compound 2, NHS and DCC was 1: (1-1.5): (1-1.5);

3) Add 5' or 3'-amino DNA solution to NaHCO ₃ buffer, add compound 3 to DMF, the molar ratio of compound 3 to 5' or 3'-amino DNA is (30-100): 1, room temperature Stir for 2h-12h, purify and column desalt to obtain compound 4 or compound 5.

When R in formula I is -(CH ₂ ) _m2 SS(CH ₂ ) _n2 -disulfide, m2=1, n2=1, the synthesis route is as follows:

Description of reagents and conditions: (i) I ₂ , MeOH; (ii) AcCl, 60-80°C reflux; (iii) PTX, Py; (iv) NHS, DCC, THF; (v) Amino-DNA, PB buffer (pH=8.0),DMF

The synthesis method includes the following steps:

1) Cool the solution of compound 6 to 0°C, add iodine dropwise, the molar ratio of compound 6 to iodine is 1: (0.2-0.45), after complete reaction, add saturated Na ₂ S ₂ O ₃ aqueous solution to quench the reaction The pH of the mixture was adjusted to about 4 by gradually adding solid NaHCO ₃ , and then the mixture was filtered and concentrated, and passed through a silica gel column to obtain compound 7;

2) Add acetyl chloride to compound 7 and heat to 60-80°C under nitrogen and reflux for 2-12 hours to obtain crude compound 8. Dissolve crude compound 8 and paclitaxel (PTX) in the solvent, and then add pyridine (Py) dropwise. Stir at room temperature for 2 days, remove the solvent in vacuo, and perform silica gel chromatography on the solid residue to obtain compound 9; wherein, the molar ratio of crude compound 8, paclitaxel (PTX) and Py is 1:1: (0.3-0.5);

3) Mix compound 9 and N-hydroxysuccinimide (NHS), the molar ratio of compound 9 and N-hydroxysuccinimide (NHS) is 1: (1-2), add dicyclohexyl carbon Diimine (DCC), stir at room temperature for 1-4 days, the solid precipitates out, after filtration, the filtrate is evaporated to obtain the crude product of compound 10;

4) Add 5' or 3'-amino DNA solution to PB buffer, add compound 10 to DMF, the molar ratio of compound 10 to 5' or 3'-amino DNA is (30-100): 1, 30- Stir at 50°C for 2h-12h, purify and column desalt to obtain compound 11 or compound 12.

When R in formula I is -(CH ₂ ) _m3 SC(CH ₃ ) ₂ S(CH ₂ ) _n3 -, m3=1, n3=1, the synthesis route is as follows:

Description of reagents and conditions: (i) H ₂ SO ₄ , acetone; (ii) PTX, DCC, DMAP, DMF; (iii) amino-DNA, DCC, HBTU, DIPEA, H ₂ O, DMF.

The synthesis method includes the following steps:

1) Cool compound 6 in acetone to 0°C, add sulfuric acid dropwise, the molar ratio of compound 6 to sulfuric acid is 1: (0.5-0.8:), stir for 2-12 hours, then dilute with water, gradually add NaOH aqueous solution to adjust the pH Adjust to 5, and then obtain compound 13 through post-processing;

2) Add compound 13 and paclitaxel (PTX), the molar ratio of compound 13 and paclitaxel (PTX) is 1: (1-2), add DMF, and then add dicyclohexylcarbodiimide (DCC) and catalytic amount to the solution The molar ratio of DMAP, DCC and compound 13 is (1-2): 1. Leave it at room temperature overnight, filter and evaporate, and perform column chromatography on silica gel to obtain compound 14;

3) Add compound 14, dicyclohexylcarbodiimide (DCC) and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU) into DMF respectively and stir. After cooling to 0°C, 5′ or 3′-amino DNA and N,N-diisopropylethylamine (DIPEA) aqueous solution were dropped into the mixture, and stirred at 30-50°C for 4-12h, in which compound 14, Dicyclohexylcarbodiimide (DCC), benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), N,N-diisopropylethylamine ( The molar ratio of DIPEA) and 5′/3′-amino DNA is (30-100): (30-100): (30-100): (30-100): 1. Add triethylamine acetate (TEAA) to The reaction mixture was quenched and then purified by post-treatment to obtain compound 15 or compound 16.

The redox-responsive nucleolin aptamer-paclitaxel conjugate obtained by the present invention has good water solubility and tumor specificity. The present invention also protects the preparation process of the redox-responsive nucleolin aptamer-paclitaxel conjugate. Application of anti-triple-negative breast cancer (TNBC) drugs.

The beneficial effects of the present invention are as follows: The present invention provides a series of new redox-responsive nucleolin aptamer-paclitaxel conjugates, their preparation methods and their applications. The conjugates are designed to respond to triple-negative breast cancer (TNBC). Redox heterogeneity, good water solubility and tumor specificity, showing redox dual responsiveness (oxidation + reduction microenvironment), rapid release of PTX and the highest anti-triple negative breast cancer (TNBC) ability.

Picture description:

Figure 1 shows the drug release and serum stability of ApDCs in response to different TMEs;

Among them, A is the drug release of ApDCs in response to different TMEs in phosphate buffered saline (PBS, 0.1x), reducing microenvironment (DTT, 10mM), and oxidative microenvironment (H2O2, 1mM); aptamer-drug conjugate ApDCs (0.15 nmol) were incubated in different buffers at 37°C and samples were taken every 3 hours. 0h normalized intact ApDC is considered 100%. Data are expressed as mean ± standard deviation of three replicates.

B is the serum stability of ApDCs with different TME stimulation conjugates; ApDCs (0.15nmol) were incubated in 90% fetal calf serum at 37°C, sampled at different time points, and imaged with 0.01% GelGreen;

In the figure, PSA is the abbreviation of compound 4, ASP is the abbreviation of compound 5, PSSA is the abbreviation of compound 11, ASSP is the abbreviation of compound 12, PTKA is the abbreviation of compound 15, and ATKP is the abbreviation of compound 16.

Figure 2 shows the cytotoxicity and apoptosis of ApDC-treated cells in response to different TME.

Among them, A is the cytotoxicity of ApDCs with different TME responses to cancer cells and normal cells. Among them, a-d are respectively the mouse triple-negative breast cancer cell line (4T1), human triple-negative breast cancer cell line (MDA-MB-231), human ovarian cancer cell line (SKOV3) and human normal liver cell line (MIHA). Cell viability after 72h treatment with free AS1411, free PTX and ApDCs at 250nM. Data are expressed as mean ± standard deviation of at least three replicates. Statistical significance was determined using the Holm-Sidak method, alpha = 0.05. ns means P>0.05; * means P<0.05; ** means P<0.01; *** means P<0.001; **** means P<0.0001. Statistical analysis was performed using GraphPad Prism 8.0.1 software.

B shows the apoptosis of MDA-MB-231 cells treated with ApDC with different TME responses. use AnnexinV-FITC/PI apoptosis detection kit, flow cytometry analysis of the percentage of normal cells, early apoptosis, late apoptosis and dead cells. ApDC and free PTX concentrations were both 250 nM. Data are expressed as mean ± standard deviation of three replicates.

Detailed ways:

The following is a further description of the present invention, rather than a limitation of the present invention.

Example 1: Synthesis of Compounds

The reaction equation is as follows:

Among them, R in compound 4 and compound 5 is -S-. Compound 4 is abbreviated as PSA, and compound 5 is abbreviated as ASP. Compound 11 is abbreviated as PSSA, compound 12 is abbreviated as ASSP, compound 15 is abbreviated as PTKA, and compound 16 is abbreviated as ATKP.

Synthesis of compound 2:

Take 1,4-glufosulfane-2,6-dione (397 mg, 3.006 mmol) in THF (20 mL) as a suspension, add Py (242 μL, 3.006 mmol) and stir for 5 min, then add it in anhydrous THF (20 mL) PTX ((2.176g, 2.548mmol) was added and stirred at room temperature for 4d. The mixture was concentrated under reduced pressure and then co-evaporated with toluene three times to remove Py. Then, the residue was purified on silica gel (PE/EA=1/1) Chromatography was performed on the reaction mixture to obtain compound 2 (1.807g, 72%) as a white solid.

MS(ESI)m/z for C ₅₁ H ₅₄ NO ₁₇ S ^- [MH] ^- , Calculated 984.3118, Found 984.3153; ¹ HNMR (400MHz, MeOD) δ8.02 (dd, J＝8.2, 6.8Hz, 2H), 7.74(dd,J＝5.3,3.2Hz,2H),7.60–7.55(m,1H),7.49(t,J＝7.5Hz,2H),7.44–7.38(m,3H),7.37–7.31(m, 4H),7.17(t,J＝7.3Hz,1H),6.35(d,J＝4.5Hz,1H),5.99(t,J＝8.6Hz,1H),5.77(d,J＝6.1Hz,1H) ,5.54(d,J＝7.3Hz,1H),5.41(d,J＝6.1Hz,1H),4.93–4.87(m,1H),4.25(dd,J＝11.0,6.7Hz,1H),4.09( s,2H),3.72(d,J＝7.2Hz,1H),3.56–3.35(m,3H),3.30–3.24(m,1H),3.16(d,J＝15.0Hz,1H),2.43–2.24 (m,4H),2.15–2.03(m,4H),1.85–1.65(m,5H),1.55(s,3H),1.04(d,J=3.5Hz,6H); ¹³ C NMR (101MHz, MeOD )δ205.24,171.33,170.24,167.71,142.40,138.37,135.48,134.90,134.66,132.93,131.34,130.14,129.97–129.47,128.87–128.4,85.93,82. 28,79.04,77.49,76.85,76.51,76.28,73.07,72.33 ,59.24,55.24,47.92,44.62,37.55,36.45,33.68,26.97,23.35,22.43,20.84,14.99,10.50.Peak overlapping was observed.

Synthesis of compound 3: Mix compound 2 (1.2g, 1.218mmol) and NHS (147mg, 1.279mmol) in anhydrous tetrahydrofuran (30mL), add DCC (276mg, 1.34mmol), and stir at room temperature for 3 days. The mixture was filtered and the filtrate was concentrated in vacuo to give a crude solid. Finally, it was cooled to 4°C with diethyl ether (100 mL) for recrystallization, and then evaporated to dryness to obtain a white solid. MS(ESI)m/z for C ₅₅ H ₅₇ N ₂ O ₁₉ S ^- [MH] ^- ,Calculated 1081.3282,Found 1081.3203.

Synthesis of compounds 4 to 5: Add 5'/3'-aminoDNA (100nmol) solution to NaHCO ₃ buffer (50mM, 0.1mL), add compound 3 (3μmol) to DMF (0.2mL), and store at 30°C Stir (800 rpm) for 2 h, and add TEAA (2M, 0.1 mL) to quench the reaction mixture. The solvent was removed in vacuo and the solid residue was redissolved in _ddH2O (1 ml). The mixture was then purified using high performance liquid chromatography (Agilent 1260). Phase A is ACN, phase B is TEAA (50mM). use Oligonucleotide BEH C18OBDTM Prep column (2.5m, 10mm×50mm), the flow rate was 1.2mL·min ^-1 , the column temperature was ambient temperature, and the gradient was run from 5% to 60% of phase A in 30 minutes. The purified aptamer-drug conjugate (ApDC) was desalted through Sephadex G25 column to obtain compound 4 or 5. The samples were further lyophilized to dry storage and confirmed by ESI mass spectrometry analysis.

Synthesis of compound 7: A solution of compound 6 (2.3g, 21.7mmol) in methanol (30mL) was cooled to 0°C, and iodine (5.634g, 11.1mmol) in methanol (30mL) was dropped into the stirring solution for 2 hours. Thin layer chromatography analysis showed complete consumption of compound 6. _The reaction mixture _was quenched by adding saturated aqueous _Na2S2O3 solution and the pH was adjusted to around 4 by gradually adding solid _NaHCO3 . The mixture was then filtered and concentrated, and chromatography was performed on silica gel (DCM/MeOH=30/1) to obtain compound 7 (2.012g, 87%) as a white solid.

MS(ESI)m/z for C ₆ H ₉ O ₄ S ₂ ^- [MH] ^- , Calculated 208.9948, Found 208.9964; ¹ HNMR (400MHz, MeOD) δ2.92 (t, J＝7.0Hz, 4H), 2.71 (t, J=7.0Hz, 4H). ¹³ C NMR (101MHz, MeOD) δ175.36, 34.76, 34.30.

Synthesis of compounds 8 and 9:

Compound 7 ((0.211 g, 1.005 mmol)) and acetyl chloride (5 mL) were added to the round bottom flask. Then, the mixture was heated to 65° C. and refluxed for 2 hours under nitrogen to obtain crude compound 8. MS(ESI)m/z for C ₆ H ₉ O ₃ S ₂ ⁺ [M+H] ⁺ ,Calculated192.9988,Found 192.9990.

All crude compound 8 and PTX (854 mg, 1 mmol)) were dissolved in anhydrous DCM (20 mL). Then, Py (0.282 mL, 3.5 mmol) was added dropwise to the mixture, and the mixture was stirred at room temperature for 2 days. The solvent was removed in vacuo and the solid residue was redissolved in DCM (20 mL). Compound 9 was obtained by silica gel (DCM/MeOH=20/1～3/1) chromatography as a white solid. MS(ESI)m/z forC ₅₃ H ₅₈ NO ₁₇ S ₂ ^- [MH] ^- , Calculated 1044.3152, Found 1044.3238; ¹ H NMR (400MHz, MeOD) δ8.02 (dd, J＝8.2, 6.8Hz, 2H) ,7.74(dd,J＝5.3,3.2Hz,2H),7.60–7.55(m,1H),7.49(t,J＝7.5Hz,2H),7.44–7.38(m,3H),7.37–7.31(m ,4H),7.17(t,J＝7.3Hz,1H),6.35(d,J＝4.5Hz,1H),5.99(t,J＝8.6Hz,1H),5.77(d,J＝6.1Hz,1H ),5.54(d,J＝7.3Hz,1H),5.41(d,J＝6.1Hz,1H),4.93–4.87(m,1H),4.25(dd,J＝11.0,6.7Hz,1H),4.09 (s,2H),3.72(d,J＝7.2Hz,1H),3.56–3.35(m,3H),3.30–3.24(m,1H),3.16(d,J＝15.0Hz,1H),2.43– 2.24(m,4H),2.15–2.03(m,4H),1.85–1.65(m,5H),1.55(s,3H),1.04(d,J=3.5Hz,6H); ¹³ C NMR(101MHz, MeOD)δ205.19,172.60,171.63,171.31,170.61,170.34,167.66,142.38,138.29,135.54,134.87,134.64,132.95,131.32,130.14,129.68,128.67 ,85.92,82.28,79.03,77.47,76.84,76.16,73.02, Peak overlapping was observed.

Synthesis of compound 10: Mix compound 9 (648 mg, 0.62 mmol) and NHS (72.8 mg, 0.633 mmol) in anhydrous THF (20 mL), add DCC (133.3 mg, 0.646 mmol), and stir at room temperature for 2 days. Solids precipitated. The mixture was filtered and the filtrate was evaporated to give crude product 10. MS(ESI)m/z for C ₅₇ H ₆₂ N ₂ NaO ₁₉ S ₂ ⁺ [M+Na] ⁺ ,Calculated1165.3280,Found 1165.3274.

Synthesis of compounds 11-12: Add 5'/3'-aminoDNA (100nmol) solution to PB buffer (50mM, 0.1mL), add compound 10 (3μmol) in DMF (0.2mL) to the solution, and add After stirring at 30°C for 2 h, TEAA (2M, 0.1 mL) was added to quench the reaction mixture. The solvent was removed in vacuo and the solid residue was redissolved in ddH2O (1 ml). The mixture was then purified using high performance liquid chromatography (Agilent 1260). Phase A is ACN, phase B is TEAA (50mM). use Oligonucleotide BEH C18OBDTM Prep column (2.5m, 10mm×50mm), the flow rate was 1.2mL·min ^-1 , the column temperature was ambient temperature, and the gradient was run from 5% to 60% of phase A in 30 minutes. The purified aptamer-drug conjugate (ApDC) was desalted using Sephadex G25 column to obtain compound 11/12. The samples were further lyophilized to dry storage and confirmed by ESI mass spectrometry analysis.

Synthesis of compound 13: Compound 6 (3.412g, 32.2mmol) was cooled to 0°C in acetone (20mL), sulfuric acid (1.882g, 19.2mmol) was added dropwise to the stirred mixture, and stirred for 2 hours. Then dilute with ddH2O (200 mL), and gradually add NaOH aqueous solution to adjust the pH to 5. Then, the mixture was evaporated to remove acetone, and the aqueous layer was extracted with ethyl acetate (100 mL×3). Finally, the crude product was chromatographed on silica gel under reduced pressure (n-hexane/EA=4/1) to obtain compound 13 (2.313g, 57%) as a white solid. MS(ESI)m/z for C ₉ H ₁₅ O ₄ S ₂ ^- [MH] ^- , Calculated 251.0417, Found251.0448; ¹ H NMR (400MHz, MeOD) δ2.76 (dd, J＝9.0, 5.4Hz, 4H), 2.49 (t, J=7.1Hz, 4H), 1.49 (s, 6H); ¹³ C NMR (101MHz, MeOD) δ 56.97, 35.33, 31.31, 26.33.

Synthesis of compound 14: Add compound 13 (0.433g, 1.718mmol) and PTX (1.547g, 1.812mmol) into a round-bottomed flask, and add DMF (20mL). DCC (355 mg, 1.722 mmol) and a catalytic amount of DMAP were added to the solution. The mixture was left at room temperature overnight. The mixture was filtered, evaporated, and chromatographed on silica gel (DCM/MeOH=50/1~30/1) to obtain compound 14 (1.064g, 57%) as a white solid. MS(ESI)m/z for C ₅₆ H ₆₄ NO ₁₇ S ₂ ^- [MH] ^- ,Calculated1086.3621,Found 1086.3747; ¹ H NMR(400MHz,MeOD)δ8.05–7.98(m,2H),7.71( dd,J＝5.2,3.3Hz,2H),7.57(td,J＝7.0,3.2Hz,1H),7.39(dddd,J＝23.6,15.3,11.1,4.7Hz,9H),7.17(t,J＝ 7.3Hz,1H),6.35(s,1H),5.97(t,J＝8.7Hz,1H),5.77(d,J＝6.5Hz,1H),5.53(d,J＝7.2Hz,1H),5.43 (d,J＝6.5Hz,1H),4.93–4.87(m,1H),4.24(dd,J＝10.9,6.7Hz,1H),4.09(s,2H),3.71(d,J＝7.2Hz, 1H),2.71(t,J＝6.8Hz,2H),2.61(t,J＝6.6Hz,2H),2.41–2.28(m,4H),2.13–2.00(m,8H),1.82(d,J ＝0.8Hz,3H),1.77–1.65(m,2H),1.55(s,3H),1.34(s,3H),1.24–1.07(m,3H),1.06–0.99(m,6H); ¹³ C NMR(101MHz,MeOD)δ205.19,172.86,171.66,171.34,170.41,170.35,167.68,142.36,138.36,135.51,134.94,132.95,131.44,131.25,130.16,129.7 6,129.60,128.75,128.72,85.91,82.29,79.07, 76.82,76.28,76.03,73.02,31.19,30.70,26.15,23.38,22.41,20.84,15.08,10.48. Peak overlapping was observed.

Synthesis of compounds 15-16: Compound 14 (3 μmol), DCC (3 μmol) and HBTU (3 μmol) were added to DMF (0.4 mL) and stirred for 10 min. After cooling to 0°C, 5′/3′-amino DNA ( 100 nmol) and DIPEA (3 μmol) in ddH ₂ O (0.1 mL) solution were dropped into the mixture, stirred at 30°C for 4 h, and TEAA (2 M, 0.1 mL) was added to quench the reaction mixture. The solvent was removed in vacuo and the solid residue was redissolved in _ddH2O (1 ml). Then, the mixture was purified using high performance liquid chromatography (Agilent 1260). Phase A is ACN, phase B is TEAA (50mM). use Oligonucleotide BEH C18 OBDTM Prep column (2.5m, 10mm×50mm), the flow rate was 1.2mL·min-1, the column temperature was ambient temperature, and the gradient was run from 5% to 60% of phase A in 30 minutes. The purified ApDC was desalted through Sephadex G25 column to obtain compound 15/16. The samples were further lyophilized to dry storage and confirmed by ESI mass spectrometry analysis.

Example 2: Drug release and stability of different tumor microenvironment (TME)-responsive aptamer-drug conjugates (ApDC).

Drug release test: ApDCs (0.15nmol) were dissolved in PBS (0.1×, 60μL), DTT (10mM, 60μL) and H ₂ O ₂ (1mM, 60μL) respectively. Incubate at 37°C and detect intact ApDC at regular intervals using an Agilent 1290 HPLC system and a 260 nm UV detector. Phase A is ACN, phase B is TEAA (50mM). In a YMC-Triart C18 column (3m, 150mm×4.6mm), the flow rate was 0.5mL·min-1, the column temperature was ambient temperature, and the gradient from 2% to 55% of phase A was run in 30 minutes. The percentage of intact ApDC was normalized to an initial amount of 100%.

In order to compare the differences in drug release of TME-stimulated linker ApDCs in different tumor microenvironments, HPLC method was used to evaluate the relatively complete aptamer after incubation in physiological buffer (PBS), reducing microenvironment (DTT) or oxidative (H ₂ O ₂ ) microenvironment. Body-drug conjugates (ApDCs), including compound 4, compound 5, compound 11, compound 12, compound 15 and compound 16.

As shown in Figure 1A, various aptamer-drug conjugates ApDC maintained high stability after incubation in physiological buffer at 37°C for 24 hours. ApDCs with disulfide linkers (ASSP, PSSA) and sulfide linkers (ATKP, PTKA) were activated using reducing microenvironment and oxidative microenvironment respectively. ApDC (ASP, PSA) with thioether linkers were co-activated simultaneously under reducing and oxidizing microenvironments. The results show that the hydrolysis efficiency of ApDCs in H ₂ O ₂ solution (1mM) is: ASP≈PSA>ATKP≈PTKA>ASSP≈PSSA. In addition, the hydrolysis efficiency of ApDCs in DTT solution (10mM) is ASSP≈PSSA>ASP≈PSA>ATKP≈PTKA. The results show that ApDC containing thioether linkers is more sensitive to the oxidative microenvironment than the reducing microenvironment. Compared with thioether-linked ApDC, thioether-linked ApDC has higher drug release efficiency. The coupling end of ApDCs has little effect on drug release.

The serum stability of ApDCs with different TME-stimulated conjugates was evaluated by degradation experiments (Figure 1B). These ApDCs (0.15 nmol) were incubated in 90% fetal calf serum at 37°C, sampled at different time points, and visualized with 0.01% GelGreen. Studies have found that 3′-end ApDC usually has higher stability than 5′-end ApDC, which may be related to the function of the 3′-end exon. Furthermore, the serum stability of ASP and ASSP is lower than that of ATKP. This may be related to the steric hindrance of the dimethyl group in the sulfhydryl linker.

Example 3: In vitro anti-triple-negative breast cancer (TNBC) ability of TME-responsive aptamer-drug conjugate ApDC in different tumor microenvironments

CCK-8 Cell Viability Assay: To identify the most potent ApDCs in tumor cells, cell viability assay will be performed using Cell Counting Kit-8 (CCK8, Dojindo). According to the provided protocol, MDA-MB-231, 4T1, SKOV-3 and MIHA cells were seeded in a 96-well plate at approximately 5000 cells per well and incubated overnight for adhesion. Solution of ApDCs and controls will be in the medium. Remove the cell culture medium, add the solution, and incubate at 37°C for 72 hours. At the end of the incubation, 100 μL of culture medium containing 10% CCK8 solution was added to each well. The plates will be read after an additional 2 hours of incubation with a spectrometer at 450nm wavelength.

Apoptosis experiment: In order to analyze the different stages of cell apoptosis, cells were seeded in 6-well plates and treated with ApDC and control group for 48 h respectively. Collect cells, rinse with cold PBS, and resuspend in 100 μL of 1× Annexin-V binding buffer. Then add 5 μL Annexin-V-FITC and 5 μL PI, incubate in the dark at room temperature for 15 min, and micro-top. Flow cytometry for quantitative determination.

The CCK-8 method was used to evaluate the anti-proliferative effects of different TME-stimulating conjugates on ApDCs in vitro. The results shown in a-b in Figure 2A show that various ApDCs can effectively inhibit the proliferation of cancer cells. Compared with free PTX, ASP inhibited 4T1 cells (24.3%±2.1% vs 12.0%±2.1%, P<0.01) and MDA-MB-231 cells (12.7%±0.5% vs 11.8%±0.4%, P<0.05 )More effective. In contrast, ASSP or ATKP had a negative effect on 4T1 cells (27.3% ± 0.5% vs 35.5% ± 2.9% vs 12.0% ± 2.1%) and MDA-MB-231 cells (14.6% ± 0.8% vs 17.9% ± 0.3% vs 11.8% ± 0.4%) had lower antiproliferative activity than ASP, providing evidence for a potential role of thioether linkers in TME-responsive chemotherapy. Importantly, ASP has more stable anti-TNBC ability than PSA. Combined with the serum stability data (Figure 1B), it was demonstrated that 3′-end coupling may be more beneficial to the design of AS1411-PTX. In contrast, the cytotoxicity of all ApDCs towards SKOV3 cells and MIHA cells was reduced (Fig. 2Ac-d).

Consistent with the anti-proliferation results, ASP was superior to ASSP and ATKP in its ability to induce apoptosis in vitro. As shown in Figure 2B, ASP increased the proportion of late apoptosis compared with ASSP and ATKP (32% ± 1% vs 22% ± 1% vs 19% ± 1%), while there was a significant difference between early apoptosis and dead cells. There is no significant difference. Overall, our in vitro data further confirm the role of thioether linkers in AS1411-PTX conjugation. Compared with PTX, ASP has better anti-proliferative ability against TNBC cells because ASP can effectively target TNBC cells and induce apoptosis, but has few biosafety issues.

Claims (7)

1. A redox-responsive nucleolin aptamer-paclitaxel conjugate of formula i:

wherein R is- (CH) ₂ ) _m1 S(CH ₂ ) _n1 -、-(CH ₂ ) _m2 SS(CH ₂ ) _n2 -、-(CH ₂ ) _m3 SC(CH ₃ ) ₂ S(CH ₂ ) _n3 -any of m1, n1, m2, n2, m3, n3 is 0 or a positive integer.

2. The conjugate according to claim 1, wherein m1, n1, m2, n2, m3, n3 are positive integers from 1 to 3.

3. A process for preparing a redox-responsive nucleolin aptamer-paclitaxel conjugate according to claim 1 of formula i, comprising the steps of: firstly, constructing oxidation-reduction reaction diacid; then, coupling the oxidation-reduction reaction diacid with taxol to obtain a carboxylic acid derivative; finally, the carboxylic acid derivative is further combined with amino DNA to obtain the redox responsive nucleolin aptamer-taxol conjugate.

4. A process according to claim 3, wherein R in formula I is- (CH) ₂ ) _m1 S(CH ₂ ) _n1 In case of monosulfide, m1=0, n1=0, the synthetic route is as follows:

the method comprises the following steps:

1) Taking 1, 4-oxasulfane-2, 6-dione and pyridine with equivalent weight as raw materials, and then adding taxol, wherein the mole ratio of the 1, 4-oxasulfane-2, 6-dione to the taxol is 1: (0.5-0.9), stirring at room temperature for 2-4d, concentrating the mixture under reduced pressure, then co-evaporating with toluene, and performing chromatography to obtain compound 2;

2) Mixing the compound 2 with N-hydroxysuccinimide, adding dicyclohexylcarbodiimide, stirring at room temperature, filtering the mixture, concentrating the filtrate in vacuum to obtain a crude solid, cooling to 4 ℃ with diethyl ether, recrystallizing, evaporating and drying to obtain a white solid compound 3; wherein, the mol ratio of the compound 2, NHS and DCC is 1: (1-1.5): (1-1.5);

3) In NaHCO ₃ Adding 5 'or 3' -amino DNA solution into buffer solution, adding compound 3 into DMF, wherein the molar ratio of compound 3 to 5 'or 3' -amino DNA is (30-100): 1, stirring for 2-12 h at room temperature, purifying and desalting by a column to obtain a compound 4 or a compound 5.

5. A process according to claim 3, wherein R in formula I is- (CH) ₂ ) _m2 SS(CH ₂ ) _n2 When disulfide, m2=1, n2=1, the synthetic route is as follows:

the synthesis method comprises the following steps:

1) The solution of compound 6 was cooled to 0 ℃, and iodine was added dropwise, the molar ratio of compound 6 to iodine being 1: (0.2-0.45), after complete reaction, by addition of saturated Na ₂ S ₂ O ₃ The reaction mixture was quenched with aqueous solution by stepwise addition of solid NaHCO ₃ Adjusting pH to about 4, filtering and concentrating the mixture, and passing through a silica gel column to obtain a compound 7;

2) Acetyl chloride is added into the compound 7, the mixture is heated to 60-80 ℃ under nitrogen and refluxed to obtain a crude compound 8, the crude compound 8 and taxol are dissolved in a solvent, pyridine is then added dropwise, the mixture is stirred for 2 days at room temperature, the solvent is removed in vacuum, and the solid residue is subjected to silica gel chromatography to obtain a compound 9; wherein, the molar ratio of the coarse compound 8 to the taxol to the pyridine is 1:1: (0.3-0.5);

3) Compound 9 was mixed with N-hydroxysuccinimide, the molar ratio of compound 9 to N-hydroxysuccinimide being 1: (1-2) adding dicyclohexylcarbodiimide, stirring at room temperature for 1-4 days, precipitating solid, filtering, and evaporating filtrate to obtain a crude product of the compound 10;

4) Adding 5 'or 3' -amino DNA solution into PB buffer solution, and adding compound 10 into DMF, wherein the molar ratio of compound 10 to 5 'or 3' -amino DNA is (30-100): stirring at 1, 30-50deg.C for 2-12 h, purifying, and desalting with column to obtain compound 11 or compound 12.

6. A process according to claim 3, wherein R is- (CH) in formula I ₂ ) _m3 SC(CH ₃ ) ₂ S(CH ₂ ) _n3 When m3=1 and n3=1, the synthetic route is as follows:

the synthesis method comprises the following steps:

1) Compound 6 was cooled to 0 ℃ in acetone and sulfuric acid was added dropwise, the molar ratio of compound 6 to sulfuric acid being 1: (0.5-0.8:) stirring for 2-12 hours, then diluting with water, gradually adding NaOH aqueous solution to adjust the pH to 5, and then carrying out post-treatment to obtain a compound 13;

2) Compound 13 and paclitaxel were added in a molar ratio of 1: (1-2) DMF was added and then dicyclohexylcarbodiimide and a catalytic amount of DMAP were added to the solution in a molar ratio of dicyclohexylcarbodiimide to compound 13 of (1-2): 1, standing at room temperature overnight, filtering, evaporating, and performing column chromatography on silica gel to obtain compound 14;

3) Adding the compound 14, dicyclohexylcarbodiimide and benzotriazol-N, N, N ', N' -tetramethylurea hexafluorophosphate into DMF respectively, stirring, cooling to 0 ℃, dropwise adding 5 'or 3' -amino DNA and N, N-diisopropylethylamine aqueous solution into the mixture, and stirring at 30-50 ℃ for 4-12h, wherein the molar ratio of the compound 14, dicyclohexylcarbodiimide, benzotriazol-N, N, N ', N' -tetramethylurea hexafluorophosphate, N, N-diisopropylethylamine and 5'/3' -amino DNA is (30-100): (30-100): (30-100): (30-100): 1, TEAA is added to quench the reaction mixture, which is then post-treated to give compound 15 or compound 16.

7. Use of a redox-responsive nucleolin aptamer-paclitaxel conjugate according to claim 1 for the preparation of a medicament against Triple Negative Breast Cancer (TNBC).