A small molecule probe based on sugar metabolism labeling and its application in improving the targeting of platinum drugs

Abstract

The present invention belongs to the field of biological probe technology, and in particular relates to a small molecule probe HBAPE-Ac3ManNAz based on sugar metabolism labeling and its application in improving the targeting of platinum drugs. The present invention provides a small molecule probe HBAPE-Ac3ManNAz based on sugar metabolism labeling. The probe can selectively label tumor cells or tissues in vivo or in vitro as a metabolic precursor, and provide new targets for refractory tumors such as triple-negative breast cancer that lack specific targeting receptors; a pharmacodynamic molecule DBCO-PEG8-Oxp(IV) complex that reacts orthogonally with the probe is synthesized. The complex reacts with an azide tag marked on the tumor cell membrane through a bioorthogonal reaction, thereby improving the targeted transport ability of platinum drugs to tumors and reducing toxic side effects, providing a new theoretical basis for the targeted treatment of tumors in organisms.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological probes, and in particular relates to a small molecule probe HBAPE-Ac3ManNAz based on sugar metabolism labeling and its application in improving the targeting of platinum drugs. Background Art

[0002] Metabolic glycan labeling (MGL) has emerged as a simple yet powerful tool. It cleverly leverages the synthetic pathways of natural sugars to metabolically label non-natural molecules containing reactive functional groups (such as azide and alkyne) onto the cell surface, thereby artificially introducing chemical receptors into tumor cells. Compared to the highly expressed receptors on the surface of conventional tumor cells, metabolic glycan labeling offers the potential to provide specific therapeutic targets for tumor cells due to its in vivo labeling capabilities, high density, low toxicity, and minimal interference. Currently, the developed tetraacetylated N-azidoacetylmannosamine (Ac4ManNAz) can efficiently introduce azide groups onto terminal sialic acids on the surface of tumor cells through enzymes in the sialic acid synthesis pathway. However, the presence of the same enzymes in normal cells catalyzes the metabolic labeling of Ac4ManNAz. Therefore, targeted labeling of tumor tissue with Ac4ManNAz is difficult to achieve in tumor-targeted therapy, and selective labeling of tumor cells in vivo remains a significant challenge. In 2000, Professor Bertozzi's research group first reported the azide-substituted N-acetylmannosamine (ManNAc) analogue N-azidoacetylmannosamine (ManNAz). This probe molecule can be converted into azide-containing sialic acid (SiaNAz) molecules through the corresponding enzymes in the sialic acid synthesis pathway to label the sugar chain structure on the cell membrane surface, thereby artificially adding azide groups to the cell membrane surface. Compared with the highly expressed receptors on the surface of conventional tumor cells, the sugar metabolism labeling strategy has the following characteristics: (1) High density: The cell surface labeling has a high coverage rate, about 10 7(1) Low toxicity: Unlike antibodies, the introduced chemical structure is less likely to trigger side effects such as immune stress in the body; (2) Low interference: Unlike large molecules such as antibodies, the azide or alkyne groups introduced into the non-natural sugar structure are small in size and will not interfere with cell-to-cell recognition and information transmission. Based on these advantages, carbohydrate metabolic labeling combined with bioorthogonal reaction strategies can target the attachment of fluorophores for imaging and observation of glycans in living cells and in vivo. It can also achieve enrichment and omics analysis of labeled glycoproteins through the specific binding of biotin and streptavidin. More importantly, the classic reactive groups, azide and alkyne, are not present in mammals and require monovalent copper ion catalysis. To avoid the toxicity of copper ion labeling in vivo, Bertozzi's group developed a cyclooctyne-based strain-promoted alkyne-azide cycloaddition (SPAAC) with dibenzocyclooctyne (DBCO), also known as copper-free click chemistry. This method can efficiently covalently link glycosylated complexes such as antibodies, folic acid, and peptides under non-copper ion catalysis conditions, providing important support for the application of glycan metabolic labeling in vivo and targeted tumor therapy. However, recent studies have found that most mammalian cells also have the same enzyme that catalyzes the post-translational modification of traditional sugar probes to cell surface glycans in the Golgi apparatus. Therefore, selectively labeling specific cell types with ManNAz without interfering with their metabolic mechanisms has proven difficult. This is particularly true for targeted tumor therapy, where targeted labeling of tumor tissues can be difficult, leading to off-target drug distribution and damage to normal cells and tissues.

[0003] Reactive oxygen species (ROS) refer to reactive small molecules derived from oxygen, including hydrogen peroxide and superoxide anions. These molecules play important roles in biological processes such as cell differentiation, cell signaling, and adaptive immunity. Studies have shown that ROS levels in most tumor cells are elevated, reaching 10-100 μM, compared to 0.001-0.7 μM in normal cells. Leveraging this characteristic, ROS-responsive vectors have been developed, potentially improving safety while achieving therapeutic goals. The ROS-responsive 4-(Hydroxymethyl)benzeneboronic acid pinacol ester (HBAPE) has been developed, which not only selectively targets tumor cells but also reduces toxicity caused by accumulation in normal cells. It is a popular prodrug protecting group in medicinal chemistry research and is often used as a protecting group in ROS-modulating prodrug molecules or detection probes.

[0004] In response to the difference in ROS content between normal and tumor systems, the present invention introduces a 4-hydroxyphenylboronic acid pinacol ester structure (HBAPE) at the 1-position of Ac4ManNAz to develop a new probe HBAPE-Ac3ManNAz that relies on ROS regulation to block the metabolic labeling pathway in healthy cells, thereby selectively chemically labeling and dynamically detecting tumor cells and tumor tissues. The pharmacodynamic molecule DBCO-PEG8-Oxp(IV) with an orthogonal reaction is synthesized to enhance the anti-tumor activity and reduce the occurrence of toxic side effects while achieving the targeting of platinum drugs. Summary of the Invention

[0005] To address the above problems, the present invention provides a small molecule probe HBAPE-Ac3ManNAz based on sugar metabolism labeling. As a metabolic precursor, the probe can selectively label tumor cells or tissues in vivo and in vitro, and provide new targets for refractory tumors such as triple-negative breast cancer that lack specific targeting receptors; a pharmacodynamic molecule DBCO-PEG8-Oxp(IV) complex that reacts orthogonally with the probe is synthesized. The complex reacts with the azide tag marked on the tumor cell membrane through a bioorthogonal reaction, thereby improving the targeted transport ability of platinum drugs to tumors and reducing toxic side effects, providing a new theoretical basis for the targeted treatment of tumors in organisms.

[0006] In order to achieve the above objectives, the HBAPE-Ac3ManNAz structure of the present invention is as follows:

[0007]

[0008] The synthetic route of the HBAPE-Ac3ManNAz is as follows:

[0009]

[0010] Reagents and conditions: (i) NaN3, NaOH, H2O, 75℃, 12h; (ii) N3CH2COOH, HOBT, EDC, MeOH, 0℃, 16h; (iii) Ac2O, P y , 0℃, 6h; (iv) 4-Hydroxyphenylboronic acid pinacolester, Sc(OTf)3, 1,2-C2H4Cl2, reflux, 2h.

[0011] In order to achieve another object of the present invention, the pharmacodynamic molecule DBCO-PEG8-Oxp that orthogonally reacts with the HBAPE-Ac3ManNAz probe of the present invention has the following structural formula:

[0012]

[0013] The synthetic route of the DBCO-PEG8-Oxp (IV) is as follows:

[0014]

[0015] Reagents and conditions: (i)NH2OH.HCl, EtOH, Py, reflux, 12h; (ii) PPA, 125℃, 1.5h; (iii) LiAlH4, Et2O, reflux, 48h; (iv) methyl 4-chloro-4-oxobutyrate, Et3N, DCM, 0℃, 4h; (v) PyHBr3, DCM, Rt, 24h; (vi) KOtBu, THF, -40℃, 4h; (vii) LiOH, H2O, rt, 12h; ( viii)NH2-PEG8-NH2, HATU, DIPEA, DMF, rt, 24h; (ix) H2O2, H2O, 60℃, 4h; (x) palmiticanhydride, DMSO, rt, 7d; (xi) succinic anhydride, DMSO, rt, 12h; (xii) HATU, DIPEA, DMF, rt, 24h.

[0016] The HBAPE-Ac3ManNAz of the present invention can be used as a metabolic precursor to selectively label tumor cells or tumor tissues in vivo or in vitro.

[0017] Preferably, the HBAPE-Ac3ManNAz of the present invention can be used as a probe to specifically mark tumors in the body, and synergize with chemotherapy drugs or immunotherapy methods (including gene therapy, antigen delivery, siRNA, etc.) to achieve the purpose of anti-tumor treatment.

[0018] Preferably, the probe HBAPE-Ac3ManNAz can synergize with platinum drugs in anti-tumor effects, wherein HBAPE-Ac3ManNAz can specifically label azide on the tumor cell membrane, thereby improving the targeted transport ability of platinum drugs to tumors and reducing toxic side effects.

[0019] The platinum drug can be DBCO-PEG8-Oxp (IV), cisplatin and other platinum drugs with anti-tumor effects.

[0020] Preferably, the tumor includes but is not limited to refractory tumors lacking specific targeting receptors, such as triple-negative breast cancer.

[0021] The DBCO-PEG8-Oxp(IV) complex of the present invention and the azide tag labeled on the tumor cell membrane with the probe HBAPE-Ac3ManNAz through a bioorthogonal reaction improve the targeted transport ability of DBCO-PEG8-Oxp(IV) to tumors and reduce toxic side effects, providing a new theoretical basis for the targeted treatment of tumors in vivo.

[0022] The significant technical effects of the present invention.

[0023] In this study, we introduced a 4-hydroxyphenylboronic acid pinacol ester structure at the 1-position of the known probe Ac4ManNAz. This enhanced the probe's selectivity for tumor cells by exploiting the high levels of ROS within tumor cells and inhibited its metabolic activity in normal cells with low ROS levels. This resulted in the development of a new ROS-dependent probe, HBAPE-Ac3ManNAz, for selective chemical labeling and dynamic detection of tumor cells and tissues. This probe, specifically for tumor types lacking effective targets, such as triple-negative breast cancer, provides a new high-density tumor target molecule. The probe HBAPE-Ac3ManNAz is a safe, efficient, and novel carbohydrate metabolism marker capable of specifically labeling tumor cells or tissues in vitro and in vivo. Furthermore, we incorporated a bioorthogonal reaction strategy to chemically link a platinum drug to DBCO. This bioorthogonal reaction then binds to azide groups on tumor cell membranes, enhancing the drug's uptake and targeting by tumor cells and tissues in vitro and in vivo, reducing the platinum drug dosage and its toxic side effects on normal cells, and enhancing the drug's anti-tumor activity. The combination of sugar metabolism labeling and bioorthogonal reactions provides a theoretical basis for the development of new targeted anti-breast cancer platinum drugs, and also provides a new reference for tumor research and the development of targeted treatment plans. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Characterization of cellular metabolic labeling with HBAPE-Ac3ManNAz. (a) Concentration gradient of HBAPE-Ac3ManNAz metabolic labeling; (b) Time gradient of HBAPE-Ac3ManNAz metabolic labeling.

[0025] Figure 2 The cellular metabolic labeling efficiency of HBAPE-Ac3ManNAz is regulated by ROS. (a) Changes in labeling levels of cells co-cultured with HBAPE-Ac3ManNAz and different concentrations of H2O2; (b) Changes in labeling levels of cells co-cultured with HBAPE-Ac3ManNAz and different concentrations of NAC.

[0026] Figure 3 HPLC analysis of the effect of H2O2 on the hydrolysis efficiency of HBAPE-Ac3ManNAz. (a) HBAPE-Ac3ManNAz incubated with varying concentrations of H2O2 for 3 h; (b) HBAPE-Ac3ManNAz incubated with the same concentration of H2O2 for varying times. HPLC injection conditions: 5% methanol (0-5 min); 15% methanol (5-10 min); 20% methanol (10-15 min).

[0027] Figure 4 Analysis of the localization and efficiency of HBAPE-Ac3ManNAz metabolic labeling on the cell membrane. (a) Confocal imaging of 4T1 cells incubated with HBAPE-Ac3ManNAz and Ac4ManNAz for 24 hours and stained with DBCO-488, WGA, and Hoechst 33342 (scale bar: 20 μm); (b) 4T1 cells were incubated with different probes (200 μM) for 24 hours, and the collected cells were coupled with DBCO-488 for flow cytometry analysis of cell membrane fluorescence intensity.

[0028] Figure 5 Selective labeling of tumors in mice with HBAPE-Ac3ManNAz. (a) Whole-body fluorescence imaging of mice injected with Ac4ManNAz, HBAPE-Ac3ManNAz, or HBAPE-Ac3ManNAz combined with NAC for seven consecutive days, 24 hours after tail vein injection of DBCO-Cy5; (b) Fluorescence images of major organs and tumors in mice labeled with different probes 24 hours after tail vein injection of DBCO-Cy5; (c) Quantification of the mean radiant efficiency of tumor tissue in mice labeled with different probes using the IVIS Spectral Imaging System (n = 2). ns indicates no statistical difference; *P < 0.05, **P < 0.01, ***P < 0.001.

[0029] Figure 6 To evaluate the anti-tumor activity of platinum drugs in vitro. (a) IC of positive control drug oxaliplatin in 4T1 cells 50 (b) IC value of DBCO-PEG8-Oxp(IV) in 4T1 cells 50 (c) IC of DBCO-PEG8-Oxp in 4T1 cells labeled with HBAPE-Ac3ManNAz 50 value.

[0030] Figure 7 ICP-MS analysis of platinum uptake in 4T1 cells. (a) Platinum content in 4T1 cells labeled with HBAPE-Ac3ManNAz for 24 hours and incubated with 1 μM oxaliplatin or DBCO-PEG8-Oxp(IV) for 2 hours (n=3); (b) Platinum content in 4T1 cells labeled with HBAPE-Ac3ManNAz for 24 hours and incubated with different concentrations of DBCO-PEG8-Oxp(IV) for 2 hours (n=3); (c) Platinum content in 4T1 cells labeled with HBAPE-Ac3ManNAz for 24 hours and incubated with 1 μM DBCO-PEG8-Oxp(IV) for different time periods (n=3). ns indicates no statistical difference; *P<0.05, **P<0.01, ***P<0.001.

[0031] Figure 8 DBCO-PEG8-Oxp(IV) significantly inhibits the migration of 4T1 breast cancer cells via a click reaction. (a) Wound healing assay investigating the inhibitory effect of DBCO-PEG8-Oxp(IV) on 4T1 cell migration before and after HBAPE-Ac3ManNAz labeling. Left: Figure, right: Statistical analysis (n=3, scale bar: 50 μm); (b) Transwell assay investigating the inhibitory effect of DBCO-PEG8-Oxp(IV) on 4T1 cell migration before and after HBAPE-Ac3ManNAz labeling. Left: Figure, right: Statistical analysis (n=3, scale bar: 100 μm); (c) Effects of DBCO-PEG8-Oxp(IV) on the expression of MMP-9 and MMP-7, proteins associated with cell growth and migration, before and after labeling. ns indicates no statistical difference; *P < 0.05, **P < 0.01, ***P < 0.001.

[0032] Figure 9Antitumor activity of DBCO-PEG8-Oxp(IV) in mice with 4T1 breast cancer based on glucose metabolism labeling. (a) Schematic diagram of dosing schedule in a 4T1 breast cancer mouse model; (b) Curves of weight changes in mice in different treatment groups during treatment; (c) Curves of tumor volume changes in mice in different treatment groups during treatment; (d) Images of tumors in mice in different treatment groups at the end of the experiment; (e) Tumor weights in mice in different treatment groups at the end of the experiment; (f) H&E staining of tumors in mice in different treatment groups at the end of the experiment (scale: 50 μm); (g) Distribution of platinum content in various tissues of mice in different treatment groups at the end of the experiment (n = 3). ns indicates no statistically significant difference; *P < 0.05, **P < 0.01, ***P < 0.001.

[0033] Figure 10 Based on glucose metabolism labeling, DBCO-PEG8-Oxp(IV) demonstrated its anti-tumor metastasis activity in 4T1 breast cancer mice. (a) Schematic diagram of the administration schedule for the 4T1 breast cancer mouse lung metastasis model; (b) Representative images of lung metastases in mice in different treatment groups at the end of the experiment (n=5); (c) Body weight changes during treatment in different treatment groups (n=5); (d) Statistical data of lung metastases in mice in different treatment groups at the end of the experiment (n=5); (e) Lung tissue weights in mice in different treatment groups at the end of the experiment (n=5); (f) H&E staining of lung tissues in mice in different treatment groups at the end of the experiment (scale bar: 50 μm); (g) Distribution of platinum content in various tissues in mice in different treatment groups at the end of the experiment (n=3). ns indicates no statistical difference; *P<0.05, **P<0.01, ***P<0.001. Specific implementation methods

[0034] The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and specific implementation methods.

[0035] Example 1.

[0036] Based on Ac4ManNAz (Structure a), this project replaced the acetyl group at position 1 with the ROS-regulated protecting group 4-(hydroxymethyl)phenylboronic acid pinacol ester (HBAPE), resulting in the chemical synthesis of a novel ROS-responsive molecular probe for glucose metabolism, HBAPE-Ac4ManNAz (Structure b). Simultaneously, a tetravalent platinum prodrug was linked to DBCO via polyethylene glycol (PEG) to further synthesize the water-soluble DBCO-PEG8-Oxp(IV) (Structure c). Building on the targeted tumor labeling of HBAPE-Ac4ManNAz, the newly synthesized platinum drug was transported to the tumor site through an efficient bioorthogonal reaction, thereby increasing its anti-tumor activity and targeting while reducing its toxic side effects.

[0037]

[0038] 1. The synthesis steps of HBAPE-Ac3ManNAz probe are as follows:

[0039] (1) Synthesis of Compound 1

[0040] 18 g of chloroacetic acid was weighed and added to a 250 mL round-bottom flask. 100 mL of distilled water was added to completely dissolve it. 8 g of NaOH solid particles were added. After stirring evenly, 15 g of NaN3 was gradually added. An N2 ball was inserted and the reaction temperature was set to 75 ° C. After reacting for 12 hours, the heating device was turned off to restore room temperature. The reaction solution in the round-bottom flask was poured into a beaker containing 500 mL of ice water and stirred continuously. 20 mL of glacial hydrochloric acid (15 mL of concentrated hydrochloric acid + 5 mL of ice water) was added gradually to the beaker to adjust the pH of the solution to 2-3. Subsequently, 300 mL of ethyl acetate was gradually added for extraction. The reaction was repeated three times. The collected ethyl acetate was dried over anhydrous sodium sulfate, filtered, and the organic solvent was evaporated under reduced pressure to obtain 15.6 g of white liquid compound 1 with a yield of 81%. 1 H NMR (300MHz, CDCl3) δ11.30 (s, 1H), 4.02 (s, 2H). 13 C NMR (75MHz, CDCl3) δ174.74,50.06.

[0041] (2) Synthesis of Compound 2

[0042] 5 g of D-mannosamine hydrochloride was weighed and added to a 250 mL round-bottom flask. 100 mL of ultra-dry methanol was added and stirred at room temperature for 20 min. Subsequently, 3.6 g of compound 1 and 9.6 mL of Et3N were added in sequence. The reaction solution was moved to an ice-water bath and stirred for 30 min. 2.2 g of HOBT and 3.2 g of EDC were added to the reaction solution. A nitrogen balloon was inserted and the reaction was allowed to proceed. After 16 h, the reaction was monitored by TCL. After the reaction was complete, the organic solvent was evaporated under reduced pressure and purified by column chromatography to obtain 3.8 g of yellow-white solid compound 2 with a yield of 62%.

[0043] (3) Synthesis of Compound 3

[0044] 2 g of compound 2 was weighed and added to a 100 mL round-bottom flask. 30 mL of ultra-dry pyridine was added and the mixture was moved to an ice-water bath with constant stirring. Subsequently, 5 mL of acetic anhydride was gradually added and an N2 ball was inserted to allow the reaction to proceed. After 6 h, the reaction was monitored using TCL. After the reaction was complete, the organic solvent was evaporated under reduced pressure. 80 mL of a 10% dilute hydrochloric acid solution (concentrated hydrochloric acid: distilled water = 50 mL:450 mL) and 120 mL of DCM were added to a 250 mL separatory funnel for extraction. The lower organic solvent was collected and the extraction operation was repeated three times. The collected DCM was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain 2.5 g of compound 3 as a white solid with a yield of 75%. 1 H NMR (300MHz, CDCl3) δ6.66(d,J=9.5Hz,1H),6.07(d,J=1.9Hz,1H),5.37(dd,J=10.2,4.2Hz,1H),5.31–5.16(m,1H),4.70–4.61(m,1H),4 .26(dd,J=8.5,3.8Hz,1H),4.16(dd,J=12.1,2.3Hz,1H),4.11(s,2H),4.09(s,1H),2.21(s,3H),2.14(s,3H),2.09(s,3H),2.03(s,3H). 13 C NMR (75MHz, CDCl3) δ170.57,170.15,169.61,168.18,166.85,91.31,70.25,68.86,65.12,61.81,52.37,49.25,20.89,20.79,20.68,20.63.

[0045] (4) Synthesis of Compound 4

[0046] 500 mg of compound 3, 1.37 g of 4-hydroxyphenylboronic acid pinacol ester and 345 mg of scandium trifluoromethanesulfonate were weighed and added to a 100 mL round-bottom flask, and 30 mL of 1,2-C2H4Cl2 was added to stir it thoroughly. Then, a condenser was inserted, vacuum was applied and an N2 ball was inserted, the temperature was raised to 90°C and the reaction was carried out for two hours. The reaction was monitored by TCL. After the reaction was complete, the organic solvent was evaporated under reduced pressure, and 30 mL of distilled water and 50 mL of DCM were added to a 100 mL separatory funnel for extraction. The lower organic solvent was collected and the extraction operation was repeated three times. The collected DCM was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain 407 mg of white solid compound 4 with a yield of 58%. 1H NMR (300MHz, CDCl3) δ7.85(d,J=7.9Hz,2H),7.39(d,J=7.8Hz,2H),6.56(d,J=9.4Hz,1 H),5.41(dd,J=10.2,4.3Hz,1H),5.18(t,J=10.1Hz,1H),4.84(d,J=1.7Hz,1H),4.76( d,J=12.2Hz,1H),4.72–4.61(m,2H),4.26(dd,J=12.3,4.4Hz,1H),4.13(dd,J=6.4,1. 9Hz, 1H), 4.06 (d, J = 10.4Hz, 3H), 2.17 (s, 3H), 2.08 (s, 3H), 2.01 (s, 3H), 1.38 (s, 12H). 13 C NMR (75MHz, CDCl3) δ170.60,170.05,169.84,166.66,139.01,135.11,127.45,97.65,83. 95,69.77,69.35,68.43,65.73,62.14,52.46,50.24,24.90,24.88,20.83,20.72,20.69.

[0047] 2. The synthesis steps of the orthogonal reaction drug molecule DBCO-PEG8-Oxp are as follows:

[0048] (1) Synthesis of compound 5

[0049] 20 g of 5-dibenzosuberenone and 13.6 g of hydroxylamine hydrochloride were weighed separately and added to a 500 mL round-bottom flask. 60 mL of ultra-dry pyridine and 240 mL of anhydrous ethanol were added in sequence and stirred evenly. A condenser was inserted, vacuum was applied, and an N2 ball was inserted. The temperature was raised to 90°C and refluxed for 12 hours. The reaction was monitored by TCL. After the reaction was complete, the organic solvent was evaporated under reduced pressure. 350 mL of icy 5% dilute hydrochloric acid was added and the mixture was stirred rapidly in an ice-water bath for 30 min. A white solid gradually precipitated during the stirring process. The precipitate was filtered and washed with distilled water several times. After drying, it was thoroughly pumped dry with an oil pump to obtain 16 g of white solid compound 5 with a yield of 74%. 1 H NMR (300MHz, CDCl3) δ7.76–7.72(m,1H),7.68–7.64(m,1H),7.52–7.40(m,6H),6.96(d,J=2.1Hz,2H). 13C NMR (75MHz, CDCl3) δ156.35,135.39,134.53,133.75,130.80,130.60,130.50,129.45,129.18,129.04,128.95,128.83,127.79,127.66.

[0050] (2) Synthesis of Compound 6

[0051] 200 mL of polyphosphoric acid was weighed and added to a 500 mL round-bottom flask. The polyphosphoric acid was heated to 125 ° C and 14 g of compound 5 was gradually added at this temperature with continuous stirring. During the reaction, it gradually turned into a viscous yellow solution. After 1.5 h, the heating device was turned off. After it returned to room temperature, it was poured into a beaker containing 800 mL of ice water and continued to stir rapidly for 30 min. During the stirring process, a gray-brown solid gradually precipitated. The precipitate was filtered and washed with distilled water several times. After drying, it was thoroughly pumped dry with an oil pump to obtain 11 g of gray-brown solid compound 6 with a yield of 79%. 1 H NMR (300MHz, DMSO) δ9.89 (s, 1H), 7.33–7.11 (m, 8H), 7.01 (d, J = 11.7Hz, 1H), 6.90 (d, J = 11.4Hz, 1H). 13 C NMR (75MHz, DMSO) δ172.20,136.76,136.59,134.92,133.91,133.11,130.66,129.44,129.39,128.54,128.26,128.23,127.87,126.85,126.68.

[0052] (3) Synthesis of Compound 7

[0053] 6 g of compound 6 and 2.6 g of LiAlH4 were weighed separately and added to a 500 mL round-bottom flask. A condenser was inserted, and the mixture was evacuated and an N2 ball was inserted. Subsequently, 200 mL of ultra-dry ether was gradually added to the flask while stirring continuously. The temperature was raised to 45°C, and ether was continuously added during the reaction to prevent the solvent from evaporating. After 48 h, the reaction was monitored by TCL. After the reaction was complete, the round-bottom flask was placed in an ice-water bath, and distilled water was gradually added to the flask until no gas was generated. Subsequently, 200 mL of DCM was added for extraction and the extraction was repeated three times. The collected DCM was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain 3 g of yellow solid compound 7 with a yield of 53%. 1H NMR (300MHz, CDCl3) δ7.37–7.22(m,4H),7.05(dd,J=7.8,1.6Hz,1H),6.96(ddd,J=8.4,7.1,1.6Hz,1H),6.69(t d,J=7.6,1.2Hz,1H),6.62(d,J=13.2Hz,1H),6.54(dd,J=8.1,1.2Hz,1H),6.43(d,J=13.1Hz,1H),4.64(s,2H). 13 C NMR (75MHz, CDCl3) δ147.15,139.29,138.20,134.79,132.81,130.23,128.96,128.05,127.72,127.48,127.43,121.87,118.04,117.83,49.64.

[0054] (4) Synthesis of Compound 8

[0055] 2.5 g of compound 7 was weighed and added to a 100 mL round-bottom flask, followed by the addition of 40 mL of ultra-dry DCM and 3.5 mL of Et3N. The mixture was evacuated and an N2 ball was inserted. The round-bottom flask was then placed in an ice-water bath with constant stirring. 2.7 mL of succinic acid monomethyl chloride was slowly added. After 4 h, the reaction was monitored by TLC. After completion of the reaction, the organic solvent was evaporated under reduced pressure, and the mixture was purified by column chromatography to obtain 2.9 g of yellow-white solid compound 8 with a yield of 75%. 1 H NMR(300MHz, CDCl3)δ7.30(s,5H),7.21–7.13(m,3H),6.83(d,J=12.9Hz,1H),6.65(d,J=12.9Hz,1H),5.55(d, J=15.0Hz,1H),4.29(d,J=15.0Hz,1H),3.65(s,3H),2.68–2.58(m,1H),2.55–2.41(m,2H),2.11–1.99(m,1H). 13 CNMR(75MHz, CDCl3)δ173.48,170.93,140.58,136.53,135.89,134.65,132.71,131.91 ,130.95,130.24,128.63,128.33,128.10,127.36,127.03,54.56,51.70,29.63,29.10.

[0056] (5) Synthesis of Compound 9

[0057] 2 g of compound 8 and 2.5 g of PyHBr3 were weighed and added to a 100 mL round-bottom flask. 40 mL of ultra-dry DCM was added, and the mixture was evacuated and inserted with an N2 ball. The reaction was allowed to react at room temperature in the dark for 24 h. The reaction was monitored by TCL. After the reaction was complete, 30 mL of 10% dilute hydrochloric acid solution and 20 mL of DCM were added to the flask and the mixture was transferred to a 100 mL separatory funnel for extraction. The lower organic solvent was collected and the extraction operation was repeated three times. The collected DCM was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain 2.1 g of gray-brown solid compound 9 with a yield of 70%. 1 H NMR (300MHz, CDCl3) δ7.76(d,J=9.0Hz,1H),7.26–7.02(m,6H),6.92(d,J=7.6Hz,1H),5.95(d,J=9.9Hz,1H),5.85(d,J=14.8 Hz,1H),5.20(d,J=9.9Hz,1H),4.23(d,J=14.9Hz,1H),3.72(s,3H),2.96–2.83(m,1H),2.69–2.58(m,2H),2.58–2.46(m,1H). 13 C NMR (75MHz, CDCl3) δ173.60,172.05,138.35,137.10,136.98,132.82,130.85,130.76,13 0.71,129.70,129.54,129.02,128.95,128.64,60.15,55.63,52.61,51.81,30.72,29.29.

[0058] (6) Synthesis of Compound 10

[0059] 2 g of compound 9 was weighed and added to a 100 mL round-bottom flask. 40 mL of ultra-dry THF was added, and the mixture was evacuated and inserted with an N2 ball. The mixture was placed at -40°C and stirred continuously. After 10 min, 8 mL of a 1 M tetrahydrofuran solution of potassium tert-butoxide was gradually added at the same temperature. After 2 h, another 3.2 mL was added and the reaction was continued for 1 h. The reaction was monitored by TLC. After the reaction was complete, 30 mL of distilled water and 50 mL of DCM were added for extraction. The lower organic solvent was collected and the extraction operation was repeated three times. The collected DCM was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain 800 mg of compound 10 as a yellow oil with a yield of 60%. 1H NMR (300MHz, CDCl3) δ7.72(d,J=8.8Hz,1H),7.60–7.49(m,1H),7.46–7.27(m,6H),5.20(d,J=13.8Hz,1H),3.67(d, J=9.6Hz,1H),3.59(s,3H),2.80–2.69(m,1H),2.66–2.58(m,1H),2.43–2.33(m,1H),1.99(dt,J=15.9,6.1Hz,1H). 13 C NMR (75MHz, CDCl3) δ173.31,171.70,151.48,148.04,132.31,129.32,128.55,128.30,128 .16,127.76,127.13,125.49,123.16,122.70,114.98,107.75,55.50,51.65,29.56,29.11.

[0060] (7) Synthesis of Compound 11

[0061] 500 mg of compound 10 was weighed and added to a 100 mL round-bottom flask. 20 mL of ultra-dry THF was added with continuous stirring. 75 mg of LiOH was dissolved in 6 mL of distilled water and then added to the round-bottom flask. The mixture was reacted in the dark at room temperature for 12 h. The reaction was monitored by TCL. After the reaction was complete, 30 mL of distilled water and 50 mL of DCM were added for extraction. The lower organic solvent was collected and the extraction operation was repeated three times. The collected DCM was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain 300 mg of yellow-white solid compound 11 with a yield of 63%. 1 H NMR (300MHz, DMSO) δ12.05(s,1H),7.65(d,J=6.1Hz,1H),7.57–7.25(m,7H),5.04(d,J=14.1Hz,1H),3.63 (d,J=14.0Hz,1H),2.68–2.52(m,1H),2.40–2.23(m,1H),2.27–2.11(m,1H),1.78(dt,J=16.3,6.4Hz,1H). 13 C NMR(75MHz,DMSO)δ174.03,171.22,151.93,148.89,132.85,130.08,129.39,128.66,1 28.48,128.14,127.29,125.63,123.02,122.02,114.76,108.51,55.41,29.74,29.45.

[0062] (8) Synthesis of Compound 12

[0063] 150 mg of compound 11 was weighed and added to a 100 mL round-bottom flask. 15 mL of anhydrous DMF was added, and the mixture was evacuated and a N2 ball was inserted with continuous stirring to completely dissolve the mixture. 280 mg of HATU was then weighed and dissolved in 6 mL of anhydrous DMF and added to the flask. After 20 minutes of reaction, 405 mg of NH2-PEG8-NH2 was weighed and dissolved in 5 mL of anhydrous DMF. 0.41 mL of DIPEA was added to the dissolved NH2-PEG8-NH2 solution. After 10 minutes, the mixed solution of NH2-PEG8-NH2 and DIPEA was added to the round-bottom flask. The mixture was allowed to react in the dark for 24 hours. The reaction was monitored by TLC. After the reaction was complete, the organic solvent was evaporated under reduced pressure and purified by column chromatography to obtain 214 mg of yellow-white milky compound 12 with a yield of 65%. HRMS (ESI+) (m / z) calculated for C 37 H 54 N3O 10 [M+H] + 700.3804,found 700.3790.

[0064] (9) Synthesis of Compound 13

[0065] 1 g of oxaliplatin was weighed into a 250 mL round-bottom flask. 30 mL of distilled water was added with continuous stirring. Subsequently, 60 mL of 30% aqueous hydrogen peroxide was added dropwise. The mixture was allowed to react at 60°C for 4 hours until completely dissolved, and the solvent was evaporated. 100 mL of distilled water was added to the round-bottom flask, and stirring was continued. The reaction temperature was adjusted to 80°C. Once the reaction solution in the flask was completely clear, heating was stopped and the flask was placed in a refrigerator at 4°C overnight. The next day, a large amount of white needle-like crystals precipitated. The solvent was poured out of the flask, and the crystals were pumped dry with an oil pump to obtain 900 mg of compound 13 as a white solid with a yield of 83%.

[0066] (10) Synthesis of Compound 14

[0067] 300 mg of compound 13 and 310 mg of palmitic anhydride were weighed into a 100 mL round-bottom flask, and the flask was vacuumed and inserted with an N2 ball. 30 mL of anhydrous DMSO solvent was added to the flask and stirred at room temperature for 7 days. After the reaction, the organic solvent was evaporated with an oil pump. Subsequently, 50 mL of acetone was added to the flask, and a large amount of white solid precipitated. The solid was filtered, washed repeatedly with acetone, and dried to obtain 350 mg of white solid compound 14 with a yield of 75%.

[0068] (11) Synthesis of Compound 15

[0069] 300 mg of compound 14 and 100 mg of succinic anhydride were weighed into a 100 mL round-bottom flask, and 30 mL of anhydrous DMSO solvent was added. The mixture was vacuum-pressed and N2 ball was inserted to react for 12 h. After the reaction, the organic solvent was evaporated under reduced pressure and purified by column chromatography to obtain 210 mg of white solid compound 15 with a yield of 61%.

[0070] (12) Synthesis of Compound 16

[0071] 100 mg of compound 15 was weighed and added to a 50 mL round-bottom flask. 10 mL of anhydrous DMF was added, and the mixture was completely dissolved by vacuum, an N2 ball was inserted, and stirring was continued. Subsequently, 75 mg of HATU was weighed and dissolved in 3 mL of anhydrous DMF and added to the flask. After reacting for 20 minutes, 174 mg of compound 12 was weighed and dissolved in 5 mL of anhydrous DMF. 0.19 mL of DIPEA was added to the dissolved compound 12 solution. After 10 minutes, the mixed solution of compound 12 and DIPEA was added to the round-bottom flask. The mixture was reacted in the dark for 24 hours and monitored by TLC. After completion of the reaction, the organic solvent was evaporated under reduced pressure, and the mixture was purified by column chromatography to obtain 89 mg of yellow-brown milky compound 16 with a yield of 47%. 1 H NMR (300MHz, MeOD) δ7.45(d,J=2.5Hz,4H),7.39–7.20(m,4H),5.11(d,J=14.0Hz,1H),3.81–3.49(m,34H),3.50–3.29(m,6H),3.24(d,J=6. 9Hz,4H),2.77–2.67(m,1H),2.67–2.45(m,2H),2.46–2.09(m,5H),1.97(dt,J=16.2,6.7Hz,2H),1.67–1.21(m,30H),0.91(t,J=5.8Hz,3H). 13 C NMR(75MHz,MeOD)δ173.40,173.17,172.96,172.52,151.31,148.11,132 .14,129.26,128.68,128.29,127.84,127.55,126.77,125.11,122.99,1 22.24,114.23,107.53,69.95,69.80,69.13,55.31,54.44,38.97,31.74 ,30.52,30.02,29.96,29.44,29.16,22.41,17.36,15.92,13.19,11.87.

[0072] Example 2 Biological Evaluation of HBAPE-Ac3ManNAz

[0073] 1.1 Metabolic labeling of living cells

[0074] To further explore the labeling efficiency of HBAPE-Ac3ManNAz, we incubated 4T1 cells with HBAPE-Ac3ManNAz at different concentrations of 50, 100, 200, and 500 μM for 24 hours to label proteins in mammalian cells. The cleaved proteins were subjected to click reaction with Biotin-PEG4-alkyne and the protein labeling efficiency of HBAPE-Ac3ManNAz was verified by Western Blot. The results are shown in Figure 2. Figure 1 As shown in a, with the increase of HBAPE-Ac3ManNAz concentration, the metabolic labeling effect of the probe becomes stronger and stronger. When the concentration reaches 200 μM, HBAPE-Ac3ManNAz can achieve efficient labeling of 4T1 cells without cytotoxicity.

[0075] Using this as a reference, 4T1 cells were incubated at this concentration for different time periods, such as 0, 8, 12, 24, 36, and 48 hours, and then the protein labeling efficiency was verified using the same method. The results are shown in the figure. Figure 1 As shown in Figure b, the labeling effect of HBAPE-Ac3ManNAz gradually increased with increasing incubation time. At 24 hours, the cell labeling effect of HBAPE-Ac3ManNAz reached an ideal intensity. Extending the incubation time further, the labeling effect became even stronger at 36 hours, and no significant difference was observed after further extensions. Therefore, based on the concentration- and time-dependent experimental results, HBAPE-Ac3ManNAz can achieve efficient cell labeling when used at a concentration of 200 μM for 24 hours.

[0076] 1.2 Cellular metabolic labeling of HBAPE-Ac3ManNAz is regulated by ROS

[0077] The metabolic labeling of HBAPE-Ac3ManNAz was basically characterized by concentration, time dependence and cell diversity experiments, which proved that the newly synthesized HBAPE-Ac3ManNAz can be used as a normal sugar metabolism labeling probe. Based on this, Western Blot was used to further explore whether the cellular metabolic labeling of HBAPE-Ac3ManNAz is regulated by ROS. 4T1 cells were continued to be used as the research object for metabolic labeling. When 200μM HBAPE-Ac3ManNAz was co-incubated with 4T1 cells for 24h, different concentrations of ROS agonist H2O2 solution were added. The protein labeling efficiency was subsequently verified by the same method. The results are as follows Figure 2As shown in a, as the H2O2 concentration continued to increase, the labeling effect of HBAPE-Ac3ManNAz gradually increased, indicating that the cell metabolic labeling of HBAPE-Ac3ManNAz was concentration-dependent on H2O2.

[0078] At the same time, in order to further illustrate that the cell metabolic labeling of HBAPE-Ac3ManNAz is regulated by ROS, different concentrations of ROS inhibitor N-acetyl-L-cysteine ​​(NAC) were co-incubated with 200 μM HBAPE-Ac3ManNAz in 4T1 cells for 24 h, and the protein verification results were as follows Figure 2 As shown in Figure b, when the NAC concentration was 0.5 mM, the labeling effect was significantly reduced and gradually weakened as the NAC concentration continued to increase. When the concentration reached 3 mM, the labeling effect was almost completely inhibited. These results indicate that the carbohydrate metabolism molecular probe HBAPE-Ac3ManNAz is regulated by ROS during the metabolic labeling process of cells.

[0079] 1.3 In vitro HPLC detection of changes in the regulation of HBAPE-Ac3ManNAz by the ROS agonist H2O2

[0080] When HBAPE-Ac3ManNAz labeled 4T1 cells, the ROS agonist H2O2 was added to change the cell metabolic labeling level of the probe to determine that its labeling process was regulated by ROS. In addition, in order to further determine that the departure of the 1-position 4-(hydroxymethyl)phenylboronic acid pinacol ester functional group in the newly synthesized HBAPE-Ac3ManNAz was affected by ROS, we used in vitro HPLC to detect the hydrolysis efficiency of the 1-position 4-(hydroxymethyl)phenylboronic acid pinacol ester functional group of HBAPE-Ac3ManNAz under the action of H2O2. HBAPE-Ac3ManNAz was incubated with different concentrations of H2O2 in a 1.5mL EP tube for 3 hours and then HPLC detection was performed. The results are as follows: Figure 3 As shown in (a), with the increase of H2O2 concentration, the hydrolysis efficiency of the 1-functional group of HBAPE-Ac3ManNAz gradually increased.

[0081] At the same time, the same concentration of HBAPE-Ac3ManNAz and H2O2 were incubated for different time periods for HPLC detection. The results are as follows: Figure 3 As shown in Figure b, the hydrolysis efficiency of the functional group 1 of HBAPE-Ac3ManNAz gradually increased with the increase of incubation time. Therefore, the above two HPLC test results further confirmed that the hydrolysis efficiency of the newly synthesized HBAPE-Ac3ManNAz was affected by the ROS agonist H2O2.

[0082] 1.4 Analysis of the localization and efficiency of HBAPE-Ac3ManNAz in cell labeling by confocal imaging and flow cytometry

[0083] Based on the chemical structure of the classic probe Ac4ManNAz, HBAPE-Ac3ManNAz, which responds to ROS, was further synthesized. Therefore, after hydrolysis under the action of high ROS content in tumor cells, its metabolic labeling mechanism is theoretically consistent with that of Ac4ManNAz. Both are converted into azide-containing sialic acid molecules through the corresponding enzymes in the sialic acid biosynthesis pathway to achieve azide group labeling of the terminal sialic acid of the sugar chain on the cell membrane surface. In order to verify this hypothesis, the cell labeling of Ac4ManNAz and HBAPE-Ac3ManNAz was compared by confocal imaging and flow cytometry. Ac4ManNAz and HBAPE-Ac3ManNAz were incubated with 4T1 cells at a concentration of 200μM in a confocal glass dish for 24 hours, and then DBCO-488 dye was added to allow azide to bind to DBCO and thus attach fluorescence to the probe labeling position. WGA dye was then used to stain the cell membrane surface glycoproteins and Hoechst 33342 dye was used to stain the cell nucleus as controls. The cells were observed under a confocal microscope, and the results are as follows: Figure 4 As shown in Figure a, the DBCO-488 fluorescence regions of HBAPE-Ac3ManNAz and Ac4ManNAz are consistent and overlap with the WGA dye-stained region, indicating that the newly synthesized HBAPE-Ac3ManNAz can achieve cell membrane surface labeling using the same metabolic mechanism as Ac4ManNAz. In addition, under a fluorescence microscope, it can be seen that the cell labeling level of HBAPE-Ac3ManNAz is slightly stronger than that of Ac4ManNAz, and after co-incubation with NAC, the fluorescence region marked by HBAPE-Ac3ManNAz is significantly weakened, further providing more sufficient evidence that the metabolic labeling of HBAPE-Ac3ManNAz is regulated by ROS.

[0084] At the same time, in order to verify that HBAPE-Ac3ManNAz can achieve efficient labeling of cell membranes, we used flow cytometry to compare the fluorescence intensity of the cell membrane surface. Ac36deoGlcNAz, Ac4GalNAz, Ac4ManNAz, HBAPE-Ac3ManNAz and other different types of probes were incubated with 4T1 cells for 24 hours. After that, the cells were collected and stained with DBCO-488 dye for on-line detection on a flow cytometer. The results are as follows: Figure 4 As shown in Figure 2, the fluorescence labeling efficiency of HBAPE-Ac3ManNAz on the cell membrane reached the intensity of Ac4ManNAz. The results of these two experiments fully demonstrate that the newly synthesized HBAPE-Ac3ManNAz is a highly efficient metabolic probe for labeling cell membrane surface glycoproteins.

[0085] 1.5HBAPE-Ac3ManNAz selectively labels tumors in mice

[0086] By verifying that HBAPE-Ac3ManNAz can be used as a non-toxic and efficient molecular probe of sugar metabolism at the cellular level, we want to further verify the labeling efficiency of HBAPE-Ac3ManNAz on tumor cells in a mouse tumor-bearing model. First, we verify the relationship between the labeling efficiency of HBAPE-Ac3ManNAz and the number of administrations. 4T1 tumor cells were subcutaneously implanted into mice to construct a tumor-bearing model. After the tumor was formed, different groups of mice were injected intraperitoneally with Ac4ManNAz, HBAPE-Ac3ManNAz, HBAPE-Ac3ManNAz and NAC solution for seven consecutive days (drug dosage was 300 mg / kg), and then 100 μg / μLDBCO-Cy5 was injected through the tail vein. After 24 hours, in vivo imaging of the small animals was performed and the fluorescence signals at the tumor location were compared. Figure 5 As shown in a, the fluorescence signal of HBAPE-Ac3ManNAz at the tumor location is stronger than the tumor labeling effect of Ac4ManNAz. At the same time, after the injection of ROS inhibitor NAC, the tumor labeling effect of HBAPE-Ac3ManNAz was significantly weakened, indicating that the tumor labeling of the new probe HBAPE-Ac3ManNAz in vivo is also regulated by ROS.

[0087] At the same time, each group of mice was dissected for tissue imaging, such as Figure 5 As shown in Figures b-c, in an in vivo mouse tumor-bearing model, Ac4ManNAz exhibited fluorescence signals in the lung and kidney tissues in addition to the tumor site. The newly synthesized HBAPE-Ac3ManNAz significantly reduced metabolic labeling in non-tumor sites, increasing the probe's selective labeling of tumor cells in vivo. Simultaneously, mice injected with NAC inhibited the normal metabolic labeling process of HBAPE-Ac3ManNAz, thereby reducing the probe's expression at the tumor site and allowing it to remain in the kidney for metabolism, resulting in a strong fluorescence signal. Based on the above in vivo imaging experimental results, it can be concluded that compared to the classic probe Ac4ManNAz, the newly synthesized HBAPE-Ac3ManNAz's in vivo metabolic labeling is regulated by ROS, thereby achieving highly selective and efficient labeling of tumor sites.

[0088] Example 3.

[0089] 1. In vitro and in vivo antitumor activity and targeting studies of DBCO-PEG8-Oxp(IV) based on HBAPE-Ac3ManNAz carbohydrate metabolism labeling

[0090] The present invention uses tetravalent oxaliplatin as the parent core, and introduces a hexadecane chain at one end of the upright bonds at both ends. The hexadecane chain-modified platinum drug is easily combined with human serum albumin (HSA) for administration, and the drug accumulates preferentially in cancer cells than in normal cells. At the same time, DBCO is introduced at the other end of the upright bond, which can undergo a click reaction with cancer cells pre-labeled with an azide group to enhance its tumor-targeting transport ability.

[0091] 1. In vitro antitumor activity test

[0092] The target compound DBCO-PEG8-Oxp(IV) was obtained by chemical synthesis, and its in vitro antitumor activity was evaluated by MTT assay. Figure 6 As shown, the positive drug bivalent oxaliplatin ( Figure 6 a) As a control, IC of the chemically synthesized tetravalent oxaliplatin complex DBCO-PEG8-Oxp(IV) in 4T1 breast cancer cells 50 ( Figure 6 b) is lower than divalent platinum, indicating that the synthesized drug has certain anti-tumor activity. At the same time, DBCO-PEG8-Oxp(IV) ( Figure 6 c) IC 50 The value reached the nanomolar level, and the inhibitory effect on tumor growth activity was significantly enhanced, indicating that click reaction-mediated drug targeted delivery may be a promising option for improving the tumor targeting ability and anti-tumor activity of platinum drugs.

[0093] 2. Platinum uptake by DBCO-PEG8-Oxp(IV) in breast cancer cells

[0094] The degree of drug accumulation in cancer cells affects the strength of its anti-tumor activity. Platinum drugs enhance the drug's targeting ability and increase the cell's drug uptake through click reactions. Therefore, ICP-MS was further used to quantitatively detect the platinum content of DBCO-PEG8-Oxp(IV) in cells based on the click reaction.

[0095] We added DBCO-PEG8-Oxp(IV) at a final concentration of 1 μM to 4T1 cells labeled with HBAPE-Ac3ManNAz for 24 hours and unlabeled cells and incubated them for 2 hours. At the same time, oxaliplatin was used as a positive drug for comparison. After the incubation, the collected cells were acidified with nitric acid and detected by ICP-MS. The results are shown in Figure 2. Figure 7 As shown in a, unlabeled cells were incubated with the same concentrations of oxaliplatin and DBCO-PEG8-Oxp(IV), and each 2×10 5There was no significant difference in the platinum content of the cells, but the platinum content in the cells labeled with HBAPE-Ac3ManNAz increased significantly, almost 4 to 5 times that of the unlabeled cells, indicating that the click reaction promoted the cell's ability to take up platinum drugs. In addition, 0.5, 1, and 2 μM DBCO-PEG8-Oxp(IV) were incubated in 4T1 cells labeled with HBAPE-Ac3ManNAz for 24 hours for 2 hours, and the results were detected by ICP-MS. Figure 7 As shown in b, every 2×10 5 The platinum content in each cell was significantly higher than that in the control group, but there was no significant difference in the platinum content in the cells between different concentrations. We speculate that this may be due to the short incubation time. In addition, 1μM DBCO-PEG8-Oxp(IV) was incubated in 4T1 cells labeled with HBAPE-Ac3ManNAz for 24 hours for 1 hour, 12 hours, and 24 hours, and the results were detected by ICP-MS. Figure 7 c, every 2×10 5 The platinum content in each cell increased with incubation time, reaching four times that of cells incubated for 24 hours compared to those incubated for 1 hour. These experimental results demonstrate that DBCO-PEG8-Oxp(IV) promotes cellular platinum uptake through a click reaction based on glucose metabolism labeling, thereby increasing drug accumulation in tumor cells.

[0096] 3. Effect of DBCO-PEG8-Oxp(IV) on the migration ability of breast cancer cells

[0097] In order to evaluate the inhibitory effect of DBCO-PEG8-Oxp(IV) on breast cancer cell migration mediated by click reaction. First, the effect of DBCO-PEG8-Oxp(IV) on the migration ability of 4T1 breast cancer cells was detected by wound healing assay. 4T1 cells labeled with HBAPE-Ac3ManNAz for 24 hours and unlabeled cells were added with DBCO-PEG8-Oxp(IV) at a final concentration of 1μM for incubation. Oxaliplatin was used as a positive drug for comparison. The cells were observed and photographed under a microscope at different time periods. The results are shown in Figure 2. Figure 8As shown in Figure a, both oxaliplatin and DBCO-PEG8-Oxp(IV) can inhibit the migration of 4T1 cells. Among them, there is a slight difference in the inhibitory effect of oxaliplatin and DBCO-PEG8-Oxp(IV) on cell migration at 12 hours for cells not labeled with HBAPE-Ac3ManNAz, but there is basically no difference in the inhibitory effect after 24 hours. However, for cells labeled with HBAPE-Ac3ManNAz, the inhibitory effect of DBCO-PEG8-Oxp(IV) on cell migration is significantly enhanced at 12 hours and 24 hours. The experimental results indicate that DBCO-PEG8-Oxp(IV) promotes the accumulation of platinum on tumor cells through click reaction, thereby enhancing the inhibitory effect on cell migration.

[0098] At the same time, in order to further prove that the click reaction promotes the inhibitory effect of DBCO-PEG8-Oxp(IV) on cell migration, the same experimental group was designed and verified in Transwell chamber. The results are as follows Figure 8 As shown in b, we can see that the effect of DBCO-PEG8-Oxp(IV) on cell migration ability was significantly enhanced in cells that were labeled with HBAPE-Ac3ManNAz for 24 h in advance.

[0099] In addition, we also used Western Blot to explore the effects of DBCO-PEG8-Oxp(IV) on matrix metalloproteinase-9 (MMP-9) and matrix metalloproteinase-7 (MMP-7), which play an important role in cell growth and migration, before and after probe labeling. The results are as follows: Figure 8 As shown in Figure c, DBCO-PEG8-Oxp(IV) significantly reduced the expression of MMP-9 and MMP-7 proteins in cells pre-labeled with HBAPE-Ac3ManNAz for 24 hours, thereby inhibiting the growth and migration of tumor cells. These experimental results fully demonstrate that DBCO-PEG8-Oxp(IV) significantly enhances the inhibitory effect on breast cancer cell migration through the click reaction.

[0100] II. Evaluation of the anti-tumor activity and anti-metastatic effects of DBCO-PEG8-Oxp(IV) in mice based on HBAPE-Ac3ManNAz carbohydrate metabolism labeling

[0101] 1. Based on glucose metabolism labeling, DBCO-PEG8-Oxp(IV) inhibits the growth of breast cancer cells in mice orthotopic solid tumors

[0102] Previous in vitro experiments have shown that DBCO-PEG8-Oxp(IV) significantly improves the killing effect of 4T1 breast cancer cells labeled with HBAPE-Ac3ManNAz. At the same time, it has been shown in vitro and in vivo that HBAPE-Ac3ManNAz has strong specificity and efficient labeling ability for tumor tissue in mice. Therefore, we injected 4T1 breast cancer cells into the armpits of Balb / C mice to create a tumor-bearing model. This experiment was divided into five groups: PBS, Oxp. (2.46 mg Pt / kg), DBCO-PEG8-Oxp(IV) (2.46 mg Pt / kg), Ac4ManNAz+DBCO-PEG8-Oxp(IV), HBAPE-Ac3ManNAz+DBCO-PEG8-Oxp(IV), with six mice in each group. The dosing cycle is as follows Figure 9 As shown in a, when the average tumor volume of mice is 50-100 mm 3 , the probe Ac4ManNAz or HBAPE-Ac3ManNAz was injected into the abdominal cavity for five consecutive days, and then the corresponding drug Oxp. or DBCO-PEG8-Oxp (IV) was injected into the tail vein of each group. The frequency of platinum drug administration was once every two days, and a total of three treatments were given. During the interval, the mice in the sugar probe-treated group were given corresponding probes every day. The weight and tumor volume of the mice were recorded every two days from the beginning of tumor implantation. After the end of the administration, the mice were observed for one day, the eyeballs of the mice were bled and the mice were dislocated and killed. The tumors and various organ tissues were then removed, photographed, and the tumor weight was recorded. The weight changes of mice in each group during the administration period ( Figure 9 b) tended to be stable. The weight of mice treated with Ac4ManNAz / HBAPE-Ac3ManNAz combined with DBCO-PEG8-Oxp(IV) on the last day was not less than the initial weight and the maximum weight did not exceed 20% of the initial weight, indicating that the drug had no obvious toxic side effects on mice. At the same time, the tumor volume change curve ( Figure 9 c) shows that on the 11th day of tumor growth, the average tumor volume of the control group mice was 920 mm 3 , Oxp. group is 711mm 3 , DBCO-PEG8-Oxp(IV) group is 655mm 3 , Ac4ManNAz+DBCO-PEG8-Oxp(IV) group is 290mm 3 , HBAPE-Ac3ManNAz+DBCO-PEG8-Oxp(IV) group is 185mm 3, we can see that compared with the control group, oxaliplatin and DBCO-PEG8-Oxp (IV) both have the effect of inhibiting tumor growth, but the inhibition rates are 23% and 29% respectively. The inhibitory effect is not obvious and the effect is not much different. When the probe is added to mark the tumor cells in advance, the inhibitory effect of DBCO-PEG8-Oxp (IV) on the tumor is significantly enhanced. Among them, under the labeling of Ac4ManNAz, the inhibition rate of DBCO-PEG8-Oxp (IV) on tumor growth reaches 68%, and when HBAPE-Ac3ManNAz with better tumor selective labeling effect is selected, the inhibition rate of DBCO-PEG8-Oxp (IV) on tumor growth reaches 80%. In addition, the dissected tumor tissue was photographed ( Figure 9 d), and then weighed ( Figure 9 e) When probe labeling was used, DBCO-PEG8-Oxp(IV) showed a more pronounced inhibitory effect on tumors, with lighter solid tumors. Compared to the control group, the average size of solid tumors in the Ac4ManNAz+DBCO-PEG8-Oxp(IV) group was approximately 21% of that in the control group, while the average size of solid tumors in the HBAPE-Ac3ManNAz+DBCO-PEG8-Oxp(IV) group was approximately 14% of that in the control group. H&E staining was performed on the tumor tissues of each group, and the results are shown in the figure below. Figure 9 As shown in figure f, the number of cells in the tumor tissue of the mice in the control group increased significantly, the volume of the cell nuclei increased, and the proliferation ability was strong, while the number of cell nuclei in the tumor tissue of the mice treated with the combination of Ac4ManNAz / HBAPE-Ac3ManNAz and DBCO-PEG8-Oxp(IV) decreased significantly, and the effect was more significant than that of the mice treated with oxaliplatin or DBCO-PEG8-Oxp(IV) alone, indicating that under the action of the probe, the inhibitory effect of DBCO-PEG8-Oxp(IV) on cancer cell proliferation became stronger.

[0103] To further verify that DBCO-PEG8-Oxp(IV) increased the drug's ability to target tumors through click reactions, we used ICP-MS to measure the platinum content in the tissues of each group of mice. The results are as follows: Figure 9As shown in Figure g, after the tumor was labeled with Ac4ManNAz or HBAPE-Ac3ManNAz, the platinum accumulation of DBCO-PEG8-Oxp(IV) in the tumor tissue was significantly increased. The platinum content in the tumors of mice in the Ac4ManNAz+DBCO-PEG8-Oxp(IV) group was approximately twice that of the mice in the groups using oxaliplatin or DBCO-PEG8-Oxp(IV) alone. The platinum content in the tumors of mice in the HBAPE-Ac3ManNAz+DBCO-PEG8-Oxp(IV) group was even higher, reaching 3500 ng Pt / g. At the same time, it can be seen from the ICP-MS results that compared with oxaliplatin, after probe labeling, DBCO-PEG8-Oxp(IV) accumulated more in the tumor site in the body, reduced accumulation in the liver and kidneys, and thus greatly reduced damage to the liver and kidneys. Furthermore, the therapeutic efficacy of DBCO-PEG8-Oxp(IV) after tumor labeling with HBAPE-Ac3ManNAz was superior to that achieved with Ac4ManNAz, indicating that HBAPE-Ac3ManNAz has a higher selectivity for tumor labeling, thereby enhancing the targeting of DBCO-PEG8-Oxp(IV). These experimental results demonstrate that in a mouse tumor-bearing model, DBCO-PEG8-Oxp(IV) enhances tumor targeting through a click reaction based on glucose metabolism labeling, thereby enhancing its anti-tumor activity while reducing side effects caused by platinum accumulation in non-tumor areas.

[0104] 2. Based on glucose metabolism labeling, DBCO-PEG8-Oxp(IV) inhibits breast cancer cell lung metastasis in mice

[0105] In vitro experiments have shown that DBCO-PEG8-Oxp (IV) has a significant inhibitory effect on the migration ability of breast cancer cells based on metabolic labeling with HBAPE-Ac3ManNAz sugar probes. For further verification, we injected mouse 4T1 breast cancer cells into Balb / C mice via the tail vein to create a mouse lung metastasis model. This experiment was divided into five groups: PBS, Oxp. (2.46 mg Pt / kg), DBCO-PEG8-Oxp (IV) (2.46 mg Pt / kg), Ac4ManNAz+DBCO-PEG8-Oxp (IV), and HBAPE-Ac3ManNAz+DBCO-PEG8-Oxp (IV), with five mice in each group. The dosing cycle is as follows: Figure 10As shown in a, the probe Ac4ManNAz or HBAPE-Ac3ManNAz was injected into the abdominal cavity for four consecutive days starting on the fifth day after tumor implantation. Subsequently, the corresponding drug Oxp. or DBCO-PEG8-Oxp(IV) was injected into the tail vein of each group. The platinum drug was injected once every two days for a total of three treatments. During the interval, the mice in the sugar probe-treated group were given the corresponding probe every day. The body weight of the mice was recorded every two days from the beginning of tumor implantation. After the end of the drug administration, the mice were observed for one day. The blood was collected from the eyeballs of the mice and the mice were dislocated and killed. The organs and tissues were then removed, and the lung tissues of the mice in each treatment group were photographed ( Figure 10 b) Lung weight and number of nodules were recorded. Body weight changes of mice in each group during the administration period ( Figure 10 c) tended to be stable, and the weight change of mice in each treatment group did not exceed 20% of the initial weight. Figure 10 d) Compared with the control group, mice treated with DBCO-PEG8-Oxp(IV) and oxaliplatin had a weak inhibitory effect on breast cancer metastasis, with inhibition rates of 27% and 36%, respectively. When the probe was added to label the tumor cells in advance, the inhibitory effect of DBCO-PEG8-Oxp(IV) on tumor metastasis was significantly enhanced. Among them, the lung nodules of mice treated with DBCO-PEG8-Oxp(IV) under Ac4ManNAz labeling were reduced by 55%, while the lung nodules of mice treated with HBAPE-Ac3ManNAz+DBCO-PEG8-Oxp(IV) were reduced by 84%. At the same time, we weighed the excised lung tissues, as shown in Figure 4. Figure 10 As shown in Figure e, the lungs of the control group, which were filled with nodules, were significantly heavier than those of the lungs of the Ac4ManNAz+DBCO-PEG8-Oxp(IV) and HBAPE-Ac3ManNAz+DBCO-PEG8-Oxp(IV) treated groups. Figure 10 As shown in Figure f, the number of cells in the lung tissue of mice with excessive nodules in the control group, oxaliplatin group, and DBCO-PEG8-Oxp(IV) group increased significantly, the size of the cell nuclei increased, and the proliferation capacity was strong. However, the number of cell nuclei in the lung tissue of mice treated with a combination of Ac4ManNAz / HBAPE-Ac3ManNAz and DBCO-PEG8-Oxp(IV) decreased significantly, indicating that under the action of the probe, the inhibitory effect of DBCO-PEG8-Oxp(IV) on tumor metastasis was significantly enhanced. It was also found that the inhibitory effect of DBCO-PEG8-Oxp(IV) on tumor metastasis after tumor labeling with HBAPE-Ac3ManNAz was better than that of tumor labeling with Ac4ManNAz, indicating that HBAPE-Ac3ManNAz has higher selectivity for tumor labeling and enhances the targeting of DBCO-PEG8-Oxp(IV).

[0106] To further verify that DBCO-PEG8-Oxp(IV) increased the inhibitory effect of the drug on tumor metastasis through click reaction, we used ICP-MS to measure the platinum content in the tissues of each group of mice. The results are as follows Figure 10 As shown in Figure g, after tumor labeling with Ac4ManNAz or HBAPE-Ac3ManNAz, DBCO-PEG8-Oxp(IV) significantly increased platinum accumulation in lung tissue, while in oxaliplatin-treated mice, platinum was primarily distributed in the liver and kidneys. These data clearly demonstrate that DBCO-PEG8-Oxp(IV) combined with glucose metabolism labeling can target tumor cells and thereby inhibit tumor metastasis.

[0107] The HBAPE-Ac3ManNAz designed in the present invention is a new type of sugar metabolism labeling molecular probe that is regulated by ROS, is safe and efficient, and can specifically label tumor cells or tissues in vivo and in vitro; based on the HBAPE-Ac3ManNAz sugar metabolism labeling, DBCO-PEG8-Oxp(IV) improves the targeting ability of platinum drugs to tumor cells, enhances the drug's anti-tumor activity and anti-tumor metastasis ability, and has good biosafety.

Claims

1. A pharmacodynamic molecule that undergoes an orthogonal reaction with a small molecule probe, characterized in that: The pharmacological molecular structural formula is as follows: 。 2. Use of the pharmacodynamic molecule that undergoes orthogonal reaction with a small molecule probe as claimed in claim 1 in the preparation of anti-breast cancer drugs.

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