CN116621901A - Small molecular probe based on sugar metabolism marker and application thereof in improving targeting of platinum drugs - Google Patents

Small molecular probe based on sugar metabolism marker and application thereof in improving targeting of platinum drugs Download PDF

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CN116621901A
CN116621901A CN202310419504.6A CN202310419504A CN116621901A CN 116621901 A CN116621901 A CN 116621901A CN 202310419504 A CN202310419504 A CN 202310419504A CN 116621901 A CN116621901 A CN 116621901A
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mannaz
hbape
tumor
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oxp
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王佳佳
李霞
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Henan University
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Abstract

The invention belongs to the technical field of biological probes, and particularly relates to a small molecular probe HBAPE-Ac based on glycometabolism markers 3 ManNAz and its application in improving platinum medicine targeting. The invention provides a small molecular probe HBAPE-Ac based on glycometabolism markers 3 ManNAz, the probe being a metabolic precursorCan selectively mark tumor cells or tissues in vitro and in vivo, and can give a new target for refractory tumors such as triple negative breast cancer and the like which lack specific targeting receptors; the pharmacodynamic molecule DBCO-PEG8-Oxp (IV) complex which is in orthogonal reaction with the probe is synthesized, and the complex and the azide tag marked on the tumor cell membrane are subjected to biological orthogonal reaction, so that the targeting transport capacity of the platinum drug to the tumor is improved, toxic and side effects are reduced, and a new theoretical basis is provided for the targeting treatment of the tumor in vivo.

Description

Small molecular probe based on sugar metabolism marker and application thereof in improving targeting of platinum drugs
Technical Field
The invention belongs to biological probesTechnical field, in particular to a small molecular probe HBAPE-Ac based on glycometabolism markers 3 ManNAz and its application in improving platinum medicine targeting.
Background
Glycan metabolism markers (metabolic glycan labeling, MGL) have become a simple but powerful tool, skillfully borrowing the synthetic pathway of natural saccharides, and metabolizing non-natural molecules containing active functional groups (azide, alkynyl, etc.) to the cell surface, thereby artificially introducing chemical receptors to tumor cells. Compared with the high-expression receptor contained on the surface of the conventional tumor cell, the glycometabolism marker can be used for in-vivo marking, and can provide a specific treatment target for the tumor cell due to the characteristics of high density, low toxicity and small interference. Currently, tetra-acetylated N-azidoacetamannonamine (Ac) 4 ManNAz) although the azide group can be introduced into the sugar chain terminal sialic acid on the surface of tumor cells by the enzyme on the sialic acid synthesis pathway in the body with high efficiency, it is inevitable that the same enzyme catalyzes Ac in normal cells as well 4 Metabolic markers of ManNAz, thus Ac 4 ManNAz is difficult to achieve targeting markers for tumor tissue in application of tumor targeting therapy, and in vivo tumor cell selection markers remain a great challenge. The professor Bertozzi problem group reported for the first time in 2000 that an analogue N-azidoacetylmannosamine (ManNAz) of azide-substituted N-acetylmannosamine (ManNAc), the probe molecule could be converted to an azide-containing sialic acid (sial) molecule via a corresponding enzyme of the sialic acid synthesis pathway to label the sugar chain structure on the cell membrane surface, thereby artificially adding an azide group to the cell membrane surface. Compared with the high-expression receptor contained on the surface of the conventional tumor cell, the sugar metabolism labeling strategy has the following characteristics: (1) high density: the cell surface markers have high coverage of about 10 7 Individual/cell 10-1000 times more than the high-expression receptor; (2) low toxicity: unlike antibodies, the introduced chemical structure is not easy to cause side reactions such as organism immunity stress and the like; (3) the interference is small: unlike macromolecules such as antibodies, azide or other molecules introduced into unnatural sugar structuresThe alkynyl group has small volume and can not interfere with the identification and information transmission among cells. Based on the advantages, the sugar metabolism mark combined with the bioorthogonal reaction strategy can be used for targeting and connecting fluorophores, and imaging observation is carried out on the polysaccharide at the level of living cells and living bodies; enrichment and histologic analysis of the labeled glycoprotein can also be achieved by specific binding of biotin to streptavidin; more importantly, classical reactive groups are azido and alkynyl groups which are not present in a mammal body, and monovalent copper ion catalysis is needed, in order to avoid toxicity caused by copper ion marking in a living body, a Bertozzi subject group develops cycloaddition (SPAAC) caused by azido and Dibenzocyclooctyne (DBCO) ring tension, which is also called copper-free click chemistry (coppers-free click chemistry), and can be efficiently and covalently connected with antibodies, folic acid, polypeptides and the like to form glycosyl ligand complexes under the non-copper ion catalysis condition, so that important guarantee is provided for application research of sugar chain metabolic marking and tumor targeted treatment in a living body. However, recent studies have found that most mammalian cells also have the same enzyme catalyzing the binding of traditional sugar molecule probes to glycans on the cell surface in the golgi apparatus through post-translational modification. Thus, the use of ManNAz to selectively label specific types of cells without interfering with their cellular metabolic mechanisms has proven difficult, particularly in application to tumor-targeted therapies where targeted labeling of tumor tissue is difficult to achieve, resulting in damage to normal cells and tissues by drug off-target distribution.
Reactive oxygen species (reactive oxygen species, ROS) refer to oxygen-derived reactive small molecules, including hydrogen peroxide, superoxide anions, etc., which play an important role in biological processes such as cell differentiation, cell signaling, and adaptive immunity. Research shows that the ROS content in most tumor cells is high, and can reach 10-100 mu M; whereas normal intracellular ROS content is typically 0.001-0.7. Mu.M. By utilizing the characteristics, ROS responsive carriers have been developed, and the purposes of treating diseases can be achieved while improving the safety of the carriers. The 4- (Hydroxymethyl) phenylboronic acid pinacol ester (4- (Hydroxymethyl) benzeneboronic acid pinacol ester, HBAPE) which is responded by ROS has been developed to selectively target tumor cells, reduce toxicity caused by normal cell accumulation, and is a popular prodrug protecting group in pharmaceutical chemistry research, and is commonly used for a prodrug molecule regulated by ROS or a protecting group of a detection probe.
The invention aims at the difference of ROS content in normal system and tumor system by Ac 4 The 1-position of ManNAz introduces 4-hydroxyphenylboronic acid pinacol ester structure (HBAPE), and a new probe HBAPE-Ac which relies on ROS regulation to block metabolic marker pathways in healthy cells, and can selectively and chemically mark and dynamically detect tumor cells and tumor tissues is developed 3 ManNAz and synthesizing a pharmacodynamic molecule DBCO-PEG8-Oxp (IV) which reacts orthogonally with the ManNAz, so that the targeting of the platinum medicine is realized, and meanwhile, the anti-tumor activity is enhanced and the generation of toxic and side effects is reduced.
Disclosure of Invention
In order to solve the problems, the invention provides a small molecular probe HBAPE-Ac based on glycometabolism markers 3 ManNAz, the probe can be used as a metabolic precursor to selectively mark tumor cells or tissues in vitro and in vivo, and can be used for endowing new targets for refractory tumors such as triple negative breast cancers and the like which lack specific targeting receptors; the pharmacodynamic molecule DBCO-PEG8-Oxp (IV) complex which is in orthogonal reaction with the probe is synthesized, and the complex and the azide tag marked on the tumor cell membrane are subjected to biological orthogonal reaction, so that the targeting transport capacity of the platinum drug to the tumor is improved, toxic and side effects are reduced, and a new theoretical basis is provided for the targeting treatment of the tumor in vivo.
To achieve the above object, the present invention provides HBAPE-Ac 3 The ManNAz formula is as follows:
the HBAPE-Ac 3 The synthetic route of ManNAz is specifically as follows:
Reagents and conditions：(i)NaN 3 ，NaOH，H 2 O，75℃，12h；(ii)N 3 CH 2 COOH，HOBT，EDC，MeOH，0℃，16h；(iii)Ac 2 O，P y ，0℃，6h；(iv)4-Hydroxyphenylboronic acid pinacol ester，Sc(OTf) 3 ，1，2-C 2 H 4 Cl 2 ，reflux，2h.
to achieve another object of the present invention, the present invention relates to a method for preparing HBAPE-Ac 3 The pharmacodynamic molecule DBCO-PEG8-Oxp of the ManNAz probe orthogonal reaction has the following structural formula:
The synthesis route of DBCO-PEG8-Oxp (IV) is specifically as follows:
Reagents and conditions：(i)NH 2 OH.HCl，EtOH，Py，reflux，12h；(ii)PPA，125℃，1.5h；(iii)LiAlH 4 ，Et 2 O，reflux，48h；(iv)methyl 4-chloro-4-oxobutyrate，Et 3 N，DCM，0℃，4h；(v)PyHBr 3 ，DCM，Rt，24h；(vi)KOtBu，THF，-40℃，4h；(vii)LiOH，H 2 O，rt，12h；(viii)NH 2 -PEG8-NH 2 ，HATU，DIPEA，DMF，rt，24h；(ix)H 2 O 2 ，H 2 O，60℃，4h；(x)palmitic anhydride，DMSO，rt，7d；(xi)succinic anhydride，DMSO，rt，12h；(xii)HATU，DIPEA，DMF，rt，24h.
HBAPE-Ac of the invention 3 ManNAz as a metabolic precursor can selectively label tumor cells or tumor tissue in vitro and in vivo.
Preferably, the HBAPE-Ac of the invention 3 ManNAz can be used as a probe, specifically marked in tumor of organism, and combined with chemotherapeutics or immunotherapy means (including gene therapy,antigen delivery, siRNA, etc.), and achieves the therapeutic purpose of anti-tumor.
Preferably, said probe HBAPE-Ac 3 Mannaz can cooperate with platinum drugs to treat tumor, wherein HBAPE-Ac 3 ManNAz can specifically mark azide in tumor cell membrane, improve the targeting transport capacity of platinum drugs to tumors, and reduce toxic and side effects.
The platinum medicine can be DBCO-PEG8-Oxp (IV), cisplatin and other platinum medicines with anti-tumor effects.
Preferably, the tumor includes, but is not limited to, refractory tumors lacking specific targeting receptors, such as triple negative breast cancer.
The DBCO-PEG8-Oxp (IV) complex and the probe HBAPE-Ac 3 The azide tag marked on the tumor cell membrane by ManNAz improves the targeting transportation capability of DBCO-PEG8-Oxp (IV) to tumors through biorthogonal reaction, reduces toxic and side effects, and provides a new theoretical basis for targeting treatment of tumors in organisms.
The invention has obvious technical effects.
In the present invention, we know probe Ac 4 The 1-position of ManNAz introduces 4-hydroxyphenylboronic acid pinacol ester structure, the selectivity of the probe to tumor cells can be improved through high-level ROS in the tumor cells, the metabolic activity of the probe in normal cells with low ROS content is inhibited, and a novel probe HBAPE-Ac which depends on selective chemical marking regulated by ROS and can dynamically detect tumor cells and tumor tissues is developed 3 ManNAz, especially provides new high-density tumor target molecules for tumor types lacking effective targets such as triple negative breast cancer and the like, and probes HBAPE-Ac 3 ManNAz has a novel carbohydrate metabolism marker molecular probe which is safe and efficient and can specifically mark tumor cells or tissues in vitro and in vivo. In addition, by combining with a bioorthogonal reaction strategy, the platinum drugs are connected to DBCO through a chemical synthesis means, and by combining with azide groups on tumor cell membranes through bioorthogonal reaction, the uptake of the platinum drugs and the targeting of the platinum drugs by tumor cells and tissues in vitro and in vivo are improved, and the dosage of the platinum drugs and the toxicity and side effects on normal cells are reducedHas effect in enhancing anti-tumor capability. The combination of the glycometabolism mark and the bioorthogonal reaction provides a theoretical basis for the development of novel targeted anti-breast cancer platinum drugs, and provides a new reference for the research of tumors and the development of targeted treatment schemes.
Drawings
FIG. 1 is HBAPE-Ac 3 ManNAz cell metabolism marker characterization. (a) HBAPE-Ac 3 Concentration gradient of ManNAz metabolic markers; (b) HBAPE-Ac 3 Time gradient of ManNAz metabolic markers.
FIG. 2 is a schematic diagram of HBAPE-Ac 3 The efficiency of the cellular metabolic markers of ManNAz is regulated by ROS. (a) HBAPE-Ac 3 ManNAz and different concentrations H 2 O 2 Co-cultured cell marker level changes; (b) HBAPE-Ac 3 ManNAz was co-cultured with NAC at different concentrations.
FIG. 3 shows the detection of HBAPE-Ac by HPLC 3 Hydrolysis efficiency of ManNAz is subject to H 2 O 2 Is controlled by the control program. (a) HBAPE-Ac 3 ManNAz and different concentrations of H 2 O 2 Co-incubating for 3h; (b) HBAPE-Ac 3 ManNAz and the same concentration H 2 O 2 Incubate for different times. HPLC sample injection conditions: 0-5min,5% methanol; 5-10min,15% methanol; 10-15min,20% methanol.
FIG. 4 is a schematic diagram of HBAPE-Ac 3 ManNAz localization and efficiency analysis of metabolic markers on cell membranes. (a) HBAPE-Ac 3 ManNAz and Ac 4 Confocal imaging (scale: 20 μm) of 4T1 cells incubated for 24h by ManNAz after staining with DBCO-488, WGA, hoechst 33342; (b) 4T1 cells were incubated with different probes (200. Mu.M) for 24 hours, and the cells collected and coupled to DBCO-488 for flow cytometry to analyze cell membrane surface fluorescence intensity.
FIG. 5 is HBAPE-Ac 3 Tumor selectable markers in ManNAz mice. (a) Ac (Ac) 4 ManNAz、HBAPE-Ac 3 ManNAz and HBAPE-Ac 3 ManNAz and NAC were used in combination for whole body fluorescence imaging of seven day continuous injection mice injected with DBCO-Cy via tail vein for 24 h; (b) Fluorescence images of major organs and tumors after 24 hours of DBCO-Cy intravenous injection of mice tail labeled with different probes; (c) UsingThe IVIS spectral imaging system quantifies the average radiant efficiency of mouse tumor tissue labeled with different probes (n=2). n.s. represents no statistical difference, P<0.05，**P<0.01，***P＜0.001。
FIG. 6 shows in vitro antitumor activity evaluation of platinum drugs. (a) Positive control oxaliplatin IC in 4T1 cells 50 A value; (b) DBCO-PEG8-Oxp (IV) IC in 4T1 cells 50 A value; (c) IC of 4T1 cells, DBCO-PEG8-Oxp, labeled with HBAPE-Ac3ManNAz 50 Values.
FIG. 7 shows the measurement of platinum uptake by 4T1 cells by ICP-MS. (a) Through HBAPE-Ac 3 Platinum content (n=3) in cells after incubation of 4T1 cells before and after 24h of ManNAz marker with 1 μm oxaliplatin or DBCO-PEG8-Oxp (IV), respectively, for 2 h; (b) Through HBAPE-Ac 3 Platinum content (n=3) in cells after incubation of 4T1 cells 24h after ManNAz labelling with DBCO-PEG8-Oxp (IV) at different concentrations for 2 h; (c) Through HBAPE-Ac 3 Platinum content in cells after 24h incubation of 4T1 cells after ManNAz labelling with 1 μmdbco-PEG8-Oxp (IV) for different times (n=3), respectively. n.s. represents no statistical difference, P <0.05，**P<0.01，***P＜0.001。
FIG. 8DBCO-PEG8-Oxp (IV) significantly inhibited the migration capacity of 4T1 breast cancer cells by click reaction. (a) Wound healing experiments to investigate HBAPE-Ac 3 Inhibition of 4T1 cell migration by DBCO-PEG8-Oxp (IV) before and after ManNAz labeling. Left, picture, right, statistical analysis (n=3, scale: 50 μm); (b) Transwell experiments to investigate HBAPE-Ac 3 Inhibition of 4T1 cell migration by DBCO-PEG8-Oxp (IV) before and after ManNAz labeling. Left, picture, right, statistical analysis (n=3, scale: 100 μm); (c) Effects of DBCO-PEG8-Oxp (IV) on cell growth migration related proteins MMP-9 and MMP-7 expression before and after labelling. S represents no statistical difference, P<0.05，**P<0.01，***P＜0.001。
FIG. 9 anti-tumor activity of DBCO-PEG8-Oxp (IV) on 4T1 breast cancer mice based on glycometabolism markers. (a) Schematic dosing time of tumor-bearing model of 4T1 breast cancer mice; (b) Body weight change curves for mice in different treatment groups during treatment; (c) Tumor growth volume change curves of mice in different treatment groups during treatment; (d) Tumor pictures of mice of different treatment groups at the end of the experiment; (e) Tumor weights of mice in different treatment groups after the experiment is finished; (f) H & E staining of tumors of mice of different treatment groups after the end of the experiment (scale: 50 μm); (g) Platinum content distribution (n=3) in each tissue of mice of different treatment groups after the end of the experiment. n.s. means no statistical difference, P <0.05, P <0.01, P < 0.001.
FIG. 10 anti-tumor metastasis activity of DBCO-PEG8-Oxp (IV) on 4T1 breast cancer mice based on glycometabolism markers. (a) Schematic dosing time of lung metastasis model of 4T1 breast cancer mice; (b) Representative images of lung metastasis nodules (n=5) of mice of different treatment groups after the end of the experiment; (c) Body weight change curves (n=5) for mice of different treatment groups during treatment; (d) Statistics of lung metastasis nodules (n=5) in mice of different treatment groups after the end of the experiment; (e) Lung tissue weight (n=5) of mice from different treatment groups after the end of the experiment; (f) H & E staining of lung tissue of mice of different treatment groups after the end of the experiment (scale: 50 μm); (g) Platinum content distribution (n=3) in each tissue of mice of different treatment groups after the end of the experiment. n.s. means no statistical difference, P <0.05, P <0.01, P < 0.001.
Detailed description of the preferred embodiments
The technical scheme of the invention is further described in detail below with reference to the accompanying drawings and the detailed description.
Example 1.
The subject is Ac 4 Based on ManNAz (structural formula a), the 1-acetyl is replaced by a protecting group 4- (hydroxymethyl) phenylboronic acid pinacol ester (HBAPE) regulated by ROS, and a novel glycometabolism molecular probe HBAPE-Ac which is responded by ROS and further realizes tumor selective marking is chemically synthesized 4 ManNAz (structural formula b). Meanwhile, the tetravalent platinum prodrug is connected to DBCO through polyethylene glycol (PEG) to further synthesize water-soluble DBCO-PEG8-Oxp (IV) (structural formula c), and the newly synthesized platinum drug is prepared in HBAPE-Ac 4 Based on ManNAz targeted tumor marking, platinum drugs are targeted and transported to tumor sites through efficient bioorthogonal reaction, so that the antitumor activity and targeting performance of the platinum drugs are improved, and the toxic and side effects of the platinum drugs are reduced.
1.HBAPE-Ac 3 The ManNAz probe synthesis procedure was as follows:
(1) Synthesis of Compound 1
18g of chloroacetic acid was weighed into a 250mL round bottom flask, added with 100mL of distilled water to dissolve completely, added with 8g of NaOH solid particles, and gradually added with 15g of NaN after stirring evenly 3 Insert N 2 Ball and set reaction temperature to 75 ℃, after 12 hours of reaction, the heating device is closed to restore to room temperature, the reaction liquid in the round bottom flask is poured into a beaker containing 500mL of ice water to be continuously stirred, 20mL of ice hydrochloric acid (15 mL of concentrated hydrochloric acid+5 mL of ice water) is prepared and gradually added into the beaker to adjust the pH of the solution to 2-3, then 300mL of ethyl acetate is gradually added for extraction, the three times of repeated steps are carried out, the collected ethyl acetate is dried by anhydrous sodium sulfate, the filtration and the reduced pressure evaporation of the organic solvent are carried out, and 15.6g of white liquid compound 1 is obtained, and the yield is 81%. 1 H NMR(300MHz,CDCl 3 )δ11.30(s,1H),4.02(s,2H). 13 C NMR(75MHz,CDCl 3 )δ174.74,50.06.
(2) Synthesis of Compound 2
5g of D-mannosamine hydrochloride was weighed into a 250mL round bottom flask, 100mL of ultra-dry methanol was added and stirred at room temperature for 20min, followed by 3.6g of Compound 1,9.6mL of Et in sequence 3 N, and the reaction solution was transferred to an ice-water bath and stirred for 30min, 2.2g HOBT and 3.2g EDC were added to the reaction solution, a nitrogen ball was inserted and the reaction was carried out, after 16h, TCL was used to monitor the reaction condition, after the reaction was completed, the organic solvent was distilled off under reduced pressure, and the purification was carried out by column chromatography to obtain 3.8g of Compound 2 as a yellowish white solid with a yield of 62%.
(3) Synthesis of Compound 3
2g of Compound 2 was weighed into a 100mL round bottom flask, 30mL of ultra-dry pyridine was added, the flask was moved to an ice-water bath and stirred continuously, then 5mL of acetic anhydride was gradually added, N2 balls were inserted and the reaction was carried out, the reaction condition was monitored by using TCL after 6 hours, the organic solvent was distilled off under reduced pressure after completion of the reaction, and 80mL of 10% diluted hydrochloric acid solution (concentrated hydrochloric acid: distilled water=50 mL:450 mL) and 120mL of DCM were added to a 250mL separating funnel to extractThe lower organic solvent was collected and the extraction was repeated three times, and the collected DCM was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to give 2.5g of compound 3 as a white solid in 75% yield. 1 H NMR(300MHz,CDCl 3 )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,CDCl 3 )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.
(4) Synthesis of Compound 4
500mg of the compound 3,1.37g of pinacol 4-hydroxyphenylborate and 345mg of scandium triflate were weighed into a 100mL round bottom flask and 30mL of 1,2-C were added 2 H 4 Cl 2 After the reaction is completed, distilled water 30mL and DCM 50mL are added into a separating funnel of 100mL to extract, the lower organic solvent is collected and the extraction operation is repeated three times, the collected DCM is dried with anhydrous sodium sulfate and concentrated, and the obtained DCM is purified by column chromatography to obtain 407mg of white solid compound 4 with the yield of 58 percent. 1 H NMR(300MHz,CDCl 3 )δ7.85(d,J＝7.9Hz,2H),7.39(d,J＝7.8Hz,2H),6.56(d,J＝9.4Hz,1H),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,CDCl 3 )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。
2. The synthesis of the drug effect molecule DBCO-PEG8-Oxp of the orthogonal reaction comprises the following steps:
(1) Synthesis of Compound 5
Respectively call forAdding 20g of 5-dibenzosuberone and 13.6g of hydroxylamine hydrochloride into a 500mL round bottom flask, sequentially adding 60mL of ultra-dry pyridine and 240mL of absolute ethanol to stir uniformly, inserting a condenser tube, vacuumizing, and inserting N 2 Ball, heating to 90 ℃ and reflux reacting for 12 hours, TCL monitoring the reaction condition, decompressing and evaporating the organic solvent after the reaction is completed, adding 350mL of 5% diluted hydrochloric acid of ice, continuously and rapidly stirring for 30 minutes in an ice water bath, gradually precipitating white solid in the stirring process, filtering, washing the precipitate for multiple times by distilled water, airing, and thoroughly pumping by an oil pump to obtain 16g of white solid compound 5 with the yield of 74%. 1 H NMR(300MHz,CDCl 3 )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). 13 C NMR(75MHz,CDCl 3 )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.
(2) Synthesis of Compound 6
200mL of polyphosphoric acid is weighed and added into a 500mL round-bottom flask, the polyphosphoric acid is heated to 125 ℃ and 14g of compound 5 is gradually added at the temperature and continuously stirred, the compound is gradually changed into a viscous yellow solution in the reaction process, a heating device is turned off after 1.5h, the compound is poured into a beaker containing 800mL of ice water after the compound is recovered to room temperature and continuously and rapidly stirred for 30min, a gray brown solid is gradually separated out in the stirring process, the compound is filtered by suction and washed and precipitated for multiple times by distilled water, and the compound is thoroughly dried by an oil pump after being dried, so that 11g of gray brown solid compound 6 is obtained, and the yield is 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.
(3) Synthesis of Compound 7
6g of Compound 6 and 2.6g of LiAlH were weighed out separately 4 Adding into 500mL round bottom flask, inserting condenser, vacuumizing, and inserting N 2 Ball, then adding 200mL ultra-dry diethyl ether into the bottle gradually, stirring continuously, heating to 45deg.C, adding diethyl ether continuously to prevent solvent from evaporating, monitoring reaction condition by TCL after 48 hr, and reacting completelyThe round bottom flask was placed in an ice-water bath, distilled water was gradually added slowly to the flask until no gas was generated, then 200mL of DCM was added for extraction and repeated three times, the collected DCM was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to give 3g of compound 7 as a yellow solid in 53% yield. 1 H NMR(300MHz,CDCl 3 )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(td,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,CDCl 3 )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.
(4) Synthesis of Compound 8
2.5g of Compound 7 was weighed into a 100mL round bottom flask, 40mL of extra dry DCM and 3.5mL of Et were added sequentially 3 N, vacuumize and insert N 2 Ball, then put round bottom flask into ice water bath and stir continuously, add 2.7mL succinic acid monomethyl ester acyl chloride slowly, after 4 hours TCL monitor the reaction, evaporate the organic solvent under reduced pressure after the reaction is complete, and purify by column chromatography to get 2.9g off-white solid compound 8, the yield is 75%. 1 H NMR(300MHz,CDCl 3 )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 C NMR(75MHz,CDCl 3 )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.
(5) Synthesis of Compound 9
2g of Compound 8 and 2.5g of PyHBr are weighed out 3 Into a 100mL round bottom flask, 40mL of extra dry DCM was added and the flask was evacuated to insert N 2 Ball, light-proof reaction for 24h at room temperature, TCL monitoring reaction condition, adding 30mL 10% diluted hydrochloric acid solution and 20mL DCM into a bottle after the reaction is completed, transferring to a 100mL separating funnel for extraction, collecting lower organic solvent, repeating the extraction operation for three times, drying the collected DCM with anhydrous sodium sulfate, concentrating, and columnAfter purification by chromatography, 2.1g of compound 9 were obtained as a tan solid with a yield of 70%. 1 H NMR(300MHz,CDCl 3 )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＝14.8Hz,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,CDCl 3 )δ173.60,172.05,138.35,137.10,136.98,132.82,130.85,130.76,130.71,129.70,129.54,129.02,128.95,128.64,60.15,55.63,52.61,51.81,30.72,29.29.
(6) Synthesis of Compound 10
2g of compound 9 is weighed into a 100mL round bottom flask, 40mL of ultra-dry THF is added, N2 balls are inserted in vacuum, the mixture is placed into the flask at the temperature of minus 40 ℃ for continuous stirring, 8mL of tetrahydrofuran solution containing 1M of potassium tert-butoxide is gradually added at the temperature after 10min, 3.2mL of tetrahydrofuran solution is added again after 2h for continuous reaction for 1h, TCL monitors the reaction condition, 30mL of distilled water and 50mL of DCM are added for extraction after the reaction is completed, the lower organic solvent is collected and three times of extraction operation are repeated, the collected DCM is dried by anhydrous sodium sulfate and concentrated, and 800mg of yellow oily compound 10 can be obtained after column chromatography is purified, and the yield is 60%. 1 H NMR(300MHz,CDCl 3 )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,CDCl 3 )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.
(7) Synthesis of Compound 11
Weighing 500mg of compound 10, adding into a 100mL round-bottom flask, adding 20mL of ultra-dry THF, continuously stirring, dissolving 75mg of LiOH in 6mL of distilled water, then adding into the round-bottom flask, carrying out light-shielding reaction at room temperature for 12h, monitoring the reaction condition by TCL, adding 30mL of distilled water and 50mL of DCM for extraction after the reaction is completed, collecting the lower organic solvent, repeating the extraction operation for three times, drying the collected DCM with anhydrous sodium sulfate, concentrating, and carrying out column chromatographyPurification gave 300mg of compound 11 as an off-white solid in 63% yield. 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)δ174.03,171.22,151.93,148.89,132.85,130.08,129.39,128.66,128.48,128.14,127.29,125.63,123.02,122.02,114.76,108.51,55.41,29.74,29.45.
(8) Synthesis of Compound 12
150mg of Compound 11 was weighed into a 100mL round bottom flask, 15mL anhydrous DMF was added, and the mixture was evacuated to N 2 The balls are dissolved completely by continuous stirring. Subsequently, 280mg of HATU was dissolved in 6mL of anhydrous DMF and added to a bottle, and after 20min of reaction, 405mg of NH was weighed 2 -PEG8-NH 2 And dissolving with 5mL anhydrous DMF, adding 0.41mL DIPEA to the dissolved NH 2 -PEG8-NH 2 After 10min in solution, NH was added to the round bottom flask 2 -PEG8-NH 2 The reaction was monitored by TLC for 24 hours in the dark from the mixed solution of DIPEA, the organic solvent was distilled off under reduced pressure after completion of the reaction, and purified by column chromatography to give 214mg of the yellowish white emulsion compound 12 in 65% yield. HRMS (ESI+) (m/z) calculated for C 37 H 54 N 3 O 10 [M+H] + 700.3804,found 700.3790.
(9) Synthesis of Compound 13
1g of oxaliplatin was weighed into a 250mL round bottom flask, 30mL of distilled water was added and stirring was continued, followed by dropwise addition of 60mL of 30% aqueous hydrogen peroxide solution, and after reacting at 60℃for 4 hours until it was completely dissolved, the solvent was distilled off. 100mL of distilled water is added into a round-bottom flask for continuous stirring, the reaction temperature is adjusted to 80 ℃, after the reaction liquid in the flask is completely clarified, heating is stopped, the flask is placed in a refrigerator at 4 ℃ for overnight, a large amount of white needle-like crystals are separated out in the next day, the solvent in the flask is poured out, and the crystals are pumped by an oil pump, so that 900mg of white solid compound 13 is obtained, and the yield is 83%.
(10) Synthesis of Compound 14
Weighing 300mg respectivelyCompound 13 and 310mg of palmitic anhydride were placed in a 100mL round bottom flask and N was inserted by applying a vacuum 2 Ball and add 30mL anhydrous DMSO solvent into bottle, then stir at room temperature for 7 days, evaporate organic solvent by oil pump after reaction, then add 50mL acetone into bottle with a large amount of white solid precipitated, filter, and repeatedly wash with acetone, dry and collect to obtain 350mg white solid compound 14 with 75% yield.
(11) Synthesis of Compound 15
300mg of Compound 14 and 100mg of succinic anhydride were weighed into a 100mL round bottom flask, respectively, added with 30mL of anhydrous DMSO solvent, and vacuum-inserted into N 2 Ball reaction for 12h, decompression evaporation of the organic solvent after the reaction, and purification by column chromatography gave 210mg of compound 15 as a white solid in 61% yield.
(12) Synthesis of Compound 16
100mg of Compound 15 was weighed into a 50mL round bottom flask, 10mL anhydrous DMF was added, and the mixture was evacuated to N 2 The balls are dissolved completely by continuous stirring. Subsequently, 75mg of HATU was dissolved in 3mL of anhydrous DMF and added to the flask, after 20min of reaction, 174mg of Compound 12 was weighed and 5mL of anhydrous DMF was dissolved, 0.19mL of DIPEA was added to the dissolved Compound 12 solution, after 10min, a mixed solution of Compound 12 and DIPEA was added to the round bottom flask, and the reaction was carried out for 24 hours in the dark, TLC was monitored for the reaction conditions, after completion of the reaction the organic solvent was distilled off under reduced pressure and purification by column chromatography was carried out to give 89mg of tan emulsion Compound 16 in 47% yield. 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)δ173.40,173.17,172.96,172.52,151.31,148.11,132.14,129.26,128.68,128.29,127.84,127.55,126.77,125.11,122.99,122.24,114.23,107.53,69.95,69.80,69.13,55.31,54.44,38.97,31.74,30.52,30.02,29.96,29.44,29.16,22.41,17.36,15.92,13.19,11.87.
EXAMPLE 2HBAPE-Ac 3 Biological evaluation of ManNAz
1.1 Metabolic markers for living cells
To further explore HBAPE-Ac 3 ManNAz labelling efficiency we will be 50, 100, 200, 500. Mu.M different concentrations of HBAPE-Ac 3 ManNAz was incubated with 4T1 cells for 24 hours, respectively, to label proteins in mammalian cells, and the cleaved proteins were click-reacted with Biotin-PEG4-alkyne and HBAPE-Ac was verified by Western Blot 3 ManNAz was efficient for protein labelling. The results are shown in FIG. 1a, with HBAPE-Ac 3 The metabolic labeling effect of the probe is stronger and stronger when the concentration reaches 200 mu M, HBAPE-Ac is increased by the concentration of ManNAz 3 ManNAz can achieve a highly efficient marker for 4T1 cells without cytotoxicity.
Taking this as a reference, the incubation with 4T1 cells at this concentration was carried out for different lengths of time such as 0, 8, 12, 24, 36, 48h, etc., respectively, and then the protein labeling efficiency was verified by the same method, and the results are shown in FIG. 1b, following HBAPE-Ac 3 The labeling effect of the increase in ManNAz incubation time was gradually increased. When the incubation time reaches 24 hours, HBAPE-Ac 3 The cell labelling effect of ManNAz reached an ideal intensity, with a longer incubation time, the 36h labelling effect was more intense, and then prolonged again without significant differences. Thus, from a combination of experimental results of concentration and time dependence, HBAPE-Ac 3 ManNAz labeled cells at 200. Mu.M concentration for 24h, high efficiency labeling of cells was achieved.
1.2HBAPE-Ac 3 The cellular metabolic markers of ManNAz are regulated by ROS
HBAPE-Ac by concentration, time dependence and cell diversity experiments 3 The metabolic markers of ManNAz are basically characterized, and can prove newly synthesized HBAPE-Ac 3 ManNAz was able to act as a normal glycometabolism marker probe. Based on this, further investigation of HBAPE-Ac by Western Blot 3 Whether the cellular metabolic markers of ManNAz are regulated by ROS. Continuing to use 4T1 cells as metabolic marker study object, at 200 mu M HBAPE-Ac 3 Different concentrations of ROS agonist H were added while ManNAz was co-incubated with 4T1 cells for 24H 2 O 2 The solution was then subjected to protein labelling efficiency verification by the same method, the results are shown in FIG. 2a, and follow H 2 O 2 The concentration is continuously increased, HBAPE-Ac 3 The labeling effect of ManNAz was gradually enhanced, indicating HBAPE-Ac 3 ManNAz cell metabolism marker pair H 2 O 2 Has concentration dependence.
At the same time, to further illustrate HBAPE-Ac 3 The cellular metabolic markers of ManNAz are controlled by ROS, and different concentrations of the ROS inhibitor N-acetyl-L-cysteine (NAC) are combined with 200. Mu. MHBAPE-Ac 3 ManNAz co-incubated in 4T1 cells for 24h showed that the labeling effect was significantly reduced at NAC concentration of 0.5mM and gradually decreased as NAC concentration increased and was substantially completely inhibited at 3mM, as shown in FIG. 2 b. The result can obtain the glycometabolism molecular probe HBAPE-Ac 3 ManNAz is regulated by ROS during metabolic labeling of cells.
1.3 in vitro HPLC detection of ROS agonist H 2 O 2 For HBAPE-Ac 3 Changes in ManNAz Regulation
In HBAPE-Ac 3 ManNAz labeled 4T1 cells by addition of ROS agonist H 2 O 2 To alter the cellular metabolic marker levels of the probe and thereby determine that the marker process is controlled by ROS. Furthermore, to further determine newly synthesized HBAPE-Ac 3 The removal of the 4- (hydroxymethyl) phenylboronic acid pinacol ester functional group at position 1 in ManNAz is affected by ROS, and in vitro HPLC detection of HBAPE-Ac is adopted 3 ManNAz at H 2 O 2 The hydrolysis efficiency of the pinacol ester functional group of the 1-position 4- (hydroxymethyl) phenylboronic acid under the action. HBAPE-Ac 3 ManNAz is respectively with different concentrations of H 2 O 2 HPLC detection after co-incubation for 3H in a 1.5mL EP tube, the results are shown in FIG. 3a, with H 2 O 2 Concentration increase, HBAPE-Ac 3 The efficiency of hydrolysis of the 1-position functional group of ManNAz gradually increases.
At the same time, HBAPE-Ac with the same concentration 3 ManNAz and H 2 O 2 HPLC detection was performed by co-incubation for various times, and the results are shown in FIG. 3b, wherein HBAPE-Ac increased with increasing incubation time 3 The efficiency of hydrolysis of the 1-position functional group of ManNAz also gradually increases. Thus, the above two HPLC detection results more evidence newly synthesized HBAPE-Ac 3 Hydrolysis efficiency of ManNAz by ROS agonist H 2 O 2 Is a function of (a) and (b).
1.4 confocal imaging and flow cytometry positioning and efficiency analysis of HBAPE-Ac3ManNAz in cell markers
In classical probe Ac 4 Further synthesis of HBAPE-Ac responsive to ROS based on chemical structure of ManNAz 3 ManNAz, therefore, its metabolic labelling mechanism and Ac after hydrolysis under high ROS content in tumor cells 4 ManNAz is theoretically consistent, and all the ManNAz is converted into an azide-containing sialic acid molecule through corresponding enzymes of a sialic acid biosynthesis pathway so as to realize azide group labeling on sialic acid at the tail end of a sugar chain on the surface of a cell membrane. To verify this hypothesis, ac was acquired by confocal imaging and flow cytometry 4 ManNAz and HBAPE-Ac 3 The cell markers of ManNAz were compared. Ac is carried out 4 ManNAz and HBAPE-Ac 3 ManNAz was incubated with 4T1 cells in confocal glass dishes at 200. Mu.M for 24h, followed by addition of DBCO-488 dye to bind azide to DBCO and attach fluorescence at the probe-labeled site, followed by staining cell membrane surface glycoprotein with WGA dye and staining cell nucleus with Hoechst 33342 dye as controls, and observation under confocal microscope, as shown in FIG. 4a, the results were obtained for HBAPE-Ac 3 ManNAz and Ac 4 The DBCO-488 fluorescence region of ManNAz was identical and overlapped with the WGA dye staining region, indicating newly synthesized HBAPE-Ac 3 ManNAz can be with Ac 4 The ManNAz metabolic mechanism also realizes the marking of the surface of the cell membrane. Furthermore, as can be seen under fluorescent microscopy, HBAPE-Ac 3 ManNAz labeled cells at slightly higher levels than Ac 4 ManNAz, and after co-incubation with NAC, HBAPE-Ac 3 The fluorescence region of the ManNAz marker was significantly reduced, and further HBAPE-Ac 3 Metabolic markers of ManNAz are regulated by ROS providing more evidence.
At the same time, to verify HBAPE-Ac 3 ManNAz can realize high-efficiency marking of cell membranes, and we use flow cytometry to measure cell membrane surfacesComparing the fluorescence intensity of the surface, and comparing Ac 3 6deoGlcNAz、Ac 4 GalNAz、Ac 4 ManNAz、HBAPE-Ac 3 Various probes such as ManNAz and the like are respectively incubated with 4T1 cells for 24 hours, then the collected cells are dyed with DBCO-488 dye and are detected on a flow machine, and the result is shown in FIG. 4b, HBAPE-Ac 3 The fluorescence labeling efficiency of ManNAz on cell membrane reaches Ac 4 ManNAz strength. The results of both experiments fully demonstrate that newly synthesized HBAPE-Ac 3 ManNAz is a metabolic probe for efficiently labeling glycoproteins on cell membrane surfaces.
1.5HBAPE-Ac 3 ManNAz in vivo tumor selective markers for mice
By verifying at the cellular level HBAPE-Ac 3 ManNAz can be used as a nontoxic and efficient glycometabolism molecular probe, so that further verification of HBAPE-Ac is desired 3 ManNAz labeling efficiency on tumor cells in a mouse in vivo tumor-bearing model. First verify HBAPE-Ac 3 Relationship between ManNAz labeling efficiency and administration frequency, subcutaneously implanting 4T1 tumor cells into mice to construct tumor-bearing model, and continuously injecting mice of different groups into abdominal cavity for seven days after tumor formation 4 ManNAz、HBAPE-Ac 3 ManNAz、HBAPE-Ac 3 ManNAz and NAC solutions (drug dose of 300 mg/kg) followed by tail vein injection of 100 μg/μLDBCO-Cy5 for 24h followed by in vivo imaging of small animals and in vivo imaging comparison of fluorescence signals at tumor sites, as shown in FIG. 5a, HBAPE-Ac 3 ManNAz gave stronger fluorescence signal at tumor site than Ac 4 Marking effect of ManNAz on tumor, after simultaneous injection of ROS inhibitor NAC, HBAPE-Ac 3 The marking effect of ManNAz on tumors is obviously weakened, which indicates that the novel probe HBAPE-Ac 3 ManNAz is also regulated by ROS in vivo tumor markers.
At the same time, each group of mice was dissected for tissue imaging, as shown in FIGS. 5b-c, ac 4 ManNAz showed fluorescence signals in both lung and kidney tissues, except tumor location, in vivo mouse tumor-bearing model, whereas newly synthesized HBAPE-Ac 3 ManNAz greatly reduces metabolic markers of non-tumor parts and increases the selectivity of the probe body to tumor cellsMarking mice injected with NAC at the same time inhibits HBAPE-Ac 3 The normal metabolic labeling process of ManNAz further reduces the expression of the probe at the tumor site, so that the probe stays at the kidney for metabolism to cause strong fluorescent signals. The result of the living body imaging experiment can be synthesized, and the probe is compared with classical probe Ac 4 Compared with ManNAz, newly synthesized HBAPE-Ac 3 The in vivo metabolic markers of ManNAz are regulated by ROS to realize high-selectivity and high-efficiency marking of tumor parts.
Example 3.
1. Based on HBAPE-Ac 3 Mannaz sugar metabolism marker, DBCO-PEG8-Oxp (IV) in-vitro and in-vivo anti-tumor activity and targeting research
The invention takes tetravalent oxaliplatin as a mother nucleus, a hexadecane chain is introduced into one end of upright bonds at two ends of the tetravalent oxaliplatin, the hexadecane chain modified platinum medicine is easy to be combined with human serum albumin (HAS) for administration, and the medicine is accumulated more preferentially in cancer cells than in normal cells; meanwhile, DBCO is introduced into the other end of the upright bond, so that click reaction can be carried out on the DBCO and cancer cells marked by the azide groups in advance to enhance the tumor targeting transportation capacity of the cancer cells.
1. In vitro antitumor Activity test
The target compound DBCO-PEG8-Oxp (IV) is obtained through chemical synthesis, firstly, the in vitro anti-tumor activity is evaluated through MTT experiment, and the result is shown in figure 6, and the positive medicine divalent oxaliplatin (figure 6 a) is used as a control, and the chemically synthesized tetravalent oxaliplatin complex DBCO-PEG8-Oxp (IV) is used as an IC in 4T1 breast cancer cells 50 (figure 6 b) is lower than bivalent platinum, which shows that the synthesized drug has a certain anti-tumor activity. At the same time, through HBAPE-Ac 3 IC of DBCO-PEG8-Oxp (IV) in 4T1 cells after 24h of Mannaz labeling (FIG. 6 c) 50 The value reaches the nanomolar level, the inhibition effect of the tumor growth activity is obviously enhanced, and the click reaction-mediated drug targeting transportation is possibly a promising choice in improving the platinum drug targeting tumor capability and the antitumor activity.
Platinum uptake of DBCO-PEG8-Oxp (IV) in breast cancer cells
The accumulation degree of the drug in cancer cells influences the intensity of the antitumor activity of the drug, and the platinum drugs enhance the targeting capability of the drug and improve the intake of the drug by cells through click reaction, so that the content of platinum in the cells is further quantitatively detected by ICP-MS (inductively coupled plasma-mass spectrometry) based on the click reaction of DBCO-PEG8-Oxp (IV).
We will go through HBAPE-Ac 3 After 24h of Mannaz labeling and 4T1 unlabeled cells were incubated with DBCO-PEG8-Oxp (IV) at a final concentration of 1. Mu.M for 2h, and at the same time, oxaliplatin was used as a positive drug, the cells collected after the end of incubation were acidified with nitric acid and detected by ICP-MS, and as a result, as shown in FIG. 7a, unlabeled cells were incubated with oxaliplatin and DBCO-PEG8-Oxp (IV) at the same concentrations, respectively, every 2X 10 5 The platinum content in individual cells was not significantly different, but was measured by HBAPE-Ac 3 The platinum content in the cells marked by ManNAz is obviously increased and almost reaches 4 to 5 times of unlabeled cells, which indicates that the click reaction promotes the uptake capacity of the cells for platinum drugs. In addition, 0.5, 1, 2. Mu.M DBCO-PEG8-Oxp (IV) was incubated with HBAPE-Ac, respectively 3 2h in 4T1 cells after 24h of ManNAz labeling, detected by ICP-MS, the results are shown in FIG. 7b, every 2X 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 the different concentrations, and we hypothesize that this might be subject to the problem of short incubation times. In addition, 1. Mu.M DBCO-PEG8-Oxp (IV) was incubated with HBAPE-Ac, respectively 3 1h,12h,24h of 4T1 cells after 24h of ManNAz labeling were examined by ICP-MS, and the results are shown in FIG. 7c, every 2X 10 5 The platinum content in individual cells increased with increasing incubation time, and the platinum content in cells after 24h incubation was up to 4 times that of 1h incubation. The above experimental results show that DBCO-PEG8-Oxp (IV) promotes the uptake of platinum into cells by click reaction based on sugar metabolism markers, thereby increasing the accumulation of drug in tumor cells.
Influence of DBCO-PEG8-Oxp (IV) on the ability of breast cancer cells to migrate
To evaluate the inhibition of breast cancer cell migration by DBCO-PEG8-Oxp (IV) under click reaction mediation. First, the effect of DBCO-PEG8-Oxp (IV) on the migration ability of 4T1 breast cancer cells was examined by wound healing experiments, and the results were obtained by HBAPE-Ac 3 ManNAz labeled 24h later and unlabeled4T1 cells were incubated with DBCO-PEG8-Oxp (IV) at a final concentration of 1. Mu.M, and compared with oxaliplatin as a positive drug, and observed under a microscope at various time periods to obtain photographs, as shown in FIG. 8a, both oxaliplatin and DBCO-PEG8-Oxp (IV) inhibited migration of 4T1 cells, wherein the cells were not subjected to HBAPE-Ac 3 ManNAz-labeled cells showed a slight difference in inhibition of cell migration between oxaliplatin and DBCO-PEG8-Oxp (IV) at 12h, but showed substantially no difference in inhibition after 24h, however, through HBAPE-Ac 3 The inhibition effect of the DBCO-PEG8-Oxp (IV) on the cell migration of the ManNAz marked cell is obviously enhanced at 12h and 24h, and experimental results show that the DBCO-PEG8-Oxp (IV) promotes the accumulation of platinum on tumor cells through click reaction so as to enhance the inhibition effect on the cell migration.
Meanwhile, in order to further prove that the click reaction promotes the inhibition effect of DBCO-PEG8-Oxp (IV) on cell migration, the same experimental group is designed and verified in a Transwell chamber, and the result is shown in FIG. 8b, we can see that HBAPE-Ac is passed in advance 3 The effect of DBCO-PEG8-Oxp (IV) on the migration ability of cells marked by ManNAz for 24h is obviously enhanced.
In addition, we have also examined the effect of DBCO-PEG8-Oxp (IV) on matrix metalloproteinase 9 (MMP-9) and matrix metalloproteinase 7 (MMP-7) that play an important role in the process of cell growth and migration before and after probe labeling by Western Blot, and the results are shown in FIG. 8c, which shows that the protein is purified by HBAPE-Ac 3 ManNAz marked 24h in advance, DBCO-PEG8-Oxp (IV) significantly reduced MMP-9 and MMP-7 protein expression, thus inhibiting tumor cell growth migration process. The experimental results fully demonstrate that DBCO-PEG8-Oxp (IV) remarkably enhances the inhibition effect on the migration capacity of breast cancer cells through click reaction.
2. Based on HBAPE-Ac 3 Mannaz glycometabolism marker, evaluation of anti-tumor Activity and anti-metastasis Effect of DBCO-PEG8-Oxp (IV) in mice
1. Experiments on inhibiting in-situ solid tumor growth of breast cancer cell mice by DBCO-PEG8-Oxp (IV) based on glycometabolism markers
Early in vitro experiments prove that DBCO-PEG8-Oxp (IV) is subjected to HBAPE-Ac 3 ManNAz instituteThe killing effect of the marked 4T1 breast cancer cells is obviously improved, and simultaneously, in vivo and in vitro HBAPE-Ac has been proved 3 ManNAz has strong specificity and high labeling capacity on tumor tissues in mice. Thus we injected 4T1 breast cancer cells into the underarm of Balb/C mice to create tumor-bearing models, which were divided into PBS, oxp (2.46 mg Pt/kg), DBCO-PEG8-Oxp (IV) (2.46 mg Pt/kg), ac 4 ManNAz+DBCO-PEG8-Oxp(IV)、HBAPE-Ac 3 ManNAz+DBCO-PEG8-Oxp (IV) was used in five groups of six mice each. The administration cycle is shown in FIG. 9a, and the average tumor volume of the mice is 50-100mm 3 Probe Ac is injected to five consecutive days in abdominal cavity 4 ManNAz or HBAPE-Ac 3 ManNAz, followed by injection of the corresponding drug Oxp or DBCO-PEG8-Oxp (IV) into the tail vein of each group, the frequency of platinum drug administration was injected once every two days for three total treatments, and the mice of the group treated with the sugar probe during the interval were correspondingly probed daily. The body weight and tumor volume of the mice were recorded every two days from the beginning of tumor inoculation, one day was observed after the end of administration, the mice were eugenoblood was taken and dislocation was killed, and then the tumors and the organ tissues were taken out for photographing and the tumor weights were recorded. The body weight changes of the mice in each group during the dosing cycle (FIG. 9 b) tended to be smooth and passed through Ac 4 ManNAz/HBAPE-Ac 3 The weight of the mice combined with DBCO-PEG8-Oxp (IV) is not lower than the initial weight and not more than 20% of the initial weight in the last day, which shows that the drug has no obvious toxic or side effect on the mice. At the same time, the tumor volume change curve (FIG. 9 c) shows that on the 11 th day of tumor growth, the tumor volume of the mice in the control group averages 920mm 3 Oxp group 711mm 3 DBCO-PEG8-Oxp (IV) group of 655mm 3 、Ac 4 ManNAz+DBCO-PEG8-Oxp (IV) group 290mm 3 ，HBAPE-Ac 3 ManNAz+DBCO-PEG8-Oxp (IV) group 185mm 3 It can be seen that oxaliplatin and DBCO-PEG8-Oxp (IV) both have the effect of inhibiting tumor growth compared with the control group, but the inhibition rates are 23% and 29%, respectively, the inhibition effects are not obvious and the effects are not quite different. When the probe is added to mark tumor cells in advance, the inhibition effect of DBCO-PEG8-Oxp (IV) on tumors is obviously enhanced, wherein the inhibition effect is shown in Ac 4 Inhibition of tumor growth by DBCO-PEG8-Oxp (IV) under the marker of MannazThe rate reaches 68 percent, and HBAPE-Ac with better tumor selective marking effect is selected 3 The inhibition rate of DBCO-PEG8-Oxp (IV) to tumor growth reaches 80% in ManNAz. In addition, the dissected tumor tissue was photographed (FIG. 9 d), subsequently weighed (FIG. 9 e), and using probe labeling, DBCO-PEG8-Oxp (IV) inhibited the tumor more significantly, solid tumors were lighter weight, ac compared to control group 4 The average tumor of solid tumors of mice in the ManNAz+DBCO-PEG8-Oxp (IV) group was about 21% of that of the control group, while HBAPE-Ac 3 The average tumor of solid tumors in mice of ManNAz+DBCO-PEG8-Oxp (IV) group was about 14% of that of control group. H for each group of tumor tissues&E staining, as shown in FIG. 9f, the control mice showed significantly increased cell number, increased cell nucleus volume, and strong proliferation capacity, while Ac 4 ManNAz/HBAPE-Ac 3 The number of tumor tissue nuclei of mice combined with DBCO-PEG8-Oxp (IV) by ManNAz is obviously reduced, and the effect is more obvious than that of mice singly using oxaliplatin or DBCO-PEG8-Oxp (IV), which shows that under the action of a probe, the inhibition effect of DBCO-PEG8-Oxp (IV) on cancer cell proliferation is enhanced.
To further verify that DBCO-PEG8-Oxp (IV) increased drug targeting transport capacity to tumor sites by click reaction, we measured the platinum content of each group of mouse tissues using ICP-MS, as shown in FIG. 9g, via Ac 4 ManNAz or HBAPE-Ac 3 After the ManNAz marks the tumor, the accumulation amount of platinum on the tumor tissue of DBCO-PEG8-Oxp (IV) is obviously improved, and Ac is remarkably increased 4 Tumor platinum content in ManNAz+DBCO-PEG8-Oxp (IV) group mice was about 2 times that of oxaliplatin or DBCO-PEG8-Oxp (IV) group mice alone, HBAPE-Ac 3 The platinum content on tumors of mice in the ManNAz+DBCO-PEG8-Oxp (IV) group is higher and can reach 3500ng Pt/g, and compared with oxaliplatin, the ICP-MS result shows that the DBCO-PEG8-Oxp (IV) is accumulated more on tumor sites in vivo after being marked by a probe, and the accumulation of sites such as liver and kidney is reduced, so that the damage to the liver and kidney is greatly reduced. And use HBAPE-Ac 3 ManNAz has better therapeutic effect on DBCO-PEG8-Oxp (IV) after tumor marking than Ac 4 Tumor markers by ManNAz, indicating HBAPE-Ac 3 ManNAz target for tumorIt was noted that the higher selectivity enhanced the targeting of DBCO-PEG8-Oxp (IV). The experimental result shows that DBCO-PEG8-Oxp (IV) improves the targeting ability of the drug to tumors through click reaction in a mouse tumor-bearing model so as to enhance the anti-tumor activity of the drug, and simultaneously reduces side effects caused by accumulation of platinum on non-tumor parts.
2. Experiments on inhibiting lung metastasis of breast cancer cells by DBCO-PEG8-Oxp (IV) based on glycometabolism markers
In vitro experiments prove that the composition is based on HBAPE-Ac 3 The metabolic marker of the ManNAz sugar probe and DBCO-PEG8-Oxp (IV) have remarkable inhibiting effect on the migration capacity of breast cancer cells. For further validation, we injected murine 4T1 breast cancer cells into Balb/C mice by tail vein injection, creating a mouse lung metastasis model. The experiments were divided into PBS, oxp (2.46 mg Pt/kg), DBCO-PEG8-Oxp (IV) (2.46 mg Pt/kg), ac 4 ManNAz+DBCO-PEG8-Oxp(IV)、HBAPE-Ac 3 ManNAz+DBCO-PEG8-Oxp (IV) was used in five groups of five mice each. The administration cycle is shown in FIG. 10a, and the abdominal cavity starts to inject the probe Ac four consecutive days on the fifth day after the tumor inoculation 4 ManNAz or HBAPE-Ac 3 ManNAz, followed by injection of the corresponding drug Oxp or DBCO-PEG8-Oxp (IV) into the tail vein of each group, the frequency of platinum drug administration was injected once every two days for three total treatments, and the mice of the group treated with the sugar probe during the interval were correspondingly probed daily. The body weight of the mice was recorded every two days from the beginning of tumor inoculation, one day was observed after the end of administration, after taking the blood of the eyeballs of the mice and killing them by dislocation, then taking the organ tissues, and photographing the lung tissues of the mice of each treatment group (fig. 10 b) and recording the lung weight and the number of nodules. The body weight change of each group of mice (fig. 10 c) tended to be smooth during the dosing period, and the body weight change of each treatment group of mice did not exceed 20% of the initial body weight. By observation of lung tissue (fig. 10 d), DBCO-PEG8-Oxp (IV) and oxaliplatin treated mice had a weak inhibition of breast cancer metastasis, compared to the control group, with inhibition rates of 27% and 36%, respectively. When the probe is added to mark tumor cells in advance, the inhibition effect of DBCO-PEG8-Oxp (IV) on tumor metastasis is obviously enhanced, wherein the inhibition effect is shown in Ac 4 DBCO-PEG8-Oxp (IV) treated Small under ManNAz labelingThe lung nodules of mice were reduced by 55%, while HBAPE-Ac 3 Mannaz+DBCO-PEG8-Oxp (IV) treated mice had 84% lower lung nodules. At the same time we weighed the dissected lung tissue and as shown in FIG. 10e, the lungs of the nodular control group were significantly heavier than Ac 4 Mannaz+DBCO-PEG8-Oxp (IV) and HBAPE-Ac 3 Mannaz+DBCO-PEG8-Oxp (IV) treated group of lungs. Subsequent H of lung tissue from each treatment group&E staining, results are shown in FIG. 10f, in which the number of cells in the lung tissue of mice with excessive numbers of nodules in the control group, oxaliplatin group and DBCO-PEG8-Oxp (IV) is significantly increased, the volume of the nuclei is increased, and the proliferation capacity is strong, while Ac 4 ManNAz/HBAPE-Ac 3 The number of cell nuclei of lung tissues of mice combined by ManNAz and DBCO-PEG8-Oxp (IV) is obviously reduced, which proves that the inhibition effect of DBCO-PEG8-Oxp (IV) on tumor metastasis is obviously enhanced under the action of a probe. At the same time, it can be found that HBAPE-Ac is used 3 The inhibition effect of ManNAz on DBCO-PEG8-Oxp (IV) after tumor marking on tumor metastasis is better than Ac 4 Tumor markers by ManNAz, indicating HBAPE-Ac 3 ManNAz has higher selectivity for tumor labeling and enhanced DBCO-PEG8-Oxp (IV) targeting.
To further verify that DBCO-PEG8-Oxp (IV) increased the inhibitory effect of the drug on tumor metastasis by click reaction, we measured the platinum content of each group of mouse tissues using ICP-MS, as shown in FIG. 10g, via Ac 4 ManNAz or HBAPE-Ac 3 After tumors are marked by ManNAz, the accumulation amount of platinum on lung tissues of DBCO-PEG8-Oxp (IV) is obviously increased, and platinum in mice treated by oxaliplatin is mainly distributed in the liver, kidney and other parts of the mice. The data fully demonstrate that DBCO-PEG8-Oxp (IV) binding to the glycometabolism marker can target tumor cells, thereby inhibiting the metastatic capacity of tumors.
HBAPE-Ac designed by the invention 3 ManNAz is a novel sugar metabolism marker molecular probe which is regulated by ROS, is safe and efficient, and can specifically mark tumor cells or tissues in vitro and in vivo; based on HBAPE-Ac 3 The ManNAz sugar metabolism marker, DBCO-PEG8-Oxp (IV) improves the targeting capability of platinum drugs to tumor cells, enhances the anti-tumor activity and anti-tumor metastasis capability of the drugs, and has the advantages ofGood biosafety.

Claims (7)

1. Small molecular probe HBAPE-Ac based on glycometabolism marker 3 ManNAz, characterized in that the small molecule probe HBAPE-Ac 3 ManNAz, its structural formula is as follows:
2. the small molecule probe based on glycometabolism marker HBAPE-Ac according to claim 1 3 ManNAz specifically labels within tumors and enhances the targeting of chemotherapeutic drugs.
3. The small molecule probe based on glycometabolism marker HBAPE-Ac according to claim 2 3 ManNAz specifically marks in tumor and improves the targeting of chemotherapeutic drugs, and is characterized in that the chemotherapeutic drugs are platinum drugs.
4. The small molecule probe based on glycometabolism marker HBAPE-Ac according to claim 2 3 ManNAz specifically labels within tumors and enhances the targeting of chemotherapeutic agents, wherein said tumors include, but are not limited to, lack of specific targeting receptors.
5. The small molecule probe based on glycometabolism marker HBAPE-Ac according to claim 2 3 The ManNAz is specifically marked in tumor and improves the targeting of chemotherapeutic drugs, and is characterized in that the probe HBAPE-Ac of the chemotherapeutic drugs 3 The azide tag on the ManNAz marked tumor cell membrane and the chemotherapeutic drug have biological orthogonal reaction, so that the targeted transport capacity of the chemotherapeutic drug to the tumor is improved.
6. A small molecule probe HBAPE-Ac according to claim 5 3 Anti-swelling agent with bioorthogonal reaction of azide tag on ManNAz labeled tumor cell membraneThe tumor active complex DBCO-PEG8-Oxp is characterized by having the following structural formula:
7. the anti-tumor active complex DBCO-PEG8-Oxp according to claim 6, wherein the DBCO-PEG8-Oxp is used as a pharmacodynamic molecule to perform biological orthogonal reaction with azide labels marked on tumor cell membranes by biological probes, so that the targeting transport capacity of DBCO-PEG8-Oxp to tumors is improved.
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CN117771184A (en) * 2023-12-27 2024-03-29 贵州医科大学 Liposome composition for treating triple negative breast cancer and application thereof

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117771184A (en) * 2023-12-27 2024-03-29 贵州医科大学 Liposome composition for treating triple negative breast cancer and application thereof

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