Twisted lanthanide metal europium supramolecular cage complex, preparation method and application

Abstract

The present invention discloses a twisted lanthanide metal europium supramolecular cage complex, a preparation method and an application. The twisted lanthanide metal europium supramolecular cage complex is synthesized by a coordination-driven self-assembly method using a benzothiadiazole derivative as a ligand and Eu(OTf)3 as a metal receptor. Metal europium is used as the "vertex" of the structure and is surrounded by the twisted benzothiadiazole derivative ligand. Compared with Eu(OTf)3, the biological toxicity of the supramolecular cage complex is greatly reduced, and the compatibility with the biological system is greatly improved. At the same time, it also has good fluorescence properties. Based on the above characteristics, the metal europium supramolecular cage complex can be used for biological labeling, and the structure can be used to stain living cancer cells, thereby realizing biological imaging.

Description

Technical Field

[0001] The invention belongs to the field of medical technology and relates to a twisted lanthanide metal europium supramolecular cage complex, a preparation method and application thereof. The metal europium supramolecular cage of the invention can realize cell imaging of cancer cells. Background Art

[0002] Because some supramolecular compounds with specific structures have excellent biocompatibility, they are widely used in bioimaging, and the preparation of sensitive and stable optical probes is the key to realizing bioimaging with optical technology. The lanthanide ion europium has special optical and magnetic properties, usually acts as a functional center in the molecular structure, and is applied in many aspects, such as: luminescent probes, analyte sensing and tissue. Due to its toxicity, europium still faces great challenges in its application in biomarkers despite its good fluorescence activity and strong penetration effect. Summary of the invention

[0003] In view of the problems existing in the prior art, the present invention provides a twisted lanthanide metal europium supramolecular cage complex, preparation method and application, a new method for combining supramolecular structure with europium, and the obtained twisted lanthanide metal europium supramolecular cage can reduce the toxicity of metal elements in biological systems and has good fluorescence properties, thereby achieving biological imaging of living cancer cells.

[0004] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0005] A twisted lanthanide metal europium supramolecular cage complex, the chemical structure of which is

[0006] C 126 H 114 F 18 N 24 O 30 S9Eu2, the cation stereostructure of the twisted lanthanide metal europium supramolecular cage complex is shown in the following formula (1):

[0007]

[0008] Among them, m=0-6.

[0009] The method for preparing the twisted lanthanide metal europium supramolecular cage complex of the present invention comprises the following steps: a benzothiadiazole derivative ligand L and a Eu(OTf)3 receptor are subjected to a coordination synergistic self-assembly reaction in a mixed solvent to form a twisted lanthanide metal europium supramolecular cage complex, wherein the benzothiadiazole derivative ligand L has the structural formula:

[0010]

[0011] Furthermore, the molar ratio of the benzothiadiazole derivative ligand L to Eu(OTf)3 is (1.4-1.6):1, and the mixed solvent is composed of acetonitrile and methanol in a volume ratio of (1-5):1; the coordination synergistic self-assembly reaction is carried out at 0-80°C for 4-8h, preferably at 30°C for 5h.

[0012] Furthermore, the preparation method of the benzothiadiazole derivative ligand L comprises the following steps:

[0013] (1) dissolving 6-[(diethylamino)carbonyl]pyridine-2-carboxylic acid and oxalyl chloride in an organic solvent for reaction, and then rotary evaporating and concentrating after the reaction to obtain an activated acyl chloride intermediate;

[0014] (2) dissolving 4,4'-(benzo[1,2,5]thiadiazole-4,7-diyl)diphenylamine and the acid chloride intermediate obtained in step (1) in an organic solvent for reaction, cooling to room temperature after the reaction, extracting, eluting, solvent precipitating, filtering, and drying.

[0015] Furthermore, in the step (1), the molar ratio of 6-[(diethylamino)carbonyl]pyridine-2-carboxylic acid to oxalyl chloride is 1:(1-1.5), the organic solvent in the step (1) is a mixed solvent of DMF and DCM; the reaction temperature in the step (1) is 20-50° C., and the reaction time is 8-12 h.

[0016] Furthermore, the molar ratio of 6-[(diethylamino)carbonyl]pyridine-2-carboxylic acid to 4,4'-(benzo[1,2,5]thiadiazole-4,7-diyl)diphenylamine is (2-3):1, and the organic solvent in step (2) is a mixed solvent of triethylamine and DCM; the reaction temperature in step (2) is 30-65° C. and the reaction time is 4-9 h.

[0017] The twisted lanthanide metal europium supramolecular cage complex of the present invention is used for preparing fluorescent imaging agents in biological imaging. The metal europium supramolecular cage complex of the present invention is subjected to optical property measurement, cytotoxicity measurement and cell confocal imaging.

[0018] The twisted lanthanide metal europium supramolecular cage complex of the present invention is synthesized by a coordination-driven self-assembly method using a benzothiadiazole derivative as a ligand and Eu(OTf)3 as a metal receptor. Metal europium is used as the "vertex" of the structure and is surrounded by the twisted benzothiadiazole derivative ligand. Compared with Eu(OTf)3, the biological toxicity of the supramolecular cage complex is greatly reduced, and the compatibility with the biological system is greatly improved. At the same time, it also has good fluorescence properties. Based on the above characteristics, the metal europium supramolecular cage complex can be used for biological labeling, and the structure can be used to stain living cancer cells, thereby realizing biological imaging. It provides a new way to better and more accurately locate cancer cells, which is helpful for medical precision treatment.

[0019] The present invention has the following beneficial effects: the Eu metal in the present invention forms a twisted lanthanide metal europium supramolecular cage complex that enters the biological system in a state of low toxicity, and on the basis of ensuring strong permeability, cell imaging is achieved through excellent fluorescence properties, providing a new way for cancer cell identification and precise positioning. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The benzothiadiazole derivative ligand L prepared in Example 1 of the present invention is 1 H NMR spectra.

[0021] Figure 2 The metal europium supramolecular cage complex prepared in Example 2 of the present invention 1 H NMR spectra.

[0022] Figure 3 This is the electrospray ionization mass spectrum (ESI-MS) of the metal europium supramolecular cage complex prepared in Example 2 of the present invention.

[0023] Figure 4 This is a photoluminescence spectrum of the metal europium supramolecular cage complex prepared in Example 2 of the present invention.

[0024] Figure 5 This is a diagram showing the cytotoxicity detection of the metal europium supramolecular cage complex prepared in Example 2 of the present invention.

[0025] Figure 6 This is a fluorescence microscope image of U87 cells stained with the metal europium supramolecular cage complex prepared in Example 2 of the present invention.

[0026] Figure 7 This is a simulation diagram of the molecular structure of the twisted lanthanide metal europium supramolecular cage complex of the present invention. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention, and those skilled in the art in this field can make some non-essential improvements and adjustments based on the content of the above invention.

[0028] Example 1

[0029] The synthesis method of benzothiadiazole derivative ligand L is as follows:

[0030] (1) At 0°C, 6-[(diethylamino)carbonyl]pyridine-2-carboxylic acid (111.05 mg, 0.5 mmol) and oxalyl chloride (76.15 mg, 0.6 mmol, 1.2 eq) were added to a reaction flask, followed by the addition of DMF (4-5 drops) and dry dichloromethane (30 mL). After 30 min, the mixture was stirred at 30°C for 12 h, and then concentrated by rotary evaporation to obtain an activated acyl chloride intermediate; the yield was 98%.

[0031] (2) 4,4'-(Benzo[1,2,5]thiadiazole-4,7-diyl)diphenylamine (53.1 mg, 0.167 mmol, 1 / 3 eq.) and the acid chloride intermediate obtained in step (1) were dissolved in dry dichloromethane (30 mL), and triethylamine (with a molar ratio of 1.5:1 to 6-[(diethylamino)carbonyl]pyridine-2-carboxylic acid) was added to react, and refluxed at 30°C for 5 h. The combined organic solution was washed with a certain amount of 1N HCl, then washed with 1N NaOH, and finally washed with saturated sodium chloride. The organic layer was separated, dried over MgSO4, and concentrated to 5 mL by rotary evaporation. The mixture was poured into diethyl ether (50 mL) to produce a precipitate, and the precipitate was collected by centrifugation to obtain a yellow powder. The yield was 54%.

[0032]

[0033] 1 H NMR (600MHz, CDCl3) δ9.95(s,2H),8.39(dd,J=7.8,0.8Hz,2H),8.05(t,J=7.7Hz,6H),7.96(d,J=8.6Hz,4H),7.82(s,2H),7.78(dd,J=7.7,0.9Hz,

[0034] 2H),3.66(q,J=7.1Hz,4H),3.37(q,J=7.0Hz,4H),1.34(dt,J=11.9,7.1Hz,

[0035] 12H). 13C NMR(151MHz, CDCl3)δ167.61(s),161.50(s),154.11(s),153.65(s),

[0036] 148.52(s),138.94(s),137.68(s),133.58(s),132.46(s),130.04(s),127.81(s),

[0037] 125.95(s),123.09(s),119.76(s),43.19(s),40.19(s),14.69(s),12.84(s).

[0038] Example 2

[0039] The preparation method of the twisted lanthanide metal europium supramolecular cage complex of this embodiment is as follows:

[0040] The benzothiadiazole derivative ligand L (13.1 mg, 0.018 mmol) and Eu(OTf)3 (7.19,

[0041] 0.012 mmol) was added to a methanol:acetonitrile mixture (v:v=600 μL:600 μL), stirred at 30°C for 5 h, the reaction mixture was poured into diethyl ether (10 mL) to obtain a precipitate, which was collected by centrifugation to obtain an orange-red solid. Yield: 81%.

[0042]

[0043] 1 H NMR (600MHz, CD3CN) δ7.86 (s, 2H), 7.66 (t, J=7.7Hz, 1H), 7.37 (d, J=

[0044] 6.6Hz,2H),7.26(s,1H),7.18(d,J=7.7Hz,1H),6.64(d,J=7.9Hz,1H),6.13

[0045] (s,1H),3.66(d,J=6.7Hz,2H),3.07(d,J=51.0Hz,2H),1.40(t,J=6.7Hz,3H),

[0046] 0.66(s,3H).

[0047] Electrospray ionization mass spectrometry (ESI-MS) Figure 3 Shown: 3378Da, consistent with the predicted chemical composition [C 126 H 114 N 24 O 30F 18 S9Eu2] are well matched.

[0048] Example 3

[0049] The preparation method of the twisted lanthanide metal europium supramolecular cage complex of this embodiment is as follows:

[0050] Add benzothiadiazole derivative ligand L (13.1 mg, 0.018 mmol) and Eu(OTf)3 (7.19, 0.012 mmol) to a methanol:acetonitrile mixture (v:v=600 μL:1200 μL), stir at 30°C for 5 h, pour the reaction mixture into ether (10 mL) to obtain a precipitate, and collect it by centrifugation to obtain an orange-red solid. Yield: 49%.

[0051] Example 4

[0052] The preparation method of the twisted lanthanide metal europium supramolecular cage complex of this embodiment is as follows:

[0053] Add benzothiadiazole derivative ligand L (13.1 mg, 0.018 mmol) and Eu(OTf)3 (7.19, 0.012 mmol) to a methanol:acetonitrile mixture (v:v=600 μL:3000 μL), stir at 30°C for 5 h, pour the reaction mixture into ether (10 mL) to obtain a precipitate, and collect it by centrifugation to obtain an orange-red solid. Yield: 36%.

[0054] Example 5

[0055] The preparation method of the twisted lanthanide metal europium supramolecular cage complex of this embodiment is as follows:

[0056] Add benzothiadiazole derivative ligand L (12.2 mg, 0.0168 mmol) and Eu(OTf)3 (7.19, 0.012 mmol) to a methanol:acetonitrile mixture (600 μL:600 μL), stir at 30°C for 5 h, pour the reaction mixture into ether (10 mL) to obtain a precipitate, and collect it by centrifugation to obtain an orange-red solid. Yield: 67%.

[0057] Example 6

[0058] The preparation method of the twisted lanthanide metal europium supramolecular cage complex of this embodiment is as follows:

[0059] Add benzothiadiazole derivative ligand L (14.0 mg, 0.0192 mmol) and Eu(OTf)3 (7.19, 0.012 mmol) to a methanol:acetonitrile mixture (v:v=600 μL:600 μL), stir at 30°C for 5 h, pour the reaction mixture into ether (10 mL) to obtain a precipitate, and collect it by centrifugation to obtain an orange-red solid. Yield: 48%.

[0060] Example 7

[0061] Photoluminescence spectroscopy, quantum yield and fluorescence lifetime determination

[0062] The optical properties of the metal europium supramolecular cage complex were measured. The absorption spectrum showed that the optimal absorption wavelength was 503nm, and the photoluminescence spectrum emission peak was centered at 589nm. The emission wavelength was red-shifted compared to the optimal absorption wavelength, and the Stokes shift reached 86 nanometers. At the optimal excitation wavelength, the absolute PL quantum yield of the metal europium supramolecular cage complex was 3.21% calculated by integrating sphere. As shown in Figure (4a), the lifetime of the metal europium supramolecular cage complex was calculated to be 1.44ns based on the decay curve fitting double exponential function as shown in Figure (4b). Therefore, it can be seen that the metal europium supramolecular cage complex has low self-absorption, can achieve efficient PL emission, and has good fluorescence properties that can be used to mark cancer cells.

[0063] Example 8

[0064] Cytotoxicity assay

[0065] The U87 cancer cell line was inoculated on a 96-well plate with a density of 104 cells per well and adhered for 24 hours. After the U87 cancer cells remained stable on the 96-well plate, they were treated with Eu-Cage DMEM solution at concentrations of 0, 12.5, 25, 50 and 100ug / mL. After culturing in a cell culture incubator (temperature 37°C, CO2 5%) for 24 hours, the 96-well plate wells were washed with DMEM and a cell counting Kit-8 (cck8) was added. After incubation for 2 hours, the number of U87 cancer cells was obtained from each well using an ELISA reader, and the variability of the detected cells was calculated using a formula. The results show that the metal europium supramolecular cage complex only shows slight cytotoxicity even at a concentration of up to 100ug / mL, ensuring its safety for use as a biomarker, such as Figure 5 .

[0066] Example 9

[0067] Confocal detection

[0068] Under a confocal microscope, the U87 cancer cell line was treated with a metal europium supramolecular cage complex at a concentration of 50ug / mL, and the cytoplasm of the entire living cell could be clearly observed under the microscope ( Figure 6 a), while no fluorescence was observed in the U87 cells of the DMEM-treated control group ( Figure 6 b) It can be seen that the metal europium supramolecular cage complex can label the living U87 cancer cell line and achieve biological imaging.

[0069] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A twisted lanthanide metal europium supramolecular cage complex, characterized in that: The chemical structural formula of the twisted lanthanide metal europium supramolecular cage complex is C 126 H 114 F 18 N 24 O 30 The cation stereostructure of S9Eu2, a twisted lanthanide metal europium supramolecular cage complex, is shown in the following formula (1): Formula (1) Among them, m=0-6.

2. The method for preparing the twisted lanthanide metal europium supramolecular cage complex according to claim 1, characterized in that The following steps are involved: The benzothiadiazole derivative ligand L and the Eu(OTf)3 receptor are subjected to a coordinated synergistic self-assembly reaction in a mixed solvent to form a twisted lanthanide metal europium supramolecular cage complex. The benzothiadiazole derivative ligand L has the structural formula: 。 3. The method for preparing the twisted lanthanide metal europium supramolecular cage complex according to claim 2, characterized in that: The molar ratio of the benzothiadiazole derivative ligand L to Eu(OTf)3 is (1.4-1.6):1, and the mixed solvent is composed of acetonitrile and methanol in a volume ratio of (1-5):1; the coordination synergistic self-assembly reaction is reacted at 0-80°C for 4-8h.

4. The method for preparing the twisted lanthanide metal europium supramolecular cage complex according to claim 2, characterized in that: The preparation method of the benzothiadiazole derivative ligand L comprises the following steps: (1) dissolving 6-[(diethylamino)carbonyl]pyridine-2-carboxylic acid and oxalyl chloride in an organic solvent for reaction, and then concentrating by rotary evaporation after the reaction to obtain an activated acyl chloride intermediate; (2) Dissolving 4,4'-(benzo[1,2,5]thiadiazole-4,7-diyl)diphenylamine and the acid chloride intermediate obtained in step (1) in an organic solvent for reaction, cooling to room temperature after the reaction, extracting, eluting, solvent precipitating, filtering, and drying.

5. The method for preparing the twisted lanthanide metal europium supramolecular cage complex according to claim 4, characterized in that: In the step (1), the molar ratio of 6-[(diethylamino)carbonyl]pyridine-2-carboxylic acid to oxalyl chloride is 1:(1-1.5), and the organic solvent in the step (1) is a mixed solvent of DMF and DCM; the reaction temperature in the step (1) is 20-50° C., and the reaction time is 8-12 h.

6. The method for preparing the twisted lanthanide metal europium supramolecular cage complex according to claim 4, characterized in that: The molar ratio of 6-[(diethylamino)carbonyl]pyridine-2-carboxylic acid to 4,4'-(benzo[1,2,5]thiadiazole-4,7-diyl)diphenylamine is (2-3):1, and the organic solvent in step (2) is a mixed solvent of triethylamine and DCM; the reaction temperature in step (2) is 30-65°C and the reaction time is 4-9 h.

7. Use of the twisted lanthanide metal europium supramolecular cage complex according to claim 1 in the preparation of fluorescent imaging agents.

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