CN106008591B - A kind of synthesis of complex, sign and active anticancer assay method - Google Patents

Abstract

The invention relates to a coordination compound whose chemical formula is [VO ₂ (tpy)]ClO ₄ . The compound has specific anticancer activity and is expected to become a low-toxicity and high-efficiency anticancer drug. The coordination compound [VO ₂ (tpy) The synthesis of ]ClO ₄ includes [VO ₂ (tpy)]ClO ₄ single crystal synthesis and [VO ₂ (tpy)]ClO ₄ microcrystal or powder synthesis, which has unique anticancer activity, especially for liver cancer cells BEL-7402 Particularly strong lethality, its IC ₅₀ =0.4±0.2µM, while the traditional anticancer drug cisplatin has an IC ₅₀ value of 11.5±1.3µM on BEL-7402 cells under the same conditions, the anti-hepatoma activity of the complex It is nearly 30 times that of cisplatin, and the drug that can achieve such anticancer activity is still very rare, which is closely related to the unique structure of the complex and the properties of multiple oxidation states of vanadium element, because the IC ₅₀ value is close to the human body. The concentration of this kind of compound is 0.3μM, and this kind of compound is expected to be developed into a new type of low-toxicity and high-efficiency anticancer drug.

Description

Synthesis, characterization and anticancer activity determination method of a coordination compound

technical field

The invention belongs to the technical field of coordination compounds, and relates to a synthesis, characterization and anticancer activity determination method of coordination compounds.

Background technique

Vanadium is one of the important trace elements in organisms. The concentration of vanadium in the human body reaches 0.3 μM. Its compounds have been confirmed to be closely related to the pathogenesis of human diseases and the maintenance of normal physiological functions ^[1-3] . Since Kieler J reported the application of cyclopentadiene complex Cp ₂ VCl ₂ in the treatment of Ehrlich ascites neoplasia in 1986 ^[4] , the anticancer activity of vanadium-containing compounds has gradually attracted widespread attention. Studies have shown that the vanadium oxy[V ^IV O] ²⁺ polypyridine complex of tetravalent vanadium is expected to become an anticancer drug with high efficiency, broad spectrum and low toxicity ^[5,6] , among which the compound Metvan (bis-4,7 -Dimethyl-1,10-phenanthroline vanadyl sulfate) has entered the stage of clinical trials ^[7] . However, there are relatively few studies on polypyridine complexes of pentavalent vanadium, and currently synthesized molecules are electrically neutral molecules or compounds containing complex anions, and polypyridine complexes containing complex cations [V ^V O ₂ ] ⁺ are also None reported.

Based on the previous research, the research significance of this work is far-reaching. On the one hand, compared with [V ^IV O] ²⁺ complexes, [V ^V O ₂ ] ⁺ complexes have more advantages as anticancer drugs: (1) In solution and physiological conditions, the complexes containing [V ^V O ₂ ] ⁺ reagents are more stable, so that they have enough time to recognize and act on target cells in vivo; (2) [V ^V O ₂ ] ⁺ has more efficient anticancer activity, and the dosage will be reduced, and Subsequently, the physical damage to the patient and the risk of chemotherapy caused by toxic side effects are reduced. This is because the anti-cancer effect of vanadium complexes is through the generation of reactive oxygen species (ROS) in the body, which triggers a series of chain reactions and signal transmission, and eventually leads to apoptosis or death of cancer cells. The more, the more significant the effect. Since the ability of [V ^V O ₂ ] ⁺ to accept electrons is stronger than that of [V ^IV O] ²⁺ , it is similar to the role of high-valent chromium compounds (chromate) in generating active oxygen in organisms ^[8] , so it will Generate more ROS in the body. On the other hand, compared with the neutral or anionic reagents of pentavalent vanadium synthesized in the past, [V ^V O ₂ (tpy)] ⁺ (tpy = 2,2':6',2''- The advantage of terpyridine) is: the monovalent cationic complex is easier to pass through the cell membrane, even the nuclear membrane, so it is easy to be absorbed by cells and produce relatively rich biochemical reactions and excellent activity. This is because the cation-π interaction in organisms is very common and the most important, and the complex cation has a stronger interaction with the π electron system of the aromatic ring with four dipoles in the transmembrane protein, so that it can be better to achieve transmembrane transport ^[9] . From another point of view, tpy is a lipophilic ligand with strong chelating ability. The complex ion containing only one molecule of tpy is small in size and has both hydrophilic and lipophilic properties, making it easy to be used as a drug by organisms. absorption and transport. Therefore, this type of compound has great potential as a new type of anticancer drug, and the research results are expected to play an inestimable role in promoting cancer chemotherapy.

At present, compounds containing [V ^V O ₂ ] ⁺ are synthesized internationally, mainly hydrazone compounds as coordination compounds of ligands. Since such ligands are negative monovalent ions L ^-1 , the synthesized compound [V ^V O ₂ L] is a neutral molecule, and cannot obtain complexes with cations ^[10-13] . In 2000, Claudia Pifferi and others synthesized [VO(tpy)SO ₄ ], although it contains tpy, but due to the strong combination of sulfate and V, only the terpyridine complex of tetravalent vanadium was obtained ^[14] , and no Compounds similar to those synthesized by this technique were obtained. There are no research papers related to the synthesis and biological activity of terpyridine vanadium (V) complexes in my country.

Attachment: References

[1] D. Rehder, The potentiality of vanadium in medicinal applications, Future Med. Chem. , 2012, 4, 1823-1837.

[2] D. Rehder, The coordination chemistry of vanadium as related to its biological functions, Coord. Chem. Rev. , 1999, 182, 297-322.

[3] MT Pepato, NM Khalil, MP Giocondo, IL Brunetti, Vanadium and its complexes: the renewed interest in its biochemistry, Lat. Amer. J. Pharm. , 2008, 27, 468-76.

[4] P. Köpf-Maier, H. Köpf, Metallocene complexes: organometallic antititumor agents. Drugs Future, 1986, 11, 297-319.

[5] A. Bishayee, A. Waghray, M.A. Patel, M. Chatterjee, Vanadium in the detection, prevention and treatment of cancer: The in vivo evidence, Cancer Letters, 2010, 294, 1-12.

[6] MW Makinen, M. Salehitazangi, The structural basis of action of vanadyl (VO ²⁺ ) chelates in cells, Coord. Chem. Rev. , 2014, 279, 1-22.

[7] OJ D'Cruz, FM Uckun, Metvan: a novel oxovanadium (IV) complexwith broad spectrum anticancer activity, Expert Investig. Drugs , 2002, 11,1829-1836.

[8] K. Jomova, M. Valko, Advances in metal-induced oxidative stress and human disease, Toxicology , 2011, 283, 65-87.

[9] DA Dougherty, Cation-π interactions in chemistry and biology: a new view of benzene, Phe, Tyr, and Trp, Science , 1996, 271, 163-167.

[10] MR Maurya, AA Khan, A. Azam, S. Ranjan, N. Mondal, A. Kumar, F. Avecilla, JC Pessoa, Vanadium complexes having [V ^IV O] ²⁺ and [V ^V O ₂ ] ⁺ coreswith binucleating dibasic tetradentate ligands: synthesis, characterization,catalytic and antiamoebic activities, Dalton Trans. , 2010, 39, 1345-1360.

[11] PIS Maia, FR Pavan, CQF Leite, SS Lemos, GF Sousa, AA Batista, OR Nascimento, J. Ellena, EE Castellano, E. Niquet, VMDeflon, Vanadium complexes with thiosemicarbazones: synthesis, characterization, crystal structures and anti -mycobacterium tuberculosis activity, Polyhedron , 2009, 28, 298-406.

[12] MR Maurya, S. Agarwal, M. Abid, A. Azam, C. Bader, M. Ebel, D. Rehder, synthesis, characterization, reactivity and in vitro antiamoebicactivity of hydrazone based oxovanadium(IV), oxovanadium(V ) and µ-bis(oxo)bis{oxovanadium(V)} complexes, Dalton Trans. , 2006, 937-947.

[13] VM Deflon, DM Oliveira, GF Sousa, AA Batista, LRDinelli, EE Castellano, Oxovanadium(IV,V) complexes with 2-acetylpyridine-2-furanoylhydrazone (Hapf) as ligand. X-ray crystal structures of [VO ₂ (apf)] and [V ₂ O ₂ (µ-O) ₂ (apf) ₂ ], Z. Anorg. Allg. Chem. , 2002, 628, 1140-1144.

[14] C. Pifferi, MP Picchi, R. Cini, Vanadium complexes as models for vanadium species of marine organisms. Synthesis, X-ray structure of oxo(O,O-sulfate)(2,2':6',2''-terpyridine) vanadium (IV) hydrate, and densityfunctional geometry optimization analysis of vanadyl complexes, Polyhedron ,2000, 19, 69-76.

Contents of the invention

In order to overcome the above-mentioned technical shortcomings, the present invention provides a method for the synthesis, characterization and anticancer activity determination of a coordination compound, which can avoid using anion ligands and anions with strong coordination ability as counter ions, and by controlling the reaction conditions, make The tetravalent vanadium complex is fully oxidized and has unique anticancer activity, especially for liver cancer cells BEL-7402.

The technical method adopted by the present invention to solve the technical problem is: a coordination compound whose chemical formula is [VO ₂ (tpy)]ClO ₄ , the coordination compound has special anticancer activity and is expected to become a low-toxicity and high-efficiency anticancer Medicine, the synthesis of the coordination compound [VO ₂ (tpy)]ClO ₄ includes [VO ₂ (tpy)]ClO ₄ single crystal synthesis and [VO ₂ (tpy)]ClO ₄ crystallite or powder synthesis, wherein [VO ₂ (tpy)] ClO ₄ single crystal synthesis steps are:

Firstly, the VOSO ₄ aqueous solution is mixed with the Ba(ClO ₄ ) ₂ solution, and the [VO(H ₂ O) ₅ ](ClO ₄ ) ₂ aqueous solution is obtained after stirring and filtering, and the [VO(H ₂ O) ₅ ](ClO ₄ ) ₂ aqueous solution was added to tpy ethanol solution to obtain [VO(H ₂ O) ₂ (tpy)](ClO ₄ ) ₂ ethanol and water mixed solution, and then the ethanol was removed by rotary evaporation to obtain [VO(H ₂ O) ₂ (tpy)] (ClO ₄ ) ₂ aqueous solution, the [VO(H ₂ O) ₂ (tpy)](ClO ₄ ) ₂ aqueous solution is oxidized by air under stirring to form [VO ₂ (tpy)]ClO ₄ aqueous solution, and finally after a slow [VO ₂ (tpy)]ClO ₄ single crystal obtained by evaporation;

In the synthesis process of [VO ₂ (tpy)]ClO ₄ crystallite or powder, the Ba(ClO ₄ ) ₂ solution is replaced by BaCl ₂ solution, and the [VO(H ₂ O) ₅ ]Cl ₂ aqueous solution is generated by stirring and filtering. Then add tpy ethanol solution to [VO(H ₂ O) ₅ ]Cl ₂ aqueous solution to obtain [VO(H ₂ O) ₂ (tpy)]Cl ₂ ethanol and water mixed solution, then remove ethanol through rotary evaporation to obtain [VO(H ₂ O) ₂ (tpy)]Cl ₂ aqueous solution, the [VO(H ₂ O) ₂ (tpy)]Cl ₂ aqueous solution is then oxidized by air under the stirring process to form [VO ₂ (tpy)]Cl aqueous solution, to [ Add saturated NaClO ₄ to VO ₂ (tpy)]Cl aqueous solution and keep stirring to prepare [VO ₂ (tpy)]ClO ₄ crystallite or powder.

The characterization of coordination compounds includes [VO ₂ (tpy)]ClO ₄ single crystal characterization and [VO ₂ (tpy)]ClO ₄ microcrystal or powder characterization, and the characterization of [VO ₂ (tpy)]ClO ₄ single crystal is done by X-ray single crystal diffraction to obtain molecular structure bond parameters and crystallographic data and generate powder diffraction simulation diagram, [VO ₂ (tpy)]ClO ₄ microcrystal or powder characterization with X-ray powder diffraction and simulation with single crystal powder diffraction Compare the graphs, determine the complex ion formula weight by electrospray mass spectrometry and determine the content of C, H, and N by elemental analysis experiments, so as to determine the composition, structure and purity of the powder sample, and determine the functional group structure by infrared spectroscopy.

The method for measuring the anticancer activity of the coordination compound, the determination steps are: firstly carry out the coordination compound [VO ₂ (tpy)]ClO ₄ cancer cell toxicity test, and measure the IC ₅₀ to determine the coordination compound [VO ₂ (tpy)] ClO ₄ is the most sensitive cell. Determine whether the complex triggers the production of active oxygen by measuring the active oxygen, determine the apoptosis rate by the apoptosis test, determine the damage degree of the line membrane by measuring the mitochondrial membrane potential, and determine the apoptosis by the comet test. Finally, the apoptosis mechanism was obtained through the above-mentioned determination.

The beneficial effects of the present invention are: firstly, the neutral terpyridine ligand is used to synthesize the solid phase and the solution phase under the conditions of near-physiological conditions (pH is about 7) by using weakly coordinated counter anions and fully oxidized conditions. stable pentavalent vanadium-terpyridine-containing cation complexes and their single crystals, which have not been reported domestically or internationally; secondly, the synthesized complexes have unique anticancer activity, especially for liver cancer cells BEL -7402 has a particularly strong lethality, its IC ₅₀ = 0.4 ± 0.2 µM, while the traditional anticancer drug cisplatin has an IC ₅₀ value of 11.5 ± 1.3 µM for BEL-7402 cells under the same conditions, so the present invention synthesized The anti-liver cancer activity of the complex is nearly 30 times that of cisplatin, and the drug that can achieve such anti-cancer activity is still very rare, which is closely related to the unique structure of the complex and the properties of multiple oxidation states of vanadium element. The IC ₅₀ value is close to the concentration allowed by the human body of 0.3 μM, and this type of compound is expected to be developed as a new type of low-toxicity and high-efficiency anticancer drug.

Description of drawings

Fig. 1 is the synthesizing complex [VO ₂ (tpy)] ClO ₄ single crystal block diagram schematic diagram;

Figure 2 is a schematic diagram of the block structure of the synthesized complex [VO ₂ (tpy)]ClO ₄ (microcrystalline or powder);

Figure 3 is a schematic diagram of the characterization of the complex [VO ₂ (tpy)]ClO ₄ single crystal and [VO ₂ (tpy)]ClO ₄ microcrystal or powder;

Figure ₄ is a schematic diagram of the assay scheme for the anticancer activity of the complex [VO2 ₍ tpy)]ClO4;

Fig. 5 is the unit structure diagram of the X-ray diffraction measurement of single crystal complex;

Figure 6 is the mitochondrial membrane potential measurement experiment of cell BEL-7402 (a) blank group, (b) (c) respectively with 0.5 µM and 1.0 µM control pictures;

Fig. 7 is the crystal data and structure refinement parameter map of the complex [VO ₂ (tpy)]ClO ₄ ;

Figure 8 is a schematic diagram of bond lengths and bond angles of coordination cations;

Fig. 9 is a powder diffraction spectrum;

Figure 10 is a schematic diagram of the IC value of the complex on selected cell lines _;

Figure 11 is the EB staining comet experiment of cell BEL-7402 (a) EB staining control group, (b) adding 0.5 µM complexes and incubating for 24 hours, the cells in b showed obvious comet tail control picture.

detailed description

The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

Example 1:

Preparation of Complex [VO ₂ (tpy)]ClO ₄ Single Crystal and Determination of Single Crystal Structure

Referring to Figure 1, the preparation process is as follows: firstly, dissolve 0.6 mmol VOSO ₄ ∙ 3H ₂ O in 25 ml of water to obtain solution A, weigh 0.6 mmol Ba(ClO ₄ ) ₂ and dissolve in 25 ml of water to obtain solution B, and dissolve the solution under stirring B was slowly added dropwise to solution A, resulting in white precipitate of BaSO ₄ , which was filtered to obtain blue solution C. Add the ethanol solution (15 ml) containing 0.5 mmol of tpy dropwise to solution C under stirring, and stir at room temperature for 30 minutes to obtain a green solution, which was evaporated to about 25 ml by rotary evaporation, and stirred at room temperature for 5 days until the solution became Turn into orange yellow, filter, and the filtrate evaporates slowly and freely at room temperature, and orange blocky crystal [VO ₂ (tpy)]ClO ₄ is obtained in about 45 days.

See Figure 3, single crystal structure determination: stick the complex single crystal on glass wool, insert the copper tube, place it on the sample stage of Bruker SMART 1000 CCD four-circle diffractometer, and use graphite monochromator to spectroscopic Mo -Kα (λ = 0.71073Å) rays are used as the light source, and the measurement is carried out at 296K. Diffraction intensity data were collected in the range of 1.78 < θ < 25.00° using ω-scan technology, and 4009 diffraction points were collected. The crystal structure was solved by the direct method of SHELXTL-97, and the anisotropy correction was carried out ^on F2 using the full matrix least squares method. Finally, the refinement of 2745 observation points ( F ≥ 4.0 σ( F )) and 235 variables was converged. After refining with anisotropic displacement parameters for all non-hydrogen atoms, an isotropic refinement is performed on the coordinates of the hydrogen atoms on the ligand and their displacement parameters. In refinement, the hydrogen atom will be relatively fixed on its parent atom, and move with the movement of the parent atom, that is, ride on the parent atom. The distance between the hydrogen atom and the carbon atom uses the preset value of the program C—H = 0.96 A. All hydrogen atoms are corrected for isotropic temperature factors. The relevant crystallographic data and structure refinement parameters are shown in Fig. 7. Partial bond lengths and bond angles of complex cations are listed in Figure 8, see Figure 5.

Example 2:

Preparation and Characterization of Complex [VO ₂ (tpy)]ClO ₄ Microcrystals

See Figure 2. Preparation: Dissolve 0.6 mmol VOSO ₄ ∙ 3H ₂ O in 25 ml water to obtain solution A, weigh 0.6 mmol BaCl ₂ and dissolve it in 25 ml water to obtain solution B, slowly add solution B dropwise to solution A while stirring , resulting in a white precipitate of BaSO ₄ , which was filtered to obtain a blue solution C. Add an ethanol solution (15 ml) containing 0.5 mmol of tpy dropwise to solution C under stirring, and stir at room temperature for 30 minutes to obtain a green solution, remove ethanol by rotary evaporation, dilute with water to a total volume of 80 ml, and store at room temperature Stir for 7 days until the solution turns orange-yellow, filter, and add saturated _NaClO4 solution dropwise to the filtrate under stirring conditions until the precipitation is complete, continue stirring at room temperature for 4 hours to obtain orange-yellow microcrystals, filter with suction, and wash with water for 3 times (5 ml each time), washed twice with ethanol (5 ml each time), and placed in a vacuum desiccator for 3 days to obtain the product with a yield of 42%.

See Figure 3, Characterization:

(1) Powder diffraction: The data and experimental spectra were measured and acquired on a Bruker D8 Advance X-ray diffractometer, in which Cu-Kα target, λ=0.154 nm, graphite monochromator diffracted beam monochromatic ratio, high pressure 50 KV, Tube flow 20 mA. The simulated diffraction pattern is calculated based on the X-ray single crystal data using the SHELXTL-XPOW program. Through the experimental spectrum (Experimental) and the simulated spectrum (Simulated), see Figure 9, the position and relative intensity of the peaks are highly consistent, which shows that the obtained microcrystal has the same structure as a single crystal;

(2) Infrared spectrum: Measured by Bruker VECTOR22 FT-IR infrared spectrometer and KBr tablet technology, each peak is assigned (cm ^-1 ): 1600, 1480 (C=C _tpy , C=N _tpy ), 1080, 953 (V=O);

(3) Electrospray mass spectrometry: Electrospray mass spectrometry (ES-MS) was recorded with an LCQ system (Finnigan MAT, USA), and DMSO was selected as the mobile phase. [DMSO, m/z]: 316.6 ([VO ₂ (tpy)] ⁺ ), the experimental value is in good agreement with the theoretical value.

(4) Elemental analysis: Elemental analysis (C, H, N) was determined by Elementar Vario EL elemental analyzer. Test results (%): C 43.37, H 2.81, N 10.05. Calculated _{( C15H11ClN3O6V} , %): C _43.34 , H 2.67, N _10.11 . The experimental value is basically consistent with the calculated value.

See Figure 4, research on the antitumor activity of the complex [VO ₂ (tpy)]ClO ₄

(1) In vitro cytotoxicity test: Collect cells in the logarithmic growth phase, adjust the concentration of the cell suspension to 5 × 10 ⁴ ~ 1.5 × 10 ⁵ cells/mL, and take 100 μL per well of a 96-well plate. Place the inoculated 96-well plate in an incubator (5% CO ₂ , 37 ^o C), and discard the culture medium when the monolayer of cells at the bottom of the well grows to about 80%, add 90 μL of 1640 culture medium and set Concentration gradient drug 10 μL, continue to incubate for 48 h, discard the culture medium, add 90 μL of 1640 culture medium and 10 μL of MTT (5 mg∙m L ^-1 ), continue to incubate for 4 h in the incubator. Take it out of the incubator, discard the culture medium, add 100 μL of DMSO to each well, shake for 10 min, measure the absorbance value at 490 nm with a microplate reader, and calculate the IC ₅₀ value as shown in Figure 10. The results show that the The synthesized compound has better activity than cisplatin, an anticancer drug widely used at present, especially for liver cancer cell BEL-7402. The anticancer activity of this compound is nearly 30 times that of cisplatin. The following experiments (2) to (5) all used BEL-7402 cells.

(2) Apoptosis detection assay by AO/EB staining: Take a 12-well plate and inoculate 1×10 ⁵ ~ 2×10 ⁵ cells per well. When the monolayer cells grow to about 90%, add the corresponding concentration of drugs. After incubation in the incubator for 24 h, the culture medium was discarded and washed twice with PBS. AO/EB staining solution (100 μg∙m L ^-1 ) was added dropwise to cover the glass slides, stained at 37 ^o C for 30 min, the staining solution was discarded, washed three times with PBS, observed under a fluorescence microscope, and photographed and recorded. The results showed that as shown in the figure below, only 0.5 μM complex acted on BEL-7402 cells for 24 hours, and then the cells were double-stained with AO/EB, the cells were stained green by AO, and the chromatin condensation staining of apoptotic cells was deepened; but EB could not penetrate Through the cell membrane (not appearing in orange), this is the characteristic of early apoptosis of cells. It shows that this complex can induce the apoptosis of liver cancer cells BEL-7402 with high efficiency.

(3) Comet experiment: Single-cell gel electrophoresis experiment. BEL-7402 cells were incubated at 37 ^o C for 24 h under the action of 0.5 μM complex, and the cells were collected by trypsinization. The gel plate was prepared by the three-layer method, the electrolyte was 300 mM NaOH, 1.2 mM EDTA, and the electrophoresis was performed at 25 V, 300 mA. After electrophoresis, the gel plate was washed to neutral with 400 mM Tris, HCl, pH 7.5 solution, stained with EB (20 μg/m L) in the dark for 20 minutes, and photographed with a fluorescent microscope. The results are shown in the figure below. Compared with the control group, the cells treated with complexes all appeared comet tails, which indicated that the complexes entered the nucleus, interacted with DNA, and broke DNA into fragments of different lengths, which is an important initiation of apoptosis. Signal.

(4) Mitochondrial membrane potential detection: Collect logarithmic phase cells and adjust the concentration of cell suspension. Take a 12-well plate and inoculate 1 × 10 ⁵ ~ 2 × 10 ⁵ per well. When the monolayer cells grow to about 80%, add the corresponding concentration of drugs. After incubating in the incubator for 24 h, add 1 μL of CCCP to the positive control group and continue to incubate for 20 min, discard the culture medium in the 12-well plate, wash with cold PBS three times, add 0.5 mL cell culture medium and 0.5 mL Mix the JC-1 staining working solution thoroughly. The cells were incubated in an incubator at 37 ^o C for 20 min, and at the same time, 3 mL of JC-1 staining buffer was added to 12 mL of double distilled water to dilute 5 times and placed on ice. After incubation, the staining solution was discarded, washed twice with JC-1 staining buffer, 1 mL of cell culture medium was added, observed under a fluorescent microscope, and photographed and recorded. The detection of mitochondrial membrane potential uses JC-1 as a fluorescent probe. When the mitochondrial membrane potential in the cell is high, JC-1 exists in the form of a polymer in the mitochondrial matrix and emits red fluorescence. When the mitochondrial membrane potential in the cell is low, JC-1 It emits green fluorescence in the form of a monomer, so the change of mitochondrial membrane potential can be detected by detecting the fluorescence of JC-1. In the early stage of apoptosis, the permeability of the inner mitochondrial membrane of cells will increase, and the mitochondrial membrane potential will decrease. Once the mitochondrial membrane potential collapses, the cells can only go to apoptosis. After the complex synthesized by this method acted on BEL-7402 cells, the cells were stained with JC-1, and the results were recorded under a fluorescence microscope as shown in the figure below. It can be seen that the mitochondrial membrane potential detection experiment results show that the complex can reduce the mitochondrial membrane potential of BEL-7402 cells. See Figure 6:

The picture shows the mitochondrial membrane potential measurement experiment of cell BEL-7402 analyzed by JC-1 staining method (a) blank group, (b) (c) were incubated with 0.5 µM and 1.0 µM complexes for 24 hours, group b, c All of them became green fluorescence, which indicated that the action of the complex decreased the mitochondrial membrane potential, which was the key to the chain reaction of mitochondria releasing cytochrome and triggering apoptosis.

(4) Detection of reactive oxygen species: Collect logarithmic phase cells and adjust the concentration of cell suspension. Take a 6-well plate and inoculate ⁴ × 105 cells per well. When the monolayer cells grow to about 90%, add the drug at the corresponding concentration, incubate in the incubator for 24 h, discard the culture medium in the 6-well plate, wash twice with PBS, digest with 1 mL trypsin, and collect the cells Centrifuge in a 2 mL EP tube (3000 rpm, 5 min), discard the supernatant, add 1 mL of serum-free culture medium to resuspend the cells, centrifuge (3000 rpm, 5 min), discard the supernatant , add 1:1000 diluted DCFH-DA, place in a cell culture incubator and incubate at 37 ^o C for 20 min, invert and mix once every 3-5 min, then wash twice with serum-free culture medium, and centrifuge ( 3000 rpm, 5 min), discard the supernatant and add 300 μL of serum-free medium to mix well and transfer to the flow tube for detection on the machine. After the complex [VO ₂ (tpy)]ClO ₄ was applied to BEL-7402 cells, the cells were stained with dichlorodihydrofluorescein-acetoacetate (DCFH-DA), and the fluorescence intensity of DCF was detected by flow cytometry, as shown in the figure below. DCFH-DA itself has no fluorescence and can enter the cell through the cell membrane. After entering the cell, it is hydrolyzed by intracellular esterase into DCFH, but DCFH cannot pass through the cell membrane. DCFH is further oxidized by intracellular reactive oxygen species into green fluorescent DCF. Therefore, the fluorescence intensity of DCF can be used to reflect the level of reactive oxygen species in cells. The experimental results show that the complex can increase the level of reactive oxygen species in BEL-7402 cells. See Figure 7:

The picture shows the determination of reactive oxygen species in BEL-7402 cells using H ₂ DCF-DA as a fluorescent probe (a) blank group, (b) Rosup positive control group, (c) incubated with 0.5 µM complex for 24 hours . Group c, like b, emits green fluorescence, which indicates that the complexes entering the cells can induce the generation of reactive oxygen species, and the generated reactive oxygen species oxidize DCFH to generate fluorescent DCF.

Based on the above anti-cancer activity experiments, it is preliminarily speculated that the anti-cancer mechanism is as follows: the complex enters the nucleus, generates active oxygen, oxidizes and breaks DNA, generates apoptotic signals, and translocates to mitochondria, causing the mitochondrial membrane potential to collapse, and further releases apoptotic signals , such as cytochrome C, and trigger a series of cascade reactions (such as caspase cascade reaction), leading to the apoptosis of cancer cells. The detailed and in-depth anti-cancer mechanism needs further systematic research.

Conclusion: The repeatability of this method is good, and the product has high purity. The synthesized [VO ₂ (tpy)]ClO ₄ has extremely high anticancer activity, and is expected to be developed as a new type of chemotherapy drug for cancer treatment with high efficiency, broad spectrum and low toxicity.

Claims (3)

1. a kind of vanadium terpyridyl complex, its chemical formula is [VO₂(tpy)]ClO₄, tpy is 2,2':6', 2''- tri- Bipyridyl, the complex [VO₂(tpy)]ClO₄Including [VO₂(tpy)]ClO₄Monocrystalline and [VO₂(tpy)]ClO₄Crystallite or Powder.

2. a kind of preparation method of vanadium terpyridyl complex as claimed in claim 1, it is characterized in that being, chooses first VOSO₄The aqueous solution and Ba (ClO₄)₂Solution is mixed, and [VO (H are obtained after agitated filtering₂O)₅](ClO₄)₂The aqueous solution the, to [VO (H₂O)₅](ClO₄)₂The aqueous solution adds tpy ethanol solutions and obtains [VO (H₂O)₂(tpy)](ClO₄)₂Ethanol, water mixed solution, then Ethanol is removed by rotary evaporation and obtains [VO (H₂O)₂(tpy)](ClO₄)₂The aqueous solution, should [VO (H₂O)₂(tpy)](ClO₄)₂Water Then oxidation by air forms [VO to solution under the process of stirring₂(tpy)]ClO₄The aqueous solution, eventually passes slow evaporation and is made [VO₂(tpy)]ClO₄Monocrystalline；

In [VO₂(tpy)]ClO₄It is then by Ba (ClO in crystallite or powder building-up process₄)₂Solution is substituted for BaCl₂Solution, through stirring Mix filtering generation [VO (H₂O)₅]Cl₂The aqueous solution, then to [VO (H₂O)₅]Cl₂The aqueous solution adds tpy ethanol solutions and obtains [VO (H₂O)₂(tpy)]Cl₂Ethanol, water mixed solution, then remove ethanol acquisition [VO (H by rotary evaporation₂O)₂(tpy)]Cl₂It is water-soluble Liquid, should [VO (H₂O)₂(tpy)]Cl₂Oxidation by air forms [VO to the aqueous solution under the process of stirring again₂(tpy)] the Cl aqueous solution, To [VO₂(tpy) saturation NaClO] is added in the Cl aqueous solution₄And be stirred continuously and finally prepare [VO₂(tpy)]ClO₄Crystallite or powder End.

3. a kind of application of vanadium terpyridyl complex as claimed in claim 1 in cancer therapy drug is prepared.