CN103936772A - Preparation method of binuclear copper complex with anti-tumor activity - Google Patents

Abstract

The invention discloses a method for preparing a binuclear copper complex with antitumor activity. The binuclear copper complex is prepared by synthesizing ligand H ₄ L with amino acid branched chains, and then using H ₄ L. Compared with the prior art, the binuclear copper complex prepared by the present invention can cause tumor cells to fall off from the adherent wall, accompanied by shrinkage and separation phenomena, reducing the number of tumor cells. The present invention is effective against human liver cancer cells (HepG2), Human cervical cancer cells (HeLa), human prostate cancer (PC3) cells have a good inhibitory effect.

Description

A kind of preparation method of binuclear copper complex with antitumor activity

technical field

The invention relates to the technical field of chemical medicines, in particular to a preparation method of a binuclear copper complex with antitumor activity.

Background technique

Cancer is currently the second leading cause of death, accounting for almost a quarter of all deaths. And clinical practice shows that among cancer patients, nearly half of them eventually die of cancer. Therefore, the development of new and efficient anticancer drugs has become the basic direction and ultimate research goal of the development of medicinal chemistry. As we all know, copper is one of the most basic elements in the normal physiological metabolism of the human body. In biological systems, most copper elements exist in the form of copper complexes; amino acids are the basic structural units of proteins, which can recognize specific base pairs in DNA sequences. Therefore, the copper complexes of amino acids are generally used as templates to study the action behavior of copper-containing biological enzymes. The complexes composed of some amino acid branched drug molecules and divalent copper have good anti-tumor activity and the function of artificial nucleases. . At the same time, studies have found that 1,10-phenanthroline (phen) also has good antitumor activity.

Therefore, it is urgent to develop an anticancer drug with antitumor activity.

Contents of the invention

The purpose of the present invention is to provide a preparation method of the binuclear copper complex with antitumor activity.

To achieve the above object, the present invention is implemented according to the following technical solutions:

A method for preparing a binuclear copper complex with antitumor activity, comprising the steps of:

1) Dissolve 1.33g of 10mmol of L-aspartic acid and 0.8g of 20mmol of NaOH in 30mL of water, and stir for 30min;

2) Add 10 mL of methanol solution containing 0.67 g of 5 mmol terephthalaldehyde, and stir at 50° C. for 5 hours, and the entire reaction system is pale yellow and clear;

3) Under cooling in an ice-water bath, slowly add 0.46 g of 12 mmol NaBH ₄ and a few drops of NaOH solution in batches, the color of the reaction solution slowly changes from yellow to colorless, and the ice-water bath is removed;

4) Stir at room temperature for 4 hours until the reaction solution has no obvious bubbles, slowly add acetic acid solution dropwise, adjust the pH to 5-6, continue stirring for a while, and then remove all solvents under reduced pressure to obtain 1.95g of white solid, which is Ligand H ₄ L with amino acid branching: C ₁₆ H ₂₀ N ₂ O ₈ : C, 52.17; H, 5.47; N, 7.61.Found: C, 52.62; H, 5.58; N, 7.81. ¹ H NMR ( D ₂ O) δ.80(q, 4H), 3.82(q, 2H), 4.41(q, 4H), 7.58(d, 4H), its chemical structure is:

5) 0.37g of 1mmol of Cu(ClO ₄ ) ₂ 6H ₂ O was added to 8mL of methanol solution, and 8mL of methanol solution containing 0.18g of 1mmol of phenanthroline was added thereto, and the reaction solution was slightly green. mix;

6) Add 20 mL of an aqueous solution containing 0.18 g of ligand H ₄ L with amino acid branch chains and 0.021 g of 0.5 mmol LiOH·H ₂ O dropwise through filter paper. After the addition is complete, the reaction solution is blue and clear. After stirring for 1.5h, a blue precipitate was formed;

7) Filter the reaction solution with blue precipitates, and keep the mother liquor to volatilize naturally. After one week, blue blocky crystals will appear, which is the binuclear copper complex C ₄₀ H ₃₂ Cu ₂ N ₆ O ₈ : C, 56.40; H , 3.79; N, 9.87. Found: C, 56.86; H, 4.01; N, 9.65.

Compared with the prior art, the binuclear copper complex prepared by the present invention can cause tumor cells to fall off from the adherent wall, accompanied by shrinkage and separation phenomena, reducing the number of tumor cells. The present invention is effective against human liver cancer cells (HepG2), Human cervical cancer cells (HeLa), human prostate cancer (PC3) cells have a good inhibitory effect.

Description of drawings

Fig. 1 is the crystal structure diagram of the binuclear copper complex that the present invention makes;

Fig. 2 is a schematic diagram of cell morphology changes before and after the binuclear copper complex interacts with cancer cells.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. The exemplary embodiments and descriptions of the present invention are used to explain the present invention, but not as a limitation to the present invention.

A kind of preparation method of the binuclear copper complex with antitumor activity of the present invention, comprises the following steps:

1) Dissolve 1.33g of 10mmol of L-aspartic acid and 0.8g of 20mmol of NaOH in 30mL of water, and stir for 30min;

2) Add 10 mL of methanol solution containing 0.67 g of 5 mmol terephthalaldehyde, and stir at 50° C. for 5 hours, and the entire reaction system is pale yellow and clear;

3) Under cooling in an ice-water bath, slowly add 0.46 g of 12 mmol NaBH ₄ and a few drops of NaOH solution in batches, the color of the reaction solution slowly changes from yellow to colorless, and the ice-water bath is removed;

4) Stir at room temperature for 4 hours until the reaction solution has no obvious bubbles, slowly add acetic acid solution dropwise, adjust the pH to 5-6, continue stirring for a while, and then remove all solvents under reduced pressure to obtain 1.95g of white solid, which is Ligand H ₄ L with amino acid branching: C ₁₆ H ₂₀ N ₂ O ₈ : C, 52.17; H, 5.47; N, 7.61.Found: C, 52.62; H, 5.58; N, 7.81. ¹ H NMR ( D ₂ O)δ.80(q, 4H), 3.82(q, 2H), 4.41(q, 4H), 7.58(d, 4H), its chemical structure is:

5) 0.37g of 1mmol of Cu(ClO ₄ ) ₂ 6H ₂ O was added to 8mL of methanol solution, and 8mL of methanol solution containing 0.18g of 1mmol of phenanthroline was added thereto, and the reaction solution was slightly green. mix;

6) Add 20 mL of an aqueous solution containing 0.18 g of ligand H ₄ L with amino acid branch chains and 0.021 g of 0.5 mmol LiOH·H ₂ O dropwise through filter paper. After the addition is complete, the reaction solution is blue and clear. After stirring for 1.5h, a blue precipitate was formed;

7) Filter the reaction solution with blue precipitates, and keep the mother liquor to volatilize naturally. After one week, blue blocky crystals will appear, which is the binuclear copper complex C ₄₀ H ₃₂ Cu ₂ N ₆ O ₈ : C, 56.40; H , 3.79; N, 9.87.Found: C, 56.86; H, 4.01; N, 9.65, its crystal structure is shown in Figure 1.

The antitumor activity of the binuclear copper complex prepared by the present invention is further tested:

cell culture

Take out the cryopreservation tube from the liquid nitrogen tank, put it into a 37-40°C water bath quickly, and shake the cryopreservation tube constantly to make the cryopreservation liquid melt quickly;

Prepare two 10×2cm culture dishes, inject 10ml of DMEM culture solution, RPMI1640 culture solution or Ham's F12 culture solution, suck out the cryopreservation solution, and pour into the culture dish;

After mixing the cell suspension with the cell culture medium, culture in a 37°C, 5% CO ₂ incubator, and replace the culture medium after 12 hours. Human liver cancer cells (HepG2), human cervical cancer cells (HeLa), and human prostate cancer ( _PC3 ) cells used in the test were placed in 10% inactivated calf serum, Subculture in 1% 10000U/mL penicillin and 10000mg/mL streptomycin DMEM medium.

cell count

Prepare the counting plate, add the above cell suspension to the counting plate, place the counting plate flat on the table, gently inject the stained cell suspension from the edge of the counting plate to fill the space between the counting plate and the cover glass, The space between the coverslip and the counting plate holds approximately 0.1 μL of cell suspension. Calculate according to the following formula with the aid of a microscope: cell suspension concentration (cells/mL) = total number of cells in 2 large grids × 10 ⁴ cells/mL.

Anti-tumor activity test:

Acid phosphatase method (AP method) to detect cell viability:

Inoculate HepG2 and other cells in a good growth state at a density of 0.8-1.0×10 ⁵ cells/ml, inoculate 100 μL per well in a 96-well culture plate, and culture at 37°C and 5% CO ₂ saturated humidity;

After culturing for 24 hours, the cells were attached to the wall, and the compounds to be tested were added to the cells, so that the concentrations of the compounds were: 10, 20, 40, 80, and 160 μmol/L, and each concentration was acted on for 24 hours. Each group has a blank control, and each well has three replicate wells;

After the effect, the supernatant culture solution was discarded, and each well was washed twice with 200 μL pH7.2 phosphate buffer solution, and 100 μL 0.1M sodium acetate buffer solution (pH5.0), 0.1% Triton X-100 (poly Ethylene glycol octylphenyl ether) and 5mM p-nitrophenyl phosphate (p-NPP) were incubated in a 37°C incubator for 2 hours, and 10 μL of 1M NaOH was added at the end of the experiment;

After culturing, use VIVTOR ³ 1420-050 microplate reader to detect the absorbance value (A) of each well at a wavelength of 405nm, and use the hydrolyzed solution of p-NPP substrate in the well without cells as a blank, and calculate according to the following formula Survival rate IC ₅₀ value, survival rate (%)=(A _drug-blank /A _{control-blank} ×100)×100%.

We tested the inhibitory rate (semi-lethal dose) IC ₅₀ of the binuclear copper complex on human liver cancer cells (HepG2), human cervical cancer cells (HeLa), and human prostate cancer (PC3) cells. Table 1 shows the semi-lethal dose data of these compounds on tumor cells.

Table 1 Anticancer activity data of binuclear copper complexes and their ligands and inorganic salts on three types of cells

All the ligands, complexes and corresponding metal salts were incubated with the above three kinds of cells for 24h. Ligands and metal salts have no inhibitory effect on the growth of tumor cells. When the ligand and copper participate in the coordination, the IC ₅₀ of the complex decreases significantly, and the participation of the metal in the coordination directly leads to the increase of the anticancer activity of the compound.

Through the observation of cell morphology under a microscope, referring to Figure 2, it can be seen that the morphology of these cells has changed significantly after being treated with drugs. Before the tumor cells interact with the drug, the cells grow well, and the adherent cells can be clearly seen; after the treatment, the number of cells in the culture medium decreases significantly, and the tumor cells fall off from the adherence, accompanied by shrinkage and separation. It shows that the binuclear copper complex prepared by the present invention has a good inhibitory effect on the above three cancer cells.

The technical solution of the present invention is not limited to the limitations of the above-mentioned specific embodiments, and any technical deformation made according to the technical solution of the present invention falls within the protection scope of the present invention.

Claims (1)

1. a preparation method with the double-core copper complex of anti-tumor activity, is characterized in that, comprises the steps:

1) get the L-Aspartic acid of 10mmol of 1.33g and the NaOH of the 20mmol of 0.8g is dissolved in the water of 30mL, stir 30min;

2) add the methanol solution containing the 10mL of the terephthalaldehyde of the 5mmol of 0.67g again, stir 5 hours at 50 ℃, whole reaction system is faint yellow clarification;

3), under ice-water bath is cooling, slowly add the NaBH of the 12mmol of 0.46g in batches ₄with several NaOH solution, the color of reaction solution is slowly become colourless by yellow, remove ice-water bath;

4) stirring at normal temperature does not have, after obvious Bubble formation, slowly to drip acetum to reaction solution for 4 hours, regulates between pH to 5-6, continues to stir decompression in a moment and extracts all solvents, obtains 1.95g white solid, is the part H with amino acid side chain ₄l:C ₁₆h ₂₀n ₂o ₈: C, 52.17; H, 5.47; N, 7.61.Found:C, 52.62; H, 5.58; N, 7.81. ¹h NMR (D ₂o) δ .80 (q, 4H), 3.82 (q, 2H), 4.41 (q, 4H), 7.58 (d, 4H), its chemical structural formula is:

5) get the Cu (ClO of the 1mmol of 0.37g ₄) ₂6H ₂o joins in the methanol solution of 8mL, adds wherein the methanol solution containing the 8mL of the phenanthroline of the 1mmol of 0.18g, and it is green micro-mixed that reaction solution is;

6) by filter paper, drip wherein the part H with amino acid side chain that 20mL contains 0.18g ₄the LiOHH of the 0.5mmol of L and 0.021g ₂the aqueous solution of O, dropwises rear reaction solution for blue clarification, after stirring at normal temperature 1.5h, has blue precipitation to generate;

7) by the reacting liquid filtering that has blue precipitation to generate, mother liquor is kept nature volatilization, occurs being double-core copper complex C by blue bulk crystals after one week ₄₀h ₃₂cu ₂n ₆o ₈: C, 56.40; H, 3.79; N, 9.87.Found:C, 56.86; H, 4.01; N, 9.65.