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

Abstract

The invention discloses a preparation method of a binuclear copper complex with anti-tumor activity. The preparation method comprises the steps of synthesizing a ligand H4L with branched-chain amino acid, and then, preparing the binuclear copper complex by using H4L. Compared with the prior art, the binuclear copper complex prepared by using the method has the advantages that tumor cells can fall off close to walls, meanwhile, shrinkage and separation phenomena are generated, and the amount of the tumor cells is reduced. The binuclear copper complex has a favorable inhibiting effect on hepatoma carcinoma cells (HepG2), human cervical carcinoma cells (HeLa) and human prostatic cancer (PC3).

Description

A kind of preparation method with the double-core copper complex of anti-tumor activity

Technical field

The present invention relates to chemicals technical field, particularly a kind of preparation method with the double-core copper complex of anti-tumor activity.

Background technology

At present, cancer is to cause dead second largest killer, in all lethal cases, almost has 1st/4th, and cancer causes.And clinical practice shows in cancer patients, there is nearly half people finally to die from cancer.Therefore, research and develop basic orientation and the final goal in research that new and effective cancer therapy drug becomes pharmaceutical chemistry development.As everyone knows, copper is one of element the most basic in human normal physiological metabolism.In living things system, copper is mostly that the complex form with copper exists; Amino acid is the basic structural unit of protein, and it can identify the particular bases pair in DNA sequence dna.Thereby amino acid whose copper complex is generally used as the function of the biological enzyme that template research contains copper, some drug molecules containing amino acid side chain and the title complex of cupric formation have the function of good anti-tumor activity and artificial nuclease.Research discovery simultaneously, 1,10-o-phenanthroline (phen) also has good anti-tumor activity.

Therefore, urgently develop a kind of cancer therapy drug with anti-tumor activity.

Summary of the invention

The object of the invention is to provide a kind of preparation method with the double-core copper complex of anti-tumor activity.

For achieving the above object, the present invention implements according to following technical scheme:

A preparation method with the double-core copper complex of anti-tumor activity, 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.

Compared with prior art, the double-core copper complex that the present invention makes, can make tumour cell come off by adherent, and be accompanied by and shrink and separation phenomenon, reduce tumour cell quantity, the present invention is to human liver cancer cell (HepG2), human cervical carcinoma cell (HeLa), and human prostata cancer (PC3) cell has good restraining effect.

Accompanying drawing explanation

Fig. 1 is the crystalline structure figure of the double-core copper complex that makes of the present invention;

Fig. 2 is that the cellular form before and after double-core copper complex and cancer cells effect changes schematic diagram.

Embodiment

Below in conjunction with specific embodiment, the invention will be further described, in illustrative examples and the explanation of this invention, is used for explaining the present invention, but not as a limitation of the invention.

A kind of preparation method with the double-core copper complex of anti-tumor activity of the present invention, 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, its crystalline structure is as shown in Figure 1.

The anti-tumor activity of double-core copper complex prepared by the present invention is for further testing:

The cultivation of cell

In liquid nitrogen container, take out cryopreservation tube, put into rapidly 37-40 ℃ of water-bath, constantly shake cryopreservation tube, frozen storing liquid speed is melted;

Be ready to the culture dish of two 10 * 2cm, inject DMEM nutrient solution, RPMI1640 nutrient solution or Ham's F12 nutrient solution 10ml, sucking-off frozen storing liquid, injects culture dish;

After cell suspension and cell culture fluid are mixed, put 37 ℃, 5%CO ₂in incubator, cultivate, after 12 hours, change nutrient solution.The human liver cancer cell (HepG2) of test use, human cervical carcinoma cell (HeLa), human prostata cancer (PC3) cell is at 37 ℃, 5%CO ₂under saturated humidity condition, be placed in the cultivation of going down to posterity of DMEM nutrient solution containing calf serum, 1%10000U/mL penicillin and the 10000mg/mL Streptomycin sulphate of 10% deactivation.

The counting of cell

Prepare tally, in tally, add above-mentioned cell suspension, tally is kept flat on desktop, from tally edge, inject gently the cell suspension of dyeing, make it to be full of between tally and cover glass in space, the space between every cover slide and tally approximately holds 0.1 μ L cell suspension.By microscope, be calculated as follows: the large lattice of concentration of cell suspension (individual/mL)=2 total cellular score * 10 ⁴individual/mL.

Anti-tumor activity test:

Acid Phosphatase Method (AP method) detects cell viability:

By the good cells such as HepG2 of growth conditions, adjustment density is 0.8-1.0 * 10 ⁵individual/ml, every hole 100 μ L are inoculated in 96 well culture plates, and 37 ℃, 5%CO ₂under saturated humidity condition, cultivate;

Cultivate 24 hour cells adherent after, in cell, add respectively test compound, compound concentration is followed successively by: 10,20,40,80,160 μ mol/L, each concentration acts on respectively 24h.Every group all has blank, and each Kong Jun does 3 repeating holes;

After effect finishes, abandon supernatant nutrient solution, wash 2 times with the phosphoric acid buffer of 200 μ LpH7.2 in every hole, every hole adds the sodium acetate buffer (pH5.0) of 100 μ L0.1M again, 0.1%Triton X-100 (Triton X-100) and 5mM p-nitrophenyl phosphate (p-NPP) are put in 37 ℃ of incubators and are cultivated 2h, and experiment adds 10 μ L1M NaOH while stopping;

After cultivation finishes, use VIVTOR ³1420-050 type microplate reader detects every hole at the absorbance (A) at 405nm wavelength place, usings the solution of p-NPP substrate hydrolysis in not celliferous hole as blank, according to following formula, calculates survival rate IC ₅₀value, survival rate (%)=(A _drug-blank/ A _{control-blank}* 100) * 100%.

We have tested double-core copper complex to human liver cancer cell (HepG2), human cervical carcinoma cell (HeLa), inhibiting rate (Lethal Dose 50) IC of human prostata cancer (PC3) cell ₅₀.These compounds to the Lethal Dose 50 data of tumour cell in Table 1.

Table 1 double-core copper complex and part thereof and the inorganic salt antitumour activity data to three kinds of cells

All parts, title complex and corresponding metal-salt are all hatched 24h with above-mentioned three kinds of cells.Part and metal-salt do not have restraining effect to the growth of tumour cell.After part and copper participation coordination, the IC of its title complex ₅₀obviously reduce, metal participates in the increase that coordination has directly caused compound anti-cancering activity.

By the observation of morphocytology under microscope, referring to Fig. 2, can find out, when with the form of later these cells of drug effect, obvious change having occurred.Before tumour cell and drug effect, Growth of Cells is good, can clearly find out attached cell; After being used as use, the cell number in nutrient solution obviously reduces, and tumour cell comes off by adherent, and is accompanied by contraction and separation phenomenon, illustrates that double-core copper complex prepared by the present invention has good restraining effect to above-mentioned three kinds of cancer cells.

Technical scheme of the present invention is not limited to the restriction of above-mentioned specific embodiment, and the technology distortion that every technical scheme according to the present invention is made, within all falling into 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.