A copper complex with anticancer activity and its preparation method and application

Abstract

The present invention belongs to the field of anticancer metal complexes, and specifically relates to a copper complex with anticancer activity, its preparation method, and application. The present invention comprises dissolving Cu(OAc)2•H2O and coumarin-3-formyl-(2-aminomethylpyridine) in a solvent, mixing the two solutions, stirring and reacting them at room temperature for a period of time, filtering the reaction solution, sealing the filtrate, and allowing it to evaporate naturally at room temperature for 3-7 days to obtain a complex. The complex has an IC value of 100% for Hela cervical cancer cells. 50 The value is 0.8964±0.09μM, and the inhibitory effect on HeLa cervical cancer cells is 3.38 times that of cisplatin. In addition, the toxicity of the complex to normal cells and HUVEC is very small, IC 50 >100μM, and has a high selectivity coefficient SI (SI=normal cell IC 50 / Cancer cell IC 50 ), SI>111, 584 times that of cisplatin (SI=0.19). This complex has the ability to induce apoptosis in cancer cells in a concentration-dependent manner. It also arrests cancer cells in the S phase, inhibiting cell proliferation, and can intercalate with HS-DNA.

Description

Technical Field

[0001] The present invention belongs to the field of anticancer metal complexes, and in particular relates to a copper complex with anticancer activity and a preparation method and application thereof. Background Art

[0002] Cancer is one of the major diseases that seriously threatens human health. On February 2, 2010, the World Health Organization warned that if people do not take serious measures to prevent cancer, the annual global cancer death toll could double to 17 million by 2030. Therefore, the research on new, highly effective, and low-toxic anti-cancer drugs has been a global focus. In my country, the annual incidence of cancer is approximately 1.2 million, and the number of cancer deaths exceeds 900,000. Cancer has become the second leading cause of death after cardiovascular disease, making the fight against cancer a pressing issue.

[0003] Metal complexes are a new area of ​​research in antibacterial and anticancer drugs. Since the discovery of the anticancer activity of cisplatin by Rosenberg in the United States in 1965 (Rosenberg B, Vancamp L, The successful regression of large solid sarcoma 180 tumors by platinum compounds[J], Cancer Research, 1970, 30(6), 1799-1802), the study of metal complex anticancer drugs has become one of the research hotspots worldwide. However, platinum drugs have significant toxic side effects and drug resistance during use. In order to overcome the limitations of platinum drugs, people have tried to develop many other more effective, less toxic, and target-specific non-platinum metal-based anticancer drugs. Summary of the Invention

[0004] In view of the above problems existing in the prior art, the object of the present invention is to provide a copper complex with anticancer activity and a preparation method and application thereof.

[0005] In order to solve the above problems, the technical solutions adopted by the present invention are as follows:

[0006] A copper complex with anticancer activity, wherein the structural formula of the copper complex is:

[0007] .

[0008] The present invention also provides a method for preparing the copper complex having anticancer activity, comprising the following steps:

[0009] S1: Coumarin 3-formyl-(2-aminomethylpyridine) is reacted with ethyl coumarin 3-formyl-(2-aminomethylpyridine);

[0010] S2: Dissolve Cu(OAc)2•H2O and coumarin-3-formyl-(2-aminomethylpyridine) in a solvent respectively, mix the Cu(OAc)2•H2O solution and coumarin-3-formyl-(2-aminomethylpyridine) solution, stir and react at room temperature for a period of time, filter, seal the filtrate and let it stand at room temperature for 3-7 days to obtain dark blue block crystals, which are the copper complex with anti-cancer activity.

[0011] Optionally, in S1, coumarin 3-formyl-(2-aminomethylpyridine) is dissolved in anhydrous ethanol, heated under reflux to react for 4-5 hours, and naturally cooled to precipitate a solid, which is then recrystallized from anhydrous ethanol to obtain coumarin 3-formyl-(2-aminomethylpyridine).

[0012] Optionally, the molar ratio of Cu(OAc)2•H2O to coumarin-3-formyl-(2-aminomethylpyridine) in S2 is 5:(0.8-1.5).

[0013] Alternatively, in S2, Cu(OAc)2·H2O is dissolved in methanol and coumarin-3-formyl-(2-aminomethylpyridine) is dissolved in dichloromethane.

[0014] Optionally, the reaction is stirred in S2 for 30-60 minutes.

[0015] The present invention also provides the use of the copper complex with anticancer activity in the preparation of anticancer drugs.

[0016] Optionally, the anticancer drug includes: an anti-cervical cancer drug.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention discloses a coumarin copper (II) complex, and a preparation method and application of the complex. The complex has an IC 50 The value is 0.8964 ± 0.09 μM, and the inhibitory effect on HeLa cervical cancer cells is 3.38 times that of cisplatin. In addition, the toxicity of the complex of the present invention to normal cells and HUVEC is very small, IC 50 >100μM, and has a high selectivity coefficient SI (SI=normal cell IC 50 / Cancer cell IC 50), SI>111, 584 times that of cisplatin (SI=0.19). This complex has the ability to induce apoptosis in cancer cells in a concentration-dependent manner. It also arrests cancer cells in the S phase, inhibiting cell proliferation, and can intercalate with HS-DNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a synthetic route diagram of the copper complex of the present invention;

[0020] Figure 2 is a crystal structure diagram of the copper complex of the present invention;

[0021] Figure 3 This is a test result diagram of the effect of the copper complex of the present invention on apoptosis of liver cancer cells HeLa;

[0022] Figure 4 This is a test result diagram of the effect of the copper complex of the present invention on the cell cycle of liver cancer cells HeLa;

[0023] Figure 5 The figure is an ultraviolet absorption spectrum of the interaction between the copper complex of the present invention and herring sperm DNA (HS-DNA);

[0024] Figure 6 The figure is a fluorescence spectrum diagram of the interaction between the copper complex of the present invention and herring sperm DNA (HS-DNA). DETAILED DESCRIPTION

[0025] The present invention will be further described below with reference to specific embodiments.

[0026] 1. Preparation Example

[0027] Example 1

[0028] Synthesis of the ligand coumarin 3-formyl-(2-aminomethylpyridine): Dissolve ethyl coumarin 3-formate (1.09 g, 5 mmol) and 2-aminomethylpyridine (0.5407 g, 5 mmol) in 20 mL of anhydrous ethanol. Heat under reflux for 5 hours. Cool naturally to precipitate a yellow solid. The crude product is recrystallized from anhydrous ethanol to obtain white crystals of coumarin 3-formyl-(2-aminomethylpyridine) in 81% yield. Mp: 156.7-158.5°C. IR(KBr)υ max (cm -1 ): 3414(-NH), 1711(-C=O), 1654(-NHC=O). 1 H NMR (600M, CDCl3, TMS) δ (ppm): 4.80~4.81 (d, J=6 Hz, 2H),7.19~7.41 (m, 4H), 7.65~7.70 (m, 3H), 8.60~8.61 (d, J =6 Hz, 1H), 8.94 (s, 1H), 9.61 (s, 1H). 13 C NMR(150MHz, CDCl3) δ(ppm): 45.38, 114.11, 116.66, 118.48,118.61, 121.70, 122.33, 125.25, 129.81, 134.07, 136.75, 148.43, 154.50,156.73, 161.24,161.69. HRMS(ESI) m / z [M+H] + calculated for C 16 H 12 N2O3: 281.0926, found: 281.0924.

[0029] Dissolve Cu(OAc)2•H2O (100 mg, 0.5 mmol) in 15 ml of methanol to obtain solution A. Dissolve coumarin-3-formyl-(2-aminomethylpyridine) (22.4 mg, 0.08 mmol) in 10 ml of dichloromethane to obtain solution B. Mix solution A and solution B and stir at room temperature for 1 hour to obtain a dark blue solution. Filter and allow the filtrate to evaporate naturally at room temperature. After one week, dark blue block crystals are obtained, which is the copper complex with anticancer activity.

[0030] Example 2

[0031] This Example differs from Example 1 in that: Cu(OAc)2•H2O (100 mg, 0.5 mmol) was dissolved in 15 ml of methanol to obtain solution A, and coumarin-3-formyl-(2-aminomethylpyridine) (28 mg, 0.1 mmol) was dissolved in 15 ml of dichloromethane to obtain solution B. Solution A and solution B were mixed and stirred at room temperature for 45 minutes to obtain a dark blue solution. The solution was filtered, and the filtrate was naturally evaporated at room temperature. After 5 days, dark blue block crystals were obtained, which were the copper complex with anticancer activity.

[0032] Example 3

[0033] This Example differs from Example 1 in that: Cu(OAc)2•H2O (100 mg, 0.5 mmol) was dissolved in 15 ml of methanol to obtain solution A, and coumarin-3-formyl-(2-aminomethylpyridine) (42 mg, 0.15 mmol) was dissolved in 15 ml of dichloromethane to obtain solution B. Solution A and solution B were mixed and stirred at room temperature for 30 minutes to obtain a dark blue solution. The solution was filtered, and the filtrate was naturally evaporated at room temperature. After 3 days, dark blue block crystals were obtained, which were the copper complex with anticancer activity.

[0034] 2. Confirmation of the complex

[0035] The complex prepared in Example 1 was subjected to infrared spectroscopy, elemental analysis and crystallography tests, and the structure was as follows:

[0036] (1) Infrared spectrum IR(KBr)υ max (cm -1 ):: 3414(=CH); 3067(Ar-H); 2926(-CH2-); 1651(-C=O); 1592, 1492(C=C); 1382(CN).

[0037] (2) Elemental analysis data: Elemental analysis is performed according to [C 18 H 18 CuN2O7], calculated value (%): C, 49.37; H, 4.14; N, 6.40, experimental value (%): C, 49.35; H, 4.11; N, 6.30.

[0038] (3) Crystal structure of the complex: Complex [C 18 H 18 The crystallographic data, main bond lengths and bond angles of CuN2O7 are listed in Table 1 and Table 2 respectively. The crystal structure of the complex is shown in Figure 2 shown.

[0039] Table 1 Crystallographic data of the complexes

[0040]

[0041] Table 2 Main bond lengths and bond angles of the complexes

[0042]

[0043] 3. MTT method was used to study the in vitro cytotoxic activity of the copper complex of the present invention on HeLa (human cervical cancer cells) and HUVEC (human umbilical vein endothelial cells)

[0044] HeLa cancer cells and HUVEC normal cells were cultured in MEM medium containing 10% fetal bovine serum and DMEM medium (containing D-glucose, L-glutamine, penicillin, and streptomycin), respectively, and cultured at 37°C and 5% CO2 in a humidified environment. MTT (5 mg / mL) was dissolved in phosphate-buffered saline (PBS, pH = 7.4), filtered, and stored at −20°C in the dark. First, the cultured cells in the logarithmic phase were digested, centrifuged, and counted. 100 μL of 5-8 × 10 cells were seeded on a 96-well plate. 3 / well of cells and cultured at 37 ° C, 5% CO2, 90% humidity for 24 hours. Different concentrations of target compounds were added to 96-well plates. After 48 hours, the old culture medium was aspirated and 100 μL of MTT (1 mg / mL) was added to each well and incubated for 4 hours. The MTT solution was completely removed and 100 μL of DMSO was added to each well and shaken for 10 minutes to dissolve the purple formazan. DMSO solution was used as a negative control, and the absorbance was read at 595 nm using a microplate reader to calculate the half-inhibitory concentration IC for cancer cells. 50 Value (results are shown in Table 3)

[0045] Table 3 Half inhibitory concentration IC of copper (II) complexes against cancer cells 50 (μM) and selectivity coefficient (SI)

[0046]

[0047] As can be seen from Table 3, the IC values ​​of the copper (II) complexes of the present invention against Hela cervical cancer cells are 50 The value is 0.8964 ± 0.09 μM, which is significantly lower than 3.03 ± 0.39 μM of cisplatin, indicating that the inhibitory effect of the copper (II) complex synthesized by the present invention on cervical cancer HeLa cells is 3.38 times that of cisplatin, and the toxicity of the complex to normal cells and HUVEC is very small, IC 50 >100μM, and has a high selectivity coefficient SI (SI=normal cell IC 50 / Cancer cell IC 50 ), SI>111, which is 584 times that of cisplatin (SI=0.19).

[0048] The present application also compares the anticancer activity and toxicity of the following three copper complexes. The test method is the same as above. The results are shown in the table below. The results show that the complex obtained in the present application has more excellent anticancer activity, and the selectivity coefficient SI of the copper complex in the present application is lower.

[0049]

[0050] The half inhibitory concentration IC of the three complexes on cancer cells 50 (μM) and selectivity coefficient (SI)

[0051]

[0052] IV. Effect of the Copper (II) Complex Synthesized by the Present Invention on the Apoptosis of Cervical Cancer Cells HeLa

[0053] HeLa cells were digested and counted, and 2 × 10 6 Cells were plated at 4% RI / well and incubated for 24 hours. The complexes were diluted in culture medium at various concentrations and incubated with the cells for 24 hours. The culture medium was discarded, and the cells were washed twice with PBS, digested with EDTA-free trypsin, harvested (2000 rpm), and rinsed twice again with PBS. After centrifugation, the supernatant was discarded, and 500 μL of binding buffer, 5 μL of Annexin V-FITC, and 5 μL of PI were added. The cells were incubated in the dark at room temperature for 10 minutes and immediately analyzed.

[0054] Figure 3 The figure shows the test results of the copper(II) complex synthesized in this invention on the apoptosis of HeLa cells. The results show that the apoptosis rate of HeLa cells gradually increases with increasing complex concentration. At a complex concentration of 10µM, the apoptosis rate increased from 2.75% to 29.13% compared to the blank control. These results demonstrate that the copper(II) complex has an effect on the apoptosis of HeLa cells and can induce apoptosis in a concentration-dependent manner.

[0055] 5. Effects of Copper(II) Complexes on the Cell Cycle of HeLa Cancer Cells

[0056] HeLa cells were digested and counted, and 2 × 10 6 Cells were incubated at 4% 4% RNase A per well for 24 hours. The complexes were diluted in culture medium at various concentrations. After incubation with the cells for 24 hours, the culture medium was discarded, the cells were washed with PBS, digested with EDTA-free trypsin, harvested (2000 rpm / 5 minutes), and washed again with PBS. The supernatant was discarded, and the cells were fixed with 75% anhydrous ethanol for at least 2 hours at 4°C, washed with PBS, and centrifuged. After discarding the supernatant, 500 μL of RNase A was added, the cells were resuspended, and incubated at room temperature in the dark for 30 minutes before detection and analysis using ModFit software.

[0057] Figure 4These are the test results for the effect of the copper (II) complex synthesized in this invention on the cell cycle of HeLa cervical cancer cells. The results show that after 24 hours of treatment with the copper (II) complex, the proportion of cells in the S phase increased with increasing drug concentration. At a drug concentration of 10 µM, the proportion of cells in the S phase increased from 25.46% to 47.62% compared to the blank control without sample addition. This indicates that the complex arrests the cell cycle in the S phase for a large proportion of HeLa cells.

[0058] VI. Study on the Interaction between Copper(II) Complexes and DNA

[0059] The interaction ability of the complex synthesized by the present invention with DNA is studied with herring sperm DNA (HS-DNA) as the research object.

[0060] (1) Ultraviolet absorption spectrum of the copper (II) complex synthesized by the present invention interacting with HS-DNA

[0061] HS-DNA was diluted to 0.2 × 10 −3 M (pH = 7.2); A260 / A280>1.8, indicating the absence of protein. The complex was diluted with DMSO to 1×10 −3 M. Add 5 µL of HS-DNA stock solution (1 × 10 –3 M) were added to 3 mL of compound solution (1 × 10 −5 M), and measure its UV absorption spectrum in the range of 200-400 nm.

[0062] Figure 5 The following is a UV absorption spectrum of the interaction between the copper(II) complex synthesized in this invention and herring sperm DNA (HS-DNA). As can be seen, as the concentration of HS-DNA increases, the complex's absorption peak at 293 nm exhibits a hypochromic effect, and the position of the absorption peak shifts slightly to the red, suggesting the presence of an intercalation interaction between the complex and HS-DNA.

[0063] (2) Fluorescence emission spectrum of the copper (II) complex synthesized by the present invention interacting with HS-DNA

[0064] Ethidium bromide (EB) was added to the HS-DNA solution (1 × 10 −4 M) to make the concentration of EB 1×10 –5 M and incubate in the dark for 2 hours. 5 μL of compound solution (1 × 10 −3M), and the fluorescence intensity was measured at an excitation wavelength of 420 nm. The quenching intensity of the fluorescence intensity of the EB-HS-DNA system was calculated using the Stern–Volmer equation as follows:

[0065] I0 / I = 1 + K q [Q],

[0066] Where I0 is the fluorescence intensity of the HS-DNA–EB system at 615 nm, I is the fluorescence intensity of the EB–HS-DNA system after the addition of the compound; [Q] is the concentration of the compound, K q is the quenching constant.

[0067] Figure 6 This is a fluorescence spectrum of the interaction between copper (II) complex and DNA. EB is a highly conjugated molecule that emits strong fluorescence when bound to DNA. The addition of copper (II) complex will cause a change in the fluorescence intensity of the EB-DNA solution, which can indirectly study the interaction between DNA and compounds that can displace EB from the EB-DNA adduct. Figure 5 It can be seen that after the addition of the copper (II) complex, the fluorescence intensity of EB–HS-DNA gradually decreased, indicating that the copper (II) complex replaced the EB bound to HS-DNA and interacted with DNA. According to the Stern-Volmer equation, the quenching constant of the copper (II) complex is 5.37×10 5 M.

Claims

1. A copper complex with anticancer activity, characterized in that: The structural formula of the copper complex is: 。 2. The method for preparing a copper complex with anticancer activity according to claim 1, wherein: The following steps are involved: S1: Coumarin 3-formyl-(2-aminomethylpyridine) is reacted with ethyl coumarin 3-formyl-(2-aminomethylpyridine); S2: Dissolve Cu(OAc)2•H2O and coumarin-3-formyl-(2-aminomethylpyridine) in a solvent respectively, mix the Cu(OAc)2•H2O solution and coumarin-3-formyl-(2-aminomethylpyridine) solution, stir and react at room temperature for a period of time, filter, seal the filtrate and let it stand at room temperature for 3-7 days to obtain dark blue block crystals, which are the copper complex with anti-cancer activity.

3. The preparation method according to claim 2, characterized in that In S1, coumarin 3-formyl-(2-aminomethylpyridine) is dissolved in anhydrous ethanol, heated under reflux to react for 4-5 hours, and naturally cooled to precipitate a solid, which is then recrystallized from anhydrous ethanol to obtain coumarin 3-formyl-(2-aminomethylpyridine).

4. The preparation method according to claim 2, characterized in that In S2, the molar ratio of Cu(OAc)2•H2O to coumarin-3-formyl-(2-aminomethylpyridine) is 5:(0.8-1.5).

5. The preparation method according to claim 2, characterized in that In S2, Cu(OAc)2•H2O was dissolved in methanol, and coumarin-3-formyl-(2-aminomethylpyridine) was dissolved in dichloromethane.

6. The preparation method according to claim 2, characterized in that Stir the reaction in S2 for 30-60 minutes.

7. Use of the copper complex with anticancer activity according to claim 1 in the preparation of anticancer drugs.

8. The use according to claim 7, characterized in that The anticancer drugs include: anti-cervical cancer drugs.

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