Copper complex with anticancer activity and preparation method and application thereof

Abstract

The invention belongs to the field of anti-cancer metal complexes, and particularly relates to a copper complex with anti-cancer activity and a preparation method and application thereof. Cu (OAc) 2H2O and coumarin-3-formyl-(2-aminomethylpyridine) are respectively dissolved in a solvent, the two solutions are mixed and stirred at room temperature to react for a period of time, the reaction solution is filtered, the filtrate is sealed and naturally volatilized at room temperature for 3-7 days, the complex can be obtained, the IC50 value of the complex to Hela cervical cancer cells is 0.8964 + / -0.09 mu M, and the IC50 value of the complex to Hela cervical cancer cells is 0.8964 + / -0.09 mu M. According to the present invention, the inhibition effect of the complex on HeLa cervical cancer cells is 3.38 times of the inhibition effect of cis-platinum, the toxicity of the complex on normal cells but HUVEC is very small, IC50 is greater than 100 [mu] M, and the complex has a high selectivity coefficient SI (SI = normal cell IC50 / cancer cell IC50), and the selectivity coefficient SI is greater than 111 and is 584 times of the selectivity coefficient of cis-platinum (SI = 0.19). The complex has the capability of inducing apoptosis of cancer cells and has concentration dependence. Meanwhile, the complex can retard cancer cells and an S phase so as to inhibit cell proliferation, and can generate an intercalation effect with HS-DNA (High-Speed Deoxyribose Nucleic Acid).

Description

Technical Field

[0001] The 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

[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 research on 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 solution adopted by the present invention is as follows: A copper complex with anticancer activity, the coordination structure diagram of the copper complex is: .

[0006] The present invention also provides a method for preparing the above copper complex with anticancer activity, comprising the following steps: 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 solvents 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 place it at room temperature for 3-7 days to obtain dark blue block crystals, which are the copper complex with anticancer activity.

[0007] Optionally, in S1, coumarin 3-formyl-(2-aminomethylpyridine) is dissolved in anhydrous ethanol, heated under reflux to react for 4-5 hours, and solid is precipitated after natural cooling, which is then recrystallized from anhydrous ethanol to obtain coumarin 3-formyl-(2-aminomethylpyridine).

[0008] Optionally, the molar ratio of Cu(OAc)2•H2O to coumarin-3-formyl-(2-aminomethylpyridine) in S2 is 5:(0.8-1.5).

[0009] Alternatively, in S2, Cu(OAc)2•H2O is dissolved in methanol and coumarin-3-formyl-(2-aminomethylpyridine) is dissolved in dichloromethane.

[0010] Optionally, the reaction is stirred in S2 for 30-60 min.

[0011] The present invention also provides the use of the copper complex with anticancer activity in the preparation of anticancer drugs.

[0012] Optionally, the anti-cancer drugs include: anti-cervical cancer drugs.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The invention discloses a coumarin copper (II) complex, and a preparation method and application of the complex. The IC of the complex to Hela cervical cancer cells 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 complex of the present invention has very low toxicity to normal cells and HUVEC. 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). The complex has the ability to induce apoptosis of cancer cells, and is concentration-dependent. At the same time, the complex can block cancer cells and S phase to inhibit cell proliferation, and can intercalate with HS-DNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is a synthetic route diagram of the copper complex of the present invention; Figure 2 is a crystal structure diagram of the copper complex of the present invention; 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; 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; 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); Figure 6 The fluorescence spectrum of the interaction between the copper complex of the present invention and herring sperm DNA (HS-DNA) is shown. DETAILED DESCRIPTION

[0015] The present invention is further described below in conjunction with specific embodiments.

[0016] 1. Preparation Example Example 1 Synthesis of ligand coumarin 3-formyl-(2-aminomethylpyridine): Dissolve coumarin 3-formyl ethyl ester (1.09 g, 5 mmol) and 2-aminomethylpyridine (0.5407 g, 5 mmol) in 20 mL of anhydrous ethanol, heat and reflux for 5 hours, and precipitate a yellow solid after natural cooling. Recrystallize the crude product with anhydrous ethanol to obtain white crystals of coumarin 3-formyl-(2-aminomethylpyridine) with a yield of 81%. Mp: 156.7-158.5℃. 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.

[0017] Dissolve Cu(OAc)2•H2O (100 mg, 0.5 mmol) in 15 ml of methanol to obtain solution A, and 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, 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 are the copper complex with anticancer activity.

[0018] Example 2 The difference between this embodiment and embodiment 1 is that Cu(OAc)2•H2O (100 mg, 0.5 mmol) is dissolved in 15 ml of methanol to obtain solution A, coumarin-3-formyl-(2-aminomethylpyridine) (28 mg, 0.1 mmol) is dissolved in 15 ml of dichloromethane to obtain solution B, solution A and solution B are mixed, stirred at room temperature for 45 minutes to obtain a dark blue solution, filtered, and the filtrate is naturally evaporated at room temperature. After 5 days, dark blue block crystals are obtained, which are copper complexes with anticancer activity.

[0019] Example 3 The difference between this embodiment and embodiment 1 is that Cu(OAc)2•H2O (100 mg, 0.5 mmol) is dissolved in 15 ml of methanol to obtain solution A, coumarin-3-formyl-(2-aminomethylpyridine) (42 mg, 0.15 mmol) is dissolved in 15 ml of dichloromethane to obtain solution B, solution A and solution B are mixed, stirred at room temperature for 30 minutes to obtain a dark blue solution, filtered, and the filtrate is naturally evaporated at room temperature. After 3 days, dark blue block crystals are obtained, which are copper complexes with anticancer activity.

[0020] 2. Confirmation of the complex The complex prepared in Example 1 was subjected to infrared spectroscopy, elemental analysis and crystallography tests, and the structure was as follows: (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).

[0021] (2) Elemental analysis data: Elemental analysis is based on [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.

[0022] (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.

[0023] Table 1 Crystallographic data of the complexes

[0024] Table 2 Main bond lengths and bond angles of the complexes

[0025] 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) HeLa cancer cells and HUVEC normal cells were cultured in MEM medium containing 10% fetal bovine serum and DMEM medium (the medium contains D-glucose, L-glutamine, penicillin and streptomycin), respectively, and cultured at 37°C and 5% CO2 under humid conditions. 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 3 / well of cells and cultured at 37°C, 5% CO2, 90% humidity for 24 hours, and different concentrations of target compounds were added to 96-well plates. After 48 hours, the old culture medium was aspirated, 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. Using the DMSO solution as a negative control, the absorbance was read at 595 nm using a microplate reader, and the half-inhibitory concentration IC for cancer cells was calculated. 50 Value (results in Table 3) Table 3 IC50 of copper(II) complexes against cancer cells 50 (μM) and selectivity coefficient (SI)

[0026] 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 50The 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).

[0027] The present application also compares the anti-cancer 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 same complex obtained in the present application has a more excellent anti-cancer activity, and the selectivity coefficient SI of the copper complex in the present application is lower.

[0028]

[0029] IC50% inhibitory concentration of three complexes on cancer cells 50 (μM) and selectivity coefficient (SI)

[0030] IV. Effect of the copper (II) complex synthesized by the present invention on apoptosis of cervical cancer cells HeLa HeLa cells were digested and counted, and 2 × 10 cells were inoculated in 2 mL in a 6-well plate. 6 / well of cells and incubate for 24 hours. The complexes were diluted in culture medium at different concentrations and incubated with cells for 24 hours. The culture medium was discarded, the cells were washed twice with PBS, digested with EDTA-free trypsin, collected (2000rpm / min), and rinsed twice with PBS again. After centrifugation, the supernatant was discarded, and 500μL binding buffer, 5μL Annexin V-FITC, and 5μL PI were added. The cells were incubated at room temperature in the dark for 10 minutes and immediately tested on the machine.

[0031] Figure 3 The test result diagram of the effect of the copper (II) complex synthesized by the present invention on the apoptosis of HeLa cells. The results show that with the increase of the concentration of the complex, the apoptosis rate of HeLa cells gradually increases. When the concentration of the complex is 10μM, compared with the blank control, the apoptosis rate increases from 2.75% to 29.13%. This result shows that the copper (II) complex has an effect on the apoptosis of HeLa cells, can induce apoptosis of HeLa cells, and has a concentration dependence.

[0032] 5. Effects of copper (II) complexes on the cell cycle of HeLa cancer cells HeLa cells were digested and counted, and 2 × 10 cells were inoculated in 2 mL in a 6-well plate. 6 / well of cells and incubate for 24 hours. The complex was diluted in the culture medium at different concentrations. After incubating the samples with cells for 24 hours, the culture medium was discarded, the cells were washed with PBS, digested with EDTA-free trypsin, collected (2000rpm / 5 minutes), and washed again with PBS. The supernatant was discarded, and the cells were fixed with 75% anhydrous ethanol at 4°C for more than 2 hours, washed with PBS, and centrifuged. After discarding the supernatant, 500μL RNase A was added, the cells were resuspended and incubated in the dark at room temperature for 30 minutes, and then detected, and the results were analyzed using ModFit software.

[0033] Figure 4 The results are the test results of the effect of the copper (II) complex synthesized by the present invention on the cell cycle of cervical cancer cells HeLa. The results show that after the copper (II) complex acts on HeLa cells for 24 hours, the proportion of cells in the S phase increases with the increase of drug concentration. When the drug concentration is 10μM, compared with the blank control without sample addition, the amount of cells in the S phase increases from 25.46% to 47.62%. This shows that the complex blocks the cycle of most HeLa cells in the S phase.

[0034] VI. Study on the interaction between copper(II) complexes and DNA The interaction ability of the complex synthesized by the present invention with DNA is studied by taking herring sperm DNA (HS-DNA) as the research object.

[0035] (1) Ultraviolet absorption spectrum of the copper (II) complex synthesized by the present invention interacting with HS-DNA 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 to 1×10 −3 M. Add 5 µL of HS-DNA stock solution (1×10 –3 M) was added with 3 mL of compound solution (1 × 10 −5 M), and measure its UV absorption spectrum in the range of 200 - 400 nm.

[0036] Figure 5 The figure is a UV absorption spectrum of the interaction between the copper (II) complex synthesized by the present invention and herring sperm DNA (HS-DNA). As can be seen from the figure, as the concentration of HS-DNA increases, the absorption peak of the complex at 293nm shows a hypochromic effect, and the position of the absorption peak has a slight red shift, which infers that there is an intercalation effect between the complex and HS-DNA.

[0037] (2) Fluorescence emission spectrum of the copper (II) complex synthesized by the present invention interacting with HS-DNA Add ethidium bromide (EB) to the HS-DNA solution (1 × 10 −4 M) to make the concentration of EB to be 1×10 –5 M and incubate it in the dark for 2 hours. Add 5 µL of compound solution (1 × 10 −3 M), and the fluorescence intensity was detected at an excitation wavelength of 420 nm. The quenching intensity of the compound on the fluorescence intensity of the EB-HS-DNA system was calculated using the Stern–Volmer equation as follows: I0 / I = 1 + K q [Q], 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.

[0038] Figure 6 This is a fluorescence spectrum of the interaction between copper (II) complexes and DNA. EB is a highly conjugated molecule that emits strong fluorescence when bound to DNA. The addition of copper (II) complexes will cause changes in the fluorescence intensity of the EB-DNA solution, which can indirectly study the interaction between DNA and compounds that can replace EB from the EB-DNA adduct. Figure 5 It can be seen that after the copper (II) complex was added, 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 having anticancer activity, characterized in that: The coordination structure diagram of the copper complex is: 。 2. The method for preparing a copper complex with anticancer activity according to claim 1, characterized in that: 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 solvents 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 place it at room temperature for 3-7 days to obtain dark blue block crystals, which are the copper complex with anticancer 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 solid is precipitated after natural cooling. Coumarin 3-formyl-(2-aminomethylpyridine) is obtained by recrystallization with anhydrous ethanol.

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: The reaction was stirred in S2 for 30-60 min.

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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