Copper (II) complex with anticancer activity as well as preparation method and application of copper (II) complex

Abstract

The invention discloses a copper (II) complex with anticancer activity as well as a preparation method and application thereof, and belongs to the technical field of complexes. According to the preparation method, CuCl22H2O, CuBr2 or Cu (NO3) 2.3 H2O is subjected to a reaction with benzoyl hydrazone 3-acetyl-7-N, N-diethylamino coumarin respectively, and the complex is obtained. The obtained complex has certain anti-proliferative activity to four cell lines, namely HeLa, MCF-7, A549 and HepG-2, and the anti-proliferative activity is obviously superior to that of a ligand, particularly, the complex shows relatively excellent anti-proliferative activity to the MCF-7 cell line, and the toxicity to normal HUVEC cells is also obviously lower than that of cis-platinum. The copper (II) complex provided by the invention has good anti-cancer activity, the preparation method is simple, the toxicity is small, and a new thought is provided for the development of anti-cancer drugs.

Description

Technical Field

[0001] This application belongs to the field of coordination technology, specifically relating to copper(II) coordination compounds with anticancer activity, their preparation methods and applications. Background Technology

[0002] Cancer is a serious threat to human health, with its incidence and mortality rates rising year by year. Traditional cancer treatments such as surgery, chemotherapy, and radiotherapy have certain limitations. For example, surgery cannot completely eliminate cancer cells, while chemotherapy and radiotherapy cause significant damage to normal cells. Therefore, developing novel, highly effective, and low-toxicity anticancer drugs is of great clinical significance.

[0003] Coumarins are a class of natural products with diverse biological activities, exhibiting anti-inflammatory, antibacterial, and antioxidant properties. Recent studies have also revealed the potential of coumarin compounds in anti-cancer applications. The structural characteristics of coumarins enable them to interact with biomolecules, thereby influencing processes such as cell proliferation, differentiation, and apoptosis.

[0004] Metal complexes have shown great potential in the field of anticancer drugs, such as classic drugs like cisplatin. By utilizing the coordination between ligands and metal ions, the physicochemical properties and biological activity of the original organic ligands can be effectively improved. Copper, as an essential trace element for the human body, can have its complexes target cancer cells through multiple mechanisms, including generating oxidative stress and inducing apoptosis, while reducing toxic side effects on normal tissues.

[0005] Therefore, combining bioactive coumarin derivatives with pharmacologically promising copper ions through coordination chemistry to construct novel metal complexes is expected to achieve synergistic effects and provide a new strategy for developing highly effective and low-toxicity novel anticancer drug candidates.

[0006] The coumarin derivatives and copper complexes involved in this application have not been reported in the prior art. Summary of the Invention

[0007] In view of the above-mentioned problems existing in the prior art, the purpose of this application is to provide a copper(II) complex with anticancer activity. Another purpose of this application is to provide a method for preparing the complex. Yet another purpose of this application is to provide the application of the complex.

[0008] To solve the above problems, the technical solution adopted in this application is as follows: Copper(II) complexes having the structure shown in Formula 1 or Formula 2, Formula 1, where X is chlorine or bromine; Formula 2.

[0009] The aforementioned method for preparing the copper(II) complex involves reacting CuCl2•2H2O, CuBr2, or Cu(NO3)2•3H2O with benzoylhydrazone 3-acetyl-7-N,N-diethylaminocoumarin, respectively, to obtain the copper(II) complex. When CuCl2•2H2O is used as a reactant, a copper(II) complex with the structure shown in Formula 1, where X is chlorine, is obtained. When CuBr2 is used as a reactant, a copper(II) complex with the structure shown in Formula 1, in which X is bromine, is obtained; When Cu(NO3)2•3H2O is used as the reactant, a copper(II) complex with the structure shown in Formula 2 is obtained.

[0010] Furthermore, the preparation method of benzoylhydrazone 3-acetyl-7-N,N-diethylaminocoumarin is as follows: 3-acetyl-7-N,N-diethylaminocoumarin and benzoylhydrazine are dissolved in a solvent, refluxed, cooled, and a solid is precipitated. After filtration and drying, benzoylhydrazone 3-acetyl-7-N,N-diethylaminocoumarin is obtained.

[0011] Furthermore, the preparation method of 3-acetyl-7-N,N-diethylaminocoumarin is as follows: 4-(diethylamino)salicylaldehyde and ethyl acetoacetate are dissolved in a solvent, refluxed, cooled, poured into ice water to precipitate solid, filtered and dried to obtain 3-acetyl-7-N,N-diethylaminocoumarin.

[0012] Furthermore, the molar ratio of 4-(diethylamino)salicylaldehyde to ethyl acetoacetate is 1:(1~3), with a preferred ratio of 1:2.

[0013] Furthermore, the molar ratio of 3-acetyl-7-N,N-diethylaminocoumarin to benzoylhydrazine is 1:(1~3), with a preferred ratio of 1:1.2.

[0014] Furthermore, the molar ratio of CuCl2•2H2O, CuBr2 or Cu(NO3)2•3H2O to benzoylhydrazone 3-acetyl-7-N,N-diethylaminocoumarin is (3~8):1, with a preferred ratio of 5:1.

[0015] Further, CuCl2•2H2O, CuBr2 or Cu(NO3)2•3H2O were dissolved with benzoylhydrazone 3-acetyl-7-N,N-diethylaminocoumarin in a solvent, respectively. After mixing, the mixture was stirred and reacted at room temperature. After filtration, the filtrate was sealed and placed at room temperature to obtain crystals, which are the copper(II) complex.

[0016] The aforementioned copper(II) complexes are used in the preparation of anticancer drugs.

[0017] Furthermore, the anticancer drugs include anti-breast cancer drugs, anti-cervical cancer drugs, anti-liver cancer drugs, or anti-lung cancer drugs.

[0018] Compared to existing technologies, the beneficial effects of this application are as follows: The copper(II) complex disclosed in this application exhibits certain cellular activity against the four cell lines studied: HeLa, MCF-7, A549, and HepG-2, and is significantly superior to the ligand. It particularly demonstrates strong anti-proliferative activity against the MCF-7 cell line, and its toxicity to normal HUVEC cells is significantly lower than that of cisplatin. In summary, the copper(II) complex provided in this application possesses good anticancer activity, is simple to prepare, and has low toxicity, offering a new approach for the development of anticancer drugs.

[0019] The remaining advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description or may be learned by practice of the embodiments of this application. Attached Figure Description

[0020] Figure 1 This is a synthetic route diagram for the copper(II) complex of this application; Figure 2 This is a crystal structure diagram of the copper(II) complexes of this application, wherein complex 1 corresponds to formula 1, and X is chlorine; complex 2 corresponds to formula 1, and X is bromine; and complex 3 corresponds to formula 2. Figure 3 A comparison chart of the selectivity index (SI) values ​​of the copper(II) complex and cisplatin in this application; Figure 4 The figure shows the test results of the effect of the copper(II) complex of this application on cell apoptosis; Figure 5 The UV absorption spectrum of the interaction between the copper(II) complex of this application and calf thymus DNA (CT-DNA); Figure 6 This is a viscosity diagram showing the interaction between the copper(II) complex of this application and calf thymus DNA (CT-DNA). Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the described embodiments of this application without creative effort are within the scope of protection of this application.

[0022] For the purposes of this application, it should be understood that various alternative variations and sequences of steps may be employed in this application unless expressly stated to the contrary. Furthermore, except in any operational instance or otherwise stated, all numerical values ​​indicating the amount of an ingredient as used, for example, in the specification and claims, should be understood to be modified in all cases by the term "about". Therefore, unless stated to the contrary, the numerical parameters listed in the following specification and appended claims are approximate values ​​that may vary depending on the desired properties to be obtained in this application. At least, without attempting to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be interpreted at least based on the reported significant figures and by applying ordinary rounding techniques.

[0023] While the numerical ranges and parameters listed in this application are approximate, the values ​​listed in the specific embodiments are reported as accurately as possible. However, any numerical value inherently contains some error that must be caused by the standard deviation found in its respective test measurements.

[0024] The following is a detailed description of this plan: This application provides a copper(II) complex with anticancer activity, a method for preparing the complex, and its application in the preparation of anticancer drugs.

[0025] The copper(II) complex has the structure shown in Formula 1 or Formula 2. Formula 1, where X is chlorine or bromine; Formula 2.

[0026] The method for preparing the copper(II) complex provided in the embodiments of this application refers to... Figure 1 This includes the following steps: Synthesis of 3-acetyl-7-N,N-diethylaminocoumarin: 4-(diethylamino)salicylaldehyde (1.9324 g, 10.0 mmol), ethyl acetoacetate (2.5314 ml, 20.0 mmol), and 1 ml piperidine were dissolved in 30 ml of anhydrous ethanol and refluxed for 6 h. After the reaction was complete, the mixture was cooled to room temperature, poured into 100 ml of ice water, stirred, and a solid precipitated. The solid was filtered and dried to give 2.32 g of a yellow powder (yield 89.6%). 1 H NMR (600MHz, Chloroform- d )δ8.43(s, 1H), 7.39(d, J =8.9Hz, 1H), 6.61(dd, J =9.0, 2.5Hz, 1H), 6.47(d, J =2.9Hz, 1H), 3.45(q,J =7.2Hz, 4H), 2.68(s, 3H), 1.24 (t, J =7.1Hz, 6H).

[0027] Ligand synthesis: 0.259 g (1 mmol) of 3-acetyl-7-N,N-diethylaminocoumarin and 0.136 g (1 mmol) of benzoylhydrazine were dissolved in 20 mL of anhydrous ethanol. The solution was refluxed for 6 h, cooled naturally to room temperature, filtered, and dried to give 0.2685 g of red solid (yield: 63.7%). mp = 212.3–213.1 °C. IR (KBr)υmax (cm⁻¹): 3423 (NH), 1686 (C=O), 1587 (C=N). 1 H NMR (600 MHz, DMSO- d 6) δ 10.70 (s, 1H), 8.09 (s, 1H), 7.90–7.85 (m, 2H), 7.61–7.57 (m, 2H), 7.52 (t, J = 7.4 Hz, 2H), 6.75 (d, J = 8.5 Hz, 1H), 6.57 (d, J = 10.0 Hz, 1H), 3.46 (q, J = 7.3 Hz, 4H), 2.32 (s, 3H), 1.14 (t, J = 7.2Hz, 6H); HRMS m / z [M+H] + Calculated for C 22 H 23 N3O3:378.1812.found:379.1848.

[0028] Synthesis of Complex 1: A methanol solution of CuCl2·2H2O (0.045 g, 0.265 mmol) was added dropwise to a methanol solution of the ligand (0.02 g, 0.053 mmol). The mixture was stirred at room temperature for 1 hour and then filtered. The filtrate was allowed to evaporate naturally at room temperature for several days to obtain crystals. After filtration, the crystals were washed with ice-cold ethanol to obtain 0.023 g of crystals (yield 35.38%, yield = (actual yield / theoretical yield) × 100%, the same below).

[0029] Synthesis of Complex 2: A methanol solution of CuBr2 (0.059 g, 0.265 mmol) was added dropwise to a methanol solution of the ligand (0.02 g, 0.053 mmol). The mixture was stirred at room temperature for 1 hour and then filtered. The filtrate was allowed to evaporate naturally at room temperature for several days to obtain crystals. After filtration, the crystals were washed with ice-cold ethanol to obtain 0.028 g of crystals (yield 35.44%).

[0030] Synthesis of Complex 3: A methanol solution of Cu(NO3)2·3H2O (0.064 g, 0.265 mmol) was added dropwise to a methanol solution of the ligand (0.02 g, 0.053 mmol). The mixture was stirred at room temperature for 1 hour and then filtered. The filtrate was allowed to evaporate naturally at room temperature for several days to obtain crystals. After filtration, the crystals were washed with ice-cold ethanol to obtain 0.034 g of crystals (yield 40.47%).

[0031] In the synthesis of 3-acetyl-7-N,N-diethylaminocoumarin, the molar ratio of 4-(diethylamino)salicylaldehyde to ethyl acetoacetate can be 1:1, 1:1.5, 1:2, 1:2.5, or 1:3. In this example, the preferred ratio is 1:2.

[0032] In the synthesis of the ligand, the molar ratio of 3-acetyl-7-N,N-diethylaminocoumarin to benzoylhydrazine can be 1:1, 1:1.2, 1:1.5, 1:1.8, 1:2, 1:2.2, 1:2.5, 1:2.8, or 1:3. In this embodiment, the preferred ratio is 1:1.2.

[0033] In the synthesis of the complexes (including complex 1, complex 2 and complex 3), the molar ratio of CuCl2•2H2O, CuBr2 or Cu(NO3)2•3H2O to benzoylhydrazone 3-acetyl-7-N,N-diethylaminocoumarin can be 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, and the preferred ratio in this embodiment is 5:1.

[0034] The prepared complex was subjected to infrared spectroscopy and crystallography, and its structure is as follows: (1) Infrared spectrum IR(KBr)υ max (cm -1 ): Complex 1: 3441(OH), 1721(C=O), 1651(C=N).

[0035] Complex 2: 3444(OH), 1726(C=O), 1654(C=N).

[0036] Complex 3: 3435(OH), 1722(C=O), 1650(C=N).

[0037] (2) Crystal structure of the coordination compounds: Crystallographic data, major bond lengths, and bond angles of the coordination compounds are listed in Tables 1 and 2, respectively. The crystal structure of the coordination compounds is as follows: Figure 2 As shown.

[0038] Table 1 Crystal data and structural parameters of the coordination compounds

[0039] Table 2. Partial bond lengths and bond angles of the coordination compounds

[0040] The in vitro cytotoxic activity of the copper(II) complex of this application against four different human cancer cells and HUVECs (human umbilical vein endothelial cells) was studied using the MTT assay, as follows: Five cell lines were cultured in MEM medium containing 10% fetal bovine serum and DMEM medium (containing D-glucose, L-glutamine, penicillin, and streptomycin), respectively, under humid conditions of 37°C and 5% CO2. 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 growth phase were digested, centrifuged, and counted. 100 μL of 5-8 × 10⁶ cells / well were seeded into 96-well plates. 3 Cells were cultured in 96-well plates at 37°C, 5% CO2, and 90% humidity for 24 hours. Different concentrations of the target compound were added to each well. 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, followed by shaking for 10 minutes to dissolve the purple formazan. DMSO solution was used as a negative control. The absorbance was read at 595 nm using a microplate reader, and the half-maximum inhibitory concentration (IC50) against cancer cells was calculated. 50 Values ​​(see Table 3 for results).

[0041] Table 3. Half-inhibitory concentration (IC50) of copper(II) complexes against cancer cells 50 (μM)

[0042] Table 3 shows that the complexes exhibited certain cellular activity against the four cell lines studied (HeLa, MCF-7, A549, and HepG-2), and were significantly superior to the ligands. In particular, they showed strong anti-proliferative activity against the MCF-7 cell line, and their toxicity to normal HUVEC cells was significantly lower than that of cisplatin. Complex 3 showed good anti-proliferative activity against A549 cells (IC50). 50=9.92μM), compared to ligand (IC) 50 The antiproliferative activity of complex 1 (60.08 μM) was increased 6-fold. Complex 1 showed good antiproliferative activity against HepG-2 cells (IC50 = 60.08 μM). 50 =8.69μM), compared to ligand (IC) 50 The antiproliferative activity was increased 11-fold at concentrations >100 μM. Complex 1 exhibited the best antiproliferative activity against MCF-7 cells (IC50). 50 =1.06μM), significantly lower than DDP (IC). 50 The concentration of the complex (9.07 μM) showed 6.97 times greater inhibitory effect on MCF-7 cancer cells than that of DDP, and the complex also exhibited lower toxicity to normal HUVEC cells (IC50). 50 =12.69μM), with a very high selectivity coefficient (SI=11.97), and a safety factor 199 times higher than that of DDP (SI=0.06). Figure 3 ).

[0043] MCF-7 cells were digested and counted, and seeded into 6-well plates at a density of 2 mL of 5 × 10⁴ cells / well, and incubated for 24 hours. Three complexes were diluted in culture medium at different concentrations and incubated with the cells for 48 hours. The culture medium was discarded, and the cells were washed twice with PBS, digested with EDTA-free trypsin, collected (2000 rpm / min), and washed 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.

[0044] Figure 4 The figure shows the test results of the effect of the copper(II) complex synthesized in this application on cell apoptosis. The results indicate that with increasing complex concentration, the number of cancer cells in early and late apoptosis also significantly increased. When the concentration of complex 1 reached 10 μM, compared with the control group, the number of MCF-7 cancer cells in early and late apoptosis increased in a dose-dependent manner to 22.7%, 29.7%, and 57.5%, respectively. When the concentration of complex 2 reached 10 μM, compared with the control group, the number of MCF-7 cancer cells in early and late apoptosis increased from 24.6% to 54.62%, 62.7%, and 82%, respectively, in a dose-dependent manner. When the concentration of complex 3 reached 10 μM, compared with the control group, the number of MCF-7 cancer cells in early and late apoptosis increased from 14% to 18.31%, 23%, and 66.5%, respectively, in a dose-dependent manner. Based on these data, it can be concluded that the complexes can act as effective anticancer agents to induce cancer cell apoptosis in a concentration-dependent manner.

[0045] The interaction between the copper(II) complex synthesized in this application and DNA was studied, using calf thymus DNA (CT-DNA) as the research object.

[0046] (1) Ultraviolet absorption spectrum of the copper(II) complex synthesized in this application reacting with CT-DNA CT-DNA was diluted to 2.882 × 10⁻⁶ with Tris-HCl–NaCl buffer. -4 M (pH=7.4); A 260 / A 280 =1.876, a ratio between 1.8 and 2.0, indicating that the purity of the CT-DNA solution meets experimental requirements. The concentration of the complex was diluted to 1×10⁻⁶ using DMSO. -5 M. Add 10 µL of CT-DNA stock solution (2.882 × 10⁻⁶). -4 Add 3 mL of compound solution (1 × 10⁻⁶) to M. -5 In M), its ultraviolet absorption spectrum in the range of 300-550 nm was measured.

[0047] Figure 5 The image shows the UV absorption spectrum of the interaction between the copper(II) complex synthesized in this application and calf thymus DNA (CT-DNA). As can be seen from the image, with increasing CT-DNA concentration, the absorption peak at 487 nm of the complex exhibits a significant hypochromic effect, and the peak position shows a slight red shift, suggesting an intercalation interaction between the complex and CT-DNA.

[0048] (2) Viscosity diagram of the interaction between the copper(II) complex synthesized in this application and CT-DNA 10 mL of Tris-HCl–NaCl buffer solution was mixed thoroughly with a certain amount of CT-DNA solution and then added to an Ubbelohde viscometer. The time required for the solution to flow through the capillary was measured. Then, a certain amount of metal complex solution was added sequentially, and the time required for the mixed solution to flow through the capillary was recorded. The time was measured using a stopwatch with an accuracy of 0.01 s. The time was measured three times after each addition of solution, and the average value was taken. The solution conditions used in this viscometry experiment were: CT-DNA concentration of 2.882 × 10⁻⁶. - 4 The molar ratios (r) of the metal complex solution to CT-DNA are 0, 0.2, 0.4, 0.6, 0.8, and 1.0, respectively. Calculate the viscosity η using the following formula.

[0049] η=(t-t0) / t0 Where t0 and t represent the time it takes for the Tris-HCl–NaCl buffer solution to flow through the capillary and the time it takes for the mixture of the complex solution and CT-DNA to flow through the capillary, respectively, and η is the viscosity of the mixture of the metal complex and CT-DNA.

[0050] Figure 6 This is a viscosity diagram of the interaction between the copper(II) complex and CT-DNA, from... Figure 6 It can be seen that as the concentration of the metal complex solution gradually increases, the relative viscosity (η / η0) of the CT-DNA solution also increases. 1 / 3 The ratio of compound to CT-DNA showed a clear upward trend, indicating that the length of the CT-DNA chain gradually increased, suggesting that the complex molecule interacts with CT-DNA through intercalation.

Claims

1. A copper (II) complex characterized in that, having a structure shown in formula 1 or formula 2, Formula 1, wherein X is chloro or bromo; Formula 2.

2. The method for preparing the copper(II) complex as described in claim 1, characterized in that, The copper (II) complex is prepared by reacting CuCl2·2H2O, CuBr2 or Cu(NO3)2·3H2O with benzoyl hydrazone 3-acetyl-7-N,N-diethylaminocoumarin, respectively. When CuCl2·2H2O is used as the reactant, the copper (II) complex having a structure shown in formula 1 with X being chlorine is obtained. When CuBr2 is used as the reactant, the copper (II) complex having a structure shown in formula 1 with X being bromine is obtained. When Cu(NO3)2·3H2O is used as the reactant, the copper (II) complex having a structure shown in formula 2 is obtained.

3. The preparation method according to claim 2, characterized in that, The benzoyl hydrazone 3-acetyl-7-N,N-diethylaminocoumarin is prepared by dissolving 3-acetyl-7-N,N-diethylaminocoumarin and benzoyl hydrazine in a solvent, refluxing, cooling, and then filtering and drying the solid precipitated to obtain the benzoyl hydrazone 3-acetyl-7-N,N-diethylaminocoumarin.

4. The production method according to claim 3, characterized by, The 3-acetyl-7-N,N-diethylaminocoumarin is prepared by dissolving 4-(diethylamino)salicylaldehyde and ethyl acetoacetate in a solvent, refluxing, cooling, pouring into ice water to precipitate a solid, and then filtering and drying the solid to obtain the 3-acetyl-7-N,N-diethylaminocoumarin.

5. The production method according to claim 4, characterized by, The molar ratio of 4-(diethylamino)salicylaldehyde to ethyl acetoacetate is 1:(1-3), and the preferred ratio is 1:

2.

6. The preparation method according to claim 3, characterized in that, The molar ratio of 3-acetyl-7-N,N-diethylaminocoumarin to benzoyl hydrazine is 1:(1-3), and the preferred ratio is 1:1.

2.

7. The preparation method according to claim 2, characterized in that, The molar ratio of CuCl2·2H2O, CuBr2 or Cu(NO3)2·3H2O to benzoyl hydrazone 3-acetyl-7-N,N-diethylaminocoumarin is (3-8):1, and the preferred ratio is 5:

1.

8. The preparation method according to claim 2, characterized in that, The copper (II) complex is prepared by dissolving CuCl2·2H2O, CuBr2 or Cu(NO3)2·3H2O and benzoyl hydrazone 3-acetyl-7-N,N-diethylaminocoumarin in a solvent, mixing, stirring at room temperature, filtering, sealing the filtrate, and then placing it at room temperature to obtain crystals.

9. Use of the copper (II) complex of claim 1 in the preparation of an anticancer drug.

10. Use according to claim 9, characterized in that, The anticancer drug includes an anti-breast cancer drug, an anti-cervical cancer drug, an anti-liver cancer drug, or an anti-lung cancer drug.

Images