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Binuclear Schiff base copper(II) complexes: Syntheses, crystal structures, HSA interaction and anti-cancer properties
Inorganica Chimica Acta 499 (2020) 119186
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Research paper
Binuclear Schiff base copper(II) complexes: Syntheses, crystal structures, HSA interaction and anti-cancer properties ⁎
Shengshi Jianga,1, Honghui Nia,1, Fen Liub, Shanshan Gua, Ping Yub, , Yi Goua,b,c,
T
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a
Nantong Hospital of Traditional Chinese Medicine, 41 Jianshe Road, Nantong 226001, China School of Pharmacy, Nantong University, Nantong, Jiangsu, China c Affiliated Hospital, Guilin Medical College, Guilin, Guangxi, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Schiff base Binuclear copper(II) complexes Anticancer activity HSA interaction
Three binuclear Schiff base copper(II) complexes: [Cu2(HL1)2(CH3OH)(NO3)]·NO3 (1), [Cu2(HL2)2(CH3OH)2]·2NO3 (2), and [Cu2(HL3)2(C2H5OH)2]·2NO3 (3) [HL1 = (E)-N′-(5-fluoro-2-hydroxybenzylidene)benzohydrazide, HL2 = (E)-N′-(5-chloro-2-hydroxybenzylidene)benzohydrazide, and HL3 = (E)N′-(5-bromo-2-hydroxybenzylidene)benzohydrazide], were synthesized and characterized employing various spectroscopic techniques. X-ray crystallography analysis revealed complexes 1–3 to have similar molecular structures. Results from the MTT assay revealed that all copper complexes displayed greater cytotoxicity toward several tumor cell types than cisplatin or the Schiff-base ligands. Further investigations revealed that complex 1 can induce mitochondrion-mediated apoptotic cell death. In addition, the interaction between these Cu(II) complexes and human serum albumin (HSA) was also investigated.
1. Introduction Metal-based drugs, especially those based on transition metals, play important biological roles and can be used in chemotherapy [1–3]. Among metal-based drugs, cisplatin was the first inorganic compound to be widely used in cancer treatment. Although platinum-based chemotherapeutic drugs are regarded as quite effective in cancer treatment, their clinical applications are restricted by the frequent development of drug resistance [4,5]. Additional limitations of platinumbased drugs include their cytotoxic activity in healthy tissues [6,7]. Therefore, compounds with greater selectivity and anti-proliferative activity than platinum-based drugs are needed for the treatment of solid tumors. In this context, copper complexes have displayed encouraging characteristics as perspective anticancer agents [8]. In contrast to platinum, copper is a trace element that is necessary to the human body. Many important enzymes and proteins require the participation and activation of trace amounts of copper [9,10]. Notably, evidence exists suggesting that a number of types of cancer cells, such as those of blood cancer and breast cancer, take up copper from the environment at higher levels than healthy cells [8,9]. Therefore, research aimed at designing and developing copper complexes for cancer treatment has been actively pursued. Since Schiff bases have a variety of metal-chelating properties,
inherent biological activity, and structural flexibility, they can be finetuned with the goal of using them for specific biological applications [11,12]. Schiff bases can act as monodentate or multidentate chelating ligands to coordinate with many transition metal cations [13–15]. Schiff base metal complexes have gained increasing attention and are being researched to develop new chemotherapeutic agents [16,17]. Interestingly, a variety of research found that the anti-cancer activity of Schiff bases copper complexes was higher than that of the free ligands [18–22]. Therefore, the study of novel Schiff base-based copper complexes seems poised to contribute to the development of a new generation of anticancer metal-based drugs. Against this background, we have designed and synthesized three binuclear Schiff base-based copper(II) complexes (Scheme 1). These complexes’ structures were characterized by way of infrared (IR) spectroscopy, elemental analysis, X-ray crystallography, and electrospray ionization-mass spectrometry. Furthermore, we investigated the cytotoxic activity of these novel copper(II) complexes against four tumor cell lines, HeLa (human epithelial carcinoma cell line), BEL-7402 (human hepatocellular carcinoma cell line), MCF-7 (human breast cancer cell line), and MGC-803 (human gastric carcinoma cell line), and a non-tumor cell line, WI38 (normal human fetal lung fibroblast cell line). We also examined the potential mechanisms of complex 1 in HeLa cells to identify promising novel chemotherapeutic drugs.
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Corresponding authors at: Nantong Hospital of Traditional Chinese Medicine, 41 Jianshe Road, Nantong 226001, China (Y. Gou). E-mail addresses: [email protected] (P. Yu), [email protected] (Y. Gou). 1 Author contributions: Honghui Ni and Shengshi Jiang contributed equally to this work. https://doi.org/10.1016/j.ica.2019.119186 Received 6 July 2019; Received in revised form 26 September 2019; Accepted 1 October 2019 Available online 03 October 2019 0020-1693/ © 2019 Elsevier B.V. All rights reserved.
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Scheme 1. Synthesis of the copper(II) complexes.
2. Experimental section
2.2. Crystal structure determination
All the chemicals and solvents were purchased from Aldrich and used without further purification. The JC-1 kit (mitochondrial membrane potential assay) and the Annexin V-FITC apoptosis detection kit were purchased from Beyotime. The elemental analyzes (C, H, and N) were carried out using a Perkin-Elmer 2400 analyzer. The infrared spectra (KBr pellets) were recorded using an Interspec 2020 FTIR spectrophotometer. The circular dichroism measurements were recorded on a Jasco-810 model spectropolarimeter.
The X-ray crystallography data of the Cu(II) complexes were collected on a Bruker SMART APEX2 with Mo-Kα radiation (λ = 0.71069). The experimental structure was obtained using the Olex-2 program [26]. Non-hydrogen atoms are refined anisotropic thermal parameters, uploading CIF format data in the Cambridge Crystal Data Center (CCDC-1922802 for 1, CCDC-1011412 for 2 and CCDC-1922803 for 3). The crystal structures of the complexes were drawn using diamond 3.1 program. The crystallographic data of complexes 1–3 are reported in Tables 1 and 2.
2.1. Synthesis
2.3. Interactions between human serum albumin and the copper(II) complexes
2.1.1. Synthesis of complexes 1–3 The (E)-N′-(5-fluoro-2-hydroxybenzylidene)benzohydrazide (HL1) ligand, the (E)-N′-(5-chloro-2-hydroxybenzylidene)benzohydrazide (HL2) ligand, and the (E)-N′-(5-bromo-2-hydroxybenzylidene)benzohydrazide (HL3) ligand were synthesized adapting the reported procedures [23–25]. The ligand (HL1, HL2, or HL3, 0.5 mmol) was dissolved in 40 mL of a methanol or ethanol solvent and Cu(NO3)2·3H2O (0.5 mmol) was added to the solution thus obtained. This solution was then refluxed for 1 h. It was then allowed to cool to room temperature and filtered. A few days later, dark green block crystals of the Cu(II) complexes were obtained from the filtrate upon slow evaporation of the solvent [Cu2(HL1)2(CH3OH)(NO3)]·NO3 (1). Yield: 73%. Elemental analysis results calculated for C29H24Cu2F2N6O11 (796.01): C, 43.67; H, 3.03; N, 10.54; found: C, 43.75; H, 3.16; N, 10.43. IR (KBr, cm−1): 3448w, 3068w, 2831w, 1998w, 1776w, 1559s, 1476w, 1352m, 1235m, 1030m, 1007w, 905m, 868w, 804w, 620w, 571w. Mass spectrometry electrospray ionization (MS ESI) m/z 639.00 ([M−2NO3−CH3OH−H]+). [Cu2(HL2)2(CH3OH)2]·2NO3 (2). Yield: 63%. Elemental analysis results calculated for C30 H28 Cl2 Cu2 N6 O12 (859.97): C, 41.77; H, 3.27; N, 9.74; found: C, 41.45; H, 3.06; N, 9.67. IR (KBr, cm−1): 3395w, 3199w, 2842w, 1910w, 1823w, 1603s, 1442w, 1372m, 1191m, 1028m, 999w, 896w, 825w. MS (ESI) m/z 670.93 ([M−2NO3−2CH3OH−H]+). [Cu2(HL3)2(C2H5OH)2]·2NO3 (3). Yield: 67%. Elemental analysis results calculated for C32H30Br2Cu2N6O12 (975.89): C, 39.32; H, 3.09; N, 8.60; found: C, 39.65; H, 2.95; N, 8.41 IR (KBr, cm−1): 3328w, 3180w, 2968w, 1993w, 1906w, 1599s, 1406w, 1035m, 886w, 824w, 795w, 699w, 651w, 571w, 522w. MS (ESI) m/z 762.83 ([M−2NO3−2C2H5OH−H]+).
The interactions between human serum albumin (HSA) and three copper(II) complexes were studied by UV–visible absorption and circular dichroism (CD) spectroscopy under physiological conditions. Absorption spectra were obtained at 298 K across 250–650 nm using a 1 cm quartz cell. The concentration of HSA and each copper(II) complex was kept at 10 µM for the UV–visible absorption measurements. The CD measurements of HSA in the absence and presence of the copper(II) complexes were carried out at 298 K under constant N2 flush over the wavelength range of 200–300 nm. The concentrations of HSA and each Table 1 Crystal data for complexes 1–3.
2
Cu complex
1
2
3
Empirical formula Formula weight Crystal system Space group a (Å) b (Å) c (Å) α (°) β (°) γ (°) V (Å3) T/K Z R1 (obs data) wR2 (all data) GOOF CCDC
C29H24Cu2F2N6O11
C30 H28 Cl2 Cu2 N6 O12 862.58 Triclinic P-1 7.4839(13) 11.0673(19) 28.910(5) 102.591(3) 95.889(2) 98.472(2) 841.1(3) 296.15 1 0.0292 0.0845 1.072 1011412
C32H30Br2Cu2N6O12
797.62 Triclinic P-1 9.253(7) 9.338(7) 11.101(9) 66.540(10) 66.118(10) 66.799(9) 773.0(10) 296.15 1 0.0480 0.1598 1.065 1922802
977.52 Monoclinic P21/c 9.4084(14) 14.848(2) 13.645(2) 90 106.503(2) 90 1827.6(5) 296.15 2 0.0366 0.0984 1.059 1922803
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BioTek Synergy HT microplate reader at a wavelength of 570 nm.
Table 2 Selected bond lengths [Å] and angles [°] in complexes 1–3.
2.6. Apoptosis detection assay
1 Cu1–O1 Cu1–N2 Cu1–O2 Cu1–O2i Cu1–O2 O2–Cu1i O1–Cu1–O2i
1.970(3) 1.925(3) 1.938(3) 1.974(3) 1.974(3) 1.974(3) 104.98(12)
O2–Cu1–O1 O2–Cu1–O2i O2–Cu1–Cu1i O2–Cu1–Cu1i N2–Cu1–O1 N2–Cu1–O2 N2–Cu1–O2i
169.68(12) 80.29(12) 39.70(8) 40.58(9) 80.14(13) 92.14(13) 161.34(15)
1.9601(14) 1.9833(13) 1.9447(14) 1.9366(16) 1.9833(13) 143.41(4) 104.59(6)
O2–Cu1i–Cu1 O2–Cu1–O1 O2–Cu1–O2i N2–Cu1–Cu1i N2–Cu1–O1 N2–Cu1–O2 N2–Cu1–O2i
39.64(4) 169.52(6) 80.24(6) 131.29(5) 80.90(6) 92.12(6) 164.40(7)
1.953(2) 1.937(2) 1.9360(19) 1.9713(19) 1.9713(19) 145.28(6) 81.01(9)
N2–Cu1–O2i O2–Cu1–Cu1i O2i–Cu1–Cu1i O2–Cu1–O1 O2–Cu1–N2 O1–Cu1–O2i N2–Cu1–Cu1i
170.34(10) 40.83(5) 39.95(6) 172.66(8) 92.31(9) 105.48(8) 132.78(7)
The annexin V/PI protocol was implemented to study the apoptosis cells [32]. Briefly, 2 × 105 HeLa cells were seeded in a 6-well plate. When the cells grew to 70–80% convergence, they were treated with complex 1 (0.5 µM and 1.0 µM) for 24 h. The cells were then collected by centrifugation, washed with cold PBS, and stained according to the instructions of the Annexin V-FITC Apoptosis Detection kit.
2 Cu1–O1 Cu–O2i Cu1–O2 Cu1–N2 O2–Cu1i O1–Cu1–Cu1i O1–Cu1–O2
2.7. Mitochondrial membrane potential detection The membrane-permeable 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) probe was used to evaluate mitochondrion-mediated apoptosis [33]. For this purpose, 2 × 105 HeLa cells were seeded in a six-well plate and allowed to grow up to 70–80% convergence. The cells were then treated with complex 1 (0.5 µM and 1.0 µM) for 24 h, collected by centrifugation, and washed with cold PBS. A 500 µL aliquot of a JC-1 staining solution was then added to each well, and the plate was incubated in the dark for 30 min. The cells were centrifuged for 5 min (1500 rpm), washed twice with cold PBS, and any change in mitochondrial membrane potential detected by flow cytometry.
3 Cu1–O1 Cu1–N2 Cu1–O2 Cu1–O2i O2–Cu1i O1–Cu1–Cu1i N2–Cu1–O1
2.8. Western blot analysis For this analysis, 1 × 106 HeLa cells were seeded in a 10 cm culture dish. Once the cells grew to 70–80% convergence, they were treated with a certain concentration (0.5 µM and 1.0 µM) of complex 1 for 48 h. The cells were collected by centrifugation and lysed in a radioimmunoprecipitation assay buffer (Beyotime, China; effective cracking components: 1% NP-40, 0.5% deoxycholate, 0.1% SDS). The proteins present in the various samples were separated on a 12% SDS-polyacrylamide gel and electrotransferred to polyvinylidene difluoride (PVDF) membranes (0.22 µm). The PVDF membranes were then blocked with 5% non-fat dry milk in TBST and probed with antibodies against β-actin (1:1000 dilution), cleaved caspase-3 (1:1000 dilution), cleaved caspase-9 (1:1000 dilution), Bcl-xl (1:1000 dilution), Bcl-2 (1:1000 dilution), Bax (1:1000 dilution), Bad (1:1000 dilution) and cytochrome c (1:1000 dilution) followed by secondary antibodies conjugated with horseradish peroxidase (1:3000 dilution). The blots were visualized using Amersham ECL Plus western blotting detection reagents.
copper(II) complex were all stabilized at 1.0 µM. In order to collect additional information on the binding of the Cu (II) complexes to HSA, docking studies were carried out using Autodock Vina [27,28]. The crystal structure of HSA was obtained from the protein data bank (1E7H). This structure was modified to include an increase in polar hydrogen atoms and removal of water molecules and ligands. The structures of the Cu(II) complexes used for docking studies were optimized in the water phase starting with the structures obtained by modifying the X-ray structures of complexes 1–3. The calculation was performed using the GAMESS program package with the hybrid functional B3LYP [29]. The LANL2DZ basis set was used for the copper atom, and the 6-31G(d, p) basis set was utilized for the other elements [30,31]. During docking studies, the single bonds of the Cu(II) complexes were permitted to rotate, whereas the HSA structure was kept rigid. 2.4. Cell lines and cell culture
3. Results and discussion
The human hepatocellular carcinoma cell line BEL-7402, the human epithelial carcinoma cell line HeLa, the human gastric carcinoma cell line MGC-803, the human breast cancer cell line MCF-7, and the normal human fetal lung fibroblast cell line WI38 were obtained from the China Center for Type Culture Collection. The cells were cultured in an incubator at 37 °C/5% CO2 in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (FBS).
3.1. Characterization of 1–3 All complexes were stable in the air at room temperature. The stability of complexes 1–3 in water was analyzed by UV–Vis absorption spectroscopy. As shown in Fig. S1, the isosbestic points at ca. 331 nm in the spectra of 1–3 were observed, indicating that ligand-exchange occurred [34]. In the solid state, although complexes 1–3 have very similar structures (Fig. 1A–C), they have different space groups, which is likely due to different solvent molecules coordinated to copper(II) centers. The Schiff-base ligand moiety of these complexes contains phenolate-O atoms that connect two neighboring copper units by way of two μ-O bridges to form binuclear structures. In all three complexes, each Cu(II) center is characterized by a distorted square-pyramidal coordination geometry, with the basal donor atoms coming from the phenolate-O (O2), amide-O (O1), and imine-N (N2) atoms of the Schiff-base ligand and a symmetry-related phenolate-O (O2i). In complex 1, the apical axis of the square-based pyramid is occupied by an NO3− anion or a methanol molecule (substitutional disorder). On the other hand, in
2.5. MTT assay The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay is used to measure the cytotoxicity of the Schiff-base ligands (HL1, HL2, and HL3) and their copper complexes. Briefly, in this assay, the cells were cultured in a 96-well plate at a concentration of 180 μL (about 5 × 103 cells) per well. Cells were allowed to grow up to 70–80% convergence, and then they were treated with different concentrations of test compounds for 48 h. Subsequently, 10 µL of MTT were added to each well, and the plate was incubated for 4 h at 37 °C. The media were then removed and to each well were added 100 µL of DMSO, and the absorbance value of each well was measured in a 3
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Fig. 1. (A) X-ray crystal structure of 1 (Symmetry code: i = 1 − x, 1 − y, 2 − z). The coordinating NO3− anion and methanol molecule are substitutional disorder. Hydrogen atoms and uncoordinated NO3− anion are omitted for clarity. (B) The coordination environment of complex 2 in the solid state (Symmetry code: i = −x, 1 − y, 1 − z). Hydrogen atoms and counter NO3− anion are omitted for clarity. (C) The coordination environment of complex 3 in the solid state (Symmetry code: i = 2 − x, 1 − y, 1 − z). Hydrogen atoms and counter NO3− anion are omitted for clarity. (D) Perspective view of a one-dimensional chain formed by π⋯π interactions in 2. (E) Perspective view of π⋯π interactions in 3.
complexes 2 and 3, the more weakly bound apical donor O atoms are supplied by a coordinating methanol or ethanol molecule, respectively. The Cu–O and Cu–N bond lengths of complexes 1–3 are within the ranges reported for other similar copper complexes [35–38]. In a similar structure, a symmetric μ2-Ophenolate bridging mode was reported [35], whereas asymmetric μ2-Ophenolate bridging modes were observed in complexes 1–3, given that the two Cu–Ophenolate bond lengths are different from each other (Cu1–O2 = 1.938(3) Å and Cu1–O2i = 1.974(3) Å in 1, Cu1–O2 = 1.9447(14) Å and Cu1–O2i = 1.9833(13) Å in 2, and Cu1–O2 = 1.9360(19) Å and Cu1–O2i = 1.9713(19) Å in 3). Owing to the Jahn–Teller effect, the lengths of the bonds linking the copper centers to the oxygen centers in the axial positions (2.189–2.307 Å) are longer than their counterparts linking the copper centers to the oxygen centers in the basal plane (1.936–1.983 Å). The Cu⋯Cu distances in complexes 1–3 are 2.9902(16), 3.0038(6), and 2.9760(8) Å respectively. These values are very close to those of copper complexes with similar coordination spheres reported in the past [35–38]. In complexes 1–3, two coordinating tridentate Schiff-base ligands are not on a plane. In all three complexes, however, two deprotonated phenoxo oxygen centers coordinate the Cu(II) center to form the bridging Cu2O2 core, which is planar. In the solid state, no π⋯π interactions were identified in complex 1. As can be evinced from Fig. 1D and E, however, complexes 2 and 3 were linked into one-dimensional zigzag chains by π⋯π interactions (face-to-face distance = 3.473 Å and center-to-center distance = 3.999 Å, in 2; face-to-face distance = 3.277 Å and center-tocenter distance = 3.794 Å, in 3). Interestingly, the π⋯π interactions in 2 involve two benzene rings in the different ligands in a binuclear molecule and two benzene rings in the different ligands in adjacent binuclear molecules. On the other hand, the π⋯π interactions in 3 involve two benzene rings in the same ligand in a binuclear molecule and two benzene rings in the same ligand in an adjacent binuclear molecule.
was found at m/z 639.00, which could be identified with isotopic envelopes corresponding to [Cu2(HL1)(L1)]+ (fit: 639.00), thus indicating that the uncoordinated NO3− and the coordinated NO3− and methanol were lost. The ESI-MS of complex 2 is characterized by an intense signal at m/z = 670.93 due to [Cu2(HL2)(L2)]+ (fit: 670.94) at 0 eV, which means that the uncoordinated NO3− and the coordinated methanol were lost. The ESI-MS of complex 3 is characterized by an intense signal at m/z = 762.83, which can be identified with isotopic envelopes to be due to [Cu2(HL3)(L3)]+ (fit: 762.83), implying that the uncoordinated NO3− and the coordinated ethanol molecules were lost. These results corroborate the structures found in solid state. 3.3. Interaction between HSA and the copper(II) complexes The binding properties of anticancer agents to HSA provide information on their uptake and distribution in tumor cells and afford an insight into their mechanism of action and metabolism [39,40]. Exploring the interactions of copper complexes 1–3 with HSA is therefore of great scientific interest. UV–visible and CD spectra are often used to probe the interaction between biological macromolecules and small molecular species. To gain an insight into the nature of HSA-copper complex interaction, the UV–visible spectra of the copper complexes, HSA and HSAcopper complex system were recorded. As can be evinced from the data reported in Fig. 2A, at 349 nm, the absorbance of complex 1 showed a hypochromism effect in the presence of HSA. This effect was also observed with complex 2 and complex 3, and reflected a complex-HSA interaction rather than a simple spectral overlap. To prove the possible influence of copper complex binding on the secondary structure of HSA, the CD spectroscopy of HSA in the presence and absence of the complexes were measured. As shown in Fig. 2B, HSA in the absence of the copper complexes dispaly negative absorption bands with maxima at 208 nm (π → π*) and 222 nm (n → π*), which is characteristic of αhelical structure of HSA [41]. The binding of each copper complex to HSA caused an intensity decrease for both 208 nm and 222 nm negative bands, indicating a decrease in the α-helical content of HSA. From the above results, we can conclude that each copper complex can interact with HSA and cause conformational change in the HSA protein. Docking studies were also conducted to afford an insight into the
3.2. Mass spectrometry The structures of the Cu(II) complexes were also established by means of electrospray ionization mass spectrometry (ESI-MS) at room temperature (Fig. S2). A peak due to the parent cluster ion of complex 1 4
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Fig. 2. (A) UV–vis absorption spectra of HSA in the presence of different copper complexes. [HSA] = 10 μM; [copper complex] = 10 μM. (B) CD spectra of HSA in the presence of different copper complexes. [HSA] = 1 μM; [copper complex] = 1 μM; T = 298 K; pH = 7.4.
Fig. 3. Molecular docked models of the copper complexes with HSA. (A) The overall structure of HSA complex. (B) The copper complexes are buried beneath the protein surface. (C) The copper complexes interacting with a variety of amino acid residues of HSA.
increased their cytotoxicity. The IC50 values of complexes 1–3 were significantly lower than that of the cisplatin. In particular, the IC50 values associated with the use of 1 were 5.65, 36.13, 23.58, and 5.36fold lower than those associated with the use of cisplatin in Bel-7402, HeLa, MCF-7, and MGC-803 cells, respectively. The IC50 values associated with the use of 2 were 3.33-, 20.47-, 19.73-, and 3.41-fold lower than those associated with the use of cisplatin in Bel-7402, HeLa, MCF7, and MGC-803 cells, respectively. Similarly, the IC50 values associated with the use of 3 were 2.67-, 14.98-, 9.29-, and 3.23-fold lower than those associated with the use of cisplatin in Bel-7402, HeLa, MCF-7, and MGC-803 cells, respectively. It is notable that the potency of the 1–3 to kill cancer cells followed the order 1 > 2 > 3. The result can probably be explained by the fact that the different halogen substituent (F, Cl or Br) led to a lipophilicitydependent decline in anticancer activity [42]. Moreover, we also determined the cytoxicity of these copper complexes toward healthy cells. The comparison between the IC50 values measured with WI-38 cells and those measured with cancer cells indicated that complexes 1–3 have low selectivity for cancer cells over healthy cells, suggesting that targeted delivery of these copper complexes by, for instance, biomolecules, liposomes, or nanoparticles would be preferable [43]. In addition, upon confirmation of the good anticancer activity displayed by complex 1, further cellular (in HeLa cells) assays were carried out to understand this complex’s mechanism of action.
potential interaction mode of these copper complexes with HSA. As depicted in Fig. 3, the copper complexes were located in the hydrophobic cavity of subdomain IIA delimited by residues His242, His288, Ala291, Lys199, Leu219, Leu260, Leu238, Arg218, Arg222, Arg257, Phe156, Phe157, and Trp214 The binding mode of these complexes suggests that they engage in hydrogen bonding interactions with Arg218. These complexes also engage in hydrophobic interactions with Trp214, Phe156, Phe157, Leu219, Leu238, and Val241. The F, Cl, and Br moieties (the closest ones to Trp214) of these complexes are located about 5.37, 4.78, and 4.74 Å away from Trp214, respectively. 3.4. Cytotoxicity of Cu(II) complexes in vitro To determine the anti-proliferative efficacy of complexes 1–3, we performed MTT assays using four cancer cell lines (Bel-7402, HeLa, MCF-7, and MGC-803) and one normal lung fibroblast cell line (WI38). The widely used metallodrug cisplatin was included as a control. The IC50 (concentrations required to induce 50% viability values obtained in these experiments are summarized in Table 3. The values of in vitro cytotoxicity measured for complexes 1–3 exceed those measured for the Schiff-base ligands by themselves, indicating that these ligands’ coordination to Cu(II) significantly Table 3 Inhibition of human cancer cell lines growth (IC50, μM) for the complexes 1–3. Compound
HL1 HL2 HL3 1 2 3 Cisplatin
3.5. Cell apoptosis
Cell growth inhibition, IC50 ± SD (μM) Bel-7402
Hela
MCF-7
MGC-803
WI-38
> 30 > 30 > 30 2.7 ± 0.2 4.5 ± 0.5 5.6 ± 0.8 15.1 ± 1.2
> 30 > 30 > 30 0.5 ± 0.2 0.9 ± 0.1 1.2 ± 0.2 18.4 ± 2.1
> 30 > 30 > 30 0.8 ± 0.1 1.0 ± 0.1 2.1 ± 0.2 19.3 ± 1.9
> 30 > 30 > 30 3.0 ± 0.4 4.8 ± 0.6 5.0 ± 0.6 16.3 ± 1.7
> 30 > 30 > 30 3.2 ± 0.6 3.4 ± 0.2 6.4 ± 0.7 14.7 ± 1.8
Most metal-based complexes exert their cytotoxicity by activating the apoptosis pathway, so we investigated features related to this pathway [44–47]. To determine whether apoptosis occurred in cancer cells, we treated HeLa cells with complex 1 and then evaluated the apoptosis of HeLa cells using the Annexin-V/PI staining protocols (Fig. 4). Evidence indicated that complex 1 efficiently induced apoptosis in HeLa cells in a concentration-dependent manner. Even at the low concentration of 1.0 µM, complex 1 induced a large proportion of 5
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Fig. 4. Apoptosis of HeLa cells treated with complex 1: (a) Control, (b) Complex 1 = 0.5 µM, and (c) Complex 1 = 1.0 µM.
HeLa cells to undergo early – (11.6%) and late – (40.7%) stage apoptosis.
mediated apoptosis in cancer cells [52]. After HeLa cells were treated with complex 1 (0.5 µM and 1.0 µM) for 24 h, evidence suggested that a decrease in the expression level of the anti-apoptotic proteins Bcl-2 and Bcl-xl and an increase in the expression level of the pro-apoptotic protein Bax had been caused (Fig. 5B). As a result, the levels of cleaved -caspase-9, -caspase-3 and cytochrome c were up-regulated, thus regulating the apoptosis pathway of HeLa cells (Fig. 5B). These results suggested the activation of mitochondrion-mediated apoptosis in HeLa cells (Fig. 5B).
3.6. Mitochondrial membrane potential Various cell apoptotic pathways are activated as a consequence of mitochondrial dysfunction (ΔΨm) [48,49]. To determine the ΔΨm induced by complex 1, we used the lipophilic fluorescent probe JC-1 and analyzed by flow cytometry. As can be evinced from the data reported in Fig. 5A, after treatment of HeLa cells with complex 1, a remarkable decrease in ΔΨm was observed, which was reflected by a decrease of Jaggregates (red fluorescence) and concurrent increase of J-monomers (green fluorescence). Members of the Bcl-2 protein family, including anti-apoptotic proteins like Bcl-2 and Bcl-xL, and pro-apoptotic proteins like Bad and Bax, have been described as key regulators of apoptosis [50,51]. Numerous metal-based anticancer complexes have been developed to target members of the Bcl-2 protein family and induce mitochondrion-
4. Conclusion In this study, three binuclear Schiff base-based copper(II) complexes (1–3) were synthesized and characterized. Single-crystal X-ray crystallography analysis results revealed each Cu(II) atom in all complexes to be characterized by a distorted square-pyramidal coordination geometry. These copper(II) complexes can interact with subdomain IIA cavity of HSA and cause conformational change in the HSA protein. All
Fig. 5. (A) Assay of HeLa cells’ mitochondrial membrane potential with JC-1 as the fluorescence probe staining method. (B) Western blot analysis of the levels of Bcl2, Bcl-xl, Bax, Bad, cleaved caspase-3, cleaved caspase-9, and cytochrome c after being treated with complex 1 (0.5 µM and 1.0 µM) for 24 h. 6
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three copper complexes synthesized were found to have significant anticancer activity (0.5–5.6 μM) as determined conducting in vitro cytotoxicity assays. The ability of complexes 1–3 to kill selected tumor cells followed the order 1 > 2 > 3. Notably, the mechanism of the cytotoxic activity of these copper complexes may consist in triggering the mitochondrion-mediated apoptosis pathway. The results of our study will be helpful in the design, development, and synthesis of new binuclear Schiff base-based copper(II) complexes with potent anticancer activity.
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Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by the projects No. 81503161, No. WKZL2018030 and Administration of Traditional Chinese Medicine of Jiangsu Province (YB201960). The authors are grateful to the Baise University for partial support of this work. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ica.2019.119186. References [1] I.E. Leon, J.F. Cadavid-Vargas, A.L. Di Virgilio, S.B. Etcheverry, Curr. Med. Chem. 24 (2017) 112–148. [2] M.R. Gill, K.A. Vallis, Chem. Soc. Rev. 48 (2019) 540–557. [3] A. Bijelic, M. Aureliano, A. Rompel, Angew. Chem. Int. Ed. Engl. 58 (2019) 2980–2999. [4] U. Ndagi, N. Mhlongo, M.E. Soliman, Drug Des. Dev. Ther. 11 (2017) 599–616. [5] K.B. Garbutcheon-Singh, M.P. Grant, B.W. Harper, A.M. Harper, Krause-Heuer, M. Manohar, N. Orkey, J.R. Aldrich-Wright, Curr. Top. Med. Chem. 11 (2011) 521–542. [6] T. Sakaeda, K. Kadoyama, Y. Okuno, Int. J. Med. Sci. 8 (2011) 487–491. [7] N.J. Wheate, S. Walker, G.E. Craig, R. Oun, Dalton Trans. 39 (2010) 8113–8127. [8] A. Gupte, R.J. Mumper, Cancer Treat. Rev. 35 (2009) 32–46. [9] F. Tisato, C. Marzano, M. Porchia, M. Pellei, C. Santini, Med. Res. Rev. 30 (2010) 708–749. [10] D. Denoyer, S. Masaldan, S. La Fontaine, M.A. Cater, Metallomics 7 (2015) 1459–1476. [11] K. Sztanke, A. Maziarka, A. Osinka, M. Sztanke, Bioorg. Med. Chem. 21 (2013) 3648–3666. [12] A. Hameed, M. Al-Rashida, M. Uroos, S. Abid Ali, K.M. Khan, Expert Opin. Ther. Pat. 27 (2017) 63–79. [13] B. Samanta, J. Chakraborty, S. Shit, S.R. Batten, P. Jensen, J.D. Masuda, S. Mitra, Inorg. Chim. Acta 360 (2007) 2471–2484. [14] G.M. Abu El-Reash, O.A. El-Gammal, A.H. Radwan, Spectrochim. Acta A: Mol. Biomol. Spectrosc. 121 (2014) 259–267. [15] N.H. Al-Shaalan, Molecules 16 (2011) 8629–8645. [16] M.A. Malik, O.A. Dar, P. Gull, M.Y. Wani, A.A. Hashmi, MedChemComm 9 (2017) 409–436. [17] C. Santini, M. Pellei, V. Gandin, M. Porchia, F. Tisato, C. Marzano, Chem. Rev. 114
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Research paper
Binuclear Schiff base copper(II) complexes: Syntheses, crystal structures, HSA interaction and anti-cancer properties ⁎
Shengshi Jianga,1, Honghui Nia,1, Fen Liub, Shanshan Gua, Ping Yub, , Yi Goua,b,c,
T
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a
Nantong Hospital of Traditional Chinese Medicine, 41 Jianshe Road, Nantong 226001, China School of Pharmacy, Nantong University, Nantong, Jiangsu, China c Affiliated Hospital, Guilin Medical College, Guilin, Guangxi, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Schiff base Binuclear copper(II) complexes Anticancer activity HSA interaction
Three binuclear Schiff base copper(II) complexes: [Cu2(HL1)2(CH3OH)(NO3)]·NO3 (1), [Cu2(HL2)2(CH3OH)2]·2NO3 (2), and [Cu2(HL3)2(C2H5OH)2]·2NO3 (3) [HL1 = (E)-N′-(5-fluoro-2-hydroxybenzylidene)benzohydrazide, HL2 = (E)-N′-(5-chloro-2-hydroxybenzylidene)benzohydrazide, and HL3 = (E)N′-(5-bromo-2-hydroxybenzylidene)benzohydrazide], were synthesized and characterized employing various spectroscopic techniques. X-ray crystallography analysis revealed complexes 1–3 to have similar molecular structures. Results from the MTT assay revealed that all copper complexes displayed greater cytotoxicity toward several tumor cell types than cisplatin or the Schiff-base ligands. Further investigations revealed that complex 1 can induce mitochondrion-mediated apoptotic cell death. In addition, the interaction between these Cu(II) complexes and human serum albumin (HSA) was also investigated.
1. Introduction Metal-based drugs, especially those based on transition metals, play important biological roles and can be used in chemotherapy [1–3]. Among metal-based drugs, cisplatin was the first inorganic compound to be widely used in cancer treatment. Although platinum-based chemotherapeutic drugs are regarded as quite effective in cancer treatment, their clinical applications are restricted by the frequent development of drug resistance [4,5]. Additional limitations of platinumbased drugs include their cytotoxic activity in healthy tissues [6,7]. Therefore, compounds with greater selectivity and anti-proliferative activity than platinum-based drugs are needed for the treatment of solid tumors. In this context, copper complexes have displayed encouraging characteristics as perspective anticancer agents [8]. In contrast to platinum, copper is a trace element that is necessary to the human body. Many important enzymes and proteins require the participation and activation of trace amounts of copper [9,10]. Notably, evidence exists suggesting that a number of types of cancer cells, such as those of blood cancer and breast cancer, take up copper from the environment at higher levels than healthy cells [8,9]. Therefore, research aimed at designing and developing copper complexes for cancer treatment has been actively pursued. Since Schiff bases have a variety of metal-chelating properties,
inherent biological activity, and structural flexibility, they can be finetuned with the goal of using them for specific biological applications [11,12]. Schiff bases can act as monodentate or multidentate chelating ligands to coordinate with many transition metal cations [13–15]. Schiff base metal complexes have gained increasing attention and are being researched to develop new chemotherapeutic agents [16,17]. Interestingly, a variety of research found that the anti-cancer activity of Schiff bases copper complexes was higher than that of the free ligands [18–22]. Therefore, the study of novel Schiff base-based copper complexes seems poised to contribute to the development of a new generation of anticancer metal-based drugs. Against this background, we have designed and synthesized three binuclear Schiff base-based copper(II) complexes (Scheme 1). These complexes’ structures were characterized by way of infrared (IR) spectroscopy, elemental analysis, X-ray crystallography, and electrospray ionization-mass spectrometry. Furthermore, we investigated the cytotoxic activity of these novel copper(II) complexes against four tumor cell lines, HeLa (human epithelial carcinoma cell line), BEL-7402 (human hepatocellular carcinoma cell line), MCF-7 (human breast cancer cell line), and MGC-803 (human gastric carcinoma cell line), and a non-tumor cell line, WI38 (normal human fetal lung fibroblast cell line). We also examined the potential mechanisms of complex 1 in HeLa cells to identify promising novel chemotherapeutic drugs.
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Corresponding authors at: Nantong Hospital of Traditional Chinese Medicine, 41 Jianshe Road, Nantong 226001, China (Y. Gou). E-mail addresses: [email protected] (P. Yu), [email protected] (Y. Gou). 1 Author contributions: Honghui Ni and Shengshi Jiang contributed equally to this work. https://doi.org/10.1016/j.ica.2019.119186 Received 6 July 2019; Received in revised form 26 September 2019; Accepted 1 October 2019 Available online 03 October 2019 0020-1693/ © 2019 Elsevier B.V. All rights reserved.
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Scheme 1. Synthesis of the copper(II) complexes.
2. Experimental section
2.2. Crystal structure determination
All the chemicals and solvents were purchased from Aldrich and used without further purification. The JC-1 kit (mitochondrial membrane potential assay) and the Annexin V-FITC apoptosis detection kit were purchased from Beyotime. The elemental analyzes (C, H, and N) were carried out using a Perkin-Elmer 2400 analyzer. The infrared spectra (KBr pellets) were recorded using an Interspec 2020 FTIR spectrophotometer. The circular dichroism measurements were recorded on a Jasco-810 model spectropolarimeter.
The X-ray crystallography data of the Cu(II) complexes were collected on a Bruker SMART APEX2 with Mo-Kα radiation (λ = 0.71069). The experimental structure was obtained using the Olex-2 program [26]. Non-hydrogen atoms are refined anisotropic thermal parameters, uploading CIF format data in the Cambridge Crystal Data Center (CCDC-1922802 for 1, CCDC-1011412 for 2 and CCDC-1922803 for 3). The crystal structures of the complexes were drawn using diamond 3.1 program. The crystallographic data of complexes 1–3 are reported in Tables 1 and 2.
2.1. Synthesis
2.3. Interactions between human serum albumin and the copper(II) complexes
2.1.1. Synthesis of complexes 1–3 The (E)-N′-(5-fluoro-2-hydroxybenzylidene)benzohydrazide (HL1) ligand, the (E)-N′-(5-chloro-2-hydroxybenzylidene)benzohydrazide (HL2) ligand, and the (E)-N′-(5-bromo-2-hydroxybenzylidene)benzohydrazide (HL3) ligand were synthesized adapting the reported procedures [23–25]. The ligand (HL1, HL2, or HL3, 0.5 mmol) was dissolved in 40 mL of a methanol or ethanol solvent and Cu(NO3)2·3H2O (0.5 mmol) was added to the solution thus obtained. This solution was then refluxed for 1 h. It was then allowed to cool to room temperature and filtered. A few days later, dark green block crystals of the Cu(II) complexes were obtained from the filtrate upon slow evaporation of the solvent [Cu2(HL1)2(CH3OH)(NO3)]·NO3 (1). Yield: 73%. Elemental analysis results calculated for C29H24Cu2F2N6O11 (796.01): C, 43.67; H, 3.03; N, 10.54; found: C, 43.75; H, 3.16; N, 10.43. IR (KBr, cm−1): 3448w, 3068w, 2831w, 1998w, 1776w, 1559s, 1476w, 1352m, 1235m, 1030m, 1007w, 905m, 868w, 804w, 620w, 571w. Mass spectrometry electrospray ionization (MS ESI) m/z 639.00 ([M−2NO3−CH3OH−H]+). [Cu2(HL2)2(CH3OH)2]·2NO3 (2). Yield: 63%. Elemental analysis results calculated for C30 H28 Cl2 Cu2 N6 O12 (859.97): C, 41.77; H, 3.27; N, 9.74; found: C, 41.45; H, 3.06; N, 9.67. IR (KBr, cm−1): 3395w, 3199w, 2842w, 1910w, 1823w, 1603s, 1442w, 1372m, 1191m, 1028m, 999w, 896w, 825w. MS (ESI) m/z 670.93 ([M−2NO3−2CH3OH−H]+). [Cu2(HL3)2(C2H5OH)2]·2NO3 (3). Yield: 67%. Elemental analysis results calculated for C32H30Br2Cu2N6O12 (975.89): C, 39.32; H, 3.09; N, 8.60; found: C, 39.65; H, 2.95; N, 8.41 IR (KBr, cm−1): 3328w, 3180w, 2968w, 1993w, 1906w, 1599s, 1406w, 1035m, 886w, 824w, 795w, 699w, 651w, 571w, 522w. MS (ESI) m/z 762.83 ([M−2NO3−2C2H5OH−H]+).
The interactions between human serum albumin (HSA) and three copper(II) complexes were studied by UV–visible absorption and circular dichroism (CD) spectroscopy under physiological conditions. Absorption spectra were obtained at 298 K across 250–650 nm using a 1 cm quartz cell. The concentration of HSA and each copper(II) complex was kept at 10 µM for the UV–visible absorption measurements. The CD measurements of HSA in the absence and presence of the copper(II) complexes were carried out at 298 K under constant N2 flush over the wavelength range of 200–300 nm. The concentrations of HSA and each Table 1 Crystal data for complexes 1–3.
2
Cu complex
1
2
3
Empirical formula Formula weight Crystal system Space group a (Å) b (Å) c (Å) α (°) β (°) γ (°) V (Å3) T/K Z R1 (obs data) wR2 (all data) GOOF CCDC
C29H24Cu2F2N6O11
C30 H28 Cl2 Cu2 N6 O12 862.58 Triclinic P-1 7.4839(13) 11.0673(19) 28.910(5) 102.591(3) 95.889(2) 98.472(2) 841.1(3) 296.15 1 0.0292 0.0845 1.072 1011412
C32H30Br2Cu2N6O12
797.62 Triclinic P-1 9.253(7) 9.338(7) 11.101(9) 66.540(10) 66.118(10) 66.799(9) 773.0(10) 296.15 1 0.0480 0.1598 1.065 1922802
977.52 Monoclinic P21/c 9.4084(14) 14.848(2) 13.645(2) 90 106.503(2) 90 1827.6(5) 296.15 2 0.0366 0.0984 1.059 1922803
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BioTek Synergy HT microplate reader at a wavelength of 570 nm.
Table 2 Selected bond lengths [Å] and angles [°] in complexes 1–3.
2.6. Apoptosis detection assay
1 Cu1–O1 Cu1–N2 Cu1–O2 Cu1–O2i Cu1–O2 O2–Cu1i O1–Cu1–O2i
1.970(3) 1.925(3) 1.938(3) 1.974(3) 1.974(3) 1.974(3) 104.98(12)
O2–Cu1–O1 O2–Cu1–O2i O2–Cu1–Cu1i O2–Cu1–Cu1i N2–Cu1–O1 N2–Cu1–O2 N2–Cu1–O2i
169.68(12) 80.29(12) 39.70(8) 40.58(9) 80.14(13) 92.14(13) 161.34(15)
1.9601(14) 1.9833(13) 1.9447(14) 1.9366(16) 1.9833(13) 143.41(4) 104.59(6)
O2–Cu1i–Cu1 O2–Cu1–O1 O2–Cu1–O2i N2–Cu1–Cu1i N2–Cu1–O1 N2–Cu1–O2 N2–Cu1–O2i
39.64(4) 169.52(6) 80.24(6) 131.29(5) 80.90(6) 92.12(6) 164.40(7)
1.953(2) 1.937(2) 1.9360(19) 1.9713(19) 1.9713(19) 145.28(6) 81.01(9)
N2–Cu1–O2i O2–Cu1–Cu1i O2i–Cu1–Cu1i O2–Cu1–O1 O2–Cu1–N2 O1–Cu1–O2i N2–Cu1–Cu1i
170.34(10) 40.83(5) 39.95(6) 172.66(8) 92.31(9) 105.48(8) 132.78(7)
The annexin V/PI protocol was implemented to study the apoptosis cells [32]. Briefly, 2 × 105 HeLa cells were seeded in a 6-well plate. When the cells grew to 70–80% convergence, they were treated with complex 1 (0.5 µM and 1.0 µM) for 24 h. The cells were then collected by centrifugation, washed with cold PBS, and stained according to the instructions of the Annexin V-FITC Apoptosis Detection kit.
2 Cu1–O1 Cu–O2i Cu1–O2 Cu1–N2 O2–Cu1i O1–Cu1–Cu1i O1–Cu1–O2
2.7. Mitochondrial membrane potential detection The membrane-permeable 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) probe was used to evaluate mitochondrion-mediated apoptosis [33]. For this purpose, 2 × 105 HeLa cells were seeded in a six-well plate and allowed to grow up to 70–80% convergence. The cells were then treated with complex 1 (0.5 µM and 1.0 µM) for 24 h, collected by centrifugation, and washed with cold PBS. A 500 µL aliquot of a JC-1 staining solution was then added to each well, and the plate was incubated in the dark for 30 min. The cells were centrifuged for 5 min (1500 rpm), washed twice with cold PBS, and any change in mitochondrial membrane potential detected by flow cytometry.
3 Cu1–O1 Cu1–N2 Cu1–O2 Cu1–O2i O2–Cu1i O1–Cu1–Cu1i N2–Cu1–O1
2.8. Western blot analysis For this analysis, 1 × 106 HeLa cells were seeded in a 10 cm culture dish. Once the cells grew to 70–80% convergence, they were treated with a certain concentration (0.5 µM and 1.0 µM) of complex 1 for 48 h. The cells were collected by centrifugation and lysed in a radioimmunoprecipitation assay buffer (Beyotime, China; effective cracking components: 1% NP-40, 0.5% deoxycholate, 0.1% SDS). The proteins present in the various samples were separated on a 12% SDS-polyacrylamide gel and electrotransferred to polyvinylidene difluoride (PVDF) membranes (0.22 µm). The PVDF membranes were then blocked with 5% non-fat dry milk in TBST and probed with antibodies against β-actin (1:1000 dilution), cleaved caspase-3 (1:1000 dilution), cleaved caspase-9 (1:1000 dilution), Bcl-xl (1:1000 dilution), Bcl-2 (1:1000 dilution), Bax (1:1000 dilution), Bad (1:1000 dilution) and cytochrome c (1:1000 dilution) followed by secondary antibodies conjugated with horseradish peroxidase (1:3000 dilution). The blots were visualized using Amersham ECL Plus western blotting detection reagents.
copper(II) complex were all stabilized at 1.0 µM. In order to collect additional information on the binding of the Cu (II) complexes to HSA, docking studies were carried out using Autodock Vina [27,28]. The crystal structure of HSA was obtained from the protein data bank (1E7H). This structure was modified to include an increase in polar hydrogen atoms and removal of water molecules and ligands. The structures of the Cu(II) complexes used for docking studies were optimized in the water phase starting with the structures obtained by modifying the X-ray structures of complexes 1–3. The calculation was performed using the GAMESS program package with the hybrid functional B3LYP [29]. The LANL2DZ basis set was used for the copper atom, and the 6-31G(d, p) basis set was utilized for the other elements [30,31]. During docking studies, the single bonds of the Cu(II) complexes were permitted to rotate, whereas the HSA structure was kept rigid. 2.4. Cell lines and cell culture
3. Results and discussion
The human hepatocellular carcinoma cell line BEL-7402, the human epithelial carcinoma cell line HeLa, the human gastric carcinoma cell line MGC-803, the human breast cancer cell line MCF-7, and the normal human fetal lung fibroblast cell line WI38 were obtained from the China Center for Type Culture Collection. The cells were cultured in an incubator at 37 °C/5% CO2 in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (FBS).
3.1. Characterization of 1–3 All complexes were stable in the air at room temperature. The stability of complexes 1–3 in water was analyzed by UV–Vis absorption spectroscopy. As shown in Fig. S1, the isosbestic points at ca. 331 nm in the spectra of 1–3 were observed, indicating that ligand-exchange occurred [34]. In the solid state, although complexes 1–3 have very similar structures (Fig. 1A–C), they have different space groups, which is likely due to different solvent molecules coordinated to copper(II) centers. The Schiff-base ligand moiety of these complexes contains phenolate-O atoms that connect two neighboring copper units by way of two μ-O bridges to form binuclear structures. In all three complexes, each Cu(II) center is characterized by a distorted square-pyramidal coordination geometry, with the basal donor atoms coming from the phenolate-O (O2), amide-O (O1), and imine-N (N2) atoms of the Schiff-base ligand and a symmetry-related phenolate-O (O2i). In complex 1, the apical axis of the square-based pyramid is occupied by an NO3− anion or a methanol molecule (substitutional disorder). On the other hand, in
2.5. MTT assay The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay is used to measure the cytotoxicity of the Schiff-base ligands (HL1, HL2, and HL3) and their copper complexes. Briefly, in this assay, the cells were cultured in a 96-well plate at a concentration of 180 μL (about 5 × 103 cells) per well. Cells were allowed to grow up to 70–80% convergence, and then they were treated with different concentrations of test compounds for 48 h. Subsequently, 10 µL of MTT were added to each well, and the plate was incubated for 4 h at 37 °C. The media were then removed and to each well were added 100 µL of DMSO, and the absorbance value of each well was measured in a 3
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Fig. 1. (A) X-ray crystal structure of 1 (Symmetry code: i = 1 − x, 1 − y, 2 − z). The coordinating NO3− anion and methanol molecule are substitutional disorder. Hydrogen atoms and uncoordinated NO3− anion are omitted for clarity. (B) The coordination environment of complex 2 in the solid state (Symmetry code: i = −x, 1 − y, 1 − z). Hydrogen atoms and counter NO3− anion are omitted for clarity. (C) The coordination environment of complex 3 in the solid state (Symmetry code: i = 2 − x, 1 − y, 1 − z). Hydrogen atoms and counter NO3− anion are omitted for clarity. (D) Perspective view of a one-dimensional chain formed by π⋯π interactions in 2. (E) Perspective view of π⋯π interactions in 3.
complexes 2 and 3, the more weakly bound apical donor O atoms are supplied by a coordinating methanol or ethanol molecule, respectively. The Cu–O and Cu–N bond lengths of complexes 1–3 are within the ranges reported for other similar copper complexes [35–38]. In a similar structure, a symmetric μ2-Ophenolate bridging mode was reported [35], whereas asymmetric μ2-Ophenolate bridging modes were observed in complexes 1–3, given that the two Cu–Ophenolate bond lengths are different from each other (Cu1–O2 = 1.938(3) Å and Cu1–O2i = 1.974(3) Å in 1, Cu1–O2 = 1.9447(14) Å and Cu1–O2i = 1.9833(13) Å in 2, and Cu1–O2 = 1.9360(19) Å and Cu1–O2i = 1.9713(19) Å in 3). Owing to the Jahn–Teller effect, the lengths of the bonds linking the copper centers to the oxygen centers in the axial positions (2.189–2.307 Å) are longer than their counterparts linking the copper centers to the oxygen centers in the basal plane (1.936–1.983 Å). The Cu⋯Cu distances in complexes 1–3 are 2.9902(16), 3.0038(6), and 2.9760(8) Å respectively. These values are very close to those of copper complexes with similar coordination spheres reported in the past [35–38]. In complexes 1–3, two coordinating tridentate Schiff-base ligands are not on a plane. In all three complexes, however, two deprotonated phenoxo oxygen centers coordinate the Cu(II) center to form the bridging Cu2O2 core, which is planar. In the solid state, no π⋯π interactions were identified in complex 1. As can be evinced from Fig. 1D and E, however, complexes 2 and 3 were linked into one-dimensional zigzag chains by π⋯π interactions (face-to-face distance = 3.473 Å and center-to-center distance = 3.999 Å, in 2; face-to-face distance = 3.277 Å and center-tocenter distance = 3.794 Å, in 3). Interestingly, the π⋯π interactions in 2 involve two benzene rings in the different ligands in a binuclear molecule and two benzene rings in the different ligands in adjacent binuclear molecules. On the other hand, the π⋯π interactions in 3 involve two benzene rings in the same ligand in a binuclear molecule and two benzene rings in the same ligand in an adjacent binuclear molecule.
was found at m/z 639.00, which could be identified with isotopic envelopes corresponding to [Cu2(HL1)(L1)]+ (fit: 639.00), thus indicating that the uncoordinated NO3− and the coordinated NO3− and methanol were lost. The ESI-MS of complex 2 is characterized by an intense signal at m/z = 670.93 due to [Cu2(HL2)(L2)]+ (fit: 670.94) at 0 eV, which means that the uncoordinated NO3− and the coordinated methanol were lost. The ESI-MS of complex 3 is characterized by an intense signal at m/z = 762.83, which can be identified with isotopic envelopes to be due to [Cu2(HL3)(L3)]+ (fit: 762.83), implying that the uncoordinated NO3− and the coordinated ethanol molecules were lost. These results corroborate the structures found in solid state. 3.3. Interaction between HSA and the copper(II) complexes The binding properties of anticancer agents to HSA provide information on their uptake and distribution in tumor cells and afford an insight into their mechanism of action and metabolism [39,40]. Exploring the interactions of copper complexes 1–3 with HSA is therefore of great scientific interest. UV–visible and CD spectra are often used to probe the interaction between biological macromolecules and small molecular species. To gain an insight into the nature of HSA-copper complex interaction, the UV–visible spectra of the copper complexes, HSA and HSAcopper complex system were recorded. As can be evinced from the data reported in Fig. 2A, at 349 nm, the absorbance of complex 1 showed a hypochromism effect in the presence of HSA. This effect was also observed with complex 2 and complex 3, and reflected a complex-HSA interaction rather than a simple spectral overlap. To prove the possible influence of copper complex binding on the secondary structure of HSA, the CD spectroscopy of HSA in the presence and absence of the complexes were measured. As shown in Fig. 2B, HSA in the absence of the copper complexes dispaly negative absorption bands with maxima at 208 nm (π → π*) and 222 nm (n → π*), which is characteristic of αhelical structure of HSA [41]. The binding of each copper complex to HSA caused an intensity decrease for both 208 nm and 222 nm negative bands, indicating a decrease in the α-helical content of HSA. From the above results, we can conclude that each copper complex can interact with HSA and cause conformational change in the HSA protein. Docking studies were also conducted to afford an insight into the
3.2. Mass spectrometry The structures of the Cu(II) complexes were also established by means of electrospray ionization mass spectrometry (ESI-MS) at room temperature (Fig. S2). A peak due to the parent cluster ion of complex 1 4
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Fig. 2. (A) UV–vis absorption spectra of HSA in the presence of different copper complexes. [HSA] = 10 μM; [copper complex] = 10 μM. (B) CD spectra of HSA in the presence of different copper complexes. [HSA] = 1 μM; [copper complex] = 1 μM; T = 298 K; pH = 7.4.
Fig. 3. Molecular docked models of the copper complexes with HSA. (A) The overall structure of HSA complex. (B) The copper complexes are buried beneath the protein surface. (C) The copper complexes interacting with a variety of amino acid residues of HSA.
increased their cytotoxicity. The IC50 values of complexes 1–3 were significantly lower than that of the cisplatin. In particular, the IC50 values associated with the use of 1 were 5.65, 36.13, 23.58, and 5.36fold lower than those associated with the use of cisplatin in Bel-7402, HeLa, MCF-7, and MGC-803 cells, respectively. The IC50 values associated with the use of 2 were 3.33-, 20.47-, 19.73-, and 3.41-fold lower than those associated with the use of cisplatin in Bel-7402, HeLa, MCF7, and MGC-803 cells, respectively. Similarly, the IC50 values associated with the use of 3 were 2.67-, 14.98-, 9.29-, and 3.23-fold lower than those associated with the use of cisplatin in Bel-7402, HeLa, MCF-7, and MGC-803 cells, respectively. It is notable that the potency of the 1–3 to kill cancer cells followed the order 1 > 2 > 3. The result can probably be explained by the fact that the different halogen substituent (F, Cl or Br) led to a lipophilicitydependent decline in anticancer activity [42]. Moreover, we also determined the cytoxicity of these copper complexes toward healthy cells. The comparison between the IC50 values measured with WI-38 cells and those measured with cancer cells indicated that complexes 1–3 have low selectivity for cancer cells over healthy cells, suggesting that targeted delivery of these copper complexes by, for instance, biomolecules, liposomes, or nanoparticles would be preferable [43]. In addition, upon confirmation of the good anticancer activity displayed by complex 1, further cellular (in HeLa cells) assays were carried out to understand this complex’s mechanism of action.
potential interaction mode of these copper complexes with HSA. As depicted in Fig. 3, the copper complexes were located in the hydrophobic cavity of subdomain IIA delimited by residues His242, His288, Ala291, Lys199, Leu219, Leu260, Leu238, Arg218, Arg222, Arg257, Phe156, Phe157, and Trp214 The binding mode of these complexes suggests that they engage in hydrogen bonding interactions with Arg218. These complexes also engage in hydrophobic interactions with Trp214, Phe156, Phe157, Leu219, Leu238, and Val241. The F, Cl, and Br moieties (the closest ones to Trp214) of these complexes are located about 5.37, 4.78, and 4.74 Å away from Trp214, respectively. 3.4. Cytotoxicity of Cu(II) complexes in vitro To determine the anti-proliferative efficacy of complexes 1–3, we performed MTT assays using four cancer cell lines (Bel-7402, HeLa, MCF-7, and MGC-803) and one normal lung fibroblast cell line (WI38). The widely used metallodrug cisplatin was included as a control. The IC50 (concentrations required to induce 50% viability values obtained in these experiments are summarized in Table 3. The values of in vitro cytotoxicity measured for complexes 1–3 exceed those measured for the Schiff-base ligands by themselves, indicating that these ligands’ coordination to Cu(II) significantly Table 3 Inhibition of human cancer cell lines growth (IC50, μM) for the complexes 1–3. Compound
HL1 HL2 HL3 1 2 3 Cisplatin
3.5. Cell apoptosis
Cell growth inhibition, IC50 ± SD (μM) Bel-7402
Hela
MCF-7
MGC-803
WI-38
> 30 > 30 > 30 2.7 ± 0.2 4.5 ± 0.5 5.6 ± 0.8 15.1 ± 1.2
> 30 > 30 > 30 0.5 ± 0.2 0.9 ± 0.1 1.2 ± 0.2 18.4 ± 2.1
> 30 > 30 > 30 0.8 ± 0.1 1.0 ± 0.1 2.1 ± 0.2 19.3 ± 1.9
> 30 > 30 > 30 3.0 ± 0.4 4.8 ± 0.6 5.0 ± 0.6 16.3 ± 1.7
> 30 > 30 > 30 3.2 ± 0.6 3.4 ± 0.2 6.4 ± 0.7 14.7 ± 1.8
Most metal-based complexes exert their cytotoxicity by activating the apoptosis pathway, so we investigated features related to this pathway [44–47]. To determine whether apoptosis occurred in cancer cells, we treated HeLa cells with complex 1 and then evaluated the apoptosis of HeLa cells using the Annexin-V/PI staining protocols (Fig. 4). Evidence indicated that complex 1 efficiently induced apoptosis in HeLa cells in a concentration-dependent manner. Even at the low concentration of 1.0 µM, complex 1 induced a large proportion of 5
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Fig. 4. Apoptosis of HeLa cells treated with complex 1: (a) Control, (b) Complex 1 = 0.5 µM, and (c) Complex 1 = 1.0 µM.
HeLa cells to undergo early – (11.6%) and late – (40.7%) stage apoptosis.
mediated apoptosis in cancer cells [52]. After HeLa cells were treated with complex 1 (0.5 µM and 1.0 µM) for 24 h, evidence suggested that a decrease in the expression level of the anti-apoptotic proteins Bcl-2 and Bcl-xl and an increase in the expression level of the pro-apoptotic protein Bax had been caused (Fig. 5B). As a result, the levels of cleaved -caspase-9, -caspase-3 and cytochrome c were up-regulated, thus regulating the apoptosis pathway of HeLa cells (Fig. 5B). These results suggested the activation of mitochondrion-mediated apoptosis in HeLa cells (Fig. 5B).
3.6. Mitochondrial membrane potential Various cell apoptotic pathways are activated as a consequence of mitochondrial dysfunction (ΔΨm) [48,49]. To determine the ΔΨm induced by complex 1, we used the lipophilic fluorescent probe JC-1 and analyzed by flow cytometry. As can be evinced from the data reported in Fig. 5A, after treatment of HeLa cells with complex 1, a remarkable decrease in ΔΨm was observed, which was reflected by a decrease of Jaggregates (red fluorescence) and concurrent increase of J-monomers (green fluorescence). Members of the Bcl-2 protein family, including anti-apoptotic proteins like Bcl-2 and Bcl-xL, and pro-apoptotic proteins like Bad and Bax, have been described as key regulators of apoptosis [50,51]. Numerous metal-based anticancer complexes have been developed to target members of the Bcl-2 protein family and induce mitochondrion-
4. Conclusion In this study, three binuclear Schiff base-based copper(II) complexes (1–3) were synthesized and characterized. Single-crystal X-ray crystallography analysis results revealed each Cu(II) atom in all complexes to be characterized by a distorted square-pyramidal coordination geometry. These copper(II) complexes can interact with subdomain IIA cavity of HSA and cause conformational change in the HSA protein. All
Fig. 5. (A) Assay of HeLa cells’ mitochondrial membrane potential with JC-1 as the fluorescence probe staining method. (B) Western blot analysis of the levels of Bcl2, Bcl-xl, Bax, Bad, cleaved caspase-3, cleaved caspase-9, and cytochrome c after being treated with complex 1 (0.5 µM and 1.0 µM) for 24 h. 6
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three copper complexes synthesized were found to have significant anticancer activity (0.5–5.6 μM) as determined conducting in vitro cytotoxicity assays. The ability of complexes 1–3 to kill selected tumor cells followed the order 1 > 2 > 3. Notably, the mechanism of the cytotoxic activity of these copper complexes may consist in triggering the mitochondrion-mediated apoptosis pathway. The results of our study will be helpful in the design, development, and synthesis of new binuclear Schiff base-based copper(II) complexes with potent anticancer activity.
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Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by the projects No. 81503161, No. WKZL2018030 and Administration of Traditional Chinese Medicine of Jiangsu Province (YB201960). The authors are grateful to the Baise University for partial support of this work. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ica.2019.119186. References [1] I.E. Leon, J.F. Cadavid-Vargas, A.L. Di Virgilio, S.B. Etcheverry, Curr. Med. Chem. 24 (2017) 112–148. [2] M.R. Gill, K.A. Vallis, Chem. Soc. Rev. 48 (2019) 540–557. [3] A. Bijelic, M. Aureliano, A. Rompel, Angew. Chem. Int. Ed. Engl. 58 (2019) 2980–2999. [4] U. Ndagi, N. Mhlongo, M.E. Soliman, Drug Des. Dev. Ther. 11 (2017) 599–616. [5] K.B. Garbutcheon-Singh, M.P. Grant, B.W. Harper, A.M. Harper, Krause-Heuer, M. Manohar, N. Orkey, J.R. Aldrich-Wright, Curr. Top. Med. Chem. 11 (2011) 521–542. [6] T. Sakaeda, K. Kadoyama, Y. Okuno, Int. J. Med. Sci. 8 (2011) 487–491. [7] N.J. Wheate, S. Walker, G.E. Craig, R. Oun, Dalton Trans. 39 (2010) 8113–8127. [8] A. Gupte, R.J. Mumper, Cancer Treat. Rev. 35 (2009) 32–46. [9] F. Tisato, C. Marzano, M. Porchia, M. Pellei, C. Santini, Med. Res. Rev. 30 (2010) 708–749. [10] D. Denoyer, S. Masaldan, S. La Fontaine, M.A. Cater, Metallomics 7 (2015) 1459–1476. [11] K. Sztanke, A. Maziarka, A. Osinka, M. Sztanke, Bioorg. Med. Chem. 21 (2013) 3648–3666. [12] A. Hameed, M. Al-Rashida, M. Uroos, S. Abid Ali, K.M. Khan, Expert Opin. Ther. Pat. 27 (2017) 63–79. [13] B. Samanta, J. Chakraborty, S. Shit, S.R. Batten, P. Jensen, J.D. Masuda, S. Mitra, Inorg. Chim. Acta 360 (2007) 2471–2484. [14] G.M. Abu El-Reash, O.A. El-Gammal, A.H. Radwan, Spectrochim. Acta A: Mol. Biomol. Spectrosc. 121 (2014) 259–267. [15] N.H. Al-Shaalan, Molecules 16 (2011) 8629–8645. [16] M.A. Malik, O.A. Dar, P. Gull, M.Y. Wani, A.A. Hashmi, MedChemComm 9 (2017) 409–436. [17] C. Santini, M. Pellei, V. Gandin, M. Porchia, F. Tisato, C. Marzano, Chem. Rev. 114
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