Synthesis, Characterization, DNA/HSA Interaction, and Cytotoxic Activity of a Copper(II) Thiolate Schiff Base Complex and Its Corresponding Water-Soluble Stable Sulfinato-O Complex Containing Imidazole as a Co-ligand.

A Cu(II) thiolato complex [CuL(imz)] (1) (H2L = o-HOC6H4C(H)=NC6H4SH-o) and the corresponding water-soluble stable sulfinato-O complex [CuL'(imz)] (2) (H2L' = o-HOC6H4C(H)=NC6H4S(=O)OH) were synthesized and characterized using physicochemical techniques. Compound 2 is found to be a dimer in the solid state as characterized using single-crystal X-ray crystallography. XPS studies clearly showed the differences in the sulfur oxidation states in 1 and 2. Both compounds are found to be monomers in solution as revealed from their four-line X-band electron paramagnetic resonance spectra in CH3CN at room temperature (RT). 1-2 were tested to assess their ability to exhibit DNA binding and cleavage activity. Spectroscopic studies and viscosity experiments suggest that 1-2 bind to CT-DNA through the intercalation mode having moderate binding affinity (Kb ∼ 104 M-1). This is further supported by molecular docking studies of complex 2 with CT-DNA. Both complexes display significant oxidative cleavage of pUC19 DNA. Complex 2 also showed hydrolytic DNA cleavage. The interaction of 1-2 with HSA revealed that they have strong ability to quench the intrinsic fluorescence of HSA by a static quenching mechanism (kq ∼ 1013 M-1 s-1). This is further complemented by Förster resonance energy transfer studies that revealed binding distances of r = 2.85 and 2.75 nm for 1 and 2, respectively, indicating high potential for energy transfer from HSA to complex. 1-2 were capable of inducing conformational changes of HSA at secondary and tertiary levels as observed from synchronous and three-dimensional fluorescence spectroscopy. Molecular docking studies with 2 indicate that it forms strong hydrogen bonds with Gln221 and Arg222 located near the entrance of site-I of HSA. 1-2 showed potential toxicity in human cervical cancer HeLa cells, lung cancer A549 cells, and cisplatin-resistant breast cancer MDA-MB-231 cells and appeared to be most potent against HeLa cells (IC50 = 2.04 µM for 1 and 1.86 µM for 2). In HeLa cells, 1-2 mediated cell cycle arrest in S and G2/M phases, which progressed into apoptosis. Apoptotic features seen from Hoechst and AO/PI staining, damaged cytoskeleton actin viewed from phalloidin staining, and increased caspase-3 activity upon treatment with 1-2 collectively suggested that they induced apoptosis in HeLa cells via caspase activation. This is further supported by western blot analysis of the protein sample extracted from HeLa cells treated with 2.

Mixed Ligand Mononuclear Copper(II) Complex as a Promising Anticancer Agent: Interaction Studies with DNA/HSA, Molecular Docking, and In Vitro Cytotoxicity Studies.

The isolated copper(II) complex [CuL(o-phen)]·H2O (1) [H2L = o-HO-C6H4C(H)=N-C6H4-SH-o, o-phen = 1,10-phenanthroline] was structurally characterized using single-crystal X-ray crystallography. 1 in CH3CN at liquid nitrogen temperature displayed a characteristic monomeric X-band electron paramagnetic resonance spectrum having a tetragonal character with g ∥ = 2.1479 and g ⊥ = 2.0691 and A ∥ ≈ 18.0 mT and A ⊥ ≤ 3.9 mT, respectively. 1 showed a strong binding affinity toward calf thymus DNA as reflected from its intrinsic binding constant (K b = 7.88 × 105 M-1), and its competitive displacement of ethidium bromide suggested an intercalative DNA-binding mode (K app = 1.32 × 106 M-1). This was confirmed from the viscosity study that showed an increase in the viscosity of DNA with an increasing concentration of 1. Complex 1 is highly efficient in promoting oxidative and hydrolytic DNA cleavage (k obs = 1.987 h-1). 1 showed a strong binding affinity with the carrier protein human serum albumin (HSA) (K a = 5.22 × 105 M-1). A high bimolecular quenching constant k q = 2.29 × 1013 M-1s-1 indicated a static quenching mechanism involved in the fluorescence quenching of HSA by 1. Fluorescence resonance energy transfer theory suggested that the distance (r = 3.52 nm) between 1 and HSA is very close. Molecular docking studies suggested that 1 primarily binds to HSA in subdomain IIA. A protein-ligand interaction profiler was used to visualize hydrophobic, hydrogen bonds, and π-cation interactions between HSA and 1. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay using HeLa and MDA-MB-231 cells showed a significant in vitro anticancer activity of 1 (IC50 2.63 and 2.68 µM, respectively). Nuclear staining assays suggested apoptotic cell death in HeLa cells treated with 1. The effect of 1 on the cytoskeletal actin filaments visualized using phalloidin staining showed extensive destruction of actin filaments. Flow cytometric analysis indicated that 1 inhibits the growth of HeLa cells through cell cycle arrest in the S phase. Western blot analysis showed upregulation in the expression of apoptotic marker proteins caspase 3, p53, and Bax. These results collectively indicate that 1 induces apoptosis by promoting DNA damage and has a high potential to act as an anticancer agent.

Synthesis, Characterization, Crystal Structure, DNA and HSA Interactions, and Anticancer Activity of a Mononuclear Cu(II) Complex with a Schiff Base Ligand Containing a Thiadiazoline Moiety.

A mononuclear Cu(II) complex [Cu(HL)(o-phen)]·H2O (1) [H3L =, o-phen = 1,10-phenanthroline] was isolated from methanol, and its X-ray single-crystal structure was determined. Frozen glass X-band EPR of 1 in dimethylformamide (DMF) at LNT showed a spectrum that is characteristic of a monomeric tetragonal character with g ∥ = 2.164, g ⊥ = 2.087, A ∥ = 19.08 mT, and A ⊥ ≤ 4 mT. Electronic spectroscopic studies using calf thymus DNA (CT-DNA) showed strong binding affinity of 1 as reflected from its intrinsic binding constant (K b) value of 2.85 × 105 M-1. Competitive behavior of 1 with ethidium bromide (EB) displayed intercalative binding of DNA (K app = 1.3 × 106 M-1). The compound displayed significant oxidative cleavage of pUC19 DNA. The interaction between HSA and complex 1 was examined by employing fluorescence and electronic absorption spectroscopic experiments. The secondary and tertiary structures of HSA were found to be altered as suggested by three-dimensional (3D) fluorescence experiments. The affinity of 1 to bind to HSA was found to be strong as indicated from its value of the binding constant (K a = 2.89 × 105 M-1). Intrinsic fluorescence of the protein was found to be reduced through a mechanism of static quenching as suggested from the k q (2.01 × 1013 M-1 s-1) value, the bimolecular quenching constant. The Förster resonance energy transfer (FRET) process may also be accounted for such a high k q value. The r value (2.85 nm) calculated from FRET theory suggested that the distance between complex 1 (acceptor) and HSA (donor) is quite close. Complex 1 primarily bound to HSA in subdomain IIA as suggested by molecular docking studies. IC50 values (0.80 and 0.43 µM, respectively) obtained from the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay with HeLa and MCF7 cells suggested remarkable in vitro anticancer activity of 1. Nuclear dual staining assays revealed that cell death occurred via apoptosis in HeLa cells and reactive oxygen species (ROS) accumulation caused apoptosis induction. On treatment with a 5 µM dose of 1 in HeLa cells, the cell population significantly increased in the G2/M phase, while it was decreased in G0/G1 and S phases as compared to the control, clearly indicating G2/M phase arrest.

Protein binding studies with human serum albumin, molecular docking and in vitro cytotoxicity studies using HeLa cervical carcinoma cells of Cu(ii)/Zn(ii) complexes containing a carbohydrazone ligand.

The interaction of two binuclear mixed ligand Cu(ii) complexes [Cu(o-phen)LCu(OAc)] (1) and [Cu(o-phen)LCu(o-phen)](OAc) (2) (H3L = o-HOC6H4C(H)[double bond, length as m-dash]N-NH-C(OH)[double bond, length as m-dash]N-N[double bond, length as m-dash]C(H)-C6H4OH-o) and a new mononuclear Zn(ii) complex [Zn(HL)(o-phen)(H2O)](OAc)·H2O (3) (H2L = o-HOC6H4-C(H)[double bond, length as m-dash]N-NH-C([double bond, length as m-dash]O)-NH-N[double bond, length as m-dash]C(H)-C6H4OH-o, o-phen = 1,10-phenanthroline, and OAc = CH3COO-) with human serum albumin (HSA) was studied using fluorescence quenching, synchronous and 3D fluorescence measurements and UV-vis spectroscopy. 3D fluorescence studies showed that the HSA structure was altered at the secondary and tertiary levels upon binding with the complexes. This was further supported by the electronic absorption spectral studies of HSA in the absence and presence of the compounds. The average binding distance (r) between HSA and the complexes was obtained by Förster's resonance energy transfer theory. Complex 3 was structurally characterized by X-ray crystallography. Molecular docking studies indicated that all three complexes primarily bind to HSA in subdomain IIA with amino acid residues such as Arg218 and Lys199 which are located at the entrance of Sudlow's site I. The in vitro cytotoxicities of complexes 1-3 against HeLa cells showed promising anticancer activity (IC50 = 3.5, 3.9 and 16.9 µM for 1, 2 and 3, respectively). Live cell time lapse imaging for 1 was done to capture the dynamic behavior of the cells upon treatment with the complex. Cell cycle analysis by flow cytometry with HeLa cells indicated that 1 and 2 induced cell cycle arrest in the G2/M phase while 3 induced arrest in the G0/G1 phase leading to cell death. Compounds 1 and 2 but not 3 induced apoptosis through the mitochondrial pathway as suggested from the relative p53, caspase3 and bcl2 mRNA levels measured by real-time quantitative PCR analysis.

Efficient hydrolytic cleavage of DNA and antiproliferative effect on human cancer cells by two dinuclear Cu(II) complexes containing a carbohydrazone ligand and 1,10-phenanthroline as a coligand.

We report the synthesis, crystal structures and biological activities of two dinuclear Cu(II) complexes [Cu(o-phen)LCu(OAc)] (1) and [Cu(o-phen)LCu(o-phen)](OAc) (2), where o-phen = 1,10-phenanthroline, H3L = o-HOC6H4C(H)=N-NH-C(OH)=N-N=C(H)-C6H4OH-o, and OAc=CH3COO-. Both compounds display strong and broad X-band EPR spectra at RT in their powder state confirming that these are paramagnetic. The intercalative DNA binding of the compounds as revealed from spectrophotometric studies was found to be consistent with the results of fluorescence spectroscopic studies for ethidium bromide displacement assay as well as enhanced viscosity of DNA in the presence of these compounds. The compounds effectively catalyze hydrolytic cleavage of supercoiled pUC19 DNA and show remarkable cytotoxicity toward human lung cancer A549 cell line (IC50 values are 4.34 and 8.46 µM for 1 and 2, respectively) and breast cancer MCF7 cell line (IC50 values are 6.50 and 8.68 µM for 1 and 2, respectively) and are found to be relatively less toxic toward keratinocyte HaCaT normal cell line (IC50 values are 11.19 and 16.01 µM for 1 and 2, respectively). Annexin-V/PI dual staining results analyzed by flow cytometry strongly suggest the induction of apoptotic pathway for the anticancer activity of these complexes. Flow cytometry experiment for cell cycle analysis showed considerable increase in the G2/M phase in both A549 and MCF7 cell lines by these two compounds. On the other hand, compounds 1 and 2 activate reactive oxygen species (ROS) level in A549 cells, but act as scavengers or inhibitors of ROS in MCF7 cell line as analyzed by DCFDA staining using flow cytometry. Two dinuclear Cu(II) complexes exhibit efficient hydrolytic cleavage of DNA and display remarkable cytotoxicity against human lung cancer A549 and breast cancer MCF7cells. The ROS level in A549 cells is activated, but the ROS level in MCF7 cells is decreased in the presence of these complexes. Cell cycle analysis by flow cytometry shows G2/M phase arrest in both these cell lines.

DNA binding, cleavage and cytotoxicity studies of three mononuclear Cu(II) chloro-complexes containing N-S donor Schiff base ligands.

We report the biological activity of three Cu(II) complexes [Cu(pabt)Cl] (1), [Cu(pma)Cl] (2), and [Cu(pdta)Cl]Cl (3) (pabt = N-(2-mercaptophenyl)-2'-pyridylmethylenimine, pma = N-(2-pyridylmethyl)-2-mercaptoaniline, pdta = 2,2'-di(pyridyl-2-methyleneimine)diphenyl disulfide). 1-3 display four-line EPR multiplet in solution at RT suggesting that these are mononuclear. DNA-binding studies using spectrophotometric titration of these complexes with calf thymus DNA showed binding through intercalation mode which was found to be consistent with the observation of increased viscosity of DNA and quenching of fluorescence of ethidium bromide bound DNA in the presence of these complexes. All three complexes were found to be efficient in bringing about oxidative and hydrolytic cleavage of DNA. The proposed mechanism of hydrolytic DNA cleavage has been discussed. MTT assay showed remarkable cytotoxicity on cervical cancer HeLa cell line and the IC50 values were 1.27, 4.13, and 3.92 µM for 1, 2 and 3, respectively, as compared to the IC50 value (13 µM) reported for cisplatin in HeLa cells. AO/PI and Annexin-V/PI assay suggest the induction of cell death primarily via apoptotic pathway. Nuclear staining using DAPI was used to assess changes in nuclear morphology during apoptotic cell death. The role of reactive oxygen species (ROS) for induction of apoptotic cell death was studied using H2DCF-DA assay and the result suggests that the generation of ROS by the complexes may be a possible cause for their antiproliferative activity. TUNEL assay showed DNA fragmentation in apoptotic cells. Cell cycle analysis using flow cytometry showed significant increase in the G2/M phase in HeLa cells by the compounds 1-3. Mononuclear Cu(II) complexes display remarkable cytotoxicity against cervical cancer HeLa cell line. The generation of ROS by the complexes may be a cause of their antiproliferative activity. Fluorescent images from DAPI staining assay revealed that the cells undergoing apoptosis displayed typical features like cell shrinkage, membrane blebbing, chromatin condensation and nuclear fragmentation. TUNEL assay showed DNA fragmentation in apoptotic cells.