Heteroleptic Copper(I) Complexes of "Scorpionate" Bis-pyrazolyl Carboxylate Ligand with Auxiliary Phosphine as Potential Anticancer Agents: An Insight into Cytotoxic Mode

Abstract

New copper(I) complexes [CuCl(PPh₃)(L)] (1: L = L_A = 4-carboxyphenyl)bis(3,5-dimethylpyrazolyl)methane; (2: L = L_B = 3-carboxyphenyl)bis(3,5-dimethylpyrazolyl)methane) were prepared and characterised by elemental analysis and various spectroscopic techniques such as FT-IR, NMR, UV-Vis, and ESI-MS. The molecular structures of complexes 1 and 2 were analyzed by theoretical B3LYP/DFT method. Furthermore, in vitro DNA binding studies were carried out to check the ability of complexes 1 and 2 to interact with native calf thymus DNA (CT-DNA) using absorption titration, fluorescence quenching and circular dichroism, which is indicative of more avid binding of the complex 1. Moreover, DNA mobility assay was also conducted to study the concentration-dependent cleavage pattern of pBR322 DNA by complex 1, and the role of ROS species to have a mechanistic insight on the cleavage pattern, which ascertained substantial roles by both hydrolytic and oxidative pathways. Additionally, we analyzed the potential of the interaction of complex 1 with DNA and enzyme (Topo I and II) with the aid of molecular modeling. Furthermore, cytotoxic activity of complex 1 was tested against HepG2 cancer cell lines. Thus, the potential of the complex 1 is promising though further in vivo investigations may be required before subjecting it to clinical trials.

Figure 1. Schematic representation of the synthesis of 1 and 2.

Figure 2. Gas phase B3LYP/DFT optimized structure of complexes 1 and 2.

Figure 3

B3LYP/DFT simulated vibrational spectra of (a) complex 1 and (b) complex 2.

Figure 4. B3LYP/TDDFT simulated electronic absorption spectra of complexes 1 and 2.

Figure 5. Frontier MOs contour plots (isovalue 0.03) of complex 1 using the B3LYP/DFT method.

Figure 6. Frontier MOs contour plots (isovalue 0.03) of complex 2 using the B3LYP/DFT method.

Figure 7

Absorption spectral traces of complexes (a) 1 and (b) 2 in 95:5% H₂O:DMSO/5 mM Tris HCl-50 mM NaCl buffer at pH 7.4 upon addition of CT DNA.

Figure 8

Emission spectra of EthBr-CTDNA in the absence and presence of compound (a) 1 and (b) 2 in 5 mMTris–HCl/50 mM NaCl buffer at pH 7.4. Arrows show the emission intensity changes upon increasing the concentration of the complexes.

Figure 9

Circular dichroism spectra of CT DNA in the absence and presence of compound (a) 1 and, (b) 2 in 5 mM Tris–HCl/50 mM NaCl buffer at pH 7.4.

Figure 10

(a) Electrophoretic pattern of pBR322 DNA (100 ng) with increasing concentration of complex 1 (5–25 μM) after 30 min incubation time in buffer (5 mMTris-HCl/50 mM NaCl, pH = 7.2 at 25 °C) (concentration dependent). (b) Cleavage pattern of pBR322 plasmid DNA (100 ng) by complex 1 (10 μM) in the presence of reactive oxygen species viz., DMSO (0.4 mM), EtOH (0.4 mM), NaN₃ (0.4 mM) and SOD (10 U), after incubation for 30 min in the buffer (5 mM Tris-HCl/50 mM NaCl, pH = 7.2 at 25 °C.

Figure 11. Gel mobility pattern for the cleavage of pBR322 DNA (100 ng) by complex 1 (10 μM) in the presence of major/minor groove recognition element: lane 1, 1 + Distamycin + DNA; lane 2, 1 + Methyl green + DNA; Lane 3, 1 + DAPI + DNA; lane 4, DNA only, control, at 25 °C after incubation for 30 min.

Figure 12

Molecular docked model of (i) complex 1 and (ii) complex 2, in the (a) cavity of minor groove of DNA (b) binding site interactions with hydrogen bonding donor (purple) and acceptor (green) surface of minor groove residues.

Figure 13

Molecular docked model of (i) complex 1 and (ii) complex 2, (a) with human-DNA-Topo-I (70 kDa) (PDB ID: 1SC7) (b) Non-covalent interaction of complex 1 with the active site residues.

Figure 14

Molecular docked model of (i) (a) complex 1 and (a’) complex 2 into the ATP binding pocket of human Topo-IIα (PDB ID: 1ZXM).(ii) (b) complex 1 and (b’) complex 2, the non-covalent interaction with the Topo IIα.

Figure 15

(A) Hoechst 33258 staining of the complex 1-induced apoptosis of HepG2 cells. The graph shows the manual count of apoptotic cells as a percentage (data are mean% ± SD% of three experiments). (B) Representative morphological changes observed in HepG2 cells treated with 1 (STR2) adopting Hoechst 33258 staining.

Figure 16

AO/EB staining of the complex 1-induced apoptosis of HepG2 cells. The graph shows the manual count of apoptotic cells as a percentage (data are mean% ± SD% of three experiments). (B) Representative morphological changes observed in HepG2 cells treated with 1 by dopting AO/EB staining.