Synthesis and crystal structure of cytotoxic copper(II) complex with 1,10-phenanthroline-5,6-dione and isothiazole derivative

Oligopyridine based copper(II) complexes are of interest to scientists as possible anticancer agents due to promising cytotoxic and DNA binding/cleaving properties. In this study, copper(II) complex [Cu(phendione)L2]·C2H5OH with 1,10-phenanthroline-5,6-dione (phendione) and 4,5-dichloro-isothiazole-3carboxylic acid (HL) was synthesized and characterized by elemental analysis, IR-spectroscopy, X-ray powder diffraction and single-crystal X-ray diffraction. According to X-ray diffraction data, obtained compound is mononuclear complex with square pyramidal coordination environment of the central atom which is surrounded by two isothiazolate molecules and one phendione ligand. The X-ray diffraction data are confirmed by IR-spectroscopy data showing the presence of characteristic stretching vibration bands of the carbonyl and carboxyl groups of oligopyridine ligand and isothiazolate ions, respectively. Density functional theory (DFT) calculations for complex were carried out using the ADF software package to perform geometry optimization and frequency calculations that were in a good agreement with experimental IR spectrum. Cytotoxicity of complex and initial reagents was tested in vitro against HepG2 (human hepatocellular carcinoma) and MCF-7 (human breast adenocarcinoma) cell lines. The complex showed high dose-dependent cytotoxic activity with the IC50 values of 0.60±0.03 μM and 0.96±0.13 μM, respectively, which is higher than the activity of cisplatin against these cell lines. The activity of the complex is due to the presence of phendione ligand, which exhibits a similar cytotoxic activity.


INTRODUCTION
Currently, mixed-ligand complexes of essential metals with 1,10-phenanthroline and its derivatives are being actively studied in vitro and in vivo for their biological properties (cytotoxic, antioxidant, antibacterial, antiviral and other activities) [1][2][3][4][5][6][7]. Essential metals include such microelemets as iron, copper, zinc, cobalt, nickel, manganese and some other metals. Among the complexes of essential metals, the most interesting as anticancer agents are 1,10-phenanthroline based copper(II) complexes, since they are capable to interact and cleave DNA/RNA backbone [8,9]. Moreover, oligopyridine and amino acid/acetylacetone based copper(II) complexes (Casiopeínas) have shown antiproliferative, genotoxic, and antineoplastic activity [10,11]. The most promising complexes from the Casiopeinas series have completed preclinical trials [12]. Besides amino acids, N-donor systems (terpyridine, imidazole, benzimidazole, tetrazole), O-donor systems (salicylic acid, dicarboxylic acids), S-donor systems (thiosemicarbazones, dithiocarbamates, thioureas), Schiff bases are often used as secondary ligand in synthesis of cytotoxic Casiopeinas-like complexes [6]. The isothiazole heterocycles and coordination compounds with isothiazoles have been shown to be bioactive substances with pesticidal, anticancer, anti-inflammatory and antiviral activities [13]. However, despite their promising properties, isothiazoles are negligibly used for the synthesis of bioactive oligopyridine based complexes with essential metals. Thus, the research aiming preparation of corresponding complexes may be indeed promising from the point of view of medicinal chemistry.
In continuation of our work [14] in order to expand the number of available cytotoxic oligopyridine based complexes with isothiazole as secondary ligand, the present study set out to synthesise new mixed-ligand copper(II) complex with 1,10-phenanthroline-5,6-dione and 4,5-dichloro-isothiazole-3carboxylic acid and evaluate its biological activity.
Quantum-chemical calculations were carried out on the computational cluster of NIIC SB RAS using the Amsterdam Density Functional (ADF) program by dint of density functional theory (DFT) 1 . The generalized gradient approximation (GGA), the density functional PBE (Perdew -Burke -Ernzerhof) [17] in combination with the all electron basis set TZ2P [18] were applied. The calculations were carried out for non-solvent system.
Bruker D8 Venture diffractometer with the graphite-monochromated MoK α radiation (λ = 0.71073 Å) was utilized to collect single-crystal XRD data for copper(II) complex. All measurements were carried out at 150 K, and the φ-and ω-scan techniques were employed. Absorption corrections were applied with the SADABS program 2 . The crystal structure was solved and refined by means of the SHELXT [19] and SHELXL [20] programs using OLEX2 GUI [21]. Atomic thermal displacement parameters for non-hydrogen atoms were refined anisotropically. Positions of hydrogen atoms were calculated according to their geometrical conditions and refined by dint of the riding model. The crystallographic data and details of the structure refinements are shown in Table 1. CCDC 2121029 contains the supplementary crystallographic data for this paper. These information can be obtained free of charge from The Cambridge Crystallographic Data Center at https://www.ccdc.cam.ac.uk/structures/.

Synthesis of [Cu(phendione)L 2 ]·C 2 H 5 OH.
[Cu(H 2 O)L 2 ] was synthesized as previously described [14]. The precipitate of [Cu(H 2 O)L 2 ] (0.048 g, 0.10 mmol) was resuspended in mixture of ethanol (2 ml) and dichloromethane (3 ml). Solid phendione (0.025 g, 0.12 mmol) was added to suspension with stirring. The reaction mixture became clear, precipitate dissolved. The solution was left for slow evaporation at room temperature. Polycrystalline phase was obtained after a week. Green crystals were filtered out, washed with ethanol and dried in air. Yield: 0.064 g (90%). Elemental analysis (%): Сalc. for Cytotoxic activity. Human hepatocellular carcinoma (HepG2) and human breast adenocarcinoma (MCF-7) cell lines were cultured in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with a 10% fetal bovine serum under a humidified atmosphere (5% CO 2 and 95% air) at 37 ºC. Cell viability was evaluated by Hoechst/PI staining as previously described [22]. Hep2 cells were cultured to 96-well plates at a density 5·10 3  Hoechst 33342 (Sigma-Aldrich) and propidium iodide (Invitrogen) for 30 min at 37 ºC. An IN Cell Analyzer 2200 (GE Healthcare, UK) was used to perform automatic imaging of four fields per well under 200×magnification, in bright field and fluorescence channels. The IN Cell Investigator image analysis software (GE Healthcare, UK) was used to determine live, apoptotic and dead cells among the whole population. All data shown are mean of three wells. The quantitative data were expressed as the mean ± standard deviation (SD). All statistical analyses were performed using the software Excel 2016 (Microsoft) and Origin 8.0. . Powder X-ray diffraction analysis have been used to demonstrate the identity of single crystal and the synthesized polycrystalline phase of complex which proved the phase purity of the bulk sample (Fig. 1).
According to single-crystal X-ray diffraction data, the copper(II) complex [Cu(phendione)L 2 ]·C 2 H 5 OH reveals square pyramidal coordination environment of central atom. The coordination sphere of Cu(II) consists of two O atoms of the isothiazoles carboxylates, one N of the isothiazole ligand and two N atoms of the phendione (Fig. 2). Additional contact (of 2.772 Å) is observed between the copper(II) and the O atom of the isothiazole moiety giving distorted octahedral 5 + 1 environment. Also there is outer-sphere ethanol molecule. Similar structure has been observed in our previous work, where bipicoline has been used instead of phendione [14]. DFT-calculations, such as IR-frequencies and energies of HOMO/LUMO, have been carried out for copper(II) complex. The highest occupied molecular orbital (HOMO) and lowest-lying unoccupied molecular orbital (LUMO) are known to be the most im-portant orbitals in a molecule. In case of copper(II) complex, the electron density of HOMO (Fig. 3) is concentrated on phendione mainly and energy of this orbital is equal to -5.978 eV. LUMO (-4,997 eV) is disposed on copper ion and donor atoms of its coordination sphere. The energy gap between HOMO and LUMO (E HOMO -E LUMO ) is -0.981 eV. A molecule with large HOMO-LUMO gap is described as a hard molecule which is much less polarizable. In contrast, the soft systems have small HOMO-LUMO gap and are highly polarizable [23]. Thus, complex refers to soft molecules due to the small value of energy gap.
Vibrational spectrum has been calculated for the complex with optimized geometry. There are no imaginary frequencies in the obtained spectrum, consequently, the geometry corresponds to local minima. The main vibrational frequencies of calculated and experimental spectra are shown in Table 2. The vibrations of characteristic groups (COO-, C-H, C-Cl, O=C-C=O) exhibit in both spectra. The slight shift and broadening of bands is observed in experimental IR-spectrum, which can be due to the presence of ethanol molecule in system. Despite these small differences spectra are in a good agreement.
The obtained IR-spectrum clearly indicates the formation of copper(II) complex. There is a disappearing of broad ν(O-H) band observed in the HL spectrum (2915 cm -1 ) in the result of ligand deprotonation during the synthesis. Moreover, the bands of asymmetrical stretching vibrations of the carboxylate group shift to the low-frequency region (1651-1614 cm -1 ) in contrast to the band in HL spectrum (ν(COO) as = 1724 cm -1 ). The cytotoxicity of copper(II) complex was tested by Hoechst/PI staining on HepG2 (human hepatocellular carcinoma) and MCF-7 (human breast adenocarcinoma) cell lines by exposing them for 48 h to the medium containing the compound in 0.1-25 µM concentration. The concentration-dependent cell viability graphs are given in Fig. 4. Calculated from these graphs IC 50 values (concentration required to reduce survival in the cell lines to 50%) are summarized in Table 3.
Earlier, copper acetate and HL were shown to be nontoxic in the concentration range of 1-50 μM [14]. At the same time, 1,10-phenanthroline-5,6dione is highly toxic to HepG2 and MCF-7 cells with IC 50 values being in the nanomolar concentration range [24].  According to cytotoxicity study of [Cu(phendione)L 2 ]·C 2 H 5 OH, complex possesses dose-depen-dent cytotoxicity against both cell lines (IC 50 = 0.96±0.13 µM (MCF-7), 0.60±0.03 µM (HepG2)), while its toxicity is higher than that of oligopyridine on HepG2 cells and vice versa on MCF-7 cells (See Table 3). Thus, the activity of the complex is due to the presence of phendione ligand, which exhibits a similar cytotoxic activity. The activity of the obtained complex exceeds the activity of cisplatin by more than an order of magnitude.
This compound is part of a series of mixed-ligand copper(II) complexes with HL and various oligopyridines, and turned out to be the most toxic in this series. So, for example, the IC 50 value against MCF-7 cells in the same conditions is 1.

CONCLUSIONS
Mononuclear copper(II) complex based on 4,5-dichloro-isothiazole-3-carboxylic acid and 1,10-phenanthroline-5,6-dione [Cu(phendione)L 2 ] has been obtained and characterized by several physicochemical methods. The results of single crystal X-ray diffraction indicate that complex reveals square pyramidal coordination environment of central atom which consist of two O atoms of the isothiazoles carboxylates, one N of the isothiazole ligand and two N atoms of the phendione. According to DFT-calculations, the value of energy gap between HOMO and LUMO of complex is small (-0.981 eV), thus, [Cu(phendione)L 2 ] refers to soft molecules. This compound causes pronounced cytotoxic effect against HepG2 and MCF-7 cells in 0.1−25 concentration range which is an order of magnitude higher than cisplatin cytotoxicity.