Novel 2-aminoimidazole-4-one complexes of copper(II) and cobalt(II): Synthesis, structural characterization and cytotoxicity

2.1 Materials and methods

All of the samples were prepared in reagent grade solvents. All of the chemicals used to synthesize the ligands were obtained from Acros or Sigma Aldrich and used as received without further purification. All of the reactions were monitored by thin-layer chromatography on Silufol precoated silica gel plates (with fluorescence indicator UV254) using a 1:1 (v/v) mixture of ethyl acetate and n-hexane as the solvent system. Melting point (mp) data were obtained in open capillaries on an SMP11 Stuart melting point apparatus and are uncorrected. Elemental analyses were conducted on a Vario MICRO cube CHNS/O Elementar system (Vario). ¹H and ¹³C NMR spectra were recorded on a Bruker Avance 400 (400 MHz) spectrometer using CDCl₃ or (CD₃)₂SO as a solvent. NMR data have been reported in the following order: chemical shift (δ) value in ppm relative to the solvent resonance, which was used as an internal reference standard; and coupling constant (J) in Hertz (Hz). X-ray diffraction analysis was conducted on a CAD4 diffractometer at 293 K (graphite monochromated radiation, $\lambda$ _(MoKα) = 0.71073 Å, ω/1.33θ scan type). The 2-methylthio-imidazole-4-one starting materials were synthesized according to previously published methods from the literature (Beloglazkina et al., 2005a,b; Majouga et al., 2004a,b; Majouga et al., 2014; Mironov et al., 2013).

Electrochemical studies were carried out on a PI-50-1.1 potentiostat in MeCN. A 0.05 M solution of Bu₄NClO₄ in MeCN was used as a supporting electrolyte, with Ag/AgCl/KCl (saturated) as a reference electrode. All of these measurements were conducted under an atmosphere of argon. The samples were dissolved in previously de-aerated solvents.

2.2 Morpholine-substituted ligands

2.3 Piperidine-substituted ligands

2.4 Coordination compounds

To the solution of ligand 1–8 in 1 ml of CH₂Cl₂ the solution of equimolar amount of CuCl₂·2H₂O or CoCl₂·6H₂O in 1 ml of n-BuOH was carefully added and the mixture was left in a closed vessel until precipitation. The forming dark green crystals were filtered off and dried in air.

3.1 Synthesis and characterization

The most commonly used approach for the synthesis to 2-amino-imidazole-4-ones involves the reaction of 2-thiohydantoins or their S-alkylated derivatives with an amine under heating (El-Barbary et al., 2000; Hu et al., 2002) or microwave irradiation (Asmaa et al., 2007). It is noteworthy that S-alkylated thiohydantoins have been reported to show better results in these reactions than 2-thiohydantoins (Carver et al., 1997; Grimmet, 1970; Lindel and Hoffmann, 1997; Roue and Bergman, 1999; Ybrahim et al., 1980). With this in mind, S-alkylated thiohydantoins were selected as starting materials for this study.

We initially investigated the reactions of 2-methylthio-substituted imidazolones with morpholine under reflux conditions. However, these conditions failed to afford the desired 2-morpholyl-substituted imidazolones 5–7 in yields greater than 10%. To increase the yields of the target amino-imidazolones we investigated the use of several Lewis acid catalysts, including AlCl₃, ZnCl₂ and Yb(OTf)₃. The results revealed that AlCl₃ and ZnCl₂ did not catalyze the reaction, whereas the use of ytterbium(III) triflate led to a significant increase in the yields of the desired amino-substituted imidazolones to 46–82% (Scheme 1).

Close

Scheme 1 Synthesis of (5Z)-2-amino-5-(pyridine-2-ylmethylene)-3,5-dihydro-4H-imidazole-4-ones 1–8.

Export to PPT

Compounds 3–6 were isolated as single geometric isomers, which were determined to the (Z)-isomers based on the chemical shifts and the coupling constants of the vinylic protons by ¹H NMR spectroscopy (Mironov et al., 2013). The (Z)-configurations of ligands 2, 5 and 6 were also confirmed based on the X-ray diffraction analysis of the corresponding coordination complexes 2b, 5b and 6b (see below).

A series of Cu(II) and Co(II) complexes were prepared using the (5Z)-2-amino-5-(pyridine-2-ylmethylene)-3,5-dihydro-4H-imidazole-4-ones prepared in the current study by the direct reaction of CH₂Cl₂ solutions of these ligands with copper(II) or cobalt(II) chloride, respectively (Scheme 2). All of the resulting complexes were characterized by elemental analysis. The coordination compounds 1a–6a, 1b, 2b, 4b, 7b and 8b were also characterized by UV/Vis spectroscopy. Characteristic low-intensity d–d transition bands were observed in the UV/Vis spectra of all of the coordination compounds prepared in the current study in the range of 550–720 nm (Fig. 1 and Supplementary information). These data were consistent with the literature data for the tetrahedral Cu(II) and Co(II) complexes of 2-substituted 5-pyridylmethylene-imidazol-4-ones (Beloglazkina et al., 2005a,b, 2006a,b; Majouga et al., 2004a,b, 2009, 2014). According to the literature (Guo et al., 2002; Rowland et al., 2000; Hennig et al., 1995, and previous references), the UV/Vis spectra of square planar copper(II) complexes should contain a broad band in the range of 550–750 nm, which would show a bathochromic shift upon tetrahedral distortion. The UV/Vis spectra of square planar cobalt complexes normally contain only one band in the visible region, whereas tetrahedral Co(II) complexes generally give three bands (or a broad band, representing the superposition of two overlapping bands) in this region (Van der Bergen et al., 1974; Beloglazkina et al., 2013). All of the complexes prepared in the current study also exhibited strong charge transfer bands in the UV region of their electronic spectra (see Supplementary information).

Close

Scheme 2 Synthesis of copper(II) and cobalt(II) complexes of 2-amino-imidazole-4-ones 1–8.

Export to PPT

Close

Figure 1 Electronic spectra of cobalt(II) complexes 2b, 4b, 5b, 7b and 8b, 550–800 nm (DMSO, 10⁻⁴ M).

Export to PPT

3.2 Molecular structures of complexes 2b, 5b and 6d

The structures of complexes 2b, 5b and 6b were confirmed by X-ray diffraction analysis, and the crystal structures are shown in Fig. 2. Selected bond lengths and bond angles from these crystal structures are shown in Tables 1 and 2. The previously reported (Majouga et al., 2004a,b) crystal structure of the cobalt complex of (5Z)-3-phenyl-2-methylthio-5-(pyridine-2-ylmethylene)-3,5-dihydro-4H-imidazole-4-one (complex A) is also shown in Fig. 1 for the sake of comparison. All of the crystallographic data and refinement parameters are shown in Tables S1, together with a list of bond lengths and bond angles. The cobalt ions in all of these complexes were tetrahedrally coordinated to the two nitrogen atoms of the pyridine and thiohydantoin rings and two chloride anions. The cobalt atoms were located in a distorted tetrahedral environment with Co—N distances of 2.032–2.040(10) Ǻ (Co—N_Py) and 1.989–1.997(10) Ǻ (Co—N_Im), which were similar to those observed in complex A (Majouga et al., 2004a,b). The phenyl substituent at the N(3) position of the ligand moiety in complex 5b was near perpendicular to the plane of the imidazolone ring. The piperidine and morpholine rings were found to exist in the chair conformations.

Close

Figure 2 Molecular structures of coordination compounds 2b, 5b, 6b in comparison with the molecular structure of (5Z)-3-phenyl-2-methylthio-5-(pyridine-2-ylmethylene)-3,5-dihydro-4H-imidazole-4-one cobalt(II) dichloride A (Majouga et al., 2004a,b). Cobalt ions are shown on blue, nitrogen atoms in light blue, chlorine atoms in green, oxygen atoms in red, and sulfur atom in yellow.

Export to PPT

Consideration of the data in Tables 1 and 2 revealed that while the differences in the Co—N and Co—Cl bond lengths of the complexes of the 2-amino- and 2-thio-substituted imidazole-4-ones were negligible, the Cl1—Co—N2 bond angles differed considerable in these complexes. The tetrahedral coordination environment of the cobalt ions in the amino-substituted ligand complexes (2b, 5b, 6b) underwent much less distortion than the corresponding sulfur-containing (A) complexes. This difference was attributed to a higher level of steric hindrance around the metal ion of the 2-methylthio substituent compared with the corresponding 2-amino group.

3.3 Electrochemistry

Electrochemical studies were conducted using ligands 1, 2 and 7, together with their complexes, as representative examples by cyclic voltammetry (CV) using glassy carbon, platinum and gold electrodes. The results showed that all three of the ligands were reduced in two steps, the first of which was reversible with the second being irreversible (see Fig. 3 and Table 3). Ligand 1 bearing a phenyl substituent on its N(3) atom was reduced much more readily (i.e., 200 mV less cathodic) than the corresponding methyl- and allyl-substituted ligands 2 and 7, most likely because of the presence of the conjugated phenyl fragment.

Close

Figure 3 CVA for ligands 1 and 2 (10⁻³ M, DMF, Bu₄NClO₄) on Au electrode.

Export to PPT

The oxidation reactions of compounds 1, 2 and 7 revealed that ligand 1 was irreversibly oxidized in a single-electron process, whereas compounds 2 and 7 were irreversibly oxidized in a two-electron process (Table 3, Fig. 3).

The cyclic voltammograms for cobalt complexes 1b and 2b contained additional peaks in the cathodic region for E_pc values in the range of −0.89 to −1.0 V for all of the electrodes, corresponding to the reduction of Co(II) to Co(I) (Table 3, Fig. 4). The initial reduction of the cobalt complexes was found to be reversible at all of the electrodes. However, the intensity of the Co(II)/Co(I) re-oxidation peak for complex 2b was equal to about half of the reduction current over the Au and Pt electrodes. This result therefore suggested that the Co(I)-containing intermediates were stable on the timescale required for their analysis by CV, and that they were less stable on metal electrodes than the GC electrode.

Close

Figure 4 CVA for cobalt complexes 1b and 2b (10⁻³ M, DMF, Bu₄NClO₄) on Au electrode.

Export to PPT

The other peaks observed in the cathode region for the Co complexes were attributed to the reduction of the ligand fragments. These peaks were shifted slightly for complexes 2b and 7b relative to the corresponding peaks of the ligand, with the shift for complex 1b being the greatest of all (approximately 200 mV). These results indicate that the complexation of the ligands with cobalt leads to a change in the mechanism with which the ligand moiety is oxidized. This was confirmed by the fact that the first anodic peak for the complexes was a two-electron process.

The copper-containing complexes 1a–3a were reversibly reduced at potentials (E_pc) in the range of 0.24–0.26 V (i.e., potentials around 200 mV). These potentials were less cathodic than those previously reported by our group for the thiohydantoin complexes of copper(II), which showed an E_pc value of 0.45 V (Beloglazkina et al., 2005a).

The cyclic voltammograms showed that there were no desorption peaks corresponding to zero-valent copper on the GC or Pt electrodes, even under the reverse potential scan conditions after the oxidation and reduction of the ligand fragments. This result therefore indicated that the Cu(I)-containing intermediates were stable as complexes with both negatively and positively charged ligands. However, sharp triangular peaks (without a diffusion loop) were observed at the Au electrode for the copper-containing complexes 1a and 2a after the oxidation or reduction of the ligand moiety (Fig. 5), which were characteristic for the desorption of metallic copper from the electrode surface. It is therefore possible that the reduction intermediates (i.e., complexes of the negatively charged ligand (L⁻) with Cu(I)) could be unstable and disproportionate to give Cu(II) and Cu(0) complexes, with the latter of these two complexes decomposing with the release of metallic copper: $$2[{\text{L}}^{-}]\text{Cu}(\text{I})\text{Cl}\to [{\text{L}}^{-}]\text{Cu}(\text{II}){\text{Cl}}_2+[{\text{L}}^{-}]\text{Cu}(0)$$ $$[{\text{L}}^{-}]\text{Cu}(0)\to [{\text{L}}^{-}]+\text{Cu}(0)\text{,}$$

Close

Figure 5 CVA for copper complex 1a on GC (left) and Au (right) electrodes (10⁻³ M, DMF, Bu₄NClO₄).

Export to PPT

In contrast, any complexes formed with the positively charged ligands would be oxidized because of the low donor ability of the cationic ligand fragments.

It is noteworthy that no desorption peaks were observed for cycling potentials in the range of 0.1–0.65 V, which indicated the cobalt(I) complex formed with the neutral ligand was stable under these conditions.

3.4 Cytotoxicity

The ligands and the corresponding copper and cobalt complexes prepared in the current study were tested in terms of their in vitro cytotoxicity against cell cancer cells lines, including human lung cancer (A549), breast adenocarcinoma (MCF-7) and human embryonic kidney (HEK293) cells using a standard MTT assay (Mosmann, 1983). The results of this assay are shown in Table 4 together with the results obtained for the clinically used drugs cisplatin and doxorubicin, which have been included for comparison.

The coordination of a metal ion was found to be essential for the cytotoxicity of the test compounds. For example, the IC₅₀ values of the free ligands were much higher than those of the corresponding copper complexes. However, the cytotoxicities of the cobalt complexes were substantially lower than those of the corresponding copper complexes, although in some cases they were higher than those of the free ligands. The nature of the amine substituent at the 2 position of the imidazolone ring was also found to be important to the cytotoxicity of these compounds. For example, compounds containing a piperidine substituent (i.e., 5–7) were generally 1.5–2 times more active than compounds containing a morpholine residue at the same position (i.e., 1–3). The nature of the substituent at the N(3) position of the imidazolone ring also had a considerable impact on the cytotoxicity, which increased in the order Ph > Allyl > Me. This increase in the cytotoxicity correlates well with the increasing lipophilicity of the complexes.

HEK293 cells were found to be the most vulnerable of all the cell lines tested in the current study toward the test compounds, with complex 5a showing the greatest cytotoxicity of all of the test complexes. Furthermore, the cytotoxicity of complex 5a toward HEK293 cells was similar to that of cisplatin. The most active cobalt complexes were found to be compounds 1b and 7b. The cytotoxicities of these complexes toward MCF7 cells were 1.5 times greater than those of cisplatin.

Although the generation of reactive oxygen species (ROS) has been cited as a major contributory factor to the high toxicity and anticancer activity of numerous copper complexes (Hajrezaie et al., 2014; Mookerjee et al., 2006; Ng et al., 2014; Guo et al., 2010; Maheswari et al., 2008), we have assumed that the compounds described in this study do not induce the generation of ROS. This suggestion is supported by the lack of a correlation between the reduction potentials of these compounds and their cytotoxicity. All of the complexes prepared in the current study showed relatively low reduction potentials compared with those reported in our previous studies (Majouga et al., 2014), where we observed potentials around 0.45 V for the copper complexes of 2-thiohydantoin. The facile reduction of these complexes would facilitate their interaction with oxygen, which would enhance their cytotoxicity. However, this outcome was not observed, which suggested that the complexes prepared in the current study exert their cytotoxicity through a different mechanism.