Copper(II) complexes of salicylaldehydes and 2-hydroxyphenones: synthesis, structure, thermal decomposition study and interaction with calf-thymus DNA and albumins

Abstract

The neutral mononuclear copper(II) complexes with substituted salicylaldehyde (X-saloH), or 2-hydroxyphenone (ketoH) ligands, having the formula [Cu(L)₂(S)_n] (where S = solvent CH₃OH or H₂O and n = 0, 1 or 2) have been prepared and characterized, and their interaction with DNA and albumins was studied. The ligands are chelated to the metal ion through the phenolate and carbonyl oxygen atoms. The crystal structures of [Cu(5-NO₂-salo)₂(CH₃OH)₂] (3), [Cu(5-Cl-salo)₂] (4), [Cu(bpo)₂] (6) and [Cu(mpo)₂]·2H₂O (7·2H₂O) have been determined by X-ray crystallography. The thermal stability of the copper complexes has been investigated by a simultaneous TG/DTG-DTA technique. Spectroscopic (UV), electrochemical (cyclic voltammetry) and physicochemical (viscosity measurements) techniques have been employed in order to study the binding mode and strength of the complexes to calf-thymus (CT) DNA while competitive studies with ethidium bromide (EB) performed by fluorescence spectroscopy have revealed the ability of the complexes to displace the DNA-bound EB. In conclusion intercalation is the most possible mode of interaction of the complexes with CT DNA. The interaction of the complexes with serum albumin proteins has been studied by fluorescence emission spectroscopy and the determined binding constants exhibit relatively high values.

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1. Introduction

The interaction of transition metal complexes with DNA has been at the center of scientific interest for many years, mainly due to their versatile applications in cancer research and molecular biology.^1,2 Copper is one of the most interesting biometals,^3,4 mainly because of its biological role and its potential synergetic activity with drugs.⁵ Moreover, copper plays a very important role in cell physiology as a catalytic cofactor in the redox chemistry of mitochondrial respiration, iron absorption, free radical scavenging and elastin cross-linking.⁶

On the other hand, the strong coordinating properties of 2-hydroxy-benzaldehyde (salicylaldehyde) and its complexes with 3d transition metals have stimulated research on these compounds, that find applications in both the pure^7,8 and applied chemistry fields.^9,10 It has been also shown that these ligands possess antimicrobial properties.^11,12 These ligands are known to coordinate in a bidentate manner with transition metals in the mono-anionic form, adopting variant geometries, from square-planar¹³ to square-pyramidal¹⁴ and octahedral geometries.¹⁵

Especially, the 2-hydroxy-3-methoxy-benzaldehyde (3-OCH₃-salicylaldehyde), commonly known as o-vanillin (o-vanH), has three oxygen donor atoms, which exhibit suitable relative positions to coordinate metal centres.^14,16 Moreover, benzophenone and its derivatives have been widely used in many commercial products, e.g. sunscreens, cosmetics and plastic surface coatings, because of their ability to absorb and dissipate ultraviolet light A (400–315 nm).^17,18 These compounds have been effective in vitro and in vivo for the treatment of anaphylaxis, androgenesis, inflammation, malaria, tuberculosis and virus;^18–26 they are also considered as inhibitors of HIV, farnesyl-transferase and reverse transcriptase.^27–29 Keeping in mind these controversial effects, it is of important significance the preparation of new salicylaldehyde or 2-OH-phenone compounds (including their complexes), which may show enhanced biological activity in comparison to the existing compounds.

The last decade we have initiated in our laboratory the synthesis and characterization of transition metal complexes with carbonyl compounds derived from salicylaldehyde and 2-OH-phenone ligands.^16,30–35 Especially, we have been focused on the synthesis of metal complexes with substituted salicylaldehydes and their interaction with DNA. Previous studies showed that zinc(II) complexes with salicylaldehydes or 2-hydroxybenzophenones presented interesting results concerning their binding to calf-thymus DNA,^33,34 while Co(II) analogues in the presence of the nitrogen-donor ligand 2,2′-dipyridylamine have showed anticancer activity.³²

As continuation of our research, seven neutral mononuclear copper(II) complexes with the ligands HL = substituted 2-hydroxy-benzaldehydes (salicylaldehyde, abbreviated as X-saloH), or with 2-hydroxy-phenones (abbreviated as ketoH) (Scheme 1), under the general formula [Cu(L)₂(S)_n], (where S = solvent CH₃OH or H₂O and n = 0, 1 or 2), have been prepared and characterized and their interaction with DNA and albumins has been studied. Five of them are novel compounds, while the crystal structures for four of them were verified by single-crystal X-ray diffraction analysis, [Cu(5-NO₂-salo)₂(CH₃OH)₂] (3), [Cu(5-Cl-salo)₂] (4), [Cu(bpo)₂] (6) and [Cu(mpo)₂]·2H₂O (7·2H₂O). The crystal structures of [Cu(o-van)₂(H₂O)]·0.25H₂O (1·0.25H₂O) and [Cu(5-CH₃-salo)₂] (2) have recently been published by our group.³⁵

Scheme 1 The ligands: (A) 3-OCH₃-saloH (=o-vanH), (B) (5-X-saloH, where X = CH₃, NO₂, Cl or Br), (C) 2-OH-benzophenone (bpoH) and (D) 2-OH-5-CH₃-acetophenone (mpoH).

The biological properties of complexes (1–7) were focused on (i) the affinity for bovine (BSA) and human (HSA) serum albumins (proteins responsible for the transport of drugs, metal ions and complexes through the bloodstream) investigated by fluorescence spectroscopy, (ii) the binding properties of the complexes to calf-thymus DNA, studied by UV spectroscopy, DNA-viscosity measurements, and cyclic voltammetry and (iii) competitive DNA-binding studies with ethidium bromide (EB) (in order to verify the intercalation to DNA) performed by fluorescence spectroscopy. Additionally, the affinity of the X-saloH and ketoH ligands used in the present study for HSA or BSA has been also evaluated since a search of the literature has not revealed any similar studies involving the interaction of substituted salicylaldehydes or their complexes, while only one study regarding benzophenones and derivatives was conducted.³⁶

2. Experimental

2.1. Materials

The substituted salicylaldehydes (X-saloH, where X = 3-OCH₃, 5-CH₃, 5-NO₂, 5-Cl and 5-Br), the ketoH ligands (bpoH and mpoH), the salts Cu(NO₃)₂·3H₂O, CH₃ONa, trisodium citrate, NaCl, CT DNA and EB were obtained as reagent grade from Sigma-Aldrich Company and used as received. Solvents for the preparation and physical measurements of “extra pure” grade were obtained from Merck without further purification. Tetraethylammonium perchlorate (TEAP) was purchased from Carlo Erba and, prior to its use, it was recrystallised twice from ethanol and dried under vacuum.

DNA stock solution was prepared by dilution of CT DNA to buffer (containing 150 mM NaCl and 15 mM trisodium citrate at pH 7.0) followed by exhaustive stirring at 4 °C for 3 days, and kept at 4 °C for no longer than a week. The stock solution of CT DNA gave a ratio of UV absorbance at 260 and 280 nm (A₂₆₀/A₂₈₀) of 1.88, indicating that the DNA was sufficiently free of protein contamination.³⁷ The DNA concentration per nucleotide was determined by the UV absorbance at 260 nm after 1 : 20 dilution using ε = 6600 M⁻¹ cm⁻¹.³⁸

2.2. Instrumentation – physical measurements

Infrared (IR) spectra (400–4000 cm⁻¹) were recorded on a Nicolet FT-IR 6700 spectrometer with samples prepared as KBr pellets. UV-visible (UV-vis) spectra were recorded as nujol mulls and in DMSO solutions at concentrations in the range 10⁻⁵ to 5 × 10⁻³ M on a Hitachi U-2001 dual beam spectrophotometer. Room temperature magnetic measurements were carried out on a magnetic susceptibility balance of Sherwood Scientific (Cambridge, UK) by the Faraday method using mercury tetrathiocyanatocobaltate(ii) as a calibrant. C, H and N elemental analyses were performed on a PerkinElmer 240B elemental microanalyzer. Molecular conductivity measurements of 1 mM DMSO solution of the complexes were carried out with a Crison Basic 30 conductometer. Fluorescence spectra were recorded in solution on a Hitachi F-7000 fluorescence spectrophotometer. Viscosity experiments were carried out using an ALPHA L Fungilab rotational viscometer equipped with an 18 mL LCP spindle and the measurements were performed at 100 rpm. Cyclic voltammetry studies were performed on an Eco chemie Autolab Electrochemical analyzer. Cyclic voltammetric experiments were carried out in a 30 mL three-electrode electrolytic cell. The working electrode was platinum disk, a separate Pt single-sheet electrode was used as the counter electrode and Ag/AgCl electrode saturated with KCl was used as the reference electrode. The cyclic voltammograms of the complexes were recorded in 0.4 mM DMSO solutions and in 0.4 mM 1/2 DMSO/buffer solutions at ν = 100 mV s⁻¹ where TEAP and the buffer solution were the supporting electrolytes, respectively. Oxygen was removed by purging the solutions with pure nitrogen which had been previously saturated with solvent vapors. All electrochemical measurements were performed at 25.0 ± 0.2 °C. The simultaneous TG/DTG-DTA curves were recorded on a SETARAM thermal analyzer, model SETARAM SETSYS-1200. The samples of approximately 10 mg were heated in platinum crucibles, in a nitrogen atmosphere at a flow rate of 50 mL min⁻¹, within the temperature range 30–1000 °C, at a heating rate of 10 °C min⁻¹.

2.3. Synthesis of the complexes [Cu(L)₂(S)_n] (1–7)

All complexes were synthesized according to the procedure published,³⁵ by the addition of a methanolic solution (15 mL) of the appropriate X-saloH or ketoH ligand (1 mmol), deprotonated with CH₃ONa (1 mmol, 54 mg), to a methanolic solution (10 mL) of Cu(NO₃)₂·3H₂O (0.5 mmol, 121 mg) at room temperature. The reaction mixture was stirred for two hours and then turned into green. The solution was filtered off and left for slow evaporation. After a few days greenish microcrystalline products were collected with filtration and were air-dried. The synthesis and characterization of two compounds (1) and (2) were reported recently in the literature.³⁵

2.3.1. Synthesis of [Cu(5-NO₂-salo)₂(CH₃OH)₂] (3). Green microcrystalline solid, yield 53.0%, 243 mg, analyzed as [Cu(5-NO₂-salo)₂(CH₃OH)₂], (C₁₆H₁₆CuN₂O₁₀) (MW = 459.8): C 41.75, H 3.48, N 6.09; found: C 41.53, H 3.42, N 6.04. IR spectrum (KBr): selected peaks in cm⁻¹: 3463 (medium, (m) v(O–H)) of coordinated methanol, 1625(strong, (s)) v(C=O), 1332(strong-to-medium (sm)) v(C–O → Cu), 536 (m) v(Cu–O); UV-vis: λ/nm (ε/M⁻¹ cm⁻¹) as nujol mull: 320, 370, 420, 470(sh), 770; in DMSO: 370(sh), 428(4560), 778(68); room temperature magnetic moment (μ_eff) = 1.75 BM, molar conductivity in 1 mM DMSO = 17.4 μS cm⁻¹.

2.3.2. Synthesis of [Cu(5-Cl-salo)₂] (4). Green crystals suitable for X-ray structure determination, yield 55.0%, 206 mg, analyzed as [Cu(5-Cl-salo)₂], (C₁₄H₈Cl₂CuO₄) (MW = 374.6): C 44.85, H 2.14; found: C 44.84, H 2.14. IR spectrum (KBr): selected peaks in cm⁻¹: 1632 (s) v(C=O), 1321(sm) v(C–O → Cu), 565 (m) v(Cu–O); UV-vis: λ/nm (ε/M⁻¹ cm⁻¹) as nujol mull: 314, 374, 420, 698; in DMSO: 315(sh), 397(6000), 698(200); μ_eff = 1.82 BM, molar conductivity in 1 mM DMSO = 5.4 μS cm⁻¹.

2.3.3. Synthesis of [Cu(5-Br-salo)₂] (5). Green microcrystalline solid, yield 56.0%, 259 mg, analyzed as [Cu(5-Br-salo)₂], (C₁₄H₈Br₂CuO₄) (MW = 463.5): C: 36.24, H: 1.72; found: C: 36.17, H: 1.74. IR spectrum (KBr): selected peaks in cm⁻¹: 1631(s) v(C=O), 1319(sm) v(C–O → Cu), 564(m) v(Cu–O); UV-vis: λ/nm as nujol mull: 318(sh), 370, 396, 450, 575(sh), 698; in DMSO: 315(sh), 395(9000), 698(200); μ_eff = 1.89 BM, molar conductivity in 1 mM DMSO = 15.1 μS cm⁻¹.

2.3.4. Synthesis of [Cu(bpo)₂] (6). Green-blue crystals suitable for X-ray structure determination, yield 54.0%, 247 mg, analyzed as [Cu(bpo)₂], (C₂₆H₁₈CuO₄) (MW = 458.0): C 68.19, H 3.93; found: C 67.91, H 4.13. IR spectrum (KBr): selected peaks in cm⁻¹: 1607(s) v(C=O), 1358(sm) v(C–O → Cu), 542(m) v(Cu–O); UV-vis: λ/nm (ε/M⁻¹ cm⁻¹) as nujol mull: 318(sh), 374, 396, 440, 695; in DMSO: 315(sh), 400(9000), 691(70); μ_eff = 1.84 BM, molar conductivity in 1 mM DMSO = 2.2 μS cm⁻¹.

2.3.5. Synthesis of [Cu(mpo)₂]·2H₂O (7·2H₂O). Olive-green crystals suitable for X-ray structure determination, yield 57.0%, 227 mg, analyzed as [Cu(mpo)₂]·2H₂O, (C₁₈H₂₂CuO₆) (MW = 397.9): C: 54.27, H: 5.52; found: C: 54.05, H: 5.54. IR spectrum (KBr): selected peaks in cm⁻¹: 1628(s) v(C=O), 1333(sm) v(C–O → Cu), 514(m) v(Cu–O); UV-vis: λ/nm (ε/M⁻¹ cm⁻¹) as nujol mulls: 315, 377(sh), 391, 690; in DMSO: 315, 377(sh), 391(5200), 697(70); μ_eff = 1.76 BM, molar conductivity in 1 mM DMSO = 1.7 μS cm⁻¹.

2.4. X-ray crystal structure determination

Single crystals of [Cu(5-NO₂-salo)₂(CH₃OH)₂] (3), [Cu(5-Cl-salo)₂] (4), [Cu(bpo)₂] (6) and [Cu(mpo)₂]·2H₂O (7·2H₂O), suitable for crystal structure analysis were obtained by slow evaporation of their mother liquids at room temperature. They were mounted at room temperature on a Bruker Kappa APEX2 diffractometer equipped with a triumph monochromator using Mo Kα radiation. Unit cell dimensions were determined and refined by using the angular settings of at least 200 high intensity reflections (>10σ(I)) in the range 11 < 2θ < 36°. Intensity data were recorded using ϕ and ω scans. All crystals presented no decay during the data collection. The frames collected for each crystal were integrated with the Bruker SAINT Software package,³⁹ using a narrow-frame algorithm. Data were corrected for absorption using the numerical method (SADABS) based on crystal dimensions.⁴⁰ All structures were solved using the SUPERFLIP package,⁴¹ incorporated in Crystals. Data refinement (full-matrix least-squares methods on F²) and all subsequent calculations were carried out using the Crystals version 14.40b program package.⁴² All non-hydrogen atoms were refined anisotropically. Hydrogen atoms were located by difference maps at their expected positions and refined using soft constraints. By the end of the refinement, they were positioned geometrically using riding constraints to bonded atoms. Illustrations with 50% ellipsoids probability were drawn by CAMERON.⁴³ Crystallographic data for the complexes are shown in Table 1.

Table 1 Crystallographic data for complexes 3, 4, 6 and 7·2H₂O

	3 [Cu(5-NO2-salo)2(CH3OH)2]	4 [Cu(5-Cl-salo)2]	6 [Cu(bpo)2]	7·2H2O [Cu(mpoH)2]·2H2O
Empirical formula	C16H16Cu1N2O10	C14H8Cl2Cu1O4	C26H18Cu1O4	C18H22Cu1O6
CCDC no.	1034140	1036592	1034138	1034139
Molecular mass	459.86	374.67	457.97	397.91
Crystal system	Triclinic	Monoclinic	Monoclinic	Monoclinic
Temperature, K	296	295	295	295
Radiation type	Mo Kα	Mo Kα	Mo Kα	Mo Kα
Wavelength λ, (Å)	0.71073	0.71073	0.71073	0.71073
Space group	P1̄	P2/c	C2/c	C2/c
Unit cell dimensions
a (Å)	5.2245(3)	13.7347(10)	18.7015(8)	23.349(4)
b (Å)	7.5079(5)	3.8442(3)	5.7571(3)	4.8974(7)
c (Å)	11.9914(7)	12.4921(9)	19.9705(10)	18.972(3)
α, deg	93.397(3)	90	90	90
β, deg	94.312(3)	102.258(3)	114.310(2)	125.037(5)
γ, deg	102.049	90	90	90
Volume, (Å^3)	457.34(3)	644.53(5)	1959.50(10)	1776.3(3)
Z	1	2	4	4
Absorption coefficient (μ) mm^−1	1.255	2.120	1.147	1.260
Crystal density, Dx, g cm^−3	1.670	1.930	1.552	1.488
Crystal size, mm	0.13 × 0.38 × 0.38	0.07 × 0.16 × 0.41	0.15 × 0.32 × 0.41	0.13 × 0.30 × 0.56
θ range for data collection, deg/completeness (%)	3.151–32.582/99.4	1.517–26.369/99.1	2.238–28.739/98.4	2.131–32.252/99.5
Range of h, k, l	−7 → 7, −11 → 11, −18 → 18	−17 → 17, −4 → 4, −15 → 15	−25 → 23, −7 → 7, −26 → 26	−34 → 34, −7 → 7, −28 → 28
Measured reflections/independent reflections/Rint	6107/3205/0.0218	12 437/1309/0.0393	17 826/2435/0.0224	14 334/3084/0.0334
No of parameters	136	97	142	115
Goodness-of-fit on F^2 (GOF)	1.000	1.000	1.000	0.998
Final R indices
R1, wR2 [I > 2σ(I)]	0.0328, 0.0772	0.0520, 0.0965	0.0467, 0.0845	0.0445, 0.0809
R1, wR2 (all data)	0.0444, 0.0804	0.0752, 0.1108	0.0667, 0.0909	0.0876, 0.0915
Largest difference peak/hole (e Å^−3)	0.35/−0.37	0.90/−0.56	0.41/−0.56	0.60/−0.59

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2.5. DNA-binding studies

In order to study the interaction of DNA with the compounds, the compounds were initially dissolved in DMSO (1 mM). Mixing of such solutions with the aqueous buffer DNA solutions used in the studies never exceeded 5% DMSO (v/v) in the final solution, which was needed due to low aqueous solubility of most compounds. All studies were performed at room temperature. The interaction of 5-NO₂-saloH, 5-Cl-saloH, 5-Br-saloH³³ and bpoH³⁴ with CT DNA was recently reported. In present study the interaction of CT DNA with o-vanH, 5-CH₃-saloH and mpoH are reported.

2.5.1. Study with UV spectroscopy. The interaction of the X-saloH, ketoH and their complexes 1–7 with CT DNA has been studied with UV spectroscopy in order to investigate the possible binding modes to CT DNA and to calculate the binding constants to CT DNA (K_b). Control experiments with 5% DMSO were performed and no changes in the spectra of CT DNA were observed.

The UV spectra of CT DNA in the presence of each compound were recorded for a constant CT DNA concentration in diverse mixing ratios (r = [compound]/[DNA]). The binding constant of the compounds with DNA, K_b (in M⁻¹), were determined by the Wolfe–Shimer equation (eqn (S1)†)⁴⁴ and the plots using the UV spectra of the compounds recorded, for a constant concentration in the presence of DNA for diverse r values.

2.5.2. Viscometry. Viscosity experiments were carried out using an ALPHA L Fungilab rotational viscometer equipped with an 18 mL LCP spindle and the measurements were performed at 100 rpm. The viscosity of DNA ([DNA] = 0.1 mM) in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0) was measured in the presence of increasing amounts of the compounds (saloH, ketoH and complexes 1–7) up to the value of r = 0.3. All measurements were performed at room temperature. The obtained data are presented as (η/η₀)^1/3 versus r, where η is the viscosity of DNA in the presence of the compound, and η₀ is the viscosity of DNA alone in buffer solution.

2.5.3. Cyclic voltammetry studies. The interaction of complexes 1–7 with CT DNA has been also investigated by monitoring the changes observed in the cyclic voltammogram of a 0.40 mM 1 : 2 DMSO : buffer solution of complex upon addition of DNA at diverse r values. The buffer was also used as the supporting electrolyte and the cyclic voltammograms were recorded at ν = 100 mV s⁻¹.

2.5.4. EB competitive studies with fluorescence spectroscopy. The competitive studies of each compound with EB have been investigated with fluorescence spectroscopy in order to examine whether the compound can displace EB from its DNA–EB complex. The DNA–EB complex was prepared by adding 20 μM EB and 26 μM CT DNA in buffer (150 mM NaCl and 15 mM trisodium citrate at pH 7.0). The possible intercalating effect of the compounds (X-saloH, ketoH and complexes 1–7) was studied by adding a certain amount of a solution of the compound step by step into a solution of the pre-treated DNA–EB complex. The influence of the addition of each compound to the DNA–EB complex solution has been obtained by recording the variation of fluorescence emission spectra with excitation wavelength at 540 nm. The compounds do not show any fluorescence emission at room temperature in solution or in the presence of DNA under the same experimental conditions; therefore, the observed quenching is attributed to the displacement of EB from its EB–DNA complex. The values of the Stern–Volmer constant (K_SV, in M⁻¹) have been calculated according to the linear Stern–Volmer equation (eqn (S2)†)⁴⁵ and the plots .

2.6. Interaction with albumins

The protein binding study was performed by tryptophan fluorescence quenching experiments using bovine (BSA, 3 μM) or human serum albumin (HSA, 3 μM) in buffer (containing 15 mM trisodium citrate and 150 mM NaCl at pH 7.0). The quenching of the emission intensity of tryptophan residues of BSA at 343 nm or HSA at 351 nm was monitored using the compounds (X-saloH, ketoH and complexes 1–7) as quencher with increasing concentration. The fluorescence spectra were recorded from 300 to 500 nm at an excitation wavelength of 295 nm.⁴⁶ Most of the compounds exhibited under the same experimental conditions (i.e. excitation at 295 nm) a low-intensity emission band in the region 395–415 nm. Therefore, the quantitative studies of the serum albumin fluorescence spectra were performed after their correction by subtracting the spectra of the compounds. The influence of the inner-filter effect⁴⁷ on the measurements was evaluated by (eqn (S3)†). The Stern–Volmer and Scatchard equations (eqn (S4)–(S6)†)⁴⁸ and graphs have been used in order to study the interaction of each quencher with serum albumins and calculate the dynamic quenching constant K_SV (in M⁻¹), the approximate quenching constant k_q (in M⁻¹ s⁻¹), the association constant K (in M⁻¹) and the number of binding sites per albumin (n) (Tables S1 and S2†).

3. Results and discussion

3.1. Synthesis-general considerations of the complexes

The reaction of Cu(NO₃)₂·3H₂O with five substituted salicylaldehydes and two 2-hydroxy-phenones in methanol (deprotonated by sodium methoxide) afforded neutral (molar conductivities in DMSO solutions were found to have values between 1.7 and 17.4 μS cm⁻¹) solid microcrystalline compounds in good yield. The obtained copper(II) complexes possess 1 : 2 metal-to-ligand composition with the general formula [Cu(X-salo)₂S_n], where X = 3-OMe (for 1), 5-Me (for 2), 5-NO₂ (for 3), 5-Cl (for 4) or 5-Br (for 5), while S = H₂O or MeOH and n = 0, 1 or 2 and are soluble in MeOH, CH₃COCH₃, CH₂Cl₂, DMF, DMSO but not in H₂O and Et₂O. Evidence of the coordination mode of the ligands in the copper compounds is also arisen from the interpretation of the IR and UV-vis data of the salicylaldehyde (X-saloH) or the 2-hydroxyphenone (ketoH) ligands and the complexes. In these compounds, the ligands behave in the normal coordination mode, as bidentate monoanionic ligands, being coordinated through the carbonyl and the phenolato oxygen atoms.

3.2. Spectroscopy (IR, UV-vis)

IR spectroscopy has been used in order to confirm the deprotonation and binding mode of X-saloH and ketoH ligands. In the spectra of the free ligands the intense bands of the stretching and bending vibrational modes of the phenolic OH around 3200 cm⁻¹ and 1410 cm⁻¹, respectively, disappear from the spectra of all complexes indicating the deprotonation of the ligands.^49,50 Additionally, the bands originating from the C–O stretching vibrations at 1245–1285 cm⁻¹, in the complexes exhibit positive shifts at 1350–1380 cm⁻¹ denoting coordination through the carbonyl oxygen of the ligand. The band at ∼1640 cm⁻¹ attributable to the aldehyde bond v(HC=O), as well as the band at ∼1660 cm⁻¹ attributable to the carbonyl bond v(R–C=O) of the free ligands, is shifted towards lower frequencies (∼1610 cm⁻¹) in the complexes, thus denoting the bidentate mono-anionic character of the ligands.⁵¹

The UV-vis spectra of the complexes have been recorded as nujol mull and in DMSO solution (0–48 h) (representatively shown for complexes 4 and 6 in Fig. S1†) and are similar, suggesting that the complexes retain their structure in solution within the timeframe used for the biological experiments. In the visible region, complex 1 exhibits a d–d transition band at 668 nm, attributed to the ²B₁ → ²E transition, which is in consistence with distorted square pyramidal geometry, while complex 3 presents a band at 778 nm, attributed to the ²E_g → ²T_2g transition expected for octahedral geometries.⁵² In the complexes 2, 4, 5, 6 and 7, the band observed in the range of 690–698 nm can be assigned as the ²B_1g → ²A_1g transition and is typical for square planar geometries.^53,54

In addition, in order to explore the stability of complexes in the presence of buffer solution used in the biological experiments, the UV-vis spectra have also been recorded in the presence of buffer solution in the pH range 6–8 (the biological experiments are performed at pH = 7) with the use of diverse buffer solutions (150 mM NaCl and 15 mM trisodium citrate at pH values regulated by HCl solution). Significant changes (shift of the λ_max or new peaks) have not been observed in the spectra; this observation combined with the non-dissociation in DMSO solution (Λ_M ≤ 17.4 μS cm⁻¹, in 1 mM DMSO solution) may indicate that complexes 1–7 keep their integrity in solution.⁵⁵ It should be also noted that complexes 1–7 exhibit similar chromophores and geometries as those found in a series of metal–oxicam complexes.^56–64 Therefore, complexes 1–7 are expected to present similar stability with their oxicam analogues,⁶⁴ which present stability constants significantly higher than the Cu–citrate.⁶⁵ Therefore, the replacement of the salo or keto ligands in complexes 1–7 by the citrate ligands is not highly anticipated, when the citrate-containing buffer solution is added in the solution of the complexes during the biological experiments, proving, thus, indirectly the stability of the complexes in the presence of citrates, being in accordance with the spectroscopic findings (representatively shown in Fig. S1 and S2†).

In order to prove the geometry around copper(II) ion, we prepared single-crystals, suitable for X-ray structural characterization for four complexes 3, 4, 6 and 7.

3.3. Description of the structures (3, 4, 6 and 7)

The molecular structures of the four complexes (3, 4, 6 and 7) with the atom numbering scheme are shown in Fig. 1–4, respectively, and selected bond distances and angles are given in Table 2.

Fig. 1 Molecular structure of [Cu(5-NO₂-salo)₂(CH₃OH)₂] (3) with the displacement ellipsoids shown at the 50% probability level.

Fig. 2 Molecular structure of [Cu(5-Cl-salo)₂] (4) with the displacement ellipsoids shown at the 50% probability level.

Fig. 3 Molecular structure of [Cu(bpo)₂] (6) with the displacement ellipsoids shown at the 50% probability level.

Fig. 4 Molecular structure of [Cu(mpo)₂]·2H₂O (7·2H₂O) with the displacement ellipsoids shown at the 50% probability level.

Table 2 Selected bond distances (Å) and angles (°) for complexes 3, 4, 6 and 7·2H₂O

Bond distance, (Å)	3	4	6	7·2H2O
Cu(1)–O(1)	1.9243(11)	1.901(4)	1.908(2)	1.9276(19)
Cu(1)–O(2)	1.9650(12)	1.934(3)	1.915(2)	1.8754(19)
Cu(1)–O(5)	2.3934(16)
Bond angle, (°)
O(1)^i–Cu(1)–O(1)	180	180	180	180
O(2)^i–Cu(1)–O(2)	180	180	180	180
O(1)^i–Cu(1)–O(2)	86.99(5)	87.13(13)	91.65(9)	87.73(8)
O(1)–Cu(1)–O(2)	93.01(5)	92.87(13)	88.35(9)	92.27(8)
O(5)^i–Cu(1)–O(5)	180
O(2)^i–Cu(1)–O(5)	85.89(6)
O(2)–Cu(1)–O(5)	94.11(6)
O(1)^i–Cu(1)–O(5)	89.29(6)
O(1)–Cu(1)–O(5)	90.71(6)
Symmetry	(i) 1 − x, 2 − y, −z	(i) 1 − x, 1 − y, 1 − z	(i) 1 − x, 2 − y, −z	(i) 0.5 − x, 1.5 − y, 1 − z

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3.3.1. Description of the structure of [Cu(5-NO₂-salo)₂(CH₃OH)₂] (3). In the molecular structure of [Cu(5-NO₂-salo)₂(CH₃OH)₂] (3), two 5-NO₂-salicylaldehyde anions are chelated through the phenolate oxygen O(1) and the carbonyl O(2) to the copper cation in trans positions and occupy the equatorial plane. In complex 3, as well as in complexes 4, 6 and 7, the Cu(1)–(O1) (phenolic oxygen) distance is shorter than the distance Cu(1)–O(2) (carbonyl oxygen), as expected, suggesting the stronger coordination ability of the ionic phenolate oxygen (Table 2). A slightly distorted octahedral coordination is achieved by the binding of two methanol molecules in the axial positions. The mean planes of the two salicylaldehyde ligands are parallel but not coplanar having a distance of 0.802 Å. The Cu(II) ion is situated in the mid-distance due to symmetry reasons. Strong intermolecular hydrogen bonding interactions arise from the methanol hydroxyl groups to the phenolate O atoms forming chains of complex planes parallel to ‘a’ crystallographic axis. Bond distances of coordinated 5-NO₂-salicylaldehyde are similar to a reported mixed-ligand 5-NO₂-salicylaldehydato copper(II) complex.⁶⁶

3.3.2. Description of the structure of [Cu(5-Cl-salo)₂] (4). In the molecular structure of [Cu(5-Cl-salo)₂] (4), two 5-Cl-salicylaldehyde anions are chelated through the phenolate oxygen O(1) and carbonyl oxygen O(2) to the copper cation in a bidentate manner. The geometry around the metal center is square planar. The torsion angle C(2)–O(1)–Cu(1)–O(2)ⁱ has a value of 166.5(5)° indicating that the salicylaldehyde ligands are not coplanar. The mean planes of the corresponding ligands of two neighboring complexes have a distance of 3.844 Å, indicating weak π–π stacking lattice interactions. This crystal structure is similar with an analogous reported Cu(II) complex, [Cu(3,5-dibromo-salo)₂].⁶⁷

3.3.3. Description of the structure of [Cu(bpo)₂] (6). The molecular structure of [Cu(bpo)₂] (6) clearly indicates that the Cu(II) atom is chelated by two bpo ligands via the phenolate and carbonyl oxygen atoms. Each ligand is singly deprotonated and the complexes are neutral. The geometry around copper is square planar. The torsion angle C(1)–O(1)–Cu(1)–O(2)ⁱ has a value of 178.1(3)° indicating that the salicylaldehyde ligands are nearly coplanar with a deviation from planarity nearly equal to the error. The two phenyl rings are parallel between them and each one is forming an angle of 62.69° with the salicylaldehyde mean planes. A review of the literature revealed that there are no similar copper complexes with 2-hydroxy-benzophenone, but its coordination mode is in agreement with previously reported mixed-ligands Co(II) and Zn(II) complexes.^16,34

3.3.4. Description of the structure of [Cu(mpo)₂]·2H₂O (7·2H₂O). The molecular structure of [Cu(mpo)₂]·2H₂O (7·2H₂O) indicates that the Cu(II) atom is chelated by two 2-OH-5-methyl-acetophenone ligands via the phenolate and carbonyl oxygen atoms. The geometry around copper is square planar. The two acetophenone ligands are nearly coplanar. The complex is similar to the reported complexes bis(2-hydroxy-4-methoxy-acetophenone)copper(II)⁶⁸ and bis(2-hydroxyacetophenone)copper(II),⁶⁹ where two molecules form a dimer via weak Cu(1)⋯O′(phenolate) interactions in contrast with the independent molecules placed in general positions in our case. Each complex moiety is accompanied by two lattice water molecules. Strong intermolecular hydrogen bonding interactions are formed between the water molecules and the phenolate oxygen atoms. They present a Y type conformation with the three branches being water to water, water to water and water to phenolate oxygen. These interactions alternate perpendicular complex molecules forming a 2D rigid lattice parallel to ‘bc’ crystallographic plane.

3.3.5. Proposed structure for complex [Cu(5-Br-salo)₂] (5). Based on the experimental data (IR, UV-vis spectroscopy, elemental analysis, molar conductivity, magnetic measurements and PXRD pattern (crystal system: orthorhombic, space group: Pbca, a = 21.0452, b = 25.5337, c = 29.5171 Å, Fig. S3†)) and after a comparison with the existing structurally characterized copper salicylaldehyde complex, [bis(3,5-dibromo-salicylaldehydato)copper(II)],⁶⁷ we could propose a structure for complex 5. This complex is expected to have similar structure with the afore-mentioned one, as well as with complex 4 in the present work, and to exhibit a square planar geometry with two deprotonated bidentate 5-Br-salicylaldehydato ligands.

3.4. Thermal studies (TG/DTG-DTA in nitrogen)

The thermal behaviour for three (3, 6 and 7) of the title compounds was studied in nitrogen atmosphere, over the temperature range ambient to 1000 °C by using the simultaneous TG/DTG-DTA technique, in order to explore their thermal stability. The temperature ranges, the determined percentage mass losses, and the thermal effects accompanying the decomposition are given in Table 3. Representative thermal curves are depicted for the complexes [Cu(5-NO₂-salo)₂(CH₃OH)₂] (3) and [Cu(mpo)₂]·2H₂O (7·2H₂O) in Fig. 5. The studied compounds are stable in air at room temperature, but unstable upon heating, decomposing in several successive stages. Their thermal decomposition profile is similar, showing firstly the elimination of the crystal water molecules (for compound 7) or the coordinated molecules of methanol (for compound 3). Upon further heating, the thermal process was found to be a multi-step decomposition related to the release of the ligand molecules for all complexes. The two decomposition stages, as it is estimated from the TG and DTG curves, could be attributed to the cleavage of coordination bonds along with the rupture of bonds inside the ligand molecules and the elimination of one ligand, which is accompanied by the release of the fragments of the second ligand{(L–O), R, RCO, Ph} (Table 3). The total mass found 75.0 ± 5% corresponds to a residue possibly attributable to a carbonaceous metal oxide (CuO + C) at 1000 °C. The complex nature of thermal decomposition for analogous transition metal complexes has also been referred,^16,70,71 while the kind of carbonaceous residue (M^IIO and un-pyrolized compounds as organic part) has been observed in other analogous complexes.⁷²

Table 3 Thermoanalytical results (TG/DTG-DTA) for [Cu(5-NO₂-salo)(MeOH)₂] (3), [Cu(bpo)₂] (6) and [Cu(mpo)₂] 2H₂O (7·2H₂O) in nitrogen atmosphere

	Stage	Trange/°C	Evolved moiety formula	DTGmax/°C	DTA(−)/°C	Mass loss exp/% (mass loss calc/%)
3	I	30–150	2MeOH	109	110	11.0 (13.5)
		150–280 (plateau)
	IIa	280–400	L + {L–O}	334	334(+)	67.5 (68.7)
	IIb	400–900	Unknown	—	—	3.0 (x)
	Residue	>900	Solid (CuO + C)			18.5 (17.3 + x)
6	I	30–350	{L + PhCO}	323	323	68.0 (66.0)
	II	350–960	Ph	—	780(br,+)	16.0 (16.8)
	Residue	>980	Solid CuO			16.0 (17.4)
7·2H2O	I	30–155	2H2O	155	100, 155	10.0 (9.0)
	II	155–300	L	214	210	38.0 (37.4.)
	IIIa	300–450	MeCHO	396	394(+)	11.0 (11.0)
	IIIb	450–900	Ph + Me	—	850(+)	12.0 (22.6)
	Residue	>900	Solid (CuO + C)			29.0 (20.0 + x)

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Fig. 5 TG/DTG-DTA curves of (A) [Cu(5-NO₂-salo)₂(CH₃OH)₂] (3) and (B) [Cu(mpo)₂]·2H₂O (7·2H₂O) in nitrogen at 10 °C min⁻¹.

3.5. Study of complexes 1–7 by cyclic voltammetry

The cyclic voltammograms of complexes 1–7 were recorded in DMSO solution (0.4 mM) in the potential region +1.5 to −1.5 V (the cyclic voltammograms of complex 2 in the region +0.5 to −1.5 V are shown representatively in Fig. 6). They are similar for all complexes and the corresponding potentials are given for complexes 1–7 in Table 4. In the cyclic voltammograms of the complexes two cathodic waves (E_pc1 and E_pc2) were initially observed showing a two-step reduction of the species; these two cathodic waves can be assigned to the process [Cu(II)] → [Cu(I)], and the formation of metallic copper,^52,73 respectively, since the corresponding free X-saloH and ketoH were electrochemically inactive in the potential range that the cyclic voltammograms were recorded. These cathodic waves were followed by two non-reversible anodic waves (E_pa2 and E_pa1) which may be attributed to the stepwise oxidation processes of metallic copper to [Cu(II)].

Fig. 6 Cyclic voltammogram of 0.4 mM DMSO solution of [Cu(5-CH₃-salo)₂], 2. Scan rate = 100 mV s⁻¹. Supporting electrolyte = TEAP, 0.1 M.

Table 4 Cathodic and anodic potentials (in mV) for the redox couples of complexes 1–7 in DMSO solution

Complex	Epc1	Epc2	Epa2	Epa1
a nd = not detected.
[Cu(o-van)2(H2O)], 1	−980	−1300	−165	+415
[Cu(5-CH3-salo)2], 2	−690	−1260	−1023	+197
[Cu(5-NO2-salo)2(CH3OH)2], 3	−615	−990	−155	+445
[Cu(5-Cl-salo)2], 4	−610	−1400	nd^a	+175
[Cu(5-Br-salo)2], 5	−595	−1200	−130	+440
[Cu(bpo)2], 6	−575	−1175	−1085	+450
[Cu(mpo)2], 7	−570	−1190	−100	+230

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3.6. Interaction with calf-thymus DNA

Metal ions and complexes can interact with DNA covalently and/or non-covalently. Covalent interactions take place upon the replacement of a labile ligand of the complex by a DNA nitrogen base (such as guanine N7) and non-covalent interactions exist in the presence of π → π stacking between the complex and DNA nucleobases (in the case of intercalation), of van der Waals or hydrogen- or hydrophobic bonds along major or minor groove of DNA helix (i.e. groove-binding) and of Coulomb forces between the complexes and the phosphate groups of DNA (electrostatic interactions).^74,75 Although the interaction of saloH and ketoH and their complexes with DNA is of increasing interest, the interaction of DNA with only the Zn(II) complexes of 5-NO₂-saloH, 5-Cl-saloH, 5-Br-saloH and bpoH have been studied and recently reported.^33,34 In this context and as a continuation of our recent research, the interaction of free o-vanH, 5-Me-saloH and mpoH and complexes 1–7 is investigated by UV spectroscopy, cyclic voltammetry, DNA viscosity measurements and ethidium bromide displacement examined by fluorescence emission spectroscopy.

3.6.1. DNA-binding study with UV spectroscopy. UV-Vis spectroscopy provides preliminary information about the DNA-binding mode of compounds and the strength of this binding. Thus, the UV spectra of a CT DNA solution (1.5–2.5 × 10⁻⁴ M) were recorded in the presence of o-vanH, 5-Me-saloH, mpoH and complexes 1–7 at increasing amounts (for different [compound]/[DNA] mixing ratios = r) as well as the UV spectra of the compounds (2.5 × 10⁻⁵ to 2 × 10⁻⁴ M) in the presence of CT DNA at increasing amounts (r′ = [DNA]/[compound] mixing ratio). The existence of any interaction between the compound and CT DNA will perturb the band of CT DNA at 258–260 nm or the intra-ligand transition bands of the compound, respectively, during the titrations with the red-shift showing stabilization of the structure and the blue-shift being evidence of structural destabilization. Furthermore, the intense hypochromism of a transition band, which is usually accompanied by a bathochromism, is evidence of an intercalative binding mode.⁷⁶

The UV spectra of DNA solution were recorded in the presence of o-vanH, 5-Me-saloH, mpoH and complexes 1–7 at increasing r values (up to 0.3). The UV spectra of a DNA solution exhibit a slight hyperchromism of the band λ_max = 258 nm followed by 2 nm red-shift in the presence of 1 at diverse r values (as shown representatively in Fig. 7). The UV spectra of the DNA solution in the presence of complexes 2–7, o-vanH, 5-Me-saloH and mpoH are quite similar exhibiting hyperchromism which may be considered as evidence of the formation of a new adduct of the compound with double-helical DNA resulting in the stabilization of the DNA duplex.⁷⁶

Fig. 7 UV spectra of CT DNA (2.25 × 10⁻⁴ M) in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0) in the absence or presence of complex 1. The arrow shows the changes upon increasing amounts of the complex.

In the UV spectra of o-vanH (2 × 10⁻⁴ M) in the presence of increasing amounts of a DNA solution, the band at 340 nm shows a ∼28% hypochromism accompanied by a 3 nm red-shift, while the absorption of the band at 400 nm increases significantly (Fig. 8(A)); an isosbestic points appears at 359 nm. In the UV spectra of 5-Me-saloH (2.5 × 10⁻⁵ M) in the presence of increasing amounts of DNA, the band at 335 nm shows a hypochromism of ∼10% and its position does not change (Fig. 8(B)); similar behaviour to 5-Me-saloH is observed for free mpoH (i.e. a ∼10% hypochromism at λ_max = 333 nm) upon addition of DNA.

Fig. 8 UV spectra of a DMSO solution of (A) o-vanH (2 × 10⁻⁴ M) and (B) 5-Me-saloH (2.5 × 10⁻⁵ M) in the presence of increasing amounts of CT DNA (r′ = 0–0.9). The arrows show the changes upon increasing amounts of CT DNA.

In the UV spectra of complex 2 (1 × 10⁻⁴ M), the band at 332 nm exhibits in the presence of DNA a slight hypochromism of ∼10% and the band at 405 nm a ∼80% hyperchromism (Fig. 9(A)); both bands are accompanied by a 3 nm bathochromism and appearance of an isosbestic point at 358 nm. In the UV spectra of complex 3 (2.5 × 10⁻⁵ M), both bands at 370 nm (band I) and 428 nm (band II) show a slight hypochromism of ∼7% and ∼10%, respectively (Fig. 9(B)); the position of band I does not shift, while band II presents a blue-shift of 10 nm. In the UV spectra of complex 6 (1 × 10⁻⁴ M), both bands at 315 nm (band I) and 400 nm (band II) present upon addition of DNA a significant hypochromism up to 30% with a 5 nm red-shift for band I and a ∼85% hypochromism which leads to gradual disappearance of band II (Fig. 9(C)); similar is the behaviour of complex 7 (Table 5). The changes in the UV spectra of complexes 1, 4 and 5 in the presence of DNA are rather more intense; the intensity of band I at 315 nm is significantly decreased (55–75%) followed be a red-shift of 17–20 nm (Table 5), while band II at ∼395 nm shows a hyperchromism up to ∼40% and a red-shift towards 415 nm (representatively shown for complex 5 (2 × 10⁻⁴ M) in Fig. 9(D)).

Fig. 9 UV spectra of DMSO solution of complex (A) 2 (1 × 10⁻⁴ M), (B) 3 (2.5 × 10⁻⁵ M), (C) 6 (1 × 10⁻⁴ M) and (D) 5 (2 × 10⁻⁴ M) in the presence of increasing amounts of CT DNA (r′ = 0–0.9). The arrows show the changes upon increasing amounts of CT DNA.

Table 5 UV Spectral features of the UV spectra of X-saloH, ketoH and complexes 1–7 upon addition of DNA (band studied in λ (nm), percentage of hyperchromism or hypochromism ΔA (%), blue- or red-shift Δλ (nm)) and the corresponding DNA binding constants (K_b)

Compound	Band (λ, nm) ((ΔA/A0) (%), Δλ (nm))	Kb (M^−1)
a “+” denotes hyperchromism (%), “−” denotes hypochromism (%).b “+” denotes red-shift (nm), “−” denotes blue-shift (nm).c “+>” denotes extreme hyperchromism (%).d “−<” denotes extreme hypochromism (%).e “nd” = not determined.f “elm” denotes elimination of the band.
o-vanH	340(−28^a, +3^b), 400(sh) (+>^c, 0)	9.84(±0.16) × 10^4
5-Me-saloH	335(−10, 0)	1.17(±0.11) × 10^6
5-NO2-saloH^33	316(−<^d, nd^e), 366(+>, +5), 430(+>, −5)	5.25(±0.25) × 10^5
5-Cl-saloH^33	336(−64, +4), 424(+>, −5)	2.59(±0.11) × 10^7
5-Br-saloH^33	336(−51, +2), 423(+>, −5)	7.13(±0.22) × 10^5
bpoH^34	319(−2.5, +1)	9.58(±0.40) × 10^4
mpoH	333(−10, 0)	9.74(±0.35) × 10^5
[Cu(o-van)2(H2O)], 1	315(sh) (−55, +20), 396(+8, +5)	1.91(±0.19) × 10^4
[Cu(5-CH3-salo)2], 2	332(−10, +3), 405(+80, +3)	4.69(±0.61) × 10^5
[Cu(5-NO2-salo)2(CH3OH)2], 3	370(−7, 0), 428(−10, −10)	9.06(±0.24) × 10^4
[Cu(5-Cl-salo)2], 4	315(−60, +17), 397(+40, +20)	1.21(±0.05) × 10^6
[Cu(5-Br-salo)2], 5	315(−75, +20), 395(+40, +17)	5.31(±0.07) × 10^6
[Cu(bpo)2], 6	315(−30, +5), 400(−85, elm^f)	1.49(±0.08) × 10^6
[Cu(mpo)2], 7	315(−15, +15), 391(−80, elm)	3.87(±0.48) × 10^5

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It is quite evident that safe conclusions concerning the interaction mode between the compounds and DNA cannot be drawn only by UV spectroscopic studies and more techniques should be combined. The data derived by the UV titration experiments suggest that all compounds can bind to CT DNA;⁷⁶ the presence of hypochromic effect which can be attributed to π → π stacking interaction between the aromatic chromophore of the X-salo or keto ligands of the complexes and DNA base pairs may be a first evidence of tight binding to CT DNA possibly by intercalation which is further pronounced upon existence of red-shift leading to a stabilization of the DNA duplex.⁷⁷

The magnitude of the binding strength a compound with CT DNA may be evaluated through the calculation of the DNA-binding constant (K_b). The K_b values can be obtained by the plots (Fig. S4 and S5†) using the Wolfe–Shimer equation⁴⁴ when monitoring the changes in the absorbance at the corresponding λ_max with increasing concentrations of CT DNA. The K_b values of complexes 1–7 (Table 5) are relatively high suggesting strong binding of the complexes to CT DNA and of similar magnitude to that of the classical intercalator EB (=1.23(±0.07) × 10⁵ M⁻¹),⁷⁸ with 5 having the highest K_b value (=5.31(±0.07) × 10⁶ M⁻¹) among the complexes; with the exception of complexes 5 and 6, the K_b of the complexes are similar or lower than that of the corresponding free X-saloH or ketoH. It is evident that the DNA-binding strength of the complexes is mainly dependant on the nature and the DNA-binding affinity of the ligands. A comparison of the K_b values with the zinc analogues of complexes 3–6 revealed that the K_b of complexes 3 and 4 are of the same magnitude with the corresponding Zn complexes,³³ and complexes 5 and 6 exhibit significantly higher K_b values than the Zn complexes with 5-Br-salo³³ and bpo ligands.³⁴ A comparison of the DNA-binding of complexes 1–7 with analogues Cu(II) complexes^{63,64,79–81} has revealed that complexes 1–7 exhibit similar or higher binding affinity for CT DNA.

3.6.2. DNA-binding study with viscosity measurements. In general, the viscosity of DNA is sensitive to DNA length changes which may occur upon the interaction of DNA with diverse compounds since the relation between the relative solution viscosity (η/η₀) and DNA length (L/L₀) is given by the equation (L/L₀) = (η/η₀)^1/3, where L₀ denotes the apparent molecular length in the absence of the compound.^82,83 Therefore, the measurement of the viscosity of DNA solution upon addition of a compound may prove a useful tool to clarify the interaction mode of a compound with DNA. In the case of intercalation, the compound inserts and is hosted between the DNA base pairs resulting in an increase of the separation distance of base pairs being at intercalation sites leading to an increase of the DNA-helix length and subsequently of the DNA viscosity. On the other hand, the binding of a compound to DNA grooves via a partial or non-classic intercalation (i.e. electrostatic interaction or external groove-binding) will induce a bend or kink in the DNA helix and subsequently a slight shortening of its effective length with the viscosity showing a slight decrease or remaining unchanged.^82–84

The viscosity measurements were carried out on CT DNA solutions (0.1 mM) upon addition of increasing amounts of o-vanH, 5-Me-saloH and mpoH and complexes 1–7 (up to the value of r = 0.30) at room temperature (Fig. 10). The relative viscosity of DNA solution exhibits an increase upon addition of all compounds and it is more pronounced in the case of the complexes. The observed behaviour of the DNA viscosity upon addition of the compounds may be considered evidence of the existence of an intercalative binding mode to DNA; a conclusion which clarifies the preliminary indications derived from UV spectroscopic studies.^33,34

Fig. 10 Relative viscosity (η/η₀)^1/3 of CT DNA (0.1 mM) in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0) in the presence of (A) o-vanH, 5-Me-saloH and mpoH and (B) complexes 1–7, at increasing amounts (r = 0–0.30).

3.6.3. Study of the DNA-interaction by cyclic voltammetry. Cyclic voltammetry is a technique that may provide useful information for the mode of interaction of metal ions or metal complexes with DNA being usually a supplement to the UV spectroscopic titrations.^85,86 In general, the negative shift of an electrochemical potential occurs in the case of an electrostatic interaction with DNA, while a shift of the potential towards a positive direction is observed in the case of intercalation.⁸⁷

The cyclic voltammograms of complexes 1–7 in a 1/2 DMSO/buffer solution were recorded in the absence and presence of CT DNA (representatively shown for 2 in Fig. S6†). The addition of DNA resulted in shift of the existing potentials without the appearance of new redox peaks, thus, suggesting an equilibrium between free and DNA-bound complex as evidence of the complex–DNA interaction.^85,86 The cathodic (E_pc) and anodic (E_pa) potentials of the redox couple Cu(II)/Cu(I) for each complex as well as their shifts upon addition of CT DNA are cited in Table 6. Upon addition of CT DNA, the cathodic and the anodic potentials of the complexes exhibit predominantly a positive shift (ΔE_pc/a = (−35) − (+74) mV). Therefore, based on the positive shifts of the cathodic and anodic potentials we may conclude the existence of intercalation as the most possible mode of interaction between the complexes and CT DNA bases;^87,88 a conclusion which may clarify the data derived from the UV spectroscopic titrations and is in accordance to the viscosity experiments.

Table 6 Cathodic and anodic potentials (in mV) for the redox couples of the complexes in DMSO/buffer solution in the absence or presence of CT DNA

Complex	Epc(f)^a	Epc(b)^b	ΔEpc^c	Epa(f)^a	Epa(b)^b	ΔEpa^c
a Epc/a in DMSO/buffer in the absence of CT DNA (Epc/a(f)).b Epc/a in DMSO/buffer in the presence of CT DNA (Epc/a(b)).c ΔEpc/a = Epc/a(b) − Epc/a(f).
[Cu(o-van)2(H2O)], 1	−728	−715	+13	−372	−355	+17
[Cu(5-CH3-salo)2], 2	−725	−735	−10	−509	−475	+34
[Cu(5-NO2-salo)2(CH3OH)2], 3	−741	−733	+8	−460	−455	+5
[Cu(5-Cl-salo)2], 4	−715	−735	−20	−521	−481	+40
[Cu(5-Br-salo)2], 5	−665	−700	−35	−421	−435	−14
[Cu(bpo)2], 6	−725	−665	+60	−499	−425	+74
[Cu(mpo)2], 7	−715	−695	+20	−425	−420	+5

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3.6.4. EB displacement studies. Ethidium bromide (EB = 3,8-diamino-5-ethyl-6-phenyl-phenanthridinium bromide) emits intense fluorescence in the presence of DNA as a result of the intercalation of the planar EB phenanthridine ring in between adjacent base pairs on the double DNA helix.⁸⁶ The presence of a compound capable to intercalate to DNA equally or more strongly than EB in a solution containing the EB–DNA complex may result in a quenching of the DNA-induced EB fluorescence emission. Solutions of free X-saloH, ketoH and their complexes 1–7 do not present any appreciable fluorescence at room temperature in solution or in the presence of CT DNA or of EB, when excited at 540 nm. Therefore, the changes observed in the fluorescence emission spectra of a solution containing the EB–DNA complex upon addition of the compounds may be useful in the investigation of the compounds' EB-displacing ability.⁸⁹

The fluorescence emission spectra of pre-treated EB–CT DNA ([EB] = 20 μM, [DNA] = 26 μM) were recorded in the presence of increasing amounts of o-vanH, 5-Me-saloH, mpoH and complexes 1–7 up to the value of r = 1.5 (representatively shown for complex 4 in Fig. 11(A)). The addition of the compounds results in a moderate (for 5 and 6) to significant quenching (Fig. 11(B) and (C)) of the fluorescence emission of the DNA–EB system at 592 nm (the final quenching is up to 54–79% of the initial EB–DNA fluorescence, Table 7) revealing the ability of the compounds to displace EB from its EB–DNA complex, being, thus, in strong competition with EB in binding to DNA via intercalation.⁴⁵

Fig. 11 (A) Emission spectra (λ_exit = 540 nm) for EB–DNA ([EB] = 20 μM, [DNA] = 26 μM) in buffer solution in the absence and presence of increasing amounts of complex [Cu(5-Cl-salo)₂], 4 (up to r = 0.8). The arrow shows the changes of intensity upon increasing amounts of [Cu(5-Cl-salo)₂], 4. (B) and (C) Plot of EB relative fluorescence intensity at λ_em = 592 nm (%) vs. r (r = [compound]/[DNA]) (150 mM NaCl and 15 mM trisodium citrate at pH = 7.0) in the presence of (B) o-vanH, 5-Me-saloH and mpoH (up to 24.9% of the initial EB–DNA fluorescence intensity for o-vanH, 23.5% for 5-Me-saloH and 28.9% for mpoH), and (C) complexes 1–7 (up to 23% of the initial EB–DNA fluorescence intensity for 1, 23.7% for 2, 26.6% for 3, 25% for 4, 45.8% for 5 and 6 and 27% for 7).

Table 7 Percentage of EB–DNA fluorescence quenching (ΔI/I_o, %) and Stern–Volmer constants (K_SV) for X-saloH, ketoH and their complexes 1–7

Compound	Quenching of EB–DNA fluorescence (% ΔI/Io)	Ksv (M^−1)
o-vanH	75.1	4.47(±0.14) × 10^4
5-Me-saloH	76.5	1.04(±0.03) × 10^5
5-NO2-saloH^33	72.5	2.22(±0.06) × 10^5
5-Cl-saloH^33	70.5	2.36(±0.05) × 10^4
5-Br-saloH^33	79.3	3.01(±0.13) × 10^5
bpoH^34	76.0	1.80(±0.08) × 10^4
mpoH	71.1	2.22(±0.08) × 10^4
[Cu(o-van)2(H2O)], 1	77.0	1.15(±0.06) × 10^5
[Cu(5-CH3-salo)2], 2	76.3	8.92(±0.31) × 10^4
[Cu(5-NO2-salo)2(CH3OH)2], 3	73.4	3.39(±0.11) × 10^5
[Cu(5-Cl-salo)2], 4	75.0	4.24(±0.11) × 10^4
[Cu(5-Br-salo)2], 5	54.2	2.07(±0.07) × 10^4
[Cu(bpo)2], 6	54.2	2.05(±0.02) × 10^4
[Cu(mpo)2], 7	73.0	4.33(±0.19) × 10^4

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The Stern–Volmer plots (Fig. S7 and S8†) indicate that the quenching of the EB–DNA fluorescence emission induced by the compounds is in good agreement (R = 0.99) with the linear Stern–Volmer equation (eqn (S2)†), thus suggesting that the observed quenching is a result of the displacement of EB from EB–DNA by each compound.^33,34 The K_SV values (Table 7) of the compounds are relatively high indicating tight binding to DNA; all complexes (with the exception of 2 and 5) present higher K_SV than the corresponding ligands and 3 exhibits the highest K_SV value (=3.39(±0.11) × 10⁵ M⁻¹) among the complexes. The K_SV values of complexes 3–6 are of the same magnitude to their zinc analogues recently reported.^33,34

3.7. Interaction with serum albumins

Serum albumins (SAs) are the major soluble protein constituents of the circulatory system having many physiological functions.⁹⁰ The most important role of SAs is that of the transporter, as they are mainly involved in the transport of drugs and other bioactive small molecules through the blood stream.^91,92 Bovine serum albumin (BSA) has been one of the most extensively albumins studied, especially because of its structural homology with human serum albumin (HSA).⁹² BSA and HSA solutions exhibit an intense fluorescence emission with λ_em,max = 342 nm and 351 nm, respectively, due to the tryptophan residues, when excited at 295 nm.⁴⁶ BSA is constituted of three homologous domains (I, II, III) and has two tryptophans, Trp-134 and Trp-212, embedded in the first subdomain IB and subdomain IIA, respectively. HSA is a globular protein composed of 585 amino acid residues in three homologous a-helices domains (I–III) bearing only one tryptophan located at position 214 along the chain, in subdomain IIA.^93,94 Most of the compounds in buffer solutions exhibit a maximum emission in the region 395–415 nm under the same experimental conditions and the SA fluorescence emission spectra have been corrected before the experimental data processing. The inner-filter effect was also taken into consideration and was calculated with eqn (S3);† it was not found so significant affecting slightly the measurements.^46,47

The addition of X-saloH, ketoH and complexes 1–7 to a SA solution (Fig. 12) results in a low to moderate quenching of the albumin fluorescence emission (i.e. at λ_em = 351 nm for HSA and at λ_em = 342 nm for BSA) which is in the case of BSA is more pronounced than for HSA (Fig. 13). The observed SA quenching in the presence of a compound can be attributed to changes in tryptophan environment of SA because of possible changes in albumin secondary structure as a result of the binding of the compound to SA.⁹⁴

Fig. 12 Fluorescence emission spectra (λ_exit = 295 nm) of (A) HSA ([HSA] = 3 μM) in the presence of increasing amounts of complex 3 (up to the value of r = [3]/[HSA] = 6) and (B) BSA ([BSA] = 3 μM), in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0) in the presence of increasing amounts of complex 5 (up to the value of r = [5]/[BSA] = 6). The arrows show the changes of intensity upon increasing amounts of the complexes.

Fig. 13 Plot of % relative fluorescence emission intensity λ_em (%) at vs. r (r = [compound]/[SA]) for the compounds in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0). (A) λ_em = 351 nm, for the free X-saloH and ketoH (up to 92% of the initial HSA fluorescence for o-vanH, 39% for 5-Me-saloH, 62.5% for 5-NO₂-saloH, 90% for 5-Cl-saloH, 79% for 5-Br-saloH, 84.5% for bpoH and 89% for mpoH), (B) λ_em = 351 nm, for complexes 1–7 (up to 84% of the initial HSA fluorescence for 1 and 2, 46% for 3, 91% for 4, 4% for 5, 60% for 6 and 95% for 7), (C) λ_em = 342 nm, for the free X-saloH and ketoH (up to 64.5% of the initial BSA fluorescence for o-vanH, 58.5% for 5-Me-saloH, 45.5% for 5-NO₂-saloH, 79% for 5-Cl-saloH, 53% for 5-Br-saloH, 73.5% for bpoH and 81% for mpoH), and (D) λ_em = 342 nm, for complexes 1–7 (up to 65% of the initial BSA fluorescence for 1, 87% for 2, 41% for 3, 80% for 4, 44.5% for 5 and 6 and 67% for 7).

The quenching constant values (k_q) for the compounds binding to the albumins were calculated from the corresponding Stern–Volmer plots (Fig. S9–S12†) using the Stern–Volmer quenching equation (eqn (S4)†) and they are cited in Table 8. The k_q values suggest moderate SA quenching ability and, being higher than 10¹¹ M⁻¹ s⁻¹, indicate the existence of static quenching mechanism.⁴⁵ In most cases, the quenching ability of complexes 1–7 is higher than their corresponding free ligand, with complex 5 exhibiting the highest k_q value for HSA (k_q(HSA) = 1.38(±0.07) × 10¹⁴ M⁻¹ s⁻¹) and 3 for BSA (k_q(BSA) = 7.11(±0.43) × 10¹² M⁻¹ s⁻¹).

Table 8 The quenching and association constants for HSA and BSA derived for the free X-saloH and ketoH and their complexes 1–7

Compound	kq(HSA) (M^−1 s^−1)	K(HSA) (M^−1)	kq(BSA) (M^−1 s^−1)	K(BSA) (M^−1)
o-vanH	2.52(±0.23) × 10^11	1.56(±0.17) × 10^5	2.37(±0.13) × 10^12	2.15(±0.10) × 10^5
5-Me-saloH	2.00(±0.15) × 10^12	8.71(±0.12) × 10^5	6.15(±0.58) × 10^11	2.35(±0.27) × 10^6
5-NO2-saloH	5.00(±0.46) × 10^12	2.41(±0.09) × 10^5	6.55(±0.17) × 10^12	1.25(±0.07) × 10^5
5-Cl-saloH	5.64(±0.40) × 10^11	2.19(±0.10) × 10^5	1.46(±0.06) × 10^12	3.11(±0.20) × 10^4
5-Br-saloH	2.31(±0.32) × 10^11	1.43(±0.09) × 10^6	4.63(±0.31) × 10^12	1.43(±0.08) × 10^5
bpoH	3.05(±0.19) × 10^11	8.33(±0.12) × 10^5	9.43(±0.36) × 10^11	3.18(±0.23) × 10^5
mpoH	7.67(±0.46) × 10^11	4.26(±0.41) × 10^4	7.41(±0.46) × 10^11	7.02(±0.17) × 10^4
[Cu(o-van)2(H2O)], 1	1.49(±0.25) × 10^12	4.39(±0.22) × 10^5	3.33(±0.23) × 10^12	9.69(±0.98) × 10^4
[Cu(5-CH3-salo)2], 2	2.81(±0.54) × 10^12	6.32(±0.48) × 10^5	8.55(±0.51) × 10^11	1.00(±0.13) × 10^5
[Cu(5-NO2-salo)2(CH3OH)2], 3	6.09(±0.27) × 10^12	1.23(±0.11) × 10^5	7.11(±0.43) × 10^12	3.03(±0.20) × 10^5
[Cu(5-Cl-salo)2], 4	4.50(±0.60) × 10^11	5.74(±0.43) × 10^5	1.52(±0.09) × 10^12	7.36(±0.81) × 10^4
[Cu(5-Br-salo)2], 5	1.38(±0.07) × 10^14	8.61(±0.36) × 10^5	6.42(±0.29) × 10^12	1.69(±0.06) × 10^5
[Cu(bpo)2], 6	5.61(±0.64) × 10^12	3.12(±0.12) × 10^5	6.56(±0.35) × 10^12	1.93(±0.10) × 10^5
[Cu(mpo)2], 7	3.79(±0.18) × 10^11	4.57(±0.68) × 10^4	2.88(±0.14) × 10^12	2.14(±0.23) × 10^3

---

The values of the binding constant (K) of the compounds to both SAs were calculated from the corresponding Scatchard plots (Fig. S13–S16†) using the Scatchard equation (eqn (S6)†) and they are given in Table 8. In most cases, the K values of the complexes are of the same magnitude to that of the corresponding free ligand, with complexes 5 and 3 exhibiting the highest association constants for HSA and BSA, respectively, (K_(HSA),5 = 8.61(±0.36) × 10⁵ M⁻¹ and K_(BSA),3 = 3.03(±0.20) × 10⁵ M⁻¹) among the complexes; among all present compounds, 5-Br-saloH and 5-Me-saloH are the strongest HSA- and BSA-binders with K_{(HSA),5-Br-saloH} = 1.43(±0.09) × 10⁶ M⁻¹ and K_{(BSA),5-Me-saloH} = 2.35(±0.27) × 10⁶ M⁻¹, respectively.

Comparing the affinity of the compounds for BSA and HSA (K values), it is obvious that most of the compounds and, especially complexes 1–7, exhibit relatively higher affinity for HSA than for BSA. In any case, the K values of the compounds are in the range 2.14 × 10³ to 2.35 × 10⁶ M⁻¹, indicating their rather tight binding to the SA in order to get transported towards their potential targets; upon their arrival, the compounds have the potential to get released, since their non-covalent binding to the SA is much less tighter than the strongest known non-covalent (irreversible) bonds, which are due to the interaction of diverse ligands to the protein avidin with K values ≈10¹⁵ M⁻¹.^95,96

4. Conclusions

Seven neutral mononuclear Cu(II) complexes with substituted salicylaldehydes or 2-hydroxy-phenones ligands (HL) have been synthesized under the general formula [Cu(L)₂(S)_n], where L is the anion of the ligand and S = solvent. In these complexes, the salicylaldehydes and the phenones behave as bidentate ligands. Five of them are novel compounds and the crystal structures for four of them ([Cu(5-NO₂-salo)₂(CH₃OH)₂] (3), [Cu(5-Cl-salo)₂] (4), [Cu(bpo)₂] (6) and [Cu(mpo)₂]·2H₂O (7·2H₂O)) were verified by single-crystal X-ray diffraction analysis, denoting for complex 3 the octahedral geometry around copper(II) ion, while for 4, 6 and 7 the square planar one. The previously reported complexes [Cu(o-van)₂(H₂O)]·0.25H₂O (1) and [Cu(5-CH₃-salo)₂] (2) possess square pyramidal and square planar geometry, respectively.

The interaction of complexes 1–7 with calf-thymus DNA and serum albumins was studied with spectroscopic, electrochemical and physicochemical techniques. UV spectroscopy studies, viscosity measurements and cyclic voltammetry were employed to investigate the ability of the complexes to bind to CT DNA. The binding constants of the complexes to CT DNA were calculated by UV spectroscopic titrations with complex [Cu(5-Br-salo)₂], 5 exhibiting the highest K_b value (K_b = 5.31(±0.07) × 10⁶ M⁻¹) among complexes 1–7, which is higher than the K_b value of EB. The DNA-binding values of the copper(II) complexes 1–7 are similar or higher that the K_b values of their zinc(II) analogues recently reported.^33,34 The complexes can displace the typical intercalator EB from the EB–CT DNA complex suggesting intercalation as the most possible interaction mode with CT DNA, a conclusion which is in accordance to the viscometry and cyclic voltammetry interaction experiments.

The complexes 1–7 exhibit more pronounced quenching of the albumins fluorescence and higher binding affinity to the albumins than the corresponding free X-saloHs or ketoHs as estimated by the values of the quenching (k_q) and the association (K) constants, respectively. The K values of the complexes (K = 2.14 × 10³ to 8.6113 × 10⁶ M⁻¹) exhibit tight binding affinity to the proteins being in a range that reveals their potential to bind, get transferred and get released upon arrival at the targets.

Abbreviations

3-OCH3-saloH	3-Methoxy-salicylaldehyde
5-Br-saloH	5-Bromo-salicylaldehyde
5-Cl-saloH	5-Chloro-salicylaldehyde
5-NO2-saloH	5-Nitro-salicylaldehyde
bpoH	2-Hydroxy-benzophenone
BSA	Bovine serum albumin
CT	Calf-thymus
DMF	N,N-Dimethylformamide
EB	Ethidium bromide, 3,8-diamino-5-ethyl-6-phenyl-phenanthridinium bromide
HSA	Human serum albumin
ketoH	Substituted 2-hydroxy-phenone
m	Medium
mpoH	2-Hydroxy-5-methyl-acetophenone
o-vanH	ortho-Vanillin, 3-methoxy-salicylaldehyde
PXRD	Powder X-ray diffraction
r	[Compound]/[DNA] mixing ratio
r′	[DNA]/[compound] mixing ratio
s	Strong
SA	Serum albumin
saloH	Salicylaldehyde, 2-hydroxy-benzaldehyde
sh	Shoulder
sm	Strong-to-medium
TEAP	Tetraethylammonium perchlorate
w	Weak
X-saloH	Substituted salicylaldehyde

Acknowledgements

This work was funded by Aristeia 2014, Aristotle University of Thessaloniki.

References

1. D. Hall, R. Holmlin and J. K. Barton, Nature, 1996, 382, 731–735.
2. X. Yang, J. Feng, J. Zhang, Z. Zhang, H. Lin, L. Zhou and X. Yu, Bioorg. Med. Chem., 2008, 16, 3871–3877.
3. G. Crisponi, V. M. Nurchi, D. Fanni, C. Gerosa, S. Nemolato and G. Faa, Coord. Chem. Rev., 2010, 254, 876–889.
4. J. A. Drewry and P. T. Gunning, Coord. Chem. Rev., 2011, 255, 459–472.
5. J. R. J. Sorenson, Prog. Med. Chem., 1989, 26, 437–568.
6. J. R. Turnlund, W. R. Keyes, H. L. Anderson and L. L. Acord, Am. J. Clin. Nutr., 1989, 49, 870–878.
7. R. N. Prasad and A. Agrawal, J. Indian Chem. Soc., 2006, 83, 75–77.
8. R. N. Prasad, M. Agrawal and R. George, J. Indian Chem. Soc., 2005, 82, 444–447.
9. S. T. Hussain, H. Ahmad, M. A. Atta, M. Afzal and M. Saleem, J. Trace Microprobe Tech., 1998, 16, 139–149.
10. K. Fujinaga, Y. Sakaguchi, T. Tsuruhara, Y. Seike and M. Okumura, Solvent Extr. Res. Dev., 2001, 8, 144–158.
11. E. Pelttaria, E. Karhumakib, J. Langshawc, H. Perakylad and H. Eloa, Z. Naturforsch., C: J. Biosci., 2007, 62, 487–497.
12. A. Jayamani, N. Sengottuvelan and G. Chakkaravarthi, Polyhedron, 2014, 81, 764–776.
13. Y. Yang, P. Lu, T. Zhu and C. Liu, Acta Crystallogr., Sect. E: Struct. Rep. Online, 2007, 63, m1613.
14. Q. Wang and D. Wang, Acta Crystallogr., Sect. E: Struct. Rep. Online, 2008, 64, m298.
15. J. C. Pessoa, I. Cavaco, I. Correira, I. Tomaz, T. Duarte and P. Matias, J. Inorg. Biochem., 2000, 80, 35–39.
16. M. Lalia–Kantouri, C. D. Papadopoulos, A. G. Hatzidimitriou, M. P. Sigalas, M. Quiros and S. Skoulika, Polyhedron, 2013, 52, 1306–1316.
17. T. Hayashi, Y. Okamoto, K. Ueda and N. Kojima, Toxicol. Lett., 2006, 167, 1–7.
18. T. Suzuki, S. Kitamura, R. Khota, K. Sugihara, N. Fujimoto and S. Ohta, Toxicol. Appl. Pharmacol., 2005, 203, 9–17.
19. J. Chen, C. Ting, T. Hwang and L. Chen, J. Nat. Prod., 2009, 72, 253–258.
20. T. D. Venu, S. Shashikanth, S. A. Khanum, S. Naveen, A. Firdouse, M. A. Sridhar and J. S. Prasad, Bioorg. Med. Chem., 2007, 15, 3505–3514.
21. K. Kohring, J. Wiesner, M. Altenkamper, J. Sakowski, K. Silber, A. Hillebrecht, P. Haebel, H. M. Dahse, R. Ortmann, H. Jomaa, G. Klebe and M. Schlitzer, ChemMedChem, 2008, 3, 1217–1231.
22. J. S. Neves, L. P. Coelho, R. S. Cordeiro, M. P. Veloso, P. M. Silva, M. H. Dos Santos and M. A. Martins, Planta Med., 2007, 73, 644–649.
23. J. Chen, C. Ting, I. Chen, C. Peng, W. Huang, Y. Su and S. Lin, Planta Med., 2008, 74, 1042–1051.
24. R. G. Ferris, R. G. Hazen, G. B. Roberts, M. H. Clair, J. H. Chan, K. R. Romines, G. A. Freeman, J. H. Tidwell, L. T. Schaller, J. R. Cowan, S. A. Short, K. L. Weaver, D. W. Selleseth, K. R. Moniri and L. R. Boone, Antimicrob. Agents Chemother., 2005, 203, 4046–4051.
25. J. Liou, C. Chang, J. Song, Y. Yang, C. Yeh, H. Tseng, Y. Lo, Y. Chang, C. Chang and H. Hsieh, J. Med. Chem., 2002, 45, 2556–2662.
26. Z. Wang, H. Lee and L. Wang, Pharm. Res.–Dordr., 2009, 26, 1140–1148.
27. R. W. Fuller, J. W. Blunt and J. L. Boswell, J. Nat. Prod., 1999, 62, 130–132.
28. M. I. Esteva, K. Kettler and C. Maidana, J. Med. Chem., 2005, 48, 7186–7191.
29. J. Ren, P. Chamberlain and A. Stamp, J. Med. Chem., 2008, 51, 5000–5008.
30. M. Lalia-Kantouri, T. Dimitriadis, C. D. Papadopoulos, M. Gdaniec, A. Czapik and A. G. Hatzidimitriou, Z. Anorg. Allg. Chem., 2009, 635, 2185–2190.
31. C. D. Papadopoulos, A. G. Hatzidimitriou, M. Quiros, M. P. Sigalas and M. Lalia-Kantouri, Polyhedron, 2011, 30, 486–496.
32. M. Lalia-Kantouri, M. Gdaniec, T. Choli-Papadopoulou, A. Badounas, C. D. Papadopoulos, A. Czapik, G. D. Geromichalos, D. Sahpazidou and F. Tsitouroudi, J. Inorg. Biochem., 2012, 117, 25–34.
33. A. Zianna, G. Psomas, A. Hatzidimitriou, E. Coutouli-Argyropoulou and M. Lalia-Kantouri, J. Inorg. Biochem., 2013, 127, 116–126.
34. E. Mrkalić, A. Zianna, G. Psomas, M. Gdaniec, A. Czapik, E. Coutouli-Argyropoulou and M. Lalia-Kantouri, J. Inorg. Biochem., 2014, 134, 66–75.
35. A. Zianna, K. Chrissafis, A. Hatzidimitriou and M. Lalia–Kantouri, J. Therm. Anal. Calorim., 2015, 120, 59–66.
36. F. Zhang, J. Zhang, C. Tong, Y. Chen, S. Zhuang and W. Liu, J. Hazard. Mater., 2013, 15, 618–626.
37. J. Marmur, J. Mol. Biol., 1961, 3, 208–211.
38. M. F. Reichmann, S. A. Rice, C. A. Thomas and P. Doty, J. Am. Chem. Soc., 1954, 76, 3047–3053.
39. Apex2, Version 2 User Manual, M86-E01078, Bruker Analytical X-ray Systems, Inc., Madison, WI, 2006.
40. SADABS: Area-Detector Absorption Correction, Siemens Industrial Automation, Inc., Madison, WI, 1996.
41. L. Palatinus and G. Chapuis, J. Appl. Crystallogr., 2007, 40, 786–790.
42. P. W. Betteridge, J. R. Carruthers, R. I. Cooper, K. Prout and D. J. Watkin, J. Appl. Crystallogr., 2003, 36, 1487.
43. D. J. Watkin, C. K. Prout and L. J. Pearce, CAMERON Program, Chemical Crystallographic, Laboratory, Oxford University, UK, 1996.
44. A. Wolfe, G. Shimer and T. Meehan, Biochemistry, 1987, 26, 6392–6396.
45. G. Zhao, H. Lin, S. Zhu, H. Sun and Y. Chen, J. Inorg. Biochem., 1998, 70, 219–226.
46. J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum Press, New York, 3rd edn, 2006.
47. L. Stella, A. L. Capodilupo and M. Bietti, Chem. Commun., 2008, 4744–4746.
48. Y. Wang, H. Zhang, G. Zhang, W. Tao and S. Tang, J. Lumin., 2007, 126, 211–218.
49. R. M. Silverstein, G. C. Bassler and G. Morvill, Spectrometric identification of organic compounds, Wiley, New York, 6th edn, 1998.
50. K. Nakamoto, Infrared and Raman Spectra of Inorganic and Coordination Compounds, Part B: Applications in Coordination, Organometallic and Bioinorganic Chemistry, Wiley, New Jersey, 6th edn, 2009.
51. A. Tarushi, C. P. Raptopoulou, V. Psycharis, A. Terzis, G. Psomas and D. P. Kessissoglou, Bioorg. Med. Chem., 2010, 18, 2678–2685.
52. B. J. Hathaway, in Comprehensive Coordination Chemistry, ed. G. Wilkinson, Pergamon Press, Oxford, 1987, vol. 5, pp. 533–773.
53. A. B. P. Lever, Inorganic Electronic Spectroscopy, Elsevier, Amsterdam, 1984.
54. R. N. Jageja, K. M. Vyas, V. K. Gupta, R. G. Joshi and C. R. Prabha, Polyhedron, 2012, 31, 767–778.
55. F. Dimiza, S. Fountoulaki, A. N. Papadopoulos, C. A. Kontogiorgis, V. Tangoulis, C. P. Raptopoulou, V. Psycharis, A. Terzis, D. P. Kessissoglou and G. Psomas, Dalton Trans., 2011, 40, 8555–8568.
56. R. Cini, G. Tamasi, S. Defazio and M. B. Hursthouse, J. Inorg. Biochem., 2007, 101, 1140–1152.
57. G. Tamasi, F. Serinelli, M. Consumi, A. Magnani, M. Casolaro and R. Cini, J. Inorg. Biochem., 2008, 102, 1862–1873.
58. D. Kovala-Demertzi, A. Koutsodimou, A. Galani, S. K. Hadjikakou, M. A. Demertzis, M. Xanthopoulou, J. R. Miller and C. S. Frampton, Appl. Organomet. Chem., 2004, 18, 501–502.
59. S. K. Hadjikakou, M. A. Demertzis, J. R. Miller and D. Kovala-Demertzi, J. Chem. Soc., Dalton Trans., 1999, 663–666.
60. M. R. Moya-Hernandez, A. Mederos, S. Dominguez, A. Orlandini, C. A. Ghilardi, F. Cecconi, E. Gonzalez-Vergara and A. Rojas-Hernandez, J. Inorg. Biochem., 2003, 95, 131–140.
61. P. Christofis, M. Katsarou, A. Papakyriakou, Y. Sanakis, N. Katsaros and G. Psomas, J. Inorg. Biochem., 2005, 99, 2197–2210.
62. A. Majumdar, S. Chakraborty and M. Sarkar, J. Phys. Chem. B, 2014, 118, 13785–13799.
63. S. Chakraborty, M. Bose and M. Sarkar, Spectrochim. Acta, Part A, 2014, 122, 690–697.
64. S. Roy, R. Banerjee and M. Sarkar, J. Inorg. Biochem., 2006, 100, 1320–1331.
65. A. Manceau and A. Matynia, Geochim. Cosmochim. Acta, 2010, 74, 2556–2580.
66. Y. M. Chumakov, L. G. Paladi, B. Y. Antosyak, Y. A. Simonov, V. I. Tsapkov, G. Bocelli, A. P. Gulea, D. Ginju and S. A. Palomares-Sanchez, Crystallogr. Rep., 2011, 56, 260–264.
67. G. Li, S. Zhang and Z. Liu, Acta Crystallogr., Sect. E: Struct. Rep. Online, 2008, 64, m52.
68. X. Xu, T. Xu, J. Gao, D. Wang and L. Lu, Acta Crystallogr., Sect. E: Struct. Rep. Online, 2006, 62, m1768–m1769.
69. W. Wang, X. Zhou, Y. Ma, Z. Zhang, R. Yuan, B. Zhao and L. Xu, J. Struct. Chem., 2011, 52, 781–784.
70. A. Zianna, S. Vecchio, M. Gdaniec, A. Czapik, A. Hatzidimitriou and M. Lalia-Kantouri, J. Therm. Anal. Calorim., 2013, 112, 455–464.
71. A. Dziewulska-Kulaczkowska, J. Therm. Anal. Calorim., 2010, 101, 19–26.
72. L. M. Dianu, A. Kriza and A. M. Musuc, J. Therm. Anal. Calorim., 2013, 112, 585–593.
73. F. Dimiza, F. Perdih, V. Tangoulis, I. Turel, D. P. Kessissoglou and G. Psomas, J. Inorg. Biochem., 2011, 105, 476–489.
74. B. M. Zeglis, V. C. Pierre and J. K. Barton, Chem. Commun., 2007, 4565–4579.
75. Q. Zhang, J. Liu, H. Chao, G. Xue and L. Ji, J. Inorg. Biochem., 2001, 83, 49–55.
76. G. Pratviel, J. Bernadou and B. Meunier, Adv. Inorg. Chem., 1998, 45, 251–262.
77. A. M. Pyle, J. P. Rehmann, R. Meshoyrer, C. V. Kumar, N. J. Turro and J. K. Barton, J. Am. Chem. Soc., 1989, 111, 3053–3063.
78. A. Dimitrakopoulou, C. Dendrinou-Samara, A. A. Pantazaki, M. Alexiou, E. Nordlander and D. P. Kessissoglou, J. Inorg. Biochem., 2008, 102, 618–628.
79. N. Selvakumaran, N. S. P. Bhuvanesh and R. Karvembu, Dalton Trans., 2014, 43, 16395–16410.
80. K. Jeyalakshmi, N. Selvakumaran, N. S. P. Bhuvanesh, A. Sreekanth and R. Karvembu, RSC Adv., 2014, 4, 17179–17195.
81. R. Loganathan, S. Ramakrishnan, E. Suresh, M. Palaniandavar, A. Riyasdeen and M. A. Akbarsha, Dalton Trans., 2014, 43, 6177–6194.
82. D. Li, J. Tian, W. Gu, X. Liu and S. Yan, J. Inorg. Biochem., 2010, 104, 171–179.
83. J. L. Garcia-Gimenez, M. Gonzalez-Alvarez, M. Liu-Gonzalez, B. Macias, J. Borras and G. Alzuet, J. Inorg. Biochem., 2009, 103, 923–934.
84. C. Tolia, A. N. Papadopoulos, C. P. Raptopoulou, V. Psycharis, C. Garino, L. Salassa and G. Psomas, J. Inorg. Biochem., 2013, 123, 53–65.
85. M. T. Carter, M. Rodriguez and A. J. Bard, J. Am. Chem. Soc., 1989, 111, 8901–8911.
86. W. D. Wilson, L. Ratmeyer, M. Zhao, L. Strekowski and D. Boykin, Biochemistry, 1993, 32, 4098–4104.
87. G. Psomas, J. Inorg. Biochem., 2008, 102, 1798–1811.
88. F. Dimiza, A. N. Papadopoulos, V. Tangoulis, V. Psycharis, C. P. Raptopoulou, D. P. Kessissoglou and G. Psomas, Dalton Trans., 2010, 39, 4517–4528.
89. S. Dhar, M. Nethaji and A. R. Chakravarty, J. Inorg. Biochem., 2005, 99, 805–812.
90. X. M. He and D. C. Carter, Nature, 1992, 358, 209–215.
91. R. E. Olson and D. D. Christ, Annu. Rep. Med. Chem., 1996, 31, 327–336.
92. C. Tan, J. Liu, H. Li, W. Zheng, S. Shi, L. Chen and L. Ji, J. Inorg. Biochem., 2008, 102, 347–358.
93. I. Petitpas, T. Grune, A. A. Bhattacharya, S. Twine, M. East and S. Curry, J. Mol. Biol., 2001, 314, 955–960.
94. V. Rajendiran, R. Karthik, M. Palaniandavar, H. Stoeckli-Evans, V. S. Periasamy, M. A. Akbarsha, B. S. Srinag and H. Krishnamurthy, Inorg. Chem., 2007, 46, 8208–8221.
95. S. Wu, W. Yuan, H. Wang, Q. Zhang, M. Liu and K. Yu, J. Inorg. Biochem., 2008, 102, 2026–2034.
96. O. H. Laitinen, V. P. Hytonen, H. R. Nordlund and M. S. Kulomaa, Cell. Mol. Life Sci., 2006, 63, 2992–3017.

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Footnote

† Electronic supplementary information (ESI) available. CCDC 1034138–40 and 1036592. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c4ra16484a

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