Copper(II) interacting with the non-steroidal antiinflammatory drug flufenamic acid: Structure, antioxidant activity and binding to DNA and albumins

Article abstract of DOI:10.1016/j.jinorgbio.2013.02.009

Copper(II) complexes with the non-steroidal antiinflammatory drug flufenamic acid (Hfluf) in the presence of N,N-dimethylformamide (DMF) or nitrogen donor heterocyclic ligands (2,2′-bipyridylamine (bipyam), 1,10-phenanthroline (phen), 2,2′-bipyridine (bipy) or pyridine (py)) have been synthesized and characterized. The crystal structures of [Cu ₂(fluf)₄(DMF)₂], 1, and [Cu(fluf)(bipyam)Cl], 2, have been determined by X-ray crystallography. Density functional theory (DFT) (CAM-B3LYP/LANL2DZ/6-31G**) was employed to determine the structure of complex 2 and its analogues (complexes [Cu(fluf)(phen)Cl], 3, [Cu(fluf)(bipy)Cl], 4 and [Cu(fluf)₂(py)₂], 5). Time-dependent DFT calculations of doublet-doublet transitions show that the lowest-energy band in the absorption spectrum of 2-5 has a mixed d-d/LMCT character. UV study of the interaction of the complexes with calf-thymus DNA (CT DNA) has shown that the complexes can bind to CT DNA with [Cu(fluf)(bipy)Cl] exhibiting the highest binding constant to CT DNA. The complexes can bind to CT DNA via intercalation as concluded by studying the cyclic voltammograms of the complexes in the presence of CT DNA solution and by DNA solution viscosity measurements. Competitive studies with ethidium bromide (EB) have shown that the complexes can displace the DNA-bound EB suggesting strong competition with EB. Flufenamic acid and its Cu(II) complexes exhibit good binding affinity to human or bovine serum albumin protein with high binding constant values. All compounds have been tested for their antioxidant and free radical scavenging activity as well as for their in vitro inhibitory activity against soybean lipoxygenase showing significant activity with [Cu(fluf)(phen)Cl] being the most active.

Full text of DOI:10.1016/j.jinorgbio.2013.02.009

Journal of Inorganic Biochemistry 123 (2013) 53–65
Contents lists available at SciVerse ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Copper(II) interacting with the non-steroidal antiinflammatory drug flufenamic acid:
Structure, antioxidant activity and binding to DNA and albumins
Charikleia Tolia a, Athanassios N. Papadopoulos b, Catherine P. Raptopoulou c, Vassilis Psycharis c,
Claudio Garino d, Luca Salassa e, George Psomas a,
⁎
a
Department of General and Inorganic Chemistry, Faculty of Chemistry, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece
Department of Nutrition and Dietetics, Faculty of Food Technology and Nutrition, Alexandrion Technological Educational Institution, Sindos, Thessaloniki, Greece
Institute of Advanced Materials, Physicochemical Processes, Nanotechnology & Microsystems, Department of Materials Science, NCSR “Demokritos”,
b
c
GR-15310 Aghia Paraskevi Attikis, Greece
d
Department of Chemistry and NIS Centre of Excellence, University of Turin, Via P. Giuria 7, 10125 Turin, Italy
CIC biomaGUNE, Paseo Miramón 182, 20009 Donostia–San Sebastián, Spain
e
a r t i c l e i n f o
a b s t r a c t
Article history:
Copper(II) complexes with the non-steroidal antiinflammatory drug flufenamic acid (Hfluf) in the presence
of N,N-dimethylformamide (DMF) or nitrogen donor heterocyclic ligands (2,2′-bipyridylamine (bipyam),
1,10-phenanthroline (phen), 2,2′-bipyridine (bipy) or pyridine (py)) have been synthesized and character-
ized. The crystal structures of [Cu2(fluf)4(DMF)2], 1, and [Cu(fluf)(bipyam)Cl], 2, have been determined by
X-ray crystallography. Density functional theory (DFT) (CAM-B3LYP/LANL2DZ/6-31G**) was employed to
determine the structure of complex 2 and its analogues (complexes [Cu(fluf)(phen)Cl], 3, [Cu(fluf)(bipy)
Cl], 4 and [Cu(fluf)2(py)2], 5). Time-dependent DFT calculations of doublet–doublet transitions show that
the lowest-energy band in the absorption spectrum of 2–5 has a mixed d–d/LMCT character. UV study of
the interaction of the complexes with calf-thymus DNA (CT DNA) has shown that the complexes can bind
to CT DNA with [Cu(fluf)(bipy)Cl] exhibiting the highest binding constant to CT DNA. The complexes can
bind to CT DNA via intercalation as concluded by studying the cyclic voltammograms of the complexes in
the presence of CT DNA solution and by DNA solution viscosity measurements. Competitive studies with
ethidium bromide (EB) have shown that the complexes can displace the DNA-bound EB suggesting strong
competition with EB. Flufenamic acid and its Cu(II) complexes exhibit good binding affinity to human or
bovine serum albumin protein with high binding constant values. All compounds have been tested for
their antioxidant and free radical scavenging activity as well as for their in vitro inhibitory activity against
soybean lipoxygenase showing significant activity with [Cu(fluf)(phen)Cl] being the most active.
© 2013 Elsevier Inc. All rights reserved.
Received 28 December 2012
Received in revised form 8 February 2013
Accepted 20 February 2013
Available online 28 February 2013
Keywords:
Copper complexes
Non-steroidal antiinflammatory drugs
Flufenamic acid
Interaction with DNA
Interaction with serum albumins
Antioxidant activity
1. Introduction
cancer cell lines including colon, breast, prostate, human myeloid leukae-
mia and stomach cell lines [15] and have a synergistic role in the activity
Copper is one of the most important biometals not only because of
its role in proteins but also due to its potential synergetic activity with
drugs [1,2]. Copper(II) complexes with drugs as ligands exhibiting in-
creased activity in comparison to free drugs have been the subject of
many research studies [2–4] and a plethora of copper(II) complexes
with diverse ligands showing potential antitumor [5,6], antioxidant
[7,8], antibacterial [9–11] and antifungal [12] activity may be found
in the literature.
Non-steroidal antiinflammatory drugs (NSAIDs) are the most fre-
quently administrated analgesic, antiinflammatory and antipyretic
drugs with known side-effects [13]. In general, the action of NSAIDs is
the inhibition of the cyclo-oxygenase (COX)-mediated production of
prostaglandins [14]. NSAIDs can also induce the apoptosis of a series of
of certain antitumor drugs [16]; these antitumorigenic properties of the
NSAIDs may be attributed to COX-independent mechanisms by modu-
lating cell proliferation and cell death in cancer cells lacking COX
[17,18], to apoptosis via activation of caspases [19] or via an unknown
molecular mechanism where free radicals may also be involved
[20,21]. Therefore, in an attempt to investigate the potential anticancer
as well as antiinflammatory activity of NSAIDs and their complexes,
their interaction with DNA as well as their antioxidant activity should
be considered of great importance and further evaluated; it should be
noted that only few relevant reports on the interaction of NSAIDs and
their complexes with DNA have been published so far [22,23]. Further-
more, numerous copper(II) complexes with NSAIDs as ligands have
been structurally characterized including those of diclofenac [8,24],
diflunisal [25], flufenamic acid [26], ibuprofen [27,28], indomethacin
[29], mefenamic acid [7], naproxen[27,28], suprofen [29,30] and
tolmentin [27].
⁎
Corresponding author. Tel.: +30 2310997790; fax: +30 2310997738.
E-mail address: gepsomas@chem.auth.gr (G. Psomas).
0162-0134/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jinorgbio.2013.02.009

54
C. Tolia et al. / Journal of Inorganic Biochemistry 123 (2013) 53–65
Phenylalkanoic acids, anthranilic acids, oxicams, salicylate derivatives,
2. Experimental
sulfonamides and furanones constitute the chemical classes of NSAIDs
[2]. The NSAID flufenamic acid (= Hfluf, Scheme 1) belongs to the
derivatives of N-phenylanthranilic acid and resembles chemically to
mefenamic and tolfenamic acids and other fenamates in clinical use
[31]. Hfluf possesses analgesic, antiinflammatory and antipyretic
properties and has been used in musculoskeletal and joint disorders
and is administered orally and topically [32]. Its channel-regulating
ability [33] has been long known since it has been used as a blocker of
calcium-dependent cationic currents [34]. Additionally, Hfluf can inhib-
it nonselective cation channels [35]. On the other hand, it activates po-
tassium channels [36] and has recently shown an interesting
modulatory effect on neuronal sodium channels [32]. The number of
the metal complexes of flufenamic acid is rather limited in comparison
to other NSAIDs; few copper(II) complexes [26,37] have been found in
the literature as well as a zinc complex recently reported by our group
[38].
Having in mind the importance of NSAIDs in medicine and the
enhanced activity of their metal complexes, we have recently initiated
the synthesis, characterization and study of biological activity of Cu(II),
Co(II) and Zn(II) with NSAIDs as ligands [7,8,25,31,38–42]. Within this
context, we present herein the synthesis of Cu(II) complexes with
the NSAID flufenamic acid in the presence of the O-donor ligand
N,N-dimethylformamide (DMF) or the nitrogen-donor heterocy-
clic ligands 2,2′-bipyridylamine (bipyam), 1,10-phenanthroline
(phen), 2,2′-bipyridine (bipy) or pyridine (py), resulting in the for-
mation of complexes [Cu2(fluf)4(DMF)2], 1, [Cu(fluf)(bipyam)Cl],
2, [Cu(fluf)(phen)Cl], 3, [Cu(fluf)(bipy)Cl], 4 and [Cu(fluf)2(py)2],
5, respectively. The complexes have been characterized with phys-
icochemical and spectroscopic techniques and their electrochemi-
cal behavior has been also investigated. The crystal structures
of [Cu2(fluf)4(DMF)2] 1 and [Cu(fluf)(bipyam)Cl] 2 have been
determined by X-ray crystallography.
2.1. Materials—instrumentation—physical measurements
All chemicals (CuCl2·2H2O, flufenamic acid, bipy, phen, bipyam, py,
KOH, CT DNA, BSA, HSA, EB, NaCl and trisodium citrate) were purchased
from Sigma-Aldrich Co and all solvents were purchased from Merck.
The chemicals and solvents were reagent grade and were used as
purchased without any further purification. Tetraethylammonium
perchlorate (TEAP) was purchased from Carlo Erba and, prior to
its use, it was recrystallized twice from ethanol and dried under
vacuum.
DNA stock solution was prepared by dilution of CT DNA to buffer
(containing 15 mM trisodium citrate and 150 mM NaCl at pH 7.0)
followed by exhaustive stirring for three 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 1.89, indicating that
the DNA was sufficiently free of protein contamination. The DNA con-
centration was determined by the UV absorbance at 260 nm after
1:20 dilution using ε = 6600 M−1 cm−1 [7,8].
Infrared (IR) spectra (400–4000 cm−1) were recorded on a Nicolet
FT-IR 6700 spectrometer with samples prepared as KBr disk. UV–visible
(UV–vis) spectra were recorded as nujol mulls and in DMSO solution at
concentrations in the range 10−5 to 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). C, H and N elemental analysis were
performed on a Perkin-Elmer 240B elemental analyzer. Molar con-
ductivity 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.
In an attempt to investigate the existence of potential anticancer
and/or anti-inflammatory activity of the resultant complexes 1–5, we
have focused on (i) the interaction of the complexes with calf-thymus
(CT) DNA studied by UV spectroscopy, cyclic voltammetry and viscosity
measurements, (ii) the ability of the complexes to displace ethidium
bromide (EB) from the EB-DNA complex in order to clarify the existence
of a potential intercalation of the complexes to CT DNA in competition
to the classical DNA-intercalator EB studied by fluorescence spectroscopy,
(iii) the antioxidant capacity of the complexes by determining their ability
to scavenge 1,1-diphenyl-picrylhydrazyl (DPPH), hydroxyl (•OH) and
2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+) radicals
as well as their inhibitory activity against soybean lipoxygenase (LOX)—
since the use of NSAIDs in medicine as anagelsics and antiinflammatories
may be related to free radicals scavenging. Furthermore, the affinity of the
complexes to bovine (BSA) and human serum albumin (HSA) has been
investigated by fluorescence spectroscopy since binding to such pro-
teins that are involved in the transport of metal ions and metal–drug
complexes through the blood stream may result in lower or enhanced
biological properties of the original drug, or new paths for drug trans-
portation [43].
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 work-
ing electrode was platinum disk, a separate Pt single-sheet electrode
was used as the counter electrode and a 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 electro-
chemical measurements were performed at 25.0
0.2 °C.
2.2. Synthesis of the complexes
2.2.1. [Cu2(fluf)4(DMF)2], 1
Potassium hydroxide (0.4 mmol, 22 mg) was added to a methanolic
solution (15 mL) of flufenamic acid (0.4 mmol, 113 mg) and the solu-
tion was stirred for 1 h. The solution was added slowly to a methanolic
solution (5 mL) of CuCl2⋅2H2O (0.2 mmol, 34 mg) followed by the
addition of 2 mL of DMF. Dark green crystals of [Cu2(fluf)4(DMF)2]
1 suitable for X-ray structure determination were collected after
twenty days. Yield: 90 mg, 65%. Anal. Calcd. for [Cu2(fluf)4(DMF)2]
(C62H50Cu2F12N6O10) (MW = 1394.19): C 54.67, H 3.70, N 6.17;
COOH
H
F
3C
N
found C 54.25, H 3.59, N 5.95. IR (KBr disk): νmax, cm−1; ν(C = O)DMF
:
1667 (very strong (vs)); νasym(CO2): 1590 (vs); νsym(CO2): 1400
(vs); Δ = νasym(CO2)–νsym(CO2) = 190 cm−1; UV-vis: λ, nm (ε,
M−1 cm−1) as nujol mull: 738, 410 (shoulder (sh)), 340, 295; in
DMSO: 735 (120), 415(sh) (150), 335 (14,200), 296 (25,000).
μeff = 1.45 BM. The complex is soluble in DMF and DMSO (ΛM
=
Scheme 1. The NSAID flufenamic acid (=Hfluf).
5 mho cm2 mol−1, in 1 mM DMSO).

C. Tolia et al. / Journal of Inorganic Biochemistry 123 (2013) 53–65
55
Table 1
2.2.2. [Cu(fluf)(bipyam)Cl], 2
methanolic (10 mL) solution containing flufenamic acid
Crystallographic data for complexes 1 and 2.
A
(0.2 mmol, 52 mg) and ΚΟΗ (0.2 mmol, 11 mg) after 1 h stirring
was added slowly, and simultaneously with a methanolic solution of
bipyam (0.2 mmol, 34 mg), to a methanolic solution (10 mL) of
CuCl2⋅2H2O (0.2 mmol, 34 mg) and stirred for 15 additional min.
Green crystals of [Cu(fluf)(bipyam)Cl] 2 suitable for X-ray structure
determination were collected after a week. Yield: 66 mg, 60%. Anal.
Calcd. for [Cu(fluf)(bipyam)Cl] (C24H20ClCuF3N4O2) (MW = 552.43):
C 52.18, H 3.65, N 10.14; found C 52.29, H 3.34, N 10.04. IR (KBr disk):
1
2
Formula
Fw
T (Κ)
Crystal system
Space group
a (Å)
C
62H50Cu2F12N6O10
C24H18ClCuF3N4O2
550.41
160(2)
Triclinic
P −1
8.5858(1)
9.0373(1)
14.9190(2)
77.038(1)
76.198(1)
82.703(1)
1092.22(2)
2
1394.16
160(2)
Monoclinic
P 21/n
12.7379(2)
9.0968(2)
26.8191(5)
90.00
104.168(1)
90.00
3013.11(10)
2
1.537
1.766
1.019
b (Å)
c (Å)
α (ο)
ν
max, cm−1; νasym(CO2): 1590 (vs); νsym(CO2): 1410 (vs); Δ =
β (ο)
180 cm−1; UV–vis: λ, nm (ε, M−1 cm−1) as nujol mull: 842(sh), 705,
421 (sh), 328, 297; in DMSO: 845(sh) (15), 702 (105), 420 (sh) (180),
325 (10,300), 300 (13,500). μeff = 1.98 BM. The complex is soluble in
DMF and DMSO (ΛM = 6 mho cm2 mol−1, in 1 mM DMSO).
γ (ο)
Volume (Å3)
Ζ
D(calc), Mg m−3
Abs. coef., μ, mm−1
GOF on F2
R1=
1.674
3.053
1.038
0.0292
2.2.3. [Cu(fluf)(B)Cl], (B = phen for 3, bipy for 4)
a
b
0.0403
0.1078
Complexes 3 and 4 have been prepared in a similar way to complex
2 with the use of the corresponding N,N′-donor ligand (B), phen
(0.2 mmol, 36 mg) for 3 and bipy (0.2 mmol, 31 mg) for 4. Green
microcrystalline product of [Cu(fluf)(phen)Cl] and [Cu(fluf)(bipy)Cl],
respectively, was collected after a week.
a
b
wR2=
0.0722
a
4618 reflections with I > 2σ(I).
3193 reflections with I > 2σ(I).
b
Data for 3: Yield: 75 mg, 66%. Anal. Calcd. for (C26H17ClCuF3N3O2)
(MW = 559.43): C 55.82, H 3.06, N 7.51; found C 55.37, H 2.65, N
8.02. IR (KBr disk): νmax, cm−1; νasym(CO2): 1585 (vs); νsym(CO2):
1395 (vs); Δ = 190 cm−1; UV–vis: λ, nm (ε, M−1 cm−1) as nujol
mull: 852, 710, 414 (sh), 314, 291; in DMSO: 850 (sh) (15), 705
(65), 409 (sh) (175), 312 (7250), 285 (10,500). μeff = 1.87 BM. The
complex is soluble in DMF and DMSO (ΛM = 5 mho cm2 mol−1, in
1 mM DMSO).
for 1: 2θmax = 130°; reflections collected/unique/used, 25717/5057
[Rint = 0.0264]/5057; 515 parameters refined; (Δ/σ)max = 0.001;
(Δρ)max/(Δρ)min = 0.902/−0.459 e/Å3; R1/wR2 (for all data),
0.0433/0.1100. Further experimental crystallographic details for
2: 2θmax = 130°; reflections collected/unique/used, 14641/3476
[Rint = 0.0275]/3476; 388 parameters refined; (Δ/σ)max = 0.000;
(Δρ)max/(Δρ)min = 0.346/−0.344 e/Å3; R1/wR2 (for all data),
0.0320/0.0737. In both structures, all hydrogen atoms were located by
difference maps and were refined isotropically, and all non-hydrogen
atoms were refined anisotropically.
Data for 4: Yield: 65 mg, 60%. Anal. Calcd. for (C24H17ClCuF3N3O2)
(MW = 535.41): C 53.84, H 3.20, N 7.85; found C 54.15, H 3.05, N
7.58. IR (KBr disk): νmax, cm−1; νasym(CO2): 1580 (vs); νsym(CO2):
1395 (vs); Δ = 185 cm−1; UV–vis: λ, nm (ε, M−1 cm−1) as nujol
mull: 843(sh), 704, 408(sh), 313, 297; in DMSO: 840(sh) (10), 701
(100), 410 (sh) (190), 315 (12,000), 301 (14,700). μeff = 1.92 BM.
2.4. Computational studies
All calculations on complexes 2–5 were performed with the Gauss-
ian 09 (G09) program [47] employing the density functional theory
(DFT) and time-dependent density functional theory (TD-DFT) methods
[48,49]. The hybrid CAM-B3LYP [50], functional was used in combina-
tion with the LANL2DZ ECP [51] (for Cu) and with the 6–31 G** basis
set (for all other atoms) [52].
Thirty-two low-lying excitation energies with the corresponding
oscillator strengths were determined for 2–5 at the ground-state
geometry by TD-DFT. The PCM solvent model [53] was adopted in
the TD-DFT calculations with DMSO as solvent. Theoretical UV–Vis
spectra were obtained using GAUSSSUM 2.2 [54]. Molecular graphics
images were produced using the UCSF Chimera package from the
Resource for Biocomputing, Visualization, and Informatics at the Univer-
sity of California, San Francisco (supported by NIH P41 RR001081) [55].
The complex is soluble in DMF and DMSO (ΛM = 8 mho cm2 mol−1
,
in 1 mM DMSO).
2.2.4. [Cu(fluf)2(py)2], 5
Flufenamic acid (0.4 mmol, 113 mg) was dissolved in methanol
(10 mL) and KOH (0.4 mmol, 22 mg) was added. After 1 h stirring,
the solution was added slowly to a methanolic solution (10 mL) of
CuCl2⋅2H2O (0.2 mmol, 34 mg) followed by the addition of 2 mL of
pyridine. Blue-green microcrystalline product of [Cu(fluf)2(py)2] 5
was collected after a month. Yield: 100 mg, 65%. Anal. Calcd. for
[Cu(fluf)2(py)2] (C38H28CuF6N4O4) (MW = 782.20): C 58.35,
H
;
3.61, N 7.16; found C 58.10, H 3.67, N 7.09. IR (KBr disk): νmax, cm−1
ν
asym(CO2): 1580 (vs); νsym(CO2): 1390 (vs); Δ = 190 cm−1; UV–vis:
λ, nm (ε, M−1 cm−1) as nujol mull: 657, 407(sh), 341 (sh), 288;
in DMSO: 660 (90), 405(sh) (205), 344 (sh) (8500), 290 (12,500).
2.5. Albumin binding studies
μeff = 1.83 BM. The complex is soluble in DMF and DMSO (ΛM
=
5 mho cm2 mol−1, in 1 mM DMSO).
The protein binding study was performed by tryptophan fluorescence
quenching experiments using BSA (3 μM) or 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
342 nm or HSA at 351 nm was monitored using Hfluf and complexes
1–5 as quenchers with increasing concentration (up to 2.2 × 10−5 M)
[56] and the fluorescence spectra were recorded from 300 to 500 nm
at an excitation wavelength of 296 nm. The fluorescence spectra of
the compounds recorded under the same experimental conditions
exhibited a maximum emission at 374 nm. Therefore, the quantita-
tive studies of the serum albumin fluorescence spectra were performed
after their correction by subtracting the spectra of the compounds.
The Stern–Volmer and Scatchard equations and graphs have been
used in order to study the interaction of a quencher with serum albumins.
2.3. X-ray structure determination
A crystal of 1 (0.36 × 0.52 × 0.55 mm) and a crystal of 2
(0.15 × 0.24 × 0.27 mm) were taken from the mother liquor and
immediately cooled to −113 °C. Diffraction measurements were
made on a Rigaku R-AXIS SPIDER Image Plate diffractometer using
graphite monochromated Cu Kα radiation. Data collection (ω-scans)
and processing (cell refinement, data reduction and Empirical absorption
correction) were performed using the CrystalClear program package [44].
The structures were solved by direct methods using SHELXS-97
[45] and refined by full-matrix least-squares techniques on F2 with
SHELXL-97 [46] (Table 1). Further experimental crystallographic details

56
C. Tolia et al. / Journal of Inorganic Biochemistry 123 (2013) 53–65
The values of the dynamic quenching constant (KSV, M−1) and the
quenching constant (kq, M−1 s−1) for the interaction of Hfluf and
complexes 1–5 with SA have been derived from the Stern–Volmer
quenching equation [57]:
compound. The obtained data are presented as (η/η0)1/3 versus r,
where η is the viscosity of DNA in the presence of a complex, and η0 is
the viscosity of DNA alone in buffer solution.
The competitive studies of each compound with EB have been in-
vestigated with fluorescence spectroscopy in order to examine
whether the compound can displace EB from its CT DNA–EB complex.
The CT 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 was
studied by adding a certain amount of a solution of the compound
step by step into a solution of the 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. Flufenamic acid and its complexes 1–5 show no fluorescence
at room temperature in solution or in the presence of CT DNA under
the same experimental conditions; therefore, the observed quenching
is attributed to the displacement of EB from its EB–DNA complex. The
Stern–Volmer plots of DNA–EB have been used in order to study the
quenching of EB bound to DNA by the compounds according to the
linear Stern–Volmer equation:
Io
I
¼ 1 þ kqτ0½Qꢀ ¼ 1 þ KSV ½Qꢀ
ð1Þ
where Io = the initial tryptophan fluorescence intensity of SA, I = the
tryptophan fluorescence intensity of SA after the addition of the
quencher, kq = the quenching rate constants of SA, KSV = the dynamic
quenching constant, τo = the average lifetime of SA without the
quencher, [Q] = the concentration of the quencher respectively,
KSV = kqτo and, taking as fluorescence lifetime (τo) of tryptophan
in SA at around 10−8 s [57], KSV (in M−1) can be obtained by the
slope of the diagram Io vs [Q], and subsequently the approximate kq
(in M−1 s−1) may beIcalculated.
From the application of the Scatchard [58] equation:
ΔI=
½Qꢀ
ΔI
Io
Io
¼ nK ꢁ K
ð2Þ
Io
I
the association binding constant K (in M−1) may be calculated from
¼ 1 þ KSV ½Qꢀ
ð4Þ
ΔI=
ΔI
Io
Io
the slope in the Scatchard plots
versus
and the number of
Q
½
ꢀ
where Io and I are the emission intensities in the absence and the pres-
ence of the quencher, respectively, [Q] is the concentration of the quench-
er (Hfluf or complexes 1–5) [7,60]. The values of the Stern–Volmer
binding sites per albumin (n) is given by the ratio of y intercept to
the slope [58].
Io
I
constant (KSV, in M−1) are obtained by the slope of the diagram vs [Q].
2.6. DNA-binding studies
The interaction of Hfluf and complexes 1–5 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 (Kb). The UV spectra of CT DNA have been recorded for a con-
stant CT DNA concentration in the presence of each compound at di-
verse r (=[compound]/[CT DNA] mixing ratios) values. The binding
constants, Kb, of the complexes with CT DNA have been determined
using the UV spectra of the compound recorded for a constant con-
centration in the absence or presence of CT DNA for diverse r values.
Control experiments with DMSO were performed and no changes in
the spectra of CT DNA were observed. The binding constant of the
complexes with CT DNA, Kb, is used in order to estimate the magni-
tude of the binding strength of their interaction. Kb (in M−1) can be
½DNAꢀ
2.7. Antioxidant biological assay
In the in vitro assays each experiment was performed at least in
triplicate and the standard deviation of absorbance was less than 10%
of the mean.
2.7.1. Determination of the reducing activity of the stable radical
1,1-diphenyl-picrylhydrazyl (DPPH)
To a solution of DPPH (0.1 mM) in absolute ethanol an equal
volume of the compounds dissolved in ethanol was added. As control
solution ethanol was used. The concentration of the solution of the
compounds was 0.1 mM. The absorbance at 517 nm was recorded
at room temperature, after 20 and 60 min in order to examine the
time-dependence of the radical scavenging activity [7,61]. The radical
scavenging activity of the compounds was expressed as the percentage
inhibition of the absorbance of the initial DPPH solution (RA%). NDGA
(nordihydroguaiaretic acid) and BHT (butylated hydroxytoluene) were
used as reference compounds.
À
Á
calculated by the ratio of slope to the y intercept in plots
sus [DNA], according to the equation [59]:
ver-
εA−εf
½DNAꢀ
½DNAꢀ
1
¼
þ
ð3Þ
ðεA−εf Þ ðεb−εf Þ Kbðεb−εf Þ
2.7.2. Competition of the tested compounds with DMSO for hydroxyl radicals
The hydroxyl radicals generated by the Fe3+/ascorbic acid system,
were detected according to Nash [7,61], by the determination of formal-
dehyde produced from the oxidation of DMSO. The reaction mixture
contained EDTA (0.1 mM), Fe3+ (167 μM), DMSO (33 mM) in phos-
phate buffer (50 mM, pH 7.4), the tested compounds (concentration
0.1 mM) and ascorbic acid (10 mM). After 30 min of incubation (37 °C)
the reaction was stopped with CCl3COOH (17% w/v) and the absorbance
at λ = 412 nm was measured. Trolox was used as an appropriate stan-
dard. The competition of the compounds with DMSO for OH•, generated
by the Fe3+/ascorbic acid system, expressed as percent inhibition of
formaldehyde production, was used for the evaluation of their hydroxyl
radical scavenging activity (•OH%).
where [DNA] is the concentration of DNA in base pairs, εf is the ex-
tinction coefficient for the free complex at the corresponding λmax
εA = Aobsd/[complex] and εb is the extinction coefficient for the com-
plex in the fully bound form.
,
The interaction of complexes 1–5 with CT DNA has been also investi-
gated by monitoring the changes observed in the cyclic voltammogram of
a 0.40 mM 1:2 DMSO:buffer solution of complex upon addition of CT 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
.
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 a DNA
solution has been measured in the presence of increasing amounts
of the compounds. The relation between the relative solution viscosity
2.7.3. Assay of radical cation scavenging activity
(η/η0) and DNA length (L/L0) is given by the equation L/L0 = (η/η0)1/3
,
ABTS was dissolved in water to a 2 mM concentration. ABTS radical
where L0 denotes the apparent molecular length in the absence of the
cation (ABTS•+) was produced by reacting ABTS stock solution with

C. Tolia et al. / Journal of Inorganic Biochemistry 123 (2013) 53–65
57
0.17 mM potassium persulfate and allowing the mixture to stand in the
dark at room temperature for 12–16 h before use. Because ABTS and po-
tassium persulfate react stoichiometrically at a ratio of 1:0.5, this will
result in incomplete oxidation of the ABTS. Oxidation of the ABTS com-
menced immediately, but the absorbance was not maximal and stable
until more than 6 h had elapsed. The radical was stable in this form
for more than 2 days when stored in the dark at room temperature.
The ABTS•+ solution was diluted with ethanol to an absorbance of
0.70 at 734 nm. After addition of 10 μL of diluted compounds or stan-
dards (0.1 mM) in DMSO, the absorbance reading was taken exactly
1 min after initial mixing [62,63]. The radical scavenging activity of
the complexes was expressed as the percentage inhibition of the absor-
bance of the initial ABTS solution (ABTS%). Trolox was used as an appro-
priate standard.
naproxen and diclofenac [8,25]. Time-dependent DFT calculations were
performed on the optimized complexes 2–5 to confirm such assign-
ment. Fig. 1(A) shows vertical electronic transitions and the resulting
theoretical spectrum in the case of complex 2. The electron density dif-
ference maps (EDDMs) reported (Fig. 1(B)) can be used to visualize the
nature of selected transitions. EDDMs highlight that the lowest-energy
band is prevalently composed by ligand-to-metal charge transfer
(LMCT) transitions (although some d–d character is present), while
the band at ~400 nm is a mixed d–d/LMCT. Similar results are obtained
for the other compounds (Tables S1–S4 and Figs. S1–S3) with 3 and 4
showing a more pronounced d–d character in the lowest-energy states.
The UV spectra of the complexes have been also recorded in the
pH range of 6–8 (since 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)
without showing (data not shown) any significant changes (shift of
the λmax or new peaks), indicating that the complexes are stable in
the pH range = 6–8. The fact that complexes 1–5 have the same
UV–Vis spectral pattern in nujol and in DMSO solution as well as in
the presence of the buffer solution (150 mM NaCl and 15 mM trisodium
citrate in the pH 7.0) used in the biological experiments and in the
pH range = 6–8 in combination to non-dissociation in DMSO solu-
tion (ΛM ≤ 8 mho cm2 mol−1, in 1 mM DMSO solution) suggests
that the compounds keep their integrity in solution [38,66].
2.7.4. Soybean lipoxygenase inhibition study in vitro
The in vitro study was evaluated as reported previously [61]. The
tested compounds dissolved in ethanol were incubated at room tem-
perature with sodium linoleate (0.1 mM) and 0.2 cm3 of enzyme so-
lution (1/9 × 10−4 w/v in saline). The conversion of sodium
linoleate to 13-hydroperoxylinoleic acid at 234 nm was recorded
and compared with the appropriate standard inhibitor caffeic acid.
3. Results and discussion
The μeff value for 1 (= 1.45 BM) is typical for dinuclear copper(II)
complexes with a paddle-wheel arrangement, in agreement with
μeff values of diclofenac [24], diflunisal [25] and naproxen [27]
structurally analogous Cu(II) complexes. The observed values of
μeff (= 1.83–1.98 BM) for complexes 2–5 are higher than the
spin-only value (= 1.73 BM) at room temperature and typical for
mononuclear Cu(II) complexes with d9 configuration (S = 1/2) [7,67].
Spin density surfaces calculated with the DFT method (Fig. 2 and
Tables S5–S9) are in agreement with such assignment.
3.1. Synthesis and spectroscopic study
The structurally characterized complexes have been synthe-
sized in high yield via the reaction of the NSAID Hfluf with KOH,
CuCl2⋅2H2O and the corresponding O-(DMF, Eq. (5)) for 1, or N,
N′-donor, e.g. (bipyam, Eq. (6)) for 2, according to the equations:
2CuCl2⋅2H2O þ 4Hfluf þ 4KOH þ 2DMF→½Cu2ðflufÞ4ðDMFÞ2ꢀ
3.2. Structure of the complexes
þ4KCl þ 8H2O
ð5Þ
ð6Þ
3.2.1. Crystal structure of [Cu2(fluf)4(DMF)2] 1
CuCl2⋅2H2O þ 1Hfluf þ 1KOH þ 1bipyam→½CuðflufÞðbipyamÞClꢀ
þ1KCl þ 3H2O
The crystal structure of complex 1 is composed of centrosymmetric
dimers. The molecular structure of the complex appears in Fig. 3, and se-
lected bond distances and angles are given in Table 2.
Four bidentate flufenamato ligands form syn-syn carboxylate brid-
ges between isolated pairs of copper atoms separated by 2.618(1) Å,
distance lying in the range observed for dinuclear copper(II) complexes
IR spectroscopy has been used in order to confirm the deproton-
ation and binding mode of flufenamic acid. In the IR spectrum of
Hfluf, the absorption band at 3436(br (broad), m (medium)) cm−1
attributed to the ν(H\O) stretching vibration has disappeared upon
binding to the metal ion, revealing the deprotonation of the carboxylato
group. The bands at 1655 (s (strong)) cm−1 and 1255(s) cm−1 attrib-
uted to the stretching vibrations ν(C = O)carboxylic and ν(C\O)carboxylic
of the carboxylic moiety (\COOH), respectively, have shifted in the
IR spectra of complexes 1–5 in the range of 1580–1590 cm−1 and
1390–1401 cm−1 assigned to antisymmetric, νasym(CO2), and sym-
metric, νsym(CO2), stretching vibrations of the carboxylato group,
respectively. The difference Δ [= νasym(CO2)–νsym(CO2)] is a character-
istic tool for determining the coordination mode of the carboxylato
ligands and has a value falling in the range of 180–190 cm−1 indicative
of bidentate binding mode of the flufenamato ligand [38,64].
of the formula [Cu2(O2CR)2(L)2] (R = Me, ClCH2, Et, C5H11, C6H13
,
C8H17, 4-HOC6H4, H, FCH2 or Ph; L = O-donor (DMSO, DMF, methanol,
H2O) or N-donor (py, urea, quinoline, methylpyridine or nicotinamide)
ligand) [25] with a paddlewheel structural motif.
The Cu\Ocarb distances are quite similar and range from
1.961(2) to 1.972(2) Å. The sum of all interatomic distances
in the CuO5 chromophore (½ of Cu…Cu distance = 1.309 Å is
included) 11.305 Å is close to 11.348 Å, average sum for a series
of copper(II) binuclear carboxylate compounds [25]. The bridging
path lengths (Cu\O(1)\C(1)\O(2)\Cu′ = 6.463 Å and Cu\O(21)\
C(21)\O(22)\Cu′ = 6.455 Å) are within the range observed for
copper(II) carboxylate dimers. The copper atom is displaced 0.195 Å to-
ward the DMF ligand from the plane containing the four co-ordinated
carboxylato oxygen atoms and is in the range of 0.19–0.22 Å known
for binuclear carboxylate complexes [25].
Assuming a five-coordinate copper chromophore, each copper atom
could be described as having a square-pyramidal geometry. The
tetragonality [67] T5 = 0.923, based on the changes in bond lengths,
along with the trigonality index [68] [τ = (φ1–φ2)/60°, where φ1
and φ2 are the largest angles in the coordination sphere; τ = 0 for
a perfect square pyramid, and τ = 1 for a perfect trigonal bipyramid]
τ = (168.64–168.60)/60 = 0.00°, show no distortion from the reg-
ular square-based pyramidal geometry.
In the visible region, complexes 2–4 exhibit a d–d transition
band in the range of 700–705 nm (ε = 65–100 M−1 cm−1) and
an additional weak broad d–d transition band at 840–850 nm
(ε = 10–15 M−1 cm−1), typical for distorted square pyramidal
geometry [65]. For complexes 1 and 5, a low-intensity band with
ε = 90–120 M−1 cm−1 observed in the Vis region (735 nm
(ε = 120 M−1 cm−1) and 660 nm (ε = 90 M−1 cm−1), respective-
ly) can be assigned to a 2Eg
→
2T2g d–d transition, typical for Cu(II)
complexes with octahedral local symmetry. The complexes also exhibit
a shoulder, at 405–420 nm (ε = 150–205 M−1 cm−1) which may be
assigned to a charge-transfer transition. Similar spectroscopic features
have been reported for the Cu(II) complexes with the NSAIDs diflunisal,

58
C. Tolia et al. / Journal of Inorganic Biochemistry 123 (2013) 53–65
Fig. 1. (A) Theoretical absorption spectra for 2 in DMSO at the CAM-B3LYP/LANL2DZ/6-31 G** level. Doublet-doublet transitions are shown as vertical bars with heights equal to
their oscillator strengths. The theoretical curves were obtained using GAUSSSUM 2.2 [54]; (B) Selected EDDMs for 2, light blue indicates a decrease in electron density, while
green indicates an increase.
Among the Cu(II)–NSAIDs complexes that have been structural-
ly characterized, 1 has similar structure with [Cu2(nap)4(DMSO)2]
[27], [Cu2(dicl)4(DMF)2] [24], [Cu2(dicl)4(acetone)2] [69] and
[Cu2(difl)4(DMF)2] [25], where Hnap = naproxen, Nadicl = sodium
diclofenac and Hdifl = diflunisal are NSAIDs.
The dimers in 1 are stabilized via four intra-molecular hydrogen
bonding interactions developed within the four flufenamato ligands
[N(1)∙∙∙O(1) = 2.653 Å, H(1)N∙∙∙O(1) = 2.057 Å, N(1)–H(1)N∙∙∙O(1) =
134.9°; N(21)∙∙∙O(22) = 2.662 Å, H(21)N∙∙∙O(22) = 2.045 Å, N(21)–
H(21)N∙∙∙O(22) =136.0°].
corresponding values for five-coordinate Cu atoms in distorted square
pyramidal geometry with Cl in apical position and chelating bipyam
and various carboxylato ligands in the basal plane [70–77].
The copper atom is displaced 0.336 Å from the basal plane towards
the chloro ligand which is 2.818 Å away from the mean basal plane.
The trans atoms system of the basal plane gives angles of O(1)\
Cu(1)\N(12) = 155.38(8)° and O(2)\Cu(1)\N(11) =155.44(8)°.
The observed N(11)\Cu(1)\N(12) bite angle of bipyam (94.16(7)°)
and those of the chelating atoms to Cu (Table 3) fall in the range of an-
gles found in similar complexes [70–77]. An interesting feature concerns
the bite angle values and planarity of bipyam chelating ligand with re-
spect to the ligands completing the coordination sphere of Cu ion. The
N\Cu\N chelating angle in 2 is higher than the corresponding angle
(91.96(7)°) in distorted octahedral CuN2O4 environment [7] and in
distorted square pyramidal CuClN2O2 environment (91.98° [78] and
90.50° [79]). This reduction in the values of N\Cu\N chelate angle is
probably related to the deviation of bipyam ligands from planarity.
In the complexes studied in references [78,79] the bipyam ligands are
not planar and the puckering of the molecule is defined by the angle
3.2.2. Crystal structure of [Cu(fluf)(bipyam)Cl]
In [Cu(fluf)(bipyam)Cl], 2, the deprotonated flufenamato is coordi-
nated to copper atom via the two carboxylato oxygen atoms on
bidentate asymmetric chelating mode (C(1)\O(1) = 1.269(2) Å and
C(1)\O(2) = 1.278(2) Å). A diagram of complex 2 and the dimers
formed in the structure are shown in Fig. 4, and selected bond distances
and angles are listed in Table 3.
The copper atom is five-coordinate and could be described
possessing a slightly distorted square pyramidal geometry. The
tetragonality T5 = 0.803 [67], along with the trigonality index
τ = (155.44°–155.38°)/60° = 0.001 [68], show slight distortion
from the regular square-based pyramidal geometry. The chelate rings
formed by nitrogen atoms N(11) and N(12) [Cu(1)\N(11) = 1.967(2)
Å and Cu(1)\N(12) = 1.959(2) Å] of the bipyam ligand (i.e. the
six-membered ring Cu\N\C\N\C\N) and by the carboxylate oxygen
atoms O(1) and O(2) [Cu(1)\O(1) = 2.029(2) Å and Cu(1\O(2) =
2.036(1) Å] (i.e. the four-membered ring Cu\O\C\O) of the
flufenamato ligand possess the basal plane, while the Cl atom
[Cu(1)\Cl = 2.488(1) Å] occupies the apical position. The values
of all bond lengths and angles mentioned above are very close to the
Fig. 2. Spin density surface for complex 4 calculated in DMSO at the CAM-B3LYP/
Fig. 3. A drawing of the molecular structure of [Cu2(fluf)4(DMF)2] 1 with only the
LANL2DZ/6-31 G** level using the PCM solvent (DMSO) method.
heteroatoms labeling.

C. Tolia et al. / Journal of Inorganic Biochemistry 123 (2013) 53–65
59
Table 2
Selected bond distances and angles for complex [Cu2(fluf)4(DMF)2].
Table 3
Selected bond distances and angles for [Cu(fluf)(bipyam)Cl] 2.
Bond distances
(Å)
1.968(2)
1.961(2)
1.972(2)
1.268(3)
1.263(3)
Bond distances
(Å)
2.029(2)
2.036(1)
2.488(1)
1.269(2)
Cu\O(1′)
Cu\O(2)
Cu\O(41)
Cu…Cu′
C(21) \O(21)
C(21) \O(22)
1.964(2)
2.131(2)
2.618(1)
1.257(3)
1.265(3)
Cu(1) \O(1)
Cu(1) \O(2)
Cu(1) \Cl
Cu(1) \N(11)
Cu(1) \N(12)
C(1) \O(2)
1.967(2)
1.959(2)
1.278(3)
Cu\O(21′)
Cu\O(22)
O(1) \C(1)
O(2) \C(1)
C(1) \O(1)
Bond angles
(°)
Bond angles
(°)
O(1) \Cu(1) \O(2)
O(1) \Cu(1) \N(11)
O(1) \Cu(1) \N(12)
O(1) \Cu(1) \Cl
64.58(6)
97.70(7)
155.38(8)
95.34(5)
94.16(7)
O(2) \Cu(1) \N(11)
O(2) \Cu(1) \N(12)
O(2) \Cu(1) \Cl
N(12) \Cu(1) \Cl
N(11) \Cu(1) \Cl
155.44(8)
96.87(6)
97.20(5)
103.41(6)
101.48(6)
O(1′)\Cu\O(2)
O(1′)\Cu\O(21′)
O(1′)\Cu\O(22)
O(1′)\Cu\O(41)
O(22) \Cu\O(41)
168.64(7)
89.38(8)
89.64(7)
91.22(7)
94.70(6)
O(2) \Cu\O(21′)
O(2) \Cu\O(22)
O(2) \Cu\O(41)
O(21′)\Cu\O(22)
O(21′)\Cu\O(41)
88.45(7)
90.29(7)
100.10(7)
168.60(7)
96.68(7)
N(11) \Cu(1) \N(12)
Primed atoms are generated by symmetry: (′) 1 − x, 1 − y, 1 − z.
bound to copper ion via the carboxylato oxygen atoms. From the
UV–Vis spectra a distorted square pyramidal environment around
copper may be suggested for complexes 3 and 4 which are expected
to have crystal structure similar to that of complex 2, with the two ox-
ygen atoms of flufenamato ligand and the two nitrogen atoms of the
1,10-phenanthroline or 2,2´-bipyridine ligand forming the basis of the
pyramid and the chlorine atom lying at the apex. On the other hand,
for complex 5 a distorted octahedron geometry of copper may be
suggested (the octahedron is formed by four oxygen atoms of the
two flufenamato ligands and two nitrogen atoms provided by two
pyridine ligands) and is similar to that of [Cu(diflunisal)2(py)2]
[25] and [Cu(diclofenac)2(py)2] [8] (Nadicl the NSAID sodium
diclofenac) which also exhibit a distorted octahedral arrangement
around Cu(II) atom and their structures are available in the literature.
DFT geometry optimization calculation were performed at the
CAM-B3LYP/LANL2DZ/6-31G** level to obtain structural insights on
3–5. Selected bond distances (Å) and angles (°) (including complex 2)
are reported in Table 4 and confirm spectroscopic observations.
of the planar pyridine rings of the bipyam ligands [73]. Puckering proba-
bly results from the stereochemical interaction developed between
bipyam ligands and the oxygen atoms of other ligands completing the co-
ordination sphere of copper atoms, which in both of the latter cases is
CuN2O2Cl distorted square pyramidal as in 2. In both of these cases the
chelating oxygens participate in five-membered rings and the bite angles
are 81.65° and 81.84° respectively, significantly greater than the analo-
gous O\Cu\O bite angle in 2 (Table 3), which is formed by oxygen
atoms participating in four-membered rings. In 2, the angle between
the planar pyridine rings of the bipyam ligands is 5.80°, slightly larger
than those in complexes studied in [70,71,73–76] which are lower than
4.0°. For the complexes studied in [72] and [77], this angle takes the
values 12.20° and 9.06° respectively. In the cases studied in [78] and
[79] where the chelating oxygen atoms participate in five-membered
rings the bending angles take significantly higher values, 20.19° and
31.30°, respectively.
The molecules of 2 are stabilized through an intra-molecular
hydrogen bonding interaction within the flufenamato ligand
[N(1)∙∙∙O(2) = 2.667 Å, H(1)N∙∙∙O(1) = 1.971 Å, N(1)–H(1)N∙∙∙O(1) =
143.2°], whereas they form dimers through two inter-molecular hydro-
gen bonds involving the NH moiety of bipyam and the chloro
ligand [N(13)∙∙∙Cl (−x, −y, −z) = 3.159 Å, H(13)N∙∙∙Cl = 2.363 Å,
N(13)–H(13)N∙∙∙Cl = 167.0°] resulting in a face to face stacking of
bipyam ligands belonging to neighboring centrosymmetrically related
complexes (Fig. 4). The interplanar distance of bipyam ligand plane is
3.286 Å a value close to the one (3.341 Å) observed in [76]. These
type of hydrogen bond interactions are observed for neighboring com-
plexes with formula [Cu(II)(bipyam)(RCO2)Cl] studied in [70–72].
With the exception of those studied in [72] and [77], all the other com-
plexes form dimers of the type observed in 2.
3.3. Interaction of the complexes with serum albumins
Serum albumin (SA), the most abundant protein in plasma, is mainly
involved in the transport of metal ions, drugs and their metal complexes
through the blood stream [80]. Human serum albumin (HSA) has a
tryptophan located at position 214, while bovine serum albumin
(BSA), the most extensively studied serum albumin due to its struc-
tural homology with HSA, has two tryptophans, Trp-134 and Trp-212
[81]. BSA and HSA solutions exhibit an intense fluorescence emission
with λem,max = 342 nm and 351 nm, respectively, due to the trypto-
phan residues, when excited at 295 nm [56]. Hfluf and its complexes
1–5 exhibit a maximum emission at 374 nm under the same experi-
mental conditions; therefore the changes and the quenching occur-
ring in the BSA or HSA fluorescence emission spectra upon addition
of Hfluf or complexes 1–5 at 343 nm and 351 nm, respectively, are
3.2.3. Proposed structures of complexes 3-5
Based on IR, UV–Vis and magnetic measurements data, complexes
3–5 are also mononuclear with the deprotonated flufenamato ligands
Fig. 4. Dimers of clusters formed through hydrogen bonds and numbering scheme for the symmetry independent complex in the unit cell of 2. Symmetry code: (′):-x,-y,-z.

60
C. Tolia et al. / Journal of Inorganic Biochemistry 123 (2013) 53–65
Table 4
A
Selected bond distances (Å) and angles (°) for the DFT-calculated structure of 2–5 at
100
80
60
40
20
0
the CAM-B3LYP/LANL2DZ/6-31G** level using the PCM solvent (DMSO) method.
2
3
4
5
Bond distances
Cu(1) \O(1)
Cu(1) \O(2)
Cu(1) \Cl
Cu(1) \N(11)
Cu(1) \N(12)
C(1) \O(1)
C(1) \O(2)
2.015
2.054
2.050
2.015
2.006
1.273
1.280
2.005 1.998 Cu(1) \O(1)
2.029 2.041 Cu(1) \O(2)
2.483 2.483 Cu(1) \N(1)
2.035 2.019 Cu(1) \O(4)
2.034 2.013 Cu(1) \O(5)
1.274 1.275 Cu(1) \N(2)
1.281 1.280
1.978
2.570
2.046
1.981
2.554
2.057
Angles (°)
Hfluf
3
1
4
2
5
O(1) \Cu(1) \O(2)
O(1) \Cu(1) \N(11)
O(1) \Cu(1) \N(12) 156.5
O(1) \Cu(1) \Cl 100.7
N(11) \Cu(1) \N(12) 91.8
O(2) \Cu(1) \N(11) 153.2
O(2) \Cu(1) \N(12)
O(2) \Cu(1) \Cl
N(12) \Cu(1) \Cl
N(11) \Cu(1) \Cl
64.4
97.3
65.2
65.1
O(1) \Cu(1) \O(2)
O(1) \Cu(1) \O(3) 179.4
O(1) \Cu(1) \O(4) 122.8
O(1) \Cu(1) \N(1)
O(1) \Cu(1) \N(2)
O(2) \Cu(1) \O(3) 124.2
O(2) \Cu(1) \O(4) 177.2
O(2) \Cu(1) \N(1)
O(2) \Cu(1) \N(2)
O(3) \Cu(1) \O(4)
O(3) \Cu(1)\N(1)
O(3)\Cu(1) \N(2)
O(4)\Cu(1) \N(1)
O(4) \Cu(1) \N(2)
56.3
100.8 100.7
156.3 157.7
102.1 101.5
0
1
2
3
4
5
6
7
90.1
90.0
r=[compound]/[HSA]
81.9
80.9
154.2 152.7
102.5 103.6
102.2 102.1
100.3
101.9 103.6
98.9
101.4
101.1
98.7
B
91.3
88.3
56.6
90.12
89.7
91.4
89.4
100
80
60
40
20
0
99.7
Hfluf
1
3
5
2
4
N(1) \Cu(1) \N(2) 179.4
primarily due to change in protein conformation, subunit association,
substrate binding or denaturation [81].
Addition of Hfluf and complexes 1–5 to SA solution results in mod-
erate to significant quenching of HSA fluorescence at λ = 351 nm
(Fig. 5(A)) (quenching up to 89% of the initial fluorescence intensity
for 1) and to a much more enhanced quenching of the BSA fluores-
cence at λ = 343 nm (Fig. 5(B)) (quenching up to 99% of the initial
fluorescence intensity for 1). The observed quenching may be due
to possible changes in protein secondary structure leading to changes
in tryptophan environment of HSA, and thus indicating the binding of
each complex to the albumins [82].
The Ksv and kq values for Hfluf and complexes 1–5 interacting with
albumins have been calculated with Stern–Volmer quenching equation
(Eq. (1)) and Stern–Volmer plots (Figs. S4 and 5), are given in Table 5
and indicate good quenching ability of the compounds with 1 exhibiting
the strongest quenching ability (kq = 3.79( 0.32) × 1013 M−1 s−1
for HSA and 5.07( 0.74) × 1014 M−1 s−1 for BSA). The kq values
(>1012 M−1 s−1) are higher than diverse kinds of quenchers for
biopolymers fluorescence (2.0 × 1010 M−1 s−1) indicating the exis-
tence of static quenching mechanism [81].
The values of the association binding constant (K) and the number of
binding sites per albumin (n), as calculated from the Scatchard equation
(Eq. (2)) and Scatchard plots [58] (Figs. S6 and S7), for all compounds
are given in Table 5. The K values are relatively high with 1 having the
highest K values for both albumins used. Comparison of the n values re-
veals that all complexes exhibit higher n values than free Hfluf. Compar-
ing the affinity of the complexes for BSA and HSA (K values), it is
obvious that all compounds with the exception of 3 show higher affinity
for BSA than HSA. In any case, the binding constant of a compound to
the albumin should be at optimum; it should be high enough to allow
binding of the compound and its possible transfer but also not too high
in order to be released upon arrival at its target. It is quite noteworthy
all K values are in accordance being within an optimum range; they are
high enough to allow the binding of the compounds to SA and they are
also quite below the association constant of one of the strongest known
non-covalent bonds, i.e. the interaction of avidin with diverse ligands
with a K value ≈ 1015 M−1, suggesting a possible release from the
serum albumin to the target cells [82].
0
1
2
3
4
5
6
7
r=[compound]/[BSA]
Fig. 5. (A) Plot of % relative fluorescence intensity at λem = 351 nm (%) vs r
(r = [complex]/[HSA]) for Hfluf and complexes 1-5 (75% of the initial fluorescence in-
tensity for Hfluf, 11% for 1, 45% for 2, 75% for 3, 52% for 4 and 49% for 5) in buffer solution
(150 mM NaCl and 15 mM trisodium citrate at pH 7.0). (B) Plot of % relative fluorescence
intensity at λem = 342 nm (%) vs r (r = [complex]/[BSA]) for Hfluf and complexes 1-5
(22% of the initial fluorescence intensity for Hfluf, 1% for 1, 23% for 2, 52% for 3, 40% for
4 and 19% for 5) in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0).
3.4. Interaction with calf-thymus DNA
The potential anticancer and the antiinflammatory activity of the
NSAIDs and their complexes may be related to their ability to interact
with DNA [22,23]. Therefore, such studies are of increasing interest
since their number so far is quite limited; complexes of oxicam
NSAIDs bind to DNA via intercalation [23], while the interaction of
DNA with the copper(II) complexes of naproxen, diclofenac (NSAIDs
of the phenylalkanoic acids' group), mefenamic acid (belongs to the
group of the anthralinic acid NSAIDs) and diflunisal (a salicylate deriva-
tive NSAID) as well as of cobalt(II) naproxenato or mefenamato com-
plexes has been recently reported by our lab [7,8,25,31,39–41]. As it is
known, transition metal complexes can bind to DNA via both covalent
(replacement of a labile ligand of the complex by a nitrogen base of
DNA, e.g. guanine N7) and/or noncovalent (intercalation, electrostatic
or groove binding) interactions [7,8].
Hfluf and complexes 1–5 exhibit similar behavior upon their addition
on CT DNA solution. The UV spectra of a CT DNA solution with standard
concentration (9 × 10−5 M) have been recorded in the presence of a
compound at different [complex]/[DNA] mixing ratios (r) and represen-
tatively for 1 are shown in Fig. 6(A). The decrease of the intensity at
λmax = 258 nm is accompanied by a red-shift of the λmax up to
263 nm for all compounds, indicating that the interaction with CT DNA

C. Tolia et al. / Journal of Inorganic Biochemistry 123 (2013) 53–65
61
Table 5
The SA binding constants and parameters derived for Hfluf complexes 1–5.
HSA
Compound
Κ
sv (M−1
)
kq (M−1 s−1
)
K (M−1
)
n
Hfluf
1.86( 0.21) × 104
3.79( 0.16) × 105
7.11( 0.21) × 104
2.07( 0.27) × 104
5.20( 0.28) × 104
5.62( 0.25) × 104
1.86( 0.42) × 1012
3.79( 0.32) × 1013
7.11( 0.41) × 1012
2.07( 0.54) × 1012
5.20( 0.55) × 1012
5.62( 0.50) × 1012
1.79( 0.17) × 105
5.53( 0.37) × 105
5.45( 0.39) × 104
1.09( 0.42) × 105
7.65( 0.10) × 104
1.11( 0.05) × 105
0.31
0.97
1.12
0.35
0.85
0.77
[Cu2(fluf)4(DMF)2] 1
[Cu(fluf)(bipyam)Cl] 2
[Cu(fluf)(phen)Cl] 3
[Cu(fluf)(bipy)Cl] 4
[Cu(fluf)2(py)2] 5
BSA
Compound
Κ
sv (M−1
)
kq (M−1 s−1
)
K (M−1
)
n
Hfluf
1.83( 0.20) × 105
5.07( 0.37) × 106
1.98( 0.09) × 105
6.09( 0.65) × 104
8.24( 0.36) × 104
2.55( 0.09) × 105
1.83( 0.40) × 1013
5.07( 0.74) × 1014
1.98( 0.18) × 1013
6.09( 1.29) × 1012
8.24( 0.71) × 1012
2.55( 0.17) × 1013
1.06( 0.04) × 106
1.61( 0.14) × 106
9.55( 0.11) × 104
3.14( 0.12) × 104
8.74( 0.08) × 104
1.75( 0.07) × 105
0.81
1.03
1.23
1.27
0.99
1.09
[Cu2(fluf)4(DMF)2] 1
[Cu(fluf)(bipyam)Cl] 2
[Cu(fluf)(phen)Cl] 3
[Cu(fluf)(bipy)Cl] 4
[Cu(fluf)2(py)2] 5
results in the direct formation of a new complex with double-helical CT
DNA [83]. The observed hypochromism could be attributed to π → π*
stacking interaction between the aromatic chromophore (either from
quinolone and/or the N,N´-donor ligands) of the complexes and DNA
base pairs consistent with the intercalative binding mode [59], while
the red-shift (bathochromism) may be considered an evidence of stabi-
lization of CT DNA duplex [84].
In the UV spectrum of 5 (Fig. 6(B)), the band centred at 344 nm
(band I) exhibits a significant hypochromism of ~40% suggesting tight
binding to CT DNA probably by intercalation. Further addition of DNA
results in a gradual elimination of this band. Additionally, the band at
290 nm (band II) presents a hyperchromism of up to 30% accompanied
by a red-shift of 12 nm (up to 312 nm), suggesting tight binding and
stabilization. A distinct isosbestic point at 336 nm appears upon addition
of CT DNA. The behavior of Hfluf (band I (at 344 nm): hypochromism of
50%; band II (at 292 nm): hyperchromism ~40% and red-shift of 10 nm)
and complex 4 (band I (at 314 nm and 302 nm): hypochromism of 75%;
band II (at 260 nm): hyperchromism ~50% and red-shift of 20 nm)
(Fig. S8) upon addition of CT-DNA is quite similar to 5.
In the UV spectrum of 2 (Fig. 6(C)), both bands centred at 300 nm
and 320 nm present a hypochromism of ~10% and ~8%, respectively,
while the first band is red-shifted towards 308 nm and the second
band is rather eliminated. Quite similar is the behavior of complexes
1 (~8% hypochromism of the band at 300 nm accompanied by a
bathochromism of 3 nm) and 3 (~6% hypochromism and a 2-nm
bathochromism of the band at 297 nm) upon addition of increasing
amounts of CT DNA (Fig. S8).
complexes 1–5 exhibit similarly the highest Kb values than the corre-
sponding Cu(II) complexes with other NSAIDs as ligands [7,8,25].
The cyclic voltammogram of 2 in DMSO solution exhibits two
cathodic waves at −680 mV (Epc1) and at −1200 mV (Epc2, of lower
current than Epc1) which can show a two-step reduction of the species
and they can be assigned to the process [Cu(II)] → [Cu(I)], and the
formation of metallic copper [7,8,25,66], respectively, since Hfluf was
found to be electrochemically inactive. The formation of Cu(I) is consis-
tent with the metal-centered character of the LUMO orbitals of 2–5
(Table S9). These waves are followed by three non-reversible anodic
waves at −550 mV (Epa1), −45 mV (Epa2) and +350 mV (Epa3
)
which may be attributed to the oxidation processes of metallic copper
to [Cu(I)] (Epa1) and [Cu(II)] (Epa2) and of [Cu(I)] to [Cu(II)] (Epa3). Com-
plexes 1 and 3–5 have similar cyclic voltammograms in DMSO solution
and the corresponding potentials are given in Table 7.
The investigation of metal–DNA interaction with electrochemical
techniques is a useful complement to spectroscopic methods and
may yield information about interactions with both the reduced and
oxidized form of the metal. In general, when the metal ion or complex
binds to DNA via intercalation, the electrochemical potential presents
a positive shift, while, in the case of electrostatic interaction with DNA,
the potential will shift to a negative direction. Moreover, if more poten-
tials than one exist, a positive shift of Ep1 and a simultaneous negative
shift of Ep2 may imply that the molecule can bind to DNA by both interca-
lation and electrostatic interaction [60,87].
The redox couple for each complex in 1/2 DMSO/buffer solution
studied upon addition of CT DNA as well as the shifts of the cathodic
It should be noted that the exact mode of binding cannot be mere-
ly proposed by UV spectroscopic titration studies and the results col-
lected from the UV titration experiments suggest that all compounds
can bind to CT DNA [85]. The observed hypochromic effect may be
considered as first evidence of tight binding to CT DNA probably by
intercalation while the stabilization of the DNA duplex may be con-
cluded as a result of the existence of a red-shift [86].
The values of the binding constant, Kb, of the compounds with CT
DNA, as calculated by Eq. (3) [59] and the plots in Fig. S9, are given in
Table 6. The Kb values are high and are of the same magnitude to that
of the classical intercalator EB (Kb = 1.23( 0.07) × 105 M−1) [7,8].
The Kb values suggest a strong binding of the compounds to CT DNA
[7,8], with complex 4 exhibiting the highest Kb values among the
compounds. It is quite obvious that in most cases the coordination of
flufenamic acid to Cu(II) results in an increase of the Kb value up to
three to ten times.
Epc and anodic Epa potentials are given in Table 7. No new redox peaks
appeared after the addition of CT DNA to each complex; nevertheless,
the observed decrease in current intensity (Fig. S10), attributed to an
equilibrium mixture of free and DNA-bound complex to the electrode
surface, may suggest the existence of an interaction between each com-
plex and CT DNA [7,8,25,60,66]. All complexes 1–5 exhibit the same
electrochemical behavior upon addition of CT DNA (Table 7) and, for
increasing amounts of CT DNA, the cathodic and the anodic potentials
exhibit predominantly a positive shift (ΔEp = (−33)–(+59) mV)
suggesting the existence of intercalation between the complexes
and CT DNA bases [7,8,25,60].
The viscosity of DNA solution is sensitive to DNA length changes
and its measurement upon addition of a compound may provide sig-
nificant aid to clarify the interaction mode of a compound with DNA
[8,39,40]. Viscosity measurements were carried out on CT DNA solu-
tions upon addition of increasing amounts of the compounds. Fig. 7
shows that the addition of the complexes results in an increase in
the relative viscosity of DNA. As known, the binding of a compound to
DNA grooves via a partial and/or non-classic intercalation may provoke
Flufenamic acid exhibits the highest Kb value among the NSAIDs
(naproxen, sodium diclofenac, mefenamic acid and tolfenamic acid)
studied by our group so far [7,8,25,40]. Additionally, the flufenamato

62
C. Tolia et al. / Journal of Inorganic Biochemistry 123 (2013) 53–65
a bend or kink in the DNA helix subsequently resulting in a slight de-
A
1.2
1.0
0.8
0.6
0.4
0.2
0.0
crease of its effective length; in such a case, the viscosity of a DNA solu-
tion may show a slight decrease or may remain unchanged [88,89]. In
the existence of classic intercalation, an insertion of the compound in
between the DNA base pairs occurs leading to an increase in the separa-
tion of base pairs at intercalation sites in order to host the bound com-
pound; therefore, the increase of the length of the DNA helix increases
and the DNA viscosity exhibits an increase, the magnitude of which is
usually in accordance to the strength of the interaction. In conclusion,
the increase of the DNA viscosity observed upon addition of the com-
pounds may be an evidence of the existence of an intercalative binding
mode between DNA and each complex [8,39,40,88,89].
Ethidium bromide (EB = 3,8-Diamino-5-ethyl-6-phenyl-phenan-
thridinium bromide) is a typical indicator of intercalation [90] due to
the intense fluorescence emission in the presence of CT DNA as a result
of strong intercalation of the planar EB phenanthridine ring between
adjacent base pairs on the double helix. Therefore, the changes observed
in the fluorescence emission spectra of a solution containing EB bound
to CT DNA may be used to study the interaction between DNA and
other compounds, such as metal complexes, since the addition of a
compound, capable to intercalate to DNA equally or more strongly
than EB, should result in a quenching of the DNA-induced EB fluores-
cence emission [91].
The emission spectra of EB bound to CT DNA in the absence and
presence of each compound have been recorded for [EB] = 20 μM,
[DNA] = 26 μM for increasing amounts of the compound. The addi-
tion of Hfluf or complexes 1–5 at diverse r values results in a signifi-
cant decrease of the intensity of the emission band of the DNA–EB
system at 592 nm (the final fluorescence is up to 21–45% of the initial
EB–DNA fluorescence intensity for the complexes) indicating the
competition of the complexes with EB in binding to DNA (Fig. 8).
The observed significant quenching of DNA–EB fluorescence from
the compounds suggests that they can displace EB from the DNA–EB
complex, thus probably interacting with CT DNA by the intercalative
mode [7,8,25,39,40].
240
260
280
300
λ (nm)
B
2.0
1.5
1.0
0.5
0.0
280
300
320
340
360
380
400
λ (nm)
C
0.8
0.6
0.4
0.2
0.0
The Stern–Volmer plots of DNA–EB (Fig. S11) illustrate that the
quenching of EB bound to DNA by the compounds is in good agree-
ment (R = 0.99) with the linear Stern–Volmer equation (Eq. (4))
[7,8,60]. The values (Table 6) of the Stern–Volmer constant (KSV) as
obtained by Stern–Volmer plots of DNA–EB (Fig. S11) show that the
compounds can bind tightly to the DNA [7,8,25,39,40]. Flufenamic
acid and its complexes 1–5 exhibit high KSV values in comparison to
those of the NSAIDs (naproxen, sodium diclofenac, mefenamic acid)
and their corresponding Cu(II) complexes studied by our group so
far [7,8,25].
280
300
320
340
360
380
λ (nm)
3.5. Antioxidant capacity
Fig. 6. (A) UV spectra of CT DNA (9 × 10-5 M) in buffer solution (150 mM NaCl and
15 mM trisodium citrate at pH 7.0) in the absence or presence of [Cu2(fluf)4(DMF)2], 1.
The arrow shows the changes upon increasing amounts of complex. (B) and (C) UV spec-
tra of a DMSO solution (2 × 10-5 M) of (B) [Cu(fluf)2(py)2], 5 and (C) [Cu(fluf)(bipyam)
Cl], 2 in the presence of CT DNA at increasing amounts. The arrows show the changes upon
increasing amounts of CT DNA.
The antioxidant activity of NSAIDs and their complexes may be re-
lated to their potential anticancer and antiinflammatory activity since
free radicals play an important role in the inflammatory process. The
NSAIDs with a broad spectrum of activity can also act either as inhib-
itors of free radical production or as radical scavengers [92]. There-
fore, compounds possessing antioxidant properties could potentially
have a crucial role against inflammation and, thus, lead to potentially
effective drugs. In this context, the potential antioxidant ability of Hfluf
and complexes 1–5 has been evaluated in regard to DPPH, ABTS and
hydroxyl radical scavenging ability and has been compared to that of
well-known antioxidant agents e.g. NDGA, BHT and trolox.
Compounds exhibiting DPPH radical scavenging activity are of in-
creasing interest since DPPH scavenging activity is closely related to
antiageing, anticancer and antiinflammatory activity [61]. Therefore,
these antioxidants may offer protection in rheumatoid arthritis and
inflammation and lead to potentially effective drugs. DPPH is a stable
free radical presenting a strong absorption band at 517 nm. When
antioxidants donate protons to DPPH radical, the initial absorbance
Table 6
The DNA binding constants (Kb) and Stern–Volmer constants (KSV) of EB–DNA fluorescence
for Hfluf and complexes 1–5.
Compound
Κb (M−1
)
Κsv (M−1
)
Hfluf
2.70( 0.11) × 105
9.44( 0.13) × 105
1.73( 0.34) × 106
2.53( 0.08) × 105
2.68( 0.40) × 106
7.80( 0.51) × 105
6.34( 0.30) × 105
5.97( 0.18) × 105
3.71( 0.11) × 105
6.97( 0.21) × 105
10.53( 0.35) × 105
3.35( 0.11) × 105
[Cu2(fluf)4(DMF)2] 1
[Cu(fluf)(bipyam)Cl] 2
[Cu(fluf)(phen)Cl] 3
[Cu(fluf)(bipy)Cl] 4
[Cu(fluf)2(py)2] 5

C. Tolia et al. / Journal of Inorganic Biochemistry 123 (2013) 53–65
63
Table 7
Cathodic and anodic potentials (in mV) for the redox couples of the complexes in DMSO solution and in DMSO/buffer solution in the absence or presence of CT DNA.
a
a
a
a
a
b
c
d
b
c
d
Complex
Εpc1
Epc2
Epa1
Epa2
Epa3
Epc(f)
Epc(b)
ΔΕpc
Epa(f)
Epa(b)
ΔΕpa
[Cu2(fluf)4(DMF)2] 1
[Cu(fluf)(bipyam)Cl] 2
[Cu(fluf)(phen)Cl] 3
[Cu(fluf)(bipy)Cl] 4
[Cu(fluf)2(py)2] 5
−590
−680
−780
−600
−650
−1370
−1200
−1220
−1130
−1210
−80
−550
−550
−390
−620
+210
−45
+35
+30
−45
+465
+350
+410
+450
+435
−736
−743
−719
−737
−788
−715
−725
−660
−722
−769
+21
+18
+59
+15
+19
−510
−512
−516
−489
−390
−528
−525
495
−483
−360
−18
−13
+21
+6
+30
a
Epc/a in DMSO solution.
Epc/a in DMSO/buffer in the absence of CT DNA (Epc/a(f)).
Epc/a in DMSO/buffer in the presence of CT DNA (Epc/a(b)).
ΔEpc/a = Epc/a(b) − Epc/a(f)
b
c
d
.
of DPPH solution decreases. The scavenging activity of all compounds
was not found to be time-dependent since no significant changes
were observed after 20-min and 60-min measurements (Table 8). In
general, the compounds present low to moderate reducing ability of
DPPH radical with the complexes showing higher DPPH radical scav-
enging activity than free flufenamic acid. The complexes present sim-
ilar scavenging activity of the DPPH radical to that found for a series of
copper(II) complexes with the NSAIDs mefenamic acid and naproxen
as ligands [7,39].
radical than free flufenamic acid, with complexes 1 and 3 being the
most active compounds.
The inhibitory activity of the compounds against soybean
lipoxygenase has been also tested. Most LOX inhibitors are antioxi-
dants or free radical scavengers [93] since lipoxygenation occurs via
a carbon centred radical. A relationship between LOX inhibition and
the ability of the inhibitors to reduce the Fe(III) at the active site to
the catalytically inactive Fe(II) has been previously reported. LOXs
contain a ‘non-heme’ iron per molecule in the enzyme active site
as high-spin Fe(III) in the native state and the high-spin in the activated
state Fe(III). Several LOX inhibitors are excellent ligands for Fe(III) and
their mechanism of action is presumably related to its coordination
with a catalytically crucial Fe(III) [93,94]. All compounds present signif-
icant inhibition against soybean lipoxygenase (Table 9), especially in
relation to the reference compound caffeic acid (IC50 = 600 μM), with
3 being the most active compound against LOX (IC50 = 24.73( 0.76)
μM). Complexes 1–5 present similar or better in vitro inhibition of
soybean lipoxygenase than the corresponding Cu(II) mefenamato com-
plexes reported by our lab [7].
In conclusion, the scavenging activity of all the compounds tested
is moderate to high in some cases. Additionally, the complexes exhib-
it slightly higher scavenging activity than free flufenamic acid against
the DPPH, hydroxyl and ABTS radicals. Furthermore, Cu(II), Co(II),
Ni(II), Zn(II) and Pd(II) complexes with drugs or Schiff bases as ligands
exhibited better antioxidant activity than the free corresponding ligands
[31,95–99].
The scavenging activity of the ABTS radical cation (ABTS•+) may
be related to the magnitude of the total antioxidant activity of the
compounds [31]. The ABTS radical scavenging activity of the compounds
was moderate to high in comparison to that of the reference compound
trolox. All complexes show higher scavenging activity of ABTS•+ than
free flufenamic acid, although it is lower than that of trolox. These results
(Table 9) are in agreement to DPPH and hydroxyl radical scavenging
studies revealing that complex 3 exhibits the highest scavenging activity
of the ABTS radical cation among the complexes. Similar scavenging
activity of the ABTS radical has been recorded for Cu(II) mefenamato
and naproxenato complexes [7,39].
Hydroxyl radicals are among the most reactive oxygen species.
During the inflammatory process, the superoxide anion radicals are
generated by phagocytes at the inflamed site and are connected to
other oxidizing species such as •OH [31]. Under these conditions,
hydroxyl radical scavengers could serve as protectors activating the
prostaglandin synthesis. The competition of Hfluf and its Cu(II) com-
plexes with DMSO for OH• generated by the Fe3+/ascorbic acid system
expressed as percent inhibition of formaldehyde production has been
used for the evaluation of their hydroxyl radical scavenging activity
and the results are given in Table 9. All compounds present higher com-
petition with DMSO (33 mM) at 0.1 mM for hydroxyl free radicals than
the reference compound trolox and is similar to previously reported
Cu(II) complexes with mefenamic acid and naproxen [7,39]. Additional-
ly, the complexes present higher competition with DMSO for hydroxyl
4. Conclusions
The synthesis and characterization of the neutral copper(II) com-
plex with the non-steroidal antiinflammatory drug flufenamic acid
100
80
1.30
Hfluf
1
2
3
4
Hfluf
2
4
1
3
5
1.25
60
40
20
0
1.20
5
1.15
1.10
1.05
1.00
0.95
0.0
0.1
0.2
0.3
0.4
r=[compound]/[DNA]
0.00
0.05
0.10
0.15
0.20
0.25
r=[compound]/[DNA]
Fig. 8. Plot of EB-DNA relative fluorescence intensity (%I/Io) at λem = 592 nm vs r
(r = [compound]/[DNA]) in buffer solution (150 mM NaCl and 15 mM trisodium
citrate at pH 7.0) in the presence of Hfluf or complexes 1-5 (quenching up to 33% of
the initial EB-DNA fluorescence for Hfluf, 45% for 1, 38% for 2, 17% for 3, 19% for 4
and 21% for 5).
Fig. 7. Relative viscosity of CT DNA (η/ηo)1/3 in buffer solution (150 mM NaCl and
15 mM trisodium citrate at pH 7.0) in the presence of Hfluf and complexes 1-5 at
increasing amounts (r).

64
C. Tolia et al. / Journal of Inorganic Biochemistry 123 (2013) 53–65
Table 8
against soybean lipoxygenase. Complex 3 is the most active scavenger
of all radicals tested and the most potent LOX inhibitor.
Interaction % with DPPH (RA%).
Compound
20 min, 0.1 mM
60 min, 0.1 mM
Abbreviations
Hfluf
7.36( 0.52)
20.64( 0.91)
13.77( 0.79)
22.46( 0.19)
9.48( 0.31)
12.53( 0.67)
81.02 ( 0.18)
31.30 ( 0.10)
9.74( 0.38)
22.38( 0.72)
14.31( 0.87)
26.52( 0.55)
10.01( 0.36)
11.39( 0.61)
82.60 ( 0.17)
60.00 ( 0.38)
ABTS
2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical
[Cu2(fluf)4(DMF)2], 1
[Cu(fluf)(bipyam)Cl], 2
[Cu(fluf)(phen)Cl], 3
[Cu(fluf)(bipy)Cl], 4
[Cu(fluf)2(py)2], 5
NDGA
cation
BHT
bipy
butylated hydroxytoluene
2,2′-bipyridine
bipyam 2,2′-bipyridylamine
BHT
BSA
COX
CT
bovine serum albumin
cyclo-oxygenase
calf-thymus
in the presence of the O-donor ligand N,N-dimethylformamide or a
N-donor heterocyclic ligand 2,2′-bipyridylamine, 1,10-phenanthroline,
2,2′-bipyridine or pyridine has been achieved. The presence of DMF
results in a dinuclear copper(II) complex with a paddlewheel arrange-
ment while the presence of the N-donor ligands leads to the formation
of mononuclear copper(II) complexes where the flufenamato ligands
are bound via carboxylato oxygen atoms. The crystal structures of the
complexes [Cu2(fluf)4(DMF)2], 1 and [Cu(fluf)(bipyam)Cl], 2 have
been determined by X-ray Crystallography.
Density functional calculations (CAM-B3LYP/LANL2DZ/6-31G**)
were employed to determine the structure of complexes 2–5. A good
agreement between the computed geometry of complex 2 and its crys-
tal structure was found. According to DFT calculations, complexes 3 and
4 have a distorted square pyramidal geometry similar to that of 2, while
complex 5 can be optimized in an octahedral structure as observed for
other related pyridine derivatives [25,28]. Time-dependent DFT calcula-
tions of doublet–doublet transitions have shown that the lowest-energy
band in the absorption spectrum of 2–5 has a mixed d-d/LMCT character.
Flufenamic acid and all complexes studied show good quenching
ability of the BSA and HSA fluorescence and tight binding affinity to
these proteins giving relatively high binding constants.
DFT
DMF
DMSO
DPPH
EB
density function theory
N,N-dimethylformamide
dimethylsulfoxide
1,1-diphenyl-picrylhydrazyl
ethidium bromide, 3,8-diamino-5-ethyl-6-phenyl-phenan-
thridinium bromide
electron density difference map
Gaussian 09
diflunisal
flufenamic acid, N-(3-[trifluoromethyl]phenyl)anthranilic
acid
naproxen
human serum albumin
ligand-to-metal charge transfer
soybean lipoxygenase
sodium diclofenac
nordihydroguaiaretic acid
non-steroidal antiinflammatory drug
1,10-phenathroline
EDDM
G09
Hdifl
Hfluf
Hnap
HSA
LMCT
LOX
Nadicl
NDGA
NSAID
phen
py
pyridine
serum albumin
SA
UV spectroscopy studies and viscosity measurements have revealed
the ability of the complexes to bind to CT DNA. The binding strength of
the complexes with CT DNA calculated with UV spectroscopic titrations
have shown that [Cu(fluf)(bipy)Cl] exhibits the highest Kb value among
the complexes examined, which is even higher than the Kb value of EB.
Competitive binding studies with EB have revealed the ability of the
complexes to displace the typical intercalator EB from the EB–CT DNA
complex indicating intercalation as a possible mode of their interaction
with CT DNA. The intercalative binding mode has been also confirmed
by viscosity measurements of CT DNA solutions in the presence of the
complexes as well as by cyclic voltammetry studies. Additionally, com-
plexes 1–5 exhibit higher Kb values than previously reported Cu(II)
complexes with the NSAIDs mefenamic acid, naproxen and diclofenac
as ligands.
sh
TEAP
Vs
shoulder
tetraethylammonium perchlorate
very strong
Δ
νasym(CO2)–νsym(CO2)
Acknowledgments
This research has been co-financed by the European Union (European
Social Fund-ESF) and Greek national funds through the Operational
Program “Education and Lifelong Learning” of the National Strategic
Reference Framework (NSRF)—Research Funding Program: Archimides
III. L.S. was supported by the MICINN of Spain with the Ramón y Cajal
Fellowship RYC-2011-07787. We thank the members of COST Action
CM1105 for stimulating discussions.
All compounds have been tested in vitro for their antioxidant,
free radical scavenging activity and inhibitory activity on soybean
lipoxygenase. They exhibit low DPPH radical scavenging activity and
significantly high scavenging activity against hydroxyl free radicals
and superoxide radicals. The compounds present significant inhibition
Appendix A. Supplementary material
CCDC-917180 and 917181 contain the supplementary crystallo-
graphic data for this paper. These data can be obtained free of charge
via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge
Crystallographic Data Centre, 12 Union Road, Cambridge CB21EZ, UK;
fax: (+44) 1223-336-033; or deposit@ccde.cam.ac.uk). Supplementary
data associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.jinorgbio.2013.02.009.
Table 9
Competition % with DMSO for hydroxyl radical (.OH%), % superoxide radical scavenging
activity (ABTS%) and in vitro inhibition of soybean lipoxygenase (LOX) (IC50, in μM).
Compound
ABTS% 0.1 mM
.OH% 0.1 mM
LOX IC50 (μM)
Hfluf
64.57( 0.43)
84.61( 0.82)
71.59( 0.79)
89.44( 0.57)
73.46( 1.23)
72.72( 0.60)
91.80( 0.17)
nta
84.51( 0.52)
93.59( 0.33)
83.92( 0.83)
93.71( 0.29)
91.48( 0.82)
82.62( 0.42)
82.80( 0.13)
42.64( 0.43)
27.51( 0.90)
38.62( 0.58)
24.73( 0.76)
39.98( 0.44)
46.09( 0.28)
nta
[Cu2(fluf)4(DMF)2], 1
[Cu(fluf)(bipyam)Cl], 2
[Cu(fluf)(phen)Cl], 3
[Cu(fluf)(bipy)Cl], 4
[Cu(fluf)2(py)2], 5
Trolox
References
[1] G. Crisponi, V.M. Nurchi, D. Fanni, C. Gerosa, S. Nemolato, G. Faa, Coord. Chem.
Rev. 254 (2010) 876–889.
[2] J.E. Weder, C.T. Dillon, T.W. Hambley, B.J. Kennedy, P.A. Lay, J.R. Biffin, H.L. Regtop,
N.M. Davies, Coord. Chem. Rev. 232 (2002) 95–126.
[3] J.A. Drewry, P.T. Gunning, Coord. Chem. Rev. 255 (2011) 459–472.
[4] B.M. Rode, Y. Suwannachot, Coord. Chem. Rev. 190–192 (1999) 1085–1099.
a
Caffeic acid
nt
600 ( 0.3)
a
nt = not tested.

C. Tolia et al. / Journal of Inorganic Biochemistry 123 (2013) 53–65
65
[5] M.E. Katsarou, E.K. Efthimiadou, G. Psomas, A. Karaliota, D. Vourloumis, J. Med.
Chem. 51 (2008) 470–478.
Raghavachari, A. Rendell, J.C. Burant, S.S. Iyengar, J. Tomasi, M. Cossi, N. Rega,
J.M. Millam, M. Klene, J.E. Knox, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R.
Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W.
Ochterski, R.L. Martin, K. Morokuma, V.G. Zakrzewski, G.A. Voth, P. Salvador,
J.J. Dannenberg, S. Dapprich, A.D. Daniels, O. Farkas, J.B. Foresman, J.V. Ortiz, J.
Cioslowski, D.J. Fox, Gaussian 09, Revision B.01, Gaussian, Inc., Wallingford
CT, 2009.
[6] E.K. Efthimiadou, H. Thomadaki, Y. Sanakis, C.P. Raptopoulou, N. Katsaros, A.
Scorilas, A. Karaliota, G. Psomas, J. Inorg. Biochem. 101 (2007) 64–73.
[7] F. Dimiza, S. Fountoulaki, A.N. Papadopoulos, C.A. Kontogiorgis, V. Tangoulis, C.P.
Raptopoulou, V. Psycharis, A. Terzis, D.P. Kessissoglou, G. Psomas, Dalton Trans.
40 (2011) 8555–8568.
[8] F. Dimiza, F. Perdih, V. Tangoulis, I. Turel, D.P. Kessissoglou, G. Psomas, J. Inorg.
Biochem. 105 (2011) 476–489.
[9] E.K. Efthimiadou, Y. Sanakis, M. Katsarou, C.P. Raptopoulou, A. Karaliota, N.
Katsaros, G. Psomas, J. Inorg. Biochem. 100 (2006) 1378–1388.
[10] G. Psomas, A. Tarushi, Y. Sanakis, E.K. Efthimiadou, C.P. Raptopoulou, N. Katsaros,
J. Inorg. Biochem. 100 (2006) 1764–1773.
[11] M. Ruiz, L. Perello, J. Server-Carrio, R. Ortiz, S. Garcia-Granda, M.R. Diaz, E. Canton,
J. Inorg. Biochem. 69 (1998) 231–239.
[12] A.M. Ramadan, J. Inorg. Biochem. 65 (1997) 183–189.
[13] C.P. Duffy, C.J. Elliott, R.A. O'Connor, M.M. Heenan, S. Coyle, I.M. Cleary, K.
Kavanagh, S. Verhaegen, C.M. O'Loughlin, R. NicAmhlaoibh, M. Clynes, Eur. J. Cancer
34 (1998) 1250–1259.
[48] M. Casida, C. Jamorski, K.C. Casida, D.R. Salahub, J. Chem. Phys. 108 (1998) 4439–4449.
[49] R. Stratmann, G.E. Scuseria, M.J. Frisch, J. Chem. Phys. 109 (1998) 8218–8224.
[50] T. Yanai, D. Tew, N. Handy, Chem. Phys. Lett. 393 (2004) 51–57.
[51] P. Hay, W.R. Wadt, J. Chem. Phys. 82 (1985) 270–273.
[52] A.D. McLean, G.S. Chandler, J. Chem. Phys. 72 (1980) 5639–5648.
[53] D.M. York, M. Karplus, J. Phys. Chem. A 103 (1999) 11060–11079.
[54] N.M. O'Boyle, A.L. Tenderholt, K.M. Langner, J. Comput. Chem. 29 (2008) 839–845.
[55] E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt, E.C. Meng,
T.E. Ferrin, J. Comput. Chem. 25 (2004) 1605–1612.
[56] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd ed. Plenum Press, New
York, 2006.
[57] J.R. Lakowicz, G. Weber, Biochemistry 12 (1973) 4161–4170.
[58] S. Wu, W. Yuan, H. Wang, Q. Zhang, M. Liu, K. Yu, J. Inorg. Biochem. 102 (2008)
2026–2034.
[14] A.R. Amin, P. Vyas, M. Attur, J. Leszczynskapiziak, I.R. Patel, G. Weissmann, S.B.
Abramson, Proc. Natl. Acad. Sci. 92 (1995) 7926–7930.
[15] D.H. Woo, I. Han, G. Jung, Life Sci. 75 (2004) 2439–2449.
[16] K. Kim, J. Yoon, J.K. Kim, S.J. Baek, T.E. Eling, W.J. Lee, J. Ryu, J.G. Lee, J. Lee, J. Yoo,
Biochem. Biophys. Res. Commun. 325 (2004) 1298–1303.
[17] W.J. Wechter, E.D. Murray, D. Kantoci, D.D. Quiggle, D.D. Leipold, K.M. Gibson, J.D.
McCracken, Life Sci. 66 (2000) 745–753.
[59] A.M. Pyle, J.P. Rehmann, R. Meshoyrer, C.V. Kumar, N.J. Turro, J.K. Barton, J. Am.
Chem. Soc. 111 (1989) 3053–3063.
[60] G. Psomas, J. Inorg. Biochem. 102 (2008) 1798–1811.
[61] C. Kontogiorgis, D. Hadjipavlou-Litina, J. Enzyme Inhib. Med. Chem. 18 (2003)
63–69.
[18] M.L. Smith, G. Hawcroft, M.A. Hull, Eur. J. Cancer 36 (2000) 664–674.
[19] L. Klampfer, J. Cammenga, H.G. Wisniewski, S.D. Nimer, Blood 93 (1999)
2386–2394.
[20] A. Inoue, S. Muranaka, H. Fujita, T. Kanno, H. Tamai, K. Utsumi, Free Radic. Biol.
Med. 37 (2004) 1290–1299.
[62] R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang, C. Rice-Evans, Free Radic.
Biol. Med. 26 (1999) 1231–1237.
[63] K.R. Prabhakar, V.P. Veerapur, P. Bansal, V. Kumar, M. Reddy, A. Barik, B.K. Reddy,
P. Reddanna, K.I. Priyadarsini, M.K. Unnikrishnan, Bioorg. Med. Chem. 14 (2006)
7113–7120.
[21] M. Barbaric, M. Kralj, M. Marjanovic, I. Husnjak, K. Pavelic, J. Filipovic-Grcic, D.
Zorc, B. Zorc, Eur. J. Med. Chem. 42 (2007) 20–29.
[22] T. Zhang, T. Otevrel, Z.Q. Gao, Z.P. Gao, S.M. Ehrlich, J.Z. Fields, B.M. Boman, Cancer
Res. 61 (2001) 8664–8667.
[23] S. Roy, R. Banerjee, M. Sarkar, J. Inorg. Biochem. 100 (2006) 1320–1331.
[24] D. Kovala-Demertzi, A. Theodorou, M.A. Demertzis, C.P. Raptopoulou, A. Terzis, J.
Inorg. Biochem. 65 (1997) 151–157.
[64] K. Nakamoto, Infrared and Raman Spectra of Inorganic and Coordination Com-
pounds, Part B: Applications in Coordination, Organometallic, and Bioinorganic
Chemistry, 6th ed. Wiley, New Jersey, 2009.
[65] B.J. Hathaway, in: G. Wilkinson (Ed.), Comprehensive Coordination Chemistry,
vol. 5, Pergamon Press, Oxford, 1987, pp. 533–773.
[66] P. Zivec, F. Perdih, I. Turel, G. Giester, G. Psomas, J. Inorg. Biochem. 117 (2012)
35–47.
[25] S. Fountoulaki, F. Perdih, I. Turel, D.P. Kessissoglou, G. Psomas, J. Inorg. Biochem.
105 (2011) 1645–1655.
[26] S. Lorinc, M. Koman, M. Melnik, J. Moncol, D. Ondrusova, Acta Crystallogr. E60
(2004) m590–m592.
[27] C. Dendrinou-Samara, D.P. Kessissoglou, G.E. Manoussakis, D. Mentzafos, A.
Terzis, J. Chem. Soc. Dalton Trans. (1990) 959–965.
[67] B.J. Hathaway, Struct. Bond. (Berlin) 14 (1973) 49–67.
[68] A.W. Addison, T.N. Rao, J. Reedijk, J. van Rijn, G.C. Verchoor, J. Chem. Soc. Dalton
Trans. (1984) 1349–1356.
[69] C. Castellari, G. Feroci, S. Ottani, Acta Crystallogr. C 55 (1999) 907–910.
[70] S. Youngme, K. Chinnakali, S. Chantrapromma, H. Fun, Acta Crystallogr. C55 (1999)
899–901.
[28] A.L. Abuhijleh, J. Inorg. Biochem. 55 (1994) 255–262.
[71] R.E. Marsh, Acta Crystallogr. B61 (2005) 359.
[29] J.E. Weder, T.W. Hambley, B.J. Kennedy, P.A. Lay, D. MacLachlan, R. Bramley, C.D.
Delfs, K.S. Murray, B. Moubaraki, B. Warwick, J.R. Biffin, H.L. Regtop, Inorg.
Chem. 38 (1999) 1736–1744;
[72] Y. Wang, N. Okabe, Inorg. Chim. Acta 358 (2005) 3407–3416.
[73] F. Ugozzoli, A.M.M. Lanfredi, N. Marsich, A. Camus, Inorg. Chim. Acta 256 (1997)
1–7.
P. Kogerler, P.A.M. Williams, B.S. Parajon-Costa, E.J. Baran, L. Lezama, T. Rojo, A.
Muller, Inorg. Chim. Acta 268 (1998) 239–248.
[30] A.E. Underhill, S.A. Bougourd, M.L. Flugge, S.E. Gale, P.S. Gomm, J. Inorg. Biochem.
52 (1993) 139–144.
[74] N. Okabe, A. Tsuji, M. Yodoshi, Acta Crystallogr. E63 (2007) m2162.
[75] N. Okabe, A. Tsuji, M. Yodoshi, Acta Crystallogr. E63 (2007) m2108–m2109.
[76] Y. Wang, G. Lin, J. Hong, T. Lu, L. Li, N. Okabe, M. Odoko, Inorg. Chim. Acta 362
(2009) 377–384.
[31] F. Dimiza, A.N. Papadopoulos, V. Tangoulis, V. Psycharis, C.P. Raptopoulou, D.P.
Kessissoglou, G. Psomas, Dalton Trans. 39 (2010) 4517–4528.
[32] H. Yau, G. Baranauskas, Marco Martina, J. Physiol. 588 (2010) 3869–3882.
[33] Y. Chi, K. Li, Q. Yan, S. Koizumi, L. Shi, S. Takahashi, Y. Zhu, H. Matsue, M. Takeda,
M. Kitamura, J. Yao, J. Pharmacol. Exp. Ther. 339 (2011) 257–266.
[34] F. Pena, M.A. Parkis, A.K. Tryba, J.M. Ramirez, Neuron 43 (2004) 105–117.
[35] P. Poronnik, M.C. Ward, D.I. Cook, FEBS Lett. 296 (1992) 245–248.
[36] M. Ottolia, L. Toro, Biophys. J. 67 (1994) 2272–2279.
[37] G. Facchin, M.H. Torre, E. Kremer, O.E. Piro, E.J. Baran, Z. Anorg. Allg. Chem. 624
(1998) 2025–2028.
[38] A. Tarushi, F. Kastanias, V. Psycharis, C.P. Raptopoulou, G. Psomas, D.P.
Kessissoglou, Inorg. Chem. 51 (2012) 7460–7462.
[39] F. Dimiza, A.N. Papadopoulos, V. Tangoulis, V. Psycharis, C.P. Raptopoulou, D.P.
Kessissoglou, G. Psomas, J. Inorg. Biochem. 107 (2012) 54–64.
[40] S. Tsiliou, L.-A. Kefala, F. Perdih, I. Turel, D.P. Kessissoglou, G. Psomas, Eur. J. Med.
Chem. 48 (2012) 132–142.
[41] A. Tarushi, X. Totta, C. Raptopoulou, V. Psycharis, G. Psomas, D.P. Kessissoglou,
Dalton Trans. 41 (2012) 7082–7091.
[77] N. Okabe, A. Tsuji, M. Yodoshi, Acta Crystallogr. E63 (2007) m1756–m1757.
[78] S. Youngme, A. Cheansirisomboon, C. Danvirutai, N. Chaichit, C. Pakawatchai, G.A.
van Albada, J. Reedijk, Inorg. Chem. Commun. 9 (2006) 973–977.
[79] R. Carballo, B. Covelo, N. Fernandez-Hermida, A.B. Lago, E.M. Vazquez-Lopez, Z.
Anorg. Allg. Chem. 633 (2007) 1791–1795.
[80] C. Tan, J. Liu, H. Li, W. Zheng, S. Shi, L. Chen, L. Ji, J. Inorg. Biochem. 102 (2008)
347–358.
[81] Y. Wang, H. Zhang, G. Zhang, W. Tao, S. Tang, J. Lumin. 126 (2007) 211–218.
[82] V. Rajendiran, R. Karthik, M. Palaniandavar, H. Stoeckli-Evans, V.S. Periasamy,
M.A. Akbarsha, B.S. Srinag, H. Krishnamurthy, Inorg. Chem. 46 (2007) 8208–8221.
[83] Q. Zhang, J. Liu, H. Chao, G. Xue, L. Ji, J. Inorg. Biochem. 83 (2001) 49–55.
[84] E.C. Long, J.K. Barton, Acc. Chem. Res. 23 (1990) 271–273.
[85] A. Jancso, L. Nagy, E. Moldrheim, E. Sletten, J. Chem. Soc. Dalton Trans. (1999)
1587–1594.
[86] G. Pratviel, J. Bernadou, B. Meunier, Adv. Inorg. Chem. 45 (1998) 251–262.
[87] M.T. Carter, M. Rodriguez, A.J. Bard, J. Am. Chem. Soc. 111 (1989) 8901–8911.
[88] J.L. Garcia-Gimenez, M. Gonzalez-Alvarez, M. Liu-Gonzalez, B. Macias, J. Borras, G.
Alzuet, J. Inorg. Biochem. 103 (2009) 923–934.
[42] P. Christofis, M. Katsarou, A. Papakyriakou, Y. Sanakis, N. Katsaros, G. Psomas, J.
Inorg. Biochem. 99 (2005) 2197–2210.
[43] C. Tan, J. Liu, H. Li, W. Zheng, S. Shi, L. Chen, L. Ji, J. Inorg. Biochem. 102 (2008)
347–358.
[44] Rigaku/MSC, CrystalClear, Rigaku/MSC Inc., The Woodlands, Texas, USA, 2005.
[45] G.M. Sheldrick, SHELXS-97: Structure Solving Program, University of Göttingen,
Germany, 1997.
[46] G.M. Sheldrick, SHELXL-97: Crystal Structure Refinement Program, University of
Göttingen, Germany, 1997.
[89] J. Liu, H. Zhang, C. Chen, H. Deng, T. Lu, L. Li, Dalton Trans. (2003) 114–119.
[90] W.D. Wilson, L. Ratmeyer, M. Zhao, L. Strekowski, D. Boykin, Biochemistry 32
(1993) 4098–4104.
[91] S. Dhar, M. Nethaji, A.R. Chakravarty, J. Inorg. Biochem. 99 (2005) 805–812.
[92] R. Cini, G. Giorgi, A. Cinquantini, C. Rossi, M. Sabat, Inorg. Chem. 29 (1990) 5197–5200.
[93] K. Muller, Arch. Pharm. 327 (1994) 3–19.
[94] J. Van der Zee, T.E. Eling, R.P. Mason, Biochemistry 28 (1989) 8363–8367.
[95] S.B. Bukhari, S. Memona, M.M. Tahir, M.I. Bhanger, J. Mol. Struct. 892 (2008)
39–46.
[47] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, G.
Scalmani, V. Barone, B. Mennucci, G.A. Petersson, H. Nakatsuji, M. Caricato, X. Li,
H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L. Sonnenberg, M. Hada, M.
Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao,
H. Nakai, T. Vreven, J.A. Montgomery Jr., J.E. Peralta, F. Ogliaro, M. Bearpark,
J.J. Heyd, E. Brothers, K.N. Kudin, V.N. Staroverov, R. Kobayashi, J. Normand, K.
[96] Z.D. Petrovic, D. Hadjipavlou-Litina, V.P. Petrovic, J. Mol. Liq. 144 (2009) 55–58.
[97] E. Pontiki, D. Hadjipavlou-Litina, A.T. Chaviara, C.A. Bolos, Bioorg. Med. Chem. Lett.
16 (2006) 2234–2237.
[98] Z. Liu, B. Wang, Z. Yang, Y. Li, D. Qin, T. Li, Eur. J. Med. Chem. 44 (2009) 4477–4484.
[99] K.C. Skyrianou, F. Perdih, A.N. Papadopoulos, I. Turel, D.P. Kessissoglou, G. Psomas,
J. Inorg. Biochem. 105 (2011) 1273–1285.